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Acute LPS inhalation in healthy volunteers induces dendritic cell maturation in vivo
Abstract
We have been studying the innate immune response of airways cells of healthy human volunteers to inhaled LPS, a Toll-like receptor 4 (TLR4) ligand, and have shown that macrophage phagocytic capacity is blunted.
Because a primary feature of dendritic cell (DC) maturation is a loss of phagocytic capacity, we sought to determine whether acute LPS inhalation in healthy volunteers promotes DC maturation in vivo.
Phagocytosis (IgG-opsonized zymosan particles) and cell-surface phenotypes were analyzed by flow cytometry of induced sputum cells obtained before and 6 hours after Clinical Center Reference Endotoxin (CCRE; 20,000 EU) inhalation in 9 healthy volunteers.
Neutrophils were elevated in the airways after CCRE inhalation (67% +/- 6% vs 37% +/- 6%; P < .05). Phagocytosis (monocytes, macrophages) was blunted (73%, 46%; P < .05) and negatively correlated with PMN influx ( R = -0.73; P < .05) after CCRE inhalation. GM-CSF and IL-1beta, potent DC maturation agents, were elevated after versus before CCRE inhalation (217 pg/mL +/- 103 pg/mL vs 722 pg/mL +/- 202 pg/mL; 83 pg/mL +/- 24 pg/mL vs 148 pg/mL +/- 37 pg/mL, respectively; P < .05). Markers of DC maturation (CD80, CD86, HLA-DR) were upregulated on monocytes and macrophages ( P < .05), and discrete populations of mature DC were observed ( P < .05) after CCRE inhalation.
Inhaled LPS, directly through TLR4 stimulation of immature DC and/or indirectly through stimulation of GM-CSF and IL-1beta, induces pulmonary DC maturation in vivo . Inhaled LPS may enhance allergic airways responses to air pollution through its ability to induce DC maturation.
... To ensure consistency of responses only papers using CCRE endotoxin (20,000 endotoxin units (equivalent to 2g) for inhaled papers and 4ng/kg for instilled papers) were included. Induced sputum cellular: neutrophils [102,122,152,[180][181][182]; macrophages [102,152,180,182]. For graphs with two x-axes, the parameters displayed on the graph should be read relative to the axis under which they are listed. ...
... To ensure consistency of responses only papers using CCRE endotoxin (20,000 endotoxin units (equivalent to 2g) for inhaled papers and 4ng/kg for instilled papers) were included. Induced sputum cellular: neutrophils [102,122,152,[180][181][182]; macrophages [102,152,180,182]. For graphs with two x-axes, the parameters displayed on the graph should be read relative to the axis under which they are listed. ...
... For example, those listed on the left hand side of the figure should be read against the left-sided x-axis. Induced sputum cytokines: IL-8 [152,180,182]; IL-6 [180][181][182]; TNF- [180,181]. BAL cellular: neutrophils [109,111,113,114,118,183]; macrophages [111,113,114,118]; lymphocytes [113,114]. ...
Inflammation is a key feature in the pathogenesis of sepsis and acute respiratory distress syndrome (ARDS). Sepsis and ARDS continue to be associated with high mortality. A key contributory factor is the rudimentary understanding of the early events in pulmonary and systemic inflammation in humans, which are difficult to study in clinical practice, as they precede the patient's presentation to medical services. Lipopolysaccharide (LPS), a constituent of the outer membrane of Gram-negative bacteria, is a trigger of inflammation and the dysregulated host response in sepsis. Human LPS models deliver a small quantity of LPS to healthy volunteers, triggering an inflammatory response and providing a window to study early inflammation in humans. This allows biological/mechanistic insights to be made and new therapeutic strategies to be tested in a controlled, reproducible environment from a defined point in time. We review the use of human LPS models, focusing on the underlying mechanistic insights that have been gained by studying the response to intravenous and pulmonary LPS challenge. We discuss variables that may influence the response to LPS before considering factors that should be considered when designing future human LPS studies.
... Like LPS challenge, O 3 inhalation induces airway neutrophilia and increases levels of cytokines . In addition, we have reported that following challenge with either O 3 or LPS, sputum monocytes and macrophages have increased expression of a number of important cell surface proteins that mediate innate and acquired immune responses, including CD11b, CD14, CD86, and human leukocyte antigen [HLA]-DR (Alexis et al., , 2005(Alexis et al., , 2008Lay et al., 2007). O 3 and LPS also enhance response to inhaled allergen in allergic subjects (Molfino et al., 1991;Ball, Folinsbee et al., 1996;Jorres et al., 1996;Eldridge and Peden, 2000;Boehlecke et al., 2003;Chen et al., 2004;Schaumann et al., 2008). ...
... The study protocol consisted of 3 consecutive days and is similar to protocols previously employed by our group (Alexis et al., 2005 The first day of each study period was for baseline measurements of spirometry, venipuncture for complete blood count (CBC) analysis, vital signs, symptom score, and induced sputum endpoints. On the second day, baseline spirometry, vital signs, and symptom scores were recorded immediately before inhalation challenge. ...
... The increased PMN response to O 3 relative to LPS noted in our study may be due to a broader range of inflammatory signaling induced by injury to the airway epithelium and its extracellular matrix, such as via the release of hyaluronic acid (Manzanares et al., 2007), which interacts with TLR4 (Taylor et al., 2007;Garantziotis et al., 2009), and through the release of potent neutrophil chemoattractants such as IL-8 and LTB4 (McKinnon et al., 1993;Devlin et al., 1994). Although LPS exposure will activate airway macrophages through its interaction with TLR4, we have not shown that challenge with 20,000 EU CCRE augments levels of IL-8 in induced sputum (Alexis et al., 2005). ...
Ozone and lipopolysaccharide (LPS) are environmental pollutants with adverse health effects noted in both healthy and asthmatic individuals. The authors and others have shown that inhalation of ozone and LPS both induce airway neutrophilia. Based on these similarities, the authors tested the hypothesis that common biological factors determine response to these two different agents. Fifteen healthy, nonasthmatic volunteers underwent a 0.4 part per million ozone exposure for 2 h while performing intermittent moderate exercise. These same subjects underwent an inhaled LPS challenge with 20,000 LPS units of Clinical Center Reference LPS, with a minimum of 1 month separating these two challenge sessions. Induced sputum was obtained 24 h before and 4-6 h after each exposure session. Sputum was assessed for total and differential cell counts and expression of cell surface proteins as measured by flow cytometry. Sputum supernatants were assayed for cytokine concentration. Both ozone and LPS challenge augmented sputum neutrophils and subjects' responses were significantly correlated (R = .73) with each other. Ozone had greater overall influence on cell surface proteins by modifying both monocytes (CD14, human leukocyte antigen [HLA]-DR, CD11b) and macrophages (CD11b, HLA-DR) versus LPS where CD14 and HLA-DR were modified only on monocytes. However, LPS significantly increased interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, with no significant increases seen after ozone challenge. Ozone and LPS exposure in healthy volunteers induce similar neutrophil responses in the airways; however, downstream activation of innate immune responses differ, suggesting that oxidant versus bacterial air pollutants may be mediated by different mechanisms.
... Recent findings in our laboratory and others suggest that exposure of lung phagocytic cells to endotoxin may alter several important phagocyte functions and increase risk for infection. We have found that inhalation of endotoxin by volunteers results in acutely increased expression of HLA-DR and other dendritic cell markers (CD80, CD86) by sputum macrophages, but reduced phagocytic capacity [21,22]. Muehlstedt et al. [23] have shown decreased HLA-DR expression in lung cells prior to acquisition of nosocomial pneumonias. ...
... Our goals were (1) to determine whether functions of cellular host defense response are altered in CF compared to other states of lung infection, and (2) to evaluate in vitro if addition of surfactant proteins can restore these alterations. The specific phagocyte functions to be studied were chosen based on our previous studies of the effects of endotoxin inhalation on sputum phagocytes [21,22,25]. ...
... Bronchoscopies were performed according to our clinical routine as has been described previously [22]. Bronchoalveolar lavage (BAL) was performed in the most affected region, as determined by infiltrates on chest radiograph or by findings seen during bronchoscopy. ...
Recent reports suggest that endotoxin exposure can blunt phagocyte functions. The aim of this study was to examine whether lung phagocytic cells have altered host defense function in young cystic fibrosis (CF) patients, and to explore the contribution of neutrophil elastase (NE) and surfactant proteins to these effects.
BALF cells from CF children (N=12) and disease controls (N=12) were analyzed by flow cytometry for mCD14 and HLA-DR expression and phagocytosis. The effects of exogenous surfactant protein A and D (SP-A,D) and proteases on BALF cells in short term culture were assessed experimentally.
Expression of the surface markers mCD14 and HLA-DR, and phagocytosis, were all blunted on CF phagocytes compared to disease controls (p<0.05). In CF phagocytes, SP-A enhanced both phagocytosis and mCD14 expression (p<0.05). Both CF BALF and NE reduced phagocytosis and expression of mCD14 and HLA-DR (p<0.05) by non-CF phagocytes; the latter effect was attenuated by protease inhibitor.
CF airway phagocytes appear to have altered host defense functions that could contribute to poor bacterial clearance. These impairments can be reproduced by incubation of non-CF cells with NE, while SP-A can partially reverse them. Decreasing protease activity and increasing collectin activity may be beneficial in early CF.
... While any of the components of organic dust could play a role in RSV-associated LRIs, a prospective study of healthy, full-term infants in Buenos Aires found an association between environmental exposure to LPS and the severity of an RSV infection for infants with the TLR4+/− genotype [31]. This genotype is found in approximately 10% of the population and is associated with increased risk of severe RSV disease [32]. The Buenos Aires study found that for this subgroup of infants with the TLR4 polymorphism, exposure to higher levels of LPS, due to being from a low-SES, rural region, provided a protective effect on the severity of RSV bronchiolitis even after adjusting for other risk factors. ...
... The innate immunity senses pathogens and through secretion of cytokines deflects the adaptive immunity into a Th1/Th2/Th17 response. It has been shown that a high-dose endotoxin challenge induces airway neutrophilia and a decrease in phagocytosis by airway monocytes, macrophages, and neutrophils [32]. However, a low-dose LPS challenge primes phagocyte function without inducing airway neutrophilia, appearing to skew airway inflammation in a Th2 direction [34]. ...
Although risk factors for hospitalization from a respiratory syncytial virus (RSV) are well known, RSV lower respiratory tract infections (LRIs) in the community are much less studied or understood, especially in developing countries. In a prospective, cohort study we studied factors predisposing Indonesian infants and children under 5 years of age to developing RSV LRIs. Subjects were enrolled in two cohorts: a birth cohort and a cross-sectional cohort of children <48 months of age. Subjects were visited weekly at home to identify any LRI, using the World Health Organization’s criteria. RSV etiology was determined through analysis of nasal washings by enzyme immunoassay and polymerase chain reaction. Risk factors for the development of the first documented RSV LRI were identified by multivariate analysis using logistic regression and Cox proportional hazard modeling. Of the 2014 children studied, 999 were enrolled within 30 days of birth. There were 149 first episodes of an RSV. Risk factors for an RSV LRI were poverty (p < 0.01), use of kerosene as a cooking fuel (p < 0.05), and household ownership of rabbits and chickens (p < 0.01). Our findings suggested that in a middle-income country such as Indonesia, with a substantial burden of RSV morbidity and mortality, lower socioeconomic status, environmental air quality, and animal exposure are predisposing factors for developing an RSV LRI.
... An alternative less invasive approach is LPS delivery to the lung by inhalation and the assessment of inflammation by analysis of induced sputum or exhaled nitric oxide. This has been done in healthy volunteers [6][7][8][9][10], in subjects with bronchial asthma [11][12][13], and recently in healthy smokers [14]. LPS inhalation has also been used to test the effect of salmeterol [15,16] and to compare the anti-inflammatory potential of a PDE4 inhibitor with a corticosteroid [17]. ...
... The available publications about low-dose LPS inhalation studies (< 5 μg) report either no cellular increases [6] or only minor effects [9]. In one study that showed a clear increase in sputum neutrophils after inhalation of 20,000 Endotoxin Units (i.e. 2 μg) [8], the baseline sputum induction was performed just prior to the LPS challenge, which could have enhanced the neutrophil response [19]. ...
Inhalation of endotoxin (LPS) induces a predominantly neutrophilic airway inflammation and has been used as model to test the anti-inflammatory activity of novel drugs. In the past, a dose exceeding 15–50 μg was generally needed to induce a sufficient inflammatory response. For human studies, regulatory authorities in some countries now request the use of GMP-grade LPS, which is of limited availability. It was therefore the aim of this study to test the effect and reproducibility of a low-dose LPS challenge (20,000 E.U.; 2 μg) using a flow- and volume-controlled inhalation technique to increase LPS deposition.
Two to four weeks after a baseline sputum induction, 12 non-smoking healthy volunteers inhaled LPS on three occasions, separated by at least 4 weeks. To modulate the inflammatory effect of LPS, a 5-day PDE4 inhibitor (Roflumilast) treatment preceded the last challenge. Six hours after each LPS inhalation, sputum induction was performed.
The low-dose LPS inhalation was well tolerated and increased the mean percentage of sputum neutrophils from 25% to 72%. After the second LPS challenge, 62% neutrophils and an increased percentage of monocytes were observed. The LPS induced influx of neutrophils and the cumulative inflammatory response compared with baseline were reproducible. Treatment with Roflumilast for 5 days did not have a significant effect on sputum composition.
