Selective accumulation of CCR4+ T lymphocytes into renal tissue of patients with lupus nephritis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 46, No. 3, March 2002, pp 735–740 DOI 10.1002/art.10112 © 2002, American College of Rheumatology Selective Accumulation of CCR4⫹ T Lymphocytes Into Renal Tissue of Patients With Lupus Nephritis Masato Yamada,1 Hideo Yagita,1 Hideko Inoue,1 Tsuyoshi Takanashi,1 Hironori Matsuda,1 Eiko Munechika,1 Yutaka Kanamaru,1 Isao Shirato,1 Yasuhiko Tomino,1 Kouji Matushima,2 Ko Okumura,1 and Hiroshi Hashimoto1 Conclusion. These results suggest that CCR4ⴙ T lymphocytes in peripheral blood, which represent Th2 cells, preferentially migrate into the renal tissue of patients with lupus nephritis. The maldistribution of CCR4ⴙ T lymphocytes might be involved in the pathogenesis of lupus nephritis. Objective. Some chemokine receptors, such as CCR5 and CCR4, are differentially expressed on Th1 and Th2 cells. To determine whether differential expression of the chemokine receptors occurs in patients with lupus nephritis, we examined the expression of CCR4 and CCR5 on peripheral blood lymphocytes and mononuclear cells infiltrated into the renal tissue of patients with lupus nephritis. Methods. The expression of CCR4 and CCR5 on CD4ⴙ,CD45ROⴙ cells was analyzed by flow cytometry and compared between patients with systemic lupus erythematosus (SLE) and healthy controls. Correlation between the absolute number of CCR4ⴙ or CCR5ⴙ cells and clinical parameters was also analyzed. Mononuclear infiltrates in the renal tissue of SLE patients were analyzed for the expression of CCR4, CCR5, and CD4 by immunohistochemical staining. Results. The absolute number of CCR4ⴙ, but not CCR5ⴙ, T lymphocytes in the peripheral blood was significantly decreased in the patients with SLE compared with that in the healthy controls, and this positively correlated with the serum levels of C3 and CH50. Most of the CD4ⴙ T lymphocytes that infiltrated into the renal tissue of the patients with lupus nephritis expressed CCR4, but not CCR5. Recently it has been proposed that the imbalance between Th1 cells and Th2 cells is associated with the pathogenesis of systemic lupus erythematosus (SLE). It has been reported that in patients with SLE, the production of Th2 cytokines is elevated, while Th1 cytokines are decreased (1). Since the Th2 cytokines promote antibody production by B cells, Th2 cells might play a pivotal role in the development of autoantibodies in SLE patients. However, some reports have contradicted this supposition, indicating that an imbalance toward Th1 predominance was associated with an acceleration of lupus-like autoimmune disease (2). It has also been suggested that Th1 and Th2 responses may play differential roles in the pathogenesis of lupus-associated tissue injury (3). The Th1 and Th2 cells are functionally defined by their cytokine secretion profiles, but recent studies have indicated that Th1 and Th2 cells express distinct sets of chemokine receptors and differentially respond to chemokines (4). It has been reported that some chemokine receptors, such as CCR3, CCR4, and CCR8, were preferentially expressed in Th2 cells (5–7), whereas CCR5 and CXCR3 were preferentially expressed in Th1 cells (8,9). Such a differential expression of chemokine receptors in Th1 and Th2 cells is important for the selective migration of a particular T cell subset, since the chemokines produced at the sites of inflammation play a major role in the recruitment of infiltrating cells in the pathologic lesions. These observations suggest that the differential Supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture and the Ministry of Health and Welfare of Japan. 