Impaired IFN-{gamma} production of V{alpha}24 NKT cells in non-remitting sarcoidosis

Seiichiro Kobayashi1, Yoshikatsu Kaneko1, Ken-ichiro Seino4,5, Yoshihito Yamada2, Shinichiro Motohashi1, Junzo Koike1, Kaoru Sugaya1, Takayuki Kuriyama2, Shigetaka Asano6, Tomiyasu Tsuda7, Hiroshi Wakao4, Michishige Harada4, Satoshi Kojo4, Toshinori Nakayama1,3 and Masaru Taniguchi1,4

Departments of 1 Molecular Immunology, 2 Respirology and 3 Medical Immunology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan 4 Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama 230-0045, Japan 5 PRESTO, Japan Science and Technology Corp., Saitama 332-0012, Japan 6 Division of Molecular Therapy, Institute of Medical Science, University of Tokyo, Tokyo 113-0033, Japan 7 Third Department of Internal Medicine, Oita Medical University, Oita 879-5593, Japan

The first two authors contributed equally to this work
Correspondence to: M. Taniguchi; E-mail: taniguti{at}med.m.chiba-u.ac.jp
Transmitting editor: M. Miyasaka


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Sarcoidosis is a systemic disorder associated with granuloma characterized by an abnormal Th1-type cytokine production and accumulation of Th1 CD4 T cells in the granuloma lesions, suggesting an importance of Th1 responses in sarcoidosis. However, the pathogenesis of sarcoidosis remains to be solved. Here, we investigated the nature of V{alpha}24 NKT cells with immunoregulatory functions in sarcoidosis. Patients with non-remitting sarcoidosis displayed a decrease in the number of V{alpha}24 NKT cells in peripheral blood, but an accumulation of these cells in granulomatous lesions. When stimulated with the specific glycolipid ligand, {alpha}-galactosylceramide, peripheral blood V{alpha}24 NKT cells from patients with non-remitting disease produced significantly less IFN-{gamma} than those from healthy volunteers, but normal levels of IL-4. The reduced IFN-{gamma} production was observed only in V{alpha}24 NKT cells and not conventional CD4 T cells, but was normal in patients with remitting disease, suggesting that non-remitting sarcoidosis involves an insufficient IFN-{gamma} production of V{alpha}24 NKT cells which is well correlated with disease activity. Thus, these results suggest that V{alpha}24 NKT cells play a crucial role in the disease status of sarcoidosis.

Keywords: cytokine, human, inflammation, lung, Th1/Th2 cell


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NKT cells constitute a unique lymphocyte subpopulation characterized by expression of NK cell markers, and an invariant antigen receptor encoded by V{alpha}14 and J{alpha}281 gene segments in mice (1) and V{alpha}24 and J{alpha}Q gene segments in humans (2,3). Activated V{alpha}14 NKT and V{alpha}24 NKT cells produce both IFN-{gamma} and IL-4 (46), for which an imbalance has previously been found to be associated with disease. V{alpha}14 NKT cells exert regulatory activity which protects against development of autoimmune diseases such as Type 1 diabetes in NOD mice (7,8) and maintains transplantation tolerance (9,10). V{alpha}14 NKT cells have also been reported to protect against microbial infections and subsequent host responses as in the granuloma formation of Mycobacterium tuberculosis infection (11).

Sarcoidosis is a systemic disorder characterized by its pathological hallmark, the non-caseating granuloma (12). Its presentation varies from an asymptomatic stage with abnormal findings on chest radiography to a progressive stage with multi-organ failure; the illness appears as both a self-limiting and a chronic progressive form, the latter showing episodic recrudescence and remission. Although the precise pathogenesis of sarcoidosis remains unclear, it is thought to involve an exaggerated cellular immune response against unknown antigens leading to granuloma formation in genetically predisposed hosts (13). Several reports have indicated elevated levels of Th1-type cytokines, such as IFN-{gamma}, in bronchoalveolar lavage fluid (BALF) of sarcoidosis patients (14) and an accumulation of Th1 CD4 T cells in the granuloma lesions (15,16), suggesting an importance of Th1 response in the pathogenesis of sarcoidosis and the elimination of unknown sarcoidosis pathogens.

