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C3a and C3a-desARG induce release of IL-6 and TNF-a from human monocytes
50
C4BPAL1, A MEMBER OF THE HUMAN REGULATOR OF COMPLEMENT ACTIVATION (RCA) GENE CLUSTER THAT RESULTED FROM THE DUPLICATION OF THE GENE CODING FOR THE uCHAIN OF C4b-BINDING PROTEIN. Pilar Shnchez-Corral, Fernando Pardo-Manuel de Villena, Javier ReyCampos and Santiago Rodriguez de C6rdoba. Unidad de Inmanologia, Centro de Investigaciones Biol6gieas (CSIC), Velfizquez 144, 28006Madrid, SPAIN. The Regulator of Complement Activation (RCA) gene cluster evolved by multiple gene duplications to produce a family of genes coding for proteins that collectively control the activation of the complement system. We report here the characterization of C4BPAL1, a member of the human RCA gene cluster that arose from the duplication of the C4BPA gene after the separation of the rodent and primate lineages. C4BPAL1 maps 20 kb downstream of the C4BPA gene and is in the same 5' to 3' orientation found for all RCA genes characterized thus far. It includes nine exon-like regions homologous to exons 2, 3, 4, 5, 6, 7, 8, 11 and 12 of the C4BPA gene. Analysis of the C4BPAL1 sequence suggests that in humans it is currently a pseudogene. However, comparisons between C4BPAL1 and the human and murine C4BPA genes show sequence conservation which strongly suggests that, for a long period of time, C4BPAL1 has been a functional gene coding for a protein with structural requirements similar to those of the o~-chain of C4b-binding protein (C4BP).
C3a A N D C 3 a - d e s A R G I N D U C E R E L E A S E O F I L - 6 A N D TNF-~ FROM HUMAN MONOCYTES S. Schermuck, R. Burger, Dept. of Immunol,, Robert Koch Inst., Berlin, Germany The anaphylatoxins are potent inflammatory mediators. Biological activities are associated mainly with the arginine-containing form of CJa and C3a. The desArg-variants generated by serum earboxypeptidasemediated cleavage the C-terminal arginine are inactive in most but not all bioassays. C3a-desArg was shown previously to induce IL-1 release. In sepsis patients and related conditions, elevated anaphylatoxin levels were found due to efficient complement activation. In these patients, increased plasma levels of cytokines were also observed and might be a consequence of anaphylatoxin-induced cellular activation processes. The capacity of purified C3a and C3a-desArg to induce IL-6 and TNF-~-release from normal human monocytes was determined. Monocytes were isolated from peripheral blood using Percoll gradient centrifugation followed by an adherence step. The cells were stimulated by incubation for defined time intervals with C3a or C3a-desArg in the presence or absence of LPS as costimulus. TNF-~x and IL-6 were measured in the culture supernatants using an ELISA system. Both C3a and C3a-desArg induced a dosedependent release of IL-6 and TNF-~. Both C3a-variants had the capacity to enhance synergistically the LPS-induced IL-6 and TNF-~-production. Maximal cytokine release was observed after 12h of incubation with C3aor C3a-desArg alone and after 24h upon costimulation with LPS. The effect of the two C3a-variants was inhibited by preineubation with mAb recognizing an epitope of C3a or C3a-desArg. Control mAb recognizing a C3b-determinant had no effect. These findings show a role for both C3a and the C3a-desArg variant in cellular activation. The pleiotropic effects of the C3a- or C3a-desArg-induced cytokines emphasize the regulatory role of the anaphylatoxins in both nonspecific and specific immune response. Supported by Bhimenthal-Foundation.
