INDEX
IL-12 and IL-10 Released from Peripheral Blood Mononuclear Cells in Sarcoidosis is Associated with Differential Modulation of B7-1 (CD80) and B7-2 (CD86) Expression on Monocytes

Haruhiko Kawaguchi*, Shigeki Sato*, Yoshiki Sugiura*, Hiroshi Tomita**, Takashi Niimi*, Yoshihiro Maeda*, Masahiko Yamamoto***, and Ryuzo Ueda*
*Second Department of Internal Medicine, Nagoya City University, Medical School, Nagoya, Japan
**Department of Internal Medicine, Toyokawa City Hospital, Toyokawa, Japan
***Nagoya Medical Examination Center, Postal Life Insurance Corporation, Nagoya, Japan

Running title: cytokine production and monocyte B7 isoform in sarcoidosis
Keywords: Sarcoidosis, Cytokines, B7 isoforms, Peripheral blood monocytes
Foot note:
Abbreviations used: PBMC, peripheral mononuclear cells; ELISA, enzyme linked immunosorbant assay; FCM, flow cytometric analysis; Th1, type 1 T helper; DC, dendritic cell; APC, antigen presenting cell; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

SUMMARY
Sarcoidosis is a systemic disorder of unknown etiology, in which noncaseating granulomas simultaneously occur in several organs and cell mediated type 1 immune responses are considered to play critical roles in the pathogenesis. The aim of this study was to ascertain whether the initial immune response developed by peripheral blood mononuclear cells (PBMCs) from patients with active sarcoidosis might induce the cell mediated type 1 immune response observed at disease sites. We evaluated IL-12 and IL-10 production in PBMCs on priming with LPS and/or INF-g using enzyme immunosorbent assay (ELISA) and the expression of the co-stimulatory molecules, CD80 and CD86, on the surfaces of monocytes by flow cytometric analysis (FCM) in 11 patients with active sarcoidosis. PBMCs from patients released the increased levels of IL-12, in contrast, the decresed levels of IL-10 as compared with those from healthy subjects. Analysis by FCM of CD14+ monocytes from patients revealed the overexpression of CD86 compared with healthy subjects, while no significant difference of CD80 expression was observed. We found that the expression of CD86 was inversely correlated with IL-10 production. The results suggest that sarcoid CD14+ monocytes may induce cell mediated immune responses under conditions of increased IFN-g concentration, such as at disease sites.

INTRODUCTION
Sarcoidosis is a disease of unknown origin characterized by formation of granulomas, composed of epithelioid cells derived from monocyte/macrophage lineage and CD4 T lymphocytes, in systemic organs. Recent investigations into the pathogenesis of sarcoidosis demonstrated CD4+CD45RO+ T cells at sites of inflammation, consistent with a conventional antigen-driven T cell immune response (1-3) with a cell mediated type 1 T helper (Th1) phenotype (4). It has also been reported that mononuclear phagocytes of affected organs, mainly alveolar macrophages, have several immunological conducive to these effector T cell induction (5-7). With regard to the immunological status of the peripheral blood from affected individuals, several investigators have demonstrated no differences as compared with normal subjects (3-7). However, some studies speculated that circulating sarcoid peripheral blood mononuclear cells (PBMCs) may be involved since monocytes may release increased levels of radical oxidants (8) and sarcoid peripheral T lymphocytes express functional IL-2 receptors (9).
Recently, it has been shown that peripheral blood monocytes represent an abundant source of precursors that can differentiate into dendritic cells (DCs), considered to be most important antigen presenting cells (APCs) for T cell stimulation, depending on external stimuli (10-12). The initial contact of host APCs with naive T cells may shape the subsequent cognate immune response orchestrated by effector CD4 T cells in vivo (13). In this context, it is opportune to evaluate the immunological status of peripheral CD14+ monocytes in diseases where APCs are thought to play important pathogenetic roles. Initial APC and T cell contact induces several factors, among which cytokines are the most investigated examples found to control the T helper cell phenotype (14-19). Signals through co-stimulatory B7 molecules have been also shown to influence the development of the effector T cell immune response. Both B7-1 (CD80) and B7-2 (CD86), which belong to a family of co-stimulatory B7 molecules, are expressed on a variety of APCs and their relative expression is known to play a critical role in directing the immune response towards cellular or humoral (20,21). Furthermore, it has been demonstrated that the provision of immunoregulatory cytokines and the levels of B7 isoform expression on APCs may influence each other in biasing T cell differentiation. Especially, IL-12 and IL-10 have been suggested to elicit biological effects via B7 molecules (22-24).
