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Departments of Clinical and Experimental Medicine, Clinical Immunology and Hematology Branches, Division of Pneumology and Institute of Pathology, Padua University School of Medicine, Padua, Italy
ABSTRACT
Rationale: Receptor expression dictates the spectrum of chemokine actions on immunocompetent cells. We have previously shown that the chemokine receptor CXCR3 is highly expressed by T-helper type 1 (Th1) cells infiltrating the lungs of patients with sarcoidosis.
Objectives: The evaluation of the role of Bonzo/CXCR6 and its ligand CXCL16 in the pathogenesis of sarcoidosis.
Methods: Immunocompetent cells infiltrating sarcoid lung have been evaluated by flow cytometry, confocal microscopy, immunohistochemical and molecular analysis, and functional assays.
Main Results: Th1 cells isolated from the bronchoalveolar lavage of patients with sarcoidosis and T-cell alveolitis coexpressed CXCR3 and CXCR6. Immunohistochemical analysis of lung specimens has shown that CXCR6+ T cells infiltrated lung interstitium surrounding the central core of the granuloma. The CXCR6 ligand CXCL16 was abundantly expressed by macrophages infiltrating sarcoid tissue and/or forming the granuloma core. From a functional point of view, sarcoid Th1 cells were able to respond to CXCL10 and CXCL16 in migratory assay. In vitro kinetic studies demonstrated that, although CXCR3 was rapidly induced by interleukin (IL)-15 and IL-18, CXCR6 induction was slow (8 d) and mainly regulated by IL-15.
Conclusions: T cells coexpressing CXCR3 and CXCR6 act coordinately with respective ligands and Th1 inflammatory cytokines in the alveolitic/granuloma phases of the disease.
Key Words: chemokines granuloma immunopathogenesis sarcoidosis T-cell alveolitis
Sarcoidosis is an immunomediated multisystemic disorder of unknown cause(s) most commonly affecting young adults, and frequently presenting with hilar lymphadenopathy, pulmonary infiltration, and ocular and skin lesions (1–3). The liver, spleen, lymph nodes, salivary glands, heart, nervous system, muscles, and bones are other frequently involved organs. The diagnosis is established when well-recognized clinicoradiographic findings are supported by histologic evidence of widespread epithelioid granulomas in more than one system (4).
Findings in recent years continue to expand our knowledge about the network of interactions between immunocompetent cells that set the stage for the pathogenesis of sarcoidosis (5–7). The antigen(s) that triggers the development of sarcoidosis favors the influx of T-helper type 1 (Th1) clones and macrophages into sites of ongoing inflammation and chemokines, and their receptors are believed to be responsible for the T-cell recruitment during sarcoid hypersensitivity reactions (8–10). In particular, it has been demonstrated that a non-ELR CXC chemokine, which is produced in response to IFN- (i.e., IFN-gamma inducible protein [IP]-10/CXCL10), is crucial in driving recruitment of Th1 cells into the pulmonary microenvironment (8). Lung macrophages are the main cell source for this molecule; they release high amounts of CXCL10 that, interacting with the specific receptor which is expressed by Th1 cells (CXCR3), favors the migratory capability of sarcoid T cells and contributes to building the granulomatous structure.
Another chemokine receptor that defines unique subsets of highly Th1-polarized T cells is Bonzo/CXCR6. Interacting with its ligand CXCL16, this receptor is involved in mediating type 1 inflammation (11–13). This study evaluated whether Th1 cells accumulating in sarcoid lung coexpress both CXCR3 and CXCR6 receptors. Using immunohistochemical studies, flow cytometry, and confocal microscopy evaluation, we have shown that sarcoid CD4+ T cells isolated from the bronchoalveolar lavage (BAL) coexpress both molecules. In addition, we have found that signaling of Bonzo/CXCR6 with CXCL16 induces a potent in vitro migratory activity of sarcoid CD4+ T cells. The ligand of CXCR6, CXCL16, has been found to be expressed by pulmonary macrophages, epithelioid cells, and epithelial cells forming the central core of the granuloma.
METHODS
Study Population
Eighteen patients with active sarcoidosis were included in this study (8 males and 10 females; age range, 35–72 yr; 3 smokers and 15 nonsmokers). According to our staging system for sarcoidosis, they were defined as having an active disease on the basis of the following characteristics: (1) lymphocytic alveolitis (> 20 x 103 lymphocytes/ml), (2) positivity to gallium citrate Ga 67 scan, (3) lung CD4/CD8 ratio more than 5.0 (14).
