Kianoosh Katchar, Department of Medicine, Division of Respiratory Medicine, Lung Research Lab L4: 01, Karolinska Hospital, S-171 76 Stockholm, Sweden (fax: +46-8-517 751 54; e-mail: email@example.com).
Objectives. The balance between Th1 and Th2 T cells, classified by virtue of their cytokine production can in an immune response influence the phenotype and progression of several clinical diseases. In this study, we examined the expression of Th1 associated chemokine and cytokine receptors CXCR3, CCR5, and interleukin (IL)-12R, IL-18R, respectively, as well as of the Th2 associated chemokine receptors CCR4 and CXCR4 on CD4+ and CD8+ T cells.
Subjects. Eighteen patients with untreated pulmonary sarcoidosis.
Materials and methods. We used monoclonal antibodies and flow cytometry to analyse the expression of chemokine receptors CXCR3, CXCR4, CCR4 CCR5 and cytokine receptors IL-12R, IL-18R in combination with anti-CD4 and anti-CD8 mAbs in bronchoalveolar lavage fluid (BAL) and peripheral blood lymphocytes (PBL) from sarcoidosis patients.
Results. There were significantly more BAL CD4+ T cells expressing CXCR3, CCR5, IL-12R and IL-18R compared with paired PBL CD4+ T cells. In contrast, the Th2 associated chemokine receptors CXCR4 and CCR4 were expressed by a fewer percentage of BAL CD4+ compared with PBL CD4+ T cells. There was a positive correlation between the percentage of BAL lymphocytes and the number of CXCR3 and CCR5 expressing CD4+ BAL T cells. Also, the number of CD4+ IL-18R+ BAL fluid cells correlated negatively with disease duration.
Conclusions. The lung accumulation of CXCR3, CCR5, IL-12R and IL-18R expressing T cells is in line with previous reports showing elevated levels in the lung of the corresponding ligands in sarcodosis. Blocking such ligands and/or receptors may develop into a future immunomodulatory therapy.
The balance between Th1 and Th2 helper cells and their associated cytokine patterns can be of critical importance for the outcome of physiological, as well as pathological, immune responses for the progression of several clinical diseases. In Mycobacterium leprae infection a Th1 response is associated with the relatively benign tuberculoid form of the disease whereas a Th2 response is related to the fatal lepromatous form . Uncontrolled Th1 and Th2 responses can cause chronic inflammatory diseases and allergies, respectively .
Sarcoidosis is a multi-organ inflammatory disorder of unknown cause, characterized by an accumulation of activated CD4 positive T cells and macrophages in the lungs, accompanied by its histopathological hallmark, the noncaseating epithelioid cell granuloma [3, 4].
Many chemokines and their receptors are important for the selective recruitment of various subsets of leukocytes. Recent studies have indicated that they may be involved in the immunopathogenesis of sarcoidosis . Among the chemokines that have shown to be involved in the recruitment of Th1 cells in sarcoidosis are regulated upon activation normal T cell expressed and secreted (RANTES) , macrophage inflammatory protein-1α (MIP-1α) and 1β (MIP-1β) , all belonging to the β-chemokine family and all sharing the specific receptor ligand CCR5 on activated T cells . Also interferon-γ (INF-γ) inducing protein 10 (IP-10), which binds to its specific receptor CXCR3, is expressed on activated T-cells and favours the migration and accumulation of Th1 lymphocytes .
The cytokine profile of sarcoidosis displays a Th1-associated pattern with increased production of IFN-γ and interleukin (IL-2) [4, 9]. One key cytokine for the induction of Th1 cells is IL-12, which is a product of activated macrophages and dendritic cells. IL-12 up-regulates the development of Th1 cells and the release of Th1 cytokines especially IFN-γ [10, 11]. IL-12 acts through the IL-12R, which is expressed on activated lymphocytes [11, 12]. Interleukin-18 is another more recently discovered cytokine, which also seems to be involved in the induction of Th1 cells. This cytokine has the ability to induce IFN-γ from Th1 cells in synergy with IL-12 and it also enhances cytotoxicity of natural killer (NK) cells . The IL-18 receptor is expressed on activated lymphocytes but it is shown to be preferentially more expressed on Th1 cell . Both of these cytokines have recently been suggested to be involved in the pathogenesis of sarcoidosis .
