ClGn. exp. Immunol. (1979) 37,436-440
Suppressive effect of IgA soluble immune complexes on neutrophil chemotaxis S. ITO, H. MIKAWA, K. SHINOMIYA & T. YOSHIDA Department of Pediatrics, Kyoto University, Kyoto, Japan
(Acceptedfor publication 2 February 1979)
SUMMARY
The effect of IgA and IgG soluble immune complexes (SIC) on neutrophil chemotaxis was investigated. Six types of SIC were prepared from ic-type IgA and IgG myeloma proteins: IgA-anti-ic chain antibody SIC in IgA excess, IgA- anti-K chain antibody in SIC in anti-IC chain antibody excess, IgA-anti-a chain antibody SIC in IgA excess, IgG-anti-ic chain antibody SIC in IgG excess, IgG-anti-ic chain antibody SIC in anti-K chain antibody excess, and IgG-anti-y chain antibody SIC in IgG excess. Three types of IgA SIC had a marked suppressive effect on neutrophil chemotaxis, while IgG SIC, free IgA, free anti-K chain antibody, and free anti-y chain antibody showed no inhibitory activity. This suppressive effect on neutrophil chemotaxis caused specifically by polymerized IgA was a cell-directed one and was expressed in a concentration dependent manner.
INTRODUCTION Some forms of immune complexes such as the rheumatoid factor are known to cause increased susceptibility to infection by inhibiting neutrophil chemotaxis (Mowat & Baum, 1971) and reducing opsonic activity (Messner et al., 1968). Van Epps & Williams (1976) showed that IgA M components from IgA myeloma sera had a cell-directed inhibitory action on neutrophil chemotaxis and that these IgA M components reduced particle ingestion and bactericidal activity probably caused by reduced phagocytosis (Van Epps, Reed & Williams, 1978). We prepared various forms of soluble immune complexes (SIC) of K-type IgA and IgG isolated from myeloma sera: IgA-anti-K chain antibody SIC in IgA excess and anti-K chain antibody excess, IgAanti-cc chain antibody SIC in IgA excess, IgG-anti-K chain antibody SIC in IgG excess and anti-K chain antibody excess, and IgG-anti-y chain antibody SIC in IgG excess. We demonstrate herein the influence ofthe SIC on neutrophil chemotaxis. Three types of SIC containing IgA have a marked suppressive effect on neutrophil chemotaxis. This suppressive effect is cell-directed and is expressed in a concentration dependent manner. MATERIALS AND METHODS Purification ofIgA. IgA myeloma sera were obtained from patients with K-type IgA myeloma. IgA myeloma proteins were isolated by Pevikon block electrophoresis followed by Sephadex G-200 gel filtration (Muller-Eberhard, 1960). IgA myeloma proteins were then added to the rabbit anti-a chain antiserum (Hoechst) coupled with CNBr-activated Sepharose 4B beads. The bound IgA was eluted from the beads with cold glycine HC1 buffer pH 2-8, immediately neutralized with glycine NaOH buffer pH 10-6, dialysed against isotonic saline for 72 hr, and finally, concentrated in a colloidion bag. Correspondence: Dr S. Ito, Department of Pediatrics, Kyoto University, Sakyo-ku, Kyoto 606, Japan. 0099-9104/79/0090-0436$02.00 (© 1979 Blackwell Scientific Publications
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Purification ofIgG. IgG myeloma sera were obtained from patients with K-type IgG myeloma. IgG myeloma proteins were isolated by DEAE-cellulose chromatography with 0 01 M phosphate buffer pH 8-0. Pure IgG was obtained from IgG myeloma proteins by utilizing an immunoadsorbent column with rabbit anti-y chain antiserum (Hoechst) coupled with CNBr activated Sepharose 4B beads following the same method as for the purification of IgA. Purification ofrabbit anti-K chain antibody. Pure anti-K chain antibody was obtained from rabbit anti-K chain antiserum (Dakopatts) by utilizing an immunoadsorbent column with the y globulin fraction of pooled sera coupled with CNBractivated Sepharose 4B beads using the same method as for the purification of IgA. Immunization and purification of rabbit anti-a chain antibody. A rabbit was immunized with pure IgA obtained by the method described above. Antiserum was absorbed with pure IgG, pure IgM, and Bence-Jones proteins (Kit). The y globulin fraction of this absorbed serum containing anti-a chain antibody was collected using 33% ammonium sulphate. Pure rabbit anti-a chain antibody was obtained from this partially purified anti-a chain antiserum utilizing an immunoadsorbent column with pure IgA coupled with CNBr activated Sepharose 4B beads using the same method as for the purification of IgA. Immunization and purification of rabbit anti-y chain antibody. Pure anti-y chain antibody was obtained using the same method as for the purification of anti-a chain antibody. Antiserum against IgG was made using pure IgG