[show abstract][hide abstract] ABSTRACT: Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role in the processing and/or secretion of procollagen. However, the knowledge on which cells are actually synthesizing HSP47 in the lung parenchyma in pulmonary fibrosis was only limited. The aim of the present study was to investigate the localization of HSP47 messenger ribonucleic acid (mRNA) in normal lung and in the lungs of mice in bleomycin-induced pulmonary fibrosis, using in situ hybridization. For the purpose, ICR mice were intravenously injected with 10 mg/kg per day of bleomycin for five consecutive days. The lung cells expressing HSP47 mRNA were identified in control (saline alone) and bleomycin-treated mice by in situ hybridization. The signal for HSP47 mRNA was markedly increased in bleomycin-treated lungs compared with that of controls. HSP47 mRNA was localized in alpha-smooth-muscle-actin-positive myofibroblasts, surfactant-protein-A-positive type II pneumocytes, and F4/80-positive macrophages in the active fibrotic areas. These results suggest that these cells may synthesize procollagen in the fibrotic process of bleomycin-treated lungs through upregulation of HSP47 mRNA and play an important role in fibrogenesis.
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 03/2010; 456(3):309-15. · 2.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neutrophils and lung fibroblasts are thought to play a role in the pathogenesis of pulmonary fibrosis. We reported previously that heat shock protein 47 (HSP47), a collagen-specific molecular chaperon, and collagen-1 synthesis were involved in pulmonary fibrosis, and that plasma levels of alpha-defensins (HNP; human neutrophil peptide), cationic proteins with antimicrobial and cytotoxic activity in neutrophils, were significantly higher in patients with idiopathic pulmonary fibrosis than in control subjects. Here, we investigated the direct effect of HNP-1 in vitro on the expression of HSP47 and collagen-1 in human lung fibroblasts (NHLF). HNP-1 at 5 microg/ml induced fibroblast proliferation but at concentrations >50 microg/ml, HNP-1 reduced cell viability. Incubation of NHLF with 10 to 25 microg/ml of HNP-1 for 24-h increased the expression of HSP47 and collagen-1 mRNAs (p<0.05). The levels of HSP47 protein also increased significantly at 50 microg/ml, and those of collagen-1 protein increased at 10 to 50 microg/ml of HNP-1 (p<0.05). The mitogen-activated protein kinase (MAPK) signaling pathway in NHLF was activated by HNP-1 stimulation, but inhibitor of MEK (PD98059) did not block HNP-1-induced HSP47 protein production. Our results suggest that alpha-defensin is a fibrogenic mediator that promotes collagen synthesis through the upregulation of HSP47 and collagen-1 in lung fibroblasts and participates in the pathogenesis of neutrophil-induced pulmonary fibrosis.
Life Sciences 05/2007; 80(20):1839-45. · 2.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interferon gamma-inducible protein (IP)-10 and epithelial neutrophil-activating peptide (ENA)-78 belong to the CXC chemokine family and are important factors in inflammatory lung diseases. In sarcoidosis, the potential role of IP-10 to regulate the migration and activation of T-cells towards sites of sarcoid activity has been suggested.
In this study, the concentrations of IP-10 and ENA-78 in the serum and BAL fluid of patients with different stages of active pulmonary sarcoidosis (n=41) and healthy subjects (n=12) were measured by enzyme-linked immunosorbent assay to evaluate the contribution of these CXC chemokines to this disease.
Serum and BAL fluid concentrations of IP-10 and BAL fluid levels of ENA-78 in patients with sarcoidosis were significantly higher than those in control subjects. The serum levels of IP-10 were significantly increased only in patients with stages I and II sarcoidosis, while BAL fluid levels of ENA-78 were increased only in stage III sarcoidosis. In addition, serum concentrations of IP-10 were elevated in patients with extrapulmonary lesions compared with those without such lesions. In patients with sarcoidosis, IP-10 concentrations in BAL fluid correlated with lymphocyte proportions in BAL fluid.
IP-10 may play an important role in regulating lymphocytes into the lung and that ENA-78 may be associated with lung parenchymal disease in pulmonary sarcoidosis.
