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Receptors for Complement on Echinoid Phagocytes. I. The Opsonic Effect of Vertebrate Sera on Echinoid Phagocytosis
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 6, pp. 423-43 1, 1982. 0145-305X/82/030423-09$03.00/0 Printe d in the USA. Copyr ight (c) 1982 Pergamon Press Ltd. All rights reserv ed.
RECEPTORS FOR COHPLEMENT ON ECHINOID PHAGOCYTES. I. THE OPSONIC EFFECT OF VERTEBRATE SERA ON ECHINOID PHAGOCYTOSIS
Kjell Berth eusse n and Rolf Selje lid Insti tute of Medic al Biolo gy Univ ersity of Troms ¢ N-900 0 Troms ¢ Norway
ABSTRACT The inges tion of sheep eryth rocyt es (SRBC) by echin oid phago cytes was great ly incre ased after treatm of SRBC with sera from mouse , human and fish Theent opson princ iple in the sera was heat labil e (56 0c ; hr) • ic Opso nizati on with mamm alian sera was studi ed ~ore exten sivel y. It was depen dent on pre-sensit on of SRBC with spec ific antib ody (IgM) I oequi redizaz~ I and was inhib ited by low temp eratu re (4 C). The Ca findi ng that serum deple ted of C 3 only opson ized very weakl y stron gly indic ates that the opson ic princ iple is the compl ement casca de C 1-4-2 -3 activ ated via the class ical pathw ay, and that the opson ic effec t 'large ly coinc ides with the coati ng of SRBC with C 3b. A role of c 5 or later compo nents was exclu ded, since the mouse AKR serum was c 5 defic ient, and the human serum was norm ally treat ed with zymos an. When varia tions known to impa ir the opson ic funct ion of C 3 was intro duced the opson izatio n proce dure, a para llel inhib ition was in recor ded on attach ment of SRBC to mouse perit onea l macro phage & and on inges tion of SRBC by echin oid cells . Thus c 3 recep tors are proba bly prese nt on echin oia phago cytes .
INTRODUCTION Recep tors for compl ement C 3 may be prese nt on phago cytes . This is indic ated by the previ ous findi ngechin oid that 423
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phagocytosis of mammalian erythrocytes by echinoid phagocytes was strongly increased when red cells were covered with specific antibodies and subsequently treated with mouse c 5 deficient serum; antibody alone, however, did not promote echinoid phagocytosis (1). In addition, a humoral complement-lik e activity nas been demonstrated in lower animals (2-4). In mammals receptors for complement on phagocytes play a crucial role in the body's defence against infectious diseases (5). Invasive microorganisms activate plasma complement components of which C 3 is a key factor. The coating of microorganisms with C 3b strongly facilitates their phagocytosis by macrophages and granulocytes. This study investigates the mechanism of opsonization of various vertebrate sera on echinoid phagocytosis, and establish that the opsonic effect in mammalian sera is dependent on complement activation and the presence of
c
3
0
MATERIALS AND 11ETHODS Animals. Phagocytes were collected from sea urchins (Strongylocent rotus droebachiensis ) held in running sea ~ater for up to 2 weeks after collection. Temperature was 2-10 c, depending on season. Mouse rnacrophages were collected from c 3o 2 mice by peritoneal lavage. Culture of echinoid phagocytes. Echinoid coelomic cells were obtained by puncture of the peristomial membrane and withdrawal of coelomic fluid with a syringe half filled with freshly made anticoagulant (50 mM mercaptoethano l, 3 ml·1 caffeine, and 2 mM TAME (p-tosyl-L-arg inine methyl ester) (final concentrations )). Phagocytes were cultured in Linbro plates (Linbro Scientific co, Inc., Hambden, Conn., USA) with 14 mm glass cover slips at 10°c in normal atmosphere according to the method of Bertheussen and Seljelid (6). One milliliter coelomic cell suspension in anti-coagulant was introduced into each well. After 1 hour, the cultures were washed to remove non-adherent cells, and the cover slips transferred to new wells containing 1 ml of Hepes-sea water (38 g/1 sea water salt with 10 mM Hepes buffer) (6). These cultures were used immediately for phagocytosis assays. Culture of mouse macrophages. Mouse macrophages were maintained in culture on 5 14 mm glass cover slips in Linbro plates. A number of 5xl0 0 cells ~e7e seeded into each well. Cells were cultured at 37 C in m~m.mum essential meaium (MEH) with 20 % fetal calf serum (FCS) and in an atmosphere of 5 % co 2 in air. After 2 hr. the cultures were washed, and new medium added. At 24 hr the cultures were washed extensively with serumfree MEM Hepes, and attachment assays were then carried out in MEM Hepes. Morphology. Echinoid phagocyte cultures were fixed in 2 % glutaraldehyde and 2 % OsO in 0.4 11 NaCl and 0.1 M cacodylate buffer, pH 5.5, for 1 hr. ~ouse macrophage cultures were fixed in 2 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, with
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0.1 M sucrose microscopy.