In this report, we examined the function of V{alpha}24 NKT cells in patients with remitting and non-remitting sarcoidosis. We used mouse V{alpha}14 NKT cells instead of relying on limited numbers of human V{alpha}24 NKT cells to measure antigen-presenting ability and performed single-cell RT-PCR to detect cytokine production. The results suggest that, in contrast to previous reports, decreased numbers of V{alpha}24 NKT cells and their abnormally low production of IFN-{gamma} are well associated with disease status in sarcoidosis.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Study population and ethical considerations
The diagnoses of pulmonary sarcoidosis were established in all patients based on clinical criteria with confirmation of non-caseating granulomas on tissue biopsies (17). The characteristics of the sarcoidosis patients and normal controls are shown in Table 1. These patients had no histories of using steroids or other anti-inflammatory drugs at the time of sampling. Staging of sarcoidosis was defined as the criteria determined in the Consensus Conference (18). The classification of patients was defined as follows. Remitting patients: abnormal findings on chest radiography have disappeared, improved or are stable over ~3 months of observation. Non-remitting patients: abnormal findings fluctuate or worsen. The disease status in individual patients was defined as follows. Stable status: abnormal findings on chest radiography have disappeared, improved or are stable over ~3 months of observation. Active status: abnormal findings fluctuate or worsen. Some muscular sarcoidosis patients were also examined for detecting NKT cell accumulation in the lesions (Fig. 5). All subjects gave consent after being informed about the nature and purpose of the study. Local ethics committee approval was obtained.


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Table 1. Characteristics of the sarcoidosis patients and normal controls
 


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Fig. 5. Accumulation of V{alpha}24 NKT cells in granuloma lesions of sarcoidosis. (A) RNA extraction and cDNA synthesis were performed using lymph node samples from sarcoidosis and lung cancer patients. cDNA was subjected to PCR with specific primers for TCR V{alpha}24–J{alpha}Q and TCR C{alpha}. PCR products were hybridized with [32P]dCTP-labeled probes. Copy numbers of rearranged V{alpha}24–J{alpha}Q DNA compared to C{alpha} in the lymph nodes of pulmonary sarcoidosis (S, n = 7: three males, four females, mean age 53.0 ± 8.7 years) and lung cancer (LC, n = 4: four samples with no metastasis confirmed; one male, three females, mean age 58.8 ± 14.2 years) patients are shown with SD; {dagger}P < 0.05. (B) Copy numbers of rearranged V{alpha}24–J{alpha}Q DNA compared to total C{alpha} in skeletal muscle granulomas of muscular sarcoidosis patients (S, n = 5: two males, three females, mean age 48.8 ± 11.8 years) and normal control (commercially available, Cont., n = 1) are shown. (C) and (D) IFN-{gamma} (C) and IL-4 (C) expression in lymph node samples from sarcoidosis and lung cancer patients shown in (A); {dagger}P < 0.05, *differences are not significant. (E and F) IFN-{gamma} (E) and IL-4 (F) expression in skeletal muscle granulomas of muscular sarcoidosis patients shown in (B). Because only one control sample (a commercial skeletal muscle sample) was used, no statistical study was performed.

 
Cell preparation
Peripheral blood and BALF samples were collected from each patient on the same day and within 6 months of disease onset, and mononuclear cells prepared as described (19).

Flow cytometric analysis
Mononuclear cells were stained with FITC-conjugated anti-TCR V{alpha}24 mAb (C15; Coulter-Immunotech, Miami, FL), phycoerythrin (PE)-conjugated anti-TCR Vß11 mAb (C21; Coulter-Immunotech) and CyChrome-conjugated anti-CD3 mAb (UCTH1; PharMingen, San Diego, CA) as described previously (19). For intracellular staining, non-adherent peripheral blood mononuclear cells (PBMC) were stimulated with 12-phorbol 13-myristate acetate (PMA) and ionomycin for 4 h as described (20). Then, the cells were permeabilized and stained with CyChrome-conjugated anti-CD4 mAb (RPA-T4), FITC-conjugated anti-IFN-{gamma} (4S.B3) and PE-conjugated anti-IL-4 mAb (8D4-8) (all from PharMingen). Lymphocytes were gated by forward and side scatter. Live lymphocytes were analyzed on an Epics-XL-MCL (Beckman Coulter, Hialeah, FL).