The purpose of this study was to investigate the phenotype of T lymphocytes in bronchoalveolar lavage (BAL) and peripheral blood (PBL) of patients with pulmonary sarcoidosis with focus on expression of markers indicating Th1 and Th2 cellular function. We examined the expression of Th1 associated chemokine receptors CXCR3 and CCR5, as well as the Th2 associated chemokine receptors CXCR4 and CCR4 in these patients. In addition, we analysed the expression of the cytokine receptors IL-12R and IL-18R, which are known to be of importance to generate responses typically seen in sarcoidosis. We studied the correlation of the investigated markers with clinical parameters such as BAL fluid parameters and disease duration and we investigated any difference between patients with Löfgren's syndrome and those without.
Materials and methods
A total of eighteen patients (mean age 34 years, range 27–57 years) diagnosed with active sarcoidosis participated in the study. Disease activity was assessed on the basis of symptoms, chest radiography, and pulmonary function tests, using previously established criteria . Eight of the patients were female and 10 were male. Five of the subjects were smokers, six were ex-smokers and seven had never smoked. All of the patients had clinical findings (symptoms such dyspnea, fatigue, fever, coughing and weight loss) and chest radiographic features typical for sarcoidosis [chest X-ray stages defined as: chest X-ray, I, bilateral hilar lymphadenopathy (BHL); II, BHL with pulmonary infiltration; III, parenchymal infiltration without BHL]. Eleven patients had positive proven biopsies and six of the patients had classical Löfgren's syndrome. One patient with negative biopsy and without Löfgren's syndrome had BAL CD4/CD8 ratio higher than 4.0, thus strongly supporting the diagnosis . None of the patients were undergoing any treatment. Disease duration was defined the time from disease onset to BAL procedure. All the patients gave their informed consent and the local ethical committee approved this study.
Bronchoalveolar lavage was performed as described before . In brief, bronchoscopy was performed under local anaesthesia and the flexible fibre-optic bronchoscope was wedged in a middle lobe bronchus for lavage. Aliquots of 50 mL sterile physiological saline (NaCl) solution at 37 °C were instilled five times and the fluid was gently aspirated and collected in a siliconized plastic bottle kept on ice.
Preparation of cells
The BAL fluid was passed through a woven pre-filter (Millipore, Cork, Ireland), centrifuged at 400 g for 10 min at 4 °C, and the pellet was re-suspended 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.
Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) gradient centrifugation was used to separate peripheral blood mononuclear cells. The separated cells were washed twice and diluted in RPMI 1640.
Immunofluorescent staining and flow cytometry
Phycoerythrin (PE)-conjugated monoclonal antibodies (mAb) specific for CXCR3, IL12R β1, and IL-18R were purchased from R & D (London, UK). CXCR4, CCR4 and CCR5 mAb all PE-conjugated were obtained from Pharmingen Becton Dickinson (Stockholm, Sweden). RPE-Cy5 conjugated anti-CD4 mAb and fluorescein isothiocyanate (FITC) anti-CD8 (Dako, Glostrup, Denmark) were used to determine lymphocyte subsets in the BAL and PBL in patients. The BAL cells and peripheral blood cells (PBL) from patients were triple stained with direct labelled mAb specific for cytokine and chemokine receptors, RPE-Cy5 anti-CD4, FITC-anti-CD8, incubated for 30 min at 4 °C, washed twice and fixed in Cell-fix (Becton Dickinson, Sweden). Cells were analysed in a flow cytometer (FACScalibur, Becton Dickinson, Mountain View, CA, USA) 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. Expression of the cytokine and chemokine receptors were evaluated on CD4+ and CD8+ T cell subsets in BAL and PBL from patients. Isotype matched negative control antibodies were used and the background stained <1% of CD4+ and CD8+ cells.
Results are presented as median values, with minimum and maximum values as the range. For comparison between BAL and PBL cell populations of patients the Wilcoxon matched pair statistical calculations were used. For comparison between BAL and PBL CD4+, CD8+ T cells subsets the Mann–Whitney statistical calculations were used. Also for comparison between different subgroups of patients (Löfgren and non-Löfgren, short or long disease duration the Mann–Whitney statistical calculations were used. For the correlation observations, the Spearman correlation test was used. A level of <0.05 was regarded significant.