for·~
425
hr. Cultures were examined by phase contrast
Sera as source of complement (C) • Mouse serum was obtained from c 5 deficient AKR mice. Human serum from healthy donors was normally treated with zymosan for 1 hour at 37°C in order to destroy terminal C components through activation of the alternative C pathway, thus leaving an intact classical pathway to opsonize sensitized erythrocytes (7). In one type of experiment human sera were depleted in C 3 or C 9 by affinity chromatography, using rabbit anti-human C 3c or anti-human C 9 (Boehringwerke AG, Marburg, W. Germany) coupled to Sepharose 4B as described by Ax~n et al. (8). The absorbeG sera were assayed by immunodiffusion for the presence of C 3 or C 9. Unless otherwise specified, human sera also were absorbed with 50 % sheep red cells (SRBC) to remove natural anti-SRBC antibodies. Fish serum was obtained from salmon (Salmo salar) blood by clot retraction for 2 hr at 4°C. Treatment of SRBC with various sera. Sheep red blood cells (SRBC) at 5 % were incubated with a 1:9 dilution of serwn in veronal buffered glucose containing ca2+, Mg 2+, and 0.1 %gelatine (9,10). Incubation was 30 min at 37°c for human serum, 20 min at 37oc for mouse serum, and 30 min at 10oc for fish serum. This SRBC preparation was used immediately after washing. Unless otherwise specified, SRBC at 1 % were sensitized with 10 pl/ml rabbit anti-SRBC IgM (Cordis Lab., Miami) prior to serum treatment. In order to inactivate C-components on the SRBC surface, some SRBC preparations were post-treated with a 1:1 dilution of ~erum in the above veronalbuffer with 10 mM EDTA for 1 hr at 37 c. Echinoid phagocytosis assay. 51 cr-labelled SRBC (2xl0 8 ) were added to 1 hr echinoid phagocyte cultures for 15 ~in of phagocytosis. Cultures were then washed and treated with NH 4Cl-rcs (10 % fetal calf serum in 0.14 M ammonium chloride) for 4 min in order to lyse attached SRBC (1). The amount of ingestion (internalization) of radiolabelled material was quantified using a gamma spectrometer. Mouse macrophaie attachment assay. SRBC (2xl0 7 ) we5e added to 24 hr cultures o mouse macrophages for 15 min at 37 C. Cultures were then washed and attachment of SRBC determined by phase-contrast microscopy. RESULTS The opsonic effect of various vertebrate sera was studied by examining the interaction of serum-treated sheep erythrocytes (SRBC) with two types of phagocytic cells. The echinoid phagocyte was the cell under study, and opsonization for this cell type was quantified on the basis of the amount of red cells internalized. In addftion, as a check that SRBC were properly opsonized with complement, binding of serum-treated SRBC to mouse peritoneal macrophages was recorded. The activation of complement via IgM antibodies excludes the possibility that SRBC attached to macrophages via Fe receptors. Complement-opsonized SRBC are not internalized by normal ~ouse macrophages (10).
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The opsonic effect of vertebrate sera. Treatment of SRBC with the two mammalian sera {mouse AKR and human) led to a strong increase in echinoid phagocytosis paralleling the extensive covering of mouse macrophages with attached red cells (Table 1) •
TABLE 1 The Oesonic Effect of Sera from Various Vertebrate Seecies No. of RBC per Phagocytec) lgM PreEchinoid Mouse treatment of T~ee of Serumb) Phagoc;ttes Macroehages Er~throc~tesa) 0.3 None 0 No 0.3 None 0 Yes 0.2 Mouse serum 0 No 4.0 Mouse serum 23.5 Yes Yes, 5>. diluted Mouse serurr; 1.2 4.2 1.0 Sx diluted mouse serum 3.6 Yes Mouse serum pretreated 0.8 0.7 Yes 56°C ! hr Mouse serum, 0.3 0 Yes opsonization at 4°C Human serum (Z) 4.8 1.7 No 0.4 0 Human serum (Z~, absorbed No Human serum (Z , absorbed 16.2 4.3 Yes Fish serum (salmon) 4.1 0 No Fish serum (salmon) 4.4 0 Yes Fish serum pretreated 1.0 0 Yes 560C 2 hr 2x108 sheep erythrocytes (SRBC) ~ere added to 1 hr echinoid cultures for 15 min of phagocytosis. 2xl0 SRBC were added to 24 hr mouse macrophage cultures for 15 min of erythrocyte rosetting. a) Treatment of rabbit IgM anti-SRBC before serum treatment. b) SRBC were treated with various sera, normally diluted 1:9, see Materials and Methods for details. Mouse serum was from c sdeficient AKR mice. Human serum (Z) was human serum treated with zymosan. c) The opsonic effect was quantitated as the mean number of SRBC internalized per echinoid phagocyte or attached per mouse macrophage. Experiments were performed in triplicates. S.D. < 12 %of quantities. This opsonic effect was dependent on the red cells being sensitized with specific antibody prior to serum treatment. Phase contrast microscopy revealed that more than 95 % of echinoid phagocytes had attached three or more red cells. similarly, t,then mouse serum was the complement source, more than 97 % of mouse macrophages had attached three or more red cells. The human serum was treated with zymosan and absorbed with SRBC in order to avoid hemolysis and remove anti-SRBC antibodies, respectively. When SRBC were treated with fish serum, a similar increase in echinoid phagocytosis was found, however, in this
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case opsonization was not dependent on pre-sensitization with specific antibodies. Fish serum did not mediate binding of red cells to mouse macrophages. Agglutination of SRBC in undiluted fish serum indicated the presence of anti-SRBC antibddies or other agglutinins in this serum. Since fish enzymic reactions in the case of the salmon are adapted to low temperatures, no attempt was made to absorb anti-SRBC antibodies out of fish serum. Opsonization with fisg serum functioned well at the lowest temperature tested: 4 c. When sera were pre-incubated at 56°C the opsonic effect was largely lost (results for human serum not shown). At this temperature, among other effects mammalian complement c 1, c 2 and factor B are inactivated. The use of a 5 x dilution of anti-SRBC IgM or AKR serum during opsonization resulted in a strong and parallel decrease in the interaction of red cells with both types of phagocytic cells. Effect of complement inhibition. Table 2 shows the results of experiments designed to inhibit complement functioning during opsonization with mammalian sera.
