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ABSTRACT: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease-modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self-maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8(+) T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7-1/CTLA4, 4-1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co-stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28(null) cells in COPD by measuring their production of proinflammatory cytokines, co-stimulatory molecules, granzyme and perforin. A smoke-exposed murine model was applied to investigate the relative expression of CD8/CD28(null) T cells in blood, lung tissue and airway. CD8/CD28(null) cells were increased in both current- and ex-smoker COPD groups; these cells expressed significantly more interferon (IFN)-γ, OX40, 4-1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28(+) T cells. There were no changes in CD4/CD28(null) T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28(null) T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co-stimulatory molecules by CD8/CD28(null) T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets.
Clinical & Experimental Immunology 10/2011; 166(1):94-102. · 3.36 Impact Factor
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ABSTRACT: Paediatric oncology patients with febrile neutropenia are usually hospitalised and treated with empirical broad-spectrum antibiotic therapy to counter the risk of infection. However, there is currently no method available to rapidly identify bacteremia in these patients. T-helper-type-1 (Th1) cytokines are required for effective immune response to many pathogenic organisms and T regulatory cells are known suppressors of Th1 cells. We hypothesized that characterization of reduced intracellular Th1 cytokines and increased T regulatory cells (Tregs) may prove useful in identifying infection in childhood oncology patients with febrile neutropenia.
Intracellular Th 1 cytokines and Tregs were enumerated in peripheral blood from a group of childhood oncology patients with febrile neutropenia using multiparameter flow cytometry.
There was a significant increase in the percentage of CD25(+) CD127(-) CD8(-) CD3(+) Tregs and a significant decrease in Th1 intracellular cytokines IFNγ, IL-2 and TNFα in the blood of culture positive patients compared with culture negative patients.
Enumeration of Tregs and intracellular Th1 cytokines may provide a sensitive, specific test for determining infection in childhood oncology patients before blood culture results become available.
Cytokine 03/2011; 53(3):286-8. · 3.02 Impact Factor
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ABSTRACT: Immunosuppression therapy following lung transplant fails to prevent chronic rejection/bronchiolitis obliterans syndrome, which we have shown is associated with lack of suppression of peripheral blood T cell granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. We hypothesized that these proinflammatory mediators may increase with time post-transplant in otherwise stable patients before clinical signs of declining lung function, and patients experiencing declining lung function would show a further increase in these mediators. Intracellular cytokine profiles and granzyme B were investigated in T cells in whole blood and airways from lung transplant patients using flow cytometry. There was a significant negative correlation between forced expiratory volume in 1 s (FEV(1) ), drug dose and time post-transplant. A significant correlation between increased granzyme B, IFN-γ, interleukin (IL)-2 and TNF-α and time post-transplant was noted in peripheral blood T cells but not lung T cells from stable patients. Patients with similar drug dose but experiencing declining FEV(1) showed a further increase in peripheral blood T cell IFN-γ, IL-2 and TNF-α. Time post-lung transplant correlates with increasing peripheral blood T cell granzyme B and proinflammatory cytokines. Declining FEV(1) is associated with a further increase in these proinflammatory mediators. Drugs that reduce these inflammatory mediators effectively may reduce the incidence of chronic graft rejection.