[show abstract][hide abstract] ABSTRACT: Epithelial neutrophil-activating peptide 78 (ENA-78) and interferon gamma-inducible protein 10 (IP10) belong to the CXC chemokine family and are considered to be important factors in idiopathic pulmonary fibrosis (IPF). Idiopathic nonspecific interstitial pneumonia (NSIP) and IPF are the two largest subsets of idiopathic interstitial pneumonias (IIP). In patients with NSIP, the prognosis is generally good compared with IPF. Therefore, the pathogenesis of NSIP seems to be different from that of IPF, but this remains unclear. The aim of the present study was to evaluate the contribution of ENA-78 and IP10 in the two diseases.
We measured the levels of ENA-78 and IP10 in serum and bronchoalveolar lavage fluid (BALF) of patients with IPF (n=17), idiopathic NSIP (n=10) and healthy subjects (n=12) by enzyme-linked immunosorbent assays.
The level of ENA-78 in BALF was significantly higher in IPF patients than in NSIP patients and controls. Serum levels of ENA-78 and BALF levels of IP10 in NSIP patients were significantly higher than in patients with IPF and controls. In BALF of patients with NSIP, IP10 level significantly correlated with the absolute number of lymphocytes. In IPF patients, BALF IP10 levels also correlated with the proportion of lymphocytes in BALF.
Our results show distinct profiles of CXC chemokines in IPF and NSIP, and suggest that these chemokines play an important role in inflammatory cell recruitment into the lung in patients with IIP.
Respiratory Medicine 10/2005; 99(9):1145-51. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Defensins are cysteine-rich cationic antimicrobial peptides that play an important role in innate immunity and are known to contribute to the regulation of host adaptive immunity. In addition to direct antimicrobial activities, it has been recently reported that alpha-defensins, mainly present in neutrophils in the lung, have a cytotoxic effect and induce IL-8 production from airway epithelial cells. Although beta-defensins are expressed in epithelial cells in various tissues, including lung, there are no reports of their effects on cytokine synthesis in airway epithelial cells. The aim of the present study was to determine the effects of both alpha- and beta-defensins on the cytokine production, transcription factor binding activity, and cytotoxicity in primary cultured human bronchial epithelial cells (HBECs). We used human neutrophil peptide-1 (HNP-1; alpha-defensin) and human beta-defensin-2 (HBD-2) to stimulate HBECs. The results showed that treatment of HBECs with HNP-1, but not HBD-2, increased IL-8 and IL-1beta mRNA expression in a dose-dependent manner and also enhanced IL-8 protein secretion and NF-kappaB DNA binding activity. The 24-h treatments with >20 microg/ml of HNP-1 or >50 microg/ml of HBD-2 were cytotoxic to HBECs. These results suggest that alpha- and beta-defensins have different effects on cytokine synthesis by airway epithelial cells, and we speculate that they play different roles in inflammatory lung diseases.
[show abstract][hide abstract] ABSTRACT: Studies from our laboratory have shown that human alveolar macrophages (AM) and bronchial epithelial cells (HBEC) exposed to ambient particles (PM10) in vitro increase their production of inflammatory mediators and that supernatants from PM10-exposed cells shorten the transit time of monocytes through the bone marrow and promote their release into the circulation.
The present study concerns co-culture of AM and HBEC exposed to PM10 (EHC-93) and the production of mediators involved in monocyte kinetics measured at both the mRNA and protein levels. The experiments were also designed to determine the role of the adhesive interaction between these cells via the intercellular adhesion molecule (ICAM)-1 in the production of these mediators.
AM/HBEC co-cultures exposed to 100 microg/ml of PM10 for 2 or 24 h increased their levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, macrophage inflammatory protein (MIP)-1beta, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and ICAM-1 mRNA, compared to exposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF, MIP-1beta and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to control cells (p < 0.05). There was synergy between AM and HBEC in the production of GM-CSF, MIP-1beta and IL-6. But neither pretreatment of HBEC with blocking antibodies against ICAM-1 nor cross-linking of ICAM-1 on HBEC blocked the PM10-induced increase in co-culture mRNA expression.
We conclude that an ICAM-1 independent interaction between AM and HBEC, lung cells that process inhaled particles, increases the production and release of mediators that enhance bone marrow turnover of monocytes and their recruitment into tissues. We speculate that this interaction amplifies PM10-induced lung inflammation and contributes to both the pulmonary and systemic morbidity associated with exposure to air pollution.
Respiratory research 02/2005; 6:87. · 3.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interleukin-18 (IL-18) is a proinflammatory cytokine that can induce interferon-gamma (IFN-gamma), and it plays an important role in T-helper 1 responses. Among idiopathic interstitial pneumonia (IIP), nonspecific interstitial pneumonia (NSIP) has an increased number of lymphocytes in bronchoalveolar lavage (BAL) fluid compared with usual interstitial pneumonia (UIP). However, the difference in their pathogenesis is unclear.
