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Perforin Expression and Cytotoxic Activity of Sputum CD8⁺ Lymphocytes in Patients With COPD^*

^* From the Department of Thoracic Medicine, University Hospital of Heraklion, Medical School, Crete, Greece.

Correspondence to: Nikolaos M. Siafakas MD, PhD, FCCP, Professor of Thoracic Medicine, Department of Thoracic Medicine, University of Crete, Medical School, PO Box 1352, 71110 Heraklion, Crete, Greece; e-mail: siafak{at}med.uoc.gr

	Abstract

TOP Abstract Introduction Materials and Methods Results Discussion References

 
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.

Key Words: CD8⁺ cells • COPD • cytotoxicity • perforin • smoking

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion References

 
COPD is characterized by a non-fully reversible airflow limitation, associated with an abnormal inflammatory response of the lungs to noxious particles or gases.¹ Smoking is the major risk factor for the development of COPD, but only a subgroup of smokers acquire severe COPD.^{2 3}

Various inflammatory cells, such as neutrophils, macrophages, and T-lymphocytes, participate in the inflammatory process leading to COPD. The activated cells release a variety of mediators, including interleukin-8, tumor necrosis factor-, leukotriene B₄, and other inflammatory molecules capable of destroying the parenchyma and/or maintaining a neutrophilic inflammation in the airways of patients with COPD.⁴

Previous reports have suggested that cytotoxic CD8⁺ lymphocytes may play a role in the pathogenesis of COPD. Studies^{5 6} based on bronchial biopsies have focused on the role of the above-mentioned cells in the central and peripheral airways of smokers with COPD. Compared to smokers who do not manifest airflow limitation, patients with COPD have increased numbers of cytotoxic CD8⁺ lymphocytes in the small and large airways.^{5 6} These cells have also been found to infiltrate the pulmonary parenchyma and pulmonary arteries, suggesting a consistent presence in the inflammatory response.⁷ The percentages of CD8⁺ cells, both in bronchial biopsy and BAL samples, seem to display a clear difference between COPD smokers and smokers without airflow limitation.

Studies of CD8⁺ cytotoxic T-lymphocytes showed that they cause lysis of target cells by two mechanisms: (1) membranolysis, in which secreted molecules, such as perforin and granzymes, form pores in the membrane of target cells⁸ ; and (2) apoptosis, mediated through the triggering of apoptosis-inducing (Fas-like) surface molecules of the target cell.⁸ Especially for alveolar epithelial cells, apoptosis is preferentially mediated through tumor necrosis factor- and is relatively insensitive to the Fas ligand pathway.⁹ It has been reported that perforin is elevated in several chronic inflammatory disorders including Takayasu arteritis¹⁰ and Crohn disease.¹¹ In patients with asthma, an inflammatory disease of the airways, it has been shown that perforin expression in peripheral blood T-lymphocytes is increased.¹² However, to our knowledge, there are no studies investigating the expression of perforin in cytolytic T-lymphocytes of patients with COPD. Nevertheless, the precise role of cytotoxic CD8⁺ lymphocytes in the inflammatory process remains unclear. Flow cytometry studies^{13 14} have shown that determination of the surface markers and functionality of sputum lymphocytes can be studied.

We have used immunofluorescence labeling and flow cytometry to assess T-lymphocyte surface marker expression in sputum from two groups of individuals with equivalent smoking history, smokers with established COPD and smokers without. In particular, we aimed to assess the cytotoxicity of CD8⁺ cells and their expression of perforin.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Subjects
Seventy-one male subjects were studied: 36 current smokers with COPD, 25 non-COPD smokers with a similar smoking history to the patients with COPD, and 10 healthy nonsmokers (Table 1 ). The diagnosis of COPD was based on the European consensus criteria.² All patients with COPD had been free of an acute exacerbation for at least 4 weeks preceding the study, and none had received antibiotics or corticosteroids (oral and inhaled) over the same period. Neither smokers nor healthy volunteers had a history of cardiopulmonary disease, and all had normal lung function. All the subjects were nonatopic (ie, they had negative skin test results for common allergen extracts) and had no history of asthma or allergic rhinitis. The hospital ethical committee approved the protocol, and all subjects gave their consent.

