Upregulation of MCAM in primary bronchial epithelial cells from patients with COPD.

An increasing body of evidence indicates that the bronchial epithelium plays a crucial role in the pathophysiology of chronic obstructive pulmonary disease (COPD). The aim of this study was to identify new genes whose bronchoepithelial expression is specifically altered in COPD patients. Primary bronchial epithelial cell (PBEC) cultures were established from exsmokers with stable airflow limitation and never smokers. Complementary deoxyribonucleic acid array technology was used to investigate the differential expression of 847 cytokine and cytokine-related genes between the two groups. Statistical analysis was performed by means of significance analysis of microarrays and Bonferroni-corrected analysis of variance on ranks. Discriminant analysis and light cycler measurements as well as flow cytometry and Western blotting were used to confirm the significance of the array results at both the messenger ribonucleic acid (mRNA) and protein expression levels. With respect to array experiments, melanoma cell adhesion molecule (MCAM) was identified as the sole gene showing highly significant upregulation in PBECs from COPD patients compared to never smokers. Light cycler measurements confirmed these results, revealing a 2.9-fold and 2.0-fold increase in MCAM mRNA expression in COPD patients compared to nonsmokers and smokers, respectively. In addition, these differences are associated with higher median protein expression levels. These results strongly suggest involvement of melanoma cell adhesion molecule in the pathophysiology of the chronic airway inflammation seen in patients with chronic obstructive pulmonary disease.     

Preferential induction of prodestructive matrix metalloproteinase-1 and proinflammatory interleukin 6 and prostaglandin E2 in rheumatoid arthritis synovial fibroblasts via tumor necrosis factor receptor-55

OBJECTIVE: To assess expression and individual functional relevance of tumor necrosis factor receptor 55 (TNF-R55) and TNF-R75 in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFB). METHODS: Seventh to 9th passage RA SFB and OA SFB were analyzed for TNF-R expression by FACS. The SFB were then stimulated with TNF-a (1-10 ng/ml) or agonistic anti-TNF-R55 (HTR-9) and anti-TNF-R75 (UTR-1) monoclonal antibodies (1-25 micro g/ml each). Matrix metalloproteinase-1 (MMP-1), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), interleukin 6 (IL-6), and prostaglandin E(2) (PGE(2)) in culture supernatants were quantified by ELISA, and DNA fragmentation by TUNEL assay. RESULTS: RA SFB variably expressed TNF-R55 (7.2 +/- 2.2% positive cells, mean +/- SEM) and TNF-R75 (0.6 +/- 0.3%), similarly to OA SFB (6.8 +/- 2.1% and 1.6 +/- 0.8%, respectively). RA SFB constitutively secreted large amounts of TIMP-1 (1700 ng/ml), but only small amounts of MMP-1 (23.7 ng/ml), IL-6 (4.4 ng/ml), and PGE(2) (0.34 ng/ml). OA SFB secreted comparable amounts of TIMP-1 (2470 ng/ml), MMP-1 (37 ng/ml), and IL-6 (5.0 ng/ml), but significantly higher amounts of PGE(2) (0.58 ng/ml; p RA) and by separate stimulation of both TNF receptors. TNF-a-induced PGE(2) release by RA SFB (92-fold) and OA SFB (56-fold) was mediated by both TNF receptors; however, predominantly by TNF-R55. DNA fragmentation was induced exclusively by high concentrations of anti-TNF-R55 Mab and only in RA SFB. CONCLUSION: These results indicate preferential induction of prodestructive and proinflammatory mediators in RA SFB by the TNF-R55, with potential implications for understanding the pathogenesis of RA and the development of more specific therapeutic strategies.     

Impaired inhibition by dexamethasone of cytokine release by alveolar macrophages from patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is characterized by inflammation of the respiratory tract in which macrophages are the predominant inflammatory cell and for which the efficacy of treatment with corticosteroids is controversial. We investigated the effect of dexamethasone on basal and interleukin (IL)-1beta or cigarette smoke media (CSM)-stimulated release of IL-8 and granulocyte macrophage-colony stimulating factor (GM-CSF) by bronchoalveolar lavage macrophages from cigarette smokers and patients with COPD (n = 15). Basal release of IL-8 was approximately fivefold greater in patients with COPD than smokers, whereas GM-CSF was similar for each group. IL-1beta and CSM increased IL-8 and GM-CSF release by macrophages from both smokers and patients with COPD. Dexamethasone did not inhibit basal or stimulated IL-8 release from macrophages from patients with COPD but inhibited release in smokers. In contrast, basal and IL-1beta-stimulated GM-CSF release, but not CSM-stimulated release, was inhibited by dexamethasone. We conclude that the lack of efficacy of corticosteroids in COPD might be due to the relative steroid insensitivity of macrophages in the respiratory tract.     