The present study aims to investigate the roles of IL-18 in patients with idiopathic UIP and idiopathic NSIP.
We measured the serum and BAL fluid (BALF) levels of IL-18 and IFN-gamma in 22 patients with IIP (12 with UIP, 10 with NSIP) and 9 healthy volunteers.
Lymphocyte proportions in BALF were significantly higher in NSIP than in UIP and healthy subjects. No significant differences were observed in the serum IL-18 levels of all subjects, while the BALF levels of IL-18 in patients with NSIP were significantly higher than in patients with UIP (p < 0.005) and in healthy subjects (p < 0.005). Among all subjects, the levels of IL-18 in BALF correlated significantly with those in serum and the lymphocyte proportions in BALF. The serum IFN-gamma levels of all subjects were below sensitivity, but there was significant reverse correlation between the levels of IFN-gamma and the lymphocyte proportions in BALF.
The lymphocytosis in BALF of patients with idiopathic NSIP and a part of idiopathic UIP might be associated with the high levels of IL-18.
[show abstract][hide abstract] ABSTRACT: Chemokines such as regulated on activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, monocyte inflammatory protein (MIP)-lalpha have been reported to play an important role in the pathogenesis of interstitial lung diseases. Among idiopathic interstitial pneumonia (IIP), nonspecific interstitial pneumonia (NSIP) has elevated percentages of Lymphocytes in bronchoalveolar lavage (BAL) fluid compared with usual interstitial pneumonia (UIP). These chemokines are candidate mediators for lymphocyte attraction to the lung in NSIR Therefore, we measured the BAL fluid levels of RANTES, MCP-1 and MIP1-alpha in 15 patients with idiopathic NSIP, 20 with idiopathic UIP, 22 with sarcoidosis and 12 healthy volunteers to evaluate the contribution of these chemokines using enzyme-linked immunosorbent assays. The levels of RANTES in BAL fluid were significantly higher in patients with NSIP compared with healthy volunteers (P < 0.01), UIP and sarcoidosis (P < 0.05). In MCP-1, the levels in BAL fluid of NSIP and UIP patients were significantly elevated compared with healthy volunteers and sarcoidosis patients (P < 0.01). These results suggest that RANTES and MCP-1 in BAL fluid may play an important role in inflammatory cell recruitment to the lung in idiopathic NSIP as well as other interstitial lung diseases.
Respiratory Medicine 11/2004; 98(10):945-51. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Amyopathic dermatomyositis (ADM) is a clinical subtype of dermatomyositis, characterized by the lack of motor weakness and the presence of normal muscle enzyme levels. ADM is sometimes accompanied by interstitial pneumonia that shows a rapid progressive course associated with a poor prognosis. We report a 49-year-old patient who presented with nonspecific interstitial pneumonia (NSIP) associated with ADM. The patient failed to respond to prednisolone and immunosuppressive therapy and died. Although idiopathic NSIP is known to have a better prognosis, NSIP in ADM could be a fatal disease. Therefore, we should appropriately treat interstitial pneumonia in ADM even if it is NSIP.
Internal Medicine 10/2004; 43(9):838-42. · 0.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Alveolar macrophages (AM) play a key role in clearing atmospheric particulates from the lung surface and stimulating epithelial cells to produce proinflammatory mediators. The present study examines the role of "acute response" cytokines TNF-alpha and IL-1 beta released by AM exposed to ambient particulate matter with a diameter of <10 microm (PM(10)) in amplifying the proinflammatory mediator expression by A549 cells and human bronchial epithelial cells (HBEC). The results showed that supernatants from human AM incubated 24 h with PM(10) (100 microg/ml) contained more TNF-alpha, IL-1 beta, granulocyte-macrophage colony stimulating factor, IL-6, and IL-8 than nonexposed AM supernatants. The 3-h treatment of A549 cells with PM(10)-exposed AM supernatants increased TNF-alpha, IL-1 beta, IL-8, regulated on activation normal T-cells expressed and secreted (RANTES), and leukemia inhibitory factor mRNA compared with the treatment with nonexposed AM supernatants and, compared with untreated A549 cells, additionally increased ICAM-1 and monocyte chemotactic protein-1 mRNA. Preincubating PM(10)-exposed AM supernatants with anti-IL-1 beta antibodies reduced all the above mediators as well as VEGF mRNA expression (P < 0.05), while anti-TNF-alpha antibodies were less effective (P > 0.05), and the combination of the two antibodies most effective. When HBEC were treated similarly, anti-TNF-alpha antibodies had the greatest effect. In A549 cells PM(10)-exposed AM supernatants increased NF-kappa B, activator protein (AP)-1 and specificity protein 1 binding, while anti-TNF-alpha and anti-IL-1 beta antibodies reduced NF-kappa B and AP-1 binding. We conclude that AM-derived TNF-alpha and IL-1 beta provide a major stimulus for the production of proinflammatory mediators by lung epithelial cells and that their relative importance may depend on the type of epithelial cell target.