TABLE 2 The Effect of Complement Inhibition on Opsonization Inhibition of Opsonizationc) Procedure for Mouse Echinoid Serum for a Macrophages Phagocytes Opsonization ) Complement Inhibition 10 mM EDTA during 100 % 100 % Mouse opsonization 10 mM EGTA and 7 mM 80 % Mouse 79 % Mg 2+ during opsonization RBC treated with EDTA85 % 92 % Mouse mouse serum after serum opsonizationb) RBC treated with EDTA0 0 Human {Z) human serum after serum opsonizationb) Serum depleted of C3 99 % 85 % Human (Control: Serum depleted of C9) Sheep erythrocytes {SRBC) were treated with IgM and mouse or human sera, and were then added to phagocyte cultures as explained in Table 1. a) See Materials and Methods for details. Serum for opsonization was diluted 1:9. b) Serum for complement inactivation after the opsonization step was diluted 1:1, and 10 mM EDTA was added (EDTA-mouse serum or EDTAhuman serum). Incubation 1 hr at 37°C. c) The opsonic effect was quantitated as in Table 1. Inhibition of opsonization was then calculated as the %reduction of the number of SRBC per phagocyte when the inhibitory procedure was introduced in the experiment. S.D. < 10 %of quantities.
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opso niza tion with seru m was inhi bite d by che latin g diva lent ca~~ons. Thu s, EDTA gave a 100 % inhi biti whi le EGTA with Mg in exce ss, whic h inhi bits clas sica l on, path way acti vati on, resu lted in a 79-8 0 % decr ease in the red cell s with phag ocyt es. According2~ sub~~quent inte ract ion of ca is need ed for prop er opso niza tion . EDTA or EGTA/Mg a sepa rate step afte r seru m opso niza tiontrea tme nt perf orm ed as effe ct (res ults of this con trol not show had no inhi bito ry n). When, how ever , seru m was pres ent duri ng this sepa rate EDTA trea tme nt, the opso nic effe ct was larg ely lost with mous e seru m, but not with human seru m (Tab le 2) • Dep letio n of C 3 by abso rptio n of colu mn decr ease d the opso nic effe ct by seru m on an imm unos orbe nt 99 % for mou se mac rophag es and by 85 % for echi noid phag ocyt dep lete d of C9 func tion ed as a pos itiv es (Tab le 2). Seru m e con trol seru m in this expe rime nt. DISCUSSION This stud y show s that vert ebra te sera con tain a hea t labi le opso nic prin cipl e whic h is reco gniz ed by echi noid phag ocyt es. The opso niza tion proc ess is temp erat ure depe nden t, bein g com plet ely inhi bite d at 4°C in mam mali that the opso nin is form ed duri ng enzy an sera . This indi cate s not of anti bod y- or lect in natu re. Thamati c reac tion s and is t the incr ease d phag ocyt ic resp onse is caus ed by the acti on of opso to a seru m enzy me mod ifyin g red cell surfnins and not mer ely due ace mol ecul es is evid ent. The seru m effe ct is depe nden t red cell s with anti body (IgM ). If red on pres ens itiz atio n of seru m were trea ted with EOTA-mouse serucell s opso nize d with mou se larg eiy lost indi cati ng the pres ence ofm.th e opso nic effe ct was prin cipl e with high turn -ove r, whe re EDTAa com plex opso nic asse rool y of the prin cipl e, whi le degr adat inhi bits synt hesi s or ion is una ffec ted. Arrl.most con sist entl y: If human seru m was dep lete d of c 3 it did not opso nize in the mous e syst em, and only a very weak opso nic effe ct pers iste d when test ed This clea rly poin ts to a cen tral role on echi noid phag ocyt es. and espe cial ly C 3 in the opso niza tion for com plem ent acti vati on proc ess. + The experim,~ts with che lato rs show ed a requ irem ent for ca 2 , but not Mg for com plete opso tion . This , toge ther with the requ irem ent for pres ensi tizaniza tion of red cell s w.i th IgM, stro ngly indi cate s that the opso nic prin seru m is the com plem ent casc ade C 1-4- cipl e in mam mali an 2-3 acti vate d via the clas sica l path way . C 5 or late r com pone sinc e the mou se seru m lack ed C 5, and nts are not nece ssar y, norm ally pret reat ed with zym osan . Zym the human seru m was osan inac tiva tes the late com pone nts in human seru m, leav ing inta opso nize via the clas sica l path way of ct the abil ity to weak opso nic effe ct of seru m with EGTAcom i!em ent (7,1 1). The /Mg may be due to some acti vati on of the alte rnat ive C path thro ugh SRBC sen sitiz ed with IgM (12, 13). When seru m was depway lete d of C 3, almo st all opso nic acti vity was lost . Thu s, the opso trea tme nt coin cide s with the dep osit ion nic effe ct of seru m surf ace. The c 3b frag men t is mod ified of C 3b on the red cell duri ng seru m opso niza tion
