European Journal of Immunology

Structurally unrelated, FK‐506 and cyclosporin (CsA) bind to and inhibit the action of distinct cytoplasmic receptors, FK‐506‐binding protein (FKBP) and cyclophilin (CyP), respectively. These receptors, termed immunophilins, share no sequence similarity, and yet both have been demonstrated to be capable of catalyzing the cis‐trans isomerization of peptidyl‐prolyl bonds (rotamase activity). Because FK‐506 and CsA bind to different intracellular target structures, we investigated the spectrum of action of FK‐506, in comparison to CsA, on T cell activation. We have shown that FK‐506, like CsA, is able to inhibit T cell activation mediated not only by the T cell receptor‐CD3 complex, but also via another surface molecule, CD2. T cell proliferation, stimulation of interleukin 2 production, and induction of apoptosis were all sensitive to inhibition by both FK‐506 and CsA. With each parameter of activation, FK‐506 is approximately 10‐100‐fold more effective than CsA. In contrast, FK‐506 did not affect T cell proliferation induced by anti‐CD28 monoclonal antibody in the presence of phorbol 12‐myristate 13‐acetate. This CD28 pathway, however, was inhibitable by a structural homolog of FK‐506, rapamycin, demonstrating that the mechanism of action of FK‐506 has specificity. These data suggest that immunophilins or the complex of drug coupled to immunophilin (i.e. FK‐506/FKBP, CsA/CyP) are involved in and regulate selective pathways of T cell stimulation.

Aberrant formation of neutrophil extracellular traps (NETs) is a key feature in rheumatoid arthritis (RA) and plays a pivotal role in disease pathogenesis. However, the mechanism through which NETs shape the autoimmune response in RA remains elusive. In this study, we demonstrate that inhibition of peptidylarginine deiminases activity in collagen‐induced arthritis (CIA) mouse model significantly reduces NET formation, attenuates clinical disease activity, and prevents joint destruction. Importantly, peptidylarginine deiminase 4 blocking markedly reduces the frequency of collagen‐specific IFN‐γ‐producing T helper 1 (Th1) cells in the draining lymph nodes of immunized mice. Exposure of dendritic cells (DCs) to CIA‐derived NETs induces DC maturation characterized by significant upregulation of costimulatory molecules, as well as elevated secretion of IL‐6. Moreover, CIA‐NET‐treated DCs promote the induction of antigen‐specific Th1 cells in vitro. Finally, NETs from RA patients show an increased potential to induce the maturation of DCs from healthy individuals, corroborating the findings obtained in CIA mouse model. Collectively, our findings delineate an important role of NETs in the induction and expansion of Th1 pathogenic cells in CIA through maturation of DCs and reveal a novel role of NETs in shaping the RA‐autoimmune response that could be exploited therapeutically.

Biểu hiện của interleukin (IL)‐1β, IL‐6 và các thụ thể tương ứng của chúng đã được nghiên cứu trong não chuột trước và trong vòng 24 giờ sau khi bị chấn thương. Các bản sao RNA thông điệp của bốn gen này đã được phát hiện bằng phương pháp lai phân tử in situ ở các cấu trúc khác nhau của não không bị tổn thương. Mẫu phân bố rất tương tự nhau đối với IL‐1β, IL‐6 và thụ thể IL‐6 (IL‐6R). Biểu hiện của IL‐1R được ghi nhận là rộng rãi hơn. Chỉ trong vài giờ sau khi chấn thương, sự gia tăng biểu hiện của IL‐1β, và sau đó là IL‐6 đã được xác nhận. Biểu hiện của IL‐1R và IL‐6R cũng đã tăng lên. Sự biểu hiện này là hai bên và không chỉ giới hạn ở khu vực bị tổn thương. Trong vòng 24 giờ, tất cả các mẫu ISH đã trở lại bình thường. Dữ liệu phân tử đã được xác nhận bởi dữ liệu protein. Thật vậy, sự phân bố của IL‐6 (được phát hiện bằng phương pháp nhuộm miễn dịch) đã đồng nhất với các mẫu ISH cho IL‐6. Hơn nữa, dịch ngoại bào đã được thu thập bằng phương pháp vi thẩm phân tại vị trí tổn thương trong 12 giờ và các phần thu được sau đó đã được kiểm tra sự hiện diện của IL‐1 và IL‐6 có hoạt tính sinh học. Sự gia tăng của mức IL‐1 và sau đó là IL‐6 đã được phát hiện. Sự biểu hiện gia tăng nhanh chóng và đồng thời của IL‐1β, IL‐6 và các thụ thể của chúng sau chấn thương nhấn mạnh vai trò có thể của chúng trong các cơ chế viêm, bao gồm cả trong não, trước khi bất kỳ sự tuyển mộ tế bào viêm từ các vùng thần kinh và không thần kinh xa xôi.