Evaluation of {alpha}-galactosylceramide ({alpha}-GalCer)-presenting ability of antigen-presenting cell (APC)
MACS-enriched CD3 human PBMC were incubated with {alpha}-GalCer (100 ng/ml; Kirin Brewery, Gunma, Japan) or its vehicle as described previously (19) and used as APC. Irradiated {alpha}-GalCer-pulsed APC (5 x 105) were co-cultured with 2 x 105 spleen cells from RAG KO/V{alpha}14TgVß8Tg mice (21) for 60 h. The proliferative responses of the mouse V{alpha}14 NKT cells were evaluated by thymidine incorporation in the last 12 h. The stimulation index was calculated from the following formula: (c.p.m. of the {alpha}-GalCer-treated group)/(c.p.m. of the vehicle-treated group).

Detection of V{alpha}24 TCR and IFN-{gamma} mRNA by single-cell RT-PCR
The detection of V{alpha}24 and IFN-{gamma} transcripts in the plate-bound anti-CD3-stimulated and sorted V{alpha}24+Vß11+ cells was performed with single-cell RT-PCR as previously described (22). PCR primer pairs used were as follows: TCR V{alpha}24 (5'-CAAA GTCGAACGGAAGATATAC-3') and TCR C{alpha} (5'-CCTCATG TCTAGCACAGTTTT-3'), and IFN-{gamma} (5'-GAGCCAAATTGTCTC CTTTTACTT-3', 5'-GTAGGCAGGACAACCATTACTGGG-3'). PCR was carried out at 94°C for 30 s, 55°C for 30 s and 72°C for 1 min for 40 cycles for TCR V{alpha}24, IFN-{gamma} and IL-4, or 25 cycles for GAPDH on a Takara PCR Thermal Cycler SP (Takara Shuzo, Shiga, Japan). An aliquot (1 µl) of the first-round PCR product was used for the second-round PCR with following primers: TCR V{alpha}24 (5'-ATGCAGACACAAAGCAAAGCAAA GCTC-3') and TCR C{alpha} (5'-GGCAGACAGACTTGTCACTGGA-3'), and IFN-{gamma} (5'-ATGACCAGAGCATCCAAAAGAGTG-3', 5'-CGCTTCCCTGTTTTAGCTGCTGGC-3').

Quantification of IFN-{gamma} and IL-4 produced by V{alpha}24 NKT cells.
The transcriptional levels of IFN-{gamma}, IL-4 and GAPDH of cultured V{alpha}24+Vß11+ NKT cells were assessed by semi-quantitative RT-PCR. PBMC were cultured with recombinant human IL-2 (50 U/ml, Immunace; Shionogi, Osaka, Japan) and {alpha}-GalCer (10 ng/ml) for 7 days. V{alpha}24+Vß11+ double-positive cells were sorted on a FACS Vantage. Cells (103) were suspended in 200 µl of complete medium in a 96-well round-bottom plate and stimulated with PMA (10 ng/ml; Sigma, St Louis, MO) and ionomycin (1 µM; Calbiochem, San Diego, CA) for 4 h. RNA extraction and reverse transcription were performed as described previously (23). cDNA samples was subjected to PCR using the following primers: IFN-{gamma}, 5'-AGTTATATC TTGGCTTTTCA-3', 5'-ACCGAATAATTAGTCAGCTT-3'; IL-4, 5'-CTTCCCCCTCTGTTCTTCCT-3', 5'-TTCCTGTCGAGCCGT TTCAG-3'; or GAPDH, 5'-CCATGGGGAAGGTGAAGGT-3', 5'-ATGACCCTTTTGGCTCCCC-3'. A 30-cycle reaction was performed for IFN-{gamma} and IL-4, and a 25-cycle reaction for GAPDH. For obtaining standardization curves, cDNAs of serial 3-fold dilutions (corresponding to 1.4~9000 copies of cDNA) were subjected to PCR with primers listed above. PCR products were hybridized with a [32P]dCTP-labeled probe and the intensity of each band quantified by densitometry (Fujix BAS2500; Fujifilm I&I, Tokyo, Japan). Copy numbers of IFN-{gamma}, IL-4 and GAPDH in each sample were estimated with the standard curves.