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and is always rapidly degraded to C 3bi by the specific serum protease C 3 inactivator. Uost C 3 on the red cell surface will be in the hemolytically inactive C 3bi form when serum is used as a complement source (14). In some sera enough proteases are present to degrade C 3bi further to the short C 3d fragment which cannot function as an opsonin. The C 3b degrading enzymes are not inhibited by EDTA and the much higher rate of opsonin inactivation in EDTA-mouse serum than in EDTA-human serum may reflect a higher content of proteases in mouse sera, or conv&rsely, mouse C 3bi is more sensitive to protease action. Opsonin inactivation by prolonged incubation of sensitized red cells in mouse serum has been described (10). Specific receptors exist on mammalian leukocytes for thefragments c 3b, c 3bi, and C 3d (14). The C 3b receptor can also bind c 4b and c 5b. The weak opsonic effect of c 3 depleted human serum may be ascribed to the presence of C 4b fragments on red cells,for it has been shown that mouse macrophage c 3b receptors do not bind human C 3b or C 4b (15); this species specificity can explain the complete lack of interaction of mouse macrophages with red cells treated with C 3 depleted human serur·., while there was a weak interaction with echinoid phagocytes. Except for this slight inconsistence all other variations in opsonization procedure with mammalian sera produced strictly parallel effects on echinoid phagocytes and mouse macrophages, thereby supporting the idea that the opsonic principle is exactly the same in both cases. From these experiments it may be inferred that echinoid phagocytes bear membrane receptors for C 3bi as is the case for their mammalian counterparts. In coherence with this is the recent finding that a complement-like activity resembling the alternative pathway of complement, and having lytic as well as opsonic properties is indeed present in echinoids. Possibly some essential structures of complement were preserved during phylogenesis, due to great complexity in funr::i.:ions with interactions between individual complement components and between components and cells. The echinoid phagocyte then recognize common C 3 structures present in most vertebrate species, while the mammalian phagocyte may recognize a structural pattern which contains both common and more species-specific traits due to the concomitant evolution of the receptors. The polyphyletic development of C 3 and C 3-receptors can explain why fish serum, if the effect was dependent on complement, only opsonized for the phylogenetically older echinoid phagocytes but not for phagocytes from mammals.
ACKNOWLEDGEMENT This work was supported in part by the Norwegian Cancer Society, Oslo, Norway.
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REFERENCES 1.
BERTHEUSSEN, K. Endocy tosis by echino id phagoc ytes in vitro. I . Recogn ition of foreign matter . Dev. Comp. Immunol . (In press 1981) •
2.
DAY, N.K.B. , GEWURZ, H., JOHANNSEN, R., FINSTAD, J., and GOOD, R.A. Comple ment and comple ment-l ike activi ty in iower verteb rates and invert ebrate s. J. £xp. ~led. 132, 941, 1970.
3. 4.
DAY 1 N., GOOD, R.A., and MOLLER-EBERHARD, H. J. "C 3 proact ivator" in starfis h hemolymph. Fed. Proc. 31, 788, 1972 • ANDERSON, R.S., DAY, N.K.B. , and GOOD, R.A. Specif ic hemag glutini n and a modula tor of comple ment in cockro ach hemolymph. Infect. lmmun. 5, 55, 1972.
s.
BIANCO, C., and NUSSENZWEIG, V. Comple ment recept ors. In: Contemporary Topics in Molecular Immunology. R.R. Porter and G.L. Ada (Eds.) New York and London : Plenum Press, 1977, vol. 6, p. 145.
6.
BERTHEUSSEN, K., and SELJELID, R. Echino id phagoc ytes in vitro. F.xp. Cell Res. 111, 401, 1978.
7.
WONG, L., and WILSON, J.D. The identi ficatio n of Fe and c 3 recept ors on human neutro phile. J. Immunol. Methods 7, 69, 1975.
8.
