２２回日本研究皮膚科学会総会・抄録

２２回日本研究皮膚科学会総会・抄録

-98- Immunology(4) 9/5 Room-A
THE ANALYSIS OF ANTINUCLEAR ANTIBODY PRODUCED BY GOLDTHIOMALATE TREATED MICE.
Kazuo Takahashi1, Takafumi Nishiyama2, Norihisa Ishii2, Hiroshi Nakajima2.
1Department of Dermatology, National Sagaihara Hospital, Sagamihara, 2Department of Dermatology, Yokohama city University, Yokohama, Japan.
It has been shown that Th2 cells played a central role in that goldthiomalate(Au) treated mice have prodced antinuclear antibody(ANA). Here we adressed the influence ofcytokines in this mouse model. Method:Mice received weekly s.c. injections of Au for 6 weeks and i.p. injections of cytokines(IL-2,αIL-2,IL-4,αIL-4,αIFN-γ) at the time of 1,3,5 week. Mouse sera were obtained every 3 weeks and tested for the presence of IgG ANA containig subclass of IgG1,IgG2a,IgG2b,IgG3 by the indirect immunofluorescence method. Result & discussion: It is thought IL-4 which can be derived from Th2 is a important cytokine because ANA production was suppressed by i.p. injection of αIL-4 in A.SW mouse. The treatment of cytokine could modify Th1/Th2 responce and effect the switching in subclass of IgG ANA since IgG2a ANA was dominantly procuced in A.SW treated with only Au whereas IgG1 ANA was dominantly procuced in A.SW treated with Au and IL4 or αIFN-γ.

-99- Immunology(4) 9/5 Room-A
EPITOPE MAPPING OF ANTI CARBONIC ANHYDRASE I ANTIBODIES IN RHEUMATIC AUTOIMMUNE DISEASES.
Keisuke Watanabe, Mariko Ono, Masashi Ono, Hiroaki Ueki.
Department of Dermatology, Kawasaki Medical School, Kurashiki, Japan.
We have previously found that the autoantibodies against carbonic anhydrase (CA) exist in sera from patients with rheumatic autoimmune diseases (RAD). For further characterization of this autoantibody,linear autoepitopes on CAI molecule were analized. CAI peptides (peptide1-21) consisted with 15 －16-mer amino acids were synthesized and coupled with bovine albumin. Reactivity of patients' sera to each peptides were detected by ELISA. We observed that anti-CAI autoantibodies showed dominant reactivity to peptide 1, 2, 7 and 8. The crystallography have shown that peptide 1 and 2 are located on the helical portion of amino-terminus and peptide 7 and 8 are located on the active portion surrounding the zinc core. The result suggests that anti-CAI antibody could have an inhibitory effect to the enzyme activity and may play pathogenic role in RAD.

-100- Immunology(4) 9/5 Room-A
IMMUNOSUPPRESSSION INDUCED BY in vivo APPLICATION OF RESTRICTION ENZYME IN LIPOSOMES TO MOUSE SKIN.
Chikako Nishigori.
Department of Dermatology, Kyoto University Kyoto Japan.
Our previous work suggested that the UV-induced DNA damage triggers immunosuppressive effects of UV radiation. The purpose of this study was to determine whether other types of DNA damage also generate such immunosuppression. To test this hypothesis, Hind III restriction enzyme, which causes double-strand breaks in DNA, was encapsulated in liposomes and applied to the skin of C3H mice in vivo. DNA damage was demonstrated by extracting DNA from skin 4 hr later, followed by electrophoresis on neutral agarose gel. Mice were immunized with non-viable Candida or FITC at 3 days after liposome application and tested for DTH and CHS responses, respectively. Hind III liposomes but not control liposomes reduced these immune responses to the same extent as UVB radiation (5 kJ/m2). Hind III-liposome treated skin showed more DNA strand breaks than control. Unlike UVB irradiation, however, neither of these suppressed responses was transferable with spleen cells. These results demonstrate that DNA damage in the form of strand breaks can cause systemic immune suppression; they also indicate that immune suppression is a separate phenomenon from suppressor cell induction and that additional events are required to generate suppressor cells.(This work has been done at the Dept Immunology, M.D. Anderson Cancer Center: Chairman; Dr. Kripke)

