INTRODUCTION

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The pathophysiology of pulmonary disorders such as tuberculosis and sarcoidosis is characterized by the development of cell-mediated immune responses, which result in the accumulation and activation of T lymphocytes and macrophages (1, 2). Recent studies have shown that these cells elaborate a number of Th1-like proinflammatory cytokines such as IL-2, IL-12, and IFN- (3). By orchestrating the inflammatory process, these Th1-like cytokines are thought to contribute to disease pathology, particularly in the development of granulomatous lesions (6, 7). Interleukin-12 is a 75 kD heterodimeric cytokine produced by dendritic cells and macrophages, which has attracted substantial interest in the pathogenesis of inflammatory diseases. Indeed, its presence during antigen presentation appears critical in the commitment of naive T lymphocytes to a Th1-type profile of cytokine expression (8). In contrast to pulmonary tuberculosis and sarcoidosis, bronchial asthma is associated with the increased expression of Th2-type cytokines, IL-4, IL-5, and IL-13 (9, 10). Commitment to a Th2-type profile of cytokine expression by naive T lymphocytes is favored by the presence of IL-4 during antigen presentation (11) and inhibited by the presence of IL-12 (12).

In eliciting its biologic activity, IL-12 acts via specific membrane-bound receptors, which have a limited distribution on activated T lymphocytes and NK cells (13). The IL-12 receptor (IL-12R) is a complex composed of two  subunits (₁ and ₂) (14) and is a member of the gp130-type subgroup of the cytokine receptor superfamily (15). The ₂ subunit of the IL-12R complex is responsible for signaling through the Jak/ STAT pathway and was recently shown to be selectively expressed on murine and human Th1 but not Th2-like cytokine producing CD4+ T cells (16). The activity of IL-4 is also dependent on the expression of specific cell surface receptors. These IL-4R consist of a high affinity  subunit that conveys specificity in conjunction with a common signaling subunit (17).

To date, there have been no studies examining the expression of Th1-type cytokine receptors in the lungs of patients with inflammatory lung disorders. We hypothesized that the presence of active pulmonary tuberculosis and active pulmonary sarcoidosis is associated with an elevated expression of IL-12R mRNA. Therefore, we investigated the expression IL-12R mRNA in cells recovered by bronchoalveolar lavage (BAL) from patients with active pulmonary tuberculosis, active pulmonary sarcoidosis, and normal nonatopic control subjects. To verify that this increase in IL-12R expression could not be attributed to the accumulation of lymphocytes within the lungs, we also examined for the expression of IL-12R mRNA in BAL cells taken from allergic asthmatic patients. The expression of the IL-4R was also performed in these patients and control subjects to support the preferential Th1- and Th2-type cytokine distribution in granulomatous diseases and allergic asthma, respectively. The phenotype of cells expressing IL-12R mRNA, in particular its colocalization to CD4+ and CD8+ T lymphocytes, was determined using combined in situ hybridization and immunocytochemistry.

	METHODS

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Study Subjects

Patients with active pulmonary tuberculosis (n = 6), active pulmonary sarcoidosis (n = 6), or allergic asthma (n = 6) were entered into the study. Ethical approval for this study was obtained from the Montreal Chest Institute and the Calmette Hospital Ethics Committees. Written informed consent was obtained from all patients and control subjects prior to bronchoscopy.

Subjects with active pulmonary tuberculosis. Patients with pulmonary tuberculosis (five men, one woman with a mean age of 41 yr; range, 20 to 82 yr) were recruited from the Montreal Chest Institute after they had been referred for bronchoscopy on the basis of an abnormal chest radiograph and the possibility of a diagnosis of pulmonary tuberculosis. All chest radiographs were reviewed by a respiratory physician who deemed that the probability of pulmonary tuberculosis was likely enough to warrant further investigation. Patients who were already receiving antituberculosis chemotherapy were excluded from the study, as were those with known HIV infection, and women who were pregnant. All subjects were nonatopic and skin-test-positive for PPD (> 10 mm). Active pulmonary tuberculosis was defined as microbiologically confirmed tuberculosis on the basis of positive cultures for M. tuberculosis (sputum or BAL fluid). Patients were followed for 6 mo, and all microbiologic and clinical data were used to establish the final diagnosis of active tuberculosis.

