Vol. 37. No. 3INFECTION AND IMMUNITY. Sept. 1982, p. 1227-12330019-9567/82/091227-07$02.00/0

Plasmodium berghei Malaria: Blockage by ImmuneComplexes of Macrophage Receptors for Opsonized Plasmodia

K. M. BROWN AND J. P. KREIER*

Depatrtnent of Microbiology, The Ohi/o Staite University, Coliumtibius, Ohio 43210

Received 19 February 1982/Accepted 17 May 1982

Immune complexes produced in vitro by mixing immune serum and solublePlasmodiiim berghei antigens and immune complexes precipitated from serum ofacutely infected rats blocked macrophage receptor sites for opsonized plasmodia.The immune complexes were precipitated from acute-phase serum, using polyeth-ylene glycol, and their composition was determined by using a Raji cell immuno-fluorescence assay. The immune complexes contained immunoglobulin G (IgG),IgM, and malarial antigens. These results indicate that immune complexes in theserum of acutely infected animals may protect the plasmodia from the activities ofmacrophages.

The means by which the host brings a plasmo-dial infection under control are still incompletelyunderstood. Defensive systems directed againstthe infected erythrocyte have been proposed(27) as have systems directed against the mero-zoite when it passes from one erythrocyte toanother (5, 8). There is of course no reason whyboth strategies could not be followed during thecourse of the infection (13). The present paperreports information on how antibodies enhancethe capacity of macrophages to recognize freeplasmodia and how soluble antigens and immunecomplexes in acute-phase serum may protect theplasmodia from phagocytosis. The compositionof the immune complexes was determined byusing a Raji cell immunofluorescence assay.

MATERIALS AND METHODS

Animals. Inbred Fisher 344 rats (CDF. CharlesRiver Breeding Laboratories, Inc., Wilmington,Mass.) were used as sources of macrophages. hyper-immune rat serum, normal rat serum, and acute-phaseserum in this study. Outbred rats (Sprague Dawley.Harlan Industries, Inc.. Cumberland, Ind.) were usedfor production of Plassmodiu,n berg/ei parasites.

Parasites. Plassmodiirn berghei berghei (WalterReed Army Institute of Research strain) was main-tained in liquid nitrogen.

Harvest of P. berghei parasites from infected erythro-cytes. Plasmodiltn berghei parasites were releasedand then isolated from infected rat erythrocytes bycontinuous flow sonication methods and by differentialcentrifugation techniques previously described (16,17). The pellet of plasmodia obtained by centrifugationwas suspended in RPMI 1640 medium to give a 10 to20% suspension.

Preparation of rat origin hyperimmune anti-P.berghei antisera. CDF rats which had recovered froman initial P. berghei infection were inoculated intra-peritoneally with 2 x 109 P. berghei-infected rat

erythrocytes at biweekly intervals for a total of fourinjections. One week after the last injection, the ratswere bled, and the sera were pooled and frozen at- 200C.Harvest of normal peritoneal macrophages. Donor

animals were normal. nonstimulated inbred CDF rats.The macrophages were collected by procedures de-scribed by Green and Kreier (8) and Brooks andKreier (2), except that RPMI 1640 medium supple-mented with 10% heat-inactivated fetal calf serum, 50,ug of gentamycin, 2.5 x 10-2 M N-2-hydroxyethylpi-perazine-N'-2-ethanesulfonic acid (HEPES) (GIBCOLaboratories, Grand Island, N.Y.), and sodium bicar-bonate to give a pH of 7.0 was substituted for medium199, and no Tris-ammonium chloride wash was done.The peritoneal macrophages were plated onto 35-mmplastic tissue culture dishes (Falcon 3001) at a concen-tration of 2 x 106 viable cells. The cells were incubat-ed at 37°C, with 7% CO, and high humidity for I h.After incubation, nonadherent cells were removed byrinsing, and the adherent cells were held at 37°C fornot more than 6 h before use.

