Mechanism of Inhibition of Immunoglobulin G-Mediated Phagocytosis by Monoclonal Antibodies That Recognize the Mac- 1 Antigen
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Mechanism of inhibition of immunoglobulin G-mediated phagocytosis by monoclonal antibodies that recognize the Mac- 1 antigen. E J Brown, … , J F Bohnsack, H D Gresham J Clin Invest. 1988;81(2):365-375. https://doi.org/10.1172/JCI113328. Research Article We have investigated the effects of the monoclonal antibodies against the cell surface molecule Mac-1 on C3bi-mediated rosetting and IgG-mediated rosetting and phagocytosis by human peripheral blood monocytes. Highly purified M1/70 F(ab')2, used in the fluid phase, inhibited both monocyte functions. Half-maximal C3bi rosette inhibition occurred at a concentration of 2 nM F(ab')2 M1/70. An equivalent decrease in IgG-mediated rosetting required 10 nM M1/70 F(ab')2, and 50% inhibition of IgG-mediated phagocytosis required 7 nM antibody. Mo-1 F(ab')2 inhibited EC3bi binding with an ID50 of 0.3 nM, whereas 50% decrease in IgG-mediated rosetting required 70 nM of this antibody. OKM1 did not inhibit rosettes of sheep erythrocytes opsonized with IgG antibody (EA) at all. F(ab')2 M1/70 did not affect the binding of monomeric human IgG to monocytes, but did substantially decrease the binding of IgG aggregates. Half-maximal inhibition of aggregated IgG binding at 0 degrees C occurred at 8 nM F(ab')2 M1/70, very close to the concentration that caused equivalent inhibition of IgG-mediated phagocytosis. Aggregated IgG inhibited the binding of radiolabeled M1/70 to monocytes by approximately 40%, suggesting that some, but not all Mac-1 molecules were associated with IgG receptors under these conditions. When cells were allowed to adhere to surfaces coated with M1/70 or Mo-1 F(ab')2, C3bi- mediated rosetting was inhibited, but IgG mediated-phagocytosis was unaffected. Moreover, the dose response of […] Find the latest version: https://jci.me/113328/pdf Mechanism of Inhibition of Immunoglobulin G-mediated Phagocytosis by Monoclonal Antibodies That Recognize the Mac-1 Antigen Eric J. Brown, John F. Bohnsack, and Hattie D. Gresham Departments ofMedicine, and Microbiology and Immunology, Washington University School ofMedicine, St. Louis, Missouri 63110 Abstract tion apparently occurs because of antibody binding to a sub- population of Mac-i molecules which are associated with IgG We have investigated the effects of the monoclonal antibodies Fc receptors and remain on the apical membrane of monocytes against the cell surface molecule Mac-i on C3bi-mediated ro- adherent to anti-Mac-l-coated surfaces. We suggest that setting and IgG-mediated rosetting and phagocytosis by there may be two functionally distinct molecules on human human peripheral blood monocytes. Highly purified M1/70 monocytes recognized by M1/70 and Mo-1 that can be distin- F(ab')2, used in the fluid phase, inhibited both monocyte func- guished by their mobility in the plane of the monocyte mem- tions. Half-maximal C3bi rosette inhibition occurred at a con- brane. The more mobile form of Mac-i is involved in C3bi centration of 2 nM F(ab')2 Mi/70. An equivalent decrease in rosettes, and does not affect IgG-mediated phagocytosis. The IgG-mediated rosetting required 10 nM M1/70 F(ab)2, and other antigen recognized by Mi/70 does not diffuse within the 50% inhibition of IgG-mediated phagocytosis required 7 nM plane of the membrane; ligation of the latter molecule by anti- antibody. Mo-i F(ab'h inhibited EC3bi binding with an ID5s of body is associated with inhibition of IgG-mediated phagocy- 0.3 nM, whereas 50% decrease in IgG-mediated rosetting re- tosis. quired 70 nM of this antibody. OKMi did not inhibit rosettes Introduction of sheep erythrocytes opsonized with IgG antibody (EA) at all. F(ab')2 M1/70 did not affect the binding of monomeric human The mAb M1/70 recognizes a two-chain, cell surface glyco- IgG to monocytes, but did substantially decrease the binding of protein on mouse and human leukocytes termed Mac- I (1, 2). IgG aggregates. Half-maximal inhibition of aggregated IgG This antigen is a member of a family of cell surface molecules binding at 0°C occurred at 8 nM F(ab)2 Mi/70, very close to which are heterodimers of noncovalently linked polypeptides. the concentration that caused equivalent inhibition of IgG-me- Although each member of the family has a unique a chain, the diated phagocytosis. Aggregated IgG inhibited the binding of 3 chains of all these molecules are apparently identical. The radiolabeled M1/70 to monocytes by 40%, suggesting that Mac- I antigen is expressed on granulocytes, monocytes, natu- some, but not all Mac-i molecules were associated with IgG ral killer cells, and some B and T lymphocytes and has been receptors under these conditions. functionally