obtained by the method described above and was absorbed with pure IgA, pure IgM, and Bence-Jones proteins. The y globulin fraction of this absorbed serum containing anti-y chain antibody was collected using 33% ammonium sulphate. Pure rabbit anti-y chain antibody was obtained from this partially purified anti-y chain antiserum utilizing an immunoadsorbent column with pure IgG coupled with CNBr-activated Sepharose 4B beads using the same method as for the purification of IgA. Preparation of immune complexes and soluble immune complexes. Pure IgA and IgG as antigens and pure anti-K chain antibody, anti-a chain antibody and anti-a chain antibody, made using the method described above, were used to make immune complexes: IgA-anti-K chain antibody, IgA-anti-y chain antibody as antibodies, IgG-anti-K chain antibody and IgG-anti-y chain antibody. Various volumes of antigen were added to a constant volume of antibody and the ratio of the volume of antigen to antibody which makes up the maximum antigen-antibody complex was determined by measuring the protein content of each insoluble immune complex, using the method of Lowry et al. (1951). The ratios of the volume of antigen to antibody used to produce the maximum antigen-antibody complex were as follows: IgA: anti-K chain antibody 1 5 : 1; IgA: anti-a chain antibody= 2-6: 1; IgG: anti-K chain antibody= 1-7: 1; and IgG: anti-y chain antibody=24: 1. Immune complexes made using antigen and antibody in the ratios described above were made soluble by adding antigen or antibody in three times the volume used to make each individual immune complex. Each supernatant containing soluble immune complex (SIC) and free antigen or antibody is abbreviated as follows: (1) IgA-anti-K chain antibody SIC in IgA excess+free IgA as IgA-anti-K SIC in IgA excess; (2) IgA-anti-K chain antibody SIC in anti-K chain antibody excess +free anti-K chain antibody as IgA-anti-K SIC in anti-K excess; (3) IgA-anti-a chain antibody in IgA excess SIC+free IgA as IgA-anti-a SIC in IgA excess; (4) IgGanti-K chain antibody in IgG excess SIC+free IgG as IgG-anti-K SIC in IgG excess; (5) IgG-anti-K chain antibody SIC in anti-K chain antibody excess+ free anti-K chain antibody as IgG-anti-K SIC in anti-K excess; (6) IgG-anti-y chain antibody SIC in IgG excess+free IgG as IgG-anti-y SIC in IgG excess. Chemotaxis. The inhibitory action of each SIC on neutrophil chemotaxis was tested using Boyden chambers. Chemotaxis was studied according to the method of Clark & Kimball (1971) with slight modifications. Samples. The samples used were pure IgA, pure IgG, pure anti-K chain antibody, pure anti-a chain antibody, pure anti-y chain antibody, IgA-anti-K SIC in IgA excess, IgA-anti-K SIC in anti-K excess, IgA-anti-a SIC in IgA excess, IgGanti-K SIC in IgG excess, IgG-anti-K SIC in anti-K excess, and IgG anti-y SIC in IgG excess. Cell preparation. Neutrophils were obtained from healthy adult donors. After sedimentation of erythrocytes using 6% dextran, mononuclear cells were removed by Ficoll-Hypaque centrifugation. A small number of erythrocytes were then removed by hypotonic haemolysis. Cell suspensions obtained by this method contained more than 95% neutrophils. Neutrophils were washed twice with Hanks' balanced salt solution (HBSS) and were then resuspended to make a concentration of 1-3 x 106 neutrophils/ml. These neutrophils were pre-incubated with 0 1 ml of each sample at various protein concentrations, washed twice with HBSS and resuspended to make a final concentration of 1 x 106 neutrophils/0-8 ml. Chemotactic factor. Endotoxin-activated fresh serum from a healthy donor was used as the chemotactic factor in the lower chamber (Snyderman, Gewurz, & Mergenhagen, 1968). Chemotactic assay. 0-8 ml of each neutrophil suspension was placed in the upper chamber, to the bottom of which a Millipore filter of 5 p of pore size (SMWP 01300, Millipore Corporation) was attached. Neutrophils pre-incubated with the donor's own serum were used as a control. Following incubation for 3 hr at 370C, the Millipore filters were fixed and stained, and the total number of cells reaching the lowest surface of each filter (chemotactic cells) was counted. The inhibitory activity of each sample on chemotaxis is described by a chemotactic index (%) which is defined as follows: chemotactic cells pre-incubated with sample control chemotactic cells (100% means that the sample has no inhibitory activity on chemotaxis.) To make it easy to compare the influence of each sample on chemotaxis, neutrophils pre-incubated with each sample containing the same total protein was studied concurrently using neutrophils from the same donor.
