Immunology 1991 74 657-660
Binding and endocytosis of erythrocytes sensitized with rabbit IgG via Fc gamma receptors of human monocytes M. KAVAI, E. GYIMESI, G. SZUCS & G. SZEGEDI 3rd Department of Medicine, University Medical School of Debrecen, Debrecen, Hungary
Acceptedfor publication 14 August 1991
SUMMARY The properties of the monocyte Fc gamma receptors (FcR) were investigated with monoclonal antibodies (mAb) against FcRI (10.1) and FcRII (IV3). mAb against FcRI inhibited partially the binding of sheep red blood cells (SRBC) sensitized with anti-SRBC rabbit IgG (EA) at 37° to monocytes pretreated with N-ethyl maleimide, which inhibits the EA ingestion. The erythrocytes (E) were sensitized with varying concentrations of anti-E rabbit IgG. The EA binding to different FcR depends on the concentration of specific antibody used to sensitize the erythrocytes. At high levels of sensitization a high proportion of rosettes form via FcRII which can be inhibited with mAb IV3. As sensitization decreases it is more difficult for FcRII to form rosettes, so an increased percentage of them is mediated via FcRI. Sensitization of SRBC with 1-1 5 x 103 anti-SRBC rabbit IgG molecules per erythrocyte is the threshold to allow FcRII to mediate rosettes. At the lowest levels of sensitization the total number of rosettes is even lower and all rosettes are mediated via FcRI, hence mAb 10.1 is fully inhibitory. In addition, our data strongly support the view that the ingestion of EA takes place mainly via FcRII. We show in this study that while binding of slightly sensitized erythrocytes was blocked efficiently by mAb 10.1, the ingestion of the equivalent EA was hardly inhibited by it. INTRODUCTION
were inhibited by monoclonal antibodies against either FcRI and/or FcRII was compared.
Substantial evidence exists indicating molecular heterogeneity of Fc receptors for IgG (FcR) on human monocytes. How these various structures promote specific biogical consequences is currently under study. Various evidence suggests that only FcRI is involved in recognition of anti-D IgG-sensitized red cells on monocytes. ' At the same time, it is known that the rabbit IgG-coated erythrocytes bind to both the high (FcRI) and low (FcRII) affinity FcR.2 An explanation for the role of the two FcR can be given by blocking of rosette formation between U937 cell and erythrocytes sensitized with rabbit IgG by monoclonal antibody against FcRI (mAb 10.1).3 Here mAb 10.1 blocked essentially all rosette formation except when highly opsonized red cells were used. The experiments reported here extend these findings to identify the FcR primarily involved in the recognition of cellbound IgG by human monocytes. The binding of erythrocytes (E) sensitized with varying concentrations of anti-E rabbit IgG to monocytes was determined at 37°. The endocytic activity of monocytes was tested with red cells sensitized by the same way. The degree to which rosette formation and endocytic function
MATERIALS AND METHODS Cell preparation Twenty-four millilitres of peripheral human blood were anticoagulated with 10 U heparin/ml. Platelet-depleted mononuclear cells (MC) were obtained by centrifugation through Ficoll-Hypaque.4 The cells were resuspended in 2 ml of medium Parker-199 containing 20 mm HEPES, pH 7-4 (Flow, Irvine, Ayrshire, U.K.) and counted in a standard haemocytometer. Thirty to forty per cent of the MC were found to be monocytes by non-specific esterase staining and by neutral red phagocytosis. Monocytes were purified by adherence to a glass surface. Briefly, 100 pi of MC suspension containing 2-4 x 105 cells were placed on round cover slides immersed into wells of a tissue culture plastic plate. After 30 min at room temperature, the non-adherent cells were removed by rigorous washing. The adherent monolayer contained more than 80% monocytes (12 x 104 cells/glass slide). Close agreement was obtained by morphological examinations with Wright's stain, esterase staining and neutral red phagocytosis.
Correspondence: Dr M. Kdvai, 3rd Dept. of Medicine, Univ. Med. School of Debrecen, 4004 Debrecen, Hungary.
Monoclonal antibodies (mAb) and polyclonal antiserum IV3 supernatant is the cell culture fluid recovered from cloned hybrodoma cells producing murine IgG2b anti-FcRII, which
657
658
M. Kavai et al.
was generous gift of C.L. Anderson (Columbus, OH). The concentration of the mAb was 20-30 Mg/ml. mAb 10.1 is a murine IgGl anti-FcRI, which was the generous gift of Nancy Hogg (London, U.K.). The mAb was isolated from ascites fluid and its concentration was 8 5 mg/ml. Other mAb used in this study as controls were murine IgG2b (220 pg/ml) and IgGl (14 mg/ml), which were generous gifts of Eva Rajnavolgyi (G6d, Hungary). Rabbit anti-sheep red blood cell (SRBC) stroma serum (haemolysin) was purchased from HUMAN, Budapest, Hungary.