[show abstract][hide abstract] ABSTRACT: Diffuse panbronchiolitis (DPB) is a chronic lower respiratory tract infection commonly associated with persistent late-stage Pseudomonas aeruginosa infection. However, low-dose long-term therapy with certain macrolides is effective in most patients with DPB. The present study was designed to examine the effects of long-term erythromycin (ERY) therapy by using our established murine model of chronic respiratory P. aeruginosa infection. ERY or saline was administered from day 80 after intubation with a P. aeruginosa-precoated tube for the subsequent 10, 20, 40, and 80 days. Bacteriologic and histologic analyses of the murine lungs and electron microscopy of the intubated tube were performed. In the murine model, treatment with ERY for 80 days significantly reduced the number of viable P. aeruginosa organisms in the lungs (P < 0.05). The biofilm formed in situ by P. aeruginosa on the inner wall of the inoculation tube placed into the murine bronchus became significantly thinner after 80 days of ERY treatment. We conclude that the clinical efficacy of macrolides in DPB may be due at least in part to the reduction in P. aeruginosa biofilm formation.
Antimicrobial Agents and Chemotherapy 07/2004; 48(6):2251-9. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human T-cell lymphotropic virus type 1 (HTLV-1)-associated bronchiolitis and diffuse panbronchiolitis might overlap. We examined whether these conditions can be differentiated by comparing their clinical features and the effect of long-term macrolide treatment.
Fifty-eight Japanese patients, including 15 with HTLV-1-associated bronchiolitis and 43 with diffuse panbronchiolitis. Both conditions were clinically compared using the clinical criteria for diffuse panbronchiolitis, including findings from CT scans and BAL fluid testing. Pulmonary function, blood gas levels, and cold hemagglutinin (CHA) levels were assessed before and after long-term treatment with macrolides. Interleukin-2 receptor (IL-2R) expression in T cells obtained from the BAL fluid of patients with HTLV-1-associated bronchiolitis or diffuse panbronchiolitis was analyzed by flow cytometry.
Clinical, laboratory, radiologic, and bacterial features were strikingly similar in both groups, except for the fact that patients with HTLV-1-associated bronchiolitis had a higher ratio of IL-2R-positive cells in the BAL fluid. The histopathologic features were also similar. Long-term treatment with macrolides improved PaO(2), FEV(1), and CHA in patients with HTLV-1-associated bronchiolitis to a lesser extent than in those with diffuse panbronchiolitis, and PaO(2) and FEV(1) in the group of patients with HTLV-1-associated bronchiolitis who had high IL-2R levels did not respond after therapy.
These findings showed that the clinicopathologic features of the two conditions are quite similar, suggesting that diffuse panbronchiolitis is a chronic pulmonary manifestation of HTLV-1 infection. However, HTLV-1-associated bronchiolitis might be associated with conditions that are distinct from those of diffuse panbronchiolitis based on the different responses to macrolide treatment and the difference in the number of activated T cells bearing IL-2R in the lungs.