Subjects with active pulmonary sarcoidosis. Patients with newly diagnosed active pulmonary sarcoidosis (two men, four women with a mean age of 44 yr; range, 27 to 64 yr) were recruited through the Department of Pneumoimmunoallergy, Calmette Hospital, Lille, France. The clinical diagnosis of pulmonary sarcoidosis was based on previously described criteria, including lung or mediastinal lymph node biopsy exhibiting histologic evidence of noncaseating epithelioid-cell granulomas. According to conventional clinical and radiologic data (18), the disease was classified as active. The criteria used to determine disease activity were: (1) recently developed or increasing cough or dyspnea; (2) and/or systemic symptoms such as cutaneous lesions, weakness, fever, arthralgia; (3) and/or increasing opacities on chest radiograph. Patients with active disease also exhibited an increased T lymphocytosis in BAL (mean percentages and range: active sarcoidosis, 44.6% [33 to 64%]; n = 6; control subjects 8.4% [3.3 to 10.4%]; n = 6; p < 0.05) and an elevated level of serum angiotensin-converting enzyme (mean values ± SEM: active sarcoidosis, 68.1 ± 6.4 U/ml; normal values < 55 U/ml). By chest-roentgenographic staging, two of these patients had Stage I and four had Stage II disease. None of the patients smoked, had any evidence of an atopic nature, as defined by the absence of clinical history and negative skin prick tests to common aeroallergens, or had taken corticosteroids during the previous year.

Subjects with bronchial asthma. Patients fulfilling the ATS criteria for asthma (19) having documented airway reversibility to inhaled ₂-agonists, and an increased airway responsiveness to methacholine (PC₂₀ < 8 mg/ml) were recruited from the Asthma Clinic at the Montreal Chest Hospital, McGill University. These asthmatic patients (four men, two women with a mean age of 31 yr; range, 19 to 47 yr) were classified as mild to moderate and had their symptoms controlled by regular usage of inhaled selective ₂-agonists. None of these patients had taken oral corticosteroids during the preceding 12 wk, were smokers, or had any other serious lung disease.

Control subjects. As control subjects, six men with a mean age of 28 yr (range, 22 to 36 yr) volunteered to participate in the study. None of these control subjects smoked, had any evidence of an atopic nature or had taken corticosteroids during the year preceding the study. The control subjects were recruited through the Asthma Clinic at the Montreal Chest Hospital.

Study Design

To examine the expression of IL-12R and IL-4R mRNA in cells recovered by BAL, fibroptic bronchoscopy was performed under local anaesthesia on patients with active pulmonary tuberculosis, active pulmonary sarcoidosis, normal control subjects, and subjects with allergic asthma. A 6-mm fibroptic bronchoscope was wedged into a segmental bronchus of the right middle lobe, which was then lavaged with successive 20-ml aliquots of sterile buffered saline (0.9%) to a total of 180 ml. The lavage fluid was aspirated using gentle suction after each aliquot, collected into sterile polypropylene tubes, and centrifuged at 300 × g for 7 min at 4° C. The cell pellet was suspended in RPMI 1640 at a concentration of 1 × 10⁶ cells/ml and used to make cytospins. The cytospins were fixed for in situ hybridization using 4% paraformaldehyde for 30 min before being washed in PBS, dried, frozen at 80° C, and shipped on dry ice to Montreal when necessary.