The controlled inhalation of 2 μg GMP-grade LPS is sufficient to induce a significant neutrophilic airway inflammation in healthy volunteers. Repeated low-dose LPS challenges potentially result in a small shift of the neutrophil/monocyte ratio; however, the cumulative response is reproducible, enabling the use of this model for “proof-of-concept” studies for anti-inflammatory compounds during early drug development.
Trial registration
Clinicaltrials.gov: NCT01400568
... LPS is a prototypic pathogen associated molecular pattern moiety which activates airway monocytes and macrophages through interaction with cell surface receptors CD14 and TLR4. Inhalation challenge studies of human volunteers demonstrate that LPS induces increased airway neutrophils (PMNs), increased pro-inflammatory cytokine levels (TNF-α, IL-6, IL-1β), increased expression of monocyte or macrophage CD14 and TLR4, and increased oxidative stress, thus serving as a model of a neutrophilic asthma exacerbation [4][5][6][7]. Clinically, neutrophilic airway inflammation is difficult to treat, as neutrophils are relatively corticosteroid resistant [8], and treatment options are limited. Inhaled LPS challenge provides a mechanism to study neutrophil-predominant airways inflammation in early phase clinical studies prior to enrolling diseased populations. ...
... Sample Size Calculations-To estimate sample size, we used previously published data from our previous LPS inhalation studies [5]. LPS induces 66% PMNs (of total cells) in sputum (SD= 19, n=9), compared to baseline samples having a mean PMN content of 37% (SD=19). ...
Epidemiologic studies suggest that dietary vitamin E is an important candidate intervention for asthma. Our group has shown that daily consumption of vitamin E (γ-tocopherol, γT) has anti-inflammatory actions in both rodent and human phase I studies. The objective of this study was to test whether γT supplementation could mitigate a model of neutrophilic airway inflammation in rats and in healthy human volunteers. F344/N rats were randomized to oral gavage with γT versus placebo, followed by intranasal LPS (20μg) challenge. Bronchoalveolar lavage fluid and lung histology were used to assess airway neutrophil recruitment. In a phase IIa clinical study, 13 nonasthmatic subjects completed a double-blinded, placebo-controlled crossover study in which they consumed either a γT-enriched capsule or a sunflower oil placebo capsule. After 7 days of daily supplementation, they underwent an inhaled LPS challenge. Induced sputum was assessed for neutrophils 6 h after inhaled LPS. The effect of γT compared to placebo on airway neutrophils post-LPS was compared using a repeated-measures analysis of variance. In rats, oral γT supplementation significantly reduced tissue infiltration (p<0.05) and accumulation of airway neutrophils (p<0.05) that are elicited by intranasal LPS challenge compared to control rats. In human volunteers, γT treatment significantly decreased induced sputum neutrophils (p=0.03) compared to placebo. Oral supplementation with γT reduced airway neutrophil recruitment in both rat and human models of inhaled LPS challenge. These results suggest that γT is a potential therapeutic candidate for prevention or treatment of neutrophilic airway inflammation in diseased populations.
... Exposure to PM as well as ozone can up-regulate the expression of airway immune cell-surface pheno types such as the major histo compatibility complex class II molecule HLA-DR and costimulatory molecules CD80 and CD86 [for a full definition, see Supplemental Material, Table 1 (doi:10.1289/ ehp.1002543)], all receptors that mediate and promote antigen presentation (Alexis et al. 2005(Alexis et al. , 2010Lay et al. 2007). Moreover, this effect is not restricted to mucosal surfaces of the airways. ...
... Furthermore, and like our findings on endothelial cell dysfunction, this association was enhanced in obese participants, in individuals with elevated HbA1c levels, and in individuals with the GSTM1-null polymorphism. The cell-surface receptors that were modified were up-regulated and reflected an enhanced antigen presentation phenotype, a finding we have reported previously in controlled exposure studies on healthy individuals for other pollutants, such as ozone and endotoxin (Alexis et al. 2005(Alexis et al. , 2008Lay et al. 2007). Relatively few studies have reported an association between exposure to ambient PM and altered innate immune activity on circulating cells in susceptible individuals. ...
Exposure of humans to air pollutants such as ozone and particulate matter (PM) may result in airway and systemic inflammation and altered immune function. One putative mechanism may be through modification of cell-surface costimulatory molecules.
We examined whether changes in expression of costimulatory molecules on circulating cells are associated with ambient levels of fine PM [aerodynamic diameter ≤ 2.5 μm (PM2.5)] in a susceptible population of diabetic individuals.
Twenty subjects were studied for 4 consecutive days. Daily measurements of PM2.5 and meteorologic data were acquired on the rooftop of the exam site. Circulating cell-surface markers that mediate innate immune and inflammatory responses were assessed by flow cytometry on each day. Sensitivity analysis was conducted on glutathione S-transferase M1 (GSTM1) genotype, body mass index, and glycosylated hemoglobin A1c (HbA1c) levels to determine their role as effect modifiers. Data were analyzed using random effects models adjusting for season, weekday, and meteorology.
We found significantly increased monocyte expression (mean fluorescent intensity) of CD80, CD40, CD86, HLA-DR, and CD23 per 10-μg/m3 increase in PM2.5 at 2- to 4-day lag times after exposure. These findings were significantly higher in obese individuals, in individuals with HbA1c > 7%, and in participants who were GSTM1 null.
Exposure to PM2.5 can enhance antigen-presenting cell phenotypes on circulating cells, which may have consequences in the development of allergic or autoimmune diseases. These effects are amplified in diabetic individuals with characteristics that are associated with insulin resistance or with oxidative stress.
... These cell surface receptors include mCD14, CD80, CD86, and human leukocyte antigen (HLA)-DR. Flow cytometry will also be used to better characterize airway inflammatory cell populations such as mature and immature macrophages, newly recruited and longer resident neutrophils, monocytes, and dendritic cells (Alexis et al., 2005; 2000; Lay et al., 2007). There is now notable interest in the public health effect of low-level O 3 exposure (≤0.08 ppm). ...
... This study extends these findings by demonstrating that even very low concentrations of O 3 can alter monocyte phenotype. We have made similar observations in healthy and allergic volunteers in airway sputum recovered after inhalation challenge with 20,000 endotoxin units (Alexis et al., 2005Alexis et al., , 2008a). These surface markers are essential to modulating key elements of both innate and acquired immunity, and may play a role in antigen presentation. ...
The effects of low-level ozone exposure (0.08 ppm) on pulmonary function in healthy young adults are well known; however, much less is known about the inflammatory and immunomodulatory effects of low-level ozone in the airways. Techniques such as induced sputum and flow cytometry make it possible to examine airways inflammatory responses and changes in immune cell surface phenotypes following low-level ozone exposure. The purpose of this study was to determine if exposure to 0.08 parts per million ozone for 6.6 h induces inflammation and modifies immune cell surface phenotypes in the airways of healthy adult subjects. Fifteen normal volunteers underwent an established 0.08 part per million ozone exposure protocol to characterize the effect of ozone on airways inflammation and immune cell surface phenotypes. Induced sputum and flow cytometry were used to assess these endpoints 24 h before and 18 h after exposure. The results showed that exposure to 0.08 ppm ozone for 6.6 h induced increased airway neutrophils, monocytes, and dendritic cells and modified the expression of CD14, HLA-DR, CD80, and CD86 on monocytes 18 h following exposure. Exposure to 0.08 parts per million ozone is associated with increased airways inflammation and promotion of antigen-presenting cell phenotypes 18 hours following exposure. These findings need to be replicated in a similar experiment that includes a control air exposure.
... Chan et al. for example, reported that short-term exposure of murine bone marrow derived DC to diesel exhaust PM increases expression of CD86 [14], de Haar et al. reported increased expression of CD86 and CD80 on DC from lymph nodes in mice after intranasal instillation of ultrafine carbon black P [11], and Ferry et al. reported increased expression of CD86 on human monocyte derived DCs incubated with combustion-derived PM [15]. The capacity of pollutants to stimulate DC maturation is not limited to PM since Alexis et al. reported increased expression of CD80 in response to either LPS or ozone [16,17]. Most recently Pfeffer et al. reported increased expression of CD83 (an alternative maturation marker) and subsequent enhanced priming of CD8 T lymphocytes by ultrafine PM exposed human DCs [18]. ...
Urban particulate matter (PM) enhances airway dendritic cell (DC) maturation in vitro. However, to date, there are no data on the association between exposure to urban PM and DC maturation in vivo. We sought to determine whether exposure of school-age children (8 to 14 y) to PM was associated with expression of CD86, a marker of maturation of airway conventional DCs (cDC). Healthy London school children underwent spirometry and sputum induction. Flow cytometry was used to identify CD86 and CCR7 expression on cDC subsets (CD1c+ cDC2 and CD141+ cDC1). Tertiles of mean annual exposure to PM ≤ 10 microns (PM10) at the school address were determined using the London Air Quality Toolkit model. Tertiles of exposure from the 409 children from 19 schools recruited were; lower (23.1 to 25.6 μg/m³, n = 138), middle (25.6 to 26.8 μg/m³, n = 126), and upper (26.8 to 31.0 μg/m³, n = 145). DC expression was assessed in 164/370 (44%) children who completed sputum induction. The proportion (%) of cDC expressing CD86 in the lower exposure tertile (n = 47) was lower compared with the upper exposure tertile (n = 49); (52% (44 to 70%) vs 66% (51 to 82%), p<0.05). There was a higher percentage of cDC1 cells in the lower tertile of exposure (6.63% (2.48 to 11.64) vs. 2.63% (0.72 to 7.18), p<0.05). Additionally; children in the lower exposure tertile had increased FEV1 compared with children in the upper tertile; (median z-score 0.15 (-0.59 to 0.75) vs. -0.21 (-0.86 to 0.48), p<0.05. Our data reveal that children attending schools in the highest areas of PM exposure in London exhibit increased numbers of “mature” airway cDCs, as evidenced by their expression of the surface marker CD86. This data is supportive of previous in vitro data demonstrating an alteration in the maturation of airway cDCs in response to exposure to pollutants.
... 6 7 Systemic exposure to LPS causes transient fever and increased levels of circulating neutrophils and inflammatory mediators. [8][9][10][11] Furthermore, inhaled LPS has been shown to increase neutrophil numbers in sputum by 22% 6 hours post challenge. 12 Hence LPS administration to the lung provides a useful tool to induce neutrophil recruitment in an experimental setting. ...
Rationale:
There is a need to develop imaging protocols which assess neutrophilic inflammation in the lung.
Aim:
To quantify whole lung neutrophil accumulation in (1) healthy volunteers (HV) following inhaled lipopolysaccharide (LPS) or saline and (2) patients with COPD using radiolabelled autologous neutrophils and single-photon emission computed tomography/CT (SPECT/CT).
Methods:
20 patients with COPD (Global initiative for chronic obstructive lung disease (GOLD) stages 2-3) and 18 HVs were studied. HVs received inhaled saline (n=6) or LPS (50 µg, n=12) prior to the injection of radiolabelled cells. Neutrophils were isolated using dextran sedimentation and Percoll plasma gradients and labelled with 99mTechnetium (Tc)-hexamethylpropyleneamine oxime. SPECT was performed over the thorax/upper abdomen at 45 min, 2 hours, 4 hours and 6 hours. Circulating biomarkers were measured prechallenge and post challenge. Blood neutrophil clearance in the lung was determined using Patlak-Rutland graphical analysis.
Results:
There was increased accumulation of 99mTc-neutrophils in the lungs of patients with COPD and LPS-challenged subjects compared with saline-challenged subjects (saline: 0.0006±0.0003 mL/min/mL lung blood distribution volume [mean ±1 SD]; COPD: 0.0022±0.0010 mL/min/mL [p<0.001]; LPS: 0.0025±0.0008 mL/min/mL [p<0.001]). The accumulation of labelled neutrophils in 10 patients with COPD who underwent repeat radiolabelling/imaging 7-10 days later was highly reproducible (0.0022±0.0010 mL/min/mL vs 0.0023±0.0009 mL/min/mL). Baseline interleukin (IL)-6 levels in patients with COPD were elevated compared with HVs (1.5±1.06 pg/mL [mean ±1 SD] vs 0.4±0.24 pg/mL). LPS challenge increased the circulating IL-6 levels (7.5±2.72 pg/mL) 9 hours post challenge.
Conclusions:
This study shows the ability to quantify 'whole lung' neutrophil accumulation in HVs following LPS inhalation and in subjects with COPD using autologous radiolabelled neutrophils and SPECT/CT imaging. Moreover, the reproducibility observed supports the feasibility of using this approach to determine the efficacy of therapeutic agents aimed at altering neutrophil migration to the lungs.
... If the latter, what would an inhaled vaccine look like and what regulatory issues would need to be addressed to assure that these approaches are safe. We have a breadth of human toxicology data with inhaled LPS that low doses are tolerated well 77 and thus the concept of inhaled vaccines may be achievable in a safe and effective manner. Another challenge to the field is actually measuring these type of immune responses. ...
IL‐17 producing cells play a critical role in mucosal immunity including the respiratory tract. This review will highlight recent advances in our understanding of these cells in mucosal immunity in the lung as well as their potential pathogenic roles in respiratory diseases. IL‐17 producing cells include γδ T‐cells, NK cells, group 3 ILCs, and Th17s. There have been recent advances of understanding these cell populations in the lung as well as emerging data on how these cells are regulated in the lung. Moreover, Th17 cells may be a key component of tissue resident memory cells that may be acquired over time or elicited by mucosal immunization that provide the host with enhanced immunity against certain pathogens.