-101- Immunology(4) 9/5 Room-A
FLOW CYTOMETRIC ANALYSIS OF INTRA CELLULAR CYTOKINE LEVEL IN HUMAN PERIPHERAL BLOOD MONOCYTES.
Akihiko Shibaki, Hiroyuki Matsue, Toshimitsu Kawashima, Norikatsu Mizumoto, Hitoshi Kobayashi, Akira Ohkawara.
Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan.
In generalized pustular psoriasis patients, serum pro inflammatory cytokine level is increased in accordance with the severity of cutaneous and systemic manifestation. To elucidate the responsible cell source of this increased cytokine level, we compared the two distinct methods, ELISA and FACS, in order to evaluate cytokine production in human mocytic cell line, THP-1. THP-1 cells were stimulated by LPS for up to 48 hours and supernatants were assayed for IL-1β, IL-6, IL-8 and TNF-α concentration by ELISA. THP-1 produced IL-1β, IL-6, IL-8 peaking at 48 hours and TNF-α peaking at 6 hours after LPS stimulation. When intracellular cytokine levels were analyzed by FACS, the expression of IL-6 and TNF-α was also upregulated at 48 hours and 6 hours, respectively. Simillary, peripheral blood monocytes increased intracellular IL-6 and TNF-α level after LPS stimulation. These results indicate the usefulness of FACS analysis in evaluating cytokine production among heterogeneous cell populations.

-102- Immunology(4) 9/5 Room-A
PHENOTYPES OF LYMPHOCYTES INFILTRATING IN B16 MELANOMA.
Naohiro Seo, Yoshiki Tokura, Fukumi Furukawa, Masahiro Takigawa.
Depertment of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
To obtain a understanding of immunologic nature of lymphocytes infiltrating in B16 melanoma. We investigated in this study the phenotypes of tumor-infiltrating lymphocytes (TILs) present at the B16 tumor site. B16 melanoma was grown for 15 days after s.c. inoculation into syngeneic B6 mice. TILs were separated from B16 tumor-cell suspension by the centrifugation using succrose gradient. TILs or cultured melanoma-specific cytotoxic TILs were phenotyped with mAbs against abTCR, NK1.1, CD4, CD8 and the several TCR Vb chains. The proportions of abTCR, NK1.1, CD4 and CD8 positive cells in TILs were 23%, 31%, 2% and 21%, respectively. Almost of abTCR positive cells bore CD8 molecule, but not CD4 molecule. Further, the Vb7 or Vb12 T cells predominantly infiltratied in B16 tumor site (27% or 39% in abTCR positive cells, respectively). On the other hand, cytotoxic TILs were generated by in vitro stimulation of TILs with chemically attenuated B16 melanoma. The percentage of abTCR, CD8 double-positive cells and NK1.1 positive cells in cytotoxic TILs were 43 and 52, respectively. In addition, the specifically expanded of cytotoxic Vb7 or Vb12 T cells were observed in the cultured TILs. These result implied that the specific or non-specific cytotoxic precursor lymphocytes against syngeneic B16 melanoma infiltratie in tumor site.

-103- Immunology(4) 9/5 Room-A
THE EFFECT OF ULTRAVIOLET-RAYS(UV) ON TUMOR REJECTION OF AN INTERLEUKIN-12 GENE-INTRODUCED CARCINOMA CELL LINE RENCA.
Hiroshi Nagai1, Tatsuya Horikawa1, Kenta Tsuru1, Osamu Ando2, Masamitsu Ichihashi1.
1Department of Dermatology, Kobe University School of Medicine Kobe, 2Hayashibara Biochemical Laboratories, Hayashibara Co. Inc., Okayama, Japan.
We investigated the effect of ultraviolet-rays(UV) on tumor rejection of the interleukin-12 (IL-12) gene introduced carcinoma cell, using mouse model. IL-12 gene was introduced into mouse renal cell carcinoma (RenCa) and an IL-12-secreting RenCa (RenCa/IL-12) cell line was selected. RenCa/IL-12 exhibited complete rejection, while parental RenCa exhibited tumor growth after the subcutaneously injection into syngenic Balb/c mice and Balb/c hairless mice. UV irradiation(UVB, total 32.7 kJ/m2) allowed transient growth of RenCa/IL-12 cells and the tumor regressed and disappeared subsequently. Histologic examination of the implanted tumors revealed that dense infiltlation of macrophages and NK cells were observed in both tumor progressive phase and tumor regressive phase. The marker of activated macrophage (158.2) was less prominent in progressive phase than in regressive phase. These findings suggest that UV-induced inhibition of tumor rejection is related to loss of macrophage activation.