1 Masato Yamada, MD, PhD, Hideo Yagita, PhD, Hideko Inoue, Tsuyoshi Takanashi, MD, Hironori Matsuda, Eiko Munechika, Yutaka Kanamaru, MD, PhD, Isao Shirato, MD, PhD, Yasuhiko Tomino, MD, PhD, Ko Okumura, MD, PhD, Hiroshi Hashimoto, MD, PhD: Juntendo University School of Medicine, Tokyo, Japan; 2Kouji Matushima, MD, PhD: University of Tokyo, Tokyo, Japan. Address correspondence and reprint requests to Masato Yamada, MD, Department of Rheumatology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku Tokyo 113-8421, Japan. E-mail: [email protected] Submitted for publication November 22, 2000; accepted in revised form October 12, 2001. 735 736 YAMADA ET AL Table 1. Patient profiles and characteristics of the systemic lupus erythematosus patients included in this study Patient/ sex/age Disease duration, years SLEDAI* CH50, units C3, mg/dl C4, mg/dl Organ involvement† Therapy (mg/day)‡ 1/M/29 2/F/64 3/F/19 4/F/27 5/F/13 6/F/24 7/F/49 8/F/23 9/F/38 10/F/29 11/F/16 12/F/17 13/F/44 14/F/18 15/F/22 16/F/42 17/F/31 18/F/42 19/F/15 20/F/23 10 33 3 0.3 0.2 7 1 3 20 5 0.1 4 1 0.6 7 3 14 0.2 0.2 0.1 15 14 23 3 17 9 10 19 4 2 24 20 19 5 6 12 4 8 29 26 13.0 18.5 24.2 36.9 27.0 28.4 19.4 34.5 27.2 34.8 42.2 39.6 27.1 29.4 39.0 19.5 13.6 31.7 16.2 12.8 24 29 38 67 30 67 53 42 78 34 63 83 45 77 92 32 20 59 44 35 11 5 9 25 15 18 5 15 23 10 9 21 14 6 22 15 5 19 17 5 Ly TH, Ly CNS, GN, TH, Ly Ly GN, TH Ly TH, Ly CNS, GN, Ly Ly, GN GN GN GN, Ly GN, TH CNS, TH, Ly GN GN GN Ly CNS, GN, TH, Ly Ly, GN PSL (40) PSL (30) PSL (30) None None PSL (22.5) None Beta (4) PSL (20) PSL (35) PSL (50) PSL (40), PP PSL (10) None None PSL (15) PSL (20) None PSL (30) None * SLEDAI ⫽ Systemic Lupus Erythematosus Disease Activity Index. † Ly ⫽ lymphocytopenia (⬍1,000/l); TH ⫽ thrombocytopenia; CNS ⫽ central nervous system; GN ⫽ glomerulonephritis. ‡ PSL ⫽ prednisolone; Beta ⫽ betamethasone; PP ⫽ plasmapheresis. expression of chemokine receptors may be useful for discriminating pathogenic T cells. In this study, we examined the expression of CCR4 and CCR5 on peripheral blood (PB) T cells from SLE patients and mononuclear cells infiltrating into the renal tissue of lupus nephritis patients. Our results showed that CCR4⫹ T cells were decreased in the PB but accumulated in the renal tissue in the SLE patients, suggesting the possible contribution of CCR4⫹ Th2 cells to the pathogenesis of lupus nephritis. PATIENTS AND METHODS Patients. This study comprised 19 women and 1 man with the diagnosis of SLE according to the 1982 revised criteria of the American College of Rheumatology (10). Characteristics of the patients are presented in Table 1. The mean age was 29.25 years (range 13–64). The mean duration of symptoms prior to this examination was 5.64 years (range 0.1–33 years). Disease activity was assessed using the SLE Disease Activity Index (SLEDAI) instruments (11), and the levels of CH50 (in units), C3 (in mg/dl), C4 (in mg/dl), anti-DNA antibodies (in IU/ml), and proteinurea were determined. Some of the patients were receiving corticosteroid therapy, and 1 patient (patient 12 in Table 1) received plasmapheresis 6 months prior to this study. None of the patients had any other autoimmune disease or infectious disease. Five milliliters of PB sample was obtained from every patient for this study after provision of informed consent. At the time of each examination, PB was also obtained from healthy donors as control subjects, who were matched for age and ethnicity. Monoclonal antibodies (mAb). Allophycocyanine (APC)–conjugated anti-CD4 (RPA-T4, mouse IgG1) or antiCD8 (RPA-T8, mouse IgG1) mAb, biotin-conjugated antiCD45RO (UCHL1, mouse IgG2a) mAb, phycoerythrin (PE)– conjugated anti-CCR5 (2D7, mouse IgG2a) mAb, and Texas