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... [48][49][50][51][52] Airborne endotoxins have been found to decrease lung function and augment an inflammatory reaction in vivo. [53][54][55] However, there is also evidence to ...
To evaluate the association between prenatal and postnatal exposure to pet ownership and lung function in children, a cross-sectional study named Seven Northeastern Cities (SNEC) study was conducted. In this study, children's lung function including the forced expiratory volume in 1 second (FEV1 ), forced vital capacity (FVC), maximal mid-expiratory flow (MMEF), peak expiratory flow (PEF) were measured by spirometers, and pet ownership situations was collected by questionnaire. Analyzed by multiple logistic regression and generalized linear modeling, we found that for all subjects, pet exposure in the first 2 years of life was significantly associated with lung function impairment of FVC<85% predicted (adjusted odds ratio [aOR]=1.28; 95% confidence interval [CI]=1.01, 1.63). For current pet exposure, the increased odds of lung function impairment ranged from 35% (aOR=1.35; 95% CI=1.12, 1.62) for FVC<85% predicted to 57% (aOR=1.57; 95%CI=1.29, 1.93) for FEV1 <85% predicted. The in utero exposure was not related to lung function impairment. Compared with other pets, higher odds were observed among children with dogs. When stratified by gender, girls with current pet exposure were more likely to have lung function impairment than boys. It implies self-reported exposures to pets were negatively associated with lung function among the children under study. This article is protected by copyright. All rights reserved.
... During the development of host responses against pathogens, monocytes are recruited to the site of infection where they differentiate into effector cells amidst crosstalk with resident tissue immune cells (35,36). After encountering an antigen, immature DCs begin their maturation process, migrate to local draining lymph nodes where they present antigen to naïve T lymphocytes to initiate, or perpetuate antigen-specific immune responses (37,38). γδ T lymphocytes are resident sentinel cells in a variety of mucosal surfaces but especially in the ileum, where infection with Map is generally assumed to initially occur (39). ...
During early Mycobacterium avium subspecies paratuberculosis (Map) infection, complex interactions occur between the bacteria, cells from the mononuclear phagocyte system (MPS) including both resident (macrophages and dendritic cells) and recruited (monocytes) cells, and other mucosal sentinel cells such as γδ T lymphocytes. Though the details of early host–pathogen interactions in cattle remain largely underexplored, our hypothesis is that these significantly influence development of host immunity and ultimate success or failure of the host to respond to Map infection. The aims of the present study were to first characterize monocyte-derived MPS cells from young calves with respect to their immunophenotype and function. Then, we set out to investigate the effects of WC1⁺ and WC1neg γδ T lymphocytes on (1) the differentiation of autologous monocytes and (2) the maturation of autologous monocyte-derived dendritic cells (MDDCs). To achieve this, peripheral blood WC1⁺ or WC1neg γδ T lymphocytes were cocultured with either autologous freshly isolated peripheral blood-derived monocytes or autologous immature MDDCs (iMDDCs). We began by measuring several markers of interest on MPS cells. Useful markers to distinguish monocyte-derived macrophages (MDMs) from MDDCs include CD11b, CD163, and CD172a, which are expressed significantly higher on MDMs compared with MDDCs. Function, but not phenotype, was influenced by WC1neg γδ T lymphocytes: viability of Map harvested from monocytes differentiated in the presence of WC1neg γδ T lymphocytes (dMonWC1neg) was significantly lower compared to MDMs and MDDCs. With respect to DC maturation, we first showed that mature MDDCs (mMDDCs) have significantly higher expression of CD11c, CD80, and CD86 compared with iMDDCs, and the phagocytic capacity of mMDDCs is significantly reduced compared to iMDDCs. We then showed that γδ T lymphocyte subsets induce functional (reduced phagocytosis) but not phenotypic (surface marker expression) iMDDC maturation. These data collectively show that γδ T lymphocytes influence differentiation, maturation, and ultimately the function of monocytes during Map infection, which has significant implications on survival of Map and success of host defense during early Map infection.
... [48][49][50][51][52] Airborne endotoxins have been found to decrease lung function and augment an inflammatory reaction in vivo. [53][54][55] However, there is also evidence to ...
Background:
Little information exists on whether gender or asthma status modifies the effects of secondhand smoke (SHS) exposure on lung function.
Objective:
To evaluate whether gender or asthma status modifies the association of SHS exposure with lung function.
Methods:
A total of 6,740 children (average 11.6 years) were recruited from 24 districts of 7 cities in northeast China in 2012. SHS exposure included exposure to environmental and maternal smoking both in utero and during early childhood (postnatal). Lung function was measured using electronic spirometers. Two-step regressions were used to analyze the association between SHS and lung function.
Results:
In utero and postnatal exposure to SHS was independently associated with decreased lung function in both genders; however, this association was greater among males. For example, when exposed to maternal smoking during pregnancy, the adjusted odds ratio (aOR) for decreased forced vital capacity (FVC) was 6.46 (95% confidence interval [CI]: 2.58-16.17) among males, while only 2.16 (95% CI: 0.96-4.88) among females. More positive associations between SHS exposure and decreased lung function were detected among nonasthmatic compared with asthmatic children. Nonasthmatics had significantly larger deficits from in utero exposure to maternal smoking, which concerned decreased lung FVC function (aOR = 2.58, 95% CI: 1.28-5.21) and decreased lung forced expiratory volume in 1 s (FEV1) function (aOR = 2.32, 95% CI: 1.01-5.33). A similar pattern was also observed for the associations between SHS exposure and continuous pulmonary function test measurements.
Conclusions:
SHS exposure was associated with decreased lung function. Males and nonasthmatics seem to be more susceptible than their respective counterparts.
... LPS is a prototypic pathogen-associated molecular pattern moiety that activates airway monocytes and macrophages. Inhalation challenge studies of human volunteers demonstrate that LPS induces increased PMNs, proinflammatory cytokine levels and oxidative stress, thus serving as a model of ALI (Alexis et al., 2005(Alexis et al., , 2008Hernandez et al., 2010Hernandez et al., , 2012. Riboflavin (vitamin B2) is a central component of the cofactors FAD and FMN and hence is required for a wide variety of cellular processes (Hung et al., 2006;Lago & Kaplan, 1981). ...
Riboflavin (vitamin B2) is an easily absorbed micronutrient with a key role in maintaining health in humans and animals. It is the central component of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and is therefore required by all flavoproteins. Riboflavin also works as an antioxidant by scavenging free radicals. The present study was designed to evaluate the effects of riboflavin against acute lungs injury induced by the administration of a single intranasal dose (20 μg/rat) of lipopolysaccharides (LPS) in experimental rats. Administration of LPS resulted in marked increase in malondialdehyde (MDA) level (p < 0.01) and MPO activity (p < 0.001), whereas marked decrease in glutathione (GSH) content (p < 0.001), glutathione reductase (GR) (p < 0.001) and glutathione peroxidase (p < 0.01) activity. These changes were significantly (p < 0.001) improved by treatment with riboflavin in a dose-dependent manner (30 and 100 mg/kg, respectively). Riboflavin (100 mg/kg, p.o.) showed similar protective effects as dexamethasone (1 mg/kg, p.o.). Administration of LPS showed marked cellular changes including interstitial edema, hemorrhage, infiltration of PMNs, etc., which were reversed by riboflavin administration. Histopathological examinations showed normal morphological structures of lungs tissue in the control group. These biochemical and histopathological examination were appended with iNOS and CAT gene expression. The iNOS mRNA expression was increased significantly (p < 0.001) and levels of CAT mRNA expression was decreased significantly (p < 0.001) in the animals exposed to LPS, while treatment with riboflavin significantly (p < 0.01) improved expression of both gene. In conclusion, the present study clearly demonstrated that riboflavin caused a protective effect against LPS-induced ALI. These results suggest that riboflavin may be used to protect against toxic effect of LPS in lungs.
... The induction or inhibition of DC maturation is a substantial immune regulatory principle. Promotion of DC maturation leads to enhancement of adaptive immunity whereas its inhibition results in suppression of specific immunity with a simultaneous enhancement of the innate scavenging functions by macrophages without the induction of a specific immune reaction (Alexis et al, 2005;Allavena et al, 1998;Corinti et al, 2001;Fiorentino et al, 1991;Fortsch et al, 2000;Steinbrink et al, 1997;Zeyda et al, 2005). Figure 3 shows the differentiation and maturation process of myeloid dendritic cells from blood monocytes in vitro. ...
Die Hämoxygenase 1 (HO-1) ist ein Stressprotein mit antientzündlichen, immunsupprimierenden und zytoprotektiven Eigenschaften, welche in vielen Tiermodellen nachgewiesen wurden. Die zugrunde liegenden Mechanismen sind wenig bekannt. Diese Arbeit demonstriert erstmalig, dass die physiologische Induktion von HO-1 wichtig für die Limitierung von T-Zell-abhängigen Hautentzündungen ist. So führt der HO-1-Inhibitor, Zinn-Protoporphyrin IX (Sn-PPIX), zu einer verstärkten Hautentzündung im Mausmodell. Die pharmakologische Induktion von HO-1 durch Kobalt-Protoporphyrin IX, Co-PPIX, hemmt dagegen die Entzündung in DNFB- bzw. TMA-induzierten murinen Kontaktallergiemodellen sowohl bei Verabreichung von Co-PPIX während der Sensibilisierung als auch vor der Auslösung. Bemerkenswerterweise hemmt eine Co-PPIX-Behandlung die Antigen-induzierte T-Zellproliferation ex vivo in Milzzellen von behandelten Mäusen und in vitro in humanen mononukleären Zellen des peripheren Blutes. Da eine HO-1-Induktion durch Co-PPIX nur in Monozyten und in aus Monozyten abgeleiteten myloischen Dendritischen Zellen (MDDC), nicht aber in T-Zellen, beobachtet wurde, fokussierten alle weiteren Untersuchungen auf Antigen-präsentierende Zellen. HO-1-Induktion durch Co-PPIX reduziert die Expression von MHC-Klasse II und akzessorischen Molekülen und steigert die Phagozytose und den oxidativen Burst von Monozyten. Die immunphänotypische Differenzierung und Maturierung von MDDC wird gehemmt. Funktionsteste zeigen eine Reduktion der Expression und Sekretion von proinflammatorischen und immunstimulatorischen Zytokinen, während die Sekretion des antientzündlichen Zytokins IL-10 gesteigert ist. Die Fähigkeit der MDDC zur Antigenpräsentation gegenüber T-Helferzellen ist für Allo- und Recallantigene stark herabgesetzt. Mittels adenoviraler HO-1-Transduktion von MDDC konnte die Spezifität der Effekte bestätigt werden. Diese Daten zeigen, dass eine verstärkte HO-1-Aktivität die Dendritischen Zellen zu einem unreifen und immunkompromittierten Phänotyp verändert und weisen darauf hin, dass die HO-1-Induktion einen wichtigen Ansatz für die Hemmung der zellulären Immunität und für die Behandlung von T-Zell-abhängigen Hautentzündungen darstellt.
... Une autre étude a rapporté que sur des cellules de souris, le LPS peut induire une activité sécrétrice de cytokines, mais pas une maturation en activatrice du lymphocyte T efficace [Nolte et al. , 2007]. Enfin, une étude menée in vivo sur patients sains a montré que la respiration de hautes concentrations de LPS induisait une maturation des cellules dendritiques [Alexis et al. , 2005]. ...
T Lymphocyte activation is a crucial event in immune responses, since it is the first and necessary step for the initiation of adaptative responses. During this activation process, the T lymphocyte forms a contact with myeloid antigen presenting cells, the only cells able to trigger the engagement of the T cell receptor. While myeloid antigen presenting cells have various shape and sizes, T cell activation requires cytoskeleton remodeling. Besides, it has been shown that T lymphocytes may exert forces on antigen presenting cells, and that T cell signaling can be triggered after purely mechanical sollicitation of the TCR : these are all clues for T cell mecanosensitivity during activation. We thus took advantage of the emerging field of cell biomechanics, for the understanding of these phenomena. We first measured the mechanical properties of antigen presenting cells, and their mo- dification upon inflammatory treatments. We discovered that the presenting cells rigidity span over a very wide range, and that it can be affected by inflammatory conditions. The evolution of rigidity upon inflammation was independent of the evolution of maturation molecules at the cell surface. We therefore identified rigidity as a new marker of inflammation. Following these results, we conducted experiments in order to decipher the underlying molecular mechanisms to these varying rigidities. We observed that while the composition of each cell type in cytoskeleton proteins is very variable, it re- mains stable throughout inflammatory treatments. This suggests that the differentiation in a myeloid type implies deep structural modifications, that are likely to be coupled to functional changes. Last, we developped a protocol for the fabrication of soft gels of controled rigidity. We then used these gels for T lymphocyte activation on substrates of varying rigidity, and the preliminary results of these experiments are presented. Overall, by providing the rigidity range to which T lymphocyte is exposed during the interaction with myeloid antigen presenting cells, this study establishes a solid basis for further investigation of T lymphocyte mecanosensitivity.