Clinical & Experimental Immunology 09/2010; 161(3):584-90. · 3.36 Impact Factor
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ABSTRACT: Bronchiolitis obliterans syndrome (BOS) is characterized by persistent alloreactive, infective and non-specific epithelial injury, loss of epithelial integrity and dysregulated repair. We have reported increased apoptosis of epithelial cells collected from the large airway in lung transplant recipients. As part of the alloreactive response, T cells induce apoptosis of target epithelial cells by secreting granzyme b. We hypothesized that granzyme b would be increased in lung transplant patients with acute rejection and BOS and that commonly used immunosuppressive agents would fail to suppress this serine protease adequately. We investigated intracellular T cell granzyme b in blood, bronchoalveolar lavage (BAL) and large airway brushing (23 controls, 29 stable transplant, 23 BOS, 28 acute rejection, 31 infection) using flow cytometry and assessed the effect of clinically relevant concentrations of cyclosporin A, tacrolimus, methylprednisolone and a protease inhibitor, gabexate mesilate, on in vitro granzyme b production. Granzyme b was increased significantly in all compartments of all transplant groups compared to controls. Surprisingly, granzyme b was even higher in patients with BOS than in patients with acute rejection. In longitudinal analysis in three patients, blood granzyme b increased prior to or at the onset of BOS. In vitro, methylprednisolone and gabexate mesilate had no effect and cyclosporin A and tacrolimus only a moderate effect on production of granzyme b by CD8(+) T cells. Increased T cell granzyme b production may contribute to BOS pathogenesis and is not curtailed by current immunosuppressants. Longitudinal investigation of granzyme b in blood may provide an adjunctive non-invasive method for predicting BOS/OB.
Clinical & Experimental Immunology 09/2009; 158(2):230-6. · 3.36 Impact Factor
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ABSTRACT: Bronchiolitis obliterans syndrome (BOS) compromises lung transplant outcomes and is characterised by airway epithelial damage and fibrosis. The process whereby the normal epithelial configuration is replaced by fibroblastic scar tissue is poorly understood, but recent studies have implicated epithelial mesenchymal transition (EMT). The primary aim of this study was to assess the utility of flow cytometry in detecting and quantifying EMT in bronchial epithelial cells. Large airway brushings were obtained at 33 bronchoscopies in 16 BOS-free and 6 BOS grade 1-3 patients at 2-120 months posttransplant. Flow cytometry was used to assess expression of the mesenchymal markers alphaSMA, S100A4 and ED-A FN and HLA-DR. TGF beta 1 and HGF were measured in Bronchoalveolar lavage (BAL). Expression of all three mesenchymal markers was increased in BOS, as was HLA-DR. BAL HGF, but not TGF beta 1 was increased in BOS. Longitudinal investigation of one patient revealed a 100% increase in EMT markers concurrent with a 6-fold increase in BAL TGF beta 1 and the diagnosis of BOS at 17 months posttransplant. Flow cytometric evaluation of bronchial epithelium may provide a novel and rapid means to assess lung allografts at risk of BOS.
American Journal of Transplantation 05/2009; 9(4):727-33. · 6.39 Impact Factor
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ABSTRACT: Current immunosuppression protocols to prevent lung transplant rejection reduce pro-inflammatory and T-helper type 1 (Th1) cytokines. However, Th1 T-cell pro-inflammatory cytokine production is important in host defense against bacterial infection in the lungs. Excessive immunosuppression of Th1 T-cell pro-inflammatory cytokines leaves patients susceptible to infection. To investigate whether pulmonary infection in lung transplant recipients is associated with reduced Th1 T-cell pro-inflammatory cytokines, whole blood and bronchoalveolar lavage (BAL) fluid from 13 stable lung transplant patients with 'culture-negative' BAL and 13 patients with 'culture-positive' BAL was stimulated in vitro, and cytokine production by CD8+ and CD4+ T-cell subsets was determined using multiparameter flow cytometry. In BAL samples, there was a significant decrease in interleukin-2 (IL2) in CD3+ T cells and tumor necrosis factor-alpha (TNF-alpha) in CD8+ T cells (but not CD4+) in 'culture-positive' compared with 'culture-negative' transplant patients. There was no difference in blood Th1 T-cell cytokines between 'culture-positive' compared with 'culture-negative' transplant patients. A decrease in Th1 cytokines IL-2 and TNF-alpha in BAL T-cell subsets is associated with isolation of potentially pathogenic organisms in the lungs in stable lung transplant patients. Excessive immunosuppression of these Th1 T-cell pro-inflammatory cytokines in stable transplant patients may leave them susceptible to infection. Modifying immunosuppression by monitoring intracellular Th1 pro-inflammatory cytokines in BAL T cells may help to improve morbidity and infection rates in stable lung transplant patients.