MMP-8, MMP-9 and Neutrophil Elastase in Peripheral Blood and Exhaled Breath Condensate in COPD

Chronic obstructive pulmonary disease (COPD) is characterised by progressive and irreversible airflow limitation associated with chronic inflammation involving cytokines and metalloproteinases (MMPs). MMP-8, MMP-9 and neutrophil elastase (NE) are known to be implicated in COPD but the factors influencing activation and suppression remain unclear. This study aimed to compare MMP-8, MMP-9 and NE in the peripheral blood of COPD patients and controls and to likewise assess exhaled breath condensate (EBC) for these MMPs. Peripheral blood micro(mi)RNA139-5p levels, which may regulate MMPs in COPD, were also measured. Blood and EBC were collected from COPD patients (stable and during exacerbations) and healthy controls. Expression of mRNA for MMP-8, MMP-9, NE and miRNA-139-5p expression in peripheral blood mononuclear cells (PBMCs) was measured using qRT-PCR. MMP-8, MMP-9 and NE protein in plasma as well as MMP-8 and MMP-9 protein in EBC were analysed by enzyme-linked immunoassays. PBMCs from COPD patients showed greater expression of mRNA for MMP-8 (p = 0.0004), MMP-9 (p = 0.0023) and NE (p = 0.0019). PBMC expression of mRNA for NE was significantly higher in COPD exacerbations compared to stable cases (p < 0.05). Expression of mRNA for MMP-9 and NE correlated negatively with spirometry in patients (p < 0.05). Plasma from COPD patients showed greater levels of protein for MMP-8 (p = 0.003), MMP-9 (p = 0.046) and NE (p = 0.018). MMP-8 protein levels were lower in the EBC of COPD patients (p < 0.0001). In PBMCs, enhanced expression of mRNA for MMP-9 and NE is associated with COPD and may correlate with disease severity and exacerbations.     

Overexpression of CD-11b and CXCR1 on circulating neutrophils: its possible role in COPD

BACKGROUND: It has been shown that the beta2-integrin molecule is up-regulated in circulating neutrophils in COPD subjects. However, little has been reported about the expression of the cell surface molecules in such patients and their relationship with pulmonary function. The aim of the present study was to investigate the surface expression of molecules in circulating neutrophils and to clarify their possible role in the airflow limitation of COPD. METHODS: The surface expression of Mac-1 cells (ie, CD-11b and CD-18 cells) and CXC chemokine receptor (CXCR) 1 and CXCR2 of circulating neutrophils obtained from COPD patients and healthy subjects (HSs) was measured by flow cytometry analysis. The serum levels of interleukin (IL)-8 were measured by enzyme-linked immunosorbent assay. RESULTS: Both CD-11b and CXCR1 expression were significantly higher in COPD patients than in HSs (mean [+/- SE] CD-11b concentration: HSs, 9.7 +/- 1.0; COPD patients, 14.2 +/- 1.8 [p < 0.05]; mean CXCR1 concentration: HSs, 9.6 +/- 0.5; COPD patients, 11.9 +/- 0.4 [p < 0.01]). Although aging was positively correlated with the expression of CXCR1 (r = 0.440; p < 0.01), none of the other background factors, including smoking and body mass index, showed a correlation with the expression of the molecules. Although serum IL-8 levels were higher in patients with COPD than in HSs, no significant correlation between serum IL-8 levels and the expression of any molecule was seen. The expression of CD-11b (r = -0.317) and CXCR1 (r = -0.383) showed a significant negative correlation with the severity of airflow limitation (both p < 0.05). CONCLUSIONS: The overexpression of CD-11b and CXCR1 in circulating neutrophils may be associated with the development of airflow limitation in COPD patients.     

Involvement of tumor necrosis factor (TNF) receptors p55 and p75 in TNF responses of acute myeloid leukemia blasts in vitro.