In sarcoidosis patients, sarcoid granulomas simultaneously occur in several organs, pointing to a systemic disorder. Therefore, it is meaningful to investigate whether the initial immune response developed by PBMCs from patients with active sarcoidosis favor the induction of a cell mediated type 1 immune response at sites of disease. We thus evaluated IL-12 and IL-10 production by PBMCs and expression of co-stimulatory molecules, CD80 and CD86, on the surfaces of monocytes from a series of affected individuals.

MATERIALS AND METHODS
Study population (Table 1)
A total of 11 sarcoidosis patients composing 6 men and 5 women with a mean (SD) age of 51.414.5years, were investigated using a cytokine production assay. Sarcoidosis was diagnosed based on the clinical picture and the presence of epithelioid cell granulomas in biopsy specimens from lung, skin, or lymph nodes (25). According to the classification system defined by DeRemee (26), there were six patients in roentgenographic stage I, four in stage II, and one in stage III. All patients were defined as having active disease since they suffered deterioration in chest radiographic findings and clinical symptoms during the past 3 months. None was receiving any corticosteroid therapy. Ten volunteers, consisting of 7 men and 3 women with a mean age of 50.615.3 years, were enrolled as normal subjects for this study. All were non-smokers, with no history of cardiopulmonary or other chronic medical illness, including tuberculosis, infection, and auto immune diseases. Informed consent was obtained from each patient and normal volunteer.
Cell preparation
Peripheral blood obtained from patients with sarcoidosis and healthy donors was anticoagulated with heparin. PBMCs were separated on a Ficoll-Hypaque (Pharmacia Biotech AB, Sweden) density gradient. To assess their cytokine production, they were cultured at 1106 cells/ml in endotoxin-tested RPMI 1640 (Gibco BRL, Paisley, UK) supplemented with 100 U/ml penicillin, 100mg/ml streptomycin, 2 mM L-glutamine, HEPES buffer, and 10 % heat-inactivated (56 for 1 h) FBS (ICN Flow, Costa Mesa, CA ), and incubated at 37 in a water-jacketed 5% CO2 incubator for 18 h with the following : LPS (from Escherichea coli, serotype 0127: B8, Sigma Chemical Co., St. Louis, MO; 10mg/ml) or IFN-g (R&D Systems, Minneapolis, MN; 1000U/ml). After cell free supernatant fluid was collected for determination of cytokine concentrations, cell suspensions were treated with trypsin-versin solution to assess the relative expression of B7 isoforms on the surfaces of monocytes. Following this multi step selection procedure, more than 95% of the cells were still viable, as judged by trypan blue exclusion. The supernatant was stored at -30 before being evaluated for the production of each cytokine. To determine the ability of peripheral monocytes to produce IL-12, PBMCs were allowed to adhere to tissue culture plastic dishes for 90 min, purified (2.5105/ml) and cultured in medium alone, or plus LPS in the presence of IFN-g(1000 U/ml ).
Measurement of IL-12 and IL-10 in cell suparnatans
Levels of IL-12 and IL-10 in culture supernatants were measured using enzyme linked immunosorbant assay (ELISA) detection kits (Quantikine Human IL-12 immunoassay ELISA kit, R&D Systems, Minneapolis, MN, and ELISA for human interleukin 10, Immunotech, Marseille, France, respectively) according to the manufacturer's recommendations. The sensitivity of the IL-10 assay was 3.0 pg/ml. The immunoassay for IL-12 was developed with a capture antibody that recognizes only the IL-12 heterodimer and not the individual subunits (p35 or p40) or dimers. A level > 3 pg/ml was considered significant.
