Involvement of CD161⁺ Vδ1⁺ γδ T cells in systemic sclerosis: association with interstitial pneumonia

Abstract

Objective. Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with high mortality and poor prognosis. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of CD161⁺ Vδ1⁺ γδ T cells in SSc patients with IP.

Methods. The proportion of CD161⁺ Vδ1⁺ γδ T cells in peripheral blood mononuclear cells (PBMCs) and serum sialylated carbohydrate antigen (KL-6) levels were determined. GeneChip analysis was performed with CD161⁻ and CD161⁺ Vδ1⁺ γδ T cells. Cytokine and chemokine expression from CD161⁺ Vδ1⁺ γδ T cells was measured and used to evaluate the effect of culture supernatant on fibroblast proliferation.

Results. The proportion of CD161⁺ Vδ1⁺ γδ T cells was significantly higher in SSc than healthy controls (HCs) and correlated negatively with serum KL-6 levels in IP-positive SSc patients. The gene and mRNA expression level of chemokine ligand 3 (CCL3) was markedly higher in CD161⁺ Vδ1⁺ γδ T cells than in CD161⁻ Vδ1⁺ γδ T cells. CD161⁺ Vδ1⁺ γδ T cells in IP-positive SSc patients showed higher production of CCL3 and lower production of IFN-γ than in HCs. Culture supernatant derived from IP-negative and IP-positive SSc patients promoted fibroblast proliferation, whereas that from HCs did not.

Conclusion. The small proportion and the altered cell functions of CD161⁺ Vδ1⁺ γδ T cells among PBMCs in SSc patients play a role in the pathogenesis of IP. These findings suggest that CD161⁺ Vδ1⁺ γδ T cells may play a regulatory role in the pathogenesis of IP in SSc patients via IFN-γ production.

interstitial pneumonia, systemic sclerosis, CD161, gamma-delta T cell, CCL3, interferon-γ, natural killer receptors, KL-6

Introduction

Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with high mortality and poor prognosis [1]. Previous studies have indicated that IP is an intractable disease induced by various factors such as autoimmune diseases, drugs and occupational and environmental exposure [2]. For example, chemotherapy with bleomycin and busulphan is reported to cause lung fibrosis in some patients [3]. Following interstitial inflammation, florid fibroblast proliferation within both the interstitium and alveolar space is often detected. However, the pathogenesis of IP remains to be elucidated.

In humans, T lymphocytes bearing the γδ T cell receptor (TCR) represent a minor population of peripheral blood mononuclear cells (PBMCs) [4]. Two main subsets of γδ T cells have been described in human PBMCs. The most dominant γδ T cells express the Vγ9-Vδ2 TCR (Vδ2⁺ γδ T cells), whereas the second one expresses TCR Vδ1 in association with various Vγ chains (Vδ1⁺ γδ T cells) [5]. The proportion of Vδ1⁺ γδ T cells among PBMCs is small, but larger in gut mucosa, spleen and several epithelial-rich tissues [6–9].

SSc is an autoimmune disease characterized by fibrosis of the skin and various internal organs. In SSc patients, IP represents the main cause of death [10]. Recent studies have described the involvement of Vδ1⁺ γδ T cells in the pathogenesis of IP, especially in SSc patients [11, 12]. Furthermore, IP is also known as a serious complication in other autoimmune diseases, such as RA and PM/DM. However, the role of Vδ1⁺ γδ T cells in IP remains elusive.

In mice, pulmonary fibrosis can be induced by the administration of bleomycin, silica particles or Bacillus subtilis. Recently the inflammatory role of γδ T cells has been reported in several IP mice models [13–15]. Furthermore, we recently reported the inflammatory role of γδ T cells that expressed a natural killer (NK) cell marker, NK1.1, in IL-2- plus IL-18-induced interstitial lung disease mice models [16]. In humans, mouse NK1.1 is homologous with CD161, a molecule that is expressed on the surface of NK cells, NK T cells and a variable proportion of both CD4⁺ and CD8⁺ αβ T cells [17]. It had been reported that the proportion of Vδ1⁺ γδ T cells expressing CD161 (CD161⁺ Vδ1⁺ γδ T cells) increased in PBMCs of patients with HIV [18]. However, there are no reports on the properties and functional characteristics of CD161⁺ Vδ1⁺ γδ T cells in patients with autoimmune diseases.

