INTRODUCTION

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Sarcoidosis is a multiorgan inflammatory disorder of unknown cause, characterized by an accumulation of activated CD4-positive T cells and macrophages in the lungs, accompanied by its histopathologic hallmark, the noncaseating epithelioid cell granuloma (1, 2). Several studies have indicated the importance of T lymphocytes in the pathogenesis of sarcoidosis (3). T-helper cells (CD4⁺) compartmentalize in the lungs of patients with active sarcoidosis resulting in an elevated CD4/ CD8 ratio in bronchoalveolar lavage (BAL) that relates to the intensity of the alveolitis (2).

The majority of T lymphocytes use an alpha/beta T-cell receptor (TCR) for antigen to contact peptides in the context of major histocompatibility complex (MHC) molecules, and the variable regions of the TCR are constructed through rearrangement of the germline V, -D, and J gene segments. Analyses of the TCR repertoire may show the existence of T cells with a restricted TCR usage, which may have a significant role in the development of the disease (4, 5). In sarcoidosis, lung compartmentalized CD4⁺ T cells are implicated in the pathogenesis of the disease because they release interleukin-2 (IL-2), proliferate at a high rate, and express activation markers (6, 7). Several groups have shown a preferential TCR variable (V) gene usage by lung T cells, suggesting that there is a specific sarcoidosis-associated antigen in the lungs of these patients (3, 5, 8).

Previous studies by our group demonstrated that CD4-positive cells compartmentalized in the lung have a preferential usage of the TCR AV2S3 gene segment in patients with sarcoidosis expressing human leukocyte-associated antigen-DR17 (HLA-DR17; DRB1*0301) or -52a (DRB3*0101) (5). Our previous findings implicate the AV2S3⁺ BAL T cells to specifically recognize a sarcoidosis-associated antigen in the lungs of these patients (5, 6), and we recently found a positive association between the relative numbers of these cells and a favorable clinical outcome (9).

The aim of this study was to characterize phenotypically the AV2S3 T cells in BAL of sarcoidosis patients with focus on activation and subset markers. The activation markers in this study were selected according to their appearance after stimulation, including the most well-established markers reflecting very early (CD69), early (CD25), and late activation marker (HLA-DR). We also investigated the two more recently found CD26 and CD27 molecules, acting both as activation markers and costimulatory molecules. The CD28 costimulatory molecule was included because of its important role in T-cell activation. Markers for naive/memory T cells (CD45RA/RO) were analyzed, as well as the subset marker CD57 which was previously associated with autoimmune disorders (10), and also the Fas receptor CD95, involved in apoptosis and therefore of interest in inflammation. We analyzed CD4⁺ T cells in BAL expressing AV2S3 versus those cells not expressing AV2S3, and CD4⁺ T cells in BAL and peripheral blood lymphocytes (PBL) in patients in comparison to CD4⁺ T cells in BAL and PBL in healthy control subjects (HC).

	METHODS

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Subjects

Sixteen patients (mean age 33 yr, range 25 to 46) diagnosed with active sarcoidosis participated in the study. All patients had AV2S3 CD4-positive T-cell expansions in their lungs, and the mean proportion of CD4⁺ cells expressing AV2S3 was 33.1% (range 23.4 to 42.0). All patients were HLA-DR17 (DRB1*0301)-positive except one who was HLA-DR52a (DRB3*0101)-positive (Patient 1). Four of the patients were female and 12 were male. One of the subjects was a smoker, three were ex-smokers, and 12 had never smoked. All of the patients had clinical and chest radiographic features typical for sarcoidosis. Eleven of the patients had classic Löfgren's syndrome and five had positive proven biopsies. The only two patients without Löfgren's syndrome and with negative biopsies had BAL CD4/CD8 ratios of 9.8 and 10.2, respectively, thus strongly supporting the diagnosis (11). One of the patients had chest X-ray Stage II and the rest had Stage I. Disease activity was judged from symptoms, chest radiography, and pulmonary function tests using the criteria established by the World Association of Sarcoidosis and other Granulomatous Disorders (WASOG) (12). Two of the patients were undergoing treatment with nonsteroidal anti-inflammatory drugs; the rest were not treated. Ten nonsmoking HC, five males and five females, mean age 30 yr (range 19 to 50) were also included in this study. All the HC had normal chest radiography and none had any sign of pulmonary disease. This study was approved by the local ethics committee.