Analysis of V{alpha}24 NKT cells in granuloma lesions
RNA extraction was performed with anterior scalene muscle lymph nodes of seven pulmonary sarcoidosis patients, hilar lymph nodes of four lung cancer patients, skeletal muscle of five muscular sarcoidosis and PBMC from three normal controls. RNA extracted from the skeletal muscle of normal controls was purchased from Clontech (K4008-1; Palo Alto, CA). Synthesized cDNA samples were subjected to PCR with following primers: TCR V{alpha}24–J{alpha}Q, 5'-GGGAGAGGTCCTG TTTCC-3', 5'-CCTCTTCCAAAGTATAGCCTCCCCAG-3'; TCR C{alpha}, 5'-GAACCCTGACCCTGCCGTGTA-3', 5'-CACTTTCAGG AGGAGGATTCG –3'. The primer pairs for IFN-{gamma} and IL-4 are described above. A 30-cycle reaction was performed for TCR V{alpha}24–J{alpha}Q and a 20-cycle reaction for TCR C{alpha}. Quantification of gene transcripts was performed as described above.

Statistical analysis
All data were expressed as the mean ± SD. Statistical analyses were performed using Student’s t-test. P < 0.05 was considered as statistically significant.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Decreased IFN-{gamma} production of V{alpha}24 NKT cells in non-remitting, but not remitting, sarcoidosis
V{alpha}24 NKT cells are known to produce abundant cytokines that regulate various immune responses. We therefore looked for functional defects in cytokine production of V{alpha}24 NKT cells in sarcoidosis. Because of limited numbers of V{alpha}24 NKT cells in patient samples (see below), we performed a sensitive analysis, semi-quantitative RT-PCR after selective expansion of V{alpha}24 NKT cells in vitro. PBMC were cultured with IL-2 and {alpha}-GalCer (4) to increase V{alpha}24 NKT cell numbers, and then FACS-sorted V{alpha}24+Vß11+ double-positive cells were stimulated with PMA and ionomycin to induce potential cytokine production. PCR products of serial dilution of control and experimental samples were hybridized with a [32P]dCTP probe, and the intensity of each band quantified by a densitometer. Copy numbers of IFN-{gamma}, IL-4 and GAPDH of experimental samples were estimated by the standard curves obtained in each experiment (Fig. 1C). The levels of IFN-{gamma} mRNA of V{alpha}24 NKT cells were revealed to be significantly reduced in patients with non-remitting sarcoidosis compared to those with remitting disease and control samples (Fig. 1A). However, no significant reduction was observed in the levels of IL-4 mRNA among the three groups (Fig. 1B), suggesting a selective dysfunction in Th1 cytokine (i.e. IFN-{gamma}) production of V{alpha}24 NKT cells in patients with non-remitting sarcoidosis.



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Fig. 1. Decreased IFN-{gamma} production of V{alpha}24 NKT cells in non-remitting sarcoidosis. (A and B) The levels of IFN-{gamma} (A) and IL-4 (B) transcripts in V{alpha}24 NKT cells induced by PMA and ionomycin were examined by semi-quantitative RT-PCR analysis. The copy numbers for IFN-{gamma} and IL-4 were estimated using standard curves generated in each assay and normalized with copy numbers of GAPDH. Mean values of remitting (R, n = 7) and non-remitting (NR, n = 8) sarcoidosis patients and normal controls (Cont., n = 8) are shown with SD; {dagger}P < 0.05, *differences are not significant. (C) Representative standard curves generated for IFN-{gamma}, IL-4 and GAPDH. Serial dilutions of the standard DNA template were amplified and hybridized with a specific [32P]dCTP probe. Radioactivity was expressed as intensity and plotted against template concentrations. (D) The IFN-{gamma}-producing potential of freshly prepared V{alpha}24 NKT cells upon CD3-stimulation for 6 h. Freshly prepared PBMC were stimulated with immobilized anti-CD3 mAb and V{alpha}24+Vß11+ double-positive cells sorted in a 96-well plate at a single cell per well. V{alpha}24 TCR and IFN-{gamma} mRNA were detected by single-cell RT-PCR. Mean values of the percentages of IFN-{gamma}-producing cells in remitting (R, n = 9) and non-remitting sarcoidosis patients (NR, n = 7) are shown with SD; {dagger}P < 0.05.