In Situ Hybridization

In situ hybridization (ISH) was performed as previously described (20). Cytospins were hybridized with a ³⁵S-labeled antisense (complementary RNA) and sense (having an identical sequence to mRNA) riboprobe coding for the human IL-12R ₁- and ₂-subunits (generous gifts from Dr. U. Gubler, Hoffman LaRoche Inc., Nutley, NJ), and the -subunit of the IL-4R (generously donated by Dr. Tony Troutt, Immunex Corp., Seattle, WA). The probes were generated from cDNA and transcribed in the presence of ³⁵S-UTP and the appropriate SP6 (for the sense probe) or T7 (for the antisense probe) RNA polymerases. Prior to the application of the probe, the cytospins were permeabilized with proteinase K (1 µg/ml) and then prehybridized with 50% formamide and 2 × standard saline citrate (SSC). For hybridization, antisense or sense probes (10⁶ cpm/section) diluted in hybridization buffer were used. Nonspecific binding was removed by posthybridization washing under high stringency conditions in decreasing concentrations of SSC (4 to 0.05 × SSC). Unhybridized single-stranded RNAs were removed by subsequent treatment with RNase A (20 µg/ ml). The autoradiographs were developed in Kodak D-19 and counterstained with hematoxylin. To ensure the specificity of our signal, we performed the ISH technique using the sense probe after pretreatment of the tissues with RNase.

Simultaneous In Situ Hybridization and Immunocytochemistry

The phenotype of cells expressing mRNA for ₁- and ₂-subunits of the IL-12R, as well as the mRNA for the -chain IL-4R was determined using the technique of simultaneous in situ hybridization and immunocytochemistry as previously described (21). Briefly, the sections were immunostained with monoclonal antibodies to CD4 (helper T cells) and CD8 (cytotoxic/suppressor T cells; Dako Diagnostics, Mississauga, ON, Canada), and subsequently underwent a modified in situ hybridization with the radiolabeled cRNA probe coding for the ₁- and ₂-subunit of the IL-12R and IL-4R. Double- stained cells were visualized by the end product of immunostaining (red) and in situ hybridization (dense silver grains).

Quantification and Data Analysis

Cytospin sections were coded, and the number of positive cells for mRNA were calculated and presented as the number of positive cells/ 1,000 BAL cells. Statistical differences in the number of positive cells between groups were assessed using a nonparametric Kruskal-Wallis ANOVA, and subsequent post hoc analyses were performed using a Mann-Whitney U test (Systat v6.1; SPSS Inc., Chicago, IL). For the colocalization studies, we examined the percentage of ₁- and ₂-subunits of the IL-12R and IL-4R mRNA positive cells that coexpressed either CD4 or CD8. A p value less than 0.05 was considered statistically significant.

	RESULTS

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It can be seen in Table 1 that there was no significant difference in T lymphocytes in asthmatic subjects when compared with patients with tuberculosis. However, in sarcoidosis, as expected, the number of T cells are much higher than those in both groups. The number of eosinophils was higher in asthmatic than in the other groups.

Using the technique of ISH, positive signals for IL-12R (₁- and ₂-subunits) and IL-4R (-subunit) mRNA were observed as specific deposits of silver grains in emulsion overlying individuals cells. There was no significant hybridization signals observed when sections were treated with RNase prior to hybridization or when sections were treated with a sense probe identical to the cytokine receptor mRNA.

Cells expressing IL-12R ₁- and ₂-subunit mRNA were demonstrated in all BAL cytospins taken from patients with active pulmonary tuberculosis or active pulmonary sarcoidosis and from normal control subjects (Figures 1A and 1B). There was, however, a significant increase in the percentage of BAL cells expressing the mRNA for both (₁- and ₂-subunits) of the IL-12R in patients with active tuberculosis compared with normal control subjects (p < 0.01, p < 0.005, respectively). Likewise, the numbers of IL-12R mRNA-positive cells (₁- and ₂-subunits) in active sarcoidosis were greater than that observed in normal control subjects (p < 0.02, p < 0.01, respectively). In contrast, there was a decrease in the numbers of cells expressing the ₁- and ₂-subunits of the IL-12R in the allergic asthmatic subjects compared to the normal control subjects (p < 0.01, p < 0.005, respectively).