... In healthy populations, increases in neutrophils, myeloperoxidase, GM-CSF, IL-1B, IL-6, and methylhistamine have been observed when subjects were exposed to DE plus O 3 , 366 DE alone, 367 O 3 alone, 368 and endotoxin. 369 Only one study of a healthy population, in which subjects cycled at both high and low pollution locations and total and differential cell counts were obtained, failed to find positive associations with this end point. 201 Thus, the majority of studies utilizing IS in relation to air pollutant exposures have found increases in inflammatory-related proteins and cells. ...
Human exposure studies, compared with cell and animal models, are heavily relied upon to study the associations between health effects in humans and air pollutant inhalation. Human studies vary in exposure methodology, with some work conducted in controlled settings, whereas other studies are conducted in ambient environments. Human studies can also vary in the health metrics explored, as there exists a myriad of health effect end points commonly measured. In this review, we compiled mini reviews of the most commonly used noninvasive health effect end points that are suitable for panel studies of air pollution, broken into cardiovascular end points, respiratory end points, and biomarkers of effect from biological specimens. Pertinent information regarding each health end point and the suggested methods for mobile collection in the field are assessed. In addition, the clinical implications for each health end point are summarized, along with the factors identified that can modify each measurement. Finally, the important research findings regarding each health end point and air pollutant exposures were reviewed. It appeared that most of the adverse health effects end points explored were found to positively correlate with pollutant levels, although differences in study design, pollutants measured, and study population were found to influence the magnitude of these effects. Thus, this review is intended to act as a guide for researchers interested in conducting human exposure studies of air pollutants while in the field, although there can be a wider application for using these end points in many epidemiological study designs.Journal of Exposure Science and Environmental Epidemiology advance online publication, 21 January 2015; doi:10.1038/jes.2014.93.
... LPS is a pathogen-associated molecular pattern moiety which activates airway monocytes and macrophages through interaction with cell surface receptors CD14 and toll-like receptor (TLR)-4. Inhalation challenge studies of human volunteers demonstrate that LPS induces increased airway neutrophils, increased pro-inflammatory cytokine levels (tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β), increased expression of monocyte or macrophage CD14 and TLR-4, and increased oxidative stress, thus serving as a model of a neutrophilic asthma exacerbation (4)(5)(6)(7). Inhaled LPS challenge provides a mechanism to study neutrophil-predominant airways inflammation in early phase clinical studies prior to enrolling diseased populations. ...
Background:
Asthma with neutrophil predominance is challenging to treat with corticosteroids. Novel treatment options for asthma include those that target innate immune activity. Recent literature has indicated a significant role for IL-1β in both acute and chronic neutrophilic asthma.
Objective:
This study used inhaled endotoxin (LPS) challenge as a model of innate immune activation to (1) assess the safety of the IL-1 receptor antagonist anakinra in conjunction with inhaled LPS and (2) to test the hypothesis that IL-1 blockade will suppress the acute neutrophil response to challenge with inhaled LPS.
Methods:
In a phase I clinical study 17 healthy volunteers completed a double-blind, placebo-controlled crossover study in which they received 2 daily subcutaneous doses of 1 mg/kg anakinra (maximum dose, 100 mg) or saline (placebo). One hour after the second treatment dose, subjects underwent an inhaled LPS challenge. Induced sputum was assessed for neutrophils 4 hours after inhaled LPS. The effect of anakinra compared with placebo on airway neutrophil counts and airway proinflammatory cytokine levels after LPS challenge was compared by using a linear mixed-model approach.
Results:
Anakinra pretreatment significantly diminished airway neutrophilia compared with placebo. LPS-induced IL-1β, IL-6, and IL-8 levels were significantly reduced during the anakinra treatment period compared with those seen after placebo. Subjects tolerated the anakinra treatment well without an increased frequency of infections attributable to anakinra treatment.
Conclusions:
Anakinra effectively reduced airway neutrophilic inflammation and resulted in no serious adverse events in a model of inhaled LPS challenge. Anakinra is a potential therapeutic candidate for treatment of asthma with neutrophil predominance in diseased populations.
... Lipopolysaccharide inhalation was shown to induce dendritic cell maturation in human airways in vivo mediated by Toll-like receptor 4 (TLR4) stimulation (Schnare et al., 2006) or through stimulation of IL-1β, tumor necrosis factor alpha (TNFα; Ning et al., 2000), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Consequently an allergic response or asthma may be initiated or potentiated (Alexis et al., 2005). concluded that exposure to endotoxins and fungal spores may have a protective effect on atopic asthma but may induce nonatopic asthma in farmers. ...
Endotoxin has been identified as important component of organic-dust exposure and is suspected as main cause of work-related adverse health effects in dusty areas. Although the determination of endotoxin levels by using the Limulus amoebocyte lysate (LAL) assay is internationally accepted, reliability and variation of values measured with this test remain a point of discussion. Therefore, the purpose of the study was to determine the influence of different parameters on endotoxin activity measured in airborne samples. This study thus analyzed: (a) dust filter extraction procedures, (b) storage of samples, (c) usage of different commercially available LAL assays, and (d) results of the whole blood assay (WBA) compared to the LAL test. Using a parallel sampler, 120 filters were loaded with dust at 4 different occupational settings and extracted in 2 labs using a standardized protocol. Parameters like Tween in the extraction medium, extraction volume, centrifugation speed, and material of tubes used for extraction were tested. The LAL test and the WBA were able to determine the differences in dust load of filters obtained from the settings investigated. In addition, results varied significantly with modifications in extraction procedures. Using Tween for filter extraction mainly influenced the resulting endotoxin activity. In addition, LAL test differences according to manufacturer of LAL test, extraction volume, and whether the samples are freshly processed or frozen also resulted in significant variations in the endotoxin levels. In conclusion, a reliable assessment of exposure to endotoxin activity is only possible if standard operation procedures (SOPs) for sampling and determination are established.
... Thus, the process of maturation of DCs is very important to initiate antitumor T cell responses. Though DC maturation can be induced by numerous pathogen-related molecules including LPS, CpG DNA, and inflammatory cytokines such as prostaglandins (Alexis et al., 2005;Hartmann, Weiner, & Krieg, 1999;Steinbrink et al., 2000), their clinical usages were limited due to the detrimental adverse effects (Holmgren, Czerkinsky, Eriksson, & Mharandi, 2003;Thiemermann, Ruetten, Wu, & Vane, 1995). In this regard, it is very important to find new DC activators having no side effects. ...
Cultivation of Cordyceps fungi is now the major means for production of Cordyceps materials. UM01 is a novel fungal strain isolated from natural Cordyceps sinensis. In this study, the chemical characters and the immunomodulatory activities of polysaccharides from cultured mycelia of UM01 (UM01 PS) were investigated. Chemical studies showed that UM01 PS had a wide MW distribution and they mainly contained glucose, mannose and galactose. (1→6)-α-D-glucosidic linkages were found in UM01 PS. Pharmacological results showed that UM01 PS promoted mouse RAW 264.7 macrophages proliferation, phagocytic activity, release of NO and multiple cytokines and chemokines. UM01 PS not only induced differentiation of RAW 264.7 macrophages into dendritic-like cells, but also promoted phenotypic and functional maturation of mouse JAWS II dendritic cells. UM01 PS has the potential to activate macrophage and dendritic cell functions. They might be explored as novel immunomodulatory foods as substitute for natural C. sinensis.
... Muller et al. [9] reported that short-term exposure of healthy young patients to organic dust in composting plants had a mild but measurable effect (increased blood neutrophilia) in eliciting an acute systemic alteration. In addition, the increase in allergic reactions was positively correlated to the composition of the dust and the duration of exposure [10][11][12][13][14]. ...
The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 102 conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5-10 μm) having higher endotoxin levels than did fine particles (0.5-2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers.
... Despite the longstanding knowledge and understanding of the adverse effects of O3 on pulmonary biology, the use of O3 as a challenge model to assess the potential of new drugs for the prevention of acute exacerbations in COPD, is relatively new. The model has been shown to be safe and to have few side effects in healthy volunteers, and in patients with asthma and COPD.24,25,28,66 Additionally, it is reliable and reproducible. ...
Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality. Current therapies confer partial benefits either by incompletely improving airflow limitation or by reducing acute exacerbations, hence new therapies are desirable. In the absence of robust early predictors of clinical efficacy, the potential success of novel therapeutic agents in COPD will not entirely be known until the drugs enter relatively large and costly clinical trials. New predictive models in humans, and new study designs are being sought to allow for confirmation of pharmacodynamic and potentially clinically meaningful effects in early development. This review focuses on human challenge models with lipopolysaccharide endotoxin, ozone, and rhinovirus, in the early clinical development phases of novel therapeutic agents for the treatment and reduction of exacerbations in COPD.
... The indication of a more antigen presentation phenotype of the macrophages (elevated level of CD86) 22 in the present study resemble patterns observed after LPS inhalation challenge. 8 After 6 h of inhaled LPS, markers of dendritic cell maturation were upregulated and discrete populations of mature dendritic cell were observed. It is also shown that the differentiation from monocytes to immature dendritic cells is driven by nonendotoxin components in organic dust, in particular grampositive bacteria. ...
Bacterial single-cell protein (BSCP) constitutes of dried bacterial mass which is used as protein enrichment in animal and fish feed. In the production of BSCP, workers are exposed to organic dust containing high levels of endotoxins (arithmetic mean 390 EU/m(3) in the moderate exposure and 5800-11 000 EU/m(3) in the high exposure groups) and have elevated levels of sputum neutrophils and cytokines associated with exposure.
The aim of the present study was to investigate if airway inflammation among the workers had declined 1 year after cessation of exposure.
Twenty-four non-smoking production workers (age 28-52) with a work history of 2-7 years were included in the study.
Markers of airways inflammation and innate immune function (using flow cytometry) were assessed in an exposure period and 1 year after cessation of exposure.
Sputum neutrophil proportion and numbers were significantly decreased 1 year after cessation of exposure to BSCP (43% vs 71%, 186 vs 598 neutrophils/mg sputum; p<0.001) as were eNO (17 ppb vs 21 ppb (p=0.01) and interleukin-1β (p<0.05) and interleukin-8 (p<0.05). Neutrophils had enhanced expression of CD11b/CR3 (p<0.01) and CD16/FcγRIII (p<0.001) and macrophages had lower expression of CD86 (p<0.01) 1 year after cessation of exposure.
One year after closure of the plant airway neutrophils and exhaled NO levels resolved to lower levels and cell surface phenotypes associated with innate immune function recovered to higher levels, indicating that these changes were partly reversible among workers who were no longer exposed to endotoxins in a BSCP plant.
... Lung macrophages that outnumber stimulatory DCs at steady state have been shown to inhibit DC antigen presentation and airway hyperresponsiveness in vivo (35)(36)(37)(38). The inversion of the ratio of lung macrophages to DCs observed in our study has been observed in a murine model of silicosis (39) and in an even more relevant human study of inhalational LPS (40), suggesting that the balance between these myeloid populations may play a critical role in the development of other environmental lung diseases. However, the associations between APCs, the persistence of proinflammatory responses, and the physiologic responses we report have not been previously described, to the best of our knowledge. ...
Little is known about the mechanisms of persistent airflow obstruction that result from chronic occupational endotoxin exposure. We sought to analyze the inflammatory response underlying persistent airflow obstruction as a result of chronic occupational endotoxin exposure. We developed a murine model of daily inhaled endotoxin for periods of 5 days to 8 weeks. We analyzed physiologic lung dysfunction, lung histology, bronchoalveolar lavage fluid and total lung homogenate inflammatory cell and cytokine profiles, and pulmonary gene expression profiles. We observed an increase in airway hyperresponsiveness as a result of chronic endotoxin exposure. After 8 weeks, the mice exhibited an increase in bronchoalveolar lavage and lung neutrophils that correlated with an increase in proinflammatory cytokines. Detailed analyses of inflammatory cell subsets revealed an expansion of dendritic cells (DCs), and in particular, proinflammatory DCs, with a reduced percentage of macrophages. Gene expression profiling revealed the up-regulation of a panel of genes that was consistent with DC recruitment, and lung histology revealed an accumulation of DCs in inflammatory aggregates around the airways in 8-week-exposed animals. Repeated, low-dose LPS inhalation, which mirrors occupational exposure, resulted in airway hyperresponsiveness, associated with a failure to resolve the proinflammatory response, an inverted macrophage to DC ratio, and a significant rise in the inflammatory DC population. These findings point to a novel underlying mechanism of airflow obstruction as a result of occupational LPS exposure, and suggest molecular and cellular targets for therapeutic development.
... Monocyte-like cells have been observed in various inflammatory lung conditions [7,10,26]. Similarly, a population of monocytes with reduced phagocytic capacity and increased expression of markers of dendritic cell maturation was described in sputum from healthy volunteers following LPS inhalation [27], and a ''small sputum macrophage'' was reported in patients with COPD [28]. However, ...
The co-ordinated recruitment of monocyte subpopulations, neutrophils and regulatory T-cells (Tregs) during the early stages of human acute lung inflammation remains poorly understood. We therefore performed a detailed characterisation of these lineages in the blood and lungs in a model of human acute lung inflammation. Healthy volunteers inhaled lipopolysaccharide (LPS) or saline (n=6 for each group). Blood was collected at 0, 2, 4, 6 and 8 h and bronchoscopy with bronchoalveolar lavage (BAL) performed at 8 h. Multiparameter flow cytometry was used to characterise monocyte subpopulations, neutrophils and Tregs in the blood and lung. Inhalation of LPS was associated with significant blood and BAL fluid neutrophilia. Blood populations of monocyte subpopulations and Tregs were unaltered by LPS. In contrast, LPS induced an accumulation of a pulmonary monocyte-like cell (PMLC) population, which was further subdivided into "inducible" CD14(++)CD16(-) and "resident" CD14(++)CD16(+) subsets. Inducible PMLCs were significantly increased following LPS inhalation (p=0.0046), whereas resident PMLCs were unchanged. In addition, we noted a significant decrease in Tregs in BAL fluid with LPS inhalation (p=0.027). The early stages of LPS-induced inflammation in humans is characterised by pulmonary accumulation of a novel inducible monocyte-like subpopulation, accompanied by significant changes in both neutrophil and Treg numbers.