In the present study we showed that the proportion of CD161⁺ Vδ1⁺ γδ T cells among PBMCs was lower, especially in SSc patients with highly active IP. On the other hand, the proportion of CD161⁺ Vδ1⁺ γδ T cells was significantly higher in IP-negative SSc patients than healthy controls (HCs) and IP-positive SSc patients. Furthermore, CD161⁺ Vδ1⁺ γδ T cells in IP-positive SSc patients showed greater production of chemokine ligand 3 (CCL3) and lower production of IFN-γ than in HCs. Thus the small proportion and the altered cell functions of CD161⁺ Vδ1⁺ γδ T cells among PBMCs in SSc patients probably play a role in the pathogenesis of IP. We also discuss the functional role of CD161⁺ Vδ1⁺ γδ T cells in the pathogenesis of SSc and IP.

Materials and methods

Patients and healthy donors

We examined 88 subjects, including 17 with RA [3 males and 14 females, mean age 62.4 years (s.d. 10.5)] diagnosed according to ACR criteria [19], 35 with SSc [6 males and 29 females, mean age 55.6 years (s.d. 12.9)] diagnosed according to ACR criteria [20], 14 with PM/DM [4 males and 10 females, mean age 52.9 years (s.d. 10.0)] diagnosed according to published criteria [21, 22] and 22 disease-free HCs [5 males and 17 females, mean age 42.9 years (s.d. 12.1)]. Patients with SSc were grouped according to the extent of skin involvement, based on the classification system [20]: 25 patients with dcSSc and 10 patients with lcSSc. The presence of IP was initially determined by radiologists, based on chest high-resolution CT findings. All subjects were examined at the University of Tsukuba Hospital and each provided signed consent to participate in this study. This study was approved by the ethics committee of the University of Tsukuba. In this study, the expression of CD161 was not influenced by age.

Staining and flow cytometry

PBMCs were isolated using Ficoll-Paque (GE Healthcare UK, Little Chalfont, UK). Cells were stained with the monoclonal antibodies (mAbs) described below. PerCP-conjugated anti-CD3, phycoerythrin-conjugated anti-CD161, fluorescein isothiocyanate-conjugated anti-IL-17A, anti-IFN-γ, anti-IL-4, anti-TNF-α, anti-mouse IgG1κ, anti-mouse IgG2bκ, rat IgG1κand Dylight 649-conjugated anti-mouse IgG1κ were obtained from BioLegend (San Diego, CA, USA). Anti-TCR Vδ1 mAb was obtained from Thermo Scientific (Waltham, MA, USA). The stained cells were analysed on an FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA, USA) and data were processed using FlowJo software (Tree Star, Ashland, OR, USA).

Gene chip analysis of Vδ1⁺ γδ T cells

Highly purified CD161⁻ Vδ1⁺ γδ T and CD161⁺ Vδ1⁺ γδ T cells were obtained from HCs (n = 3). The purity of FACS-sorted cells was >99%. Total RNA was extracted from each cell using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. cDNA was amplified from total RNA using the Ovation Pico WTA System (NuGEN, San Carlos, CA, USA). After amplification, hybridization with SurePrintG3 Human GE (Agilent, Santa Clara, CA, USA) was performed. Analysis was performed by GeneSpring GX gene expression software (Agilent). The data discussed in this article have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE43402.

Quantitative PCR

Total RNA was extracted from isolated CD161⁻ Vδ1⁺ γδ T and CD161⁺ Vδ1⁺ γδ T cells (see above) and reverse transcribed into cDNA using PrimeScript RT Master Mix (Takara, Otsu, Japan) according to manufacturer’s protocol. Quantitative PCR was performed using a 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with SYBR Premix EX Taq (Takara) and the following primers: human GAPDH (HA067812), KLRB1/CD161 (HA162351), CCL2/monocyte chemoattractant protein-1 (HA145737), CCL3/macrophage inflammatory protein-1α (HA175954), CCL4L1//macrophage inflammatory protein-1β like-1 (HA097607) and CCL5/regulated upon activation, normal T cell expressed and secreted (HA156658).