COMPLEMENT SYNTHESIS REGULATING FACTORS IN T H E C O N D I T I O N E D MEDIUM OF ACUTE MYELOID LEUKEMIA (AML) BLAST CELLS Schmidt, B., Gyapay, G., Kramer, J., Ftist, G. Natl. Inst. Haemat. Blood Transf. & Immunol., Budapest, Hungary We have previously reported (Compl. Intl. 8(5-6), 370-377, 1991.) that AML blast cells produce (a) factors(s) that had regulating effects on the complement protein synthesis by human monocyte and HepG2 cultures. These factors may account for the hypercomlementemia observed in AML. The effects of the AML blast cell conditioned medium (CM-AML) has been further investigated. The new findings are as follows: (1) CM-AML increased the CI-INH, C3, C4 and factor B synthesis by HepG2 cells. (2) The effect of CM-AML was inhibited either by Actinomycin D or Cycloheximide. (3) Fractionation by gel filtration revealed a factor B synthesis increasing fraction and a decreasing one of smaller molecular weight. (4) Both anti-TNF-alpha and anti-IL-6 antibodies could partially block the CI-1NH and factor B increasing effect of CM-AML. (5) CMAML has also been tested on fibroblasts: it substantially increased the factor B synthesis as measured by biosynthetic labeling. Despite the marked elevation in the amount of released factor B, no increase could bc observed intracellularly. Interestingly, CM-AML did not affect the C1INH synthesis in fibroblasts.
EXPRESSION OF MEMBRANE CO-FACTOR PROTEIN(MCP) IN GAETRO-INTEETINAL
MUCOSA
N.Seto, S.Takemura, M.Ueda, S.Nakanishi, N.Ichio, K.Ashihara, R.Nakahara, T.Doi, Y.kasamatsu, M.Okamoto, K.Yanagida, W.Fukuda, H.Onodera, M.Deguchi, S.Sugino and M.Kondo Department of Internal Medicine, Kyoto Prefectural university of Medicine, Kyoto 602, Japan Membrane co-factor protein(MCP;CD46) is a membrane protein with molecular weight of the two species of 63kD and 55kD. The function of MCP is a co-factor of factor I, which inactivates C3b bound on autologous cell membrane to produce C3bi. It is reported that MCP is expressed on all blood cells except erythrocytes, epidermis, mesangial ceils and juxtaglomerular cells, but has not been examined in the gastro-intestinal mucosa as yet. We studied the expression of MCP on normal mucosa and cancer lesions of gastro intestinal tract obtained by surgery. Four-micrometer thick sections were fixed with acetone and used for immuno-histochemical study. Monoclonal antibody against MCP(MCP75, MCPI60) was used and stained immuno-enzymatically. MCP was stained in the epithelium of esophagus, stomach and colon both in normals and cancer lesions.
C4BPAL1, A MEMBER OF THE HUMAN REGULATOR OF COMPLEMENT ACTIVATION (RCA) GENE CLUSTER THAT RESULTED FROM THE DUPLICATION OF THE GENE CODING FOR THE uCHAIN OF C4b-BINDING PROTEIN. Pilar Shnchez-Corral, Fernando Pardo-Manuel de Villena, Javier ReyCampos and Santiago Rodriguez de C6rdoba. Unidad de Inmanologia, Centro de Investigaciones Biol6gieas (CSIC), Velfizquez 144, 28006Madrid, SPAIN. The Regulator of Complement Activation (RCA) gene cluster evolved by multiple gene duplications to produce a family of genes coding for proteins that collectively control the activation of the complement system. We report here the characterization of C4BPAL1, a member of the human RCA gene cluster that arose from the duplication of the C4BPA gene after the separation of the rodent and primate lineages. C4BPAL1 maps 20 kb downstream of the C4BPA gene and is in the same 5' to 3' orientation found for all RCA genes characterized thus far. It includes nine exon-like regions homologous to exons 2, 3, 4, 5, 6, 7, 8, 11 and 12 of the C4BPA gene. Analysis of the C4BPAL1 sequence suggests that in humans it is currently a pseudogene. However, comparisons between C4BPAL1 and the human and murine C4BPA genes show sequence conservation which strongly suggests that, for a long period of time, C4BPAL1 has been a functional gene coding for a protein with structural requirements similar to those of the o~-chain of C4b-binding protein (C4BP).