AXEN, R., PORATH, J., and ERNBACK, S. Chemic al couplin g of peptid es and protein s to polysa ccharid es by means of cyanog en halide s. Nature 214, 1302, 1967.
9.
RAPP, H.J., and BORSOS, T. !1olecular basis of complement action. New York: Appelt on-Cen tury-C rofts, 1970, p. 75.
10.
GRIFFIN, F.M., BIANCO, C., and SILVERSTEIN, S.C. Charac terizat ion of the macrop hage recept or for comple ment and demon stratio n of its functio nal indepe ndence from the recept or for the Fe portio n of immun oglobu lin G. J. Exp.
Med. 141, 1269, 1975.
11.
LACHMANN, P.J., and HOBART, M.J. Comple ment techno logy. In: Handbook of Experimental Immunology. D.M. Weir, (Ed.) oxford : Blackw ell, 1978, chapte r SA.
12.
POLHILL, R.B., NEWMAN, S.L., PRUITT, K.l-1., and JOHNSTON, R.B. Kineti c assessm ent of altern ative comple ment pathwa y activi ty in a hemoly tic system . II. Influen ce of antibod y on altern ative pathwa y activa tion. J. I11111unol. 121, 371, 1978.
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13.
EDWARDS, M.S., NICHOLSON-WELLER, A., BAKER, C.J., and KASPER, D.L. The role of specific antibody in alternative complement pathway mediated opsonophagocytosis of type III, group B Streptococcus. J. Exp. Med. 151, 1275, 1980.
14.
ROSS, G.D. Analysis of the different types of leukocyte membrane complement receptors and their interaction with the complement system. J. Immunol. Methods 37, 197, 1980.
RABELLINO, E.M., ROSS, G.D., and POLLEY, M.J. Membrane receptors of rno~se leukocytes. I. Two types of complement receptors for different regions of C 3. J. Immunol. 120, 879, 1978. Received : June 1981 Accepted : November 1981 15.
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RECEPTORS FOR COHPLEMENT ON ECHINOID PHAGOCYTES. I. THE OPSONIC EFFECT OF VERTEBRATE SERA ON ECHINOID PHAGOCYTOSIS
Kjell Berth eusse n and Rolf Selje lid Insti tute of Medic al Biolo gy Univ ersity of Troms ¢ N-900 0 Troms ¢ Norway
ABSTRACT The inges tion of sheep eryth rocyt es (SRBC) by echin oid phago cytes was great ly incre ased after treatm of SRBC with sera from mouse , human and fish Theent opson princ iple in the sera was heat labil e (56 0c ; hr) • ic Opso nizati on with mamm alian sera was studi ed ~ore exten sivel y. It was depen dent on pre-sensit on of SRBC with spec ific antib ody (IgM) I oequi redizaz~ I and was inhib ited by low temp eratu re (4 C). The Ca findi ng that serum deple ted of C 3 only opson ized very weakl y stron gly indic ates that the opson ic princ iple is the compl ement casca de C 1-4-2 -3 activ ated via the class ical pathw ay, and that the opson ic effec t 'large ly coinc ides with the coati ng of SRBC with C 3b. A role of c 5 or later compo nents was exclu ded, since the mouse AKR serum was c 5 defic ient, and the human serum was norm ally treat ed with zymos an. When varia tions known to impa ir the opson ic funct ion of C 3 was intro duced the opson izatio n proce dure, a para llel inhib ition was in recor ded on attach ment of SRBC to mouse perit onea l macro phage & and on inges tion of SRBC by echin oid cells . Thus c 3 recep tors are proba bly prese nt on echin oia phago cytes .
INTRODUCTION Recep tors for compl ement C 3 may be prese nt on phago cytes . This is indic ated by the previ ous findi ngechin oid that 423
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phagocytosis of mammalian erythrocytes by echinoid phagocytes was strongly increased when red cells were covered with specific antibodies and subsequently treated with mouse c 5 deficient serum; antibody alone, however, did not promote echinoid phagocytosis (1). In addition, a humoral complement-lik e activity nas been demonstrated in lower animals (2-4). In mammals receptors for complement on phagocytes play a crucial role in the body's defence against infectious diseases (5). Invasive microorganisms activate plasma complement components of which C 3 is a key factor. The coating of microorganisms with C 3b strongly facilitates their phagocytosis by macrophages and granulocytes. This study investigates the mechanism of opsonization of various vertebrate sera on echinoid phagocytosis, and establish that the opsonic effect in mammalian sera is dependent on complement activation and the presence of
c
3
0
MATERIALS AND 11ETHODS Animals. Phagocytes were collected from sea urchins (Strongylocent rotus droebachiensis ) held in running sea ~ater for up to 2 weeks after collection. Temperature was 2-10 c, depending on season. Mouse rnacrophages were collected from c 3o 2 mice by peritoneal lavage. Culture of echinoid phagocytes. Echinoid coelomic cells were obtained by puncture of the peristomial membrane and withdrawal of coelomic fluid with a syringe half filled with freshly made anticoagulant (50 mM mercaptoethano l, 3 ml·1 caffeine, and 2 mM TAME (p-tosyl-L-arg inine methyl ester) (final concentrations )). Phagocytes were cultured in Linbro plates (Linbro Scientific co, Inc., Hambden, Conn., USA) with 14 mm glass cover slips at 10°c in normal atmosphere according to the method of Bertheussen and Seljelid (6). One milliliter coelomic cell suspension in anti-coagulant was introduced into each well. After 1 hour, the cultures were washed to remove non-adherent cells, and the cover slips transferred to new wells containing 1 ml of Hepes-sea water (38 g/1 sea water salt with 10 mM Hepes buffer) (6). These cultures were used immediately for phagocytosis assays. Culture of mouse macrophages. Mouse macrophages were maintained in culture on 5 14 mm glass cover slips in Linbro plates. A number of 5xl0 0 cells ~e7e seeded into each well. Cells were cultured at 37 C in m~m.mum essential meaium (MEH) with 20 % fetal calf serum (FCS) and in an atmosphere of 5 % co 2 in air. After 2 hr. the cultures were washed, and new medium added. At 24 hr the cultures were washed extensively with serumfree MEM Hepes, and attachment assays were then carried out in MEM Hepes. Morphology. Echinoid phagocyte cultures were fixed in 2 % glutaraldehyde and 2 % OsO in 0.4 11 NaCl and 0.1 M cacodylate buffer, pH 5.5, for 1 hr. ~ouse macrophage cultures were fixed in 2 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, with
COMPLEMENT IN ECHINODERMS
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0.1 M sucrose microscopy.
