Xenoantiserum to human C3 receptors: its preparation andeffect on the C3b and C3d receptors of tonsil cells and the
C3b receptors of erythrocytes and neutrophils
J. GERDES, UTE KLATT & H. STEIN Institute ofPathology, University ofKiel, Kiel, West Germany
Acceptedfor publication 9 August 1979
Summary. Antisera directed against complement (C3)receptors on human tonsil cells were prepared andtested for their capacity to block specifically C3 recep-tors on various types ofhuman cells. The antisera werecapable of blocking both membrane-bound and solu-bilized C3 receptors of human tonsil cells. The C3breceptors of human erythrocytes and granulocyteswere also blocked by the anti-C3 receptor sera. Sheep
Abbreviations:AET, 2-amino-ethyl-isothiouronium bro-mide hydrobromide; C1,2,3,4,5,6, first six complement com-ponents; C3 receptor, membrane receptor for conversionproducts of the third component of complement; CLL,chronic lymphocytic leukaemia; E, erythrocyte; EAET, sheeperythrocyte treated with AET; Ehu, human erythrocyte; E.,ox erythrocyte; E,, sheep erythrocyte; EA, erythrocyte-anti-body complex; EAC, erythrocyte-antibody-complementcomplex; EACm, sheep erythrocytes coated with rabbit anti-bodies and rabbit complement derived from rabbit normalserum; EACra.c6def, sheep erythrocytes coated with rabbitantibodies and C 1-5 derived from C6-deficient rabbit serum;EAC3b, sheep erythrocytes coated with rabbit antibodiesand purified human complement components Cl, C4, C2,and C3; EAC3d, C3b inactivator-treated EAC3b; GVB, ver-onal-buffered saline containing gelatine; HEPES, N-2-hyd-roxyethylpiperazine-N'-2-ethane sulphonic acid; HL-B,human B lymphocyte; Ia-like antigen, human glycoproteincomplex resembling murine Ia-antigen; IgG-Eo0A, ox eryth-rocytes coated with antibodies of the IgG type; KHB;HEPES buffer containing 2 M potassium bromide; RHITC,rhodamine isothiocyanate; SHB, sucrose HEPES buffer;TC- 199, tissue culture medium 199.
erythrocyte rosette formation was not affected. IgG-E0xA rosette formation was only slightly reduced bythe anti-C3 receptor sera. Immunofluorescent stainingwith anti-C3 receptor sera revealed only a faint ornegative staining of T cells and a distinct staining ofEAC-reactive tonsil cells, lymphocytic leukaemiacells, and granulocytes. Absorption of the antiserawith human serum proteins, brain, thymus, liver,EU-1 cell line cells, or trypsinized tonsil cells did notinfluence the capacity of the anti-C3 receptor sera toinhibit C3 receptors, whereas absorption with splenictissue or tonsil cells completely removed the blockingactivity of the anti-C3 receptor sera. Absorption withhuman erythrocytes or kidney removed only the in-hibitory effect of the antisera on C3b receptors oftonsil cells, human erythrocytes, and granulocytes, butnot on C3d receptors of tonsil cells. The results indi-cate that (a) the antisera prepared with the describedprocedure contained significant amounts of antibodyagainst C3 receptors, (b) the receptors for C3b andC3d differ in antigenicity, and (c) the C3b receptors oftonsil cells, human erythrocytes, granulocytes, andprobably glomerular cells have common antigenicsites.
INTRODUCTION
A number of human cell populations, namely, lym-phoid cells (Bianco, Patrick & Nussenzweig, 1970),human erythrocytes (Ehu) (Nishioka & Linscott,
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J. Gerdes, Ute Klatt & H. Stein
1963), neutrophils (Lay & Nussenzweig, 1968), mono-cytes (Huber, Polley, Linscott, Fudenberg & Muller-Eberhard, 1968), liver cells (Hopf, Meyer zum Busch-enfelde & Dierich, 1976), and renal glomerular cells(Gelfand, Frank & Green, 1975), have receptors forC3b and/or C3d. With the exception of C3b receptor-mediated phagocytosis by neutrophils and monocytes,little is known about the function of C3 receptors ofthe various types of cells. A useful tool for elucidatingthe biological role ofC3 receptors might be a blockadeof the C3 receptors by specific antibodies. Antibodiesdirected against C3 receptors might (a) also shed lighton the antigenic relationship between the C3 receptorsof different cellular origin, (b) improve the demon-stration ofC3 receptors, e.g. on tissue sections, and (c)help to isolate solubilized C3 receptors.