C3a A N D C 3 a - d e s A R G I N D U C E R E L E A S E O F I L - 6 A N D TNF-~ FROM HUMAN MONOCYTES S. Schermuck, R. Burger, Dept. of Immunol,, Robert Koch Inst., Berlin, Germany The anaphylatoxins are potent inflammatory mediators. Biological activities are associated mainly with the arginine-containing form of CJa and C3a. The desArg-variants generated by serum earboxypeptidasemediated cleavage the C-terminal arginine are inactive in most but not all bioassays. C3a-desArg was shown previously to induce IL-1 release. In sepsis patients and related conditions, elevated anaphylatoxin levels were found due to efficient complement activation. In these patients, increased plasma levels of cytokines were also observed and might be a consequence of anaphylatoxin-induced cellular activation processes. The capacity of purified C3a and C3a-desArg to induce IL-6 and TNF-~-release from normal human monocytes was determined. Monocytes were isolated from peripheral blood using Percoll gradient centrifugation followed by an adherence step. The cells were stimulated by incubation for defined time intervals with C3a or C3a-desArg in the presence or absence of LPS as costimulus. TNF-~x and IL-6 were measured in the culture supernatants using an ELISA system. Both C3a and C3a-desArg induced a dosedependent release of IL-6 and TNF-~. Both C3a-variants had the capacity to enhance synergistically the LPS-induced IL-6 and TNF-~-production. Maximal cytokine release was observed after 12h of incubation with C3aor C3a-desArg alone and after 24h upon costimulation with LPS. The effect of the two C3a-variants was inhibited by preineubation with mAb recognizing an epitope of C3a or C3a-desArg. Control mAb recognizing a C3b-determinant had no effect. These findings show a role for both C3a and the C3a-desArg variant in cellular activation. The pleiotropic effects of the C3a- or C3a-desArg-induced cytokines emphasize the regulatory role of the anaphylatoxins in both nonspecific and specific immune response. Supported by Bhimenthal-Foundation.
COMPLEMENT SYNTHESIS REGULATING FACTORS IN T H E C O N D I T I O N E D MEDIUM OF ACUTE MYELOID LEUKEMIA (AML) BLAST CELLS Schmidt, B., Gyapay, G., Kramer, J., Ftist, G. Natl. Inst. Haemat. Blood Transf. & Immunol., Budapest, Hungary We have previously reported (Compl. Intl. 8(5-6), 370-377, 1991.) that AML blast cells produce (a) factors(s) that had regulating effects on the complement protein synthesis by human monocyte and HepG2 cultures. These factors may account for the hypercomlementemia observed in AML. The effects of the AML blast cell conditioned medium (CM-AML) has been further investigated. The new findings are as follows: (1) CM-AML increased the CI-INH, C3, C4 and factor B synthesis by HepG2 cells. (2) The effect of CM-AML was inhibited either by Actinomycin D or Cycloheximide. (3) Fractionation by gel filtration revealed a factor B synthesis increasing fraction and a decreasing one of smaller molecular weight. (4) Both anti-TNF-alpha and anti-IL-6 antibodies could partially block the CI-1NH and factor B increasing effect of CM-AML. (5) CMAML has also been tested on fibroblasts: it substantially increased the factor B synthesis as measured by biosynthetic labeling. Despite the marked elevation in the amount of released factor B, no increase could bc observed intracellularly. Interestingly, CM-AML did not affect the C1INH synthesis in fibroblasts.
EXPRESSION OF MEMBRANE CO-FACTOR PROTEIN(MCP) IN GAETRO-INTEETINAL
MUCOSA
N.Seto, S.Takemura, M.Ueda, S.Nakanishi, N.Ichio, K.Ashihara, R.Nakahara, T.Doi, Y.kasamatsu, M.Okamoto, K.Yanagida, W.Fukuda, H.Onodera, M.Deguchi, S.Sugino and M.Kondo Department of Internal Medicine, Kyoto Prefectural university of Medicine, Kyoto 602, Japan Membrane co-factor protein(MCP;CD46) is a membrane protein with molecular weight of the two species of 63kD and 55kD. The function of MCP is a co-factor of factor I, which inactivates C3b bound on autologous cell membrane to produce C3bi. It is reported that MCP is expressed on all blood cells except erythrocytes, epidermis, mesangial ceils and juxtaglomerular cells, but has not been examined in the gastro-intestinal mucosa as yet. We studied the expression of MCP on normal mucosa and cancer lesions of gastro intestinal tract obtained by surgery. Four-micrometer thick sections were fixed with acetone and used for immuno-histochemical study. Monoclonal antibody against MCP(MCP75, MCPI60) was used and stained immuno-enzymatically. MCP was stained in the epithelium of esophagus, stomach and colon both in normals and cancer lesions.