BAL and Cell Preparation

BAL was performed under local anesthesia, the flexible fiber-optic bronchoscope was wedged in a middle lobe bronchus, and sterile phosphate-buffered saline (PBS) at 37° C was instilled in five aliquots of 50 ml. After each instillation the fluid was gently aspirated and collected in a siliconized plastic bottle kept on ice. The BAL fluid (BALF) was strained through a Dracon net (Millipore, Cork, Ireland), centrifuged at 400 × g for 10 min at 4° C, and the pellet was resuspended in RPMI 1640 medium (Sigma Aldrich, Irwine, UK). Cells were counted in a Bürker chamber and total cell amount was determined by trypan blue exclusion. Smears for differential counts were prepared by centrifugation (Cytospin 2; Shandon, Runcorn, Cheshire, UK) at 22 × g for 3 min, after which cells were stained with May-Grünwald-Giemsa. Heparinized peripheral blood was separated by using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient centrifugation to obtain peripheral blood mononuclear cells. The separated cells were washed twice and diluted in RPMI 1640.

Immunofluorescent Staining and Flow Cytometry

Antibodies for a detailed phenotypic characterization of AV2S3⁺ and AV2S3 lymphocytes are listed in Table 1. To determine lymphocyte subsets in the BALF in patients, cells were labeled with unconjugated anti-human TCR V alpha 2.3-specific monoclonal antibody (Mab) clone F1, purchased from Serotec (Oxford, UK). Cells were incubated for 30 min in the dark at 4° C and washed twice before adding F (ab')₂ fragments of rabbit anti-mouse immunoglobulin (Ig), conjugated with either fluorescein isothiocyanate (FITC) or R-phycoerythrin (RPE), from Dako (Glostrup, Denmark). After incubation with secondary antibodies, cells were washed twice. Normal mouse serum (NMS) from BALB/c mice diluted 1:500 was used to block inappropriate binding of rabbit anti-mouse immunoglobulin to Mabs used in the subsequent staining step. Direct-labeled Mabs against HLA class II (RPE), CD69 (FITC), or CD57 (FITC) (from Becton Dickinson, San José, CA) CD45RA (RPE), CD45R0 (RPE), or CD25 (RPE) (from Dako, Denmark), CD28 (FITC) (from Coulter Immunotec, Marseille, France), CD26 (FITC), CD27 (FITC), or CD95 (FITC) (from Pharmingen Becton Dickinson Lab, San Diego, CA) were added to the cells together with RPE-Cy5-conjugated anti-CD4 Mab (Dako) and incubated for 30 min at 4° C. Irrelevant mouse antibodies of the same isotype and concentration were used as background controls (Dako). After incubation, cells were washed twice and fixed in PBS with 1% formaldehyde. BAL cells and PBL from HC and PBL from patients were double-stained with direct-labeled Mab specific for the various activation and subset markers (Table 1) and RPE-Cy5 anti-CD4, incubated for 30 min at 4° C, washed twice and fixed in PBS with 1% formaldehyde. Cells were analyzed in a flow cytometer (FACScalibur; Becton Dickinson, Mountain View, CA) within the next 24 h.

Lymphocytes were easily distinguished on the basis of forward and side scatters, and a gate was set around these lymphocytes. In patients' BAL, CD4⁺ lymphocytes were gated and CD4⁺AV2S3⁺ and CD4⁺AV2S3 cells were separately analyzed for expression of the various activation markers (Table 1). Expression of the activation markers was evaluated on CD4⁺ T cells in BAL and PBL from HC and patients. Isotype-matched negative control antibodies stained less than 1% of CD4⁺ AV2S3⁺, AV2S3, BAL and PBL CD4⁺ cells, and were used to set markers to delineate positive and negative cells.