Flowcytometric analysis
For evaluation of B7 isoform expression on surfaces of monocytes of freshly isolated and cultured PBMCs, the frequency of positive cells was determined by flow-cytometric analysis (FCM). Briefly, freshly isolated and cultured cell suspensions were washed three times with PBS (-) containing 2% FCS and 0.1% sodium azide and stained with either fluorescein isothiocyanate (FITC)-labeled anti-CD80 or FITC-labeled anti-CD86 mAb and phycoerythrin (PE)-labeled anti-CD14 mAb on ice. These conjugated mAbs were from Pharmingen (San Diego, CA ). Peripheral blood monocytes were gated in FCM using two different approaches: 1) physical characteristics of cells and 2) expression of CD14. For direct fluorescence analysis, FITC- or PE-conjugated control isotype-matched nonrelevant anti mouse mAbs were used to set the negative controls (IgM and IgG1, Pharmingen, San Diego, CA). The gates to define the number of positive cells were chosen in order to have <1% positive cells with isotype matched negative control mAbs. Cells were scored using a FACScan analyzer (Becton Dickinson, Mountain View, CA) and data were processed using the Macintosh CELLQuest software program (Becton Dickinson). When values are also given for mean fluorescence intensity (MFI), the proportions from CD80 or CD86 conjugated antibodies relative to those from control antibodies are supplied (sample MFI/control MFI).
Statistical analysis
Data are presented as meanSD values unless otherwise indicated. Comparison of cytokine production was accomplished using the unpaired two-tailed t test. Analysis of expression of B7 isoforms was performed using the Mann-Whitney U test for comparison of two groups, and the Kruskal-Wallis test for three groups. The Spearman rank correlation coefficient was used to calculate the statistical significance of correlation coefficients (27). p < 0.05 was considered significant .

RESULTS
Cytokine production by PBMC
IL-12 production by PBMCs cultured with LPS or medium alone was not detectable or at very low levels both in patients with active sarcoidosis and normal subjects. In contrast, when stimulated with LPS and IFN-g, PBMCs from both groups produced significant amounts of IL-12 dependent on the concentration of IFN-g (data not shown ). On priming LPS with IFN-g, the mean IL-12 level in supernatants from active sarcoid PBMCs was significantly higher than that from normal volunteers (276.2184.5 pg/ml v.s. 52.724.4 pg/ml; p<0.001, Fig. 1). The production of IL-12 by monocytes primed with LPS and IFN-g from sarcoid patients was also significantly increased compared with those from normal volunteers (485.1267.8 pg/ml v.s. 250.8452.54 pg/ml; p < 0.05), whereas those from both groups produced no detectable levels of IL-12 without stimulation (Fig. 2).
In contrast to the IL-12 production, with LPS alone, PBMCs from both groups produced significant levels of IL-10 with no significant difference between patients and healthy volunteers (1370.8487.3 pg/ml and 1582.5577.4 pg/ml, respectively). On priming with LPS and IFN-g (1000 U/ml), however, PBMCs from active sarcoidosis patients demonstrated significantly lower levels of IL-10 production than normal controls (340.7169.1 pg/ml and 843.8 490.4 pg/ml, respectively, p<0.005) (Fig. 3).