Interleukin 6 (IL6) là một trong những cytokine chính liên quan đến viêm. Mức độ IL 6 trong huyết thanh hoặc mô bị tăng cao đã được báo cáo xảy ra trong một số bệnh ở người, bao gồm các bệnh nhiễm trùng hệ thần kinh trung ương (CNS), nhưng không phải trong viêm CNS không nhiễm trùng, ví dụ như bệnh xơ cứng đa dịch. Trong khi nghiên cứu bệnh viêm não tự miễn thí nghiệm (EAE) như một mô hình động vật cho viêm tự miễn ở CNS, chúng tôi phát hiện nồng độ IL 6 tăng cao trong CNS của chuột mắc phải một dạng bệnh nguy hiểm. Mức độ IL 6 trong lách và huyết thanh không tăng đáng kể. Những phát hiện này cho thấy sự sản xuất IL6 tại chỗ trong CNS trong quá trình EAE, và đại diện cho sự chứng minh đầu tiên về sản xuất IL6 trong bệnh viêm CNS không nhiễm trùng.

T cell receptor (TcR) α and β nucleotide sequences involved in the human autoreactivity to myelin basic protein (MBP) were studied by screening cDNA libraries derived from 11 independent T lymphocyte clones (TCC) established from multiple sclerosis patients and healthy donors. The TCC with defined MBP peptide specificity and HLA‐DR restriction expressed multiple TcR. Even TCC recognizing the same human MBP peptide [amino acids (aa) 139–153] in identical or very similar HLA‐DR context expressed diverse TcR. Two TCC which recognized peptide aa 139–153 equally well in the context of both HLA‐DR2a and ‐DR1 molecules used distinct TcR α but identical β chains.

The knowledge of TcR β and TcR α chain sequences of human MBP‐specific T cells will allow studies correlating structure and function of TcR and their targets in MBP autoreactivity. This may have an impact on the development of immunotherapies in multiple sclerosis.

Plasma cells secreting IgG, M, and A abound in the synovium of patients with rheumatoid arthritis, yet their immunoglobulin repertoire and clonal relationship remain to be elucidated. Locally synthesized immunoglobulins probably contribute to the chronic joint inflammatory processes which are characteristic of these patients. To determine whether B lymphocyte proliferation contributes to the synovial plasma cell infiltrate, the clonality of IgG mRNA in individual synovial biopsies from an actively inflamed joint of patients with rheumatoid arthritis was investigated by a combination of cDNA length analysis and DNA sequencing. Particular sizes of immunoglobulin cDNA, detectable in subclasses 1, 3, or 4, were expressed in most synovial biopsies from one patient, suggesting their origin from expanded clones present in each biopsy. To prove a clonal relationship between recurrent cDNA lengths, immunoglobulin cDNA was cloned from three regions of synovium in three patients. The sequence of clones with a recurrent cDNA length was determined. An IgG3 clone found in most synovial biopsies of one patient was encoded by an unmutated copy of the V_H1 gene, DP7. In contrast, IgG3 clones encoded by mutated versions of the V_H3 gene DP49 or the V_H4 gene DP63 were expanded in the other two patients. Different somatic mutants of these clones were isolated from different sites in these patients. The ratio of replacement/silent somatic mutations in these two families of clones suggests that the selective clonal expansion in the synovium of patients with rheumatoid arthritis is due to an antigen‐driven immune response.