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RESULTS Effect ofpure IgA, pure IgG, pure anti-K chain antibody, pure anti-a chain antibody, and pure anti-y chain antibody Pure IgA, pure IgG, pure anti-K chain antibody, pure anti-a chain antibody, pure anti-y chain antibody had no effect on neutrophil chemotaxis at least at the low protein concentration of 31 Lg. Chemotactic indexes of each sample at 31 gg/106 neutrophils were as follows: pure IgA 103-3%, pure IgG 102-7%, pure anti-K chain antibody 100%, pure anti-a chain antibody 98.8%, and pure anti-y chain antibody 97 3%.
Effect ofIgA-anti-a SIC in IgA excess and IgG-anti-y SIC in IgG excess on chemotaxis IgA-anti-a SIC in IgA excess had a marked concentration-dependent suppressive effect on chemotaxis (Fig. 1). More than 50% inhibition was seen using a concentration of as low as 1V8 jig/106 neutrophils. IgG-anti-y SIC in IgG excess had almost no suppressive effect on chemotaxis.
Effect ofIgA-anti-K SIC and IgG-anti-K SIC on chemotaxis IgA-anti-K SIC in both IgA excess and anti-K excess showed a marked concentration dependent suppressive effect on chemotaxis (Figs 2 & 3). More than 50% inhibition was seen using a concentration of as low as 3d1 gg/106 neutrophils. On the other hand, IgG-anti-K SIC in IgG excess and anti-K excess had no suppressive effect on chemotaxis. !00 _
._0~~~~~~ X
50 0
E
0-18
1.8
18
jig/106 neutrophi Is 0) and IgG-anti-y SIC in IgG excess (a-9) on FIG. 1. Influence of IgA-anti-a SIC in IgA excess (0 chemotaxis. IgA-anti-a SIC in IgA excess had a marked concentration-dependent suppressive effect on chemotaxis. IgG-anti-y SIC in IgG excess had almost no suppressive effect on chemotaxis. 100 0_
.y
0
_
50
0
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3X1 0.31 ig/i106 neutrophi Is
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a) and IgG-anti-K SIC in IgG excess (-@*) on FIG. 2. Influence of IgA-anti-K SIC in IgA excess (a chemotaxis. IgA-anti-K SIC in IgA excess had a marked concentration-dependent suppressive effect on chemotaxis. IgG-anti-K SIC in IgG excess had no suppressive effect on chemotaxis.
Suppressive effect ofIgA SIC on chemotaxis 100
439
_
0)~~~~~~~~ 50
-
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E
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0-31 3-1 tLg /I 06 neutrophils
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FIG. 3. Influence of IgA-anti-K SIC in anti-K excess (0 ) 0) and IgG-anti-K SIC in anti-K excess ( on chemotaxis. IgA-anti-K SIC in anti-K excess had a marked concentration-dependent suppressive effect on chemotaxis. IgG-anti-K SIC in anti-K excess had no suppressive effect on chemotaxis.