Tumor necrosis factor (TNF)-alpha and TNF-beta have multiple effects on human acute myeloid leukemia (AML) cells in vitro, including (1) synergistic stimulation of proliferation with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and upregulation of interleukin-3 (IL-3) and GM-CSF receptors; (2) inhibition of granulocyte-CSF (G-CSF)-induced growth and rapid downmodulation of G-CSF receptors; and (3) induction of autocrine growth. Recently, two distinct TNF receptors (TNF-Rs), TNF-R(p55) and TNF-R(p75), have been identified. In this study, we show that both receptor types may be expressed by AML blasts. It has been investigated whether the different effects of TNF on AML blasts can be explained by differential activation of the distinct TNF-R structures. For this purpose, we used the monoclonal antibodies HTR-1 and HTR-9, specifically recognizing TNF-R(p55), and UTR-1, specific for TNF-R(p75). TNF-(alpha and -beta) mediated synergistic activation with IL-3/GM-CSF, upregulation of IL-3/GM-CSF receptors, inhibition of G-CSF-induced growth, and rapid downmodulation of G-CSF receptors exclusively result from activation of TNF-R(p55). In certain cases in which TNF-alpha, rather than TNF-beta, induces AML growth through an autocrine mechanism, both TNF-R(p55) and (p75) are involved. These data indicate that the variety of TNF responses observed in AML can only be partially explained by differential activation of the TNF-R(p55) and (p75) structures, and that TNF-R(p55) on AML blasts can transduce both positive (synergism with IL-3/GM-CSF) and negative regulatory signals (inhibition of G-CSF-induced proliferation) following TNF activation.     

Airway inflammation and bronchial microbial patterns in patients with stable chronic obstructive pulmonary disease.

The effect of bacterial colonization of the bronchi on the progress of airflow limitation is not well known. Therefore, the pattern of airway inflammation in smokers and patients with stable chronic obstructive pulmonary disease (COPD) and its relation to bronchial microbial colonization was assessed. Eight nonsmoking and 18 smoking controls as well as 52 patients with COPD (28 mild, 11 moderate and 13 severe) were studied. All subjects were investigated by means of flexible bronchoscopy including protected specimen brush and bronchoalveolar lavage (BAL) sampling. Differential cell counts, cytokine (interleukin (IL)-1beta, IL-6, IL-8, IL-10 and tumour necrosis factor-alpha(TNF-alpha) concentrations and microbial patterns were determined in BAL fluid. Forced expiratory volume in one second (FEV1) % of the predicted value was inversely correlated with pack-yrs of cigarette smoking (r=-0.47, p<0.0001), the percentage of neutrophil (p=-0.56, p<0.0001) and IL-6 (p=-0.37, p=0.01) and IL-8 concentration (p=-0.43, p=0.004) in BAL fluid. Accordingly, pk-yrs of cigarette smoking (p=0.39, p=0.01) and IL-8 (p=0.69, p<0.0001) and TNFalpha (p=0.4, p<0.005) were positively correlated with the percentage of neutrophils in BAL fluid. Smoking controls and COPD patients were mainly colonized in the bronchial tree (33%) by community endogenous potentially pathogenic micro-organisms (PPMs). Colonization rates and patterns of PPMs were not affected by severity of airflow obstruction. The presence of PPMs was significantly associated with higher percentages of neutrophils (33.2+/-10.4% versus 10.1+/-3.5%, p=0.02) and TNF-alpha concentration (29.9+/-10.8 versus 6.3+/-2.1 pg x mL(-1), p=0.01) in BAL fluid. In conclusion, bronchial neutrophilia is a key inflammatory pattern in chronic obstructive pulmonary disease patients. Bronchial colonization with potentially pathogenic micro-organisms may represent an independent stimulus for additional airway inflammation.     