Transplant Infectious Disease 05/2008; 10(2):99-105. · 2.22 Impact Factor
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ABSTRACT: The role of T cells in the pathophysiology of chronic obstructive pulmonary disease (COPD) is not yet certain, although varying reports have shown increases in T helper 1 (Th1) and/or Th2 cytokines in peripheral blood and bronchoalveolar lavage (BAL). No studies have examined cytokine production by intraepithelial T cells obtained by bronchial brushing (BB). Intracellular cytokine analysis of T cell subsets from peripheral blood, BAL and BB from smoker and ex-smoker COPD patients, COPD patients receiving inhaled corticosteroids and smoker and non-smoker control subjects was studied using multi-parameter flow cytometry. CD4 : CD8 inversion was noted in the peripheral blood of smoker and ex-smoker COPD groups, in BAL and BB from smoker controls and BAL of COPD smokers. There was an increase in intracellular CD8(+) T cell Th1 proinflammatory cytokines in some COPD groups in the peripheral blood and in CD8(+) T cell tumour necrosis factor (TNF)-alpha in some COPD groups and smoker controls in BAL and BB. There was an increase in proinflammatory cytokines in COPD smokers compared with ex-smokers and a decrease in COPD smokers receiving inhaled corticosteroids in the airways. There was a negative correlation between forced expiratory volume in 1 s (FEV(1)) and the percentage of BAL and intraepithelial CD8(+) T cells producing TNF-alpha. COPD patients exhibit systemic inflammation as evidenced by increased intracellular Th1 proinflammatory cytokines in blood, BAL and intraepithelial CD8(+) T cells, whereas smoker controls showed localized Th1 response in the lung only. Systemic therapeutic targeting of TNF-alpha production by CD8(+) T cells may improve morbidity in COPD patients while targeting of TNF-alpha in the lung may prevent smokers progressing to COPD.
Clinical & Experimental Immunology 11/2007; 150(1):22-9. · 3.36 Impact Factor
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ABSTRACT: Chronic obstructive pulmonary disease (COPD) is associated with increased apoptosis and defective phagocytosis in the airway. As uncleared cells can undergo secondary necrosis and perpetuate inflammation, strategies to improve clearance would have therapeutic significance. There is evidence that the 15-member macrolide antibiotic azithromycin has anti-inflammatory properties. Its effects may be increased in the lung due to its ability to reach high concentrations in alveolar macrophages (AMs). The present study investigated the effects of low-dose (500 ng x mL(-1)) azithromycin on the phagocytosis of apoptotic bronchial epithelial cells and neutrophils by AMs. Flow cytometry was applied to measure phagocytosis and receptors involved in AM recognition of apoptotic cells. Cytokines were investigated using cytometric bead array. Baseline phagocytosis was reduced in COPD subjects compared with controls. Azithromycin significantly improved the phagocytosis of epithelial cells or neutrophils by AMs from COPD subjects by 68 and 38%, respectively, often up to levels comparable with controls. The increase in phagocytosis was partially inhibited by phosphatidylserine, implicating the phosphatidylserine pathway in the pro-phagocytic effects of azithromycin. Azithromycin had no effect on other recognition molecules (granulocyte-macrophage colony-stimulating factor, CD44, CD31, CD36, CD91, alphavbeta3 integrin). At higher doses, azithromycin decreased levels of pro-inflammatory cytokines. Thus, low-dose azithromycin therapy could provide an adjunct therapeutic option in chronic obstructive pulmonary disease.