DISCUSSION All of the three kinds of IgA SIC, i.e. IgA-anti-K SIC in IgA excess, IgA-anti-K SIC in anti-K excess and IgA-anti-a SIC in IgA excess showed a concentration-dependent suppressive effect on neutrophil chemotaxis as shown in Figs 1-3. More than 50% inhibitory activity was seen at a concentration as low as 3-1 gg/106 neutrophils for IgA-anti-K SIC in IgA excess and anti-K excess, and as low as 1P8 ig/106 neutrophils for IgA-anti-a SIC in IgA excess. This inhibitory activity is caused by SIC and not by free IgA, free anti-iK chain antibody or free anti-a chain antibody because these free proteins alone had no inhibitory activity on neutrophil chemotaxis, at least at these low concentrations. On the contrary, three types of IgG SIC, i.e. IgG-anti-K SIC in IgG excess and anti-K excess and IgG-anti-y SIC in IgG excess had almost no suppressive effect on chemotaxis. Four conclusions can be drawn from these data in relation to the inhibitory activity of IgA SIC on chemotaxis. (1) This inhibitory effect is not due to the cytotoxic effect of IgA SIC as far as testing with trypan blue is concerned: more than 99% of the cells pre-incubated with IgA SIC were viable. (2) This effect is a cell-directed one as the effect of SIC on chemotaxis was examined after washing the cells to remove free SIC. (3) This effect is not due to enzymatic change induced by the binding of IgA SIC to neutrophils. Although IgG1, IgG3, IgAl and IgA2 are cytophilic for neutrophils (Spiegelberg, Lawrence & Henson, 1974) and insoluble aggregates of IgG and IgA induce a release of f glucuronidase following phagocytosis (Henson, Johnson & Spiegelberg, 1972), soluble complexes of IgA and IgG do not induce liberation of this enzyme unless these immune complexes are bound to a non-phagocytosable surface, such as Millipore filters, prior to incubation with neutrophils (Henson et al., 1972). (4) This inhibitory effect is caused by a polymerized type of IgA, because free IgA, free anti-K chain antibody, free anti-K chain antibody and IgG SIC containing anti-K chain antibody had no inhibitory effect on chemotaxis. We used two types of IgA and IgG polymerized through light chains and heavy chains. Polymerized IgA had much greater inhibitory activity on neutrophil chemotaxis than did polymerized IgG. The inhibitory activity on neutrophil chemotaxis of polymer IgA can be attributed to the stereotypic change of IgA induced by polymerization, as monomer IgA had no inhibitory activity on chemotaxis. This activity was probably the result of interactions between IgA and cell membranes of neutrophils. This is supported by the data of Van Epps et al. (1976) who demonstrated that polymer IgA formed by heat aggregation also showed inhibitory activity on neutrophil chemotaxis and that the activity was greatly reduced by both pepsin digestion and reduction and alkylation with dithiothreitol and iodoacetamide. These workers also showed that polymer IgA had only a slight inhibitory activity on monocyte chemotaxis. Lawrence, Weigle & Spiegelberg (1975) demonstrated that monocytes did not have IgA receptors but that neutrophils had both IgG and IgA receptors, and proved that these two receptors were not identical as demonstrated by a cross inhibition study using 125I-labelled human immunoglobulins.
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All these data suggest that the depressed chemotaxis seen in our study was caused by the polymer IgA attached to the neutrophil IgA receptors. Changes in the neutrophil membrane near the IgA receptors induced by the attachment of polymer IgA to the receptors rather than occupancy of IgA receptors seems to play an important role in the suppression of chemotaxis, as monomer IgA which also occupies IgA receptors has no inhibitory activity on chemotaxis. It is probable that IgA SIC bind to IgA receptors and blocks the chemotactic receptors or induces metabolic change and thus a depressed chemotaxis. We are grateful to Dr K. Takatsuki, First Division of the Department of Internal Medicine, for the provision of sera of IgA and IgG multiple myeloma and to M. Ohara for assistance in preparing this manuscript.