identified with the type 3 complement receptor When cells were allowed to adhere to surfaces coated with (CR3)' and the iC3b receptor (3). M1/70 and certain other M1/70 or Mo-i F(ab32, C3bi-mediated rosetting was inhib- antibodies that recognize Mac-i inhibit the binding of iC3b- ited, but IgG mediated-phagocytosis was unaffected. More- coated erythrocytes (EC3bi) to leukocytes. Moreover, cells over, the dose response of inhibition of phagocytosis by fluid- from patients lacking this membrane antigen fail to rosette phase F(ab')2, of anti-Mac-i monoclonals was similar on with EC3bi. monocytes adherent to albumin-coated and antibody-coated Some studies have attributed other inhibitory properties to surfaces. Kinetic experiments showed that even prolonged in- monoclonal antibodies against Mac-1. Arnaout et al. (4) cubation of monocytes on M1/70 coated surfaces did not lead showed that the IgG2a mAb Mo-I (which recognizes Mac-i) to inhibition of EA binding nor did these incubations alter the and its Fab fragment inhibited IgG-Fc receptor-mediated pha- dose response for inhibition of EA binding by fluid-phase gocytosis as well as EC3bi rosetting (4). We have shown that M1/70 F(ab')2. This suggested that not all molecules recog- M 1/70 inhibited the binding of fibronectin-coated micro- nized by M1/70 are freely mobile in the plasma membrane. spheres to monocytes and PMN (5), and Ezekowitz et al. (6) Indeed, only 60% of 125I-Ml/70-biding sites were lost even have shown that Mo- I and M 1/70 inhibited zymosan binding after 4 h when monocytes were adherent to Ml/70-coated sur- to monocytes and macrophages. Similar inhibition of zymo- faces. We conclude that some anti-Mac-i antibodies can in- san phagocytosis has been found by Ross et al. (7), who hibit EA binding because of their epitope specificity, indepen- showed that some anti-Mac-I mAb also could inhibit the in- dent of any direct interaction with monocyte Fc receptors. This gestion of unopsonized rabbit erythrocytes. These studies sug- interference with IgG-Fc receptor-mediated binding and inges- gest that Mac- I plays an important role in the general adhesive properties of leukocytes, since it is unlikely that each of these inhibitory properties of the anti-Mac-I antibodies can be Dr. Bohnsack's current address is Department ofImmunology, Scripps a interaction. Clinic and Research Foundation, La Jolla, CA 92037. Address reprint ascribed to disruption of single receptor-ligand requests to Dr. Brown, Campus Box 8051, Washington University This hypothesis is supported by studies in patients deficient in School of Medicine, 660 South Euclid Avenue, St. Louis, MO 631 10. Mac-1. These patients' cells exhibit impaired chemotaxis, ag- Received for publication 23 October 1986 and in revised form 11 gregation, and substrate adherence in addition to their inabil- May 1987. ity to rosette with iC3b-coated particles. J. Clin. Invest. 1. Abbreviations used in this paper: AI, attachment index; CR3, com- © The American Society for Clinical Investigation, Inc. plement receptor 3; EA, erythrocytes (sheep) opsonized with IgG anti- 0021-9738/88/02/0365/11 $2.00 body; EC3bi, erythrocytes (sheep) opsonized with iC3b receptor; PI, Volume 81, February 1988, 365-375 phagocytotic index. Functionally Different Mac-1 Molecules 365 In this study we have examined the mechanism by which 2 anti-Mac-i antibodies interfere with cellular functions not obviously dependent on soluble ligand interactions with the 20)C- - Mac-i molecule. We have studied the inhibition of IgG-me- - diated rosetting and phagocytosis by M 1/70 and Mo-I in de- 42 a- tail. We have shown that both Mo-I and M 1/70 F(ab')2, which Figure 1. F(ab')2 M1/70. Lane 1, IgG Ml/70 do not interact at all with monocyte Fc receptors, inhibit IgG- after Biogel AO.5M chromatography with in- mediated erythrocyte binding and phagocytosis. Binding stud- tact IgG and one major contaminant. Lane ies with monomeric and multimeric IgG showed that M 1/70 2, the final preparation of F(ab')2 M 1/70, inhibited multimeric IgG binding while having no effect on containing no visible intact IgG. Proteins monomeric IgG binding to the Fc receptor. Aggregated IgG were electrophoresed on 5-15% gradient was able to inhibit the binding of '251-M1/70 to monocytes by SDS-PAGE under nonreducing conditions. almost 40%, while it had no effect on the binding of mono- clonal anti-CRi . This suggested that some, but not all Mac- I chromatography on Ultragel ACA 54 (Spectrum Medical, Los Angeles, molecules were associated with IgG Fc receptors. By adhering CA). Mo- I F(ab')2 showed a single band on SDS-PAGE. IgG of the monoclonal anti-CR 1 3D9 was purified as described M1/70 F(ab')2 to surfaces, we have been able to distinguish (14). OKM 1 was purchased from Ortho Diagnostics, Raritan, NJ.