Generation of polyclonal cell lines from CD161⁺ Vδ1⁺ γδ T cells

Highly purified CD161⁺ Vδ1⁺ γδ T cells were obtained fromHCs (n = 3), IP-negative SSc patients (n = 3) and IP-positive SSc patients (n = 4). To obtain polyclonal cell lines, cells were seeded at 300 cells/well in the presence of 1% phytohaemagglutinin (PHA) in 96-well U-bottom microplates (Nunc, Naperville, IL, USA). These cells were cultured in complete RPMI 1640 medium supplemented with 5% fetal calf serum (FCS), 5% human serum and 100 U/ml recombinant human IL-2 (rhIL-2). Ten days later the cells were co-cultured with irradiated allogeneic PBMCs as feeder cells (1 × 10⁵ cells/well) and 1 μg/ml PHA. Cells were then expanded with rhIL-2 and restimulated every 3 weeks with PHA and irradiated feeder cells according to standard procedure [23].

Stimulation of γδ TCR on CD161⁺ Vδ1⁺ γδ T cell lines

Before adding CD161⁺ Vδ1⁺ γδ T cells, 96-well flat-bottom microplates (Nunc) were coated for 16 h at room temperature with 10 µg/ml anti-TCR Vδ1 mAbs (Thermo Scientific). After washing twice, CD161⁺ Vδ1⁺ γδ T cell lines (5 × 10⁵ cells/well) were cultured for 72 h at 37°C.

Detection of chemokines and cytokines in culture supernatants

Human FlowCytomix (eBioscience, San Diego, CA, USA) was used to examine the expression of CCL2, CCL3, CCL4, CCL5, IFN-γ, TNF-α, IL-4, IL-17 and TGF-β1 in culture supernatants using a FACS Calibur flow cytometer (Becton Dickinson).

RT-PCR for CCR1, CCR5 and IFN-γR

Total RNA was extracted from WI-38 fibroblast and reverse transcribed into cDNA using PrimeScript RT Master Mix (Takara) according to the manufacturer’s protocol. For amplification of cDNA, after an initial denaturation step at 94°C for 7 min, 35 cycles were conducted each at 94°C for 30 s followed by 60°C for 30 s and 72°C for 30 s, with a further extension at 72°C for 4 min. After amplification, the PCR products were separated by electrophoresis in 2.0% agarose gels. The primer sequences (5′–3′) were as follows: CCR1, 5′-TTTGGTGTCATCACCAGCAT, 3′-GCCTGAAACAGCTTCCACTC; CCR5, 5′-GGCAAAGACAGAAGCCTCCA, 3′-AACCTTCTGCAACACCAACC; IFN-γR1, 5′-GGCAGCATCGCTTTAAACTC, 3′-GGAGGTGGGGGCTTTTATTA; GAPDH, 5′-GAAGGTGAAGGTCGGAGTC, 3′-GAAGATGGTGATGGGATTTC.

Fibroblast proliferation assay

WI-38 lung fibroblast was provided by the Riken Bioresource Center (Tsukuba, Japan) through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. These cells were cultured in minimum essential medium (MEM; Sigma, St Louis, MO, USA) supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were fed twice weekly and used for experiments from three to eight passages. One hundred microlitres (1 × 10³ cell/ml) of WI-38 resuspended in MEM with 1% FCS were placed in a 96-well flat-bottom plate (Nunc) and several doses of culture supernatant were applied from CD161⁺ Vδ1⁺ γδ T cells, recombinant human IFN-γ, CCL3, anti-human IFN-γ mAbs (BioLegend) and anti-human CCL3 mAbs (R&D Systems, Minneapolis, MN, USA) or normal vehicle. After 24 h incubation, WI-38 proliferations were measured by BrdU ELISA (Roche, Basel, Switzerland).

Statistical analysis

Data are expressed as the median or mean (s.e.m.). Differences between groups were examined for statistical significance using the Mann–Whitney U-test and Spearman’s correlation. A P-value of <0.05 denoted the presence of a statistically significant difference. For multiple group comparisons, one-way analysis of variance (ANOVA) was performed, followed by the Bonferroni test. A P-value of <0.01 denoted the presence of a statistically significant difference. All data were analysed using the Statistical Package for Social Sciences (SPSS version 21, Chicago, IL, USA).