 
In order to further investigate whether the dysfunction of IFN-{gamma} production is a specific event in V{alpha}24 NKT cells of non-remitting patients, we performed a single-cell RT-PCR analysis. Non-adherent PBMC were stimulated with immobilized anti-CD3 mAb and then V{alpha}24+Vß11+ double-positive cells sorted in a 96-well plate at a single cell per well. Then, V{alpha}24 PCR-positive wells were further assayed for IFN-{gamma} gene expression by another RT-PCR. The percentages of IFN-{gamma}-producing cells among V{alpha}24 NKT cells in patients with non-remitting and remitting sarcoidosis are shown (Fig. 1D). This single-cell-level analysis demonstrated a significant decrease in the number of IFN-{gamma}-producing V{alpha}24 NKT cells in non-remitting sarcoidosis patients as compared to that in remitting patients (Fig. 1D). These results indicate that V{alpha}24 NKT cells in non-remitting, but not in remitting, sarcoidosis patients have a decreased potential to produce IFN-{gamma}.

We then investigated cytokine production in conventional CD4 T cells. In contrast to the dysfunction of V{alpha}24 NKT cells, CD4 T cells demonstrated normal production of IFN-{gamma} and/or IL-4 in the three groups as demonstrated by an intracellular cytokine staining (Fig. 2). Indeed, no significant difference was detected in the proportion of IFN-{gamma}- (Fig. 2A), IL-4- (Fig. 2B), and IFN-{gamma}/IL-4 double-producing cells (Fig. 2C). The results indicate that cytokine production by CD4 T cells is entirely normal and that the decreased IFN-{gamma} production is therefore specific for V{alpha}24 NKT cells in non-remitting sarcoidosis.



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Fig. 2. Normal production of IFN-{gamma} and IL-4 in CD4 T cells from sarcoidosis patients. Non-adherent PBMC were stimulated with PMA and ionomycin for 4 h, and intracellular cytokine staining (IFN-{gamma}/IL-4) carried out using anti-CD4 staining. The percentages of IFN-{gamma}- (A), IL-4- (B) and (IFN-{gamma} + IL-4)-producing (C) CD4 T cells in remitting (R, n = 10) and non-remitting (NR, n = 11) sarcoidosis patients and normal controls (Cont., n = 10) were determined. Mean values with SD are shown; *differences are not significant.

 
Reduced number of V{alpha}24 NKT cells in non-remitting sarcoidosis
We also examined the overall number of V{alpha}24 NKT cells and CD3+ cells in PBMC of patients with remitting and non-remitting sarcoidosis. The proportion of V{alpha}24 NKT cells in the CD3+ population was significantly reduced in patients with non-remitting sarcoidosis compared with that of remitting sarcoidosis and normal controls (Fig. 3A). The absolute number of V{alpha}24 NKT cells was also significantly reduced in non-remitting patients (Fig. 3B). In contrast, the percentage of CD3+ T cells of non-remitting sarcoidosis is not significantly different from that of remitting sarcoidosis and normal controls (Fig. 3C). Thus, the reduction of V{alpha}24 NKT cell numbers seems to be most prominent in patients with non-remitting sarcoidosis. When paying attention to the disease progress in individual patients, the levels of V{alpha}24 NKT cell numbers were lower at the active stage compared to that in the stable stage in all cases (Fig. 3D). However, CD3+ T cell numbers were unchanged in most cases regardless of the disease status (Fig. 3E). When V{alpha}24 NKT cell numbers in PBMC were compared with those in BALF of six patients with remitting or non-remitting sarcoidosis, there was a significant correlation in their numbers in PBMC and BALF in all except one patient (Fig. 3F). The results indicate that V{alpha}24 NKT cell numbers in PBMC are at a similar level to that in the lung.



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Fig. 3. Frequency of V{alpha}24 NKT cells in the peripheral blood of sarcoidosis patients. (A–C) Percentages of V{alpha}24+Vß11+ NKT cells in peripheral blood lymphocytes (PBL) (A), the absolute numbers of V{alpha}24+Vß11+ NKT cells per 1 ml of PBMC (B) and percentages of CD3+ T cells in PBMC (C) from remitting (R, n = 20) and non-remitting (NR, n = 10) sarcoidosis patients and normal controls (Cont., n = 17) were assessed, and mean values with SD are shown; {dagger}P < 0.05, *differences are not significant. (D and E) Percentages of V{alpha}24+Vß11+ NKT cells (D) and CD3+ T cells (E) in PBMC from patients with stable and active sarcoidosis (n = 8). (F) Proportion of V{alpha}24+Vß11+ NKT cells in lymphocytes in both PBMC and BALF determined in six patients.