Statistical Analysis

Results are presented as median values, with minimum and maximum values as the range. For comparison of AV2S3⁺ and AV2S3 cells Wilcoxon matched pair statistical calculations were used and for comparisons between BAL and PBL cell populations of patients and HC, the Mann-Whitney U test was performed. Considering multiple comparisons, a p value of less than 0.01 was regarded as significant.

	RESULTS

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BAL cells from 16 patients, all with lung accumulated CD4⁺ T-cell expansions expressing TCR AV2S3 (mean 33.0%, range 23.4 to 42.0%), and from 10 HC were analyzed in this study. In addition, PBL from six of the patients and from the 10 HC were included. All of the patients except one (DR52a-positive, Patient 1) were HLA-DR17-positive. An individual characterization from the analyses of patients' BAL cells is presented in Table 2.

Phenotypic Evaluation

Flow cytometry was performed to study the expression of markers for activation, costimulation, and subsets of BAL and PBL T cells in patients and HC. The results are presented as the percentage of cells in each subset that express the respective activation marker. Surface markers studied for T-cell activation were CD69 (very early activation), CD25 (early activation, IL-2 receptor [IL-2R], and HLA-DR (late activation). Activation markers with costimulatory functions CD26 (also a proposed T helper cell, type 1 [Th1] marker), CD27 (a member of the tumor necrosis factor-R [TNF-R] family), and CD28 were also investigated. Finally, the expression of CD95 (Fas, involved in apoptosis), CD57 (subset marker), CD45RA and CD45R0 (markers for naive and memory T cells, respectively) were also included (Table 1).

Patients with Sarcoidosis

In general, patient's BAL CD4⁺ cells showed a higher expression of activation markers CD26, CD69, and HLA-DR compared with paired PBL CD4⁺ T cells (Table 3). All of these markers were in addition expressed by more BAL CD4⁺ AV2S3⁺ compared with BAL CD4⁺ AV2S3 T cells (Table 3, Figure 1). In contrast, the expression of early activation markers CD25 and CD27, which is also a costimulatory molecule, was reduced in BAL CD4⁺ T cells compared with PBL CD4⁺ cells, and also less expressed by BAL CD4⁺AV2S3⁺ compared with BAL CD4⁺ AV2S3 T cells (Table 3).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Expression of activation markers CD25, CD26, CD27, CD57, CD69, and HLA-DR in AV2S3-positive and AV2S3-negative CD4+ BAL lymphocytes. p Values for comparison between the cell subsets are indicated.

The costimulatory molecule CD28 was expressed more by PBL CD4⁺ compared with BAL CD4⁺ T cells, although the difference was not significant. CD28 was expressed more by BAL CD4⁺ AV2S3⁺ compared with BAL CD4⁺ AV2S3 T cells. The subset marker CD57 was significantly higher expressed by CD4⁺ BAL than PBL T cells and even more by the CD4⁺ AV2S3⁺ cells (Table 3). A representative dot-plot analysis comparing the expression of these activation and subset markers on AV2S3-positive and -negative BAL T cells of patients is shown in Figure 2.

View larger version (37K): [in this window] [in a new window]   Figure 2.   Representative flow cytometry dot plots of CD4-positive BAL lymphocytes. Cells were stained with antibodies against AV2S3 (x-axis) and against various activation/subset markers (y-axis). The number in each upper quadrant denotes the percentage of CD4+AV2S3+ (right upper quadrant) or CD4+ AV2S3 (left upper quadrant) lymphocytes expressing the respective marker.

CD95 was also relatively higher expressed by BAL CD4⁺ compared with PBL CD4⁺ cells, but there was no difference between CD4⁺AV2S3⁺ and CD4⁺AV2S3 cells. Nearly all BAL lymphocytes (CD4⁺AV2S3+ as well as CD4⁺ AV2S3) were positive for the memory marker CD45R0 (> 95%), whereas in contrast almost all were negative for the naive marker CD45RA (< 1.5%). The expression of CD45R0 and CD45RA on CD4⁺ cells in PBL was 74.5% (median; range 54.0 to 81.3) and 39.5% (median; range 27.3 to 49.0), respectively.