View larger version (13K): [in this window] [in a new window]   Figure 1.   The percentage of IL-12 receptor mRNA positive cells in BAL fluid from active pulmonary tuberculosis (n = 6), active pulmonary sarcoidosis (n = 6), normal non-atopic controls (n = 6), and allergic asthmatics (n = 6). Results are shown for both the 1 (A) and 2 subunits (B). There was a significant increase in the percentage of cells expressing IL-12R in active tuberculosis and active sarcoidosis compared to normal controls (p < 0.05). There was also a significant decrease in the percentage of cells expressing IL-12R in patients with allergic asthma compared to normal controls (p < 0.05).

As shown in Figure 2, the numbers of cells expressing mRNA for the -subunit of the IL-4R were significantly increased in the allergic asthmatic subjects compared with the normal subjects. There was no difference in the expression of this -subunit between patients with either active tuberculosis or active sarcoidosis and normal control subjects (p > 0.05).

View larger version (9K): [in this window] [in a new window]   Figure 2.   The percentage of IL-4 receptor mRNA positive cells in BAL fluid from active pulmonary tuberculosis (n = 6), active pulmonary sarcoidosis (n = 6), normal non-atopic controls (n = 6), and allergic asthmatics (n = 6). There was a significant increase in the percentage of cells expressing the  subunit mRNA of the IL-4R in allergic asthma compared to normal controls (p < 0.05). There were no significant differences in the percentage of cells expressing IL-4R in patients with active tuberculosis or sarcoidosis compared to normal controls (p > 0.05).

When simultaneous in situ hybridization and immunocytochemistry was performed, it was observed that the majority of IL-12R ₂-subunit mRNA was associated with CD4+ and CD8+ cells in pulmonary sarcoidosis, as shown in Table 3. In tuberculosis, it was demonstrated that the majority of IL-12R ₁- and ₂-subunits were associated with CD8+ cells, as shown in Tables 2 and 3.

In asthmatic subjects, 62% (± 6.7) of the IL-4R mRNA-positive cells were CD4 positive and only 12% (± 5) of the IL-4R mRNA-positive cells were CD8 positive.

	DISCUSSION

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To extend our understanding into the pathogenesis of disease activity in granulomatous disorders such as pulmonary tuberculosis and sarcoidosis, we examined the expression of IL-12R mRNA in patients with active tuberculosis or sarcoidosis compared with asthmatics and normal subjects. We also examined the expression of IL-4R mRNA in these patients, and the expression of both IL-4R and IL-12R in asthmatic subjects with a Th2-type driven pulmonary immune response. This study provides evidence that BAL fluid from subjects with active pulmonary tuberculosis or sarcoidosis exhibits an increased percentage of cells positive for IL-12R mRNA (both ₁- and ₂-subunits) compared with normal control subjects. Furthermore, we demonstrated that the majority of IL-12R ₂-subunit mRNA is associated with CD4+ and CD8+ cells in pulmonary sarcoidosis and tuberculosis, respectively.

A current paradigm in immunology is that the nature of an immune response to an antigenic stimulus is largely determined by the profile of cytokines produced by activated lymphocytes. In this respect, IL-12 appears to be critical in the commitment of naive CD4+ T cells to a Th1-type profile of cytokine production (8, 12). In addition to the capacity of IL-12 to promote the differentiation and proliferation of CD4+ lymphocytes, this cytokine also acts to enhance the cytolytic activity of CD8+ T cells and natural killer (NK) cells (22, 23), and to augment their production of IFN- (24). The fundamental role of IL-12 in providing innate immunity is exemplified by experimental studies on mice infected with Mycobacterium tuberculosis. These have demonstrated that IL-12 p40 knockout mice are defective in their ability to induce IFN- production, and are unable to generate a protective Th1-type of immunologic response (25). Furthermore, exogenous administration of IL-12 increases host resistance to tuberculosis infection via the induction of IFN- production from T cells and NK cells (26).