 
Normal APC function in non-remitting sarcoidosis patients
Since the V{alpha}24 NKT cell number in patients with non-remitting sarcoidosis was significantly reduced, it is important to investigate whether the reduction is due to a functional defect in either APC or V{alpha}24 NKT cells. We thus examined the CD1d-dependent antigen-presenting ability of patient’s APC using {alpha}-GalCer as a surrogate antigen. CD3 PBMC were incubated with {alpha}-GalCer or control vehicle, irradiated and used as APC. To avoid the influence of variability of V{alpha}24 NKT cell numbers in individual patients, we used mouse V{alpha}14 NKT cells as responders against patient’s APC (19). We found no significant difference observed among the three groups in the ability of patient’s APC to stimulate mouse V{alpha}14 NKT cells, indicating that these were functioning normally (Fig. 4).



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Fig. 4. Evaluation of {alpha}-GalCer antigen-presenting ability of patient APC. Murine V{alpha}14 NKT cells were cultured with irradiated CD3 human APC pulsed with {alpha}-GalCer or control vehicle. The 3H uptake of responder NKT cells was measured and the stimulation index calculated. Mean values of remitting (R, n = 6) and non-remitting (NR, n = 5) sarcoidosis and normal controls (Cont., n = 6) are shown with SD; *differences are not significant.

 
Accumulation of V{alpha}24 NKT cells in granuloma lesion of sarcoidosis
We assessed an accumulation of V{alpha}24 NKT cells within the granulomas of sarcoidosis by measuring the copy numbers of V{alpha}24–J{alpha}Q and C{alpha} gene transcripts by semi-quantitative RT-PCR. We first compared anterior scalene muscle lymph nodes of pulmonary sarcoidosis patients with hilar lymph nodes of lung cancer patients as a control sample. The proportion of V{alpha}24 NKT cells (V{alpha}24–J{alpha}Q) among total T cells (C{alpha}) was significantly higher in lymph nodes of sarcoidosis than those of lung cancer (Fig. 5A). We also found the proportion of V{alpha}24 NKT cells in skeletal muscle granulomas of muscular sarcoidosis patients to be extremely high compared to normal control tissues (Fig. 5B). These data suggest that sarcoidosis involves an accumulation of V{alpha}24 NKT cells in granulomatous lesions.

Cytokine expression in granuloma lesion of sarcoidosis
We assessed cytokine expression in the granuloma of sarcoidosis by semi-quantitative RT-PCR. The same samples as Fig. 5(A and B) were used. As shown in Fig. 5(C and D), the expression of IFN-{gamma} is significantly higher in sarcoidosis lymph node granulomas compared to that of lung cancer. As for IL-4, no significant difference was detected. Figure 5(E and F) shows the results with skeletal muscle granulomas of muscular sarcoidosis. A similar increase in IFN-{gamma} expression was detected as compared to normal muscle samples. These results suggest that a substantial amount of IFN-{gamma} expression exists in sarcoidosis granulomas. Since ~100 times more T cells existed in the lymph node granuloma and double the numbers in the muscle granuloma (see Fig. 5A and B, ratios) compared to V{alpha}24 NKT cells, the source of IFN-{gamma} would be from Th1-type T cells as reported (1416). It is not known about the contribution of V{alpha}24 NKT cells to the detected amounts of IFN-{gamma}.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Despite the uncertainty regarding etiology, much evidence supports the notion that sarcoidosis involves a severe immunologic dysfunction (12,24). Although V{alpha}24 NKT cells are known to produce large amounts of cytokines and control Th1/Th2 responses, there have been few reports on their role in the pathogenesis of sarcoidosis in humans. On the other hand, in a murine study, granuloma formation induced by a phosphatidyl inositolmannoside fraction of M. tuberculosis was impaired in V{alpha}14 NKT cell-deficient (J{alpha}281–/–) mice (11), suggesting that V{alpha}14 NKT cells play a primary role in the granulomatous responses. Moreover, dysfunction of NKT cells leads to development of autoimmune diseases, such as Type 1 diabetes in NOD mice or experimental encephalomyelitis in mice (25,26). We thus hypothesized that human V{alpha}24 NKT cells contribute to the modulation of disease progression and the formation of non-caseating granulomas in sarcoidosis. Here, we found a reduction in both V{alpha}24 NKT cell number and IFN-{gamma} production in the PBMC of non-remitting, but not remitting, sarcoidosis patients. These results strongly suggest a role for V{alpha}24 NKT cells in the disease progression of sarcoidosis.