Relative expression of B7 isoforms on surfaces of CD14 positive monocytes (Table 2)
Data for expression of CD80 and CD86 on the surfaces of CD14+ monocytes of freshly isolated and cultured PBMCs are shown in Table 2. In patients with active sarcoidosis, CD80 on surfaces of freshly isolated CD14+ monocytes was present on only 1.81.7% by FCM, CD86 being found on 85.810.0%. These values were essentially similar for healthy individuals. In both groups, when PBMCs were cultured and primed with LPS in the presence or absence of IFN-g, analysis of surface expression of B7 isoforms on CD14+ monocytes revealed an increase in CD80 with LPS alone, and greater elevation with additional IFN-g. However, with each culture condition, there was no significant difference in expression between the two groups. With regard to CD86 on cultured CD14+ cells, in healthy volunteers, expression was down-regulated with medium alone, compared with those of freshly isolated monocytes. This inhibitory effect on the CD86 expression was markedly enhanced by LPS in the absence or presence of IFN-g. In patients with active sarcoidosis, however, these inhibitory effects were reduced as compared with those of healthy subjects. Stimulation of both LPS alone and LPS with IFN-g gave significant differences in expression between the two groups. Regarding the association between B7 isoform expression and production of cytokines, significant inverse correlations were observed between the percentage of CD86 positive monocytes and the production of IL-10 both with LPS alone and LPS with IFN-g (Fig. 4).

DISCUSSION
Our results indicate that PBMCs from the patients with sarcoidosis would induce cell mediated immunity as the initial response to stimulation with LPS in the presence of IFN-g. Because IFN-g has been shown to play a pivotal role in the pathogenesis of sarcoidosis (4, 5,7,28) and contribute to the induction of a cell mediated type 1 immunoresponse (14-18), we investigated PBMCs cultured with IFN-g. IFN-g and LPS synergistically stimulated the IL-12 production in PBMCs, including CD14+ monocytes, both in contorl and sarcoidosis groups. This enhanced production could have been due to amplification of the Th1 immunoresponse by a positive-feedback mechanism between IFN-g and IL-12. It is known that IL-12 produced by phagocytic cells is one of the factors inducing T cells and NK cells to produce IFN-g (29,30), which in turn enhances the ability of phagocytes to produce IL-12 (31). It is thus possible that PBMCs and CD14+ monocytes from active sarcoidosis patients may be capable of causing a stronger positive-feedback than those from healthy controls. The biological activity of IL-12 has been shown to be dependent on its heterodimeric structure (p70), which is composed of two disulfide-linked subunits (p35 and p40) encoded by two separate genes (32). In this study, we evaluated IL-12 protein levels by an immunoassay with a capture antibody that recognizes only the bioactive IL-12 heterodimer. It has been reported that IL-10 serves to down-modulate IFN-g production by affecting APCs so that IL-12 production is inhibited (14,19). Recently Moller et al reported that , in patients with active sarcoidosis, chronic overproduction IL-12 was observed in affected sites with suppressed expression of IL-10 mRNA in PBMCs (not stimulated) (4). In agreement with the report by Moller, our findings suggested that suppressed production of IL-10 in PBMCs from the patients with sarcoidosis leads to uninhibited production of cytokines such as IL-12. These process may induce a Th1 type cell mediated immunoresponse when these cells are exposed IFN-g.
This alteration in the production of cytokines observed in PBMCs from the patients with sarcoidosis was accompanied with the different expression of B7 isoforms on the surface of activated cultured CD14+ monocytes as compared with normal volunteers. It has been demonstrated that the provision of cytokines and the expression of B7 isoform on APCs may influence the differentiation of T cell. Cytokines which predominantly induce the development of Th2 cells, IL-10 and IL-4, have been shown to moderately enhance the expression of CD80 and to inhibit the expression of CD86, while IFN-g enhances the expression of both CD80 and CD86 (24). In contrast, it has been reported that IL-12 does not modulate the expression of B7 isoform, while IL-12 and endogenous B7 on mononuclear cells cooperate in inducing IFN-g production (21). A preliminary study using stimulation with LPS in the presence of IFN-g revealed no significant difference in IL-4 production by PBMCs between normal and disease groups (data not shown). We observed an inverse correlation between the positivity of CD86+ monocytes and production of IL-10. Considering that IL-10 inhibits macrophage co-stimulatory activity by selectively inhibiting the up-regulation of B7 expression (22), the relative lack of inhibitory effects on expression of CD86 observed in sarcoid monocytes might be due to suppressed production of IL10 production in PBMCs.