The histopathology of pulmonary sarcoidosis and tuberculosis share a number of common features such as the recruitment of activated lymphocytes and monocytes to the lungs, the activation of alveolar macrophages, and the formation of granulomatous lesions. In keeping with these common characteristics, we and others have recently reported that in both disorders there is an increased expression of cytokines reflecting the activation of Th1-type lymphocytes (IFN- and IL-2) and cells of the monocyte/macrophage lineage (IL-1, IL-6, TNF-, IL-12) (5, 27, 28). Notably, IL-12 mRNA expression in both tuberculosis and sarcoidosis was indicative of disease activity, and this may reflect the overwhelming activation of macrophages within the lungs of these patients. Indeed, the ability of IL-12 to direct Th1-type lymphocyte development and the subsequent enhancement of IFN- production by alveolar macrophages may contribute to the formation of granulomatous lesions (29). Our results suggest that the IL-12 found within the lungs of these patients acts on specific receptors present on T lymphocytes and that disease activity involves a perpetuated macrophage-lymphocyte interaction within the lungs.

The increase in IL-12R within the BAL fluid of patients with either active sarcoidosis or tuberculosis could not be attributed to a specific recruitment of T lymphocytes since such an increase in IL-12R was not evident in asthmatic subjects, and the number of T cells in patients with tuberculosis was not significantly different from those in asthmatics and control subjects. However, in sarcoidosis, the number of T cells was very high, and we cannot exclude the possibility that the increase in IL-12R+ve cells is due to an increase in the number of T cells present in the BAL fluid. Indeed, fewer BAL cells derived from these patients expressed the IL-12R ₁- or ₂-subunits than those from the normal control subjects. These results are consistent with the Th2-type profile of cytokine expression associated with allergic asthma and the decreased expression of IL-12 demonstrated previously (10). The increase in IL-4R mRNA expression in this disorder supports the commitment to a Th2-type profile of cytokine expression observed in allergic asthma. In contrast, the lack of increase in IL-4R mRNA expression in active sarcoidosis and tuberculosis is consistent with these disorders exhibiting a preferential Th1-type reaction within the airways.

Studies investigating the in vitro regulation of the IL-12R suggest it is under the control of the local cytokine milieu. IFN- has been shown to assist in this T-cell switching by promoting the ability of naïve T cells to express the ₂-subunit of the IL-12R (16). The ₂-subunit of the IL-12R is induced upon antigen activation via stimulation of the T-cell receptor and appears critical for eliciting biologic activity and signal transduction. Thus, the extinction of IL-12 signaling in early Th2 cells results from a selective loss of ₂- but not ₁-subunit expression (16, 30). Cytokines, including IL-4, IL-10, and IFN-, have previously been shown in vitro to modulate the expression of the ₂-subunit and so influence T-cell commitment (31). IL-12 can also induce the expression of its own receptor ₂ mRNA (26). The selective expression and regulation of the IL-12R ₂-subunit may improve our understanding of the basis of Th1/Th2 differentiation and may provide therapeutic options for altering the Th1/Th2 balance in several immunopathologic conditions.

In summary, these data demonstrate increased numbers of cells expressing the ₁- and ₂-subunits of the IL-12R in active pulmonary sarcoidosis and tuberculosis when compared with those in normal control subjects. This cytokine receptor mRNA was localized preferentially to the infiltrating CD4 and CD8 T lymphocytes, respectively. Conversely, a reduced expression of the IL-12R was evident in BAL cells from allergic asthmatic subjects. This was consistent with the increase in cells expressing IL-4R mRNA in this disease and its predominantly Th2-type cytokine profile. With respect to the pathogenesis of granulomatous diseases, these findings support the switching of CD4+ and CD8+ T lymphocytes to a Th1-type phenotype in vivo.