INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Sarcoidosis is a systemic, granulomatous disease of unknown etiology. Although frequently affecting intrathoracic organs, it may affect virtually any organ. The disease is considered to develop in genetically predisposed individuals who are exposed to an as yet undescribed antigen. Studies of the immune response in sarcoidosis, including the typical formation of noncaseating epithelioid granulomas, as well as epidemiologic data and case reports on patients who have undergone transplantion all indicate the presence of a sarcoidosis-specific, transmissible agent in patients with the disease (1).

The immune response in sarcoidosis is believed to involve presentation of a presumed antigen to T lymphocytes by antigen-presenting cells (APCs). Antigens are normally presented as peptides in the context of human leukocyte antigen (HLA) class II molecules (HLA-DR, -DP, -DQ) that are expressed by APCs, and recognized by the T cell through its T-cell receptor (TCR) for antigen. Following the interaction between APCs and T lymphocytes in sarcoidosis, an exaggerated inflammation develops, characterized by an accumulation in the lungs of CD4⁺ T cells (2) that mainly produce T-helper type 1 (Th1) cytokines such as interferon (IFN)- and interleukin (IL)-2 (3, 4). Subsequently, granulomas are formed and in a subset of patients, a severe condition with fibrosis may develop.

The variable parts of the TCR are constructed through a random combination of variable (V), diversity (D; only for the  chain), and joining (J) gene segments of the TCR  (A) and  (B) genes, respectively, during maturation of the lymphocyte in the thymus. The resulting diversity of the TCR is impressive, including up to 10¹⁵ variants (5). However, when recognizing a particular antigen, T cells have in many cases been shown to preferentially use a specific TCR AV or BV gene segment (6).

Analyzing T cells obtained by bronchoalveolar lavage (BAL), we previously described a strict correlation between lung accumulated CD4⁺ and AV2S3⁺ T cells and the HLA-DR17 haplotype (i.e., the DRB1*0301 allele; previously named DR17 [3]) in Scandinavian sarcoidosis patients who had active disease (10, 11). In a recent study, we found this association to also include the HLA-DRB3*0101 allele, which is expressed by most HLA-DR17-positive and by a subpopulation of HLA-DR13-positive individuals (J. Grunewald and colleagues, submitted manuscript). We also sequenced TCR  chains of AV2S3-expressing, lung-accumulated T cells, and the results indicated that these cells had been stimulated by a yet unidentified sarcoidosis-associated antigen (12).

In the present study we related the relative numbers of lung-accumulated TCR AV2S3⁺ T cells to clinical data in a group of 51 Scandinavian patients with active pulmonary sarcoidosis, who had varying degrees of lung-restricted AV2S3⁺ T cell expansion. We found associations between the relative numbers of AV2S3⁺ T cells in bronchoalveolar lavage fluid (BALF) and several clinical features of the disease, including a short disease duration. These results strongly link AV2S3⁺ T cells in BALF to the acute immune response in sarcoidosis, and also suggest that these cells may have a protective role in the response to a presumed sarcoidosis antigen.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

The study group consisted of 51 HLA-typed Scandinavian pulmonary sarcoidosis patients who had been included in a recent previous study of individual TCR AV2S3 expression on their BALF and paired peripheral blood lymphocytes (J. Grunewald and colleagues, submitted manuscript). All of the patients were Caucasian and had signs of more or less pronounced accumulations of lung-restricted T-cell populations expressing TCR AV2S3.

All patients had clinical presentations and chest radiographic findings compatible with sarcoidosis. Twenty-one of the patients had positive biopsies for the disease, 38 of the patients had a BAL CD4⁺-to-CD8⁺ cell ratio > 3.5 (13), and 41 of the patients had classical Löfgren's syndrome. All 51 patients included in this study were classified as having active disease, defined on the basis of results of chest radiography, lung function tests, and the presence of symptoms at the time of the study or within 3 mo before the study began. Disease onset was classified as acute or nonacute, and the time from first presentation of symptoms to BAL was determined. Airway symptoms and extrathoracic manifestations were recorded, and Löfgren's syndrome was diagnosed through the presence of such additional symptoms as arthritis and/or erythema nodosum and fever. The chest-radiographic stage of disease was determined for each patient (Table 1). Thirty-nine of the patients were followed for more than 2 yr, and were classified according to changes in chest radiographs and/or symptoms as having a short disease duration (i.e., recovery within 2 yr) or a long disease duration (i.e., with signs of disease activity for more than 2 yr) (Table 1) (14).