Recent studies demonstrated that sarcoid lung macrophages express relatively high levels of CD80 and CD86, while their counterparts in healthy subjects are usually ineffective in presenting antigen to T cells because of reduced expression of B7 isoforms (33, 34). They also demonstrated that CD86, but not CD80, functions as a co-stimulatory molecule for T lymphocyte activation. Alveolar macrophages, resident phagocytic cells in the lung, are derived from CD14+ peripheral blood monocytes which differentiate into either DCs or macrophages depending upon the influence of different cytokines (10-12, 35), so that our study may provide interesting clues concerning the differentiation of alveolar macrophages in patients with sarcoidosis.
In conclusion, our study demonstrated that PBMCs from active patients, accompanied by the overexpression of CD86 on the surfaces of monocytes, released the increased levels of IL-12, in contrast, decreased levels of IL-10 as compared with those from healthy subjects. Considering immunological fanction of these molecules, PBMCs from patients with active sarsoidosis may induce the type 1 immune response under conditions of increased concentrations of IFN-g, such as found in disease sites, although the cellular events that trigger pathogenesis of this disease remain unclear. In patients with sarcoidosis, circulating PBMCs have the opportunity to contact several organs which involve active sarcoid granulomas. In this context, this accessibility of PBMCs to granulomatous inflammation at the disease sites might provide the possible effect that skews the subsequent cognate immune response into the type 1 immune response in the initial contact of host APC with naive T cell.

ACKNOWLEDGEMENTS
The writers thank Dr. Morishita for his kind advice.

REFERENCES
1. Klein JT, Horn TD, Forman JD, Silver AL. Selection of oligoclonal Vb-specific T cells in the intradermal response to Kveim-Siltzbach reagent in individuals with sarcoidosis. J Immunol 1994; 153: 1450-1460.
2. Forman JD, Klein JT, Silver RF, Liu MC, Greenlee BM, Moller DR. Selective activation and accumulation of oligoclonal Vb-specific T cells in active pulmonary sarcoidosis. J Clin Invest 1994; 94: 1533-1542.
3. Hudspith BN, Flint KC, Geraint-James D, Brostoff J., McI Johonson N. Lack of immune deficiency sarcoidosis: compartmentalisation of the immune response. Thorax 1987; 42: 250-255.
4. Moller DR, Forman JD, Liu MC, Noble PW, Greenlee BM, Vyas P et al. Enhanced expression of IL-12 associated with Th1 cytokine profiles in active pulmonary sarcoidosis. J Immunol 1996; 156: 4952-4960.
5. Robinson BWS, McLemore TL, Crystal RG. Gamma interferon is spontaneously released by alveolar macrophages and lung T-lymphocytes in patients with pulmonary sarcoidosis. J Clin Invest 1985; 75: 1488-1495.
6. Theodore JS, Rolfe MW, Kunkel SL, Lymch III JP, Burdick MD, Gilbert AR et al.
Macrophage inflammatory portein-1a expression in interstitial lung disease. J Immunol 1993; 151: 2852-2863.
7. Muler-Quernheim J, Pfeifer S, Manel D, Strausz J, Ferlinz R. Lung-restricted activation of the alveolar macrophage/monocyte system in pulmonary sarcoidosis. Am Rev Respir Dis 1991; 145: 187-192.
8. Kanegasaki K, Homma JY, Homma H, Washizaki M. Enhanced chemiluminescence response of phagocytosing monocytes from sarcoidosis patients. Int Archs Allergy Appl Immun 1981; 64: 72-79.
9. Konishi K, Moller DR, Saltini C, Kirby M, Crystal RG. Spontaneous expression of the interleukin 2 receptor gene and presence of functional interleukin 2 receptors on T lymphocytes in the blood of individuals with active pulmonary sarcoidosis. J Clin Invest 1988; 82:755-781.
10. Cella M, Sallusto F, Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol 1997; 9: 10-6.
11. Sallusto F, Lazavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte / macrophage colony -stimulating factors plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994; 179: 1109-1118.