for·~
425
hr. Cultures were examined by phase contrast
Sera as source of complement (C) • Mouse serum was obtained from c 5 deficient AKR mice. Human serum from healthy donors was normally treated with zymosan for 1 hour at 37°C in order to destroy terminal C components through activation of the alternative C pathway, thus leaving an intact classical pathway to opsonize sensitized erythrocytes (7). In one type of experiment human sera were depleted in C 3 or C 9 by affinity chromatography, using rabbit anti-human C 3c or anti-human C 9 (Boehringwerke AG, Marburg, W. Germany) coupled to Sepharose 4B as described by Ax~n et al. (8). The absorbeG sera were assayed by immunodiffusion for the presence of C 3 or C 9. Unless otherwise specified, human sera also were absorbed with 50 % sheep red cells (SRBC) to remove natural anti-SRBC antibodies. Fish serum was obtained from salmon (Salmo salar) blood by clot retraction for 2 hr at 4°C. Treatment of SRBC with various sera. Sheep red blood cells (SRBC) at 5 % were incubated with a 1:9 dilution of serwn in veronal buffered glucose containing ca2+, Mg 2+, and 0.1 %gelatine (9,10). Incubation was 30 min at 37°c for human serum, 20 min at 37oc for mouse serum, and 30 min at 10oc for fish serum. This SRBC preparation was used immediately after washing. Unless otherwise specified, SRBC at 1 % were sensitized with 10 pl/ml rabbit anti-SRBC IgM (Cordis Lab., Miami) prior to serum treatment. In order to inactivate C-components on the SRBC surface, some SRBC preparations were post-treated with a 1:1 dilution of ~erum in the above veronalbuffer with 10 mM EDTA for 1 hr at 37 c. Echinoid phagocytosis assay. 51 cr-labelled SRBC (2xl0 8 ) were added to 1 hr echinoid phagocyte cultures for 15 ~in of phagocytosis. Cultures were then washed and treated with NH 4Cl-rcs (10 % fetal calf serum in 0.14 M ammonium chloride) for 4 min in order to lyse attached SRBC (1). The amount of ingestion (internalization) of radiolabelled material was quantified using a gamma spectrometer. Mouse macrophaie attachment assay. SRBC (2xl0 7 ) we5e added to 24 hr cultures o mouse macrophages for 15 min at 37 C. Cultures were then washed and attachment of SRBC determined by phase-contrast microscopy. RESULTS The opsonic effect of various vertebrate sera was studied by examining the interaction of serum-treated sheep erythrocytes (SRBC) with two types of phagocytic cells. The echinoid phagocyte was the cell under study, and opsonization for this cell type was quantified on the basis of the amount of red cells internalized. In addftion, as a check that SRBC were properly opsonized with complement, binding of serum-treated SRBC to mouse peritoneal macrophages was recorded. The activation of complement via IgM antibodies excludes the possibility that SRBC attached to macrophages via Fe receptors. Complement-opsonized SRBC are not internalized by normal ~ouse macrophages (10).