x co
0
53 0
2
-
0
Rosette-forming cell (RFC) assays The adherent cells (1-2 x 104) on the cover slides were treated with N-ethyl maleimide (NEM) of 2 5 mm in 200 p1 of H-Parker at room temperature for 30 min.5 After rigorous washing the cells were incubated with 2-5% sensitized SRBC (EA) in 200 pi H-Parker at 370 for 20 min. The erythrocytes were appropriately sensitized with rabbit IgG isolated from haemolysin. After washing, the cells were stained with 0-2% crystal violet and the percentage of RFC as well as the numbers EA bound to one monocyte, i.e. the binding index (BI), were determined.6 EA endocytosis by monocytes The adherent cells were incubated with the appropriately sensitized SRBC of 2-5% pl H-Parker at 37° for 20 min. After washing, the EA bound to the monocyte surface were lysed by water for 30 seconds. After washing and staining the percentage of monocytes ingesting three of more EA, as well as the numbers of EA internalized per one monocyte, i.e. the phagocytic index (PI), were determined. Determination of red cell bound IgG '251-protein-A was used to measure anti-SRBC IgG bound to the surface of sheep erythrocytes sensitized with varying dilutions of anti-SRBC rabbit IgG.7
Effect of anti-FcRI and anti-FcRII on monocyte FcR In the inhibition experiments the adherent cells (1-2 x 104/slide) were preincubated with mAb at room temperature for 10 min and then with phagocytic targets (EA) at 370 for 20 min. AntiFcRI mAb 10. 1, 17 pg/200 p1,3 anti-FcRII mAb IV3, 0-20 yg/ 200 pl and control mAb mouse IgGl, 21 pg/200 pi, and mouse IgG2b, 0-22 pg/200 pl, were used. This concentration of antiFcRII antibody was sufficient to stain 106 monocytes in an indirect immunofluorescence assay.8 For testing the saturating conditions of mAb IV3, rosette formation was inhibited -430 x 10-2 pg/200 pl mAb Ig2b. The EA binding and endocytosis by monocytes were tested in presence of these mAb. -
RESULTS Effect of anti-FcRI on binding and ingestion of EA by monocytes
The binding of anti-SRBC rabbit IgG to sheep erythrocytes depends on the amount of IgG added to the cells and incubated with them (Fig. l). Sensitized sheep erythrocytes (EA) bind to monocytes treated with N-ethyl maleimide (NEM) and form rosettes with them at 370 for 20 min. 2-5 mM NEM completely blocks the ingestion of EA by monocytes, whereas it has no
64 128 256 512 32 Reciprocal dilution of rabbit IgG added to erythrocytes
Figure 1. Binding of IgG to erythrocytes sensitized with various amounts of rabbit IgG. The bound IgG was measured with 1251-protein-A and expressed as molecules. Values represent mean of triplicate determinations + SD.
Table 1. Interactions of sensitized erythrocytes with monocytes pretreated with 2-5 mm N-ethyl maleimide (NEM)
Medium
Binding + ingestion
Ingestion
H-Parker 2 5 mm NEM
2 34 + 0 21 (BI + PI) 2 28+0-18 (BI)
2-07 + 0 16 (PI) 0
The adherent cells (1-2 x 104) were treated with H-Parker or NEM in H-Parker at room temperature for 30 min. After washing the cells were incubated with SRBC sensitized with rabbit IgG of 1:32 dilution at 370 for 20 min. The bound and ingested erythrocytes per adherent cell were determined (BI + PI). In parallel plates, after incubation at 37°, the cell-bound erythrocytes were lysed with water and the ingested erythrocytes per adherent cell determined (PI). Values represent mean of triplicate determinations + SD.
effect on binding of EA (Table 1). The binding of EA depends on the density of the IgG coating the erythrocytes (Fig. 2). The presence of anti-FcRI (mAb 10.1) in the incubation medium results in an effective inhibition of EA binding, which depends slightly on the sensitization of erythrocytes. Control murine IgGl had no inhibitory effect. The EA are almost completely ingested by untreated monocytes at 370 for 20 min. The EA bound to erythrocyte surface were lysed; in this way only the EA ingested by monocytes were detected. The internalization depended entirely on the concentration of specific antibody used to sensitize the red cells (Fig. 3). Preincubation of monocytes with anti-FcRI inhibited EA ingestion not depending on the magnitude of sensitization. The inhibition of EA binding via FcRI was higher than that of EA ingestion.
Binding and endocytosis via Fc gamma receptors a E
Control mAb
0 o
0
70 60-
-'
50.
