Increased CD4+ T cell infiltrates in rheumatoid arthritisassociated interstitial pneumonitis compared with idiopathic interstitial pneumonitis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 1, January 2005, pp 73–79 DOI 10.1002/art.20765 © 2005, American College of Rheumatology Increased CD4⫹ T Cell Infiltrates in Rheumatoid Arthritis–Associated Interstitial Pneumonitis Compared With Idiopathic Interstitial Pneumonitis Carl Turesson,1 Eric L. Matteson,2 Thomas V. Colby,3 Zvezdana Vuk-Pavlovic,2 Robert Vassallo,2 Cornelia M. Weyand,4 Henry D. Tazelaar,2 and Andrew H. Limper2 Objective. To study lymphocyte markers in rheumatoid arthritis (RA)–associated interstitial pneumonitis (IP) compared with idiopathic IP. Methods. Paraffin-embedded lung biopsy specimens from patients with RA (n ⴝ 15) and from those without RA (n ⴝ 16), all of whom had a diagnosis of either nonspecific IP or usual IP, were studied. Tissue sections from each patient were reviewed by a pathologist, who was blinded to the clinical data. Age and pulmonary function test results were similar in RA and non-RA patients. After high-temperature antigen unmasking, sections were incubated with mouse monoclonal antibodies directed against CD3, CD4, CD8, CD16, and CD20. All slides were coded, and digital images (100ⴛ magnification) of the entire tissue area were obtained. Staining was quantified using computerassisted image analysis. Results. Staining for CD4 was more prominent in patients with RA than in the non-RA comparison group (median 9.3 cells/mm2, interquartile range [IQR] 5.5– 27.3 versus 0.6 cells/mm2, IQR 0.2–1.9; P ⴝ 0.002). CD4ⴙ cell counts were increased in RA patients with nonspecific IP as well as in RA patients with usual IP, with no major difference between these groups. Results were similar for quantification of CD3 (P ⴝ 0.012). There was a less striking trend toward more CD8ⴙ cells in RA patients (P ⴝ 0.27 versus those with non-RA lung disease). Conclusion. IP lesions in patients with RA are characterized by an increased number of CD4ⴙ cells, as compared with that in patients with idiopathic IP. This finding suggests that CD4ⴙ T cells are critical for the development of pulmonary manifestations in RA, and may have implications for the treatment of RAassociated lung disease. Rheumatoid arthritis (RA) is associated with a number of pulmonary manifestations, including vascular disease, pleuritis (1), airway disorders (2,3), and interstitial pneumonitis (IP) (4–6). The frequency of lung disease in patients with RA depends on the definitions used for screening (3) and the criteria used for selection of the patient sample (7). In a survey of a communitybased RA cohort, in which the case definition relied on clinical diagnosis and the results of pulmonary function tests (PFTs), the 10-year cumulative incidence of IP after diagnosis of RA was estimated to be 6% in patients diagnosed during the period 1975–1995 (8). Based on histopathologic findings, IP has been subclassified into categories, which include usual IP and nonspecific IP (9). A wide variety of pathologic patterns in RA-associated IP have been reported (6), and IP in patients with RA has been described as clinically indistinguishable from idiopathic IP (5,6). Some authors have reported a more stable disease course (10) and a lower mortality (11–13) in patients with IP in the setting of connective tissue disease (CTD) compared with those with idiopathic IP, but this has not been a consistent finding (14,15). Guidelines issued jointly by the Ameri- Supported by NIH grant K24-AR-47578-01A1, the Swedish Rheumatism Association, the Swedish Society for Medicine, and the Robert N. Brewer Family Foundation. 1 Carl Turesson, MD, PhD: Mayo Clinic College of Medicine, Rochester, Minnesota, and Malmö University Hospital, Malmö, Swe2 den; Eric L. Matteson, MD, MPH, Zvezdana Vuk-Pavlovic, PhD, Robert Vassallo, MD, Henry D. Tazelaar, MD, Andrew H. Limper, MD, FCCP: Mayo Clinic College of Medicine, Rochester, Minnesota; 3 Thomas V. Colby, MD, FCCP: Mayo Clinic College of Medicine, Scottsdale, Arizona; 4Cornelia M. Weyand, MD, PhD: Emory University, Atlanta, Georgia. Address correspondence and reprint requests to Carl Turesson, MD, PhD, Department of Rheumatology, Malmö University Hospital, Södra Förstadsgatan 101, S-205 02 Malmö, Sweden. E-mail: email@example.com. Submitted for publication April 13, 2004; accepted in revised form September 30, 2004. 