Another 14 BAL samples were obtained from patients in the inactive phase of the disease, either spontaneously or after therapy. Six BAL control samples from healthy adults were selected (from four men and two women; average age, 32.5 ± 4.3 yr; two were nonsmoking, healthy subjects, and four subjects without lung disease who were evaluated for complaints of cough).
Preparation of Cell Suspensions
Alveolar macrophages and BAL T cells were enriched from the entire mononuclear cell suspensions as previously described (15).
Flow Cytometry Analysis
The commercially available conjugated or unconjugated monoclonal antibodies (mAbs) used included the following: CD3, CD4, CD8, CD45R0, interleukin (IL)-12R2), isotype-matched controls. Anti–IL-4 and anti–IFN- mAbs were purchased from PharMingen (San Diego, CA). Purified or phicoerytrin-conjugated antihuman CXCR6 mAb (clone 56811; R&D Systems, Inc., Minneapolis, MN) and a purified goat antihuman CXCL16 (R&D Systems, Inc.) were also used for immunohistochemistry.
The frequency of BAL cells positive for the above reagents was determined by flow cytometry as previously described (15).
Confocal Microscopy
Purified T cells from BAL of patients with active sarcoidosis were stained at 4°C both with purified anti-CXCR6 (1:150) and fluorescein isothiocyanate–conjugated anti-CXCR3 antibodies (1:150). Then samples were analyzed by confocal microscope (Biorad 2100 Multiphoton; Bio-Rad Laboratories, Hercules, CA) with a 60x objective lens (Nikon), using laser excitation at 488 nm. Methods used are described in the online supplement.
RNA Purification and Real-Time Polymerase Chain Reaction for the Quantification of CXCR6 and CXCL16 Expression by Sarcoid Lung Cells
Total RNA was prepared from (1) purified T lymphocytes obtained from patients with sarcoid T-cell alveolitis or normal T cells or (2) purified alveolar macrophages from patients with active sarcoidosis and control subjects. Methods used for the real-time polymerase chain reaction are detailed in the online supplement.
Migration Activity of Pulmonary T Cells in Response to CXCL16
T-cell migration was measured in a 48-well, modified Boyden chamber (AC48; Neuro Probe, Inc., Gaithersburg, MD) as previously reported (15).
Immunohistochemical Analysis of CXCR6+ Cells and CXCL16-producing Cells
Expression of CXCR6 and CXCL16 was measured by permanent section immunohistochemistry with anti-CXCR6 and anti-CXCL16 antibodies (R&D Systems, Inc.) (15). Immunoassay was detected by using digital quantitative analysis (Image Pro Plus; Media Cybernetics, Silver Spring, MD). Quantification of positive cells was restricted to the alveolar wall. Images were acquired with a 40x lens. In all experiments, negative control samples were included and showed negative staining (data not shown).
Effect of Sarcoid Cytokines on the Expression of CXCR3 and CXCR6 in Peripheral T Lymphocytes from Patients with Sarcoidosis
To test the effects of cytokines that are released in a sarcoid microenvironment on the expression of CXCR3 and CXCR6, a time-course experiment was performed using highly purified T cells. Details are reported in the online supplement.
RESULTS
BAL cell recovery (normal range, 115,500–195,500 cells/ml of BAL) was significantly higher in patients with active sarcoidosis compared with patients with inactive disease (mean, 237,446 ± 57,216 vs. 102,042 ± 27,380; p < 0.001). Regarding differential count of BAL cells, the absolute number of lymphocytes was higher in patients with active disease (62,926 ± 11,060) than in the inactive group (8,343 ± 1,467; p < 0.001). As a consequence of the increase in the absolute number of CD4+ T cells, the BAL CD4/CD8 ratio was significantly increased in patients with active disease (6.47 ± 1.28) with respect to patients with inactive disease (2.1 ± 0.29; p < 0.001) and control subjects (1.7 ± 0.34; p < 0.001).