Results

Increased CD161⁺ Vδ1⁺ γδ T cells in IP-negative SSc patients

We examined CD161⁺ Vδ1⁺ γδ T cells in PBMCs by flow cytometry (FCM; Fig. 1A). The proportion of CD161⁺ Vδ1⁺ γδ T cells in PBMCs from the HCs [0.23% (s.e.m. 0.09), P < 0.05] was significantly lower than in SSc patients [0.55% (s.e.m. 0.13), P < 0.05], but was similar to that from RA [0.38% (s.e.m. 0.13)] and PM/DM patients [0.23% (s.e.m. 0.11) (Fig. 1B). Conversely, the proportion of CD161⁻ Vδ1⁺ γδ T cells was not different among the groups (Fig. 1B). Similar results were found with regard to the absolute number of CD161⁻ Vδ1⁺ T and CD161⁺ Vδ1⁺ γδ T cells in PBMCs (Fig. 1C). The proportion of CD161⁺ Vδ1⁺ γδ T cells was significantly higher in IP-negative SSc patients [1.03% (s.e.m. 0.30)] compared with IP-positive SSc patients [0.28% (s.e.m. 0.07), P < 0.01] and HCs [0.23% (s.e.m. 0.09), P < 0.005; Fig. 1D]. However, CD161⁺ Vδ1⁺ γδ T cells were not altered by the presence of IP in patients with RA and PM/DM. Similar results were found with regard to the absolute number of CD161⁺ Vδ1⁺ γδ T cells in PBMCs (Fig. 1E). In contrast, the proportion and number of CD161⁻ Vδ1⁺ γδ T cells were not affected in patients with IP (Fig. 1D and E).

Fig. 1

High proportion of CD161⁺ Vδ1⁺ γδ T cells in IP-negative SSc patients

(A) Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls (HCs) and patients with RA, PM/DM and SSc, as described in Materials and methods. Cells were stained with anti-CD161 and anti-TCR Vδ1 monoclonal antibodies (mAbs). (B) Proportion (%) of CD161⁻ Vδ1⁺ γδ T cells (left) and CD161⁺ Vδ1⁺ γδ T cells (right) in PBMCs from HCs and RA, PM/DM and SSc patients. (C) Density of CD161⁻ Vδ1⁺ γδ T cells (left) and CD161⁺ Vδ1⁺ γδ T cells (right) in HCs and RA, PM/DM and SSc patients. (D) Proportion (%) of CD161⁻ Vδ1⁺ γδ T cells (left) and CD161⁺ Vδ1⁺ γδ T cells (right) in PBMCs from HCs and autoimmune disease patients with and without IP. (E) Density of CD161⁻ Vδ1⁺ γδ T cells (left) and CD161⁺ Vδ1⁺ γδ T cells (right) in PBMCs from HCs and autoimmune disease patients with and without IP. (F) Proportion of CD161⁺ Vδ1⁺ γδ T cells in HCs and patients with lcSSc and dcSSc. (G) Proportion of CD161⁺ Vδ1⁺ γδ T cells in patients with lcSSc and dcSSc with and without IP. Bars represent the group mean values. P < 0.05.

Unaltered proportion of CD161⁺ Vδ1⁺ γδ T cells between lcSSc and dcSSc patients

The proportion of CD161⁺ Vδ1⁺ γδ T cells was similar in HCs, lcSSc and dcSSc (Fig. 1F). Furthermore, the proportion of CD161⁺ Vδ1⁺ γδ T cells was significantly higher in IP-negative lcSSc patients [1.03% (s.e.m. 0.59)] than in HCs [0.31% (s.e.m. 0.08), P < 0.01; Fig. 1G].