European Respiratory Journal 10/2006; 28(3):486-95. · 5.89 Impact Factor
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ABSTRACT: Allograft rejection remains a major cause of morbidity and mortality following lung transplantation and is associated with an increased expression of T cell proinflammatory cytokines. We have shown that CD4(+) T cell proinflammatory cytokine production was significantly reduced in peripheral blood and bronchoalveolar lavage (BAL) of stable lung transplant patients, consistent with immunosuppression therapy. However, analysis of inflammatory cytokine profiles of intraepithelial T cells in bronchial brushing (BB) may be more relevant than peripheral blood or BAL T cells for assessing immune graft status. To investigate the immunomodulatory effects of currently used immunosuppressive regimens on bronchial intraepithelial T cell cytokine production, whole blood, BAL and BB from stable lung transplant patients and control volunteers were stimulated in vitro and cytokine production by CD8(+) and CD4(+) T cell subsets determined using multi-parameter flow cytometry. In bronchial intraepithelial T cell subsets in control subjects and transplant patients there was compartmentalization of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha production, a decrease in interleukin (IL)-2 production by CD4(+) T cells and CD4 : CD8 inversion compared with blood and BAL. Although there was a decrease in T cell proinflammatory cytokine production in blood of transplant patients, this was not found in BAL or bronchial intraepithelial CD8 T cell subsets, suggesting that the same level of immunosuppression may not occur in the lung of transplant recipients. Drugs that effectively reduce CD8 T cell proinflammatory cytokine production in the lung compartment may improve current protocols for reducing graft rejection in these patients.
Clinical & Experimental Immunology 10/2006; 145(3):413-9. · 3.36 Impact Factor
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British Journal of Haematology 02/2006; 132(2):247-8. · 4.94 Impact Factor
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ABSTRACT: Intracytoplasmic detection of leucocyte cytokines has become a powerful tool for the characterisation of cytokine-producing cells in heterogeneous cell populations, however the effect of specimen storage conditions is unknown. The aim of this study was to determine the effect of whole blood stored at room temperature (RT) or 4 degrees C, on intracellular cytokine production by T cells and monocytes. In cell cultures stored at RT or 4 degrees C for 24h, significant changes in several leucocyte cytokines/chemokines were shown compared to blood cultures stimulated at time=0. There was a significant decrease in IL-2, IL-4 and TNFalpha production by CD4+ T cells in blood cultures stored at RT but an increase in IL-2 in cultures at 4 degrees C. There was a significant decrease in TGFbeta production by CD4+ and CD8+ T cells in cultures kept at RT or 4 degrees C. There was a significant increase in MCP-1 and MCP-3 production by monocytes in blood cultures kept at RT or 4 degrees C. There was a decrease in IL-12 production by monocytes in cultures kept at 4 degrees C, whereas IL-10 production was decreased at RT and increased in cultures kept at 4 degrees C. Blood stored at 4 degrees C showed less immunomodulatory changes than blood kept at RT although overall a possible Th1 bias at 4 degrees C.
Cytokine 11/2005; 32(1):7-11. · 3.02 Impact Factor
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ABSTRACT: There is heterogeneity in the propensity of smokers to develop chronic obstructive pulmonary disease (COPD), and improved treatment strategies are hindered by limited understanding of COPD pathogenesis, especially as distinct from the effects of smoking per se. Although apoptosis is essential for tissue homeostasis, increased apoptosis may cause tissue damage and inflammation. This study addressed whether airway T-lymphocytes and airway epithelial cells (AEC) show an increased likelihood of undergoing apoptosis in COPD and if this was related to smoking. Apoptosis (7-amino-actinomycin D, Annexin, single-stranded DNA and caspase), Bcl-2, Bax and p53 were assessed in cells obtained from bronchial bushing and bronchoalveolar lavage from ex- and continuing smokers with COPD, and nonsmoking controls, using flow cytometry. A mean 87% increase in apoptosis of AEC and a 103% increase in T-lymphocyte apoptosis were found in COPD. There were no significant differences in apoptosis of AEC between current and ex-smokers with COPD. Apoptosis may contribute to chronic obstructive pulmonary disease pathogenesis, and continued excess apoptosis after smoking cessation may offer a new target for therapeutic interventions. Whether the persistence of increased apoptosis after smoking cessation results from changes in the pulmonary milleau after years of noxious insult, or whether some individuals have a natural predisposition toward increased apoptosis and possible development of chronic obstructive pulmonary disease remains to be determined.