Flow Cytometry and Confocal Analysis of the Expression of CXCR6 by Sarcoid and Normal Pulmonary T Cells
Flow cytometry analysis of BAL T lymphocytes obtained from patients with sarcoidosis who presented high-intensity T-cell alveolitis showed that more than 98% of T lymphocytes recovered from the BAL of patients with sarcoidosis were CD4+/CD45R0+/IL-12R2+ cells expressing CXCR3+ (Figure 1). The mean percentage of CXCR6+ T cells was 49.25 ± 3.06% in patients with the active form of the disease, whereas the percentage was 20.53 ± 2.90% in the inactive disease (p < 0.01). Pulmonary T cells of normal donors showed a very low expression of CXCR6 (mean percentage, 5.68 ± 0.81%).
Interestingly, BAL T cells of patients with active sarcoidosis showed an intensity of CXCR6 expression that paralleled the degree of T-cell alveolitis (Figure 2). BAL T cells of patients with active disease, patients with inactive sarcoidosis, and normal subjects showed a mean fluorescence intensity of 12.86 ± 3.65, 2.17 ± 0.50, and 1.47 ± 0.55, respectively (p < 0.001, as determined by the Kolgomorov-Smirnov analysis). However, it is interesting to note that the small number of CD8+ T cells found in the BAL of patients with sarcoidosis expressed CXCR6, even if at a lower density then CD4+ BAL T cells (Figure 3).
To evaluate whether CXCR6 and CXCR3 are coexpressed by sarcoid T cells, confocal analysis of was performed on highly purified BAL T cells. As shown in Figure 4, BAL T cells with evident expression of superficial CXCR3 (Figure 4A, panel with green fluorescence) also presented a homogeneous high intensity of CXCR6 expression at the surface of the cells (panel with red fluorescence). The colocalization of the two receptors was confirmed by the confocal microscopy in merged modality and by flow cytometric analysis (Figure 4B).
Immunohistochemical Analysis of CXCR6 and CXCL16 Expression at Sites of Disease Activity
Immunohistochemical analysis confirmed the high-intensity expression of CXCR6 by sarcoid T-cell–infiltrated pulmonary biopsy specimens obtained from patients with active sarcoidosis (Figure 5A). Interestingly, T cells surrounding the central core of the granuloma were also CXCR6 positive in lung specimens obtained from patients with chronic, refractory sarcoidosis (Figure 5B).
Immunohistochemical analysis was also performed using an antibody that recognizes CXCL16 to investigate the cell sources of the chemokine in sarcoid tissues (Figures 6A and 6B). The central core of sarcoid granuloma was made up of a number of monocyte/macrophages at various states of activation and differentiation, as well as epithelioid cells and multinucleated giant cells. CXCL16 was preferentially expressed by macrophages, multinucleated giant cells, and epithelioid cells localized inside the granuloma (Figure 6A). Interestingly, immunohistochemical analysis of native lungs obtained from three patients with refractory sarcoidosis and pulmonary fibrosis who underwent lung allograft transplantation showed that the fibrotic phases of the disease macrophages were mainly nonreactive for CXCL16, whereas epithelial cells along the alveolar wall were strongly reactive for the chemokine (Figure 6B).
Molecular Analysis of CXCR6 and CXCL16 Expression by Sarcoid Lung Cells
Real-time polymerase chain reaction evaluation of CXCR6 mRNA expression by highly purified BAL T lymphocytes demonstrated that mRNA expression of CXCR6 was higher in lung T lymphocytes of active sarcoidosis (mean, 20.65 ± 5.11) than in normal T cells (mean, 0.67 ± 0.30; p < .01; Figure 5C). In parallel, a real-time polymerase chain reaction evaluation of CXCL16 mRNA expression by highly purified BAL macrophages demonstrated that mRNA expression of CXCL16 was higher in lung macrophages of active sarcoidosis (mean, 72.26 ± 8.02) than in normal lung macrophages (mean, 30.40 ± 1.75; p < .05; Figure 6C).
CXCR6 Mediates the Migration of Sarcoid T Cells
To characterize biological properties of CXCR6 and CXCR3, highly purified T cells isolated from BAL of patients with sarcoidosis with T-cell alveolitis were assessed for their migratory capabilities in response to CXCL16 and CXCL10. The evaluation of the migratory capabilities of pulmonary T cells of subjects with sarcoidosis with no sign of T-cell alveolitis (inactive disease) and of normal subjects was prevented by the low recovery of pulmonary T cells from the BAL. As shown in Figure 7, CXCR6+ lung T cells exhibited a strong, definite migration in response to CXCL16, suggesting that pulmonary T cells infiltrating sarcoid lung express a functional CXCR6 receptor able to signal migration in response to its ligand. The migratory capability in response to CXCL16 overlapped the response to CXCL10 (p = not significant).