0
am
659
2-5 -2 020, ^ 0. -
1)-5
._1 40 r-
mAb IV3
30-
Binding and endocytosis of erythrocytes sensitized with rabbit IgG via Fc gamma receptors of human monocytes M. KAVAI, E. GYIMESI, G. SZUCS & G. SZEGEDI 3rd Department of Medicine, University Medical School of Debrecen, Debrecen, Hungary
Acceptedfor publication 14 August 1991
SUMMARY The properties of the monocyte Fc gamma receptors (FcR) were investigated with monoclonal antibodies (mAb) against FcRI (10.1) and FcRII (IV3). mAb against FcRI inhibited partially the binding of sheep red blood cells (SRBC) sensitized with anti-SRBC rabbit IgG (EA) at 37° to monocytes pretreated with N-ethyl maleimide, which inhibits the EA ingestion. The erythrocytes (E) were sensitized with varying concentrations of anti-E rabbit IgG. The EA binding to different FcR depends on the concentration of specific antibody used to sensitize the erythrocytes. At high levels of sensitization a high proportion of rosettes form via FcRII which can be inhibited with mAb IV3. As sensitization decreases it is more difficult for FcRII to form rosettes, so an increased percentage of them is mediated via FcRI. Sensitization of SRBC with 1-1 5 x 103 anti-SRBC rabbit IgG molecules per erythrocyte is the threshold to allow FcRII to mediate rosettes. At the lowest levels of sensitization the total number of rosettes is even lower and all rosettes are mediated via FcRI, hence mAb 10.1 is fully inhibitory. In addition, our data strongly support the view that the ingestion of EA takes place mainly via FcRII. We show in this study that while binding of slightly sensitized erythrocytes was blocked efficiently by mAb 10.1, the ingestion of the equivalent EA was hardly inhibited by it. INTRODUCTION
were inhibited by monoclonal antibodies against either FcRI and/or FcRII was compared.
Substantial evidence exists indicating molecular heterogeneity of Fc receptors for IgG (FcR) on human monocytes. How these various structures promote specific biogical consequences is currently under study. Various evidence suggests that only FcRI is involved in recognition of anti-D IgG-sensitized red cells on monocytes. ' At the same time, it is known that the rabbit IgG-coated erythrocytes bind to both the high (FcRI) and low (FcRII) affinity FcR.2 An explanation for the role of the two FcR can be given by blocking of rosette formation between U937 cell and erythrocytes sensitized with rabbit IgG by monoclonal antibody against FcRI (mAb 10.1).3 Here mAb 10.1 blocked essentially all rosette formation except when highly opsonized red cells were used. The experiments reported here extend these findings to identify the FcR primarily involved in the recognition of cellbound IgG by human monocytes. The binding of erythrocytes (E) sensitized with varying concentrations of anti-E rabbit IgG to monocytes was determined at 37°. The endocytic activity of monocytes was tested with red cells sensitized by the same way. The degree to which rosette formation and endocytic function
MATERIALS AND METHODS Cell preparation Twenty-four millilitres of peripheral human blood were anticoagulated with 10 U heparin/ml. Platelet-depleted mononuclear cells (MC) were obtained by centrifugation through Ficoll-Hypaque.4 The cells were resuspended in 2 ml of medium Parker-199 containing 20 mm HEPES, pH 7-4 (Flow, Irvine, Ayrshire, U.K.) and counted in a standard haemocytometer. Thirty to forty per cent of the MC were found to be monocytes by non-specific esterase staining and by neutral red phagocytosis. Monocytes were purified by adherence to a glass surface. Briefly, 100 pi of MC suspension containing 2-4 x 105 cells were placed on round cover slides immersed into wells of a tissue culture plastic plate. After 30 min at room temperature, the non-adherent cells were removed by rigorous washing. The adherent monolayer contained more than 80% monocytes (12 x 104 cells/glass slide). Close agreement was obtained by morphological examinations with Wright's stain, esterase staining and neutral red phagocytosis.
Correspondence: Dr M. Kdvai, 3rd Dept. of Medicine, Univ. Med. School of Debrecen, 4004 Debrecen, Hungary.
Monoclonal antibodies (mAb) and polyclonal antiserum IV3 supernatant is the cell culture fluid recovered from cloned hybrodoma cells producing murine IgG2b anti-FcRII, which
657
658
M. Kavai et al.
was generous gift of C.L. Anderson (Columbus, OH). The concentration of the mAb was 20-30 Mg/ml. mAb 10.1 is a murine IgGl anti-FcRI, which was the generous gift of Nancy Hogg (London, U.K.). The mAb was isolated from ascites fluid and its concentration was 8 5 mg/ml. Other mAb used in this study as controls were murine IgG2b (220 pg/ml) and IgGl (14 mg/ml), which were generous gifts of Eva Rajnavolgyi (G6d, Hungary). Rabbit anti-sheep red blood cell (SRBC) stroma serum (haemolysin) was purchased from HUMAN, Budapest, Hungary.