Patients with Sarcoidosis versus HC

PBL CD4⁺ cells in patients with sarcoidosis had a significantly higher expression of activation markers CD25, CD26, and HLA-DR compared with HC (Figure 3). For the costimulatory molecule CD28, the subset marker CD57, and the early activation marker CD69, no significant differences in PBL of patients and HC were found. Patients' BAL CD4⁺ cells showed a statistically significant higher expression of CD26 (p < 0.01) and HLA-DR (p < 0.0002) compared with BAL CD4⁺ cells of HC (Figure 3). A similar tendency, although not significant, was found for CD28, CD57, and CD69, whereas no difference was observed for CD25 (Figure 3).

View larger version (19K): [in this window] [in a new window]   Figure 3.   Expression of activation markers CD25, CD26, CD28, CD57, CD69, and HLA-DR in BAL and PBL CD4+ T cells in patients and HC. p Values for comparison between the patients and HCs are indicated. Note the different scales on the y-axis of the CD28 and CD69 graphs.

	DISCUSSION

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The aim of this study was to phenotypically analyze and determine the activation state of CD4⁺ BAL T cells from patients with sarcoidosis with active disease. In particular, we wanted to investigate BAL T cells expressing the TCR AV2S3 gene segment because we previously have found such T cells to accumulate in the lungs of patients with sarcoidosis, provided they are HLA-DR17- or HLA-DR52a-positive (5). In this specific subgroup of patients, making up one-third of all Swedish sarcoidosis patients and having distinct clinical manifestations such as Löfgren's syndrome and good prognosis (13), the lung accumulated AV2S3-positive T cells are believed to recognize a sarcoidosis-associated antigen. A close characterization of such AV2S3-positive lung T cells may therefore increase our understanding of key pathogenic events in the disease, such as T-cell activation after antigen presentation.

T-cell subsets of a total of 16 patients with sarcoidosis were studied with respect to percentage of cells expressing various surface activation markers, costimulatory molecules, and subset markers. Ten healthy individuals were also investigated. We detected an altered expression of several of the molecules in patients' PBL and BAL CD4⁺ T cells compared with HC. More importantly, most of these markers were even more differentially expressed by the patients' BAL CD4 AV2S3⁺ T cells. Thus, the different expression of the well-established activation markers CD25, CD69 (both reflecting early activation), and HLA-DR (late activation), that was found in patients' compared with control subjects' BAL CD4⁺ T cells, was even more pronounced in the CD4 AV2S3⁺ subset (Figure 1).

Soluble CD25 (IL-2R) is a parameter used for estimating the inflammatory activity of sarcoidosis, and activated immune cells of patients with sarcoidosis release CD25 in BALF and serum (1). In line with previous reports, we found that CD25 was expressed to a higher degree by PBL of patients compared with HC, but to normal levels by patients' BAL T cells (14). Interestingly, there was a significantly reduced expression of CD25 especially on CD4⁺AV2S3⁺ BAL T cells. One mechanism explaining this result, and the lower expression of CD25 in BAL T cells versus PBL, could be the shedding of this marker after prolonged antigen stimulation. High concentrations of soluble CD25 have also been reported in patients compared with control subjects (14). This result is therefore in agreement with a specific stimulation of AV2S3⁺ T cells in the lungs. CD69 is an early activation marker for T cells both in vivo and in vitro (15) and cannot be detected on resting lymphocytes (14). Earlier reports have shown a high expression of CD69 on BAL cells of healthy control subjects, with even higher levels in patients with sarcoidosis (14, 16), which is in agreement with our findings although they were not statistically significant. The elevated CD69 expression especially on AV2S3 BAL T cells may again indicate a preferential stimulation of this specific cell subset. Another activation marker, HLA-DR, further indicated a strong activation of BAL cells of patients with sarcoidosis and in particular the AV2S3⁺ T cells. HLA-DR is considered as a late activation marker, and it has been demonstrated that HLA class II positive T cells accumulate in tissues affected by autoimmune disorders and that they may reflect a low grade of inflammation or autoimmune reactions in the lungs (17). Afeltra and coworkers (18) found an unusual CD69⁺CD25 HLA-DR⁺ T-cell subset in synovial fluid of patients with rheumatoid arthritis and suggested them to be important in the pathogenesis of this disease. Interestingly, especially the AV2S3-positive BAL T cells were to a high degree of that particular phenotype, i.e., CD69⁺CD25HLA-DR⁺.