Sputum
Sputum was induced via inhalation of a hypertonic saline solution aerosol, generated by an ultrasonic nebulizer (Ultraneb 2000; DeVilbiss; Somerset, PA) as previously described.¹⁴ The viscid portions of the expectorated sample were separated from the sputum and processed within 15 min after termination of the induction.¹⁴

Sputum Processing, Total Cell Counting, and Differential Cell Counting
The weight of the plugs was determined, and dithiothreitol 0.1% (Sputolysin; Calbiochem; La Jolla, CA) in phosphate-buffered saline solution (PBS) was added at a ratio of 2 mL to 1 g of the sputum plugs. The samples were agitated on a vortex mixer in a wide-bore plastic test tube and placed in a shaking water bath for 15 min at 37°C to ensure complete homogenization. Subsequently, RPMI-1640 plus 10% fetal calf serum (FCS) was added in a volume twice that of the homogenized sample. The samples were then filtered through 48-µm nylon gauze (Thompson; ON, Canada) and agitated on a vortex mixer (initial suspension). A total cell count of the filtered sample was performed and viability was tested by means of the trypan blue exclusion staining (Sigma-Adrich Corporation; St. Louis, MO) in a Neubauer hematcytometer. The suspension was then centrifuged at 400g for 5 min, and the pellet was resuspended with 500 µL RPMI-1640 plus 10% FCS. The supernatant was aspirated and stored in Eppendorf cups at 80°C.

The samples were adjusted to a concentration of 0.35 x 10⁶ cells per milliliter. Cytospins were made by putting 50 µL of the cell suspension in the funnels of an aerospray cytocentrifuge (Wescor; Claremont, ON, Canada) at 300 revolutions per minute with low deceleration for 5 min. Two slides were stained with May-Giemsa-Grunwald stain for the differential cell counts. Five hundred nonsquamous cells in each coded May-Giemsa-Grunwald cytospin were counted in a blinded fashion by two independent investigators and averaged. Cell differential counts were expressed as percentage of nonsquamous cells and as absolute number of cells per gram of selected sputum sample. Absolute cell numbers were calculated by multiplying the cell percentage by the total (nonsquamous) cell number in the sputum, divided by the weight of the selected sputum sample.

Antibody Labeling
The following mouse anti-human monoclonal antibodies were used for labeling sputum lymphocyte cells: peridinin chlorophyll protein (PerCP)-conjugated anti-CD3, phycoerythrin-cyanine (PCy-5)–conjugated anti-CD4 and PCy-5–conjugated anti-CD8, fluorescein isothiocyanate (FITC) conjugated anti-perforin (Serotec; Raleigh, NC), as well as unlabeled anti-human-perforin for intracellular perforin detection. Mouse anti-mouse isotype matched PerCP-conjugated, PCy-5–conjugated, or FITC-conjugated Ig were used as control antibodies (Immunotech; Marseille, France).

The method used for labeling cell surface antigens has been previously described.¹⁴ Briefly, 1 x 10⁶ cells in suspension were incubated first with normal bovine serum to block the unspecific binding (blocking reagent), and then the monoclonal antibody/antibodies was added in excess (according to the instructions of the manufacturer) for 45 min at 10°C in the dark and washed three times with PBS plus 10% FCS.

For intracellular perforin staining, CD8⁺ cells were subsequently incubated in a permeabilization reagent (Intraprep; Beckman Coulter; Fullerton, CA) for 10 min. As a control, unlabeled antiperforin at molar excess was used after permeabilization and then antiperforin FITC was added without washing and incubated for 30 min at room temperature (competitive labeling). After staining, the samples were washed with PBS plus 10% FCS and were immediately analyzed using flow cytometry.