Preparation of soluble P. berghei antigen-antibodycomplexes. Soluble P. berghei antigens were preparedby taking 4.8 x 1010 free parasites in 2 ml of RPMI andsonicating them three times for 10 s. The debris waspelleted by centrifugation at 3,300 x g for 15 min. andthe supernatant was collected. Each time complexeswere needed, a 0.5-ml volume of soluble P. berg/ieiantigens was mixed with a 1.5-ml volume of heat-inactivated normal or hyperimmune rat serum.

Polyethylene glycol precipitation of acute-phase se-rum from P. berghei-infected animals. Sera were col-lected from CDF rats infected with P. berg/ei whentheir parasitemias were about 60% (4 to 7 days afterinfection). The acute-phase sera were pooled andfrozen. When complexes were desired. a 10-ml sampleof the acute-phase serum was mixed with 10 ml of3.5% polyethylene glycol (molecular weight 6,000;Sigma Chemical Co., St. Louis. Mo.) in 0.1 M boratebuffer, pH 8.5 (3). This mixture was allowed to reactfor 18 h at 4°C, after which time the precipitated

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1228 BROWN AND KREIER

immune complexes were collected by centrifugationand suspended and dialyzed in RPMI 1640 medium.

Raji cells. Raji cells were maintained in RPMI 1640medium supplemented with 2.5 x 10-2M HEPES, 50Fg of gentamycin, 10% newborn calf serum, andenough sodium bicarbonate to give a pH of 7.0.

Assay of complement receptors on Raji cells. The Rajicells were tested for functional complement receptorsby using sheep erythrocytes coated with anti-sheeperythrocyte antiserum (Gibco Laboratories) and freshmouse complement (7).

Preparation of fluorescein isothiocyanate-conjugatedrabbit anti-P. berghei globulins. Samples (1 ml) offreshly isolated P. berghei parasites (10% suspension[vol/vol]) were injected intravenously into New Zea-land rabbits. The rabbits were injected three additionaltimes with a mixture containing free P. berghei (1 ml ofa 10% suspension [vol/vol]) and 1 ml of Freund com-plete adjuvant at biweekly intervals. The rabbits werebled 2 weeks after the last injection, and the sera werecollected and stored at -20°C. The rabbit anti-P.berghei serum was precipitated with ammonium sul-fate (4) and subsequently conjugated with fluoresceinisothiocyanate (FITC) (6). The labeled globulins wereadsorbed with rat liver spleen powder and rat erythro-cytes until they did not react with rat macrophages.

Raji cell immunofluorescence assay for detection ofimmune complexes. The acute-phase serum and thepolyethylene glycol precipitates from acute-phase se-rum were examined for immune complexes containingimmunoglobulin G (IgG), IgM, and P. berghei antigensby absorbing 0.5 ml of control or test serum with 5 x106 Raji cells at 37°C for 45 min (24, 25). In somecases, fresh mouse serum was added along with thetest or control serum as a source of complement. Thecells were washed three times with RPMI. The im-mune complexes bound to the Raji cells were subse-quently stained with FITC-conjugated rabbit anti-ratIgG (Miles Pharmaceuticals, Elkhart, Ind.), rabbitanti-rat IgM (Miles), and rabbit anti-P. ber-ghei for 30min at 4°C. The cells were washed and suspended in10% bovine serum albumin in phosphate-buffered sa-line. The cells were observed for fluorescence, using aZeiss microscope adapted for dark field and fluores-cence illumination, using transmitted light from anOsram-halogen lamp.

Procedures for testing the effects of complexes onmacrophage recognition of free P. berghei. One andone-half milliliters of a suspension of the in vitro-produced immune complexes or polyethylene glycol-precipitated immune complexes mixed with eitherhyperimmune or normal serum, or antisera alone, wasadded to each macrophage monolayer, and the cul-tures were incubated for 30 min at 4°C. After incuba-tion, 3 x 109 intact, free parasites in 0.5 ml of RPMIwere added to each monolayer, and the cells werereincubated at 4°C for 15 min. The cultures were thenwashed with RPMI containing 10 -1 M sodium azide toprevent internalization of the bound parasites by themacrophages (19). A drop of fetal calf serum wasplaced on each plate, and the plates were air dried.The cells were fixed with absolute methanol andstained with Giemsa.