73 74 TURESSON ET AL can Thoracic Society and the European Respiratory Society have separated IP in patients with CTD from idiopathic cases of IP, on the basis of potential differences in etiology and pathogenesis which may affect management and outcome (16). Prominent nodular aggregates of lymphocytes in the parenchymal interstitial tissue have been described in patients with RA-associated usual IP and in those with RA-associated nonspecific IP (15). Studies of bronchoalveolar lavage (BAL) samples obtained from RA patients in comparison with healthy controls have yielded somewhat contradictory results (17,18), although differences in T cell subpopulations have been suggested (17). Previous studies of patients with IP with and without associated CTD have revealed a poor correlation of CD4⫹ and CD8⫹ cell counts in samples obtained at BAL with the number of CD4⫹ and CD8⫹ cells in corresponding lung biopsy samples (19). There have been no quantitative studies of lymphocyte populations in lung biopsy tissue from patients with IP and RA. T cells are considered to be important in the pathogenesis of RA (20), and specific T cell abnormalities may be of particular relevance to extraarticular disease manifestations, including lung disease (21). The purpose of the present study was to describe T cell subpopulations in lung biopsy tissue from patients with RA-associated IP, and to use quantitative data for a comparison of RA-associated IP with idiopathic IP. PATIENTS AND METHODS Patients. Samples from patients with a clinical and histopathologic diagnosis of RA-associated lung disease (n ⫽ 23) were obtained from the tissue archives at the Mayo Clinics in Rochester, Minnesota and Scottsdale, Arizona. The selection of biopsy specimens was based on an ongoing survey of RA-associated lung disease at the Mayo Clinic and the consultation files of one of the authors (TVC). The basis for inclusion was a histopathologic diagnosis of inflammatory lung disease by an experienced lung pathologist (HDT or TVC) in a patient with documented RA. The medical records were reviewed in accordance with a structured protocol. One patient diagnosed with RA did not fulfill the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 criteria for RA (22), and was therefore excluded. Slides of hematoxylin and eosin–stained sections from each patient were reviewed by one of the pathologists (TVC), who was blinded to the clinical data. Ten of the RA patients were classified as having nonspecific IP, and 5 as having usual IP. Seven cases were not classified as IP at either of the 2 reviews. At the second review, 2 patients originally classified as having usual IP were reclassified as nonspecific IP, and 1 nonspecific IP case was relabeled as usual IP. Patients without RA who had a clinical and histopathologic diagnosis of idiopathic nonspecific IP (n ⫽ 10) or idiopathic usual IP (n ⫽ 8) were identified from a survey of Mayo Clinic–Rochester interstitial lung disease cases, and these were designated as controls. Slides of specimens from these patients underwent the same review as described above. Two patients originally classified as having usual IP were not considered to have IP at the second review, and 1 case of usual IP was reclassified as nonspecific IP. In total, 11 patients were classified as having nonspecific IP, and 5 as having usual IP. Control patients were compared with the RA patients with nonspecific IP or usual IP. Most of the patients with nonspecific IP had a histopathologic picture mainly compatible with fibrotic nonspecific IP, with the exception of 2 patients (both non-RA controls) who had prominent features of cellular nonspecific IP. All specimens from both groups were obtained by openlung biopsies. All medical records for the cases and controls were reviewed, and demographic data, the presence of other autoimmune diseases, and PFT results at the time of diagnosis of interstitial lung disease were recorded. For patients with RA, data on rheumatoid factor and antinuclear antibody tests as well as the presence of rheumatoid nodules and severe extraarticular manifestations, determined according to predefined criteria (7,23), were noted. The study was approved by the Mayo Clinic Institutional Review Board. Immunohistochemical