BALF and paired peripheral blood lymphocytes were analyzed for expression of TCR AV2S3 on CD4⁺ T cells (J. Grunewald and associates, submitted manuscript), as well as for CD4⁺-to-CD8⁺ cell ratios, and differential counts were performed on BALF cells (Table 2). Pulmonary function was evaluated through determination of VC, FEV₁, and diffusing capacity of carbon monoxide (DL_CO) according to procedures previously reported (14), and the results were expressed as percents of predicted values (Table 2).

All subjects gave their informed consent to participate in the study, and the study was approved by the local ethics committee.

BAL Procedure and Handling of Cells

Two hundred and fifty milliliters of sterile saline solution at 37° C were instilled into a middle-lobe bronchus in five aliquots of 50 ml each. The fluid was gently aspirated after each instillation and was collected in a siliconized bottle kept on ice. The BALF was strained through Dacron nets and the collected cells were pelleted at 400 × g for 10 min at 4° C and resuspended in RPMI-1640 medium (Gibco, Paisley, UK). After cell counting and viability determination by trypan blue exclusion (mean = 95%), smears for differential counts were prepared by cytocentrifugation (Cytospin 2; Shandon, UK) at 22 × g for 3 min and staining with May-Grünwald-Giemsa. Paired peripheral blood mononuclear cells were separated from heparinized peripheral blood by gradient centrifugation through Ficoll Hypaque (Pharmacia, Uppsala, Sweden), after which the cells were washed twice in RPMI-1640 medium and diluted to appropriate concentrations.

Monoclonal Antibodies, Development of Immunofluorescence, and Flow Cytometry

Determination of TCR AV2S3 expression (J. Grunewald and colleagues, submitted manuscript) and of CD4⁺-to-CD8⁺ cell ratios was performed as follows: Cells were incubated with an unlabeled TCR AV2S3-specific monoclonal antibody (mAb) and washed twice, and fluorescein isothiocyanate-conjugated F(ab')₂ fragments of rabbit antimouse Ig were added for the detection of bound antibodies. Normal mouse serum, diluted 1:500, was used to block remaining rabbit antimouse Ig before the addition of a cocktail of RPE-Cy5-conjugated CD4-specific and RPE-conjugated CD8-specific mAbs (DAKO, Glostrup, Denmark). Cells were analyzed with a FACS Calibur flow cytometer (fluorescence-activated cell sorter; Becton-Dickinson) and a Hewlett-Packard 300 computer (Hewlett-Packard, Palo Alto, CA). Lymphocytes were gated out by forward and side scatter, and the percentage of positive cells in each subset was determined. Isotype-matched negative control antibodies always stained fewer than 1% of cells.

Statistical Analyses

The nonparametric Mann-Whitney U test was used for calculation of statistically significant differences in AV2S3 expression among clinically defined groups of patients, and the chi-square test was used for calculating associations between AV2S3⁺ cells and prognosis, as noted. For calculating associations between AV2S3 expression and clinical parameters, we used the nonparametric spearman's rank correlation (R) test. All results are presented as median values, with minimum and maximum values as the range.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Every patient enrolled in the study was either HLA-DRB1* 0301 and/or -DRB3*0101 positive, and all had signs of various degrees of lung-accumulated CD4⁺ T cells expressing TCR AV2S3 (J. Grunewald and colleagues, submitted manuscript). The patients included in the study had a median age of 36 yr, and consisted of 30 males and 21 females (Table 1). All had active sarcoidosis, and AV2S3⁺ BALF T cells constituted 29.0% (median) of the patients' BALF CD4⁺ T cells, as compared with only 4.1% of the paired CD4⁺ peripheral blood T cells (Table 2). For comparison, previous studies found that 3.5% (median) of CD4⁺ peripheral blood T cells of healthy controls expressed AV2S3 (15), that 7.6% (median) of CD4⁺ BALF T cells from six HLA-DR17-positive healthy controls expressed AV2S3 (11), and that 5.0% (median) of BALF T cells from four HLA-DR17-positive patients with allergic alveolitis expressed AV2S3 (16). Since the number of lung-accumulated AV2S3⁺ T cells in the present study varied from a minimum value of 8.6% to a maximum of 41.6%, we set out to determine whether this variation correlated in any way with other features of the patients. We therefore evaluated each patient's clinical characteristics, BALF data, and results of pulmonary function tests (Tables 1 and 2), and analyzed any relation of these measures to the relative numbers of AV2S3⁺ BALF T cells.