Flow Cytometric Analysis
The sputum samples prepared as described above were analyzed on an fluorescence activated flow cytometer (EPICS ELITE; Coultronics; Louton, UK). The lymphocytes were tightly gated by volume and complexity on a forward (0°) and side-light scattering (90°) mode. At least 10⁵ cells were analyzed in each session. PCy-5–conjugated anti-human CD45 monoclonal antibodies (Dako; Ely, UK) were used as pan-leukocyte stain to exclude nonleukocyte events by logical gating. The percentage of one-color, two-color, and three-color positive cells were measured and the mean channel value as well as the relative fluorescence intensity (RFI) corresponding to the antigen density was estimated. The QC-Combo Kit (FCSC; San Juan, Puerto Rico) was used for quantification of antibody binding and day-to-day instrument calibration (amplification and compensation settings of the flow cytometer) was routinely carried out. The number of positive cells for PCy5-conjugated anti-CD4 or PCy5-conjugated anti-CD8 in gated cell populations that were stained with anti-CD3 PerCP was measured and expressed as a percentage of CD3⁺ cells. We also measured the number of positive cells for FITC-conjugated antiperforin in gated cell populations that were stained with anti-CD8 PCy-5 plus anti-CD3 PerCP. The number of positive cells for perforin was expressed as a percentage of CD8⁺ T cells.

Detection of Cytotoxic Activity
Sputum lymphocytes were isolated for measurement of their cytotoxic activity, and were then treated with mycostatin for 1 to 2 days and before being resuspended in RPMI-1640 plus 10% FCS supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.02 mg/mL fluconazole. Lymphocytes were subsequently isolated on the Lymphoprep Density Gradient (Nycomed; Oslo, Norway). After isolation on the density gradient, the cells were washed, counted, and adjusted to a range of concentrations in RPMI plus 2% human albumin serum. Target cells (K562 human erythroleukemia cell line) were co-cultured with mononuclear cells at 3:1 to 50:1 ratios in round-bottom tissue culture plates in 200 µL culture medium for 4 h at 37°C under CO₂. Supernatants were collected and the lactate dehydrogenase was measured using a Boerhinger-Mannheim cytotoxicity detection kit (Boerhinger-Mannheim Biochemical; Indianapolis, IN) according to the instructions of the manufacturer. Triton X-100 was used to determine the maximum lysis and target cells alone to determine spontaneous lysis. Percentage of lysis was calculated according to the formula: (experimental optimal density [OD] - spontaneous OD - spontaneous effector OD)/(maximal OD - spontaneous targets OD).¹⁶ All measurements were repeated three times.

Statistics
Clinical characteristics are presented as mean ± SD. Cell parameters were nonnormally distributed and expressed as median (range). Therefore, differences between the three groups of subjects were tested using the Kruskal-Wallis test with post hoc pairwise comparisons made by the Conover-Inman method.¹⁷ All analyses were performed using StatsDirect for Windows version 2.2.3 (Camcode; Cambridge, UK); p < 0.05 was considered statistically significant.

	Results

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Demographic and spirometric data of the three groups of subjects are shown in Table 1 . The patients with COPD exhibited significantly lower values of FEV₁ (percentage of predicted) and FEV₁/FVC (percentage) than non-COPD smokers, although the smoking history was similar. There were no significant differences in FEV₁/FVC (percentage) and FEV₁ (percentage) between non-COPD smokers and normal subjects. Change in FEV₁ (percentage of predicted) after salbutamol was similar in the three groups (Table 1) . The induced-sputum procedure was well tolerated by all subjects. The viability of the cells (percentage of total) in induced sputum did not differ between COPD (median, 75%; range, 71 to 82%), non-COPD smokers (median, 74%; range, 65 to 81%), and healthy control subjects (median, 70%; range 62 to 79%) [p = 0.23]. The total cell count (x 10⁷ cells per gram) was higher in patients with COPD (median, 8.2; range, 1 to 18) than in non-COPD smokers (median, 4.4; range, 0.7 to 8.0; p = 0.001) and control subjects (median, 1.1; range, 0.2 to 2.0; p = 0.0001). There were significant differences (p = 0.001) in the percentages of sputum neutrophils between COPD, non-COPD smokers, and control subjects (Table 2 ). The same was true for the percentages of macrophages (p = 0.0001; Table 2 ). The percentages of lymphocytes were significantly different between COPD and control subjects (p = 0.02), but not between COPD and non-COPD smokers COPD (Table 2) . The percentages of eosinophils in COPD did not differ compared to the non-COPD smokers or to the control subjects (Table 2) .