REFERENCES morphonuclear leukocytes from patients with rheumatoid CLARK, R.A. & KIMBALL, H.R. (1971) Defective granulocyte chemotaxis in the Chediak-Higashi syndrome. arthritis.]. clin. Invest. 50, 2541. 3. clin. Invest. 50, 2645. MULLER-EBERHARD, H.J. (1960) A new supporting medium HENSON, P.M., JOHNSON, H.B. & SPIEGELBERG, H.L. (1972) for preparative electrophoresis. Scand. a. clin. Lab. The release of granule enzymes from human neutrophils Invest. 12, 33. stimulated by aggregated immunoglobulins of different SNYDERMAN, R., GEWURZ, H. & MERGENHAGEN, S.E. (1968) Interactions of the complement system with endotoxic classes and subclasses.J7. Immunol. 109, 1182. lipopolysaccharide.J. exp. Med. 128,259. LAWRENCE, D.A., WEIGLE, W.O. T SPIEGELBERG, H.L. (1975) Immunoglobulins cytophilic for human leuko- SPIEGELBERG, H.L., LAWRENCE, D.A. & HENSON, P. (1974) cytes, monocytes, and neutrophils.J. clin. Invest. 55, 368. Cytophilic properties of IgA to human neutrophils. Adv. exp. Med. Biol. 45, 67. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951) Protein measurement with the Folin phenol VAN Epps, D.E., REED, K. & WILLIAMS, R.C., JR. (1978) reagent. J. biol. Chem. 193, 265. Suppression of human PMN bactericidal activity by MESSNER, R.P., LAXDAL, T., QUIE, P.G. & WILLIAMS, R.C., human IgA paraproteins. Cell. Immunol. 36, 363. JR. (1968) Serum opsonin, bacteria, and polymorpho- VAN Epps, D.E. & WILLAMs, R.C., JR. (1976) Suppression nuclear leukocyte interactions in subacute bacterial endoof leukocyte chemotaxis by human IgA myeloma comcarditis. J. clin. Invest. 47, 1109. ponents.]. exp. Med. 144,1227. MOWAT, A.G. & BAUM, J. (1971) Chemotaxis of poly-
Suppressive effect of IgA soluble immune complexes on neutrophil chemotaxis S. ITO, H. MIKAWA, K. SHINOMIYA & T. YOSHIDA Department of Pediatrics, Kyoto University, Kyoto, Japan
(Acceptedfor publication 2 February 1979)
SUMMARY
The effect of IgA and IgG soluble immune complexes (SIC) on neutrophil chemotaxis was investigated. Six types of SIC were prepared from ic-type IgA and IgG myeloma proteins: IgA-anti-ic chain antibody SIC in IgA excess, IgA- anti-K chain antibody in SIC in anti-IC chain antibody excess, IgA-anti-a chain antibody SIC in IgA excess, IgG-anti-ic chain antibody SIC in IgG excess, IgG-anti-ic chain antibody SIC in anti-K chain antibody excess, and IgG-anti-y chain antibody SIC in IgG excess. Three types of IgA SIC had a marked suppressive effect on neutrophil chemotaxis, while IgG SIC, free IgA, free anti-K chain antibody, and free anti-y chain antibody showed no inhibitory activity. This suppressive effect on neutrophil chemotaxis caused specifically by polymerized IgA was a cell-directed one and was expressed in a concentration dependent manner.
INTRODUCTION Some forms of immune complexes such as the rheumatoid factor are known to cause increased susceptibility to infection by inhibiting neutrophil chemotaxis (Mowat & Baum, 1971) and reducing opsonic activity (Messner et al., 1968). Van Epps & Williams (1976) showed that IgA M components from IgA myeloma sera had a cell-directed inhibitory action on neutrophil chemotaxis and that these IgA M components reduced particle ingestion and bactericidal activity probably caused by reduced phagocytosis (Van Epps, Reed & Williams, 1978). We prepared various forms of soluble immune complexes (SIC) of K-type IgA and IgG isolated from myeloma sera: IgA-anti-K chain antibody SIC in IgA excess and anti-K chain antibody excess, IgAanti-cc chain antibody SIC in IgA excess, IgG-anti-K chain antibody SIC in IgG excess and anti-K chain antibody excess, and IgG-anti-y chain antibody SIC in IgG excess. We demonstrate herein the influence ofthe SIC on neutrophil chemotaxis. Three types of SIC containing IgA have a marked suppressive effect on neutrophil chemotaxis. This suppressive effect is cell-directed and is expressed in a concentration dependent manner. MATERIALS AND METHODS Purification ofIgA. IgA myeloma sera were obtained from patients with K-type IgA myeloma. IgA myeloma proteins were isolated by Pevikon block electrophoresis followed by Sephadex G-200 gel filtration (Muller-Eberhard, 1960). IgA myeloma proteins were then added to the rabbit anti-a chain antiserum (Hoechst) coupled with CNBr-activated Sepharose 4B beads. The bound IgA was eluted from the beads with cold glycine HC1 buffer pH 2-8, immediately neutralized with glycine NaOH buffer pH 10-6, dialysed against isotonic saline for 72 hr, and finally, concentrated in a colloidion bag. Correspondence: Dr S. Ito, Department of Pediatrics, Kyoto University, Sakyo-ku, Kyoto 606, Japan. 0099-9104/79/0090-0436$02.00 (© 1979 Blackwell Scientific Publications