Relative Numbers of TCR AV2S3⁺ BALF Cells and Clinical Characteristics

A negative correlation was found between the number of AV2S3⁺ lung T cells and patient age, with older patients having smaller numbers of AV2S3⁺ BALF T cells (p = 0.002, R = 0.41). There was also a negative correlation with time from presentation of disease symptoms to BAL. Thus, the longer the time that passed from disease onset to BAL, the lower the numbers of AV2S3⁺ BALF T cells found (p = 0.0001, R = 0.517; Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Percent CD4+ BALF T cells expressing TCR AV2S3 (y axis) correlated negatively with the duration from disease onset to BAL (months) (x axis); p = 0.0001, R = 0.51.

An acute form of disease onset was associated with significantly higher proportions of AV2S3⁺ BALF T cells (30.2%; range: 12.1 to 41.6%) than was a nonacute form of onset (21.7%; range: 8.6 to 40.0%) (p = 0.018) (Figure 2A), and patients with Löfgren's syndrome had a tendency to have higher percentages of AV2S3⁺ BALF T cells (p = 0.059).

View larger version (12K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 2.   (A) Percent CD4+ BALF T cells expressing TCR AV2S3 (y axis) in patients with a nonacute (median: 30.2%) and with an acute (median: 21.7%) disease onset (x axis); p = 0.018. (B) Percent CD4+ BALF T cells expressing TCR AV2S3 (y axis) in patients with a long (> 2 yr) (median: 30.0%) and a short (< 2 yr) (18.6%) disease duration (x axis); p = 0.003.

We followed 39 of the patients for more than 2 yr, and classified them into two groups according to disease outcome. We found that patients with a short disease duration (< 2 yr) had significantly higher percentages of AV2S3⁺ BALF T cells (30.0%; range: 12.1 to 41.4%) than did patients with a long (> 2 yr) disease duration (18.6%; range: 9.0 to 30.4%) (p = 0.003) (Figure 2B). In fact, only one of 10 patients with a disease duration > 2 yr had  30% AV2S3⁺ BALF T cells, as compared with 15 of 29 patients with a short disease duration (p = 0.02, chi-square test). Thus, any patient with  30% AV2S3 BALF T cells has a very good prognosis. There were no significant associations between AV2S3⁺ BALF T cells and gender, smoking habit, airway/extrathoracic symptoms, or chest-radiographic staging.

Relative Numbers of TCR AV2S3⁺ BALF Cells, Other BALF Cell Findings, and Pulmonary Function

As expected, the BALF CD4⁺-to-CD8⁺ cell ratio was increased in this group of patients (median: 6.7) as compared with the CD4⁺-to-CD8⁺ cell ratio in peripheral blood (median: 2.0) (Table 2). The proportion of AV2S3⁺ lung T cells showed a strong positive association with the BALF CD4⁺-to-CD8⁺ cell ratio (p = 7 × 10⁶; R = 0.59) (Figure 3). It also showed an association with signs of a lymphocytic alveolitis. Thus, high numbers of AV2S3⁺ BALF cells had a tendency to correlate with high BALF cell concentrations (p = 0.04, R = 0.29), with high relative numbers of BALF lymphocytes (p = 0.02, R = 0.30), and with low relative numbers of alveolar macrophages (p = 0.04, R = 0.28) and basophilic granulocytes in BALF (p = 0.02, R = 0.32).

View larger version (13K): [in this window] [in a new window]   Figure 3.   Percent CD4+ BALF T cells expressing TCR AV2S3 (y axis) correlated with the BALF CD4+-to-CD8+ cell ratio (x axis); p = 7 × 106, R = 0.59.

There were no associations between the proportions of AV2S3⁺ BALF T cells and measures of pulmonary function (VC, FEV₁, or DL_CO; Table 2), or with s-angiotensin converting enzyme (ACE) levels (median: 29.4 units/L; range: 9.7 to 63.1 units/L; reference values in our laboratory < 32 units/L).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In previous studies of BALF T cells of Scandinavian sarcoidosis patients, we established an association between DRB1*0301 (HLA-DR17) and lung-accumulated T cells expressing TCR AV2S3 (10, 11), and extended this association to also comprise the HLA-DRB3*0101 allele (J. Grunewald and colleagues, submitted manuscript). The strict association between a certain TCR V gene segment, expressed selectively by T cells located at the inflammatory focus, and particular HLA alleles is quite unique in human autoimmune immunology research. A closer study of possible associations between the relative numbers of AV2S3⁺ BALF T cells and various clinical features of our sarcoidosis patients was therefore of interest as an effort to elucidate a role for these BALF T cells in sarcoidosis. The present data appear to link AV2S3⁺ BALF T cells to signs reflecting the acute inflammatory response in sarcoidosis, and moreover suggest that these cells have a protective role against a postulated sarcoidosis antigen.

