BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 268-272
Vol. 180, No. 1, 1991 October 15, 1991
MACROPHAGE HYDROGEN PEROXIDE PRODUCTION AND PHAGOCYTIC FUNCTION ARE DECREASED FOLLOWING PHAGOCYTOSIS MEDIATED BY F¢ RECEPTOR8 BUT NOT COMPLEMENT RECEPTORS Daniel J. Loegering and Martin G. Schwacha Department of Physiology and Cell Biology Albany Medical College 47 New Scotland Avenue Albany, New York 12208 Received September 4, 1991
Previous in vivo and in vitro studies have shown that the phagocytosis of IgG-coated erythrocytes results in a depression of macrophage function. The present study compared the effect of phagocytosis mediated by Fc receptors with that mediated by complement receptors. The phagocytosis of IgG-coated erythrocytas by elicited peritoneal macrophages depressed their capacity to produce hydrogen peroxide as well as phagocytic function. Phagocytosis of erythrocytes coated with IgM and complement had neither of these effects. These results implicate the intracellular signaling that results from Fc receptor mediated phagocytosis in the depression of macrophage function that is caused by phagocytosis. ©1991AcademicP. . . . . "rnc.
Challenge of macrophages by the phagocytosis of IgG-coated erythrocytes (EIgG) in vitro has been shown to decrease macrophage phagocytic function, respiratory burst capacity and bactericidal activity (1-3). These effects probably contribute to the increase in mortality rate with bacterial infection that has been observed following the phagocytosis of EIgG by Kupffsr cells in vivo (4,5). The mechanism for the depression of macrophage function following phagocytosis is unknown, but the intracellular signalling from the receptor mediating the phagocytosis could be important in causing this effect. The present study compared the effect of a phagocytic challenge mediated by Fc receptors with a challenge mediated by complement receptors.
It was found that
macrophage H202 production and phagocytic function were depressed following Fc receptor-mediated phagocytosis but not with complement receptor-mediated phagocytosis. This finding implicates the intracsllular signaling that results from Fc receptormediated phagocytosis in the depression of macrophage function that is caused by a phagocytic challenge. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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METHODS MacrophaQes. Rat peritoneal macrophages elicited with casein were isolated as previously described (1). Briefly, macrophagas were obtained by peritoneal lavage of Sprague-Dawley rats (250-275 g). The exudate cells were suspended in Dulbecco's modified Eagle's media (DMEM) containing 100 units/ml of penicillin and =100pg/ml of streptomycin and plated in 24-well tissue culture plates at a density of 2xl0"/well. Nonadherent cells were removed by washing after incubation for 2 hr at 37°C in 5% CO2 in air and after incubation overnight in DMEM containing 10% bovine calf serum. The resulting monolayers contained about 6x105 cells/well and were 95% macrophages as determined by Wright-stained morphology. •Phagocytic challenae. Macrophages were challenged with phagocytosis mediated by Fc or complement receptors. Fc receptor-mediated phagocytosis was induced by incubating the macrophages with sheep erythrocytes coated with anti-erythrocyte IgG (EIgG), and complement receptor-mediated phagocytosis was induced with erythrocytes coated with anti--erythrocyte IgM (EIgM) that were incubated in serum (EIgMC). EIgG and EIgM were prepared using the procedure previously described for the preparation of EIgG (1). EIgMC were prepared by incubating EIgM in 10% mouse serum at 37°C for 30 rain. The EIgMC were washed twice and used within 2 hrs. Mouse serum was obtained from Swiss-Webster mice and stored at -70°C. Macrophages were incubated with EIgG or EIgMC for 1 hr to allow phagocytosis. Controls were incubated with an equal number of unopsonized erythrocytes. Then the macrophages were washed to remove non-adherent erythrocytes and the adherent, noninternalized erythrocytes were hypotonically lysed. Following this phagocytic challenge, macrophagas were incubated for 1 hr before determining H202 production or Fc receptor phagocytic function. The phagocytosis of EIgG and EIgMC was determined using erythrocytas labeled with 51Cr, and the radioactivity associated with the macrophages after the hypotonic lysis was taken as the amount of phagocytosis. Assay of H20__2 production. Macrophage H202 production, triggered by phorbol myristate acetate (PMA, 50 ng/ml), was determined as previously described (6). Assay of Fc receptor phagocytic function. Macrophage Fc receptor phagocytic function was determined from the percent of a lx107 dose of EIgG that was phagocytized in 1 hr as previously described (1). Statistics. All measurements were made on triplicate monolayers using at least 3 separate macrophage isolations. Data are expressed as the mean + SEM. Comparisons were analyzed using a paired t-test or analysis of variance. The level of confidence is placed at 95% for all experiments.