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The opsonic effect of vertebrate sera. Treatment of SRBC with the two mammalian sera {mouse AKR and human) led to a strong increase in echinoid phagocytosis paralleling the extensive covering of mouse macrophages with attached red cells (Table 1) •
TABLE 1 The Oesonic Effect of Sera from Various Vertebrate Seecies No. of RBC per Phagocytec) lgM PreEchinoid Mouse treatment of T~ee of Serumb) Phagoc;ttes Macroehages Er~throc~tesa) 0.3 None 0 No 0.3 None 0 Yes 0.2 Mouse serum 0 No 4.0 Mouse serum 23.5 Yes Yes, 5>. diluted Mouse serurr; 1.2 4.2 1.0 Sx diluted mouse serum 3.6 Yes Mouse serum pretreated 0.8 0.7 Yes 56°C ! hr Mouse serum, 0.3 0 Yes opsonization at 4°C Human serum (Z) 4.8 1.7 No 0.4 0 Human serum (Z~, absorbed No Human serum (Z , absorbed 16.2 4.3 Yes Fish serum (salmon) 4.1 0 No Fish serum (salmon) 4.4 0 Yes Fish serum pretreated 1.0 0 Yes 560C 2 hr 2x108 sheep erythrocytes (SRBC) ~ere added to 1 hr echinoid cultures for 15 min of phagocytosis. 2xl0 SRBC were added to 24 hr mouse macrophage cultures for 15 min of erythrocyte rosetting. a) Treatment of rabbit IgM anti-SRBC before serum treatment. b) SRBC were treated with various sera, normally diluted 1:9, see Materials and Methods for details. Mouse serum was from c sdeficient AKR mice. Human serum (Z) was human serum treated with zymosan. c) The opsonic effect was quantitated as the mean number of SRBC internalized per echinoid phagocyte or attached per mouse macrophage. Experiments were performed in triplicates. S.D. < 12 %of quantities. This opsonic effect was dependent on the red cells being sensitized with specific antibody prior to serum treatment. Phase contrast microscopy revealed that more than 95 % of echinoid phagocytes had attached three or more red cells. similarly, t,then mouse serum was the complement source, more than 97 % of mouse macrophages had attached three or more red cells. The human serum was treated with zymosan and absorbed with SRBC in order to avoid hemolysis and remove anti-SRBC antibodies, respectively. When SRBC were treated with fish serum, a similar increase in echinoid phagocytosis was found, however, in this
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case opsonization was not dependent on pre-sensitization with specific antibodies. Fish serum did not mediate binding of red cells to mouse macrophages. Agglutination of SRBC in undiluted fish serum indicated the presence of anti-SRBC antibddies or other agglutinins in this serum. Since fish enzymic reactions in the case of the salmon are adapted to low temperatures, no attempt was made to absorb anti-SRBC antibodies out of fish serum. Opsonization with fisg serum functioned well at the lowest temperature tested: 4 c. When sera were pre-incubated at 56°C the opsonic effect was largely lost (results for human serum not shown). At this temperature, among other effects mammalian complement c 1, c 2 and factor B are inactivated. The use of a 5 x dilution of anti-SRBC IgM or AKR serum during opsonization resulted in a strong and parallel decrease in the interaction of red cells with both types of phagocytic cells. Effect of complement inhibition. Table 2 shows the results of experiments designed to inhibit complement functioning during opsonization with mammalian sera.
TABLE 2 The Effect of Complement Inhibition on Opsonization Inhibition of Opsonizationc) Procedure for Mouse Echinoid Serum for a Macrophages Phagocytes Opsonization ) Complement Inhibition 10 mM EDTA during 100 % 100 % Mouse opsonization 10 mM EGTA and 7 mM 80 % Mouse 79 % Mg 2+ during opsonization RBC treated with EDTA85 % 92 % Mouse mouse serum after serum opsonizationb) RBC treated with EDTA0 0 Human {Z) human serum after serum opsonizationb) Serum depleted of C3 99 % 85 % Human (Control: Serum depleted of C9) Sheep erythrocytes {SRBC) were treated with IgM and mouse or human sera, and were then added to phagocyte cultures as explained in Table 1. a) See Materials and Methods for details. Serum for opsonization was diluted 1:9. b) Serum for complement inactivation after the opsonization step was diluted 1:1, and 10 mM EDTA was added (EDTA-mouse serum or EDTAhuman serum). Incubation 1 hr at 37°C. c) The opsonic effect was quantitated as in Table 1. Inhibition of opsonization was then calculated as the %reduction of the number of SRBC per phagocyte when the inhibitory procedure was introduced in the experiment. S.D. < 10 %of quantities.