In one set of experiments the macrophage monolay-ers were rinsed with RPMI after incubation with thecomplexes and before the free plasmodial suspensionswere added.

All of the macrophage monolayers were examinedunder the 10OX objective lens of a bright-field micro-scope from one edge of the culture dish to the other;the macrophages in every other field were counted.More than 500 macrophages were examined on eachplate. The number of macrophages with parasitesattached and the numbers of parasites per macrophagewere recorded for each field counted. The data werecalculated as the mean of the percentage ± the stan-dard deviation based on duplicate, triplicate, or qua-druplicate cultures. Statistical comparisons were doneby the Student t test.

RESULTS

Effect on macrophage recognition of intact freeP. berghei of adding soluble components of P.berghei to mixtures of P. berghei, serum, andmacrophages. In these experiments, in which theserum and soluble antigen mixtures were placedon the macrophage monolayers and then theintact free parasites were added, the percentageof macrophages with attached plasmodia wasgreatest in the presence of immune serum (im-mune serum groups significantly different fromall other treatment groups, P < 0.05). In thepresence of immune serum and soluble antigen,the percentage of macrophages with attachedplasmodia was about the same as or lower thanthe percentage binding plasmodia in the pres-ence of normal serum (immune serum plus anti-gen groups not significantly different from nor-mal serum groups, P > 0.05). The percentage ofmacrophages binding plasmodia in the presenceof normal serum and soluble antigen was alsosimilar to or lower than the percentage of macro-phages binding plasmodia in the presence ofnormal serum alone (groups of normal serumplus antigen were not significantly different fromgroups of normal serum alone, P > 0.05). Solu-ble antigens not only interfered with the abilityof serum to increase the proportion of macro-phages capable of binding plasmodia, they alsoreduced the numbers of plasmodia bound. Theeffect of soluble antigens was greatest on im-mune serum-mediated binding (interference in-dex 6.5) and less on normal serum-mediatedbinding (interference index 2.9) (Table 1).An analysis indicated that the immune serum-

mediated increase in plasmodia bound was notsimply a result of more macrophages bindingplasmodia but the many of the macrophageswhich bound plasmodia bound more plasmodia(Data not shown).

Effect of macrophage-bound P. berghei anti-gen-antibody complexes on macrophage recogni-tion of intact free P. berghei. In these experi-ments, the macrophages were exposed tonormal serum or immune serum or to mixturesof normal serum or immune serum and solubleantigens and then rinsed clear of unbound mate-rials before they were exposed to the free plas-

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IMMUNE COMPLEX EFFECT ON MACROPHAGE RECEPTORS 1229

TABLE 1. The effect of mixtures of soluble P. berghei antigens and serum upon the percentage ofmacrophages binding free P. berghei asnd on the number of P. berghei bound to macrophages

Treatment of macrophages % of macrophages with No. of plasmodia per 250 Interferenceadherent plasmodia' macrophages' index'

Normal rat serum 17 ± 8 53 ± 26 2.9Normal rat serum + soluble antigens 7 ± 3 18 ± 9Hyperimmune rat serum 44 ± 6 168 ± 32Hyperimmune rat serum + soluble antigens 10 ± 1 26 ± 4 6.5

a Mean ± the standard deviation of quadruplicate tests.b Interference index = the number of plasmodia bound in the absence of soluble P. berghei antigens divided by

the number of plasmodia bound in the presence of soluble P. berghei antigens.

modia. Exposure to normal serum-antigen mix-tures had no effect on the percentage ofmacrophages that bound nonopsonized, freeplasmodia (no significant difference at P > 0.05).Soluble antigens interfered with immune serum-mediated adherence whether the adherence was

by cytophilic or by opsonic mechanisms (differ-ence significant at P < 0.05). In the latter case,

exposure to the soluble antigen-serum mixturescaused a reduction in the total numbers ofplasmodia bound compared with the total num-

bers bound by macrophages not exposed toantigens (Table 2). In general, among thosemacrophages which bound plasmodia, those ex-

posed to serum-antigen mixtures bound fewerplasmodia to their surfaces than did those ex-

posed to serum alone (data not shown).Characterization of antigen-antibody complex-