Bronchoalveolar lavage and paired PBL cells from in total of 18 patients with untreated sarcoidosis were analysed in this study. Total and differential cell counts for BAL fluid and CD4/CD8 ratio is presented in Table 1. As expected there was an elevated percentage of BAL lymphocytes (18.0; 3.8–65.6) .
Chemokine receptor expression in BAL and paired peripheral blood T lymphocytes
Three-colour flow cytometry was performed to study the expression of the two Th1 associated CXCR3 and CCR5 and the two Th2 associated CXCR4 and CCR4 chemokine receptors. The CXCR3 and CCR5 receptors were expressed on the majority of BAL CD4+ (median, minimum–maximum) (97.4, 88.6–99.2; 50.5, 30.5–81.2, respectively) compared with PBL CD4+ T cells (30.9, 22.7–57.0; 13.3, 2.6–27.4, respectively) p < 0.005; p < 0.01, respectively (Fig. 1a). In contrast, there were significantly lower numbers of BAL CD4+ T cells that expressed the Th2 associated chemokine receptors CXCR4 and CCR4 (38.1, 15.7–51.6; 14.5, 10.1–38.8, respectively) compared with PBL CD4+ T cells (63.6, 46.2–85.6; 36.4, 19.0–40.4, respectively) (p < 0.005; p < 0.01, respectively) (Fig. 1a).
CXCR3 and CCR5 were both expressed also by excessive numbers of CD8+ BAL (97.1, 83.1–99.9; 59.0, 38.3–83.3, respectively) compared with CD8+ peripheral blood T cells (56.6, 21.8–63.5; 35.0, 2.3–44.7, respectively) (p < 0.005; p < 0.01, respectively), while the expression of the Th2 associated chemokine receptors CXCR4 and CCR4 were not as distinct (p < 0.05; p = not significant; ns, respectively) (Fig. 1b).
Comparing CD4+ and CD8+ BAL T cells, both subsets in general showed a clear Th1 pattern and CXCR4 and CCR4 were expressed by significantly fewer CD4+ compared with CD8 T cells (p < 0.01 in both cases).
In PBL, there were more cells positive for CXCR3 and CCR5 among CD8+ cells than among CD4+ T cells and there were significantly more CD4+ T cells expressing the Th2 associated receptors (p < 0.001 for both CXCR4 and CCR4).
IL-12 and IL-18 receptor expression in BAL and paired PBL T lymphocytes
We examined the expression of cytokine receptors IL-12R and IL-18R in BAL and paired PBL T cells. The number of CD4+ cells expressing IL-12R in BAL (72.8, 23.0–85.7) was significantly higher compared with CD4+ T cells in PBL (16.8, 0.2–38.1) (p < 0.005). Also the IL-18R was expressed by more BAL CD4+ (92.4, 63.9–96.4) compared with PBL CD4+ (54.7, 21.0–81.1) T cells (p < 0.01) (Fig. 2).
Both IL-12R and IL-18R were expressed by significantly more BAL CD8+ cells (40.8, 19.5–68.3; 55.7, 34.1–81.1, respectively) compared with PBL CD8+ cells (19.6, 0.2–60.6; 37.1, 17.4–67.0, respectively) (p ≤ 0.01 in both cases) (Fig. 2).
We further examined the difference between the CD4+ and CD8+ T cell subsets in BAL and in peripheral blood. In BAL there were significantly more CD4+ cells that expressed the IL-12 receptor (p < 0.01) as well as IL-18R (p < 0.005) compared with CD8+ T cells.
Comparison between BAL parameters and receptor expression
There was a positive correlation between the relative numbers of CD4+ CXCR3+ and CD4+ CCR5+ T cells and the percentage of lymphocytes in BAL (r = 0.64, p < 0.05), respectively (r = 0.70, p < 0.05). There was also a tendency towards a positive correlation between relative numbers of CD4+ CCR5+ T cells and the BAL CD4/CD8 ratio. In line with this, there was a negative correlation (not significant) between the number of Th2 receptor expressing BAL CD4+ T cells and the BAL CD4/CD8 ratio.