European Respiratory Journal 04/2005; 25(3):447-54. · 5.89 Impact Factor
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ABSTRACT: Allograft rejection remains a major cause of morbidity and mortality following lung transplantation and is associated with an increase in T-cell pro-inflammatory cytokine expression. Systemic levels of immunosuppressive drugs used to reduce pro-inflammatory cytokine expression are closely monitored to their 'therapeutic range'. However, it is currently unknown if levels of these drugs correlate with pro-inflammatory cytokine expression in peripheral blood T cells. To investigate the immunomodulatory effects of currently used immunosuppressive regimes on peripheral blood T-cell cytokine production, whole blood from stable lung transplant patients and control volunteers were stimulated in vitro and cytokine production by CD8+ and CD4+ T-cell subsets determined using multiparameter flow cytometry. T-cell IL-2 and TNFalpha production was significantly reduced from lung transplant patients compared to controls. CD4+ T-cell production of IFNgamma was also significantly reduced from lung transplant patients but production of IFNgamma by CD8+ T cells remained unchanged. There was an excellent correlation between the percentage of CD8+ T cells and the percentage of CD8+ T cells producing IFNgamma from transplant patients. T-cell IL-4 and CD8+ T-cell production of TGFbeta was significantly increased from lung transplant patients. We now provide evidence that current immunosuppression protocols have limited effect on peripheral blood IFNgamma production by CD8+ T-cells but do up-regulate T-cell anti-inflammatory cytokines. Drugs that effectively reduce IFNgamma production by CD8+ T cells may improve current protocols for reducing graft rejection in these patients. Intracellular cytokine analysis using flow cytometry may be a more appropriate indicator of immunosuppression than drug levels in these patients. This technique may prove useful in optimizing therapy for individual patients.
Clinical & Experimental Immunology 02/2005; 139(1):159-64. · 3.36 Impact Factor
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ABSTRACT: Early diagnosis of neonatal infection has proved problematic due to the inadequacy of currently available laboratory tests. Neonatal sepsis is associated with an increase in plasma-derived cytokine levels, but an increase of a single cytokine cannot identify neonatal sepsis specifically and multiple cytokine levels are required. The time constraints and relatively large volume of plasma required to measure multiple cytokines from newborn infants by conventional enzyme-linked immunosorbent assay (ELISA) techniques is prohibitive. We therefore applied cytometric bead array (CBA) technology for simultaneous measurement of multiple cytokines from a group of 18 term neonates with infection confirmed by culture and a control group. 'Normal' ranges were established for each cytokine from 1-7-, 8-14- and 15-21-day-old newborns. There was no significant change in the levels of cytokines from infants in different control age groups, suggesting that basal cytokine levels are unchanged in the first 3 weeks of life. In the patient groups, however, there was a significant difference in several cytokines between the different age groups. Interleukin (IL)-6, IL-10 and IL-12 were increased significantly in the 1-7-day-old patient group compared to either the 8-14 and 15-21 age group, suggesting that infection in utero is associated with increased levels of these cytokines compared to infection acquired following birth. When individual patient cytokine levels were compared to normal control reference ranges, two patients failed to show significant elevation of any cytokine tested. All other patients showed elevated levels of between one and nine cytokines tested (mean of 4.6). There was no correlation between elevated cytokine levels and types of infective organism or patient age. In conclusion, neonatal sepsis is associated with the elevation of multiple plasma cytokines. The use of CBA kits is a rapid, easy, low sample volume and sensitive method to measure multiple plasma cytokines.