es precipitated from acute-phase serum. Materi-als present in and precipitated from acute-phaseserum bound on Raji cells. After staining with

fluorescent-labeled rabbit origin antibodies,highly fluorescent, large-to-medium-sized aggre-gates were observed on a high proportion of theRaji cells in positive tests. In negative tests,most of the Raji cells did not have fluorescentaggregates on their surfaces, but a few Raji cellsappeared as uniformly fluorescent disks (deadcells) (Fig. 1).The immune complexes present in acute-

phase serum and polyethylene glycol precipi-tates from acute-phase serum were shown bystaining with fluorescent-labeled anti-rat IgG,anti-rat IgM, and anti-P. berghei antibodies tocontain IgG, IgM, and P. berghei components.The Raji cells did not bind rabbit origin fluo-

rescent-labeled anti-rat IgG, anti-rat IgM, oranti-P. berghei in the absence of bound com-

plexes. The Raji cells bound heat-aggregatednormal rat globulin in the presence of comple-ment (fresh mouse serum), but not in its ab-sence. Staining of the bound normal globulin

TABLE 2. The effect of pretreatment of macrophages with soluble P. berghei antigens and serum mixturesupon the percentage of macrophages binding free P. berghei and on the number of P. berghei bound to

macrophagesPretreatment of Pretreatment of % of macrophages with No. of plasmodia per 250 Interferencemacrophages plasmodia adherent plasmodia" macrophages" index'

Normal rat serum Normal serum 14 ± 0.5 34 ± 1Normal rat serum + Normal serum 13 ± 1 29 ± 1 1.2

soluble antigensNormal rat serum Hyperimmune serum 40 ± 7 155 ± 25 1.4Normal rat serum + Hyperimmune serum 29 ± 2 107 ± 7

soluble antigensHyperimmune rat Normal serum 30 ± 6 98 ± 21 1.8serum

Hyperimmune rat Normal serum 19 ± 1 54 ± 6serum + solubleantigens

Hyperimmune rat Hyperimmune serum 37 ± 3 136 ± 23 1.8serum

Hyperimmune rat Hyperimmune serum 23 ± 7 77 ± 26serum + solubleantigensa Mean ± the standard deviation of duplicate cultures.b Interference index = the number of bound plasmodia in the absence of soluble P. berghei antigens divided by

the number of plasmodia bound in the presence of soluble P. berghei antigens.

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FIG. 1. A representative positive and negative Raji cell immunofluorescence assay for immune complexes.(A) The appearance under fluorescent illumination of Raji cells which had been incubated with polyethyleneglycol precipitates from acute-phase serum and then incubated with FITC-rabbit anti-rat IgM (a positivereaction). A high proportion of the Raji cells have large fluorescent aggregates on their surfaces. Theseaggregates are immune complexes containing IgM. (B) The appearance under dark-field illumination of the samefield shown in (A). A high proportion of the Raji cells in (B) can be seen to have bound complexes in (A) above.(C) The appearance of Raji cells under fluorescence illumination after treatment with normal rat serum andsubsequent staining with FITC-rabbit anti-P. berghei (a negative reaction). (Bar, 10 pm). The uniformlyfluorescent cells are dead cells which nonspecifically bound the FITC antiserum. (D) The appearance underdark-field illumination of the same field shown in (C). Only a very small proportion of the Raji cells in (D) can beseen in (C).

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IMMUNE COMPLEX EFFECT ON MACROPHAGE RECEPTORS 1231

TABLE 3. The effect of polyethylene glycol-precipitated immune complexes, isolated from acute-phaseserum, upon the percentage of macrophages binding free P. berghei and on the number of P. berghei bound

to macrophages

Treatment of macrophages 5% of macrophages with No. of plasmodia per 250 Interferenceadherent plasmodia" macrophages" indexb