12. Akagawa K, Takasuka N, Nozaki Y, Komura I, Azuma M, Ueda M et al. Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multrinucleated giant cells from human monocytes. Blood 1996; 88: 4029-4039.
13. Medzhitov R, Janeway Jr CA. Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 1997; 9: 4-9.
14. Locksley RM. Interleukin 12 in host defense against microbial pathogens. Proc Natl Acad Sci USA 1993; 90: 5879-5880.
15. Trinchieri G. Interleukin-12 and its role in the generation of Th1 cells. Immunol Today 1993; 14: 335-337.
16. Romagnani S. Biology of human TH1 and TH2 cells. J Clin Immunol 1995; 15: 121-129.
17. Scott P. IL-12: Initiation cytokine for cell-mediated immunity. Science 1993; 260: 496-497.
18. Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Development of Th1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 1993; 260: 547-549.
19. D'Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Interleukin 10 inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med 1993; 178: 1041-1048.
20. Thompson CB. Distinct roles for the co-stimulation ligands B7-1 and B7-2 in T helper cell differentiation ?. Cell 1995; 81: 979-982.
21. Kubin M, Kamoun M, Trinchieri G. Interleukin 12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells. J Exp Med 1994; 180: 211-222.
22. Ding L, Linsley PS, Huang LY, Germain RN, Shevach EM. IL-10 inhibits macrophage co-stimulatory activity by selectively inhibiting the up-regulation of B7 expression. J Immunol 1993; 151: 1224-1234.
23. Freeman GJ, Boussiotis VA, Anumanthan A, Bernstein GM, Ke XY, Rennert PD et al. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially co stimulates the initial production of IL-4. Immunity 1995; 2: 523-532.
24. Creery WD, Diaz-Mitoma F, Filion L, Kumar A. Differential modulation of B7-1 and B7-2 isoform expression on human monocytes by cytokines which influence the development of T helper cell phenotype. Eur J Immunol 1996; 26: 1273-1277.
25. Thomas PD, Hunninghake GW. Current concepts of pathogenesis of sarcoidosis. Am Rev Respir Dis 1987; 135: 747-60.
26. DeRemee RA. The chest roentgenology of sarcoidosis. In: Lieberman J eds. Sarcoidosis. Orlando: Grune and Stratton, 1985; 117-135.
27. Woolson RF. Statistical Methods for the Analysis of Biomedical Data. New York: John Wiley and Sons, 1987.
28. Bergerom A, Bonay M, Kambouchner M, Lecossier D, Riquet M, Soler P et al. Cytokine patterns in tuberculous and sarcoid granulomas: correlations with histopathologic features of the granulomatous response. J Immunol 1997; 159(6): 3034-3043.
29. Trinchieri G. Cytokines acting on or secreted by macrophages during intracellular infection (IL-10, IL-12, IFN-g). Curr Opin Immunol 1997; 9: 17-23.
30. Emil RU. Inter-relationship among macrophages, natural killer cells and neutrophils in early stages of Listeria resistance. Curr Opin Immunol 1997; 9: 35-43.
31. Kubin M, Chow JM, Trinchieri G. Differential regulation of interleukin-12 (IL-12), tumor necrosis factor alpha, and IL-1 beta production in human leukemia cell lines and peripheral blood mononuclear cells. Blood 1994; 83: 1847-1855.
32. Gluber UA, Chua O, Schoenhaut DS, Dwyer CM, McComas W, Motyka R et al. Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor. Proc Natl Acad Sci USA 1991; 88: 4143-4147.
33. Laurent PN, Isler P. Alveolar macrophages in sarcoidosis coexpress high levels of CD86 (B7.2), CD40, and CD30L. Am J Respir Cell Mol Biol 1997; 17: 91-96.
34. Chelen CJ, Fang Y, Freeman GJ, Secrist H, Marshall JD, Hwang PT et al. Human alveolar macrophages present antigen ineffectively due to defective expression of B7 co-stimulatory cell surface molecules. J Clin Invest 1995; 95: 1415-1421.
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