Clinical & Experimental Immunology 09/2004; 137(2):402-7. · 3.36 Impact Factor
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ABSTRACT: Diagnosis of congenital or neonatal infection is often based on clinical signs. However, clinical symptoms of infections may not be specific, and for this reason early diagnosis is often determined on results of laboratory tests, which may not currently be adequate. A more reliable method of detection of infection may be the demonstration of activated lymphocytes, which can be conducted rapidly and before the isolation of the infected organism. We have shown that detection of up-regulation of CD45RO, an activated/memory isoform of CD45 present on T cells, provides a reasonably sensitive screening test for neonatal infection. We also showed that dual expression of CD45RA/CD45RO was up-regulated early during the infective process in neonates with documented infection. However, other leucocytes are also activated during the infective process. To improve the sensitivity of the neonatal infection screening test and to identify the types of leucocytes involved in the immune response to the infective organism, we studied further the up-regulation of a comprehensive range of surface activation markers on T cells, monocytes and natural killer (NK) cells from a group of 17 newborn patients with positive culture, a group of 40 possibly infected patients based on clinical signs and a control group. 'Normal' ranges were established for each activation marker for each leucocyte subset from 1 to 7 and 7-14-day-old newborns <35 weeks' gestation and 35-40 weeks' gestation. There was a significant increase in the percentage of T cells expressing CD25 in the peripheral blood from infants at 2 weeks of age. Expression of HLA-DR on T cells, CD25 and CD69 on monocytes and HLA-DR on NK cells was also increased significantly in the peripheral blood from infants at 2 weeks of age and may reflect a maturation of these functional surface molecules. Up-regulation of CD69 on NK cells was the most sensitive marker for neonatal sepsis (positive in 13/16 patients). CD69 and CD25 expression was increased significantly on T cells in 11/17 and 10/17 patients, respectively. A combination of CD45RA/CD45RO and CD45RO identified 11/16 infected patients. Measurement of CD69 expression on NK cells with CD45RA, CD45RO, CD25 and CD69 expression on T cells resulted in a significant increase in at least two leucocyte activation markers from infected patients. In conclusion, this is the first report of the up-regulation of CD69 on NK cells as a sensitive marker of neonatal infection. A combination of this marker with CD45RA, CD45RO, CD25 and CD69 expression on peripheral blood derived T cells is the most sensitive and specific for neonatal infection.
Clinical & Experimental Immunology 01/2004; 135(1):125-9. · 3.36 Impact Factor
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ABSTRACT: Human bronchial epithelial cells are known to secrete an array of inflammatory cytokines including tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-4, which may play a role in immune responses in lung diseases such as chronic obstructive pulmonary disease (COPD). However, the regulatory mechanisms governing cytokine production in bronchial epithelia in COPD are largely unknown. Transforming growth factor-beta (TGF-beta) is an anti-inflammatory cytokine and is involved in airway repair. The purpose of this study was to study the effect of TNF-alpha and IL-4 (pro-inflammatory cytokines known to be up-regulated in COPD), on the production of TGF-beta (a negative regulator of inflammation) by epithelial cells.
A bronchial epithelial cell line was used as an in vitro culture model (16HBE). Cell cultures were stimulated with various combinations of TNF-alpha and IL-4 (20 ng/mL) for 24 h. Transforming growth factor-beta production was measured by flow cytometry, enzyme-linked immunosorbent assay and immunohistochemistry.
Exposure to TNF-alpha significantly up-regulated production of IL-4 from cultured epithelial cells. Unstimulated cells spontaneously released TGF-beta. Exposure to TNF-alpha and IL-4 significantly inhibited production of TGF-beta. The inhibitory effects of TNF-alpha and IL-4 on TGF-beta synthesis were summative.