CD161⁺ Vδ1⁺ γδ T cells correlate negatively with KL-6 values

We next examined the correlation between CD161⁺ Vδ1⁺ γδ T cells in PBMCs and serum sialylated carbohydrate antigen (KL-6) values, a laboratory test that reflects the activity of IP in IP-positive SSc patients. As shown in Fig. 2A, the proportion of CD161⁺ Vδ1⁺ γδ T cells in PBMCs correlated negatively with serum KL-6 values among IP-positive SSc patients (r = −0.511, P < 0.05), whereas that of total Vδ1⁺ γδ T cells (r = −0.339) and CD161⁻ Vδ1⁺ γδ T cells (r = −0.286) did not. Furthermore, we performed follow-up analysis over a mean duration of 15.8 months (s.e.m. 1.27). As shown in Fig. 2B, the change in the proportion of CD161⁺ Vδ1⁺ γδ T cells correlated negatively with that in serum KL-6 (r = −0.883, P < 0.05), whereas the change in total Vδ1⁺ γδ T cells (r = −0.637) and CD161⁻ Vδ1⁺ γδ T cells (r = −0.553) did not.

Fig. 2

CD161⁺ Vδ1⁺ γδ T cells correlate negatively with KL-6

(A) Correlation of serum KL-6 and proportion of total Vδ1⁺ γδ T cells (left), CD161⁻ Vδ1⁺ γδ T cells (middle) and CD161⁺ Vδ1⁺ γδ T cells (right) among peripheral blood mononuclear cells (PBMCs) from IP-positive SSc patients. (B) Correlation between changes in serum KL-6 levels and proportion of total Vδ1⁺ γδ T cells (left), CD161⁻ Vδ1⁺ γδ T cells (middle) and CD161⁺ Vδ1⁺ γδ T cells (right) among PBMCs from IP-positive SSc patients over a period of time. ΔKL-6: time-related changes in KL-6 level. The ordinate represents the ratio calculated using the following formula: proportion of cells at the second evaluation/proportion of the same cells at the first evaluation. Serum KL-6 levels and the proportion of each Vδ1⁺ γδ T cells among PBMCs were determined simultaneously. P < 0.05.

Th1 cytokine secretion from CD161⁺ Vδ1⁺ γδ T cells

Vδ1⁺ γδ T cells can secrete several kinds of cytokine, such as IFN-γ, IL-4 and IL-17 [18, 24]. To determine whether CD161⁺ Vδ1⁺ γδ T cells have the potential to secrete cytokines, PBMCs from HCs, IP-negative SSc patients and IP-positive SSc patients were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin and stained for intracellular cytokines, IFN-γ, TNF-α, IL-4 and IL-17 (Fig. 3A). The secretion of IFN-γ was significantly greater by CD161⁺ Vδ1⁺ γδ T cells than by CD161⁻ Vδ1⁺ γδ T cells in three groups (P < 0.05, P < 0.05 and P < 0.05; Fig. 3B). In contrast, similar production levels of IL-4, TNF-α and IL-17 were noted for CD161⁻ and CD161⁺ Vδ1⁺ γδ T cells. Furthermore, the secretion levels of IFN-γ, IL-4, TNF-α and IL-17 for CD161⁺ Vδ1⁺ γδ T cells were not different among three groups (Fig. 3C).

Fig. 3

Cytokine secretion from CD161⁺ Vδ1⁺ γδ T cells

(A) Intracellular staining for IFN-γ, TNF-α, IL-4 and IL-17 was performed after the in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs), IP-negative SSc patients and IP-positive SSc patients by PMA/ionomycin for 6 hr and analysed among gated Vδ1⁺ cells by FCM. (B) Comparison of cytokine production by CD161⁻ and CD161⁺ Vδ1⁺ γδ T cells from HCs, IP-negative SSc patients and IP-positive SSc patients. (C) Comparison of cytokine production of CD161⁺ Vδ1⁺ γδ T cells among HCs, IP-negative SSc patients and IP-positive SSc patients. Data are mean (s.e.m.). P < 0.05.

High expression of CCL3 and CCL4 in CD161⁺ Vδ1⁺ γδ T cells

To elucidate the difference between CD161⁻ and CD161⁺ Vδ1⁺ γδ T cells, we performed GeneChip analysis of these two types of cells in PBMCs from HCs. Such analysis identified 192 genes that were expressed at 2-fold or higher levels on CD161⁺ Vδ1⁺ γδ T cells than on CD161⁻ Vδ1⁺ γδ T cells (see supplementary Table S1, available at Rheumatology Online). Furthermore, among the 192 genes, the expression levels of CCL3/MIP-1α and CCL4/MIP-1β genes on CD161⁺ Vδ1⁺ γδ T cells were markedly higher than those on CD161⁻ Vδ1⁺ γδ T cells (Fig. 4A and B). Genes of other CC chemokines, CCL2/MCP-1 and CCL5/RANTES were not up-regulated.

