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Enhancement of macrophage complement production by factors from cloned T cells
1372
ABSTRACTS
MOLECULAR BASIS OF C2 (C2D) and C4 (C4D) DEFICIENCIES IN GUINEA PIGS. Goldberger G.‘, D.. Co/ten H.R. Harvard Medical School, Boston, MA., University of Sakivama Y. Bitter-Suermann Mainz, West’Germany. In order to further define the molecular basis of C2 and C4 deficiencies in guinea pigs Cell free translation oroducts of liver mRNA from normal and deficient animals were analyzed. Poly A ( + 1 mRNA was isolated by phenol/chloroform extraction and oligo-dT chromatography. Translations were performed in a rabbit reticulocyte lysate system optimized for K +, Mg+ + and mRNA. Specific prodircts were identified by SDS PAGE analysis of immunoprecipitates. Translation of normal mRNA produced C2 and C4 proteins with Mr of 78 Kd and 180 Kd; proteins similar in size to the underglycosylated C2 and pro-C4 found in macrophages cultured with tunicamycin, a giycosylation inhibitor, or generated from fully glycosylated intracellular proteins by Endo&N-acetylglucosaminidase-H. Kinetic studies of translation of C2 mRNA demonstrated initial production of the 78 Kd product then generation of an antiC precipitable protein of Mr 110 Kd. C2 protein was also detected in the translation of mRNA from a C2D auinea Dia. Three bands with Mr of 110 Kd. 77 and 72 Kd were observed. suaaestinc that (a) functional 52 mRNA is present in the C2 deficient’and (b) that a structural defeci>s expressed ‘in the primary translation product. Analysis of cell free translation products of mRNA isolated from C4D guinea pigs showed no anti-C4 precipitable bands whereas a previous study showed apparent synthesis of nascent C4 fragments by an endogenous (S-20) cell free translation system from C4D liver. Thus in a heterologous translating system there was no evidence for functional C4 mRNA in C4D.
STUDIES WITH MONOCLONAL ANTIBODIES TO AN UNUSUAL ~URINE COMPLEMENT-RELATED ACTIVITY. John N. Goldman*, Frank W. Fitch and Margaret B. Goldman. Michael Reese Medical Center and the University of Chicago, Chicago, IL. We have previously described an unusual complement-related activity present in mouse blood that is clearly distinct from individual early classical components but that leads to lysis of EA on further exposure to C-EDTA. Other major characteristics of this activity are: a) It is mediated by a single molecule or complex with an apparent molecular weight of 150,000 d on gel filtration. b) It forms a stable intermediate with EA but not with E. c) It loses hemolytic function upon exposure to EDTA but remains bound to the cell as evidenced by full recovery of activity upon exposure to Mg+ + containing buffers. d) 1 mM DFP blocks > 95% of the cell bound activity. e) Binding to and decay from the cell follow first order kinetics. and f) Functional levels of the activity are controlled by genes within the S region of the H-2 complex. We have developed a panel of monoclonal antibodies derived from rat-mouse hybridomas selected on the basis of their inhibition of this activity in mouse serum. Two of the monoclonal antibodies lead to immunoprecipitat~on in the presence of PEG-4000. These are both lQGpb immunoglobulins and show lines of identity when run against mouse serum or plasma on Ouchterlony analysis but they are not derived from identical clones since one precipitates the antigen optimally at low ionicity and blocks < % of the hemolytic function while the other precipitates optimally at high ionicity and can block > 95% hemolytic function. Affinity chromatography performed with this latter antibody coupled to Sepharose 48 removed > 95% of the activity from mouse serum or plasma while leaving almost all of the classical complement activity (measured by a standard Cl-l50 assay) in the efflUent. Control columns to which an irrelevant lgG2b monoclonal antibody was coupled removed no activity. The above data indicate that this is an unprecedented complement function that is not mediated by the known classical complement components.
ENHANCEMENT OF MACROPHAGE COMPLEMENT PRODUCTION BY FACTORS FROM CLONED T CELLS. Margaret 8. Goldman*, Mjchael P~ystowksy, John Eiy, Frank W. Fitch and John N. Goldman. Michael Reese Medical Center and the University of Chicago, Chicago, IL. It had previously been shown that products of antigen stimulated T cells increased C2 synthesis by cultured human monocytes (Littman and Ruddy, J. Exp. Med. 145:1344, 1977). Other recent studies have focused on lymphokine production and cytolytic activity of cloned T cell lines to facilitate elucidation of mechanisms of action, biochemistry and genetic control of T cell functions. We have recently initiated experiments examining the effects of products of cloned murine T cell lines on the synthesis of C4, C2,P -glucuronidase (a_glu.) and other products of cultured guinea pig peritoneal macrophages. Production of C4, C2 and p-glu. were enhanced by factor(s) obtained from a T cell line of C57BL16 origin (termed L2). L2 cells restimulated by irradiated DBA/2 spleen cells produce enhancing factors maximally between days 2 and 4, falling to low levels by day 7. A variant cell line derived from L2 (termed L2V) does not produce several lymphokines that are made by L2 and L2V cells produced no enhancing activity in this system. Complement stimulating activity was assessed by comparing activity released into the media by macrophages exposed to T cell supernatants vs control supernatants without T-cells. The first signs of stimulation were seen on day 2 of culture (17% stimulation) and increased to between days 7 and 14 (290% stimulation) when the cells began to die. Similar though less marked changes are seen with C2 and&glu. In addition, the L2 supernatants caused an increase in the number of surviving adherent cells, especially at the later time points. These experiments therefore suggested that in this xenogeneic system, the complement stimulatory activity is part of a more global process of macrophage stimulation by cloned T cell factors. Present experiments are aimed at determining the natureof the factor or factors responsible.