Modulation of CXCR3 and CXCR6 Expression by Peripheral T Cells Exposed In Vitro to Sarcoid Cytokines
To determine mechanisms leading to the upmodulation of the expression of CXCR3 and CXCR6 by sarcoid T cells, T cells purified from the peripheral blood of four patients with active sarcoidosis were cultured in the presence of cytokines that are actively released in sarcoid lung, including IL-2, IL-12, IL-15, IL-18, and IFN- (16, 17); the intensity of CXCR3 and CXCR6 expression was evaluated by flow cytometry after culture in the presence of the above cytokines for 2, 18, and 24 h and after a long-term culture (8 d). CXCR3 expression on peripheral T cells increased after 18 h of incubation with IL-15 and with IL-18, with respect to T cells cultured with medium alone (Table 1 and Figure 8). CXCR3 expression persisted after 24 h and 8 d with the two cytokines.
CXCR6 expression was very low on peripheral T cells at resting conditions and was not increased after exposure of T cells to all the above cytokines in a short-duration experiment (2–24 h; Table 2). By contrast, an upregulation of CXCR6 by T cells was demonstrated after stimulation for 8 d to 400 U/ml IL-2 (Table 2). When peripheral T cells were cultured with IL-15 for 8 d, a high expression of CXCR6 was induced; the effect of IL-15 on CXCR6 expression was more robust than effects shown by IL-2 (Table 1 and Figure 9). However, CXCR6 expression by sarcoid T cells was not further increased by the coculture of IL-2, suggesting that IL-15 is per se able to obtain the maximal functional activation of sarcoid T cells, as previously reported by our group (data not shown). A weak, but significant upregulation of CXCR6 expression has been observed after incubation with IL-18 and IFN- for 8 d (Table 2). By contrast, no effect or a slight effect on CXCR6 expression has been observed when peripheral T cells were cultured in the presence of IL-12 alone. Interestingly, the intensity of CXCR6 expression was further increased when IL-2–stimulated T cells were costimulated with IL-12, IL-18, or IFN- (mean fluorescence intensity, 70.4, 75.4, and 78.3, respectively).
DISCUSSION
This study provides evidence that CXCR6/CXCL16 interactions are critical for the localization of Th1 cells in sarcoid tissues. We also suggest that CXCR6 is expressed on long-term, stimulated Th1-accumulating cells, acting coordinately with CXCR3 in the homing process of effector cells at sites of sarcoid inflammation.
It is known that CXCR6 is expressed by subsets of Th1 or T-cytotoxic type 1 (Tc1) cells, but not by Th2 or Tc2 cells, establishing Bonzo as a differential marker of polarized type 1 T cells in vitro and in vivo (11–13). Priming of naive T cells by dendritic cells induces expression of CXCR6 on T cells, and IL-12, a cytokine that is actively released in sarcoid lung, enhances the dendritic cell–dependent upregulation (11). We have observed that lung T cells infiltrating the lungs of patients with acute sarcoidosis overexpress Bonzo. Furthermore, the receptor is functionally active, because pulmonary T cells responded to the ligand in chemotaxis assay. The phenomenon of the attraction of Bonzo+ T cells into sites of inflamed tissues cannot be considered as specific for sarcoidosis; in fact, CXCR6+ T cells are also dramatically enriched among T cells in tissue sites involved by chronic inflammation, including rheumatoid joints (11), cardiac valves during inflammatory valvular heart disease (18), and inflamed livers (19), suggesting that Bonzo is a general trafficking factor for Th1/Tc1 lymphocytes.
Interestingly, CXCR6+ T cells were localized in the area surrounding the central core of the granuloma indicating that this receptor is crucial in the assembly of the granuloma structure (Figure 5C). Unutmaz and colleagues (20), tracing mouse Bonzo expression in EGFP knockin mice, demonstrated that green fluorescent protein expression on CD4+ murine T cells is restricted to the CD44+ T-cell subset, suggesting that Bonzo is primarily expressed in this memory subset of mouse CD4+ T cells. Because there are data indicating that CD44–osteopontin interaction is crucial in the development of Th1 granuloma (21), both in animal models and sarcoidosis, studies are in progress in our lab to determine whether the persistent cytokine stimulation taking place in sarcoid lung (and partially reproduced in our in vitro 8-d culture assay) favors CD44+/CXCR6+ T-cell recruitment and helps granuloma assembly.