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 1.. Cytotoxicity (mean ± SD) as percentage of lysis of target cells in COPD, non-COPD smokers, and normal subjects.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 2.. Expression of perforin (mean ± SD) in COPD, non-COPD smokers, and normal subjects.

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion References

 
In agreement with other studies,^{13 18} we also showed that it is possible to analyze sputum samples for lymphocyte cell surface markers by flow cytometry. The main findings of the present study were that CD8⁺ lymphocytes of patients with COPD showed increased cytotoxic activity and increased expression of intracellular perforin compared to smokers without COPD, but with similar smoking history, and healthy nonsmokers (normal subjects). It is well known that it is difficult to induce sputum samples in normal subjects. However, in accordance with another report,¹³ we succeeded in obtaining representative sputum samples from our normal subjects. In addition, patients with COPD have increased numbers of CD8⁺ T-lymphocytes in sputum samples with a concomitant imbalance between CD4⁺ and CD8⁺ (decreased CD4⁺/CD8⁺ ratio).

Several studies^{19 20 21 22 23} have shown that smoking is the major cause of the inflammatory process that characterizes COPD. However, it is not yet known why only a minority of smokers acquire clinically important disease.^{3 23 24} It is of critical importance to study smokers who acquire COPD and to compare them with smokers who have a similar smoking history but do not have COPD. Differences in cellular composition between smokers with COPD and smokers without COPD have been reported in bronchial biopsy and BAL specimens.^{5 6} In one reported study,¹³ sputum CD4⁺:CD8⁺ analysis using flow cytometry showed a decrease in patients with COPD compared to normal subjects and mild asthmatics, although it was not nonstatistically significant. All these reports suggest that an imbalance between cytotoxic CD8⁺ lymphocytes and helper CD4⁺ may contribute to this abnormal inflammatory process in the airways of patients with COPD and that COPD is a lymphocyte-driven inflammatory condition, in particular, a CD8⁺-type response. This is a study that extends the findings to sputum samples of patients with COPD.

The sputum-derived T-lymphocytes in the present study were activated more in COPD smokers, since their cytotoxic capability was higher in comparison to that of smokers without COPD (p < 0.01) and to nonsmokers (p < 0.001). It is very likely that CD8⁺ cells are responsible for the observed lysis, as they are the main cytotoxic lymphocyte subpopulation. These finding are in agreement with a recent study by Leckie et al,¹³ who reported that sputum T-lymphocytes of patients with COPD are of activated intraepithelial phenotype expressing CD103+. To further investigate the mechanism of cytotoxic activity of CD8⁺ lymphocytes, we measured their perforin expression. Perforin is a 60-kd, pore-forming protein stored intracellulary; it is produced mainly by cytotoxic CD8⁺ cells, natural killer cells, / cells, and it has been reported in elevated concentrations in chronic inflammatory disorders with autoimmune features, such as Takayasu arteritis and Crohn disease.^{10 11} Arnold and colleagues¹² reported increased perforin expression in peripheral blood CD4⁺ and CD8⁺ cells in patients with bronchial asthma, which is also an inflammatory airway disease. The findings of increased cytotoxicity of CD8⁺ cells support the recently reported hypothesis that the increased numbers of activated CD8⁺ cells in the sites of inflammation (airway epithelium) may cause acute tissue damage via the release of lytic substances such as perforin and granzyme.⁴ This may imply also that perforin is the main mediator of the membranolytic action of cytotoxic CD8⁺ lymphocytes and that it is implicated in the apoptotic and destructive process leading to the development of COPD.

There are several possible pathways in which the increased cytotoxicity and perforin expression of CD8⁺ cells might be associated with inflammation in COPD. A hypothesis concerning the natural history of the disease could be that the potent cytotoxic activity of CD8⁺ lymphocytes in COPD contributed initially to the defense against viral infections, which are common causes of COPD exacerbations.^{25 26} A study²⁷ of antiviral activity of CD8⁺ cells show that this cytotoxic activity is both necessary and sufficient to affect the lysis of viral infected target cells. However, this beneficial function may become an abnormal process for the lungs if excess CD8⁺ cells are present and with inappropriate activation.^{28 29} Indeed, the presence of activated CD8⁺ cells found in this study in patients with COPD without acute exacerbation could be a result of previous viral infections, and this excessive response may damage the lungs of susceptible smokers.⁵