[show abstract][hide abstract] ABSTRACT: Human T-lymphotropic virus type 1 (HTLV-1) carriers are known to develop pulmonary complications characterized by T-lymphocytic alveolitis. The aim of this study was to determine the profile and role of soluble Fas (sFas) and sFas ligand (sFasL) in the lung of asymptomatic HTLV-1 carriers. We measured sFas and sFasL levels in serum and bronchoalveolar lavage fluid (BALF) of 16 seropositive asymptomatic HTLV-1 carriers and 32 healthy subjects. The serum levels of both sFas and sFasL were significantly higher in HTLV-1 carriers than in the control. In BALF, the percentage of lymphocytes and CD4 positive T-cells, and the levels of sFasL were also significantly higher in asymptomatic carriers than the control, but there were no significant differences in sFas levels between the two groups. There was a significant correlation between BALF sFasL levels and serum sFasL levels and percentage of CD4 positive T-cells in BALF. Our results suggest that the increased levels of sFasL in the lung of asymptomatic HTLV-1 carriers are associated with accumulation of CD4 positive T-cells, and that resistance to apoptosis in HTLV-1 infected T-cells and overproduction of sFasL could contribute to T-lymphocytic alveolitis by down-regulating Fas-FasL mediated apoptosis.
Respiratory Medicine 04/2004; 98(3):213-9. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: The 47-kDa heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role during the processing and/or secretion of procollagen. Expression of HSP47 has been reported to increase in parallel with expression of collagens during the progression of various fibrosis models. The aim of the present study was to investigate the association between HSP47 expression and collagen accumulation in bleomycin (BLM)-induced murine fibrosis. We investigated the expression of HSP47 protein and mRNA using immunohistochemical analysis and semi-quantitative RT-PCR in murine BLM-induced pulmonary fibrosis. Immunohistochemical analysis showed that higher expression of HSP47 protein was present in BLM-induced pulmonary fibrosis compared with controls. HSP47 was localized predominantly in alpha-smooth muscle actin-positive myofibroblasts, F4/80 negative, surfactant protein-A-positive type II pneumocytes, and F4/80-positive macrophages. RT-PCR also demonstrated an increase of HSP47 mRNA expression in BLM-treated lungs. Moreover, the relative amounts of HSP47 mRNA correlated significantly with the lung hydroxyproline content as an indicator of pulmonary fibrosis in BLM-treated lungs (r = 0.406, P <0.05). Our results suggest that these cells may play a role in the fibrotic process of BLM-treated lungs through upregulation of HSP47.
[show abstract][hide abstract] ABSTRACT: We examined the hypothesis that ambient particulate matter with a diameter of <10 microm (PM(10))-induced lung inflammation is amplified by latent adenovirus infection. Inflammatory mediator expression in response to PM(10) exposure was compared between adenovirus E1A-transfected A549 alveolar epithelial cells and cells transfected with control plasmid. Messenger RNA was measured by the RNase protection assay and protein by ELISA or immunocytochemistry. Intercellular adhesion molecule-1 and IL-8 mRNA and protein were increased in E1A-positive cells exposed to 500 microg/ml PM(10). Monocyte chemoattractant protein-1 mRNA and protein were unchanged in E1A-positive cells but increased in E1A-negative cells after 100 and 500 microg/ml PM(10) exposure. Electrophoretic mobility shift assays showed increased NF-kappaB and decreased specificity protein 1 nuclear binding in E1A-positive cells exposed to PM(10). These results indicate that E1A modulates cytokine and adhesion molecule expression in epithelial cells in a manner that could amplify PM(10)-induced lung inflammation. We suggest that this amplified inflammatory response may contribute to the pathogenesis of exacerbations of chronic obstructive pulmonary disease associated with exposure to particulate matter air pollution.
[show abstract][hide abstract] ABSTRACT: Exposure to ambient air pollution particles with a diameter of < 10 microm (PM(10)) has been associated with increased cardiopulmonary morbidity and mortality. We postulate that these adverse health effects are related to proinflammatory mediators produced in the lung and released into the circulation where they initiate a systemic inflammatory response. The present study was designed to determine if alveolar macrophages (AMs) and primary human bronchial epithelial cells (HBECs) interact to amplify the production of certain cytokines when exposed to ambient PM(10) (EHC-93). Candidate cytokines were measured at the mRNA level using a RNase protection assay and at the protein level by enzyme-linked immunosorbent assay (ELISA). When AM/HBEC cocultures were exposed to 100 microg/ml of PM(10), levels of tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (IL)-1beta, IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), and IL-8 mRNA increased within 2 h (P < 0.05) and 8 h following exposure compared with control cells. GM-CSF mRNA expression was more rapidly induced in cocultured cells compared with HBECs or AMs alone. The concentrations of TNF-alpha, GM-CSF, IL-1beta, IL-6, and IL-8 in the cocultured supernatants collected after 24 h PM(10) exposure increased significantly compared with control cells. There was a significant synergistic effect between AMs and HBECs in the production of GM-CSF and of IL-6 (P < 0.05). Instillation of supernatants from HBECs cultured with PM(10) into lungs of rabbits failed to increase circulating band cell counts or stimulate the bone marrow. However, those from AM/HBEC cocultures exposed to PM(10) increased circulating band cell counts (P < 0.05) and shortened the transit time of polymorphonuclear leukocytes (PMNs) through the bone marrow compared with control co-cultures (P < 0.01). These results suggest that the interaction between AMs and HBECs during PM(10) exposure contributes to the production of mediators that induce a systemic inflammatory response.