Red–conjugated avidin were purchased from PharMingen (San Diego, CA). Anti-human CCR4 (KM2160, mouse IgG1) mAb (6) and anti-human CD4 (Leu 3a, mouse IgG1) mAb were conjugated with Alexa 488 or Alexa 594, respectively, according to the manufacturer’s instructions (Molecular Probes, Eugene, OR). Isotype-matched control mAb were also obtained from PharMingen. Immunofluorescence and flow cytometry. Immunofluorescent staining for surface markers, flow cytometry, and data analysis were performed as described previously (12). Briefly, aliquoted samples (500 l of heparinized whole blood) were stained with biotin-conjugated anti-CD45RO mAb first. After washing twice, the cells were stained with APC-conjugated anti-CD4 or anti-CD8 mAb, PE-conjugated anti-CCR5 mAb, Alexa 488–conjugated anti-CCR4 mAb, and Texas Red– avidin. After washing twice again, red blood cells were lysed using ammonium chloride solution. The remaining mononuclear cells were analyzed on a FACScan (Becton Dickinson, San Jose, CA). Immunohistochemical staining. Serial sections were prepared from formalin-fixed, paraffin-embedded renal tissues from 13 patients with lupus nephritis that was classified as World Health Organization (WHO) class IV, and stained with anti-CD4 (1F6, mouse IgG1; Nichirei, Tokyo, Japan) mAb, SELECTIVE ACCUMULATION OF CCR4⫹ T LYMPHOCYTES IN LUPUS NEPHRITIS 737 CH50, C3, C4, anti-DNA antibodies, and proteinurea) was determined by linear regression and Spearman’s rank correlation. P values less than 0.05 were considered significant. RESULTS Selective loss of CCR4ⴙ cells from CD45ROⴙ, CD4ⴙ T cells in PB from SLE patients. We first examined the surface expression of CCR4 and CCR5 on PB CD45RO⫹ (memory) CD4⫹ or CD8⫹ T cells. Four-color flow cytometric analysis of PB from 20 SLE patients and 10 healthy controls was performed. As represented in Figure 1A, the absolute number of CCR4⫹,CD4⫹ T cells in PB was significantly decreased Figure 1. A, The absolute cell numbers of CCR4⫹ T cells in peripheral blood (PB) from patients with systemic lupus erythematosus (SLE) were significantly lower than those from healthy controls. B, The absolute cell numbers of CCR5⫹ T cells in PB from SLE patients were not significantly (NS) different from those in PB from healthy controls. anti-CCR4 (KM2160) mAb, or anti-CCR5 (2D7) mAb using the avidin–biotin–peroxidase complex system (Vector, Burlingame, CA) for CD4 and the catalyzed signal amplification system (Dako, Kyoto, Japan) for CCR4 and CCR5. Human tonsil was used as a positive control and isotype-matched mouse IgG was used as a negative control. In some experiments, frozen sections were stained with Alexa 594–conjugated anti-CD4 (Leu 3a) mAb and biotin-conjugated anti-CCR4 (KM2160) mAb followed by Alexa 488–conjugated streptavidin, and observed under confocal laser microscopy. Statistical analysis. Comparison between SLE patients and healthy controls was made by the nonparametric MannWhitney U test. Correlation between the absolute number of CCR4⫹ or CCR5⫹ cells and clinical parameters (SLEDAI, Figure 2. Correlation of blood CH50 (A) and C3 (B) levels with the absolute numbers of CCR4⫹ T cells in peripheral blood from systemic lupus erythematosus patients. 