Normal rat serum 37 ± 6 141 ± 36Normal rat serum + polyethylene 34 ± 10 135 ± 70 1.0

glycol-precipitated immunecomplexesc

Hyperimmune rat serum 52 ± 7 237 ± 80Hyperimmune rat serum + 18 ± 4 70 ± 27 3.4

polyethylene glycol-precipitatedimmune complexes'a Mean ± the standard deviation of triplicate cultures.b Interference index = the number of plasmodia bound in the absence of soluble P. berghei antigens divided by

the number of plasmodia bound in the presence of soluble P. berghei antigens.' Immune complexes isolated from acute-phase serum by polyethylene glycol precipitation.

complexes with the FITC-labeled antisera indi-cated that the heat-aggregated normal rat globu-lin contained IgG, but not IgM.The Raji cells bound sheep erythrocytes coat-

ed with antibody and complement but did notbind noncoated sheep erythrocytes or sheeperythrocytes coated with antibody alone; thus,the complexes also contained complement.

Effect on macrophage recognition of intact freeP. berghei of adding antigen-antibody complexesfrom acute-phase serum to mixtures of P. berghei,serum, and macrophages. Immune complexesfrom acute-phase serum had no significant effecton the proportion of macrophages binding freeP. berghei in the presence of normal serum (notsignificant, P > 0.05), on the numbers of plas-modia bound in the presence of normal serum(Table 3), or on the distribution of plasmodiabound on macrophages in the presence of nor-mal serum (data not shown).Immune complexes from acute-phase serum

reduced the proportion of macrophages thatbound free plasmodia in the presence of immuneserum (significant at P < 0.05) and reduced thenumbers of plasmodia bound in the presence ofimmune serum (Table 3). In the presence ofimmune complexes, few macrophages boundmore than one plasmodium (data not shown).

DISCUSSIONEvidence has been accumulating which indi-

cates a crucial role for macrophages, both ininduction and in effector functions in immunityto malaria (13). Any mechanism by which mac-rophage functions are inhibited could prevent ordelay the ability of the host to clear the orga-nism.

In earlier studies in this laboratory, weshowed that sera from rats recovered from ma-laria facilitated macrophage recognition of freeplasmodia (5, 8, 10). Macrophages picked up

free trophozoites in the presence of normalserum, but merozoites were bound to macro-phages only in the presence of immune serum(2). The present studies confirm a role for anti-body in macrophage-plasmodial interaction anddemonstrate that antigen-antibody complexesproduced in the laboratory and those present inthe plasma of rats with acute malaria can inhibitthe ability of macrophages to bind antibody-coated plasmodia. Whether phagocytosis is nec-essary to prevent the opsonized merozoite fromentering an erythrocyte is still a matter of de-bate. The apparatus for recognition of erythro-cytes by merozoites and for erythrocyte pene-tration is apparently not blocked by the antibodywhich coats the merozoites (14), but if thesurface coats of the merozoites are cross-linkedby antibody in the late-stage schizonts, mero-zoite infectivity is reduced (9). Thus, phagocyto-sis of effectively agglutinated merozoites may beonly a scavenging function; however, phagocy-tosis of weakly agglutinated merozoites may beimportant in their elimination before the mero-zoites break loose from the clumps. If the latteris the case, interference with phagocytosis byimmune complexes would affect plasmodial sur-vival and, thus, the course of the infection.Takayanagi and Nakatake (23) demonstrated

that the soluble fraction of a homogenate ofTrypanosoma gambiense was capable of block-ing the attachment of T. gambiense to macro-phages, and Rao et al. (18), using L1210 leuke-mia cells, demonstrated in vivo inhibition ofmacrophage receptors for cytophilic antibody,using soluble immune complexes. In these re-ports the authors considered that the immunecomplexes containing IgG blocked the Fc recep-tors on the macrophages. Thus, the type ofblockage we describe here may be a generaldefense that parasites mount against the immuneresponses of the host.