We observed a significant negative correlation between relative numbers of BAL CD4+ IL-18R+ T cells and disease duration (r = −0.78, p < 0.01) (Fig. 3). Also, we noticed that fewer BAL CD4+ cells expressed CCR4 in patients with shorter disease duration, although not statistically significant.
Finally, there was a tendency of more peripheral blood CD4+ cells expressing CXCR3 in patients with a shorter disease duration. No statistical difference in BAL CD4+ cells for any receptor expression between patients with Löfgren versus those without was observed. However, we did notice a significant difference in BAL CD8+ cells expressing the CCR4 receptor. Thus in patients with Löfgren's syndrome, significantly more BAL CD8+ cells (10.7, 8.0–16.5) expressed CCR4 versus those without (5.3, 2.3–10.6) (p < 0.05).
In the present study, we have analysed the expression of chemokine receptors CXCR3, CXCR4, CCR4 and CCR5 and the two cytokine receptors IL-12R and IL-18R in BAL and PBL in patients with sarcoidosis. We studied the expression of these receptors on CD4+ and CD8+ T cell subsets separately. In addition, we investigated any correlations between differential cell counts, BAL parameters and the expression of these receptors in BAL cells, and found a significant positive correlation between the relative numbers of Th1 cells (expressing CXCR3 and CCR5) and percentage of lymphocytes in BAL. We also found a significant a negative correlation between relative numbers of BAL CD4+ IL-18R+ T cells and disease duration.
Previous studies have demonstrated that accumulated T lymphocytes at the sites of inflammation have a Th1 dominant cytokine production [20, 21]. This altered production of cytokines such as IL-2, tumour necrosis factor (TNF)-α and IFN-γ is believed to contribute to disease pathology and granuloma formation [4, 9, 15, 22]. Our findings demonstrate an increased expression of all investigated Th1 associated receptors on BAL T cell subsets compared with PBL, both in CD4+ and CD8+ T cells. Concordantly there was a significantly reduced expression of the Th2 associated chemokine receptors CXCR4 and CCR4 on BAL CD4+ compared with PBL CD4+ T cells. Our results, therefore, highlight the role of the Th1 cells in sarcoidosis. This finding is in agreement with others and our previous study investigating the cytokine profile of T cell subsets in BAL fluid and PBL in patients with sarcoidosis, which showed a predominant Th1 cytokine production in BALF T cells [9, 10, 20, 22].
Chemokines and their receptors are believed to be involved in recruitment of immune cells to the site of inflammation and they provide a central concept for understanding the mechanisms regulating the tissue-specific recruitment of Th1 and Th2 cells . Agostini et al. have earlier demonstrated elevated levels of IP-10 (interferon-γ inducing protein 10) in the lungs of patients with pulmonary sarcoidosis and also shown a positive correlation between the levels of IP-10 and the degree of lymphocytosis in BAL . IP-10 interacts with its specific receptor CXCR3, expressed on activated T cells and favouring the migration and accumulation of T-helper cells at sites of disease activity . It has been suggested that the CXCR3 receptor is important for a Th1 inflammatory response, and numbers of CD4+ CXCR3+ T cells have been shown in the submucosa of patients with Crohn's disease . These findings are in agreement with our present study, which shows an accumulation of CD4+ CXCR3+ cells in BAL fluid and also a positive correlation between the expression of this receptor and the percentage of BAL lymphocytes.