Fig. 4

Gene expression profiling in CD161⁻ and CD161⁺ Vδ1⁺ γδ T cells

(A) Heat map showing CCL3, CCL4 and CCL5 gene expression in CD161⁻ and CD161⁺ Vδ1⁺ γδ T cells in three healthy control subjects (HC1, HC2 and HC3). (B) Comparison of log fold change in CCL3, CCL4 and CCL5 gene expression between CD161⁻ and CD161⁺ Vδ1⁺ γδ T cells. The CD161 gene expression showed positive control for the GeneChip array. (C) CCL2, CCL3, CCL4, CCL5 and CD161 mRNA levels in CD161⁻ and CD161⁺ Vδ1⁺ γδ T cells measured by quantitative PCR. These cells were freshly isolated from peripheral blood mononuclear cells of HCs (n = 3) and IP-negative (IP−) (n = 4) and IP-positive (IP+) (n = 4) SSc patients. (D) CCL2, CCL3, CCL4, CCL5 and CD161 mRNA levels measured by quantitative PCR. CD161⁺ Vδ1⁺ γδ T cells were freshly isolated from HCs (n = 3) and IP-negative (IP−) (n = 4) and IP-positive (IP+) (n = 4) SSc patients. Data are mean (s.e.m.). P < 0.05.

The mRNA expression levels of CCL3, CCL4 and CD161 in CD161⁺ Vδ1⁺ γδ T cells of HCs were significantly higher than those in CD161⁻ Vδ1⁺ γδ T cells (P < 0.05, P < 0.05, P < 0.05; Fig. 4C). Furthermore, the mRNA expression levels of CCL3 and CCL4 were higher in CD161⁺ Vδ1⁺ γδ T cells of IP-negative SSc patients, whereas the levels of CCL4 and CCL5 were significantly higher in IP-positive SSc patients (P < 0.05 and P < 0.05; Fig. 4C). The mRNA expression levels of CCL2, CCL3, CCL4 and CCL5 in CD161⁺ Vδ1⁺ γδ T cells were not different between the HCs and SSc patients (Fig. 4D).

Production of CCL3 and IFN-γ by CD161⁺ Vδ1⁺ γδ T cells through TCR stimulation

To analysis chemokine and cytokine production, polyclonal CD161⁺ Vδ1⁺ γδ T cell lines were derived from HCs, IP-negative SSc patients and IP-positive SSc patients and stimulated by TCR. The production of CCL3 from CD161⁺ Vδ1⁺ γδ T cell lines of IP-positive SSc patients was significantly higher than in HCs (Fig. 5A). On the other hand, the production of IFN-γ from CD161⁺ Vδ1⁺ γδ T cell lines of IP-negative and IP-positive SSc patients was significantly lower than in HCs. However, the production of CCL2, CCL4, CCL5, IL-4, TNF-α, IL-17 and TGF-β1 was not significantly different between HCs, IP-negative SSc patients and IP-positive SSc patients.