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1232 BROWN AND KREIER

By adding 3.5% polyethylene glycol to serum(3) we obtained a precipitate from acute-phaseserum, as have other researchers (11, 12). Thisprecipitate bound to Raji cells. The Raji cells weused bind rat antigen-antibody complexesthrough C3b, C3d, Clq, and other complementreceptors, but not through Fc receptors. Rajicells have been reported to have Fc receptorsfor human IgG but not for rodent IgG (24, 25).The complexes in the acute-phase serum thuscontained complement, since an external sourceof complement was not needed for binding ofthese complexes to the Raji cells.The fluorescent-antibody studies of the com-

plexes bound on the Raji cells showed that thecomplexes contained IgG, IgM, and plasmodialcomponents. As we had no reagent to test forautoantigens in the complexes and, thus, did nottest for such complexes, we cannot preclude thepossibility that there are autoantibodies andautoantigens in the complexes.The macrophages used in this study were

nonelicited, nonactivated, peritoneal lavagecells. Immune complexes, isolated from the seraof P. berghei-infected animals and those pre-pared by mixing soluble P. ber-ghei antigens andantibody to P. berghei, were shown in this studyto block adherence of opsonized plasmodia tomacrophages. Thus, these complexes in theshort term interfered with a macrophage func-tion. After more prolonged exposure to immunecomplexes, macrophages are activated (15, 22).Inhibition of a function in the short term andlater activation by the same factor is an exampleof a common physiological response. With thepassage of time, the system adjusts to the ad-verse situation in such a way that it may dealwith it more effectively.The presence of soluble antigens (20, 21) and

antigen-antibody complexes (11, 12) in the plas-ma of humans and other animals with acutemalaria is well known. The roles these solubleantigens play in the pathogenesis of the infectionare still a matter of conjecture. The antigens, orcomplexes they form with antibody, may precip-itate out in the kidneys and, along with comple-ment, cause glomerulonephritis (1, 26). Thisresult would not seem to benefit either the hostor the parasite. The results of this study showthat these complexes can inhibit the binding ofplasmodia to macrophages. Thus, it may providean advantage to the parasite to justify the effortit must expend to synthesize and release thesoluble plasmodial components.

ACKNOWLEDGMENTSThis work was supported in part by a World Health Organi-

zation grant (M2/181/151).We thank R. Whisler for providing a stock culture of Raji

cells. We are grateful to Donna Bucci and Ann Perry for theirassistance in preparing this manuscript and to Kenneth Mil-

liser, Jacob Carpenter, and Kenneth Woody for maintainingthe animal colony.

LITERATURE CITED

1. Boonpucknavig, S., S. Wongsawang, V. Boonpucknavig,and N. Bhamarapravati. 1976. Serum-soluble malarialantigens and immune complex nephritis in Plasinodiumnberghei berghei infected mice. J. Trop. Med. Hyg.79:116-119.

2. Brooks, C., and J. P. Kreier. 1978. Role of the surfacecoat in in vitro attachment and phagocytosis of Plasmnodi-irm berghei by peritoneal macrophages. Infect. Immun.20:827-835.

3. Creighton, W. D., P. H. Lambert, and P. A. Miescher.1973. Detection of antibodies and soluble antigen-anti-body complexes by precipitation with polyethylene gly-col. J. Immunol. 111:1219-1227.

4. Cherry, W. B., M. Goldman, and T. R. Carski. 1960.Fluorescent antibody techniques in the diagnosis of com-municable diseases. In U.S. Public Health Service publi-cation no. 729. U.S. Public Health Service, Washington,D.C.

5. Chow, J. S., and J. P. Kreier. 1972. Plasmnodiumn be-ghei:adherence and phagocytosis by rat macrophages ini vitro.Exp. Parasitol. 31:13-18.

6. Goldman, M. 1968. Fluorescent antibody methods. Aca-demic Press, Inc., New York.

7. Gravely, S. M., J. Hamburger, and J. P. Kreier. 1976. Tand B cell population changes in young and in adult ratsinfected with Plasmnodilun berghei. Infect. Immun.14:178-183.

8. Green, T. J., and J. P. Kreier. 1978. Demonstration of therole of cytophilic antibody in resistance to malaria para-sites (Plasmnodiirn berghei) in rats. Infect. Immun.19:138-145.

9. Green, T. J., M. Morhardt, R. G. Brackett, and R. L.Jacobs. 1981. Serum inhibition of merozoite dipersal fromPlasmodiiut1 falciparurn schizonts: indicator of immunestatus. Infect. Immun. 31:1203-1208.