The inhibitory effect of IL-4 and TNF-alpha on production of the regulatory cytokine TGF-beta in a bronchial epithelial cell line suggests that such mechanisms may contribute to the progression of the inflammatory response and compromise repair processes in inflammatory lung diseases such as COPD.
Respirology 10/2001; 6(3):205-11. · 2.42 Impact Factor
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ABSTRACT: The cellular immune system of the newborn infant is immature and hypo-responsive when compared with adults. The extent to which immaturity of the leucocyte function underlies hyporesponsiveness in the newborn is incompletely understood. In this study flow cytometric techniques were applied to investigate the concurrent expression of a range of surface and intracellular leucocyte functional molecules and cytokines in resting and stimulated cord and adult blood. Production of interleukin (IL)-2 and expression of the components of its receptor, IL-2R alpha/beta/gamma, were investigated. No differences in the proportion of leucocytes producing IL-2R alpha and IL-2R gamma were observed for newborns and adults. A lower proportion of T cells and natural killer (NK) cells from newborns expressed IL-2R beta and upregulation of expression was slower. We hypothesize that reduced IL-2R beta may curtail early autocrine IL-2 activation of immune responses in the newborn. This hypothesis was supported by the observation that an increased proportion of stimulated T cells from newborns produced IL-2 at 4 h poststimulation, but at 24 h the proportion was lower than for adult T cells. The very low levels of interferon (IFN)-gamma produced by neonatal T cells and NK cells may also be partly explained by a curtailment of early autocrine activation of T cells. Expression and kinetics of upregulation for other functional molecules were studied. CD71, HLA-DR, tissue factor and CD152 levels were not significantly different for adults and newborns, suggesting that cord blood leucocytes, in some respects, may demonstrate functional maturity. IL-6 secretion by stimulated monocytes was also comparable in cord and adult blood. However, IL-1 alpha and IL-1 beta were produced by a lower proportion of monocytes from newborns than adults. Similarly, tumour necrosis factor (TNF)-alpha production for monocytes and T cells was lower in cord blood. The mean fluorescence intensity for IL-1 alpha, IL-1 beta and TNF-alpha was also lower for leucocytes from cord blood. These findings are significant in relation to the inability of newborn infants to mount a febrile response to infection. The findings of lower expression of IL-2R beta and lower production of inflammatory cytokines IL-1 alpha, IL-1 beta and TNF-alpha is a basis for improved understanding of the immunological immaturity of leucocytes in the newborn.
Scandinavian Journal of Immunology 02/2001; 53(1):72-8. · 2.23 Impact Factor
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ABSTRACT: Most of the investigatory studies of cytokine production by cells have been performed on purified cells or cell lines by measuring the secreted cytokine levels in the bulk culture supernatant. However, results of cytokine production from isolated peripheral blood mononuclear cells (PBMCs) cultivated in synthetic media, have been reported to be inaccurate and of low reproducibility. Isolation procedures have been shown to be toxic to certain cells. We hypothesised that purified cell culture techniques may result in increased levels of apoptosis of cells compared with whole blood culture techniques. To compare the effects on cell viability between PBMCs and whole blood techniques, an Annexin V binding assay was utilised. The effect of different cell concentration and serum/plasma concentrations on apoptosis levels in the various leukocyte subsets in PBMC and whole blood cultures following stimulation was investigated. There were significantly increased levels of apoptosis of cells in PBMC compared to whole culture at similar plasma concentrations, suggesting that cell viability was plasma concentration-dependent. There were significantly increased levels of apoptosis in PBMC cultures at the same cell concentration to whole blood techniques, suggesting that interaction between all cellular elements (as in whole blood techniques) is important in maintaining cell viability. These results suggest that whole blood culture techniques provide the best conditions for study of leukocyte cytokine production. If PBMC culture is performed, similar plasma and cell concentration to whole blood will best preserve cell viability.