staining. Formalin-fixed, paraffin-embedded sections of 5-m–thick tissue were deparaffinized through 2 exchanges of xylene, each of 5 minutes’ duration. The tissues were then rehydrated using a graded series of alcohol washes and incubated for 30 minutes in 0.5% H2O2 to quench endogenous peroxidase activity. Hightemperature antigen unmasking was performed as part of all staining protocols. Before incubation with antibodies against CD8, the slides were incubated in preheated Copeland jars containing 10 mM sodium citrate buffer, pH 6.0, in a 98°C water bath for 40 minutes. Before incubation with antibodies against CD3, CD4, CD16, and CD20, slides were heated at high pressure in 10 mM citrate buffer, pH 6.0 (for CD3, CD16, and CD20 protocols), or 1 mM EDTA buffer, pH 8.0 (for CD4 protocols), in a pressure cooker for 60 seconds, in accordance with the recommendations of the antibody manufacturer (Novocastra, Newcastle upon Tyne, UK). Pretreated slides were incubated for 30 minutes with 1% blocking horse serum before application of primary antibodies. For all experiments, the following mouse monoclonal antibodies were used: anti-CD8 (clone C8/144B; Dako, Glostrup, Denmark), anti-CD3 (clone PS1, dilution 1:100), anti-CD4 (clone 4B12, dilution 1:20), anti-CD16 (clone 2H7, dilution 1:20), and anti-CD20 (clone L26, dilution 1:200) (all from Novocastra). Antibody dilutions and incubation times (60 minutes at room temperature plus 10 hours at ⫺20°C for anti-CD8; 60 minutes at room temperature for all other antibodies) were applied uniformly in parallel across all tissues studied. Slides of specimens from patients with a clinical and histopathologic diagnosis of nonspecific IP, incubated with a universal mouse negative reagent containing a cocktail of mouse IgG and IgM (Dako), were used as negative controls. Primary antibody binding was detected using the avidin–biotin–immunoperoxidase method (Vectastain Elite ABC Kit; Vector, Burlingame, CA) with 3-amino-9-ethyl-carbazole as the colorimet- ASSOCIATION OF CD4⫹ T CELLS WITH INTERSTITIAL PNEUMONITIS IN RA 75 Table 1. Demographic variables and pulmonary function test results at diagnosis* Sex, no. female/male Age, years Ever smoker, % Current smoker, % Current steroid treatment, % VC, % of predicted FVC, % of predicted FEV1, % of predicted DLCO, % of predicted RA ILD Non-RA ILD P 9/6 64 (57–72) 54† 31† 60 8/8 64 (57–69) 44 0 31 0.58 0.80 0.58 0.03 0.11 67 (51–91) 67 (46–80) 69 (51–91) 48 (42–58) 69 (51–89) 66 (53–83) 76 (54–95) 57 (39–75) 0.71 0.83 0.62 0.61 * Except where indicated otherwise, values are the median (interquartile range). RA ⫽ rheumatoid arthritis; ILD ⫽ interstitial lung disease; VC ⫽ vital capacity; FVC ⫽ forced vital capacity; FEV1 ⫽ forced expiratory volume in 1 second; DLCO ⫽ diffusion capacity of the lung for carbon monoxide. † Data available on 13 patients. ric substrate. The sections were counterstained with 1% Meyer’s hematoxylin. Quantification and statistical analysis. All slides were coded before quantification. Using an Axioplan 2 microscope and an AxioCam camera connected to KS400 software (all from Zeiss, Oberkochen, Germany), digital images (⫻100 magnification) of the entire tissue area of each slide were obtained. A macro specifically designed for the quantification of these stains was developed using the KS400 software, and a total of 11,412 images were analyzed in a blinded manner. The total lung tissue area, the area stained, and the number of stained cells were quantified for each image, and the proportion of the total area stained and the total number of stained cells per mm2 were calculated for each slide. Because the studied variables did not have a normal distribution, the RA and non-RA groups were compared using the Mann-Whitney U test. Statistical analysis was performed using SAS software, version 8.2 (SAS, Chicago, IL). Figure 1. A, CD4⫹ cells (red stain) in a patient with rheumatoid arthritis (RA) and nonspecific interstitial pneumonitis (IP). B, Scarcity of CD4⫹ cells in a patient with nonspecific IP and no RA. C, CD4⫹ cells in a patient with RA and usual IP. D, Scarcity of CD4⫹ cells in a patient with usual IP and no RA. (Original magnification ⫻ 100.) In the RA group, 8 of 12 patients for whom laboratory data were available were seropositive for rheumatoid factor, and 5 of the 11 patients tested were positive for antinuclear