Presentation of alloantigens by host cells has been examined in vivo by means of a murine cell transfer system. Primary (1°) hosts were activated by the i.p. administration of allogeneic spleen cells and their spleen or peritoneal cells were transferred into syngeneic secondary (2°) hosts 3 days later. Sensitization of 2° hosts was assessed by their ability to reject donor strain skin grafts prematurely. The transferred cells were routinely depleted of T lymphocytes. We show that (a) 5 × 10⁷ spleen and 3 × 10⁶ peritoneal cells consistently caused marked accelerated graft rejection; (b) this effect was antigen specific and observable in all strain combinations studied; (c) it was caused by the active sensitization of 2° hosts, but not by contaminating donor strain cells; (d) the cells involved were plastic adherent and viability was not a requirement; and (e) both class I and II, but not minor, histocompatibility antigens played a role in this model. We conclude that presentation of alloantigens by host antigen‐presenting cells can be a potent route of allosensitization.

Cytokines have been suggested to act as intermediates between the immune and the central nervous system, but little is known about the type of cells synthesizing them in the brain. We have immortalized with oncogenic retroviruses primary brain cell cultures from mouse embryos and have generated clones of microglial cells that have been characterized. Three of the clones studied produce interleukin 1 (IL 1), IL 6 and tumor necrosis factor‐α as assessed by biological assays and by Northern blot analysis. Our data raise the question on the role of these cytokines in the brain and suggest that early resident microglial cells might play an important role in developmental processes and in the adult brain.

Myeloma is one of the interleukin (IL)‐6‐related diseases to which abnormal expression of IL‐6 has been reported to be linked. We examined the in vivo inhibitory effect of anti‐human IL‐6 receptor (IL‐6R) antibody on human myeloma cell growth in mice. SCID mice were subcutaneously inoculated with solid tumor of the myeloma cell line S6B45 in which human IL‐6 was acting as an autocrine growth factor. Ten intraperitoneal administrations of 100 μg of the anti‐human IL‐6R antibody PM1 at 48‐h intervals strongly inhibited the growth of S6B45 cells when the administration started 24 h after tumor inoculation. The tumor growth inhibition in vivo was also observed by administration of the anti‐human IL‐6 antibody MH166 using the same procedure as for PM1. The inhibitory effect of PM1 was not significant when the administration started 5 or more days after tumor inoculation. This work indicates that anti‐human IL‐6R antibody, as well as anti‐human IL‐6 antibody inhibits human myeloma growth in vivo, and provides an animal model for testing the therapeutic value of agents such as antibodies to human IL‐6, IL‐6R and gp130, an IL‐6R‐associated signal transducer, in the treatment of human myelomas.

The characteristics of soluble interleukin‐6 receptor (sIL‐6R) in murine sera were examined. To investigate a relationship between serum sIL‐6R level and autoimmune diseases, quantitative analysis of serum sIL‐6R in MRL/lpr mice was performed by an enzyme‐linked immunosorbent assay. The serum sIL‐6R level in MRL/lpr mice of both sexes was below the detection limit (< 1.0 ng/ml) at 8 weeks of age, but it increased in accordance with age and reached 42 ± 9.3 ng/ml in female and 31 ± 13 ng/ml in male mice at 30 weeks of age. In MRL/+ mice, although an age‐associated increase in serum sIL‐6R level was observed, it was much less extensive than that in MRL/lpr mice. Elevated serum sIL‐6R level at the age of 30 weeks was observed in female and male (NZB × NZW)F1 mice (32 ± 10 ng/ml and 17 ± 5.0 ng/ml, respectively), and male BXSB/Mpj Yaa mice (42 ± 18 ng/ml), suggesting that elevated serum sIL‐6R in aged mice is one of the characteristics of autoimmune‐prone mice. Quantitative analysis of serum IL‐6 in MRL/lpr revealed that the serum sIL‐6R level correlated well with the serum IL‐6 level. We also showed that sIL‐6R in the sera from MRL/lpr mice could mediate the IL‐6 functions through the IL‐6 signal‐transducing receptor component gpl30, suggesting that elevated production of sIL‐6R may partly contribute to development of autoimmune disease in MRL/lpr mice.