CXCL16 is the first chemokine identified as having a scavenger receptor activity (22). CXCL16 may be expressed in soluble and in transmembrane forms on dendritic cells and macrophages; in addition, it ligates CXCR6 chemokine receptor and drives migration of activated Th1 and Tc1 cells (23). It is identical to the scavenger receptor SR-PSOX, which mediates uptake of oxidized low-density lipoprotein. As a soluble form, CXCL16 is a chemoattractant for activated CD4+ and CD8+ T cells through binding with its receptor, CXCR6. The activity of soluble CXCL16 is keenly regulated by a member of the disintegrin and metalloproteinase (ADAM) family protease ADAM10 (24). Furthermore, recent data indicate that SR-PSOX/CXCL16 not only attracts T cells and natural killer T cells toward dendritic cells but also supports their firm adhesion to cells expressing CXCR6 (22). From a molecular point of view, CXCL16 is a potent and direct activator of nuclear factor (NF)-B and induces B-dependent proinflammatory gene transcription (25). Because T-cell inflammation in sarcoidosis is associated with local NF-B activation (26, 27), it is possible that, rather than mediating T-cell recruitment, CXCL16 may participate in the local mechanisms leading to the immune activation of the T-cell compartment.
Our immunohistologic data suggest that sarcoid epithelial cells could represent a cell source of CXCL16. Whether epithelial expression has positive or detrimental effects on the local microenvironment still remains to be established. Because transmembrane CXCL16 has antimicrobial activity (28), one possibility is that CXCL16-expressing epithelial cells may participate in the innate immunity against the unknown etiologic agent(s) of sarcoidosis. A transmissible infectious agent has been involved in the pathogenesis of the disease (29). Most studies have reported that at least a few specimens are positive for Mycobacterium tuberculosis, and the findings prompted the suggestion that some patients with sarcoidosis have a disease that could have been initiated by a mycobacterial infection (30). Swedish researchers have suggested that Rickettsiae may contribute to the pathogenesis of sarcoidosis (31). In addition, Japanese researchers suggest that Propionibacterium acnes, a bacterial commensal that causes a granulomatous reaction when injected experimentally into sensitized rats and rabbits, may by involved in the disease development (32–34). Studies should be planned to define whether sarcoid epithelial cells that express CXCL16 exhibit antibacterial activities against these pathogens. Furthermore, we need to know whether pulmonary epithelial cells may express chemokine receptor and if there are differences in chemokine/chemokine receptor expression between distal and proximal epithelium.
Another possibility is that transmembrane and soluble CXCL16 released by local antigen presenting cells (APCs) and sarcoid epithelial cells could have deleterious effects on the surrounding environment. A consistent observation in patients with sarcoidosis evolving to fibrosis is the progressive loss of epithelial cells, which undergo programmed cell death. Whether CXCL16 expression is relevant in inducing the epithelial apoptotic process is another topic that deserves further investigation. Almost all CXCR6+ Tc1 cells contain preformed granzyme A and display cytotoxic effector phenotype. Thus, there is a possibility that epithelial cells that provide continuous stimulation for the recruitment of CXCR6+ Tc1 cells might contribute to the creation of a local milieu that favors epithelial apoptosis.
In conclusion, our data suggest the importance of interactions of CXCR6 with its ligand in the migration of Th1 cells in sarcoid tissues. CXCR6 is preferentially expressed on long-term–stimulated sarcoid Th1 cells and is under the influx of cytokines. However, CXCR6 likely cooperates with CXCR3 in homing mechanisms leading to the accumulation of effector cells at sites of granuloma formation. New agents are currently undergoing clinical trials in patients with sarcoidosis (35) and it is likely that, in the future, agents able to interfere with Th1 chemokine receptors could be investigated as potential therapeutic targets for sarcoidosis.
FOOTNOTES
Supported by a PRIN grant (MIUR) and by Progetto di Ateneo CPDA038833 (Padua University).
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200501-142OC on August 11, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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