436
Suppressive effect of IgA SIC on chemotaxis
437
Purification ofIgG. IgG myeloma sera were obtained from patients with K-type IgG myeloma. IgG myeloma proteins were isolated by DEAE-cellulose chromatography with 0 01 M phosphate buffer pH 8-0. Pure IgG was obtained from IgG myeloma proteins by utilizing an immunoadsorbent column with rabbit anti-y chain antiserum (Hoechst) coupled with CNBr activated Sepharose 4B beads following the same method as for the purification of IgA. Purification ofrabbit anti-K chain antibody. Pure anti-K chain antibody was obtained from rabbit anti-K chain antiserum (Dakopatts) by utilizing an immunoadsorbent column with the y globulin fraction of pooled sera coupled with CNBractivated Sepharose 4B beads using the same method as for the purification of IgA. Immunization and purification of rabbit anti-a chain antibody. A rabbit was immunized with pure IgA obtained by the method described above. Antiserum was absorbed with pure IgG, pure IgM, and Bence-Jones proteins (Kit). The y globulin fraction of this absorbed serum containing anti-a chain antibody was collected using 33% ammonium sulphate. Pure rabbit anti-a chain antibody was obtained from this partially purified anti-a chain antiserum utilizing an immunoadsorbent column with pure IgA coupled with CNBr activated Sepharose 4B beads using the same method as for the purification of IgA. Immunization and purification of rabbit anti-y chain antibody. Pure anti-y chain antibody was obtained using the same method as for the purification of anti-a chain antibody. Antiserum against IgG was made using pure IgG obtained by the method described above and was absorbed with pure IgA, pure IgM, and Bence-Jones proteins. The y globulin fraction of this absorbed serum containing anti-y chain antibody was collected using 33% ammonium sulphate. Pure rabbit anti-y chain antibody was obtained from this partially purified anti-y chain antiserum utilizing an immunoadsorbent column with pure IgG coupled with CNBr-activated Sepharose 4B beads using the same method as for the purification of IgA. Preparation of immune complexes and soluble immune complexes. Pure IgA and IgG as antigens and pure anti-K chain antibody, anti-a chain antibody and anti-a chain antibody, made using the method described above, were used to make immune complexes: IgA-anti-K chain antibody, IgA-anti-y chain antibody as antibodies, IgG-anti-K chain antibody and IgG-anti-y chain antibody. Various volumes of antigen were added to a constant volume of antibody and the ratio of the volume of antigen to antibody which makes up the maximum antigen-antibody complex was determined by measuring the protein content of each insoluble immune complex, using the method of Lowry et al. (1951). The ratios of the volume of antigen to antibody used to produce the maximum antigen-antibody complex were as follows: IgA: anti-K chain antibody 1 5 : 1; IgA: anti-a chain antibody= 2-6: 1; IgG: anti-K chain antibody= 1-7: 1; and IgG: anti-y chain antibody=24: 1. Immune complexes made using antigen and antibody in the ratios described above were made soluble by adding antigen or antibody in three times the volume used to make each individual immune complex. Each supernatant containing soluble immune complex (SIC) and free antigen or antibody is abbreviated as follows: (1) IgA-anti-K chain antibody SIC in IgA excess+free IgA as IgA-anti-K SIC in IgA excess; (2) IgA-anti-K chain antibody SIC in anti-K chain antibody excess +free anti-K chain antibody as IgA-anti-K SIC in anti-K excess; (3) IgA-anti-a chain antibody in IgA excess SIC+free IgA as IgA-anti-a SIC in IgA excess; (4) IgGanti-K chain antibody in IgG excess SIC+free IgG as IgG-anti-K SIC in IgG excess; (5) IgG-anti-K chain antibody SIC in anti-K chain antibody excess+ free anti-K chain antibody as IgG-anti-K SIC in anti-K excess; (6) IgG-anti-y chain antibody SIC in IgG excess+free IgG as IgG-anti-y SIC in IgG excess. Chemotaxis. The inhibitory action of each SIC on neutrophil chemotaxis was tested using Boyden