x co
0
53 0
2
-
0
Rosette-forming cell (RFC) assays The adherent cells (1-2 x 104) on the cover slides were treated with N-ethyl maleimide (NEM) of 2 5 mm in 200 p1 of H-Parker at room temperature for 30 min.5 After rigorous washing the cells were incubated with 2-5% sensitized SRBC (EA) in 200 pi H-Parker at 370 for 20 min. The erythrocytes were appropriately sensitized with rabbit IgG isolated from haemolysin. After washing, the cells were stained with 0-2% crystal violet and the percentage of RFC as well as the numbers EA bound to one monocyte, i.e. the binding index (BI), were determined.6 EA endocytosis by monocytes The adherent cells were incubated with the appropriately sensitized SRBC of 2-5% pl H-Parker at 37° for 20 min. After washing, the EA bound to the monocyte surface were lysed by water for 30 seconds. After washing and staining the percentage of monocytes ingesting three of more EA, as well as the numbers of EA internalized per one monocyte, i.e. the phagocytic index (PI), were determined. Determination of red cell bound IgG '251-protein-A was used to measure anti-SRBC IgG bound to the surface of sheep erythrocytes sensitized with varying dilutions of anti-SRBC rabbit IgG.7
Effect of anti-FcRI and anti-FcRII on monocyte FcR In the inhibition experiments the adherent cells (1-2 x 104/slide) were preincubated with mAb at room temperature for 10 min and then with phagocytic targets (EA) at 370 for 20 min. AntiFcRI mAb 10. 1, 17 pg/200 p1,3 anti-FcRII mAb IV3, 0-20 yg/ 200 pl and control mAb mouse IgGl, 21 pg/200 pi, and mouse IgG2b, 0-22 pg/200 pl, were used. This concentration of antiFcRII antibody was sufficient to stain 106 monocytes in an indirect immunofluorescence assay.8 For testing the saturating conditions of mAb IV3, rosette formation was inhibited -430 x 10-2 pg/200 pl mAb Ig2b. The EA binding and endocytosis by monocytes were tested in presence of these mAb. -
RESULTS Effect of anti-FcRI on binding and ingestion of EA by monocytes
The binding of anti-SRBC rabbit IgG to sheep erythrocytes depends on the amount of IgG added to the cells and incubated with them (Fig. l). Sensitized sheep erythrocytes (EA) bind to monocytes treated with N-ethyl maleimide (NEM) and form rosettes with them at 370 for 20 min. 2-5 mM NEM completely blocks the ingestion of EA by monocytes, whereas it has no
64 128 256 512 32 Reciprocal dilution of rabbit IgG added to erythrocytes
Figure 1. Binding of IgG to erythrocytes sensitized with various amounts of rabbit IgG. The bound IgG was measured with 1251-protein-A and expressed as molecules. Values represent mean of triplicate determinations + SD.
Table 1. Interactions of sensitized erythrocytes with monocytes pretreated with 2-5 mm N-ethyl maleimide (NEM)
Medium
Binding + ingestion
Ingestion
H-Parker 2 5 mm NEM
2 34 + 0 21 (BI + PI) 2 28+0-18 (BI)
2-07 + 0 16 (PI) 0
The adherent cells (1-2 x 104) were treated with H-Parker or NEM in H-Parker at room temperature for 30 min. After washing the cells were incubated with SRBC sensitized with rabbit IgG of 1:32 dilution at 370 for 20 min. The bound and ingested erythrocytes per adherent cell were determined (BI + PI). In parallel plates, after incubation at 37°, the cell-bound erythrocytes were lysed with water and the ingested erythrocytes per adherent cell determined (PI). Values represent mean of triplicate determinations + SD.
effect on binding of EA (Table 1). The binding of EA depends on the density of the IgG coating the erythrocytes (Fig. 2). The presence of anti-FcRI (mAb 10.1) in the incubation medium results in an effective inhibition of EA binding, which depends slightly on the sensitization of erythrocytes. Control murine IgGl had no inhibitory effect. The EA are almost completely ingested by untreated monocytes at 370 for 20 min. The EA bound to erythrocyte surface were lysed; in this way only the EA ingested by monocytes were detected. The internalization depended entirely on the concentration of specific antibody used to sensitize the red cells (Fig. 3). Preincubation of monocytes with anti-FcRI inhibited EA ingestion not depending on the magnitude of sensitization. The inhibition of EA binding via FcRI was higher than that of EA ingestion.
Binding and endocytosis via Fc gamma receptors a E
Control mAb
0 o
0
70 60-
-'
50.
0
am
659
2-5 -2 020, ^ 0. -
1)-5
._1 40 r-
mAb IV3
30-