antibodies. Six patients with RA had current or previous rheumatoid nodules, and 4 had a history of nonpulmonary, severe extraarticular disease manifestations consisting of pericarditis (n ⫽ 2) or major cutaneous vasculitis (n ⫽ 2). The median duration of RA was 7.5 years (interquartile range [IQR] 0.5–12.0 years) RESULTS Demographic data and PFT results are shown in Table 1. Age distributions and results of all PFTs were similar between the 2 groups. The female:male ratio was 1:1 among patients without RA and 1.5:1 among patients with RA. Of the patients for whom data on smoking at the time of lung biopsy were available, 4 of 13 in the RA group and none of 16 in the non-RA group were current smokers, whereas 7 of 13 in the RA group and 7 of 16 in the non-RA group had been smokers at any time. Among the patients without RA, 1 had psoriasis, 1 had ulcerative colitis, and 1 had a prior diagnosis of extrapulmonary sarcoidosis. However, after a thorough clinical and histopathologic evaluation, in none of these cases was the presence of these inflammatory conditions considered to be the cause of the IP. Figure 2. CD4⫹ cell counts per tissue area in patients with rheumatoid arthritis (RA)–associated interstitial lung disease (ILD) versus idiopathic ILD. Bars show the median and interquartile range. 76 TURESSON ET AL Table 2. Quantification of immunohistochemical staining in patients with RA-associated ILD compared with those with idiopathic ILD* RA ILD Non-RA ILD CD4 Cells/mm2 9.3 (5.5–27.3) 0.6 (0.2–1.9) % of area stained 0.029 (0.013–0.083) 0.001 (0–0.006) CD3 Cells/mm2 9.8 (2.9–17.5) 1.5 (0.4–5.2) % of area stained 0.019 (0.005–0.035) 0.004 (0.001–0.011) CD8 Cells/mm2 60.4 (10.9–112.5) 23.4 (7.9–53.8) % of area stained 0.171 (0.029–0.270) 0.049 (0.016–0.148) P 0.002 0.003 0.012 0.065 0.27 0.19 * Except where indicated otherwise, values are the median (interquartile range) of measured cell counts per tissue area or measured stained area as a proportion of the total tissue area, both quantified by computer-assisted image analysis. See Table 1 for definitions. Figure 3. CD4⫹ cell counts per tissue area, by rheumatoid arthritis (RA) diagnosis and nonspecific interstitial pneumonitis (NSIP) or usual interstitial pneumonitis (UIP) classification. Each circle denotes an individual patient. at the time of lung biopsy. Two of the patients had received treatment with methotrexate before the onset of pulmonary symptoms. The clinical course and the histopathologic picture on lung biopsy were not considered compatible with methotrexate-induced pneumonitis in either of these cases. Nine of the 15 patients in the Figure 4. A, Perifollicular CD3⫹ cells (red stain) in a patient with RA and nonspecific IP, with most cells in the aggregate being negative for CD3. B, Subpleural infiltrate of CD8⫹ cells in a patient with RA and nonspecific IP. C, Scattered peribronchiolar cells positive for CD16 in a patient with RA and usual IP. D, Aggregate of mononuclear cells positive for CD20 in a follicular structure, with scattered positive cells in the surrounding lung tissue. See Figure 1 for definitions. (Original magnification ⫻ 100.) RA group were receiving glucocorticosteroids at the time of the biopsy, as were 5 of 16 in the non-RA group. Mononuclear cells staining for CD4 were present in peribronchial infiltrates as well as in fibrotic interstitial lung tissue in patients with RA and IP (Figures 1A and C). In most sections from patients with idiopathic IP, only scattered CD4⫹ cells were seen (Figures 1B and D). When quantified by computer-assisted image analysis, the number of CD4⫹ cells per tissue area was significantly higher in patients with RA-associated IP compared with control patients with non-RA IP (Figure 2), with a more than 10-fold increase, on average, in CD4⫹ cell counts in the RA group. The median number of CD4⫹ cells/mm2 was 9.3 (IQR 5.5–27.3) in the RA group compared with 0.6 (IQR 0.2–1.9) in the non-RA group (P ⫽ 0.002). When subdivided by histopathologic classification, the increase in CD4⫹ cell counts among RA patients, compared with controls, was similar between those with nonspecific IP and those with usual IP (Figure 3). Major infiltrates of CD3⫹ cells were seen in most patients with RA-associated IP. Structures resembling lymphoid follicles were identified, with CD3⫹ cells seen mainly in a perifollicular