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opso niza tion with seru m was inhi bite d by che latin g diva lent ca~~ons. Thu s, EDTA gave a 100 % inhi biti whi le EGTA with Mg in exce ss, whic h inhi bits clas sica l on, path way acti vati on, resu lted in a 79-8 0 % decr ease in the red cell s with phag ocyt es. According2~ sub~~quent inte ract ion of ca is need ed for prop er opso niza tion . EDTA or EGTA/Mg a sepa rate step afte r seru m opso niza tiontrea tme nt perf orm ed as effe ct (res ults of this con trol not show had no inhi bito ry n). When, how ever , seru m was pres ent duri ng this sepa rate EDTA trea tme nt, the opso nic effe ct was larg ely lost with mous e seru m, but not with human seru m (Tab le 2) • Dep letio n of C 3 by abso rptio n of colu mn decr ease d the opso nic effe ct by seru m on an imm unos orbe nt 99 % for mou se mac rophag es and by 85 % for echi noid phag ocyt dep lete d of C9 func tion ed as a pos itiv es (Tab le 2). Seru m e con trol seru m in this expe rime nt. DISCUSSION This stud y show s that vert ebra te sera con tain a hea t labi le opso nic prin cipl e whic h is reco gniz ed by echi noid phag ocyt es. The opso niza tion proc ess is temp erat ure depe nden t, bein g com plet ely inhi bite d at 4°C in mam mali that the opso nin is form ed duri ng enzy an sera . This indi cate s not of anti bod y- or lect in natu re. Thamati c reac tion s and is t the incr ease d phag ocyt ic resp onse is caus ed by the acti on of opso to a seru m enzy me mod ifyin g red cell surfnins and not mer ely due ace mol ecul es is evid ent. The seru m effe ct is depe nden t red cell s with anti body (IgM ). If red on pres ens itiz atio n of seru m were trea ted with EOTA-mouse serucell s opso nize d with mou se larg eiy lost indi cati ng the pres ence ofm.th e opso nic effe ct was prin cipl e with high turn -ove r, whe re EDTAa com plex opso nic asse rool y of the prin cipl e, whi le degr adat inhi bits synt hesi s or ion is una ffec ted. Arrl.most con sist entl y: If human seru m was dep lete d of c 3 it did not opso nize in the mous e syst em, and only a very weak opso nic effe ct pers iste d when test ed This clea rly poin ts to a cen tral role on echi noid phag ocyt es. and espe cial ly C 3 in the opso niza tion for com plem ent acti vati on proc ess. + The experim,~ts with che lato rs show ed a requ irem ent for ca 2 , but not Mg for com plete opso tion . This , toge ther with the requ irem ent for pres ensi tizaniza tion of red cell s w.i th IgM, stro ngly indi cate s that the opso nic prin seru m is the com plem ent casc ade C 1-4- cipl e in mam mali an 2-3 acti vate d via the clas sica l path way . C 5 or late r com pone sinc e the mou se seru m lack ed C 5, and nts are not nece ssar y, norm ally pret reat ed with zym osan . Zym the human seru m was osan inac tiva tes the late com pone nts in human seru m, leav ing inta opso nize via the clas sica l path way of ct the abil ity to weak opso nic effe ct of seru m with EGTAcom i!em ent (7,1 1). The /Mg may be due to some acti vati on of the alte rnat ive C path thro ugh SRBC sen sitiz ed with IgM (12, 13). When seru m was depway lete d of C 3, almo st all opso nic acti vity was lost . Thu s, the opso trea tme nt coin cide s with the dep osit ion nic effe ct of seru m surf ace. The c 3b frag men t is mod ified of C 3b on the red cell duri ng seru m opso niza tion
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and is always rapidly degraded to C 3bi by the specific serum protease C 3 inactivator. Uost C 3 on the red cell surface will be in the hemolytically inactive C 3bi form when serum is used as a complement source (14). In some sera enough proteases are present to degrade C 3bi further to the short C 3d fragment which cannot function as an opsonin. The C 3b degrading enzymes are not inhibited by EDTA and the much higher rate of opsonin inactivation in EDTA-mouse serum than in EDTA-human serum may reflect a higher content of proteases in mouse sera, or conv&rsely, mouse C 3bi is more sensitive to protease action. Opsonin inactivation by prolonged incubation of sensitized red cells in mouse serum has been described (10). Specific receptors exist on mammalian leukocytes for thefragments c 3b, c 3bi, and C 3d (14). The C 3b receptor can also bind c 4b and c 5b. The weak opsonic effect of c 3 depleted human serum may be ascribed to the presence of C 4b fragments on red cells,for it has been shown that mouse macrophage c 3b receptors do not bind human C 3b or C 4b (15); this species specificity can explain the complete lack of interaction of mouse macrophages with red cells treated with C 3 depleted human serur·., while there was a weak interaction with echinoid phagocytes. Except for this slight inconsistence all other variations in opsonization procedure with mammalian sera produced strictly parallel effects on echinoid phagocytes and mouse macrophages, thereby supporting the idea that the opsonic principle is exactly the same in both cases. From these experiments it may be inferred that echinoid phagocytes bear membrane receptors for C 3bi as is the case for their mammalian counterparts. In coherence with this is the recent finding that a complement-like activity resembling the alternative pathway of complement, and having lytic as well as opsonic properties is indeed present in echinoids. Possibly some essential structures of complement were preserved during phylogenesis, due to great complexity in funr::i.:ions with interactions between individual complement components and between components and cells. The echinoid phagocyte then recognize common C 3 structures present in most vertebrate species, while the mammalian phagocyte may recognize a structural pattern which contains both common and more species-specific traits due to the concomitant evolution of the receptors. The polyphyletic development of C 3 and C 3-receptors can explain why fish serum, if the effect was dependent on complement, only opsonized for the phylogenetically older echinoid phagocytes but not for phagocytes from mammals.
ACKNOWLEDGEMENT This work was supported in part by the Norwegian Cancer Society, Oslo, Norway.
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