738 YAMADA ET AL in the SLE patients compared with that in the healthy controls. In contrast, the absolute number of CCR5⫹, CD4⫹ T cells was not significantly different between the SLE patients and the healthy controls (Figure 1B). In CD8⫹ T cells, no significant difference between the SLE patients and the healthy controls was observed in the absolute number of CCR4⫹ or CCR5⫹ cells (data not shown). Correlation between CCR4ⴙ cell number and clinical parameters. We analyzed the correlation between the absolute number of CCR4⫹ or CCR5⫹ cells in the PB of SLE patients and clinical parameters (SLEDAI, CH50, C3, C4, anti-DNA antibodies, and proteinurea). As represented in Figures 2A and B, positive correlations were observed between the absolute number of CCR4⫹ cells in PB and the levels of CH50 (P ⬍ 0.05) or C3 (P ⬍ 0.02). No significant correlation was observed between the CCR4⫹ T cell number and the other clinical parameters or between the CCR5⫹ cell number and any of the clinical parameters analyzed. Accumulation of CCR4ⴙ,CD4ⴙ T cells in the renal tissue of patients with lupus nephritis. We examined the expression of CCR4 and CCR5 on the infiltrating CD4⫹ T cells in the renal tissue of patients with lupus nephritis whose disease was classified as WHO class IV. Biopsy samples from 13 patients with lupus nephritis were included in this study. As represented in Figures 3A–D, a large part of the infiltrating mononuclear cells were strongly stained with anti-CD4 and anti-CCR4 mAb, but only weakly with anti-CCR5 mAb. A double-immunofluorescent staining showed that CCR4 was exclusively coexpressed in CD4⫹ T cells (Figure 3E). In 10 of the 13 renal tissues examined, a similar high expression of CCR4, but not CCR5, in infiltrating CD4⫹ T cells was observed (data not shown). DISCUSSION In the present study, we demonstrated that 1) the absolute number of CCR4⫹,CD4⫹ T cells in PB was greatly decreased in the SLE patients compared with the healthy controls, while the absolute number of CCR5⫹, CD4⫹ T cells was not significantly different between the SLE patients and the controls; 2) the absolute number of CCR4⫹ T cells in PB was positively correlated with the serum C3 and CH50 levels in the SLE patients; and 3) infiltrating CD4⫹ T cells in the renal tissues of the patients with lupus nephritis mostly expressed CCR4, but not CCR5. Recent studies have indicated that CCR5 was preferentially expressed on Th1 cells (8), while CCR4 was preferentially expressed on Th2 cells (6). Although D’Ambrosio et al reported that CCR4 was expressed not only on Th2 cells, but also on activated Th1 cells (13), we could not detect interferon-␥ (IFN␥)–producing cells but could detect interleukin-4 (IL-4)–, IL-5–, and IL-13– producing cells in the CCR4⫹,CD4⫹ T cell population from the PB of normal individuals (Yamada M, et al: unpublished observations). This indicated that CCR4 is a useful marker for Th2 cells in PB CD4⫹ T cells. The actual contribution of the Th1:Th2 imbalance to the pathogenesis of SLE is still a matter of debate. Funauchi et al showed that Th1-like cells producing IL-2 and IFN␥ were decreased and Th2-like cells producing IL-4 and IL-10 were increased in the PB of SLE patients (1). Akahoshi et al showed, by measurement of intracellular IL-4 and IFN␥, a predominance of Th1 cells in the SLE patients with WHO class IV lupus nephritis, but no predominance of Th1 or Th2 cells in the patients without nephritis (2). In this study, we analyzed CCR4⫹ or CCR5⫹ T cells in the PB of 13 patients with nephritis and 7 patients without nephritis, but no significant difference between these 2 groups was observed. Although SLE is a systemic disease, the tissues damaged by SLE vary among individual patients, and it is possible that the circulating lymphocytes in PB and the infiltrating lymphocytes in the target organs may represent different T cell subsets. We showed here that CCR4⫹,CD4⫹ T cells were selectively decreased in the PB of SLE patients, and the infiltrating CD4⫹ T cells in the renal tissue of the SLE patients were mostly CCR4⫹. This suggests that CCR4⫹,CD4⫹ T cells in PB might preferentially migrate into the renal tissue of the patients with lupus nephritis. Given that CCR4 is