distribution (Figure 4A). CD3⫹ cell counts per tissue area were significantly increased in patients with RA and IP compared with non-RA patients with IP (P ⫽ 0.012). CD8⫹ cells, including prominent peribronchial and subpleural infiltrates (Figure 4B), were also seen, with a trend toward increased CD8⫹ cell counts in patients with RA. However, in contrast to the scores on CD4 and CD3 image analysis, the corresponding quantification of CD8⫹ cells did not reveal a significant difference between interstitial lung disease patients with and those without RA (P ⫽ 0.27). Staining for CD4, CD3, and CD8 was also ASSOCIATION OF CD4⫹ T CELLS WITH INTERSTITIAL PNEUMONITIS IN RA quantified as the proportion of the total tissue area stained for each of these cell types. These results were similar to those of cell counts per tissue area (Table 2). Scattered CD16⫹ cells were seen in tissue sections from patients with RA (Figure 4C) as well as from patients without RA. Notably, aggregates of mononuclear cells in follicular structures were negative for CD16, indicating that natural killer cells do not make up a major part of these cell populations. Staining for CD20 revealed major aggregates of positive cells in structures resembling lymphoid follicles with germinal centers (Figure 4D). DISCUSSION In this study of lung biopsy tissue from patients with RA-associated IP, we found a significantly increased number of CD4⫹ cells when compared with patients with idiopathic IP. CD3⫹ cells were also significantly increased, whereas there was a less striking difference for the CD8⫹ subset. The prominent increase of CD4⫹ cell counts in patients with RA was similar between those classified as having nonspecific IP and those classified as having usual IP. These findings may be of major importance to our understanding of the nature of RA-associated lung disease. RA is characterized by increased production of proinflammatory cytokines of the Th1 subset (24,25). Based on observations of synovial pathologic patterns (20) and the strong association between HLA–DRB1 genotypes and RA (26,27), CD4⫹ T cells are considered important in the pathogenesis of RA, although the role of T cell antigen recognition in arthritis is unclear (28). T cell abnormalities in RA include a marked contraction of the T cell repertoire, with less diversity and emergence of dominant T cell clonotypes (29). In addition, clonal expansion of CD4⫹ cells lacking the costimulatory molecule CD28 (21,30) is observed most markedly in patients with severe extraarticular RA manifestations (31). Associations between some extraarticular manifestations and class II major histocompatibility complex (MHC) genes (in particular the HLA–DRB1*0401/0401 genotype) (32) and class I MHC genes (in particular HLA–C3) (33) may be due to effects on the T cell repertoire and on T cell activation. The observed increased pleural fluid concentrations of soluble interleukin-2 receptor in patients with RA-associated pleuritis, compared with patients with pleural exudates of other origins (34), also support the importance of T cell activation in RA-associated lung disease. The present finding of more abundant CD8⫹ 77 cells than CD4⫹ cells in IP lesions is consistent with the findings in previous studies of idiopathic nonspecific IP (35). The findings by other authors (35,36) of S100 protein–positive dendritic cells (DCs) in areas of lymphoid aggregates in interstitial lung disease specimens support a role of T cell activation in interstitial lung disease. Furthermore, in these studies, DCs were more abundant in idiopathic nonspecific IP (35) and RAassociated usual IP (36) than in idiopathic usual IP. Important immunopathologic differences in IP between patients with RA and patients with idiopathic pulmonary fibrosis may thus include several interacting cell populations. The role of the B cell aggregates observed in our patients should also be further explored. Patients with RA were more likely to be receiving current treatment with glucocorticosteroids at the time of lung biopsy than were patients with idiopathic IP. Steroids would be expected to reduce lymphoid infiltrates, so this would, if anything, tend to diminish differences in the number of cells positive for CD4 and other markers between the RA group and the non-RA group. Thus, treatment differences cannot explain our results. Our