The expression of activation markers with costimulatory functions, i.e., CD26, CD27, and CD28, was also studied. The notable increase of CD4⁺ cells positive for CD26 in BALF of patients has also been reported previously and suggests chronic T-cell stimulation in the lung (19, 20). On human T cells, CD26 expression is preferentially restricted to the CD4⁺ helper/memory population and can deliver a potent costimulatory T-cell activation signal (21). That the expression of CD26 was significantly enhanced on BAL CD4⁺AV2S3⁺ in comparison to BAL CD4⁺AV2S3 T cells could be due to a prolonged, chronic stimulation of this cell subset in particular. In contrast, we observed a dramatically decreased expression of the CD27 molecule in BAL CD4⁺ T cells with even more reduced expression by the CD4 AV2S3⁺ BAL T cells. The CD27 molecule that belongs to a family of TNF-receptors is considered as a costimulatory molecule and was recently defined also as an early activation marker (14). In vitro studies have shown that activation via the TCR/CD3 complex causes an increased CD27 expression on T cells, with a maximum level after 4 d, and a declined expression thereafter probably because of shedding of the receptor (14). Lung CD4⁺ T cells of patients with sarcoidosis were previously shown to lack CD27 expression (14), in agreement with our findings, suggesting a continuous and chronic stimulation of these T cells, and especially of the AV2S3⁺ T cells. A tendency to a reduced expression of CD28 by BAL CD4⁺ cells compared with CD4⁺ cells in PBL was observed, in congruence with previous studies (16). The expression of the costimulatory molecule CD28 diminishes after multiple cell divisions and it has been suggested that a reduced expression of CD28 indicates T-cell senescence (22, 23). Our finding of a slightly higher expression of CD28 by BAL CD4⁺AV2S3⁺ in comparison to BAL CD4⁺AV2S3 T cells may therefore seem intriguing, although it is in agreement with the tendency to a higher CD28 expression of BAL CD4⁺ T cells of patients compared with HC. An enhanced expression of CD28 might indicate that new and "fresh" AV2S3⁺ T cells are recruited to the lungs.

The subset marker CD57 was expressed more by patients' BAL T cells compared with PBL and even more by CD4 AV2S3⁺ T cells in BAL. CD57 delineates subsets of T cells that have been associated with autoimmune disorders (10). Not only the induction of an immune response but also its dissolution is important in maintaining homeostasis. CD95, also known as Fas, has the ability to activate the cellular death program "apoptosis," which can be critical for limiting an inflammatory response (24). Indeed, more CD4⁺ T cells were positive for CD95 in BAL than PBL. The significance of this is not clear, however, because apoptosis of BAL lymphocytes in vitro does not seem to be mediated by Fas, and CD95 expression is associated with stimulation of the T cells (24, 25).

In this study, we have found a significantly increased expression of activation markers CD26, CD69, and HLA-DR in the CD4 BAL AV2S3⁺ T cells from patients with sarcoidosis. At the same time, we detected a significantly reduced expression of activation markers CD25 and CD27, likely because of a shedding of these molecules after long-standing inflammation. The strength of our results is the consistent pattern of significant differences between patients' PBL and BAL T cells as well as between BAL T cells of patients and HC, that were even more pronounced when specifically analyzing the disease-associated AV2S3⁺ BAL T cells. Lymphocytes are found in different compartments of the lung. The BAL T-lymphocyte pool is regulated by an influx of lymphocytes mainly from blood, by a local proliferation or apoptosis, and by migration back to the blood via the lymphoid system (26, 27). Our finding of a high expression of both early and late activation markers on patients' BAL T cells, and especially on the CD4⁺ AV2S3⁺ cells, may suggest a continuous influx of AV2S3⁺ T cells as well as a local proliferation of already lung accumulated AV2S3 T cells, most likely in response to a locally expressed antigen. Such a scenario is also congruent with our previous finding of an oligoclonal nature of the AV2S3⁺ BAL T cells (6).