Similar gene expression profiles in smokers and patients with moderate COPD

Chronic obstructive pulmonary disease (COPD) is characterized by multiple cellular and structural changes affecting the airways, lung parenchyma and vasculature, some of which are also identified in smokers without COPD. The molecular mechanisms underlying these changes remain poorly understood. With the aim of identifying mediators potentially implicated in the pathogenic processes that occur in COPD and their potential relationship with cigarette smoking, we evaluated the mRNA expression of genes involved in inflammation, tissue remodeling and vessel maintenance. Lung tissue samples were obtained from 60 patients who underwent lung resection (nonsmokers, n=12; smokers, n=12; and moderate COPD, n=21) or lung transplant (severe-to-very severe COPD, n=15). PCR arrays containing 42 genes coding for growth factors/receptors, cytokines, metalloproteinases, adhesion molecules, and vessel maintenance mediators were used. Smoking-induced changes include the up-regulation of inflammatory genes (IL-1β, IL-6, IL-8, CCL2, and CCL8) and the decreased expression of growth factor/receptor genes (BMPR2, CTGF, FGF1, KDR and TEK) and genes coding for vessel maintenance factors (EDNRB). All these genes exhibited a similar profile in moderate COPD patients. The up-regulation of MMP1 and MMP9 was the main change associated with COPD. Inflammatory genes as well as the endothelial selectin gene (SELE) were down-regulated in patients with more severe COPD. Clustering analysis revealed a closer relationship between moderate COPD and smokers than between both subsets of COPD patients for this selected set of genes. The study reveals striking similarities between smokers and COPD patients with moderate disease emphasizing the crucial role of cigarette smoking in the genesis of these changes, and provides additional evidence of the involvement of the matrix metalloproteinase's in the remodeling process of the lung in COPD.     

Administration of recombinant human granulocyte-macrophage colony- stimulating factor after chemotherapy regulates the expression and secretion of monocyte tumor necrosis factor (TNF) and TNF receptors p55 and p75

Monocyte expression and secretion of tumor necrosis factor (TNF) and TNF receptors (TNF-R) p55 and p75 was studied in patients receiving granulocyte-macrophage colony-stimulating factor (GM-CSF) after intensive chemotherapy. TNF expression and secretion of biologically active TNF was increased at regeneration compared with that of patients who had received chemotherapy alone. This effect persisted for several weeks after cessation of growth factor therapy. GM-CSF restored the responsiveness of monocytes to bacterial lipopolysaccharide (LPS), which appeared to be diminished after chemotherapy alone. Expression and secretion of TNF-R p55 and p75 by monocytes was augmented by GM-CSF therapy in association with the increase in TNF protein. We propose that GM-CSF administration after chemotherapy restores the normal responsiveness of monocytes to a secondary stimulus such as LPS and primes monocytes to respond to LPS with increased expression and secretion of TNF and TNF-R.     

Expression of muscarinic receptors by human macrophages

Macrophages increase in number and are highly activated in chronic obstructive pulmonary disease (COPD). Muscarinic receptor antagonists inhibit acetylcholine-stimulated release of neutrophilic chemoattractants, suggesting that acetylcholine may regulate macrophage responses. Therefore, expression and function of components of the non-neuronal cholinergic system in monocyte-macrophage cells was investigated. RNA was isolated from monocytes, monocyte-derived macrophages (MDMs), lung and alveolar macrophages from nonsmokers, smokers and COPD patients, and expression of the high-affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter and muscarinic receptors (M(1)-M(5)) ascertained using real-time PCR. M(2) and M(3) receptor expression was confirmed using immunocytochemistry. Release of interleukin (IL)-8, IL-6 and leukotriene (LT)B(4) were measured by ELISA or EIA. All monocyte-macrophage cells expressed mRNA for components of the non-neuronal cholinergic system. Lung macrophages expressed significantly more M(1) mRNA compared with monocytes, and both lung macrophages and alveolar macrophages expressed the highest levels of M(3) mRNA. Expression of M(2) and M(3) protein was confirmed in MDMs and lung macrophages. Carbachol stimulated release of LTB(4) from lung macrophages (buffer 222.3 ± 75.1 versus carbachol 1,118 ± 622.4 pg · mL(-1); n = 15, p<0.05) but not IL-6 or IL-8. LTB(4) release was attenuated by the M(3) antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; half maximal effective concentration 5.2 ± 2.2 nM; n = 9). Stimulation of macrophage M(3) receptors promotes release of LTB(4), suggesting that anti-muscarinic agents may be anti-inflammatory.     

