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on soluble material, and reports the following two observations. First, in contrast .... 906. THE EFFECT OF COMPLEMENT ON SOLUBLE COMPLEXES. TAnLE I.
THE EFFECT OF COMPLEMENT

ON THE INGESTION OF

SOLUBLE ANTIGEN-ANTIBODY COMPLEXES AND IgM A G G R E G A T E S BY M O U S E P E R I T O N E A L M A C R O P H A G E S * By J. L. VAN SNICK:~ aNt~ P. L. MASSON From the Unit of Experimental Medicine, International Institute of Cellular and Molecular Pathology, Universitk Caiholique de Louvain, Brussels, Belgium

In vivo, the elimination of soluble antigen-antibody (AgAb) 1 complexes by the reticuloendothelial system is apparently independent of complement (1). This contrasts with the promoting effect of complement on the phagocytosis of Ab-coated particles. In this case, C3 and IgG act in synergy. IgG, by reacting with Fc-receptors, stimulates particle ingestion, but is relatively inefficient at inducing particle binding. C3 mediates the binding of the particle to the phagocyte without direct stimulation of the ingestion (2). The present work deals with the opsonie activity of complement on soluble material, and reports the following two observations. First, in contrast to what has been observed in vivo, complement markedly promotes the ingestion by mouse peritoneal macrophages (MPM) of soluble AgAb complexes containing IgG antibodies. However, with t25I-labeled complexes, this effect is evident only when the release of degradation products is considered. Using 59Fe-labeled human transferrin (Tf) as Ag in the complexes, it is possible to demonstrate directly the increase of uptake induced by complement because, as it will be shown, ~9Fe is not released from the macrophage~ after digestion of its carrier. Second, contrasting with the results obtained with IgM-coated particles (2), soluble heat-aggregated human IgM is ingested in significant amounts in the presence of complement, indicating that the endocytosis of soluble material, in contrast to particles, does not necessarily require the involvement of Fc receptors. Materials a n d M e t h o d s Nonstimulated macrophages were collected from the peritoneal cavity of normal female N M R I mice. In some experiments, 129/Sv mice maintained under specific pathogenfree conditions were used. The cells were processed as previously described (3) with slight modifications. Briefly, a 10-ml vol of basal medium of Eagle (BME) containing 20 U of heparin per milliliter was injected into the peritoneal cavity, and reaspirated after abdominal kneading. Cells.

After centrifugation, the cells were resuspended in BME containing 20% heat-decomplemented fetal calf serum (FCS), 50 U/ml penicillin, and 50 ~g/ml streptomycin. About 3.10s cells were * Supported by a grant from the Cancer Research Fund of the Caisse G~n~rale d'Epargne et de Retraite and grant 3.4503.75 from the Fonds National de la Recherche Scientifique M6dicale, Brussels, Belgium. :~ Aspirant at the Fonds National de la Recherche Scientifique, Brussels, Belgium. 1Abbreviations used in this paper: Ab, antibody; Ag, antigen; ATf, mouse antiserum against human transferrin; BME, basal medium of Eagle; E-IgM, e/'ythrocytes coated with IgM, E-IgM-C, E-IgM and complement; FCS, fetal calf serum; FMS, fresh mouse serum; HMS, mouse serum decompiemented by heating; MPM, mouse peritoneal maerophages; PBS, phosphate-buffered saline; TCA, trichloroacetic acid; Tf, human transferrin. J. ExP. Men. ©The Rockefeller University Press - 0022-1007/78/1001-090351.00 Volume 148 October 1978 903-914

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THE EFFECT OF COMPLEMENT ON SOLUBLE COMPLEXES

seeded per well in Sterilin tissue culture plates (Teddington, Middlesex, Great Britain). After about a l-h incubation at 37°C in a 10% CO2 atmosphere, the adherent macrophages (5-7.105 cells per well) were washed free of other cells with BME, and reincuhated with the appropriate medium. Reagents. Human iron-free Tf was from Behring Institute (Marburg/Lahn, West Germany). Antiserum against T f (ATt) was obtained from NMRI mice after weekly subcutaneous injections of 100/~g of antigen suspended in complete Freund's adjuvant. For labeling Tf, the protein, dissolved in phosphate-buffered saline (PBS) containing 0.05 M NaHCO3, was mixed with [59Fe]citrate in sufficient amounts to saturate the protein up to 80% of its iron-binding capacity. Tf-ATf complexes were prepared in 3-, 5-, and 10-fold Ag excess by adding the appropriate amount of heat-decomplemented mouse antiserum to labeled Tf. After a l-h incubation at 37°C, the complexes were left for 3 days at 4°C before use. Even, after 3 wk, no precipitate was visible. The amount of Tf bound by the anti-Tf antibodies was measured for each preparation of immune complexes by density gradient ultracentrifugation (4). The distribution of complexes was as follows. For the preparation in threefold Ag excess, 13% of the complexes sedimented as a 13.5 S peak, 30% as a polydisperse fraction (13.5-30 S), and 57% were concentrated in the pellet (>30 S). For the preparation in 5- and 10-fold Ag excess, respectively, 27 and 34% were in the 13.5 S peak, 45 and 50% in the polydisperse fraction, and 30 and 16% in the pellet. A human IgM monoclonal component was kindly provided by Dr. J. P. Vaerman (Universit~ Catholique de Louvain, Brussels, Belgium), and aggregated by heating at 63°C for 30 min. After such treatment, the preparation was slightly opalescent, but no precipitate was visible. In gradient ultracentrifugation, all the aggregates were recovered in the pellet corresponding to material of >30 S. No IgG (