Enelow and colleagues³⁰ have demonstrated that recognition of a lung "autoantigen" by T-cytotoxic cells may directly produce a marked lung injury. A review³¹ proposed that the accumulation of cytotoxic CD8⁺ lymphocytes observed in COPD could be the result of a response to an autoantigenic stimulus caused by smoking and not necessarily to recurrent viral infections. The findings of the present study are in accordance with this hypothesis, since CD8⁺ lymphocytes of non-COPD smokers showed increased cytotoxic activity and perforin expression when compared to healthy nonsmoking individuals. It seems that smoking alone is a sufficient activating factor for CD8⁺ lymphocytes. However, our findings could not clarify whether this activation remains under control in non-COPD smokers. The abnormal inflammation leading to COPD probably depends on a combination of factors, such as genetic susceptibility related to the regulation of CD8⁺ lymphocytes, repeated infections, and environmental agents.

In conclusion, our findings show that the inflammation seen in COPD could be mediated by T-lymphocyte lysis (increased cytotoxic activity) especially by CD8⁺ cells (increased perforin expression). This procedure is less intense in smokers without COPD. Further investigations are required to clarify the exact pathogenetic mechanism that leads some smokers to acquire COPD while others do not.

	Footnotes

 
Abbreviations: FCS = fetal calf serum; FITC = fluorescein isothiocyanate; OD = optimal density; PBS = phosphate-buffered saline solution; PerCP = peridin in chlorophyll protein; PCy-5 = phycoerythrin-cyanine; RFI = relative fluorescence intensity

This study was supported by an unrestricted grant from ASTRA Hellas.

Part of this study has been presented at the 11th Hellenic Thoracic Society annual meeting, Thessalonica, December 6–9, 2001, and as prize winner an extensive summary has been published in the Greek journal Pneumon (2002; 15:78).

This work has been awarded the national prize during the 12th European Respiratory Society meeting, Stockholm, Sweden, September 14–18, 2002.

Received for publication March 6, 2003. Accepted for publication July 1, 2003.

	References

TOP Abstract Introduction Materials and Methods Results Discussion References

 