Fig. 5

Fibroblast proliferation in the presence of CCL3 and IFN-γ

(A) Comparison of IFN-γ, TNF-α, IL-4, IL-17, TGF-β1, CCL2, CCL3, CCL4 and CCL5 production by CD161⁺ Vδ1⁺ γδ T cell lines among healthy controls (HCs) and IP-negative and IP-positive SSc patients in the presence of anti-TCR Vδ1 and anti-mIgG1 mAbs. Data are mean (s.e.m.). P-values were calculated by the Mann–Whitney U test. (B) CCR1, CCR5 and IFN-γ receptor (IFN-γR) expressions on WI-38 fibroblast were determined by RT-PCR. (C) WI-38 fibroblasts were cultured in 1% FCS MEM with 0, 0.1, 0.3, 1 or 3% culture supernatant from CD161⁺ Vδ1⁺ γδ T cell lines after TCR Vδ1 stimulation. The cell proliferation values were measured by BrdU ELISA. (D) WI-38 fibroblasts were cultured with IFN-γ (0, 10, 100, 1000 and 3000 ng/ml) and CCL3 (0, 10, 100, 1000 and 3000 ng/ml). The cell proliferation values were measured by BrdU ELISA. (E) WI-38 fibroblasts were cultured with IFN-γ (0 and 1000 ng/ml), CCL3 (0 and 1000 ng/ml), anti-IFN-γ mAbs (0, 10, 100 and 1000 ng/ml) and anti-CCL3 mAbs (0, 10, 100 and 1000 ng/ml). The cell proliferation values were measured by BrdU ELISA. Data are mean (s.e.m.). P < 0.05.

Effects of IFN-γ and CCL3 on fibroblast proliferation

Since IFN-γ and CCL3 are known to be crucial molecules for the fibrotic process, we analysed the influence of IFN-γ and CCL3 on fibroblast proliferation in vitro. WI-38 cells are known to be established from fetal lung fibroblast and these cells expressed IFN-γ receptor (IFN-γR) and CCL3 receptor, CCR1 and CCR5 (Fig. 5B). To examine WI-38 proliferation with culture supernatant from CD161⁺ Vδ1⁺ γδ T cell lines, we checked the dose response. WI-38 proliferation was promoted dose-dependently with culture supernatant from HCs, IP-negative SSc patients and IP-positive SSc patients (Fig. 5C). WI-38 proliferation was promoted with a high dose of culture supernatant derived from IP-negative SSc and IP-positive SSc patients compared with that from HCs (P < 0.05 and P < 0.05, respectively; Fig. 5C). As shown in Fig. 5D, IFN-γ suppressed WI-38 cell proliferation in a concentration-dependent manner, whereas CCL3 did not affect WI-38 cell proliferation. Interestingly, in the presence of CCL3, WI-38 cell proliferation was not inhibited by IFN-γ. Thus CCL3 cancelled the suppressive effect of IFN-γ on WI-38 proliferation. Furthermore, the effect of IFN-γ and CCL3 on fibroblast proliferation was clearer when using neutralizing antibodies for these cytokines (Fig. 5E).

Discussion

IP is the main cause of death in patients with SSc [10]. Recent studies have shown that T, B, NK, fibroblast and other cells play crucial roles in the pathogenesis of SSc and IP [25]. However, the exact pathomechanisms of SSc and IP remain elusive. In the present study we demonstrated that CD161⁺ Vδ1⁺ γδ T cells play a regulatory role in the pathogenesis of IP in SSc patients via IFN-γ production.

In humans, although Vδ1⁺ γδ T cells are known to be a minor population in PBMCs [5], the proportion of Vδ1⁺ γδ T cells is reported to increase in PBMCs, skin and bronchoalveolar lavage fluid (BALF) in SSc patients [26]. In the present study we showed that CD161⁺ Vδ1⁺ γδ T cells increased in IP-negative SSc patients compared with HCs and IP-positive SSc patients. The finding of significantly higher levels of CD161⁺ Vδ1⁺ γδ T cells in SSc patients, especially IP-negative subjects, suggests that these cells play a pathogenic role in the involvement of several organs other than the lung in SSc. KL-6 has been used as a serological maker to estimate the severity and activity of IP in SSc patients [27]. Our results showed that the proportion of CD161⁺ Vδ1⁺ γδ T cells correlated negatively with serum KL-6 levels in IP-positive SSc patients. These results also suggest that CD161⁺ Vδ1⁺ γδ T cells play a regulatory role in the pathogenesis of IP in SSc patients.