ABSTRACTS
MOLECULAR BASIS OF C2 (C2D) and C4 (C4D) DEFICIENCIES IN GUINEA PIGS. Goldberger G.‘, D.. Co/ten H.R. Harvard Medical School, Boston, MA., University of Sakivama Y. Bitter-Suermann Mainz, West’Germany. In order to further define the molecular basis of C2 and C4 deficiencies in guinea pigs Cell free translation oroducts of liver mRNA from normal and deficient animals were analyzed. Poly A ( + 1 mRNA was isolated by phenol/chloroform extraction and oligo-dT chromatography. Translations were performed in a rabbit reticulocyte lysate system optimized for K +, Mg+ + and mRNA. Specific prodircts were identified by SDS PAGE analysis of immunoprecipitates. Translation of normal mRNA produced C2 and C4 proteins with Mr of 78 Kd and 180 Kd; proteins similar in size to the underglycosylated C2 and pro-C4 found in macrophages cultured with tunicamycin, a giycosylation inhibitor, or generated from fully glycosylated intracellular proteins by Endo&N-acetylglucosaminidase-H. Kinetic studies of translation of C2 mRNA demonstrated initial production of the 78 Kd product then generation of an antiC precipitable protein of Mr 110 Kd. C2 protein was also detected in the translation of mRNA from a C2D auinea Dia. Three bands with Mr of 110 Kd. 77 and 72 Kd were observed. suaaestinc that (a) functional 52 mRNA is present in the C2 deficient’and (b) that a structural defeci>s expressed ‘in the primary translation product. Analysis of cell free translation products of mRNA isolated from C4D guinea pigs showed no anti-C4 precipitable bands whereas a previous study showed apparent synthesis of nascent C4 fragments by an endogenous (S-20) cell free translation system from C4D liver. Thus in a heterologous translating system there was no evidence for functional C4 mRNA in C4D.
STUDIES WITH MONOCLONAL ANTIBODIES TO AN UNUSUAL ~URINE COMPLEMENT-RELATED ACTIVITY. John N. Goldman*, Frank W. Fitch and Margaret B. Goldman. Michael Reese Medical Center and the University of Chicago, Chicago, IL. We have previously described an unusual complement-related activity present in mouse blood that is clearly distinct from individual early classical components but that leads to lysis of EA on further exposure to C-EDTA. Other major characteristics of this activity are: a) It is mediated by a single molecule or complex with an apparent molecular weight of 150,000 d on gel filtration. b) It forms a stable intermediate with EA but not with E. c) It loses hemolytic function upon exposure to EDTA but remains bound to the cell as evidenced by full recovery of activity upon exposure to Mg+ + containing buffers. d) 1 mM DFP blocks > 95% of the cell bound activity. e) Binding to and decay from the cell follow first order kinetics. and f) Functional levels of the activity are controlled by genes within the S region of the H-2 complex. We have developed a panel of monoclonal antibodies derived from rat-mouse hybridomas selected on the basis of their inhibition of this activity in mouse serum. Two of the monoclonal antibodies lead to immunoprecipitat~on in the presence of PEG-4000. These are both lQGpb immunoglobulins and show lines of identity when run against mouse serum or plasma on Ouchterlony analysis but they are not derived from identical clones since one precipitates the antigen optimally at low ionicity and blocks < % of the hemolytic function while the other precipitates optimally at high ionicity and can block > 95% hemolytic function. Affinity chromatography performed with this latter antibody coupled to Sepharose 48 removed > 95% of the activity from mouse serum or plasma while leaving almost all of the classical complement activity (measured by a standard Cl-l50 assay) in the efflUent. Control columns to which an irrelevant lgG2b monoclonal antibody was coupled removed no activity. The above data indicate that this is an unprecedented complement function that is not mediated by the known classical complement components.
ENHANCEMENT OF MACROPHAGE COMPLEMENT PRODUCTION BY FACTORS FROM CLONED T CELLS. Margaret 8. Goldman*, Mjchael P~ystowksy, John Eiy, Frank W. Fitch and John N. Goldman. Michael Reese Medical Center and the University of Chicago, Chicago, IL. It had previously been shown that products of antigen stimulated T cells increased C2 synthesis by cultured human monocytes (Littman and Ruddy, J. Exp. Med. 145:1344, 1977). Other recent studies have focused on lymphokine production and cytolytic activity of cloned T cell lines to facilitate elucidation of mechanisms of action, biochemistry and genetic control of T cell functions. We have recently initiated experiments examining the effects of products of cloned murine T cell lines on the synthesis of C4, C2,P -glucuronidase (a_glu.) and other products of cultured guinea pig peritoneal macrophages. Production of C4, C2 and p-glu. were enhanced by factor(s) obtained from a T cell line of C57BL16 origin (termed L2). L2 cells restimulated by irradiated DBA/2 spleen cells produce enhancing factors maximally between days 2 and 4, falling to low levels by day 7. A variant cell line derived from L2 (termed L2V) does not produce several lymphokines that are made by L2 and L2V cells produced no enhancing activity in this system. Complement stimulating activity was assessed by comparing activity released into the media by macrophages exposed to T cell supernatants vs control supernatants without T-cells. The first signs of stimulation were seen on day 2 of culture (17% stimulation) and increased to between days 7 and 14 (290% stimulation) when the cells began to die. Similar though less marked changes are seen with C2 and&glu. In addition, the L2 supernatants caused an increase in the number of surviving adherent cells, especially at the later time points. These experiments therefore suggested that in this xenogeneic system, the complement stimulatory activity is part of a more global process of macrophage stimulation by cloned T cell factors. Present experiments are aimed at determining the natureof the factor or factors responsible.