1. Pauwels, RA, Buist, AS, Ma, P, et al (2001) Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: National Heart, Lung, and Blood Institute and World Health Organization Global Initiative for Chronic Obstructive Lung Disease (GOLD); executive summary. Respir Care 46,798-825[Medline]
2. Siafakas, NM, Vermeire, P, Pride, NB, et al Optimal assessment and management of chronic obstructive pulmonary disease (COPD). The European Respiratory Society Task Force. Eur Respir J 1995;8,1398-1420[CrossRef][ISI][Medline]
3. Siafakas, NM, Tzortzaki, EG Few smokers develop COPD: Why? Respir Med 2002;96,615-624[CrossRef][ISI][Medline]
4. Barnes, PJ Chronic obstructive pulmonary disease. N Engl J Med 2000;343,269-280[Free Full Text]
5. O’Shaughnessy, TC, Ansari, TW, Barnes, NC, et al Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV₁. Am J Respir Crit Care Med 1997;155,852-857[Abstract]
6. Saetta, M, Di Stefano, A, Turato, G, et al CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998;157(3 pt 1),822-826
7. Peinado, VI, Barbera, JA, Abate, P Inflammatory reaction in pulmonary muscular arteries of patients with mild chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;159(5 pt 1),1605-1611
8. Berke, G The binding and lysis of target cells by cytotoxic lymphocytes: molecular and cellular aspects. Annu Rev Immunol 1994;12,735-773[CrossRef][ISI][Medline]
9. Liu, AN, Mohammed, AZ, Rice, WR, et al Perforin-independent CD8(+) T-cell-mediated cytotoxicity of alveolar epithelial cells is preferentially mediated by tumor necrosis factor-: relative insensitivity to Fas ligand. Am J Respir Cell Mol Biol 1999;20,849-858[Abstract/Free Full Text]
10. Seko, Y, Minota, S, Kawasaki, A, et al Perforin-secreting killer cell infiltration and expression of a 65-kD heat-shock protein in aortic tissue of patients with Takayasu’s arteritis. J Clin Invest 1994;93,750-758[ISI][Medline]
11. Muller, S, Lory, J, Corazza, N, et al Activated CD4+ and CD8+ cytotoxic cells are present in increased numbers in the intestinal mucosa from patients with active inflammatory bowel disease. Am J Pathol 1998;152,261-268[Abstract]
12. Arnold, V, Balkow, S, Staats, R, et al Increase in perforin-positive peripheral blood lymphocytes in extrinsic and intrinsic asthma. Am J Respir Crit Care Med 2000;161,182-186[Abstract/Free Full Text]
13. Leckie, MJ, Jenkins, GR, Khan, J, et al Sputum T lymphocytes in asthma, COPD and healthy subjects have the phenotype of activated intraepithelial T cells (CD69+ CD103+). Thorax 2003;58,23-29[Abstract/Free Full Text]
14. Tsiligianni, J, Tzanakis, N, Kyriakou, D, et al Comparison of sputum induction with bronchoalveolar lavage cell differential counts in patients with sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis 2002;19,205-210[ISI][Medline]
15. Quanjer, PH, Tammeling, GJ, Cotes, JE, et al Lung volumes and forced ventilatory flows. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J Suppl 1993;16,5-40[Medline]
16. Vukmanovic-Stejic, M, Vyas, B, Gorak-Stolinska, P, et al Human Tc1 and Tc2/Tc0 CD8 T-cell clones display distinct cell surface and functional phenotypes. Blood 2000;95,231-240[Abstract/Free Full Text]
17. Conover, WJ Practical nonparametric statistics. Conover, WJ eds. Practical nonparametric statistics. 1999,491-510 Wiley. New York, NY:
18. Loppow, D, Bottcher, M, Gercken, G, et al Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers. Eur Respir J 2000;16,324-329[Abstract]
19. Jeffery, PK Comparison of the structural and inflammatory features of COPD and asthma: Giles F. Filley lecture. Chest 2000;117(5 Suppl 1),251S-260S
20. Mullen, JB, Wright, JL, Wiggs, BR, et al Reassessment of inflammation of airways in chronic bronchitis. BMJ (Clin Res Ed) 1985;291,1235-1239[ISI][Medline]
21. Niewoehner, DE, Kleinerman, J, Rice, DB Pathologic changes in the peripheral airways of young cigarette smokers. N Engl J Med 1974;291,755-758[ISI][Medline]
22. Diener, CF, Burrows, B Further observations on the course and prognosis of chronic obstructive lung disease. Am Rev Respir Dis 1975;111,719-724[ISI][Medline]
23. Fletcher, C, Peto, R The natural history of chronic airflow obstruction. BMJ 1977;1,1645-1648[ISI][Medline]
24. Doll, R, Peto, R Mortality in relation to smoking: 20 years’ observations on male British doctors. BMJ 1976;2,1525-1536[ISI][Medline]
25. Hogg, JC Viral infection and exacerbations of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001;164,1555-1556[Free Full Text]
26. Hogg, JC Role of latent viral infections in chronic obstructive pulmonary disease and asthma. Am J Respir Crit Care Med 2001;164(10 pt 2),S71-S75
27. Ramsay, AJ, Ruby, J, Ramshaw, IA A case for cytokines as effector molecules in the resolution of virus infection. Immunol Today 1993;14,155-157[CrossRef][ISI][Medline]
28. Isaacs, D, Bangham, CR, McMichael, AJ Cell-mediated cytotoxic response to respiratory syncytial virus in infants with bronchiolitis. Lancet 1987;2,769-771[ISI][Medline]
29. Cannon, MJ, Openshaw, PJ, Askona, BA Cytotoxic T cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus. J Exp Med 1988;168,1163-1168[Abstract/Free Full Text]
30. Enelow, RI, Mohammed, AZ, Stoler, MH, et al Structural and functional consequences of alveolar cell recognition by CD8(+) T lymphocytes in experimental lung disease. J Clin Invest 1998;102,1653-1661[ISI][Medline]
31. Saetta, M, Turato, G, Maestrelli, P, et al Cellular and structural bases of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001;163,1304-1309[Free Full Text]

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