The factors that initiate sarcoidosis or modulate its progression have remained unknown. Similarly, it remains impossible to predict whether patients will develop an indolent self-limiting illness or progress to severe tissue destruction. Several pieces of evidence suggest that sarcoidosis is initiated by exposure of genetically susceptible hosts to inhaled environmental agents. Both infectious and non-infectious agents, ranging from simple metals to infectious organisms, have been implicated (17). In this context, it has recently been reported that Gram-positive anerobic bacteria, Propionibacterium acnes and Propionibacterium granulosum, are probable antigens triggering granuloma formation in sarcoidosis (13).

Elevated expression of Th1-type cytokines in BALF of sarcoidosis patients and an accumulation of CD4 T cells in granuloma lesions have been reported, suggesting the involvement of Th1 CD4 T cells in disease pathogenesis (1416). However, our study revealed that conventional CD4 T cells in the peripheral blood of sarcoidosis patients produced normal (or relatively larger) amounts of IFN-{gamma} compared to those from normal controls (Fig. 2). Thus, the decreased IFN-{gamma} production seemed to be a specific feature of V{alpha}24 NKT cells in non-remitting sarcoidosis. Furthermore, the normal levels of IFN-{gamma} production seen in V{alpha}24 NKT cells from remitting patients (Fig. 1), together with the accumulation of these cells in granuloma lesions, suggest that IFN-{gamma} production by V{alpha}24 NKT cells may control the disease activity. Because IFN-{gamma} produced by NKT cells substantially affects the total Th1/Th2 response (27), the dysfunction of V{alpha}24 NKT cells in non-remitting patients might lead to an insufficient Th1 response, resulting in the non-remitting state of the disease.

Mempel et al. have reported an absence of V{alpha}24 NKT cells in cutaneous lesions of sarcoidosis (28). In contrast, our experiments showed a significant accumulation of V{alpha}24 NKT cells in the granuloma lesions of pulmonary and muscular sarcoidosis (Fig. 5). The reason for the discrepancy is unknown, but it may be due to the difference in the stage of sarcoidosis and tissues sampled or the sensitivity of detection of V{alpha}24 NKT cells. In this regard, we have recently found that activation of V{alpha}14 NKT cells rapidly induces a down-regulation of their V{alpha}14 receptor expression (Harada et al., submitted). Thus, it is possible that repeated exposure to unknown pathogens triggers V{alpha}24 NKT cells in sarcoidosis patients to down-regulate their receptor expression. The down-regulated V{alpha}24 receptor of NKT cells can be detected by PCR, but not by staining with antibody. The use of antibodies or an {alpha}-GalCer/CD1d tetramer may therefore fail to detect the existence of V{alpha}24 NKT cells.

In any event, the reduced number of V{alpha}24 NKT cells and their IFN-{gamma} production appear to be involved in the disease progression of sarcoidosis. A practical benefit of these observations is that analysis of V{alpha}24 NKT cell number and function in sarcoidosis may be useful in evaluating disease prognosis. The present findings implicate a new therapeutic strategy for sarcoidosis, such as an activation of V{alpha}24 NKT cells with a glycolipid ligand or adoptive transfer of expanded V{alpha}24 NKT cells. Further clinical studies are needed to clarify these possibilities.


    Acknowledgements
 
We thank Dr Robert Triendl for reading and Ms Hiroko Tanabe for preparation of this manuscript. This work was supported by the following: Ministry of Education, Culture, Sports, Science and Technology, Japan, Grants-in-Aid for Scientific Research (A) # 13307011 (M. T.), Scientific Research, Priority Areas Research #13218016 (T. N.), Scientific Research B # 14370107 (T. N.), and Special Coordination Funds for Promoting Science and Technology (T. N.), Ministry of Health, Labor and Welfare, Japan, Organization for Pharmaceutical Safety and Research (Project ID #MF-24) (M. T.), Human Frontier Science Program Research Grant (RG00168/2000-M206) (M. T.), and Kirin Brewery Co. Ltd.


    Abbreviations
 
{alpha}-GalCer—{alpha}-galactosylceramide

APC—antigen-presenting cell

BALF—bronchoalveolar lavage fluid

PBL—peripheral blood lymphocyte

PBMC—peripheral blood mononuclear cell

PE—phycoerythrin

PMA—phorbol 12-myristate 13-acetate


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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