... At this dose, we also observed increased expression of CD80, CD86, HLA-DR on macrophages, decreased phagocytic function of monocytes and macrophages, and influx of a discrete population of dendritic cells into airway sputum. We also observed increased levels of IL-1b and granulocyte macrophage colony-stimulating factor (GM-CSF) (69,70). ...
Ozone (O(3)) and endotoxin are common environmental contaminants that cause asthma exacerbation. These pollutants have similar phenotype response characteristics, including induction of neutrophilic inflammation, changes in airway macrophage immunophenotypes, and ability to enhance response to inhaled allergen. Evoked phenotyping studies of volunteers exposed to O(3) and endotoxin were used to identify the response characteristics of volunteers to these pollutants. New studies support the hypotheses that similar innate immune and oxidant processes modulate response to these agents. These include TLR4 and inflammasome-mediated signaling and cytokine production. Innate immune responses are also impacted by oxidative stress. It is likely that continued discovery of common molecular processes which modulate response to these pollutants will occur. Understanding the pathways that modulate response to pollutants will also allow for discovery of genetic and epigenetic factors that regulate response to these pollutants and determine risk of disease exacerbation. Additionally, defining the mechanisms of response will allow rational selection of interventions to examine. Interventions focused on inhibition of Toll-like receptor 4 and inflammasome represent promising new approaches to preventing pollutant-induced asthma exacerbations. Such interventions include specific inhibitors of innate immunity and antioxidant therapies designed to counter the effects of pollutants on cell signaling.
... Endotoxine-inhalatiestudies bij vrijwilligers vonden bijvoorbeeld een toename in DC-ontwikkeling, neutrofiel-infiltratie en een afname van macrofaagfagocytose, terwijl een tegengestelde respons werd gevonden bij endotoxineniveaus beneden de drempel voor een waarneembare neutrofiel-influx. 75,76 Inhalatiestudies bij muizen laten ook zien dat hoge blootstelling aan endotoxinen geassocieerd is met een verminderde Th2-en versterkte Th1-respons. Inhalatie van endotoxinen door gesensibiliseerde muizen die vooraf blootgesteld waren aan allergenen, leidde bijvoorbeeld tot een verminderde bronchiale hyperreactiviteit en lagere eosinofiele (Th2) niveaus in de luchtwegen. ...
n 1873 werd er al een verband gelegd tussen het opgroeien en werken op een boerderij en een verminderd risico op allergieën, zoals hooikoorts. Recenter onderzoek bevestigt deze vroege observatie en laat een duidelijk verband zien tussen het leven op een boerderij en een verlaagd risico op atopie, hooikoorts en allergische astma bij boerenkinderen. Een verminderd risico op allergische aandoeningen is ook aangetoond voor volwassen boeren. Blootstelling aan landbouwhuisdieren, consumptie van melk direct van de boerderij, en inhalatie van microbiële agentia zoals bacterieel endotoxine zijn herhaaldelijk in verband gebracht met de lagere prevalentie van allergieën in boerenfamilies. De onderliggende immuunmechanismen zijn niet volledig duidelijk, maar het aangeboren immuunsysteem lijkt een belangrijke rol te spelen. In dit overzichtsartikel wordt de meest recente literatuur met betrekking tot allergie en astma bij boerenkinderen en volwassenen besproken. De hygiënehypothese, specifieke beschermende blootstellingen, de onderliggende immunologische processen, en de rol van dosis en moment van blootstelling komen ook aan de orde. De bevindingen in boerenpopulaties zijn van groot belang, omdat ze kunnen bijdragen tot betere preventie en behandelmethoden voor allergie en astma in de algemene populatie.
... 3×10 5 PBMCs were used for measurement of CD40 protein expression. Flow cytometry was performed with a FACSORT (Becton Dickinson, Miami, FL) using an Argon-ion laser (wavelength 488nm) [15]. The FACSORT was calibrated with Calibrite beads before each use, and 10,000 events were counted for all sample runs. ...
CD40 plays important roles in cell-mediated and humoral immune responses. In this study, we explored mechanisms underlying lipopolysaccharide (LPS)-induced CD40 expression in purified human peripheral blood monocytic cells (PBMCs) from healthy volunteers. Exposure to LPS induced increases in CD40 mRNA and protein expression on PBMCs. LPS stimulation caused IkappaBalpha degradation. Inhibition of NFkappaB activation abrogated LPS-induced CD40 expression. LPS stimulation also resulted in phosphorylation of mitogen-activated protein kinases, however, only Jun N-terminal kinase (JNK) was partially involved in LPS-induced CD40 expression. In addition, LPS exposure resulted in elevated interferon gamma (IFNgamma) levels in the medium of PBMCs. Neutralization of IFNgamma and IFNgamma receptor using specific antibodies blocked LPS-induced CD40 expression by 44% and 37%, respectively. In summary, LPS-induced CD40 expression on human PBMCs through activation of NFkappaB and JNK, and partially through the induction of IFNgamma production.
... 9 To determine whether BMI may be a factor in modulating the response to inhaled endotoxin, we re-examined data from our previous studies in which patients with mild allergic asthma and normal volunteers underwent inhaled challenge with either 5 ug endotoxin obtained from Sigma Chemical Co (Milwaukee, Wis) or (more recently) 20,000 EU Clinical Center Reference Endotoxin (CCRE) obtained from the National Institutes of Health. 10 The postendotoxin PMN response (PMNs/mg sputum, mean 6 SEM) was similar across each of the groups studied (P 5 .33, ANOVA), with 1810 6 277 PMNs/mg recovered from patients with asthma challenged with Sigma LPS (n 5 22), 1507 6 666 PMNs/mg from normal volunteers challenged with Sigma LPS (n 5 7), and 1045 6 244 PMNs/mg from normal volunteers challenged with CCRE (n 5 9). ...
Background
The eosinophil (Eo) activation is a crucial factor evoking allergic rhinitis (AR) attacks; factors; the mechanism of triggering Eo activation remains to be further investigated. The interaction of antigen (Ag) and antibody plays a critical role in evoking allergy attacks. This study aims to elucidate the role of FcγRI, the high affinity receptor of IgG, in the Ag-mediated Eo activation.
Methods
Nasal lavage fluids (NLF) were collected from AR patients and healthy control (HC) subjects. Eos were isolated by flow cytometry cell sorting and analyzed by pertinent immunological approaches.
Results
Eos composed more than 60% of the cellular components in AR NLF. Exposure to specific Ags (sAgs) in the culture triggered Eos to release inflammatory mediators. High levels of FcγRI were detected on the surface of AR NLF Eos. Exposure to lipopolysaccharide markedly increased the FcγRI expression in naive Eos, which could be bound by Ag-specific IgG (sIgG) to form complexes on the surface of Eos; this made Eos at the sensitized status. Eos bore with the sIgG/FcγRI complexes could be activated upon exposure to sIgG in the culture; these Eos can be designated as Ag-specific Eos. Passive transfer of Ag-specific Eos resulted in profound AR response in mice upon sAg challenge. Depletion of FcγRI on Eos efficiently abolished AR response in mice.
Conclusions
AR Eos express high levels FcγRI, that can be bound by sIgG to make Eos sensitized. Re-exposure to specific Ags can activate the sensitized Eos.
How very high exposure levels to endotoxin in a farming environment provide protection against respiratory allergic symptoms and low-to-moderate levels of endotoxin in urban homes promote allergic response is unclear. Dose-specific bacterial endotoxin or LPS-induced tolerance mechanisms can affect lung inflammations, coupled with the Th2 immune responses. Here, we explored the effects of intranasal exposure of LPS at two different doses (based on occupational exposures during handling of agricultural wastes) in OVA-sensitized allergic wild type (WT) and TLR4-KO mice, particularly, with respect to Th2 cytokines and Tregs level. Low-dose LPS (100 ng) exposure prohibited airway tolerance and failed to generate T-cell-dependent protection against lung inflammations in allergic mice. Furthermore, low Tregs at the inflammatory site and induced Th2 cytokines, as well as IL-6 and IL-25, suggested that low-dose LPS might be associated with the suppression of tolerance mechanisms. In contrast, high-dose LPS (20 µg) favored the suppression of Th2 cytokines, IL-6 and IL-25, but failed to induce Th1 cytokines (e.g. IFN-γ). Our results suggest that low-dose LPS can enhance airway allergic inflammation through failing of antigen-dependent immune regulatory homeostasis. The exposure levels of LPS can determine the generation of inflammatory responses in airway allergy.
Background:
The relationship between airborne particulate matter (PM) and pulmonary function in children has not been consistent among studies, potentially owing to differences in the inflammatory response to PM, based on PM types and sources. The objective of this study was to investigate the effect of airborne PM on pulmonary function in schoolchildren and its potential for an inflammatory response.
Methods:
Daily morning peak expiratory flow (PEF) was measured in 339 schoolchildren in February 2015. Interleukin (IL)-8 production was assessed in THP1 cells stimulated by airborne PM collected every day during the study period, and these IL-8 concentrations are described as the daily IL-8 levels. A linear mixed model was used to estimate the association between PEF values and the daily levels of suspended PM (SPM), PM diameters smaller than 2.5 μm (PM2.5), and IL-8.
Results:
The daily IL-8 levels were significantly associated with those of SPM and PM2.5. A 0.83 μg/mL increase in IL-8 levels was significantly associated with a -1.07 L/min (95% confidence interval, -2.05 to -0.08) decrease in PEF. A 12.0 μg/m(3) increase in SPM and a 10.0 μg/m(3) increase in PM2.5 were associated with a -1.36 L/min (-2.93 to 0.22) and -1.72 L/min (-3.82 to 0.36) decreases in PEF, respectively. There were no significant relationships between PEF, SPM, and PM2.5.
Conclusions:
These findings suggest that the effects of airborne PM on pulmonary function in schoolchildren might depend more on the pro-inflammatory response than the mass concentration of the PM.
Respiratory mucosal surfaces are constantly exposed to a broad range of non-pathogenic environmental antigens. In the absence of proinflammatory signals, inhalation of harmless antigens results in immunological tolerance. Indeed, lung dendritic cells stimulate the development of antigen-specific regulatory T cells. Nevertheless, epidemiological studies have shown that ambient air contains not only inert antigens but also immunostimulatory molecules of microbial origin. Of particular interest are endotoxins, a cell wall component of gram-negative bacteria that is ubiquitous in the environment. In spite of the fact that high levels of endotoxin exposure in early life protect against allergic sensitization, most evidence indicates that exposure to house-dust endotoxin is a significant risk factor for increased asthma prevalence and severity. When the respiratory tract is stimulated with airborne endotoxins, lung dendritic cells lose their tolerogenic properties and rather promote the development of an allergic response directed against concomitant aeroantigens. Although endotoxins are omnipresent in the environment and favour airway allergy, only a minority of people develops asthma. These contradictory observations imply the existence of unknown mechanisms capable of preventing endotoxin-triggered allergic responses to inhaled antigens.
Endotoxins are commonly found at workplaces where large amounts of bioaerosols are generated. In Germany, especially since the Ordinance on Safety and Health Protection related to work involving biological agents (Biostoff-Verordnung) became effective (1999), threshold limit values are widely discussed. Up to the present, endotoxin values are measured with non-uniform methods and therefore values are of limited benefit for classification of exposure groups. In Germany there is no threshold limit value for endotoxin.
Relevant literature of the last 20 years was selected from Medline and discussed.
In this review we focused on the impact of endotoxin exposure on human health with special respect to the measurements on workplace and methodological aspects of endotoxin determination. Methods for sampling and endotoxin determination have to be validated, optimized, and standardized first.
The adverse health effects of endotoxins are known, standardization of measurements is a necessary goal and protection measures should be established immediately.
Jellyfish has recently attracted much attention for various applications, including functional foods due to its abundance of collagen. Jellyfish collagen, extracted from Nemopilema nomurai, was found to stimulate murine macrophage-like J774.1 cells. However, few reports have determined the immunostimulatory effects of jellyfish collagen on the innate immune response. We herein demonstrate the effect of jellyfish collagen on mouse bone marrow-derived dendritic cells (BMDCs). Jellyfish collagen stimulated TNF-α, IL-6, IL-1β and IL-12 production by BMDCs as the result of the elevation of gene expression level of these cytokines. In addition, jellyfish collagen-treated BMDCs have more wrinkles and longer pseudopodia on the cell surface compared with the control cells. Jellyfish collagen also stimulated cell-surface MHC-II expression level. Furthermore, jellyfish collagen downregulated phagocytosis capacity of BMDCs. Thus, our findings suggest that JC has the potential to activate DCs and thereby contribute to health promotion.
