Makoto Katagiri

Association between HLA and Japanese patients with rheumatoid arthritis.

Japanese patients with rheumatoid arthritis (RA) were observed to have a statistical association with HLA-DR4, MT3. Strong association between the clinical severity of RA and HLA was also observed. Male patients had a stronger association with HLA than female patients. Males are more resistant to RA than females. This suggested that the threshold of liability for RA is higher in males than in females. Japanese patients with RA with systemic vasculitis were negative for HLA-Bw44 and had antilymphocytotoxic autoantibody, indicating that RA with systemic vasculitis is different in etiology from RA without systemic vasculitis.

Evidence for two B-cell alloantigen loci in theHLA-D Region

Antigenic determinants recognizable by human antisera (Hon 7 and 2075abs sera) were found in a partially purified antigen preparation obtained from an HLA-D and -DR homozygous cell line (EBV-Wa). Sequential coprecipition tests showed that two determinants detectable with Hon 7 and 2075abs sera (Hon 7 and 2075abs determinants) were present on different molecules. These two antigenic determinants were shown to be allotypic and were expressed predominantly in the B-cell-rich fraction. Family studies showed that both antigenic determinants segregated concordantly with respectiveHLA haplotypes. In the population study, the 2075abs and Hon 7 determinants were shown to be in strong linkage disequilibrium and the 2075abs determinant perfectly correlated with the HLA-DRw4 specificity. The results indicate that the Hon 7 determinant is coded for by a gene distinct from alleles at theHLA-DR locus. Furthermore, the locus (Hon 7) coding for the Hon 7 determinant is suggested to be very closely linked with theHLA-DR locus.

Tyrosinase epitope recognized by an HLA-DR-restricted T-cell line from a Vogt-Koyanagi-Harada disease patient

Abstract Human T-cell-mediated autoimmune diseases are often genetically linked to particular alleles of HLA class II genes. Vogt-Koyanagi-Harada’s (VKH) disease, which is regarded as an autoimmune disorder in multiple organs containing melanocytes, has been found to be associated with HLA-DR4 (DRB1*0405) and HLA-DR53 (DRB4*0101). Tyrosinase is a melanoma antigen (Ag) expressed by normal melanocytes as well as melanoma cells against which responses by autologous T cells have been detected. We established a T-cell line from the peripheral blood of a patient with VKH disease which responded to synthetic peptides corresponding to tyrosinase. The T-cell line was generated which recognized the tyrosinase p188 – 208 peptide when presented by the HLA-DR4 (DRB1*0405) molecule on the surface of HLA class II-expressing L-cell transfectants. The minimal antigenic peptide which induced T-cell responses was an 11-amino-acid sequence and located at tyrosinase p193 – 203 (E-I-W-R-D-I-D-F-A-H-E). This peptide contained the DRB1*0405-binding peptide motif (hydrophobic residues (Y, F, W) at position 1 as an anchor residue, and negatively charged residues (D, E) at position 9), which corresponded to the W at p195 and the D at p203. These observations demonstrate that tyrosinase peptides are immunogenic, and may be a candidate for an autoantigen in VKH disease, suggesting that probing the T-cell responses against synthetic peptides is a productive approach for identifying the autoantigenic peptides associated with autoimmune diseases including VKH disease.

The Role of the DAP12 Signal in Mouse Myeloid Differentiation

DAP12 is a recently cloned, immunoreceptor tyrosine-based activation motif-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. Previous reports showed that the DAP12 message could be detected not only in NK cells but also in granulocytes, monocytes, dendritic cells, and macrophages. In this study we found a significant level of DAP12 protein expression in macrophage-related cell lines and organs. Additionally, we observed increased expression of DAP12 after LPS-induced differentiation of M1 cells into macrophages. To examine the role of DAP12 in the myeloid cell lineage, we established M1 FLAG-DAP12 transfectants (FDAP-M1) and demonstrated the marked morphological changes in FDAP-M1 cells caused by signaling through DAP12. Cell surface phenotypic analysis showed up-regulation of macrophage markers CD11b, 2.4G2, and adhesion molecule B7-2. Additionally, after stimulation through DAP12, phosphorylated FLAG -DAP12 could be immunoprecipitated using anti-phosphotyrosine mAbs. Collectively, these findings indicate that direct DAP12 signaling has an important role in macrophage differentiation.

Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules

DAP12 (also known as KARAP) is a novel ITAM-bearing transmembrane adapter molecule that is expressed on the cell surface of natural killer cells, monocytes, dendritic cells, and macrophages. Several myeloid cell-specific DAP12-associating receptors, such as TREM receptor family, SIRP-beta1, and MDL-1 have been identified. The in vivo function of DAP12 and its associating molecules in inflammation has remained primarily unknown. To investigate DAP12 signaling during chronic inflammation, we constructed two adenoviral gene transfer vectors to express FLAG/DAP12 (Ad-FDAP12) and the extracellular domain of mouse TREM-1 and the Fc portion of human IgG1 (Ad-TREM-1 Ig), respectively, and observed their modulatory activities in a mouse model of hepatic granulomatous inflammation elicited by zymosan A. Mice were injected with zymosan A intravenously and 24 hours after zymosan A, they were injected with Ad-FDAP12 or Ad-TREM-1 Ig. Zymosan A-induced hepatic granuloma formation peaked at day 7 and markedly declined by day 10. Although adenoviral-mediated DAP12 gene transfer did not enhance granuloma formation by day 7, it sustained and enhanced granuloma formation beyond day 7. However, an anti-FLAG monoclonal antibody used to potentiate the signaling of adenoviral-derived DAP12, enhanced granuloma formation at day 7. In sharp contrast to the effect by Ad-FDAP12, transgene expression in the liver of soluble form of extracellular domain of TREM-1 as an antagonist of DAP12 signaling, remarkably inhibited zymosan A-induced granuloma formation at all time points examined. Our findings thus suggest that both DAP12 and TREM-1 are involved in the development of granulomatous responses in the liver.

Peptide motifs of HLA-DR4/DR53 (DRB1*0405/DRB4*0101) molecules

HLA-DR4(DRBI*0405) is frequently observed among Japanese, Koreans, Singapore Chinese, Sardinians, and Highlanders in Papua New Guinea. It has been shown that HLA-DR4(DRBI*0405) is positively associated with rheumatoid arthritis (Tsuchiya et al. 1991) and VogtKoyanagi-Haradas syndrome (Ohno 1992). The elucidation of the characteristics of naturally processed peptides bound to HLA-DR4(DRBl*0405) molecules may help in understanding the pathogenesis of these diseases. Peptides were isolated and sequenced from HLA-DR4/ DR53 (DRBI*0405/DRB4*0101) molecules according to the method used by Van Bleek and Natbenson (1990), with minor modifications. Briefly, HLA-DR molecules were solubilized by Nonidet-P40 from 3 x 1010 cells of EBVWa cell line (homozygous for DR4(DRBI*0405) and DR53(DRB4*0101); Katagiri et al. 1979) and immunopurified by L243 antibody specific for HLA-DR (Deboreh et al. 1981). The isolated HLA-DR molecules were treated with 10% acetic acid at 70 ° C for 15 min and the released peptides were separated from HLA-DR molecules by filtration through Centricon 10 (Amicon, Danvers, MA). They were fractionated by reverse phase high pressure liquid chromatography (RP-HPLC), using C2/C18 RPC column (Pharmacia, Uppsala, Sweden). The amino acid sequence of each peptide peak was determined by automated Edman microsequencing (477A; Applied Biosysterns, Foster City, CA). The source proteins of peptides were identified by searching the PRF-SEQDB and the GenBank databases, using the JOIS-F sequence search system. Nineteen peptides were identified, and their amino acid sequences and source proteins were determined (Table 1). Table 2 summarizes the core sequences of eluted peptides. The sequences contain conserved Tyr, which has been shown to be an anchor residue of DR binding peptides

Successful treatment of immunoblastic lymphadenopathy-like T-cell lymphoma with cyclosporin A.

Immunoblastic lymphadenopathy (IBL)-like T-cell lymphoma is considered to belong to peripheral T-cell lymphoma. Its prognosis is grave and effective treatments have not been established. Recently, we gave oral cyclosporin A (CsA) to a patient with IBL-like T-cell lymphoma, and succeeded in achieving dramatic remission. In this case, serum levels of interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF alpha) were elevated and decreased or returned to normal after achieving remission. Since CsA is a potent suppressor of the immune system and most notably T-cells, the immunosuppression of T-cell function might have played an important role in achieving remission in this case, although the precise mechanism still remains to be elucidated. The present case indicates that administration of CsA may be a very effective and safe selection of therapy for IBL-like T-cell lymphoma, as well as analogous disorders such as IBL and angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), thereby will contribute to improving the prognosis of patients with these diseases.

