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Immunology and Molecular Genetics

  • Katsuhiko Ishihara, M.D., Ph.D. Professor

Main Areas and Themes of Research

Autoimmune diseases develop through breakdown of self-tolerance resulted from immune deregulation under the combined influences of genetic factors and environmental factors. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by the synovial hyperplasia leading to destruction of the bones and joints. The incidence of RA is approximately 1 % of the population. Correlation of the incidence with certain HLA-DR haplotypes and the presence of autoantibodies, such as rheumatoid factor and anti-cyclic citrullinated peptide antibody strongly suggest the involvement of the deregulated immune system in its etiology and/or pathophysiology. Although, various molecules, such as type II collagen, gp39, citrullinated peptides, glucose-6-phosphoisomerase, have been reported as potential, pathogenetic autoantigens, their involvements explained only a portion of RA cases. Among the T-cell cytokine systems, Th17, a novel helper T-cell subset producing IL-17, has become a topic as a pivotal player in the local inflammation driven by acquired immunity. Pathologically, the joints are infiltrated with T cells, B cells, and plasma cells, characteristic cells for chronic inflammation. The cartilage and bone are destroyed by the invasion of pannus formed with proliferating fibroblast-like synoviocyte (FLS) and multi-nucleated osteoclasts. Cytokines increased in the joints, such as bFGF, PDGF, TGFβ, TNFα, and IL-1β are considered to be responsible for the hyperplasia of FLS. Activated FLS produce TNFα, IL-1β, IL-6, chemokines, and matrix metalloproteinases, establishing the chronic and destructive inflammatory circuit. The critical roles of these inflammatory cytokines have been evidenced by the effectiveness of cytokine-blockade therapies using anti-TNFα or anti-IL-6 receptor antibodies for the treatment of RA. However, details of the pathophysiology of RA are not completely understood, yet.

To clarify the etiology and pathophysiology of rheumatoid arthritis, we are planning the integrated analyses of human RA and a murine autoimmune arthritis model, gp130F759; a knock-in mouse having a mutant gp130. The analyses include two main themes;

1) Molecular and genetic characterization of human FLS from RA.

To identify the cell intrinsic abnormalities of FLS from RA (RA-FLS), we established transformed cell lines from the synovium of RA or OA cases. Characterization of FLS from two types of arthritis revealed no significant differences in surface molecules, growth, tyrosine phosphorylation of proteins, and expression of genes related to inflammation (IL-1β, IL-6, MMP-1, MMP-3, etc.). Since expression levels of these genes are variable (ranges exceeding 1,000 folds) among FLS lines from both types of arthritis, suggesting there is a very wide diversity of properties on FLS regardless the disease. Thus, we tentatively categorized them into two subtypes, resting(r) and active(a), depending on the expression levels of IL-1β, and MMP-1. Then we performed micro DNA assay to obtain the gene expression profile between four representative cell lines, i.e. r-OA-FLS, a-OA-FLS, r-RA-FLS, a-RA-FLS, and obtained ten gene clusters. Through the efforts to search the candidates for disease-related genes, we found two of those related to the pathophysiology of RA. The one is AID (activation-induced cytidine deaminase) and another is A20/ABIN families.

A novel role for AID (activation-induced cytidine deaminase) in the acquisition of the tumor-like characteristics of RA-FLS

AID is known to have essential roles in class switch recombination and somatic hypermutation of variable regions in immunoglobulin genes in B-lymphocytes. Recently several reports showed ectopic expression of AID in the organs with inflammation, such as the colon, and the liver, and possible tumorigenic roles by inducing mutations in p53, a well-known tumor suppressor gene and one of targets for AID-induced mutations. We found that the transcription of AID was detected in more than half of RA-FLS (5 out of 9 lines) but not in OA-FLS, and induced by TNFα stimulation in both OA-FLS and RA-FLS. Interestingly, the frequencies of mutation in p53 were significantly increased in AID+-RA-FLS compared with AID--RA-FLS and OA--FLS. Most mutations localize within the DNA binding domain and dominant base substitution was transition type, which is characteristic of AID-dependent mutations. Furthermore, immunohistochemical analysis revealed the production of AID in the fibroblasts in the synovium of RA. These results suggest the novel pathophysiologic mechanism in RA; inflammation in the synovium of RA induces the ectopic expression of AID, which induces mutations in p53, leading to generation of tumor-like FLS. (Igarashi H et.al. TP53 mutations coincide with the ectopic expression of activation-induced cytidine deaminase in the fibroblast-like synoviocytes derived from a fraction of patients with rheumatoid arthritis. (Clinical and experimental immunology, 2010, 161:71-80). We extended this study and identified p53R248Q mutation provides the apoptosis-resistance against oxidative stress to MH7A RA-FLS cell line by suppressing the expression of pro-apoptotic molecule p53AIP1(p53-regulated Apoptosis- Inducing Protein 1).