Background:
In healthy nonsmokers, inhaled endotoxin [lipopolysaccharide (LPS)] challenge induces airway neutrophilia and modifies innate immune responses, but the effect on mucociliary clearance (MCC), a key host defense response, is unknown. Although smokers are chronically exposed to LPS through inhaled tobacco smoke, the acute effect of inhaled LPS on both MCC and airway inflammation is also unknown. The purpose of this study was to determine the effect of inhaled LPS on MCC in nonsmokers and mild smokers with normal pulmonary function.
Methods:
We performed an open-label inhalational challenge with 20,000 endotoxin units in healthy adult nonsmokers (n=18) and young adult, mild smokers (n=12). At 4 hr post LPS challenge, we measured MCC over a period of 2 hr, followed by sputum induction to assess markers of airway inflammation.
Results:
No significant changes in spirometry occurred in either group following LPS challenge. Following LPS, MCC was significantly (p<0.05) slowed in nonsmokers, but not in smokers [MCC=10±9% (challenge) vs. 15±8% (baseline), MCC=14±9% (challenge) vs. 16±10% (baseline), respectively]. Both groups showed a significant (p<0.05) increase in sputum neutrophils 6 hr post LPS challenge versus baseline. Although there was no correlation between the increased neutrophilia and depressed MCC post LPS in the nonsmokers, baseline neutrophil concentration predicted the LPS-induced decrease in MCC in the nonsmokers, i.e., lower baseline neutrophil concentration was associated with greater depression in MCC with LPS challenge (p<0.05).
Conclusions:
These data show that a mild exposure to endotoxin acutely slows MCC in healthy nonsmokers. MCC in mild smokers is unaffected by mild endotoxin challenge, likely due to preexisting effects of cigarette smoke on their airway epithelium.
Major distinctive features of avian lungs are the absence of draining lymph nodes and alveoli and alveolar macrophages (MPhs). However, a large network of MPhs and dendritic cells (DCs) is present in the mucosa of the larger airways and in the linings of the parabronchi. For the modulation of respiratory tract immune responses, for example, by vaccination, a better understanding of Ag uptake in the chicken respiratory tract is needed. In this study, we provide detailed characterization of APCs in chicken lungs, including their functional in vivo activities as measured by the uptake of fluorescently labeled 1-μm beads that are coated with either LPS or inactivated avian influenza A virus (IAV) mimicking the uptake of bacterial or viral Ag. We identified different subsets of MPhs and DCs in chicken lungs, based on the expression of CD11, activation markers, and DEC205. In vivo uptake of LPS- and IAV-beads resulted in an increased percentage MHC class II(+) (MHC II(+)) cells and in the upregulation of CD40. The uptake of LPS-beads resulted in the upregulation of CD80 and MHC II on the cell surface, suggesting either uptake of LPS- and IAV-beads by different subsets of phagocytic cells or LPS-mediated differential activation. Differences in phagosomal acidification indicated that in chicken lungs the MHC II(+) and CD80(+) bead(+) cell population includes DCs and that a large proportion of beads was taken up by MPhs. LPS-bead(+) cells were present in BALT, suggesting local induction of immune responses. Collectively, we characterized the uptake of Ags by phagocytes in the respiratory tract of chickens.
To determine if the GSTM1 null genotype is a risk factor for increased inflammatory response to inhaled endotoxin.
35 volunteers who had undergone inhalation challenge with a 20 000 endotoxin unit dose of Clinical Center Reference Endotoxin (CCRE) were genotyped for the GSTM1 null polymorphism. Parameters of airway and systemic inflammation observed before and after challenge were compared in GSTM1 null (n=17) and GSTM1 (n=18) sufficient volunteers.
GSTM1 null volunteers had significantly increased circulating white blood cells (WBCs), polymorphonuclear neutrophils (PMNs), platelets and sputum PMNs (% sputum PMNs and PMNs/mg sputum) after CCRE challenge. GSTM1 sufficient volunteers had significant, but lower increases in circulating WBCs, PMNs and % sputum PMNs, and no increase in platelets or PMNs/mg sputum. Linear regression analysis adjusted for baseline values of the entire cohort revealed that the GSTM1 null genotype significantly increased circulating WBCs, platelets and % sputum PMNs after challenge.
These data support the hypothesis that the GSTM1 null genotype is a risk factor for increased acute respiratory and systemic inflammatory response to inhaled CCRE. These data are consistent with other observations that the GSTM1 null genotype is associated with increased respiratory, systemic and cardiovascular effects linked to ambient air particulate matter exposure and indicate that the GSTM1 null genotype should be considered a risk factor for adverse health effects associated with exposure to environmental endotoxin.
Small sputum macrophages represent highly active cells that increase in the airways of patients with inflammatory diseases such as chronic obstructive pulmonary disease (COPD). It has been reported often that levels of cytokines, chemokines and pro-teases are increased in sputum supernatants of these patients. In COPD, the small sputum macrophages may contribute to these supernatant proteins and recruit additional cells via specific chemokine expression patterns. We therefore investigated the expression profile of chemokines in sputum macrophages obtained from COPD patients in comparison to cells from healthy donors and cells isolated after inhalation of lipopolysaccharide (LPS). We used the minimally invasive procedure of sputum induction and have purified macrophages with the RosetteSep technology. Using macrophage purification and flow cytometry we show that in COPD small sputum macrophages account for 85.9% ± 8.3% compared with 12.9% ± 7.1% of total macrophages in control donors. When looking at chemokine expression we found, for the small macrophages in COPD, increased transcript and protein levels for CCL2, CCL7, CCL13 and CCL22 with a more than 100-fold increase for CCL13 mRNA (P < 0.001). Looking at active smokers without COPD, there is a substantial increase of small macrophages to 60% ± 15% and, here, chemokine expression is increased as well. In a model of airway inflammation healthy volunteers inhaled 20 μg of lipopolysaccharide (LPS), which resulted in an increase of small sputum macrophages from 18% ± 19% to 64% ± 25%. The pattern of chemokine expression was, however, different with an upregulation for CCL2 and CCL7, while CCL13 was downregulated three-fold in the LPS-induced small macrophages. These data demonstrate that sputum macrophages in COPD show induction of a specific set of CCL chemokines, which is distinct from what can be induced by LPS.
Endotoxin or lipopolysaccharide (LPS) is a cell wall component of Gram-negative bacteria. Like aeroallergens, LPS is ubiquitous in our living environment. Epidemiology studies in young children have found that LPS exposure at home is inversely correlated with the development of atopic diseases, thus the ‘hygiene hypothesis’ for allergic diseases. However, positive association has also been found between indoor LPS exposure and the development of wheezing or asthma in children. In humans, experimental exposure to LPS in the airways can cause inflammatory responses and lung function changes directly or modulate responses to allergens indirectly, particularly in those with asthma. In animal studies, experimental exposure to LPS has generated some conflicting, sometimes opposite, results in host responses to allergen stimulation. In this article, we will review recent advances in our understanding of the immunomodulating effects of LPS on allergen-induced responses and analyse some of the possible reasons for the inconsistent findings.
Cite this as: Z. Zhu, S. Y. Oh, T. Zheng and Y-K. Kim, Clinical & Experimental Allergy, 2010 (40) 536–546.
The respiratory tract is continuously exposed to both innocuous airborne antigens and immunostimulatory molecules of microbial origin, such as LPS. At low concentrations, airborne LPS can induce a lung DC-driven Th2 cell response to harmless inhaled antigens, thereby promoting allergic asthma. However, only a small fraction of people exposed to environmental LPS develop allergic asthma. What prevents most people from mounting a lung DC-driven Th2 response upon exposure to LPS is not understood. Here we have shown that lung interstitial macrophages (IMs), a cell population with no previously described in vivo function, prevent induction of a Th2 response in mice challenged with LPS and an experimental harmless airborne antigen. IMs, but not alveolar macrophages, were found to produce high levels of IL-10 and to inhibit LPS-induced maturation and migration of DCs loaded with the experimental harmless airborne antigen in an IL-10-dependent manner. We further demonstrated that specific in vivo elimination of IMs led to overt asthmatic reactions to innocuous airborne antigens inhaled with low doses of LPS. This study has revealed a crucial role for IMs in maintaining immune homeostasis in the respiratory tract and provides an explanation for the paradox that although airborne LPS has the ability to promote the induction of Th2 responses by lung DCs, it does not provoke airway allergy under normal conditions.
Millions of dollars are spent annually on research to advance the scientific basis for setting air quality standards and implementing programs to protect public health and the environment. It is necessary for the U.S. Environmental Protection Agency’s Office of Research and Development (ORD) to document and evaluate their accomplishments in order to communicate the value of their research to the public and government leaders. This report is intended to highlight the major research accomplishments of EPA’s Clean Air Research Program since 2002. Among the most notable achievements are near roadway studies, understanding emissions from wild and prescribed fires, advancing characterization of emissions, improving source apportionment and characterization, and control of secondary PM2.5 from coal combustion. Significant progress has also been made in understanding secondary organic aerosol formation, development of a new secondary organic aerosol model, updating emissions inventories, and improving measurement and modeling techniques. Additionally, health research has investigated target doses of pollutants to various human lung sites, identified risk factors for susceptible subpopulations, uncovered biological mechanisms for cardiopulmonary effects, identified new target tissues from air pollution exposure, and explored co-pollutants and other mediating factors. Research generated by ORD directly affects the National Ambient Air Quality standards setting process through health and environmental research, supports the evaluation of environmental conditions with monitoring and modeling research, provides information to states for regulation and management via technical products and support, and allows EPA to track progress. This report, in addition to summarizing the major research accomplishments of ORD, evaluates the research accomplishments against the framework for air quality management, detailing which efforts directly support the framework and how future research will support the framework. With the support of research generated by the Clean Air Research Program, EPA can continue to protect human health and the environment from the adverse effects of air pollution.
Dendritic cells are professional antigen presenting cells linking innate and adaptive immune responses. Different dendritic cell subsets were identified in human lung, each with their own functional characteristics. As innate and adaptive immune responses are activated in Chronic Obstructive Pulmonary Disease (COPD), dendritic cells could play a role in the pathogenesis of this disease. Indeed, cigarette smoke appears to modulate dendritic cell function in vitro and alters dendritic cell numbers and function in cigarette smoke exposed mice. The number of pulmonary dendritic cells differs between COPD patients, smokers and non-smokers. Moreover, the number of Langerhans-type dendritic cells increases with the severity of the disease. In this review we will discuss the scientific evidence regarding the role of dendritic cells in COPD and we will put forward the concept of modulation of dendritic cell differentiation and function as a crucial step in the pathogenesis of COPD.
Asthma is a complex disorder and evidence now suggests that it is not solely attributable either to allergy or eosinophilia. Non-eosinophilic asthma accounts for up to one half of all cases and a large proportion have a non-allergic aetiology. The innate immune system responds to a variety of triggers, including viral and bacterial components which are known non-allergic triggers of asthma. The innate immune response may be involved in both the development of and protection against asthma. Factors which are likely to determine the nature of the response include the timing of the exposure (childhood or adulthood), baseline asthma inflammatory subtype (eosinophilic or non-eosinophilic) and the dose of the exposure. Gene-environment interactions are likely to modify the response. Further research is required to elucidate the specific mechanisms involved in the innate immune response in asthma and will be important in the identification of new targets for therapy and management.
Although respiratory infections cause significant morbidity and mortality throughout the world, the immunologic factors that mediate host susceptibility to these infections remain poorly understood. The lung contains a vast surface at the host-environment interface and acts as a crucial barrier to invading pathogens. The lung is equipped with specialized epithelial and hematopoietic cells, which express pattern recognition receptors that act as both sentinels and mediators of pulmonary innate immunity. Toll-like receptors (TLRs) mediate a particularly critical role in pathogen recognition and subsequent initiation of the host immune response. In this review, we will summarize current knowledge of TLRs and their bacterial ligands and explore their role in respiratory infections. Moreover, we will highlight recent advances in the role of TLRs in pulmonary infections from a human immunogenetics perspective.
A low allergy rate in coal and wood heated homes has been described in the small villages in the Alpine foothills and subsequently found to be associated with the farming environment. This was interpreted within the framework of the hygiene hypothesis but there are also alternative explanations. Lower air pollution could be one reason, which is, however, unlikely since the differences between the Bavarian countryside and the Munich municipal area were only weak. There could be genetic differences between the urban and rural population by previous isolation or by self-selection. The potential drop-out of allergy genes, however, will also not explain the absent increase of allergies in two generations. More likely, other lifestyle factors are important. Dietary habits are different in farmers and a less frequent vitamin D supplementation of newborns (otherwise expected to be allergy promoting) has been shown recently. The underlying cause for the "non-allergic farm child" remains speculative until the transfer of any farm-associated factor is leading to a similar risk reduction in the general population.
A developing area of interest regarding the relationship between the adverse health effects associated with particles suspended in the troposphere is an understanding of how particle chemical composition influences different biological outcomes. Described is the development and application of an apparatus and methodology wherein a known number of particles of tropospherically relevant chemical composition can be designed and levitated in an alternating current (ac) trap followed by their controlled deposition directly from the ac trap onto air-liquid interface cultured lung cells. A downstream biological response, differential upregulation of intercellular adhesion molecule (ICAM)-1, was measurable using fluorescence microscopy in the air-liquid interface human lung cell cultures even though the dose per culture was 0-100 lipopolysaccharide (LPS)-containing elemental carbon particles (52 pg LPS per 6.3 microm diameter particle). Fluorescence emission intensity data measured from a 1 mm2 area centered over the site of particle deposition were fitted using a least squares linear regression line. Because the total mass of each different compound comprising each of the particles delivered to the culture was known, the data generated with this methodology can be expressed as a pro-inflammation potential (in this case ICAM-1 expression) per particle number and composition. Also described is how this methodology affords opportunities to quantitatively study pro-inflammatory intercellular signaling leading to ICAM-1 expression at sites distal to the site of particle deposition.