ANALYSIS OF ANCHOR RESIDUES IN A NATURALLY PROCESSED HLA-DR53 LIGAND

The peptide motif of the HLA-DR53 (DRB4*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Haradas syndrome, was determined by peptide binding assay using human L plastin p581–595 peptide and its substituted analogues. L plastin p581–595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1*0901/DRB4*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylate peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. binding peptides in combination with functional analyses, by alignment of sequenced endogeneous peptides, and by the use of an M13 display library (Rammensee et al. 1995; Hammer et al. 1993, 1992). No sequence information has been reported for naturally occurring HLA-DR53 (DRB4*0101)-associated peptides partly because their expression on the cell surface is relatively low for sequencing endogeneous self-peptides (Kinouchi et al. 1995). It has been shown that HLA-DR53 is positively associated with Vogt-Koyanagi-Haradas Syndrome in Japanese subjects (Moriuchi et al. 1979). The identification of a peptide motif for HLA-DR53 may help in understanding the mechanisms of this disease. We have previously reported that naturally processed peptides bound to HLA-DR9/DR53 molecules (Futaki et al. 1995). In this report, we determined the peptide which could bind to the HLA-DR53 molecule and we identified a putative HLA-DR53-specific binding motif by a peptide binding assay using L-cell transfectants expressing HLA-DR molecules.

EXON-INTRON ORGANIZATION OF XENOPUS MHC CLASS II BETA CHAIN GENES

The ampibian Xenopus laevis is the most primative vertebrate in which the major histocompatibility complex (MHC) has been defined at the biochemical, functional, and molecular genetic levels. We previously described the isolation and characterization of cDNA clones encoding X. laevis MHC class II β chains. In the present study, genomic clones encoding class II β chains were isolated from X. laevis homozygous for the MHC f haplotype. Three class II β chain genes, designed Xela-DAB, Xela-DBB, and Xela-DCB, were identified. Seqeunce analysis of these genes showed that Xela-DBB and Xela-DCB corresponding to the previously characterized cDNA clones F3 and F8, respectively, whereas Xela-DAB encodes a third, hitherti unidentified class II β chain of the MHC f haplotype. As a representative of X. laevis class II β chain genes, the Xela-DAB gene underwent detailed structural analysis. In addition,the nucleotide sequence of Xela-DABf cDNA clones was determined. The Xela-DAB gene is made up of a least six exons, with an exon-intron organization similar to that of a typical mammalian class II β chain gene. The 5′-flanking region of the Xela-DAB gene contains transcriptional control elements known as X1, X2, and Y, but lacks typical TATA or CCAAT boxes. A notable feature of the X. laevis class II β chain genes is that sizes of the introns are larger than those of their mammalian counterparts. As assessed by northern blot analysis, the three class II β chain genes had similar expression patterns, with the highest level of transcription detected in the intestine. Identification of the Xela-DAB,-DBB, and -DCB genes is consistent with our previous observations, which suggested that the MHC of the tettraploid frog X. laevis is diploidized at the genomic level and contains three class II β chain genes per haplotype that cross-hybridize to one another under reduced stringency conditions.

Identification of metallopanstimulin-1 as a member of a tumor associated antigen in patients with breast cancer

The immunological screening of breast cancer was performed with IgG autoantibodies by the serological analysis of recombinant cDNA expression library methods to explore novel tumor associated antigens. We have focused on a small zinc finger protein metallopanstimulin-1 (MPS-1). MPS-1 mRNA was ubiquitously expressed in normal human tissues except the brain and the placenta. In Western blot analysis, MPS-1 was easily detected strongly in actively proliferating cells and three breast cancer cell lines. In the tissue the protein of MPS-1 in cancer cells was more abundant than that of surrounding normal cells. Screening of tissue specimens by immunohistochemistry revealed 50.4% positive for MPS-1 in 125 cancer patients. These data suggest that MPS-1 could be applicable to the immunotherapy of breast cancer.