American Journal of Respiratory Cell and Molecular Biology 07/2002; 27(1):34-41. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Studies from our laboratory have shown that exposure to air pollution particles smaller than 10 microm (PM10) induced a systemic inflammatory response that includes the release of granulocytes from the bone marrow. In the present study we tested the hypothesis that mediators released from human alveolar macrophages (AM) exposed to PM10 accelerate the maturation of granulocyte precursors. Human myeloid precursor cells (HL60 cells) were incubated with the supernatant from AM exposed to PM10. Phagocytosis of PM10 by AM resulted in the production of cytokines, particularly interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < .05). The supernatant from AM exposed to PM10 did not influence myeloid cell proliferation but promoted cell differentiation as measured by surface GD11b and CD14 expressions compared to control supernatant (P < .05). This effect of exposed-AM supernatants on myeloid cell differentiation was blocked by anti-IL-6 monoclonal antibodies (CD11b and CD14; P < .05) and anti-GM-CSF monoclonal antibodies (CD14, P < .01). We conclude that human AM exposed to PM10 produce mediators, particularly IL-6 and GM-CSF that promote the differentiation of bone marrow myeloid cells and we speculate that these cytokines are involved in the release of granulocytes from the bone marrow associated with exposure to air pollution particulates.
Experimental Lung Research 28(1):1-18. · 1.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study was designed to determine cytokines pro- duced by primary human bronchial epithelial cells (HBECs) ex- posed to ambient air pollution particles (EHC-93). Cytokine messenger RNA (mRNA) was measured using a ribonuclease protection assay and cytokine protein production by enzyme- linked immunosorbent assay. Primary HBECs were freshly iso- lated from operated lung, cultured to confluence, and exposed to 10 to 500 � g/ml of a suspension of ambient particulate matter with a diameter of less than 10 � m (PM 10 ) for 2, 8, and 24 h. The mRNA levels of leukemia inhibitory factor (LIF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 � , and IL-8 were increased after exposure to PM 10 , and this increase was dose-dependent between 100 ( P � 0.05) and 500 ( P � 0.05) � g/ml of PM 10 exposure. The con- centrations of LIF, GM-CSF, IL-1 � , and IL-8 protein measured in the supernatant collected at 24 h increased in a dose- dependent manner and were significantly higher than those in the control nonexposed cells. The soluble fraction of the PM 10 (100 � g/ml) did not increase these cytokine mRNA lev- els compared with control values and were significantly lower compared with HBECs exposed to 100 � g/ml of PM 10 (LIF, IL-8, and IL-1 � ; P � 0.05), except for GM-CSF mRNA ( Pnot sig- nificant). We conclude that primary HBECs exposed to ambi- ent PM 10 produce proinflammatory mediators that contribute to the local and systemic inflammatory response, and we speculate that these mediators may have a role in the patho- genesis of cardiopulmonary disease associated with particu- late air pollution. Recent epidemiologic studies have shown an association between cardiopulmonary morbidity and mortality and levels of ambient particulate matter with a diameter of less than 10 � m (PM 10 ) (1, 2). Residents of communities exposed to high compared with low levels of air pollution have faster rates of decline in lung function, more chronic respiratory and cardiovascular disease, and hospital admissions for pneu- monia, chronic obstructive pulmonary disease (COPD), myocardial infarction, and heart failure after adjusting for several individual risk factors, including cigarette smoking (3). Previous studies in our laboratory showed that small in- ert carbon particles instilled into the lungs of rabbits in- duced a systemic inflammatory response that includes stim- ulation of the bone marrow (4). This observation was supported by a report from Tan and colleagues demon- strating a leukocytosis in young military recruits during an episode of acute air pollution in Southeast Asia (5). This leukocytosis was associated with bone-marrow stimulation and greater release of granulocytes from the bone marrow into the circulation (5). Stimuli such as acute pneumonia