RESULTS A greater percentage of the challenge doses of EIgG were phagocytized than EIgMC. For challenge doses of lx107, 5x107 and lx108, the number of EIgG phagocytized was 2.6¢0.2, 8.9+0.5, and 22.4¢1.9 x 10S/Well, respectively; and the number of EIgMC phagocytized was 1.0+0.2, 3.3+0.5, and 5.8+0.4 x 106/well, respectively. Macrophages 269
VOI. 180, NO. 1, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
120. ~ ~o
O--O EIgMC , - - @ EIgG
110-
110 ,
Vol. 180, No. 1, 1991 October 15, 1991
MACROPHAGE HYDROGEN PEROXIDE PRODUCTION AND PHAGOCYTIC FUNCTION ARE DECREASED FOLLOWING PHAGOCYTOSIS MEDIATED BY F¢ RECEPTOR8 BUT NOT COMPLEMENT RECEPTORS Daniel J. Loegering and Martin G. Schwacha Department of Physiology and Cell Biology Albany Medical College 47 New Scotland Avenue Albany, New York 12208 Received September 4, 1991
Previous in vivo and in vitro studies have shown that the phagocytosis of IgG-coated erythrocytes results in a depression of macrophage function. The present study compared the effect of phagocytosis mediated by Fc receptors with that mediated by complement receptors. The phagocytosis of IgG-coated erythrocytas by elicited peritoneal macrophages depressed their capacity to produce hydrogen peroxide as well as phagocytic function. Phagocytosis of erythrocytes coated with IgM and complement had neither of these effects. These results implicate the intracellular signaling that results from Fc receptor mediated phagocytosis in the depression of macrophage function that is caused by phagocytosis. ©1991AcademicP. . . . . "rnc.
Challenge of macrophages by the phagocytosis of IgG-coated erythrocytes (EIgG) in vitro has been shown to decrease macrophage phagocytic function, respiratory burst capacity and bactericidal activity (1-3). These effects probably contribute to the increase in mortality rate with bacterial infection that has been observed following the phagocytosis of EIgG by Kupffsr cells in vivo (4,5). The mechanism for the depression of macrophage function following phagocytosis is unknown, but the intracellular signalling from the receptor mediating the phagocytosis could be important in causing this effect. The present study compared the effect of a phagocytic challenge mediated by Fc receptors with a challenge mediated by complement receptors.
It was found that
macrophage H202 production and phagocytic function were depressed following Fc receptor-mediated phagocytosis but not with complement receptor-mediated phagocytosis. This finding implicates the intracsllular signaling that results from Fc receptormediated phagocytosis in the depression of macrophage function that is caused by a phagocytic challenge. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
268
Voh 180, No. 1, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
METHODS MacrophaQes. Rat peritoneal macrophages elicited with casein were isolated as previously described (1). Briefly, macrophagas were obtained by peritoneal lavage of Sprague-Dawley rats (250-275 g). The exudate cells were suspended in Dulbecco's modified Eagle's media (DMEM) containing 100 units/ml of penicillin and =100pg/ml of streptomycin and plated in 24-well tissue culture plates at a density of 2xl0"/well. Nonadherent cells were removed by washing after incubation for 2 hr at 37°C in 5% CO2 in air and after incubation overnight in DMEM containing 10% bovine calf serum. The resulting monolayers contained about 6x105 cells/well and were 95% macrophages as determined by Wright-stained morphology. •Phagocytic challenae. Macrophages were challenged with phagocytosis mediated by Fc or complement receptors. Fc receptor-mediated phagocytosis was induced by incubating the macrophages with sheep erythrocytes coated with anti-erythrocyte IgG (EIgG), and complement receptor-mediated phagocytosis was induced with erythrocytes coated with anti--erythrocyte IgM (EIgM) that were incubated in serum (EIgMC). EIgG and EIgM were prepared using the procedure previously described for the preparation of EIgG (1). EIgMC were prepared by incubating EIgM in 10% mouse serum at 37°C for 30 rain. The EIgMC were washed twice and used within 2 hrs. Mouse serum was obtained from Swiss-Webster mice and stored at -70°C. Macrophages were incubated with EIgG or EIgMC for 1 hr to allow phagocytosis. Controls were incubated with an equal number of unopsonized erythrocytes. Then the macrophages were washed to remove non-adherent erythrocytes and the adherent, noninternalized erythrocytes were hypotonically lysed. Following this phagocytic challenge, macrophagas were incubated for 1 hr before determining H202 production or Fc receptor phagocytic function. The phagocytosis of EIgG and EIgMC was determined using erythrocytas labeled with 51Cr, and the radioactivity associated with the macrophages after the hypotonic lysis was taken as the amount of phagocytosis. Assay of H20__2 production. Macrophage H202 production, triggered by phorbol myristate acetate (PMA, 50 ng/ml), was determined as previously described (6). Assay of Fc receptor phagocytic function. Macrophage Fc receptor phagocytic function was determined from the percent of a lx107 dose of EIgG that was phagocytized in 1 hr as previously described (1). Statistics. All measurements were made on triplicate monolayers using at least 3 separate macrophage isolations. Data are expressed as the mean + SEM. Comparisons were analyzed using a paired t-test or analysis of variance. The level of confidence is placed at 95% for all experiments.
RESULTS A greater percentage of the challenge doses of EIgG were phagocytized than EIgMC. For challenge doses of lx107, 5x107 and lx108, the number of EIgG phagocytized was 2.6¢0.2, 8.9+0.5, and 22.4¢1.9 x 10S/Well, respectively; and the number of EIgMC phagocytized was 1.0+0.2, 3.3+0.5, and 5.8+0.4 x 106/well, respectively. Macrophages 269
VOI. 180, NO. 1, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
120. ~ ~o
O--O EIgMC , - - @ EIgG
110-
110 ,