吸烟患或未患慢性阻塞性肺疾病患者肺组织中巨噬细胞移动抑制因子表达

 目的 通过检测吸烟患或未患慢性阻塞性肺疾病（COPD）患者肺组织中巨噬细胞移动抑制因子（MIF）表达水平,为进一步了解COPD易患机制提供基础研究资料。方法 肺组织来源于因肺癌行肺叶切除术的41例患者,分吸烟患COPD组（16例）、吸烟未患COPD组（13例）、无吸烟对照组（12例）3组。所有受试者均于术前1周做肺功能检查［第1秒用力呼气容积（FEV₁）,用力肺活量（FVC）］,采用实时定量PCR和免疫组化检测肺组织中MIF mRNA和蛋白表达水平,并行相关分析。结果 (1）实时定量PCR检测肺组织内MIF mRNA表达水平,吸烟患COPD组（4.87±1.79）高于吸烟未患COPD组（2.16±0.72）,而吸烟未患COPD组又高于无吸烟对照组（1.09±0.48;P<0.01）。（2）免疫组化显示,肺组织内MIF蛋白表达吸光度值吸烟患COPD组（0.277±0.025）高于吸烟未患COPD组（0.199±0.034）,而吸烟未患COPD组高于无吸烟对照组（0.130±0.021;P<0.01）。（3）相关性分析：肺组织内MIF mRNA表达与FEV₁占预计值百分比、FEV₁／FVC呈负相关(r分别为-0.578、-0.607,P值均<0.01)。结论 吸烟患COPD者肺组织中MIF表达明显增加,且与气流受限程度呈正相关,提示MIF可能在吸烟致COPD发病机制中起重要作用。     

Increased expression of nuclear factor-kappaB in bronchial biopsies from smokers and patients with COPD.

The expression of nuclear factor (NF)-kappaB is an indicator of cellular activation and of inflammatory mediator production. The aim of the present study was to characterise the expression and localisation of p65, the major subunit of NF-kappaB, in the bronchial mucosa of patients with chronic obstructive pulmonary disease (COPD), and to examine the relationship between p65 expression and disease status. Bronchial biopsies were obtained from 14 smokers with COPD, 17 smokers with normal lung function and 12 nonsmokers with normal lung function. The number of p65 positive (+) cells was quantified by immunohistochemistry and the expression of p65 in bronchial biopsies from the three groups was examined by Western blotting (WB). Smokers with normal lung function and patients with COPD had increased numbers of p65+ cells in the epithelium and increased p65 nuclear expression. In COPD patients the number of epithelial p65+ cells correlated with the degree of airflow limitation. WB analysis showed an increase in p65 in smokers with normal lung function and COPD patients (p<0.05). Bronchial biopsies in smokers with normal lung function and chronic obstructive pulmonary disease patients show increased expression of p65 protein, predominantly in the bronchial epithelium. Disease severity is associated with an increased epithelial expression of nuclear factor-kappaB.     

Localization of tumor necrosis factor receptors in the synovial tissue and cartilage-pannus junction in patients with rheumatoid arthritis. Implications for local actions of tumor necrosis factor alpha.

OBJECTIVE. We have previously described the location of tumor necrosis factor alpha (TNF alpha)-producing cells in synovial tissue and cartilage-pannus junction in rheumatoid arthritis (RA). To further understand the local actions of TNF alpha, we investigated the expression of TNF receptors (TNF-R) on cells in the same compartments in patients with RA. METHODS. The expression of both p55 TNF-R and p75 TNF-R was determined using alkaline phosphatase-conjugated mouse anti-alkaline phosphatase (APAAP) and double immunofluorescence staining techniques with monoclonal antibodies. RESULTS. In RA synovial membrane, both p55 TNF-R and p75 TNF-R were detectable in up to 90% of the cells in the lining layer, and were demonstrated on cells in deeper layers of the membrane, including vascular endothelial cells. Cells in lymphoid aggregates expressed both TNF-R, but with a predominant expression of p75 receptor. At the cartilage-pannus junction, the majority of pannus cells, especially those invading cartilage, expressed both the p55 and the p75 TNF-R. Sequential section and double immunofluorescence staining showed that the TNF-R-expressing cells were in the vicinity of TNF alpha-containing cells, and some TNF alpha-containing cells also expressed TNF-R. TNF-R-expressing cells were also detected in osteoarthritic and normal synovial tissue, but in smaller numbers and at a lower intensity. CONCLUSION. These results provide histologic evidence that both p55 TNF-R and p75 TNF-R are expressed by a variety of cell types in RA synovial tissue, reflecting the fact that a wide range of cells are potential targets for TNF alpha in this tissue. This study further supports the hypothesis that TNF alpha plays a major role in the pathogenesis of RA.     