10. Hamburger, J., and J. P. Kreier. 1976. Plasmnodiumnberghei: use of free blood stage parasites to demonstrateprotective humoral activity in the serum of recoveredrats. Exp. Parasitol. 40:158-169.

11. Houba, V., P. H. Lambert, A. Voller, and M. A. 0.Soyanwo. 1976. Clinical and experimental investigation ofimmune complexes in malaria. Clin. Immunol. Immu-nopathol. 6:1-12.

12. June, C. H., C. E. Contreras, L. H. Perrin, P. H. Lam-bert, and P. A. Miescher. 1979. Circulating and tissue-bound immune complex formation in murine malaria. J.Immunol. 122:2154-2161.

13. Kreier, J. P., and T. J. Green. 1980. The vertebrate host'simmune response to plasmodia, p. 111-162. In J. P.Kreier (ed.), Malaria, vol. 3. Academic Press, Inc., NewYork.

14. Miller, L. H., M. Aikawa, and J. A. Dvorak. 1975. Malar-ia (Plasmodiirm kzowlesi) merozoites: immunity and thesurface coat. J. Immunol. 114:1237-1242.

15. Pestel, J., M. Joseph, J.-P. Dessaint, and A. Capron. 1981.Macrophage triggering by aggregated immunoglobulins. I.Delayed effect of IgG aggregates or immune complexes. J.Immunol. 126:1887-1891.

16. Prior, R., and J. P. Kreier. 1972. Plasmodjiisn bergheifreed from host erythrocytes by a continuous-flow ultra-sonic system. Exp. Parasitol. 32:239-243.

17. Prior, R., and J. P. Kreier. 1972. Isolation of Plasmtiodjuiintberghei by use of a continuous-flow ultrasonic system: amorphological and immunological evaluation. Proc. Hel-minthol. Soc. Wash. 39:563-574.

18. Rao, V. S., R. L. Grodzicki, and M. S. Mitchell. 1979.Specific in vivo inhibition of macrophage receptors forcytophilic antibody by soluble immune complexes. Can-cer Res. 39:174-182.

19. Romans, D. G., L. Pinteric, R. E. Falk, and K. J. Dorring-

INFECT. IMMUN.

IMMUNE COMPLEX EFFECT ON MACROPHAGE RECEPTORS 1233

ton. 1976. Redistribution of the Fc receptor on humanblood monocytes and peritoneal macrophages induced byimmunoglobulin G-sensitized erythrocytes. J. Immunol.116:1473-1481.

20. Seitz, H. M. 1972. Demonstration of malarial antigens inthe sera of Plasmodium berghei-infected mice. J. Parasi-tol. 58:179-180.

21. Seitz, H. M. 1976. The Plasmodium berghei-infection inisogenic F1(C57B1 x DBA)-mice. II. Antibodies andantigen in the serum. Tropenmed. Parasitol. 27:33-43.

22. Shear, H. L., R. S. Nussenzweig, and C. Bianco. 1979.Immune phagocytosis in murine malaria. J. Exp. Med.149:1288-1298.

23. Takayanagi, T., and Y. Nakatake. 1975. Trypanosomagambiense: blocking ability of parasite and macrophagehomogenates on attachment during phagocytosis. Exp.

Parasitol. 37:218-222.24. Theofilopoulos, A. N., F. J. Dixon, and V. A. Bokisch.

1974. Binding of soluble immune complexes to humanlymphoblastoid cells. I. Characterization of receptors forIgG Fc and complement and description of the bindingmechanism. J. Exp. Med. 140:877-894.

25. Theofilopoulos, A. N., C. B. Wilson, V. A. Bokisch, andF. J. Dixon. 1974. Binding of soluble immune complexesto human lymphoblastoid cells. II. Use of Raji cells todetect circulating immune complexes in animal and hu-man sera. J. Exp. Med. 140:1230-1244.

26. Ward, P. A., and J. W. Kibukamusoke. 1969. Evidencefor soluble immune complexes in the pathogenesis of theglomerulonephritis of quartan malaria. Lancet i:283-285.

27. Zuckerman, A. 1960. Autoantibody in rats with Plasmodi-um berghei. Nature (London) 185:189-190.

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