A novel inflammatory pathway mediated by NF-кB inhibitors, A20 and ABIN families in RA-FLS

Among a cluster of genes down-regulated in a subtype of RA-FLS, we picked up TNFAIP3 (A20) interacting protein 1 (TNIP-1/ABIN-1), a negative regulator for activation of NF-кB, because aberrant down-regulation of ABIN-1 could cause the development of the activated, pro-inflammatory RA-FLS subtype with increased transcription of inflammatory cytokines by deregulated activation of NF-кB. However, the analysis of TNFα-high responder RA-FLS revealed unexpected character; the enhanced expression of both pro-inflammatory cytokines (TNFα, IL-1β and IL-6) and A20-related NF-кB inhibitors (A20, ABIN-1 and ABIN-3) in the same kinetics upon TNFα stimulation. To verify the emerging hypothesis that A20 and ABINs might be a positive regulator for the pro-inflammatory cytokines, we performed the forced expression of each NF-кB inhibitors in RA-FLS. Although single transfection of each NF-кB inhibitor suppressed the canonical NF-кB pathway as reported previously, it rather promoted the induction of pro-inflammatory cytokines independent of TNFα stimulation. This finding suggests the presence of alternative pathways toward inflammation mediated by NF-кB inhibitors (Igarashi H et.al. A pro-inflammatory role for A20 and ABIN family proteins in human fibroblast-like synoviocytes in rheumatoid arthritis. Immunology Letters, 2012, 141:246-253). We are on investigating the molecular mechanism of this new inflammatory signals by searching associated molecules with A20/ABINs and genetic changes when these NF-кB inhibitors over-expressed in RA-FLS.

2) Profiling the spacio-temporal sequence of arthritis in gp130F759.
Basic features of gp130F759

gp130 is a common, signal transducing subunit for IL-6 family cytokines. IL-6 family cytokines include IL-6, IL-11, IL-27, Leukemia-inhibitory factor, Oncostatin M, Ciliary Neurotropic Factor, Cardiotrophin-1, and NNT/BSF-3/CLC, which regulate variety of biological functions, such as the development of hematopoietic as well as neuronal systems, bone homeostasis, acute-phase responses, inflammation, and immune responses. The ligand binding to gp130 leads to its dimerization, activation of tyrosine kinase Jaks, and tyrosine phosphorylation of its cytoplasmic tail, which is followed by operation of 2 major signaling pathways dependent on the sites of tyrosine-phosphrylation; YXXQ/STAT3 pathway and Y759/PHP2/Gab/MAPK pathway. To know the roles for these 2 signaling pathways in vivo, the series of knock-in mouse lines are generated. In order to selectively block each signaling pathways, mutant cDNAs of gp130 whose tyrosine (Y) residues are converted to phenylalanine (F) were knocked in (Immunity, 12: 95-105, 2000). Among them, gp130F759 mice having a Y759F mutation apparently grow normal but spontaneously developed autoimmune arthritis like human RA (J Exp Med 196: 979-990, 2002).

Currently we are focusing to analyze the natural course of the arthritis in terms of local and systemic immunity.

Educational Features

We have conducted the course of the Immunology and Allergy block with 23 lectures, and 2 days’ practice hours for the second grade medical students. Main features of this course are as follows;

  1. Essential and important knowledge of immunology for clinicians based on the model core curriculum.

    • i ) Important organs, cells, molecules and genes of the immune system (T, B cells, antigen presenting cells, antibody, antigen receptor, MHC).
    • ii ) How effective biological responses are induced by the immune system, as a defense mechanism, to fight against each pathogen having various life cycle.
  2. The basic concepts in modern immunology ( innate and acquired immunity, the clonal selection theory, variability of antigen recognition by lymphocytes, immunological tolerance, immunological memory ). Episodes of breakthrough, latest topics, and relation to clinical practice are also introduced to evoke interest of students.
  3. The etiology, pathophysiology, and the rationale for therapies for major immune diseases.
  4. In the practice hours, through the actual experience of flow cytometry analysis and ELISA, the students learn the basis for immunological techniques used in clinical fields as well as in basic research. This facilitates the understanding of immunology learned in the lectures or literatures.