The CD161 molecule is a cell surface marker of human Th17 [28]. It has also been reported that in human γδ T cells, IL-17-producing γδ T cells express CD161 [18, 29]. However, CD161⁺ Vδ1⁺ γδ T cells secreted large amounts of IFN-γ, but not IL-17, through PMA/ionomycin stimulation. Furthermore, up-regulated expression of Th17-related genes (RORC, IL-23R and CCR6) was not detected in CD161⁺ Vδ1⁺ γδ T cells by GeneChip analysis. On the other hand, the CD161⁺ Vδ1⁺ γδ T cell line of HCs produced larger amounts of IFN-γ upon TCR stimulation than IP-negative SSc and IP-positive SSc patients. Previous reports [30, 31] have indicated that IFN-γ inhibits fibroblast proliferation. Furthermore, IFN-γ reduced inflammation and the extent of fibrosis in mice with bleomycin-induced pulmonary fibrosis [32]. Thus IFN-γ seems to prevent the progression of pulmonary fibrosis. In the present study we found that CD161⁺ Vδ1⁺ γδ T cell lines produced large amounts of IFN-γ and the proportion of these cells was lower in IP-positive SSc patients compared with IP-negative SSc patients. Thus CD161⁺ Vδ1⁺ γδ T cells seem to have a regulatory effect on the pathogenesis of IP in SSc patients mediated through IFN-γ production.

CCL3, CCL4 and CCL5 are members of the CC chemokines [33] and all three play pathogenic roles in SSc through their chemoattraction properties for monocytes and T cells [34]. Previous studies have shown significantly high levels of CCL3, CCL4 and CCL5 in BALF and sera of IP-positive SSc patients compared with HCs and IP-negative SSc patients [35–37]. Furthermore, these three chemokines also act as inflammatory mediators, as demonstrated in various IP and SSc mouse models [38–40]. GeneChip analysis showed up-regulation of CCL3 and CCL4 gene expression in CD161⁺ Vδ1⁺ γδ T cells of HCs. Similar results were also observed in quantitative CCL3 and CCL4 mRNA expression analysis in normal subjects and IP-negative SSc patients. Interestingly, in IP-positive SSc patients, the mRNA expression levels of CCL4 and CCL5, but not CCL3, were significantly higher in CD161⁺ Vδ1⁺ γδ T cells than in CD161⁻ Vδ1⁺ γδ T cells. However, the mRNA expression levels of CCL3, CCL4 and CCL5 in CD161⁺ Vδ1⁺ γδ T cells were similar in the three groups. On the other hand, in the chemokine secretion assay, CD161⁺ Vδ1⁺ γδ T cell lines of IP-positive SSc patients produced a significantly greater amount of CCL3 upon TCR stimulation than those of HCs.

In the present study, we found that high-dose CCL3 cancelled the suppressive effect of IFN-γ on fibroblast proliferation in vitro. These results indicate that CCL3 blocks IFN-γ signal transduction molecules through direct or indirect mechanisms in fibroblasts. Thus the balance between IFN-γ and CCL3 concentration might affect fibroblast proliferation. Indeed, culture supernatant from CD161⁺ Vδ1⁺ γδ T cells derived from IP-negative and IP-positive SSc patients promoted fibroblast proliferation, whereas that from HCs did not. TGF-β1 is known as a fibrotic factor [41]. However, the production of TGF-β1 was not significantly different between HCs, IP-negative SSc patients and IP-positive SSc patients. Thus we hypothesized that CD161⁺ Vδ1⁺ γδ T cells of IP-negative and IP-positive SSc patients show the imbalance between IFN-γ and CCL3 secretion compared with HCs and consequently promote fibroblast proliferation.

In conclusion, the present study demonstrated the small proportion and the altered cell function of CD161⁺ Vδ1⁺ γδ T cells in SSc patients with IP. These results suggest that these cells play a regulatory role in the pathogenesis of IP in SSc patients via IFN-γ production. However, further studies are needed to examine the exact roles of CCL3 in IP associated with SSc.

Rheumatology key messages

• CD161⁺ Vδ1⁺ γδ T cells increased in SSc patients compared with healthy controls.

• CD161⁺ Vδ1⁺ γδ T cells correlated negatively with serum KL-6 in SSc patients with interstitial pneumonia (IP).

• CD161⁺ Vδ1⁺ γδ T cells might play a regulatory role in the pathogenesis of IP.

Acknowledgements

We thank Dr F. G. Issa for critical reading of the manuscript.

Funding: This work was supported by the Research Program for Intractable Diseases, Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare and the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Disclosure statement: The authors have declared no conflicts of interest.