: Francisella tularensis is the etiologic agent of the zoonotic disease, tularemia. An inoculum as small as 10 bacteria can cause a flulike disease with substantial morbidity and mortality among infected humans. Human tularemia presents in ulceroglandular, oculoglandular, oropharyngeal, pneumonic, and septic forms. Rapid administration of antibiotics prevents mortality in the majority of human cases if exposure doses are low and nonaerosol. Without early diagnosis and administration of antibiotics, high-dose aerosol exposure progresses rapidly to life threatening pleuropneumonitis and systemic infection. The relative abundance of F tularensis in nature and the relative ease with which it may be administered raise concerns over its exploitation as a biothreat agent.
The signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor–dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.
Exposure to endotoxin and to its purified derivative lipopolysaccharide (LPS) is related to several occupational pulmonary diseases and to severe domestic asthma. An inhalation of a given dose of pure LPS produces both a systemic and a bronchial inflammatory response. Information on the dose-response relationship to inhaled LPS in normal subjects is a prerequisite to define the safety threshold of exposure. In the present study, the clinical and inflammatory responses to rising doses of inhaled LPS was evaluated. Nine normal volunteers were challenged weekly by inhalation with saline, 0.5, 5, and 50 microg LPS (Escherichia coli). The response determinators are the clinical symptoms, fever, FEV1, blood polymorphonuclear neutrophils (PMNs) with their level of activation (measured by luminol enhanced-chemiluminescence), and both the blood and the urine concentrations of the C-reactive protein (CRP). To assess the bronchial inflammatory response, an induced sputum was obtained 6 h after each dose of LPS, and the total and differential cell counts as well as the MPO, ECP, and TNF-alpha concentrations were measured. Compared with the saline, an inhalation of 0.5 microg LPS induces a significant decrease in the PMN luminol-enhanced chemiluminescence (p < 0.01), which could reflect a process of margination and/or extravascular sequestration of activated PMN. Inhalation of 5 microg LPS is associated with a significant rise in blood CRP (p < 0.01) and PMNs (p < 0.001) and in sputum PMNs (p < 0.05), monocytes (p < 0.05), and MPO (p < 0.05). Inhalation of 50 microg LPS was characterized by a significant increase in temperature (p < 0.01), blood PMNs (p < 0.001), blood and urine CRP (p < 0.01 and < 0.01), and sputum PMNs (p < 0.001), monocytes (p < 0.05), lymphocytes (p < 0.05), MPO (p < 0.01), TNF-alpha (p < 0.01), and ECP (p < 0.01) while five subjects develop symptoms. In normal subjects, the response to inhaled LPS is dose-related, the most sensitive markers of LPS-induced inflammation being the blood PMNs count with their level of activation, the blood CRP concentration, and the sputum PMNs count. The no-response threshold to an acute inhalation of LPS is less than 0.5 microg.
Dendritic cells (DCs) express several receptors for the Fc portion of immunoglobulin (Ig)G (FcγR), which mediate internalization
of antigen–IgG complexes (immune complexes, ICs) and promote efficient major histocompatibility complex (MHC) class II–restricted
antigen presentation. We now show that FcγRs have two additional specific attributes in murine DCs: the induction of DC maturation
and the promotion of efficient MHC class I–restricted presentation of peptides from exogenous, IgG-complexed antigens. Both
FcγR functions require the FcγR-associated γ chain. FcγR-mediated MHC class I–restricted antigen presentation is extremely
sensitive and specific to immature DCs. It requires proteasomal degradation and is dependent on functional peptide transporter
associated with antigen processing, TAP1-TAP2. By promoting DC maturation and presentation on both MHC class I and II molecules,
ICs should efficiently sensitize DCs for priming of both CD4+ helper and CD8+ cytotoxic T lymphocytes in vivo.
Identification of precursor dendritic cells as the natural interferon-producing
cells reveals their role in connecting two branches of the immune system (page
919).
Little is known about the functional capabilities of bronchial macrophages (BMs) and their relationship to airway disease such as asthma. We hypothesize that BMs from asthmatics may be modulated in their function compared with similar cells from healthy individuals. BMs obtained by induced sputum from mild asthmatics (n = 20) and healthy individuals (n = 20) were analyzed using flow cytometry for CD16, CD64, CD11b, CD14, and human leukocyte antigen-DR expression, phagocytosis of IgG opsonized yeast, and oxidant production. Asthma status was assessed by lung function [percent predicted forced vital capacity and forced expiratory volume in 1 s (FEV(1))], percent sputum eosinophils, and nonspecific airway responsiveness [provocative concentration that produces a 20% fall in FEV(1) (PC(20,FEV1))]. Asthmatics with >5% airway eosinophils (AEo+) had decreased BM CD64 expression and phagocytosis compared with asthmatics with <5% eosinophils (AEo-). Among asthmatics, a significant correlation was found between CD64 expression and BM phagocytosis (R = 0.7, P < 0.009). Phagocytosis was also correlated with PC(20,FEV1) (R = 0.6, P < 0.007), lung function (%predicted FEV(1), R = 0.7, P < 0.002) and percent eosinophils (R = -0.6, P < 0.01). In conclusion, BM from asthmatics are functionally modulated, possibly by Th2 cytokines involved in asthma pathology.
B and T lymphocytes are the mediators of immunity, but their function is under the control of dendritic cells. Dendritic cells in the periphery capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. They not only activate lymphocytes, they also tolerize T cells to antigens that are innate to the body (self-antigens), thereby minimizing autoimmune reactions. Once a neglected cell type, dendritic cells can now be readily obtained in sufficient quantities to allow molecular and cell biological analysis. With knowledge comes the realization that these cells are a powerful tool for manipulating the immune system.
Bacterial endotoxin has been suggested as responsible for the development of subjective symptoms and transient or chronic lung function impairment seen after exposure to organic dusts in cotton mills, poultry houses, swine confinement buildings and saw mills. Animal experiments have demonstrated bronchoalveolar neutrophilia being the most prominent cell response in animals following bacterial lipopolysaccharide (LPS) inhalation. The present study was conducted to obtain information on some aspects of the early inflammatory response to inhaled LPS in man. Eight healthy nonsmoking subjects, 23-27 yrs old, underwent bronchoalveolar lavage (BAL), 3 h after a provocation test with 100 micrograms LPS from E. coli dissolved in 2 ml isotonic NaCl. The solution was aerosolized with a jet nebulizer and inhaled. The calculated dose delivered to the lung was approximately 25 micrograms, which equals exposure in some occupational settings. The BAL data for each individual subject were compared with data from a control BAL performed at least 6 weeks prior to the LPS challenge. The major cellular response to LPS, reflected in BAL fluid, was an approximately hundredfold increase in neutrophils. The total number of lymphocytes was on average tripled. The alveolar macrophage phagocytosis of opsonized yeast particles in vitro was significantly reduced. A further indicator of an ongoing inflammation was an increase in fibronectin. No changes were seen in the levels of BAL albumin, indicating that the elevated level of fibronectin could not be explained by an increased permeability, but rather by a local production. The results correspond with data from animal studies and further supports the hypothesis that bacterial LPS is important in the pulmonary reaction induced by organic dusts.
Eight healthy volunteers were exposed to purified lipopolysaccharide (LPS) by controlled inhalation. Bronchoalveolar lavage (BAL) 3 hours after exposure revealed a pronounced neutrophilia, increase in lymphocytes, fibronectin concentration, and decrease in alveolar macrophage phagocytosis, as compared to a reference BAL.
Using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 we have established dendritic cell (DC) lines from blood mononuclear cells that maintain the antigen capturing and processing capacity characteristic of immature dendritic cells in vivo. These cells have typical dendritic morphology, express high levels of major histocompatibility complex (MHC) class I and class II molecules, CD1, Fc gamma RII, CD40, B7, CD44, and ICAM-1, and lack CD14. Cultured DCs are highly stimulatory in mixed leukocyte reaction (MLR) and are also capable of triggering cord blood naive T cells. Most strikingly, these DCs are as efficient as antigen-specific B cells in presenting tetanus toxoid (TT) to specific T cell clones. Their efficiency of antigen presentation can be further enhanced by specific antibodies via FcR-mediated antigen uptake. Incubation of these cultured DCs with tumor necrosis factor alpha (TNF-alpha) or soluble CD40 ligand (CD40L) for 24 h results in an increased surface expression of MHC class I and class II molecules, B7, and ICAM-1 and in the appearance of the CD44 exon 9 splice variant (CD44-v9); by contrast, Fc gamma RII is markedly and sometimes completely downregulated. The functional consequences of the short contact with TNF-alpha are in increased T cell stimulatory capacity in MLR, but a 10-fold decrease in presentation of soluble TT and a 100-fold decrease in presentation of TT-immunoglobulin G complexes.
The combination of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) induces the growth of antigen-presenting cells (APC) from adherent peripheral blood leukocytes. These cells have been characterized as dendritic cells (DC), yet many questions exist regarding their relationship to other DC populations and the nature of their progenitors. To address these issues, we utilized a combination of immunomagnetic depletion, cell sorting, and cell culture to isolate four distinct APC populations; macrophages expressing high levels of CD14 (CD14bright macrophages), DC produced by culturing adherent cells in GM-CSF and IL-4 (cultured DC), and two different subsets of fresh DC that express low levels of CD14 (CD14dim DC). Each population exhibited a unique morphology and a unique profile of cell surface markers. In contrast to macrophages,all three DC populations expressed the DC marker CD83, as well as highlevels of MHC molecules and the costimulatory molecules B7-1 (CD80) and B7-2 (CD86). In addition. all three DC populations presented soluble tetanus toxin antigen and stimulated T cell proliferation to levels far superior to that of macrophages. Blocking studies demonstrated a costimulatory role for B7-1, B7-2, and CD40 in antigen presentation, although B7-2 expression was the single most important factor. To identify the progenitors of cultured DC, we sorted the adherent fraction of PBMC into discrete subpopulations prior to exposure to GM-CSF and IL-4. DC activity derived entirely from CD14+ precursors and was equally demonstrable using either the CD14dim or CD14bright subsets. Although these DC precursors lost expression of CD14 in culture, they maintained most of their other myeloid features. We conclude that human CD14+ leukocytes acquire the phenotype and function of DC when cultured in GM-CSF and IL-4.
We have investigated an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The procedure uses 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or 'priming' phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or 'differentiation' phase requires the exposure to macrophage conditioned medium. This medium cannot be replaced by several known cytokines such as TNF-alpha, IL-1, IL-6, IL-12 and IL-15, and cannot be inhibited with neutralizing antibodies to IL-1, TNF-alpha, IL-6 or IL-12 alone, or in combination. Using this two-step approach, we obtain substantial yields. About 1-3 x 10(6) mature dendritic cells are generated from 40 ml of blood vs. < 0.1 x 10(6) from noncytokine treated blood. The dendritic cells derive from progenitors found primarily in a radioresistant population of CD14+ and adherent blood mononuclear cells and have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. Strong APC function was evident for both the proliferation of allogeneic T cells in the MLR, and the generation by syngeneic T cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers is also expressed, including CD83, p55, and perinuclear CD68. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. We suggest that these cells will be effective in vivo as adjuvants for active immunotherapy.
The main function of dendritic cells (DC) is to induce the differentiation of naive T lymphocytes into helper cells producing a large array of lymphokines, including interleukin (IL)-2; interferon-gamma (IFN-gamma), IL-4, IL-5 and IL-10. The potent immunostimulatory properties of DC develop during a process of maturation that occurs spontaneously in vitro. Since IL-10 has been shown to inhibit Th1 responses, we determined its effect on DC maturation and accessory function. Our data show that DC that have undergone maturation in vitro in the presence of IL-10, have an impaired capacity to induce a Th1-type response in vivo, leading to the development of Th2 lymphocytes. Their inability to promote the synthesis of IFN-gamma seems to correlate with a decreased production of IL-12, an heterodimeric cytokine necessary for optimal generation of Th1-type cells. These results suggest that IL-10 skews the Th1/Th2 balance to Th2 in vivo by selectively blocking IL-12 synthesis by the antigen-presenting cells that play a role of adjuvant of the primary immune response. The cytokines present in the environment at the presentation step may, therefore, determine the class of the immune response induced by DC in vivo, i.e. Th0, Th1 and/or Th2.
Dendritic cells have the remarkable property of presenting any incoming antigen. To do so they must not only capture antigens with high efficiency and broad specificity, but must also maximize their capacity to load class II molecules of the major histocompatibility complex (MHC) with antigenic peptides in order to present a large array of epitopes from different proteins, each at a sufficient copy number. Here we show that formation of peptide-MHC class II complexes is boosted by inflammatory stimuli that induce maturation of dendritic cells. In immature dendritic cells, class II molecules are rapidly internalized and recycled, turning over with a half-life of about 10 hours. Inflammatory stimuli induce a rapid and transient boost of class II synthesis, while the half-life of class II molecules increases to over 100 hours. These coordinated changes result in the rapid accumulation of a large number of long-lived peptide-loaded MHC class II molecules capable of stimulating T cells even after several days. The capacity of dendritic cells to load many antigenic peptides over a short period of initial exposure to inflammatory stimuli could favour presentation of infectious antigens.