Our original observation of AV2S3⁺ BALF T-cell expansions in HLA-DR17-positive patients, although they still had normal AV2S3⁺ T cells in paired peripheral blood, connected these cells to the site of disease activity (i.e., where these patients had signs of an active inflammatory process). We report here a tendency of these patients' AV2S3⁺ BALF cells to normalize with the time from disease onset to BAL. The disappearance of the AV2S3⁺ BALF T cells parallels a decline in lympocytic alveolitis as well as a trend toward clinical improvement in most of these patients with the passage of time. Therefore, this finding also links AV2S3⁺ BALF T cells to the time of an acute local immune response in sarcoidosis, and the relative numbers of AV2S3⁺ BALF T cells may therefore be used as a marker for the degree of inflammation in patients with these sarcoidosis-associated HLA-DR alleles.

Large T-cell populations expressing a certain TCR V gene segment (T-cell expansions) are considered to be the consequence of a previous immune reaction against a specific antigenic peptide. The AV2S3⁺ BALF T cells may therefore be implicated in an immune response against a specific antigen that is located in the lungs of sarcoidosis patients. Likewise, a preferential TCR V gene segment expression by T cells infiltrating the affected organ also has been reported in other autoimmune diseases, such as multiple sclerosis (17). Nevertheless, T-cell expansions can also be detected in seemingly healthy individuals (18), and such normally occurring T-cell expansions have been suggested to be more frequent with increasing age (19). Although these latter findings were made only on peripheral blood T cells, they may still be relevant for lung T cells, since previous studies comparing T-cell repertoires in peripheral blood and BALF have not found any distinct differences (20, 21). An increased frequency of BALF T-cell expansions in elderly healthy individuals might therefore be expected. In contrast, our finding that elderly sarcoidosis patients have reduced numbers of AV2S3⁺ BALF T cells suggests that these cells are linked to the pathology of sarcoidosis.

Although the function of the lung-accumulated AV2S3⁺ T cells in sarcoidosis has been investigated, their exact role in the immune response in sarcoidosis is still not known. Generating T-cell clones from the lungs of patients and using them in stimulatory proliferation tests has so far not been fruitful. However, the significant association of AV2S3⁺ T cells with a short disease duration, as we found in the present study, indicates a beneficial role for these cells. One beneficial mode of these cells' action could be efficient eradication of a presumed sarcoidosis-related antigen; another could be the production of certain effector molecules, such as cytokines, that subsequently lead to a more effective immune response. The close association between higher relative numbers of AV2S3⁺ BALF T cells and a good prognosis in sarcoidosis also opens the possibility of using this information when clinically evaluating patients for treatment, follow-up, and other reasons. Notably, patients having AV2S3⁺ BALF T-cell expansions exceed one third of all Scandinavian sarcoidosis patients, since HLA-DR17 is markedly overrepresented in this patient group (14).

The relative numbers of AV2S3⁺ BALF T cells in our sarcoidosis patients were also linked to several markers of a lymphocytic alveolitis, and particularly to the BALF CD4⁺-to-CD8⁺ cell ratio. Interestingly, an increased BALF CD4⁺-to-CD8⁺ cell ratio has been reported to be specific for sarcoidosis, provided the patient has a clinical picture typical of the disease (13). The disease specificity of AV2S3⁺ BALF T cells is also suggested by findings in our previous study on another pulmonary inflammatory disease (allergic alveolitis), in which we could not find accumulated AV2S3⁺ BALF T cells in the lungs of HLA-matched patients (16). A similar investigation of BALF T cells of HLA-matched patients with berylliosis, which more closely resembles sarcoidosis, would be of great interest to further establish the disease specificity of AV2S3⁺ BALF T cells. Unfortunately in this regard, berylliosis seems to be a very rare disorder in Sweden.

In conclusion, we have presented data that link AV2S3⁺ BALF T cells, found in HLA-DR17 (and a subset of HLA-DR13)-positive Scandinavian sarcoidosis patients, with the acute inflammatory response seen in these patients' lungs. Our data suggest a beneficial role for these cells in the immune response to a postulated sarcoidois-associated antigen. An estimation of relative numbers of AV2S3⁺ BALF T cells in HLA-DR17 (or DRB3*0101)-positive sarcoidosis patients, who make up one third of all Scandinavian sarcoidosis patients, may even be used clinically as a marker of inflammation, contributing to the prognostic and diagnostic evaluation of patients with sarcoidosis.