Cytokine 01/2001; 12(12):1763-8. · 3.02 Impact Factor
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ABSTRACT: The kinetics of assembly of the high-affinity interleukin-2 receptor (IL-2R)alpha/beta/gamma were investigated by studying intracellular and surface expression of IL-2Ralpha, beta and gamma by T cells, monocytes and natural killer (NK) cells. IL-2Ralpha and IL-2Rgamma were expressed by small numbers of resting T cells. These numbers increased following stimulation, to maximal expression at 48 h and 72 h, respectively. This observation was consistent with de novo synthesis of the receptor protein in response to the stimulus. The proportion of T cells producing IL-2Rbeta was smaller and up-regulated later than the proportion of cells producing IL-2Ralpha or IL-2Rgamma. IL-2Rbeta may therefore slow the assembly of the high-affinity IL-2R on T cells. A small number of resting NK cells expressed IL-2Ralpha, both on the cell surface and intracellularly, but this increased over 72 h on stimulated NK cells. IL-2Rbeta was constitutively expressed, both on the cell surface and intracellularly, by monocytes and NK cells. An increased proportion of NK cells and monocytes produced IL-2Rbeta, 24 h and 4 h post-stimulation, respectively. Maximal or plateau expression occurred at 72 h and 24 h post-stimulation, for NK cells and monocytes, respectively. The early up-regulation of intracellular IL-2Rbeta for monocytes may facilitate the up-regulation of surface IL-2Rbeta, and early assembly of the high-affinity IL-2R, accelerating monocyte activation and function. High constitutive intracellular IL-2Rgamma expression (> 80%) in all types of leucocyte investigated, decreased over the 72 h following stimulation with a concurrent increase in surface expression. IL-2Rgamma was expressed by increased proportions of T cells, monocytes and NK cells, 4 h following stimulation. The intracellular storage of IL-2Rgamma may accelerate translocation to the cell surface after stimulation. The early translocation of IL-2Rgamma may reflect its usage as a signal transduction molecule by other cytokine receptors - IL-4, IL-7, IL-9 and IL-15. This study delineated the potential expression of the high-affinity IL-2Ralpha/beta/gamma on various stimulated leucocytes. The differential kinetics of assembly of the high-affinity IL-2Ralpha/beta/gamma on different leucocyte subsets suggests that IL-2 may regulate the inflammatory cellular responses in a sequential manner, paralleling the timed expression of IL-2Ralpha/beta/gamma on the monocytes, NK cells and T cells.
Scandinavian Journal of Immunology 02/2000; 51(1):67-72. · 2.23 Impact Factor
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ABSTRACT: Immune abnormalities have been reported in recipients of cellular and plasma blood products. To document the effect of current transfusion practices, we performed ex vivo lymphocyte immunophenotypic studies on patients with thalassaemia major who had received multiple (leucocyte-depleted) transfusions and patients with haemophilia A and B who had received heat viral-inactivated factor concentrates. Patients with thalassaemia major showed a significant lymphocytosis, with mainly B-cell changes consistent with ongoing B-cell stimulation associated with chronic exposure to red cell antigens. Reduced T-cell IL-2Ralpha expression would be consistent with inhibition by desferrioxamine chelation therapy. In contrast, patients with haemophilia showed predominantly T-cell changes. Patients with haemophilia A showed significantly elevated activated CD8+ cytotoxic T lymphocytes whereas those with haemophilia B showed an increase in CD8+CD11adim and CD4+CD45RA+ suppressor T cells. Several of the immune abnormalities found may be due to the presence of cytokines not removed by leucocyte filtration or destroyed by factor concentrate production (e.g. TGF-beta) causing a T-helper-2-like response. The extensive lymphocyte characterization in this study has not previously been performed and has enabled a closer examination of the functional lymphocyte immunophenotypes seen in patients treated according to current transfusion practices.
British Journal of Haematology 07/1999; 105(3):817-25. · 4.94 Impact Factor