chambers. Chemotaxis was studied according to the method of Clark & Kimball (1971) with slight modifications. Samples. The samples used were pure IgA, pure IgG, pure anti-K chain antibody, pure anti-a chain antibody, pure anti-y chain antibody, IgA-anti-K SIC in IgA excess, IgA-anti-K SIC in anti-K excess, IgA-anti-a SIC in IgA excess, IgGanti-K SIC in IgG excess, IgG-anti-K SIC in anti-K excess, and IgG anti-y SIC in IgG excess. Cell preparation. Neutrophils were obtained from healthy adult donors. After sedimentation of erythrocytes using 6% dextran, mononuclear cells were removed by Ficoll-Hypaque centrifugation. A small number of erythrocytes were then removed by hypotonic haemolysis. Cell suspensions obtained by this method contained more than 95% neutrophils. Neutrophils were washed twice with Hanks' balanced salt solution (HBSS) and were then resuspended to make a concentration of 1-3 x 106 neutrophils/ml. These neutrophils were pre-incubated with 0 1 ml of each sample at various protein concentrations, washed twice with HBSS and resuspended to make a final concentration of 1 x 106 neutrophils/0-8 ml. Chemotactic factor. Endotoxin-activated fresh serum from a healthy donor was used as the chemotactic factor in the lower chamber (Snyderman, Gewurz, & Mergenhagen, 1968). Chemotactic assay. 0-8 ml of each neutrophil suspension was placed in the upper chamber, to the bottom of which a Millipore filter of 5 p of pore size (SMWP 01300, Millipore Corporation) was attached. Neutrophils pre-incubated with the donor's own serum were used as a control. Following incubation for 3 hr at 370C, the Millipore filters were fixed and stained, and the total number of cells reaching the lowest surface of each filter (chemotactic cells) was counted. The inhibitory activity of each sample on chemotaxis is described by a chemotactic index (%) which is defined as follows: chemotactic cells pre-incubated with sample control chemotactic cells (100% means that the sample has no inhibitory activity on chemotaxis.) To make it easy to compare the influence of each sample on chemotaxis, neutrophils pre-incubated with each sample containing the same total protein was studied concurrently using neutrophils from the same donor.
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RESULTS Effect ofpure IgA, pure IgG, pure anti-K chain antibody, pure anti-a chain antibody, and pure anti-y chain antibody Pure IgA, pure IgG, pure anti-K chain antibody, pure anti-a chain antibody, pure anti-y chain antibody had no effect on neutrophil chemotaxis at least at the low protein concentration of 31 Lg. Chemotactic indexes of each sample at 31 gg/106 neutrophils were as follows: pure IgA 103-3%, pure IgG 102-7%, pure anti-K chain antibody 100%, pure anti-a chain antibody 98.8%, and pure anti-y chain antibody 97 3%.
Effect ofIgA-anti-a SIC in IgA excess and IgG-anti-y SIC in IgG excess on chemotaxis IgA-anti-a SIC in IgA excess had a marked concentration-dependent suppressive effect on chemotaxis (Fig. 1). More than 50% inhibition was seen using a concentration of as low as 1V8 jig/106 neutrophils. IgG-anti-y SIC in IgG excess had almost no suppressive effect on chemotaxis.
Effect ofIgA-anti-K SIC and IgG-anti-K SIC on chemotaxis IgA-anti-K SIC in both IgA excess and anti-K excess showed a marked concentration dependent suppressive effect on chemotaxis (Figs 2 & 3). More than 50% inhibition was seen using a concentration of as low as 3d1 gg/106 neutrophils. On the other hand, IgG-anti-K SIC in IgG excess and anti-K excess had no suppressive effect on chemotaxis. !00 _
._0~~~~~~ X
50 0
E
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1.8
18
jig/106 neutrophi Is 0) and IgG-anti-y SIC in IgG excess (a-9) on FIG. 1. Influence of IgA-anti-a SIC in IgA excess (0 chemotaxis. IgA-anti-a SIC in IgA excess had a marked concentration-dependent suppressive effect on chemotaxis. IgG-anti-y SIC in IgG excess had almost no suppressive effect on chemotaxis. 100 0_
.y
0
_
50
0
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0.031
3X1 0.31 ig/i106 neutrophi Is
31
a) and IgG-anti-K SIC in IgG excess (-@*) on FIG. 2. Influence of IgA-anti-K SIC in IgA excess (a chemotaxis. IgA-anti-K SIC in IgA excess had a marked concentration-dependent suppressive effect on chemotaxis. IgG-anti-K SIC in IgG excess had no suppressive effect on chemotaxis.