The aim of this study was to better define how glucocorticoids influence primary human T cell responses. Dendritic cells (DC*) are the most effective antigen presenting cells able to activate naive T cells. Previous studies have shown that dexamethasone impaired the function of murine DC. Here, we analyzed how methylprednisolone (MP) might affect the function and maturation of human DC.
Human DC were generated from peripheral blood mononuclear cells cultured in granulocyte macrophage-colony stimulating factor and interleukin (IL)4. DC maturation was induced either by lipopolysaccharide (LPS) or by fibroblast transfected with the CD40-ligand gene (3T6-CD40L). DC phenotype was characterized by flow cytometric analysis, their cytokine production by ELISA. The ability of DC to activate naive T cells was evaluated in mixed leukocyte reactivity.
Although MP did not affect viability of DC, it enhanced their antigen uptake and down-regulated their basal expression of CD86. The expression of CD80 and CD54 by DC was slightly decreased and HLA-DR expression was not modified. MP prevented LPS-induced DC maturation as assessed by the inhibition of CD86, CD80 and CD54 up-regulation, CD83 induction and production of TNF-alpha, IL-6, and IL-12. In contrast, when DC were stimulated by 3T6-CD40L, MP prevented only the synthesis of IL-12. Moreover, MP-treated DC were deficient in their ability to elicit proliferative responses of CD4+CD45RA+ allogeneic T cells as well as their synthesis of interferon (IFN)-gamma, IL-5, and IL-13. CONCLUSION. Glucocorticoids exert potent suppressive effects on human DC and thereby inhibit the induction of primary T cell responses.
Epidemiological and in vivo studies suggest that inhaled endotoxin may be an important environmental factor associated with the increases in asthma-related morbidity and mortality. Recent studies by our group and others provide a rationale for the hypothesis that airway exposure of atopic asthmatics to both allergen and endotoxin might result in greater inflammatory responses than those observed with either stimulus alone. Moreover, these studies may provide further evidence that concomitant exposure to allergen and endotoxin is an important factor in asthma pathogenesis.
Inhaled endotoxin (LPS) is associated with airway neutrophilic (PMN) inflammation in both asthmatic and control subjects, with asthmatic subjects demonstrating possibly higher sensitivity. CD14 is the principal receptor mediating LPS responses in vivo. It is unknown whether constitutive CD14 can predict the magnitude of the PMN response after LPS inhalation and whether atopy plays a role in this response.
We sought to examine associations between constitutive airway CD14 expression and LPS-induced PMNs after 5 microg of LPS inhalation and to examine associations between markers of atopy (eosinophils and eosinophil cationic protein) and CD14 expression and LPS-induced PMNs.
Ten atopic asthmatic subjects and 8 healthy control subjects inhaled 0.9% saline and LPS (Escherichia coli 026:B6, 5 microg) separated by 3 weeks. Induced sputum was collected at 24 hours before and 6 hours after inhalation. Induced sputum was analyzed for total and differential cell counts and soluble markers (soluble [s]CD14, eosinophil cationic protein, IL8, and total protein). Flow cytometry was used to analyze membrane-bound CD14 expression.
Significant associations were found between the LPS-induced PMN response (PMNs per milligram of sputum) and both constitutive sCD14 (R = 0.7, P =.005) and membrane-bound CD14 (R = 0.9, P =.01). Asthmatic subjects demonstrated significantly higher levels of constitutive sCD14 compared with control subjects, and baseline eosinophils were significantly associated with baseline sCD14 (R = 0.7, P =.01) and LPS-induced PMNs (R = 0.6, P =.03).
Constitutive airway CD14 expression can predict the magnitude of the PMN response after inhaled LPS. Atopy appears to play a role in the level of CD14 expression and may contribute to LPS sensitivity in asthmatic subjects.
In a cohort of 8 normal and 10 allergic asthmatic volunteers, we previously reported that inhalation of 5 microg of endotoxin (LPS) induced airway inflammation that correlated with CD14 expression that was, in turn, correlated with eosinophil numbers in the airway. Macrophage and neutrophil functions have been reported to be modified by endotoxin in vitro and in vivo, and response to endotoxin is mediated largely by airway phagocytes and related to allergic inflammation.
We sought to examine functional and cell-surface phenotype changes in phagocytes recovered from atopic asthmatic subjects after endotoxin challenge.
Sputum and peripheral blood from 10 allergic asthmatic subjects was recovered after saline and LPS challenge. Assessment of phagocytosis and cell-surface phenotype (CD11b, CD14, and CD64) was performed on phagocytes obtained from sputum (n = 7) and blood samples (n = 10).
Phagocytosis of blood and sputum phagocytes was blunted after LPS challenge in a fashion that correlated with the increase in airway neutrophils after LPS challenge. Cell-surface expression of CD14 (membrane-bound CD14) was increased in sputum cells, whereas CD11b was decreased in sputum and circulating phagocytes. Baseline expression of CD11b in blood correlated with the magnitude of the neutrophil response after LPS inhalation, as well as (inversely) with baseline airway eosinophil levels.
Inhalation of endotoxin at levels adequate to induce a neutrophil influx to the airways (but not systemic symptoms) results in decreased phagocytosis in both airway and circulating cells and modifies CD11b expression in a way that implicates its involvement in phagocyte responsiveness to inhaled LPS.
There is a breakdown of tolerance to neutrophil components during systemic vasculitis, which is marked by autoantibodies and T cells with specificity for proteinase 3 or myeloperoxidase, expressed on the surface of apoptotic neutrophils. This study was undertaken to investigate the effects of human apoptotic and necrotic neutrophils on human dendritic cell (DC) phenotype and ability to stimulate allogeneic T cell proliferation.
DCs were generated from human peripheral blood mononuclear cells and allowed to interact with human apoptotic and necrotic neutrophils in the presence or absence of tumor necrosis factor alpha (TNFalpha). Effects on DC phenotype and ability to stimulate T cell proliferation were observed.
Immature DCs engulfed apoptotic and necrotic neutrophils, resulting in up-regulation of CD83 and class II major histocompatibility complex molecules, but down-regulation of CD40, CD80, and CD86, and a decreased ability to stimulate T cell proliferation. When TNFalpha was added in combination with apoptotic neutrophils, the inhibitory effects were overcome to some extent.
Our results suggest that DC uptake of apoptotic or necrotic neutrophils alone does not shift the immune response from tolerance to autoimmunity in systemic vasculitis. However, cytokines found at sites of inflammation in vasculitis patients may act as maturation factors for DCs, and in combination with apoptotic neutrophils, may lead to an autoimmune phenotype.
The response to lipopolysaccharide exposure is highly variable and might be a result of genetic diversity between individuals. The toll-like receptor 4 (TLR-4) is the principal receptor for lipopolysacharide.
We investigated the association between single-nucleotide polymorphisms in the TLR4 locus and levels of systemic inflammatory markers in response to lipopolysaccharide.
Healthy subjects (n = 116) were genotyped for the most frequent polymorphisms found in the promoter and coding region of the TLR4 gene (-2026A/T, -1607T/C, +896A/G, and +1196C/T relative to the translation start site). Subjects were challenged with 20 microg lipopolysaccharide by inhalation.
Polymorphisms at +896 and +1196 were in complete linkage disequilibrium, and no homozygotes for the less common allele, G and T respectively, were found. After lipopolysaccharide inhalation, subjects heterozygous for either TLR-4/+896 or TLR4/+1196 had significantly lower numbers of white blood cell counts and lower levels of C-reactive protein and lipopolysaccharide-binding protein compared with homozygotes with the common allele. None of the heterozygous subjects (n = 18) except 1 were high responders to lipopolysaccharide (defined as a rise in C-reactive protein > 10 mg/L), whereas 36 of 98 homozygous subjects were high responders (P <.02). No association was observed between the TLR-4/-2026 and TLR-4/-1607 polymorphisms and lipopolysaccharide responsiveness.
The single-nucleotide polymorphisms at position +896 or +1196 in the TLR-4 gene is associated with systemic inflammatory hyporesponsiveness to inhaled lipopolysaccharide.
Immature dendritic cells (iDCs) can ingest apoptotic cells, which do not lead to maturation of the iDCs. In this paper we examine the phagocytosis of apoptotic cells by iDCs in the absence of stimuli for the maturation of iDCs and the subsequent cytokine production. Phagocytosis was observed by confocal microscopy, and it increased as apoptosis proceeded. The coculturing of iDCs with apoptotic cells did not induce the maturation of iDCs even after the subsequent LPS treatment, as assessed as to the expression of MHC class II, CD80, CD86, and CD40. Moreover, IL-6 and IL-12p40 among the cytokines examined were specifically up-regulated by the coculturing at the mRNA and protein levels. The coculturing decreased the expression of MHC class II on iDCs and allogenic T cell proliferation induced by iDCs. Although anti-IL-6 antibodies only partially reversed the effect of coculturing with apoptotic cells, exogenous IL-6 decreased significantly the expression of MHC class II on iDCs and allogenic T cell proliferation induced by iDCs, raising the possibility that IL-6 may be partly involved in maintaining the immature status of iDCs in an autocrine manner.
We recently reported that baseline expression of circulating CD11b is associated with the magnitude of the neutrophil response following inhaled endotoxin. In this study, we examined whether circulating CD11b plays a similar role in the inflammatory response following inhaled ozone exposure. Twenty-two volunteers underwent controlled exposure to ozone (0.4 ppm, 2 h) and to clean air on two separate occasions. Induced sputum and peripheral blood were collected before and after exposure. Induced sputum collected from subjects exposed to ozone revealed marked neutrophilia and increased expression of mCD14 on airway macrophages and monocytes. Baseline CD11b expression on blood phagocytes correlated positively with ozone-induced neutrophil influx into the airways. In conclusion, in human volunteers, circulating CD11b predicts the magnitude of the airway neutrophil response following inhaled ozone exposure. Consequently, CD11b may be a useful biomarker for predicting susceptibility to airway neutrophilic inflammation caused by pollutants.
We previously reported that inhalation of 5 mug of endotoxin (30,000 endotoxin units [EU]) induced airway neutrophilia and decreased phagocytosis by airway monocytes, macrophages, and neutrophils. Conversely, we recently reported that very low doses of endotoxin, which are not associated with neutrophil influx, enhance response to allergen in the nasal and bronchial airway.
We sought to determine whether endotoxin (0-10,000 EU) at doses that do not induce airway neutrophilia prime airway phagocyte function, alter expression of relevant cell-surface receptors (membrane-bound CD14 [mCD14] and CD11b/CR3), and cause induction of a T(H)2 cytokine profile in the airway.
Thirteen nonallergic healthy volunteers were challenged on separate occasions with escalating doses of Clinical Center Reference Endotoxin (CCRE; 0, 2500, 5000, and 10,000 EU), with 9 volunteers completing the entire dose range. Sputum cells and fluid-phase components were recovered 6 hours after challenge. Sputum inflammatory cells were analyzed by means of flow cytometry for mCD14 and CD11b expression and immune function (phagocytosis of IgG-opsonized zymosan particles).
At all doses of CCRE, there was no increase in airway neutrophils relative to that caused by saline. However, inhalation of 10,000 EU enhanced phagocytosis (monocytes and macrophages), upregulated expression of CD11b and mCD14 (monocytes and neutrophils), and increased IL-13 levels, whereas IFN-gamma levels were significantly decreased.
The 10,000-EU dose of CCRE is subthreshold for inducing airway neutrophilia but primes phagocyte function and cell-surface receptor expression in the presence of increased IL-13 and decreased IFN-gamma levels. We speculate that low-dose endotoxin challenge skews airway inflammation in a T(H)2 direction in vivo .
Type I inflammatory cytokines are essential for immunity to many microbial pathogens, including Toxoplasma gondii. Dendritic cells (DC) are key to initiating type 1 immunity, but neutrophils are also a source of chemokines and cytokines involved in Th1 response ignition. We found that T. gondii triggered neutrophil synthesis of CC chemokine ligand (CCL)3, CCL4, CCL5, and CCL20, chemokines that were strongly chemotactic for immature DC. Moreover, supernatants obtained from parasite-stimulated polymorphonuclear leukocytes induced DC IL-12(p40) and TNF-alpha production. Parasite-triggered neutrophils also released factors that induced DC CD40 and CD86 up-regulation, and this response was dependent upon parasite-triggered neutrophil TNF-alpha production. In vivo evidence that polymorphonuclear leukocytes exert an important influence on DC activation was obtained by examining splenic DC cytokine production following infection of neutrophil-depleted mice. These animals displayed severely curtailed splenic DC IL-12 and TNF-alpha production, as revealed by ex vivo flow cytometric analysis and in vitro culture assay. Our results reveal a previously unrecognized regulatory role for neutrophils in DC function during microbial infection, and suggest that cross-talk between these cell populations is an important component of the innate immune response to infection.
Airway response to concomitant exposure with
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Eldridge MW, Peden DB. Airway response to concomitant exposure with
Inhalation of low dose endotoxin by human volunteers favors a local TH2 response profile and primes airway phagocytes in vivo
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Alexis N, Lay JC, Almond M, Peden DB. Inhalation of low dose endotoxin by human volunteers favors a local TH2 response profile and primes airway phagocytes in vivo. J Allergy Clin Immunol 2004;114: 1325-31.
Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection
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Bennouna S, Bliss SK, Curiel TJ, Denkers EY. Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection. J Immunol 2003;171: 6052-8.
Dendritic cells and the control of immunity
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