Association of IL-6 gene polymorphisms and COPD in a Spanish population

Interleukin-6 (IL-6) is a potential mediator of systemic effects of Chronic Obstructive Pulmonary Disease (COPD). In the present case-control study we investigated the association of promoter polymorphisms of this gene and COPD in a cohort of 191 patients, smokers without COPD (n=75) and a healthy control population (n=296). Besides spirometry, exercise capacity (6MWD, 6 min walking distance) and body mass index (BMI) were measured in COPD patients. Genotyping of the IL-6 polymorphisms at positions -174, -572 and -597 was performed. The -597G/A and -174G/C polymorphisms were not associated with the disease. However, the -572G/C polymorphism was significantly associated with COPD susceptibility under a dominant model of inheritance. The frequency of the genotypes containing the C allele was significantly lower in the COPD cases (9.9%) compared with the healthy control group (16.9%) and smokers (23.1%), (OR=0.46, p=0.032 and OR=0.28, p=0.012, respectively). The GCG (-597/-572/-174) haplotype was significantly associated with the disease (OR=0.37, p=0.022, COPD cases vs. healthy subjects and OR=0.17, p=0.011, COPD cases vs. smokers). Moreover, a borderline association was also found for the -572G allele and hypoxemia (PaO(2)<60 mmHg) (p=0.05). Our data suggest that the IL-6 -572C allele may confer a diminished risk of developing COPD.     

Proinflammatory cytokines and chemokine production and expression by human osteoblasts isolated from patients with rheumatoid arthritis and osteoarthritis

OBJECTIVE: To evaluate whether subchondral osteoblasts (OB) are involved in the production of cytokines and chemokines in rheumatic diseases. METHODS: OB were isolated from subchondral bone of rheumatoid arthritis (RA), osteoarthritis (OA) and post-traumatic (PT) patients, cultured in vitro in the presence or absence of interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), and assessed for the production, immunolocalization, and mRNA expression of proinflammatory cytokines (IL-1alpha, IL-1beta, TNF-alpha) and alpha and beta chemokines [IL-8, growth related gene product (GRO-alpha), monocyte chemoattractant protein 1 (MCP-1), RANTES, and macrophage inflammatory proteins MIP-1alpha, MIP-1beta]. RESULTS: Cultured OB from different patients did not release IL-1alpha, IL-1beta, or TNF-alpha, and constitutively secreted IL-8, GRO-alpha, and MCP-1, while RANTES, MIP-1alpha, MIP-1beta were undetectable or near the lower level of sensitivity of the immunoenzymatic assay. GRO-alpha was significantly higher in RA than in OA and PT patients. IL-1beta and TNF-alpha alone or in combination strongly stimulated chemokine release by OB. Only RANTES production was not increased by the combination of the 2 cytokines. IL-1alpha, IL-1beta, and TNF-alpha were expressed as cytoplasmic proteins and were not secreted by OB even after stimulation. CONCLUSION: OB from subchondral bone release chemokines that could be involved in the mechanisms that directly or indirectly cause bone remodelling and cartilage destruction.     

Perforin expression and cytotoxic activity of sputum CD8+ lymphocytes in patients with COPD

BACKGROUND: Previous studies have shown that the inflammatory response to cigarette smoking differs between smokers who acquire COPD and those who do not, and the CD8(+) T- lymphocytes have been identified as a key player in this response. OBJECTIVE: To investigate the cytotoxic activity and perforin expression of CD8(+) lymphocytes in the airway lumen of patients with COPD. METHODS: Thirty-six male smokers with COPD, 25 male smokers without COPD, and 10 healthy nonsmokers participated in the study. T-lymphocytes of induced sputum samples were labeled with appropriate monoclonal antibodies and measured using flow cytometry. The cytotoxic activity of CD8(+) cells was defined by incubating them with specific target cells (K562). RESULTS: The percentage and the total number of CD8(+) lymphocytes were significantly higher in COPD smokers compared to non-COPD smokers (p = 0.01 and p = 0.005, respectively) or to healthy nonsmokers (p = 0.02 and p = 0.01, respectively). Perforin expression in CD8(+) cells was significantly higher in smokers with COPD compared to the other two groups (p = 0.001). Increased cytotoxic activity of T cells was also observed in induced sputum of patients with COPD in comparison to the other two groups. CONCLUSION: CD8(+) cells are not only increased in number in sputum samples of smokers with COPD but are highly activated, expressing high levels of perforin. These findings suggest that CD8(+) T-lymphocytes play a significant role in the inflammatory process of COPD.