Suppressive effect ofIgA SIC on chemotaxis 100
439
_
0)~~~~~~~~ 50
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0-31 3-1 tLg /I 06 neutrophils
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FIG. 3. Influence of IgA-anti-K SIC in anti-K excess (0 ) 0) and IgG-anti-K SIC in anti-K excess ( on chemotaxis. IgA-anti-K SIC in anti-K excess had a marked concentration-dependent suppressive effect on chemotaxis. IgG-anti-K SIC in anti-K excess had no suppressive effect on chemotaxis.
DISCUSSION All of the three kinds of IgA SIC, i.e. IgA-anti-K SIC in IgA excess, IgA-anti-K SIC in anti-K excess and IgA-anti-a SIC in IgA excess showed a concentration-dependent suppressive effect on neutrophil chemotaxis as shown in Figs 1-3. More than 50% inhibitory activity was seen at a concentration as low as 3-1 gg/106 neutrophils for IgA-anti-K SIC in IgA excess and anti-K excess, and as low as 1P8 ig/106 neutrophils for IgA-anti-a SIC in IgA excess. This inhibitory activity is caused by SIC and not by free IgA, free anti-iK chain antibody or free anti-a chain antibody because these free proteins alone had no inhibitory activity on neutrophil chemotaxis, at least at these low concentrations. On the contrary, three types of IgG SIC, i.e. IgG-anti-K SIC in IgG excess and anti-K excess and IgG-anti-y SIC in IgG excess had almost no suppressive effect on chemotaxis. Four conclusions can be drawn from these data in relation to the inhibitory activity of IgA SIC on chemotaxis. (1) This inhibitory effect is not due to the cytotoxic effect of IgA SIC as far as testing with trypan blue is concerned: more than 99% of the cells pre-incubated with IgA SIC were viable. (2) This effect is a cell-directed one as the effect of SIC on chemotaxis was examined after washing the cells to remove free SIC. (3) This effect is not due to enzymatic change induced by the binding of IgA SIC to neutrophils. Although IgG1, IgG3, IgAl and IgA2 are cytophilic for neutrophils (Spiegelberg, Lawrence & Henson, 1974) and insoluble aggregates of IgG and IgA induce a release of f glucuronidase following phagocytosis (Henson, Johnson & Spiegelberg, 1972), soluble complexes of IgA and IgG do not induce liberation of this enzyme unless these immune complexes are bound to a non-phagocytosable surface, such as Millipore filters, prior to incubation with neutrophils (Henson et al., 1972). (4) This inhibitory effect is caused by a polymerized type of IgA, because free IgA, free anti-K chain antibody, free anti-K chain antibody and IgG SIC containing anti-K chain antibody had no inhibitory effect on chemotaxis. We used two types of IgA and IgG polymerized through light chains and heavy chains. Polymerized IgA had much greater inhibitory activity on neutrophil chemotaxis than did polymerized IgG. The inhibitory activity on neutrophil chemotaxis of polymer IgA can be attributed to the stereotypic change of IgA induced by polymerization, as monomer IgA had no inhibitory activity on chemotaxis. This activity was probably the result of interactions between IgA and cell membranes of neutrophils. This is supported by the data of Van Epps et al. (1976) who demonstrated that polymer IgA formed by heat aggregation also showed inhibitory activity on neutrophil chemotaxis and that the activity was greatly reduced by both pepsin digestion and reduction and alkylation with dithiothreitol and iodoacetamide. These workers also showed that polymer IgA had only a slight inhibitory activity on monocyte chemotaxis. Lawrence, Weigle & Spiegelberg (1975) demonstrated that monocytes did not have IgA receptors but that neutrophils had both IgG and IgA receptors, and proved that these two receptors were not identical as demonstrated by a cross inhibition study using 125I-labelled human immunoglobulins.
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All these data suggest that the depressed chemotaxis seen in our study was caused by the polymer IgA attached to the neutrophil IgA receptors. Changes in the neutrophil membrane near the IgA receptors induced by the attachment of polymer IgA to the receptors rather than occupancy of IgA receptors seems to play an important role in the suppression of chemotaxis, as monomer IgA which also occupies IgA receptors has no inhibitory activity on chemotaxis. It is probable that IgA SIC bind to IgA receptors and blocks the chemotactic receptors or induces metabolic change and thus a depressed chemotaxis. We are grateful to Dr K. Takatsuki, First Division of the Department of Internal Medicine, for the provision of sera of IgA and IgG multiple myeloma and to M. Ohara for assistance in preparing this manuscript.
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