preferentially expressed on Th2 cells, Th2 cells might be selectively recruited from the circulation to the renal tissue in the lupus nephritis patients. There is a considerable number of observations in SLE that circulating DNA–anti-DNA autoantibody immune complexes are deposited in the glomeruli, in association with C3, and this is involved in the pathogenesis of lupus nephritis. Moreover, it has been known that the activity of lupus nephritis was correlated with decreased C3 and CH50 levels in the sera of SLE patients. In keeping with this notion, the correlation between the serum C3 and CH50 levels and the absolute number of CCR4⫹,CD4⫹ T cells in the PB of SLE patients suggests that the decrease in CCR4⫹, CD4⫹ T SELECTIVE ACCUMULATION OF CCR4⫹ T LYMPHOCYTES IN LUPUS NEPHRITIS 739 Figure 3. Accumulation of CCR4⫹,CD4⫹ T cells in renal tissues of lupus nephritis patients. Serial paraffin sections of renal tissue from a lupus nephritis (World Health Organization [WHO] class IV) patient were stained with anti-CD4 monoclonal antibodies (mAb) (A), anti-CCR4 mAb (B), anti-CCR5 mAb (C), or control mouse IgG (D) by the avidin–biotin–peroxidase complex method (A) or the catalyzed signal amplification method (B–D). A large part of infiltrating mononuclear cells showed strong staining with anti-CD4 and anti-CCR4 mAb but only weak staining with anti-CCR5 mAb. The staining of renal duct epithelial cells observed in B and C was nonspecific, since it was also observed with control mouse IgG (D). E, Frozen section of renal tissue from a lupus nephritis (WHO class IV) patient was immunofluorescently double-stained with anti-CCR4 (green) and anti-CD4 (red) mAb. Note that CCR4 was exclusively coexpressed in CD4⫹ T cells. cells in the PB may be a useful marker for the activity of SLE. It is well known that the renal dysfunction of lupus nephritis is mainly caused by glomerulus damage. Leukocyte infiltration is an indicator of the disease and implicated in the progression to chronic renal failure. Infiltration of leukocytes into the tissue is regulated by the local expression of adhesion molecules in concert with the release of chemokines. In this study, we found a prominent expression of CCR4 on the infiltrating CD4⫹ T cells in the renal tissues of SLE patients. This suggests that some chemokines specifically attracting 740 YAMADA ET AL CCR4⫹ cells, such as thymus- and activation-regulated chemokine and macrophage-derived chemokine, are produced in the renal tissue of lupus nephritis patients. Further studies are needed to identify the chemokine responsible for the selective recruitment of CCR4⫹, CD4⫹ T cells and the cells producing this chemokine in the lupus nephritis lesions. Our present observations suggest that CCR4⫹, CD4⫹ T cells preferentially migrated into the renal tissue of the patients with lupus nephritis and thus the maldistribution of CCR4⫹ T cells might be involved in the pathogenesis and progression of lupus nephritis. This may provide a new molecular target for therapeutic interventions in lupus nephritis. ACKNOWLEDGMENTS We thank Drs. Sachiko Hirose, Masaaki Abe, Takafumi Uchida, Hajime Sudo, Akiko Komiya, Hirokazu Yamada, and Koichi Hirai for helpful suggestions. REFERENCES 1. Funauchi M, Ikoma S, Enomoto H, Horiuchi A. Decreased Th1-like and increased Th2-like cells in systemic lupus erythematosus. Scand J Rheumatol 1998;27:219–24. 2. Akahoshi M, Nakashima H, Tanaka Y, Kohsaka T, Nagano S, Ohgami E, et al. Th1/Th2 balance of peripheral T helper cells in systemic lupus erythematosus. Arthritis Rheum 1999;42:1644–8. 3. Klinman DM, Steinberg AD. Inquiry into murine and human lupus. Immunol Rev 1995;144:157–93. 4. 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