selection of patients was done without matching, but the age distribution and PFT results were similar between the RA group and the control group. Although our sample size was small, the ratio of women to men of 1.5:1 in the RA group is compatible with an overall 2–3-fold increase in the incidence of RA in women compared with men (37), and is consistent with a suggested association between male sex and lung disease in patients with RA (38). Current smoking was more frequent among patients with RA than among controls. Smoking is a risk factor for RA (39), and, in patients with established RA, is a predictor of severe extraarticular manifestations (40), including IP (41). In transbronchial biopsy specimens from patients with chronic obstructive pulmonary disease, CD8⫹ cells, but not CD4⫹ cells, were more abundant in smokers (42–44). Therefore, the observed increase in CD4⫹ cells in patients with RA cannot be explained by the frequency of smoking. There are no effective therapies for idiopathic pulmonary fibrosis. Although many patients with idiopathic pulmonary fibrosis still receive corticosteroids, cyclophosphamide, and azathioprine, there are no data demonstrating efficacy of these agents on lung function or survival. Randomized controlled trials have failed to demonstrate a benefit from cyclophosphamide (45) or azathioprine (46) in idiopathic pulmonary fibrosis. There are no controlled trials demonstrating a benefit of 78 immunosuppressive therapies in RA-associated IP, but studies of case series have suggested that interstitial lung disease in the setting of RA and other CTDs may respond better to immunosuppression (47). Individual cases of RA-associated pulmonary fibrosis (with radiographic characteristics of usual IP) have been reported to respond to cyclosporine (48–50) and infliximab (51). The present results, which highlight the importance of CD4⫹ T cells in RA-associated IP, suggest that treatment with drugs that suppress T cell activation or new agents that interfere with T cell functions may be useful in the treatment of RA-associated IP. Our data also suggest that despite similarities in the radiographic and histopathologic appearance, there are fundamental differences in the pathophysiologic patterns between RA-associated IP and idiopathic usual and nonspecific IP. Furthermore, the distinction between usual IP and nonspecific IP may not have the same significance in patients with RA and other CTDs as it does in patients with idiopathic IP, since we found markedly increased CD4⫹ cell counts in the RA with usual IP subset. Although the number of patients with usual IP in this study is too small for any final conclusions on this subset, our findings are consistent with the observations by Nakamura et al, who found no difference in survival between usual IP and nonspecific IP subgroups in a cohort of patients with CTD, in contrast to a marked decrease of survival in idiopathic IP patients with usual IP as compared with those with nonspecific IP (12). The major strength of this study is the thorough quantification of T cell markers, which was done using computer-assisted image analysis. This method enables standardized assessment of all stained tissue, reducing the risk of sampling error. Differences in the extent of staining may thus be more reliably assessed than is possible by semiquantitative methods. In our opinion, a measure of the total tissue area by computer-assisted image analysis should be included in any quantification of immunohistochemical staining, and related to either manual- or image-analysis–based cell counts in the same tissue area. In conclusion, we have demonstrated an increased number of CD4⫹ T cells in lung biopsy tissue from patients with RA-associated IP compared with patients having IP without RA. This difference seems to be independent of the nonspecific versus usual IP classifications, supports the concept that RA-associated lung disease is different from idiopathic pulmonary fibrosis, and may have implications for the treatment of these disorders. Further studies should explore the character- TURESSON ET AL istics and functional properties of CD4⫹ T cells in RA-associated IP. ACKNOWLEDGMENTS The authors thank James Tarara for excellent technical help with microscopic photography and image analysis, Drs. 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