Regulated upon activation normal T cells expressed and secreted is another chemokine, which has been reported to be involved in the recruitment of Th1 cells to lung of patients with sarcoidosis . Also, increased levels of macrophage inflammatory protein-1α and protein-1β (MIP-1α and MIP-1β) in BAL fluid of patients affected by different stages of sarcoidosis have been reported . All of these chemokines have the CCR5 receptor in common . This receptor has been associated with Th1-type immune responses and has been shown to be expressed on accumulated T cells at site of inflammation in organ specific autoimmune diseases such as multiple sclerosis and rheumatoid arthritis (RA) . Suzuki et al. have shown that CD4+ CCR5+ synovial fluid mononuclear cells (SFMC) produce high amounts of IFN-γ but a low level of IL-4 . An increased number of lymphocytes expressing the CCR5 receptor has also been shown in the gut of patients suffering from inflammatory bowel disease (IBD) . We have previously shown that a high IFN-γ/IL-4 ratio was detected in both CD4+ and CD8+ BAL fluid T cells . We here show that the vast majority of both CD4+ and CD8+ T cells in BAL fluid express the CXCR3 as well as the CCR5 receptor. Although CD4+ cells are more numerous, BAL CD8+ cells therefore also contribute to a Th1-type inflammatory process. Capelli and coworkers have recently shown an increased expression of the CCR5 receptor on nonseparated lymphocytes in patients with sarcoidosis from all of the stages of the disease although the highest expression was observed in patients with stage I and a downregulation of CCR5 expression in fibrotic stages of sarcoidosis was observed . Also, an enhanced expression of CCR5 mRNA on nonseparated BAL cells in patients with pulmonary sarcoidosis has previously been shown by Petrek et al. [29, 30]. In our study, we observed no difference in the chemokine and cytokine receptor expression in BAL CD4+ T cells between patients with Löfgren versus non-Löfgren, although the number of patients was too small to draw any firm conclusions. However, IL-18R was expressed by significantly more CD4+ BAL T in patients with a short disease duration, implicating a more pronounced Th1 response early in disease.
CCR4 is a receptor for thymus and activation-regulated chemokine (TARC) and associated with Th2 cells [7, 31]. CXCR4 is a receptor for SDF-1 (stromal–cell-derived factor 1) and expressed on naïve T cells . Recently, CXCR4 expression was also shown to be upregulated by IL-4 and downregulated by IFN-γ in naïve T cells [33, 34]. Panina et al. have in a recent study demonstrated that after allergen challenge, all T cells infiltrating airways express CCR4 and also the specific ligand TARC is strongly upregulated on airway epithelial cells, suggesting an involvement of this receptor in the regulation of lymphocyte recruitment into the asthmatic bronchi . The reduction of CCR4 and CXCR4 expressing BAL compared with peripheral blood T cells may, therefore, reflect not only a pronounced Th1 immune response but also the dominance of memory T cells in the BAL fluid. Interestingly, in the BAL CD8 subset, a less pronounced T cytotoxic cell type 1 (Tc1) profile was noticed in patients with Löfgren's syndrom.
Interleukine 12 is primarily produced by macrophages and it plays a key role in promoting Th1 responses. It exerts it biological activity by binding to the IL-12 receptor, which is composed of two β subunits (β1 and β2) . Increased levels of IL-12 in the BAL fluid as well as an increased percentage of BAL cell expressing the mRNA for both subunits of IL-12R (β1 and β2) in active sarcoidosis was demonstrated [13, 36]. IL-18 acts synergistically with IL-12 to promote a Th1-immune response, and they are together potent inducers of IFN-γ production by T cells . The IL-18 receptor is considered to be preferentially expressed on Th1 cells, while Th2 cells do not express this receptor . The pattern of IL-12 and IL-18 receptor expression by BALF CD4+ and CD8+ T cells in patients with sarcoidosis again implies that the majority of both subsets are committed to Th1-type cytokine production and that the enhanced expression of these receptors in the lung can explain the greater inducibility of IFN-γ production [36, 38].
In summary, these data demonstrate increased numbers of BAL CD4+ and CD8+ cells expressing the Th1 associated chemokine receptors CXCR3, CCR5 together with cytokine receptors which are considered to be associated to Th1 immune responses, in patients with pulmonary sarcoidosis. We thus identified lung localized T cell subsets that most likely have been attracted to the lungs by elevated levels of the recently reported chemokines [5, 8, 11]. Interference with such ligands or their corresponding receptors may prove to be a fruitful way to block the typical Th1 immune response in sarcoidosis.
Conflict of interest statement
No conflict of interest was declared.
This work was supported by The Swedish Medical Research Council (grant-74X-14782-01A), The Swedish Heart Lung Foundation, the King Oscar II Jubilee Foundation, the Swedish Society For Medical Research (SSMF), the Swedish Society of Medicine and the Karolinska Institutet.
The authors highly appreciate the excellent technical assistance of research nurses Gunnel de Forest, Margita Dahl and the laboratory technician Benita Dahlberg.