Ross, Polley, Rabellino & Grey (1973) investigatedantisera raised in rhesus monkeys by immunizationwith whole spleen cells, chronic lymphocytic leukae-mia (CLL) cells, or lymphoblastoid cell line cells, andantisera raised in rabbits with Ehu for their capacity toinhibit the binding of EAC3b and EAC3d to varioustypes of target cells. The rhesus monkey anti-spleencell sera and anti-lymphoblastoid cell line cell serainhibited both EAC3b and EAC3d rosette formation;the rhesus monkey anti-CLL sera inhibited onlyEAC3d rosette formation; and one out of three rabbitanti-Ehu sera inhibited EAC3b, but not EAC3d rosetteformation after absorption with papain-treated Eh0.These anti-whole cell sera reacted not only with C3receptors, however, but also with other membraneproteins. We have performed similar experiments, butfailed to obtain clear-cut results with rabbit antiseraraised with whole human tonsil cells, CLL cells, or EhubTo obtain antisera containing significant amounts
of antibody against C3 receptors, we decided to loadEAC, prepared with rabbit antibodies and rabbit com-plement, with C3-binding protein prepared fromhuman tonsil cells, and to immunize rabbits with theEAC-C3 receptor complexes. In this report, we pre-sent the results of our approach and describe the effectof the obtained antisera on C3 receptors of varioustypes of cells.
MATERIALS AND METHODS
Buffers and mediumVeronal-buffered saline, pH 7-4, containing 0-10% gela-tine, 1 5 x 10-4 M calcium chloride, and 5 x 10-4 M
magnesium chloride (GVB) was used during the prep-aration of EAC intermediates.
Sucrose N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES; Serva, Heidelberg, Germany)buffer (SHB) was prepared by mixing equal volumes of0-02 M HEPES buffer, pH 7-4, containing 0 13 M NaCl,and 4 x 10-4 M MgCl2. SHB was used as buffer duringthe preparation of cell fragments. 0-01 M HEPESbuffer, pH 7.4, containing 0-065 M NaCl, 2-5 x 10-3 MMgCI2, and 2 M KBr (KHB) was used for the solubili-zation of C3 receptors. During the preparation ofwhole cells, tissue culture medium 199 (TC- 199; DifcoLaboratories, Detroit, Michigan, U.S.A.) was used.
CellsLeucocytes were separated from heparinized peri-pheral blood from healthy blood donors by 1 g sedi-mentation of the erythrocytes in the presence of Hae-maccel (Behringwerke, Marburg, Germany) (I partHaemaccel to 4 parts blood). After 1 h, the superna-tant was layered on a Ficoll-sodium metrizoate (Phar-macia, Uppsala, Sweden) gradient and centrifugedaccording to B0yum (1968). The granulocytes col-lected at the bottom of the centrifuge tubes were separ-ated from contaminating erythrocytes by applyingtwo hypo-osmolar shocks. The lymphocytes collectedtogether with monocytes at the interphase were iso-lated by removal of the monocytes by incubation in aglass chamber at 370 for 2 h in the presence of 20%foetal calf serum (Seromed, Munich, Germany).
Cells from normal and neoplastic lymphoid tissuewere prepared by mincing the tissue and passing itthrough a nylon mesh, followed by Ficoll-sodiummetrizoate gradient centrifugation (B0yum, 1968).For the preparation of B-cell enriched tonsil cells,sheep erythrocytes (Es) treated with 2-amino-ethyl-isothiouronium bromide hydrobromide (AET; Sigma,Munich, Germany) (EAET) were prepared according toKaplan & Clark (1974). One volume of tonsil cellsuspension (5 x 106 cells/ml) was mixed with fourvolumes of the EAET suspension (5 x 108 EAET/mlGVB), incubated at 370 for 15 min, centrifuged at 200g for 5 min at 40, and incubated again at 40 for 60 min.The cells were carefully resuspended and layered on aFicoll-sodium metrizoate gradient. After centrifuga-tion, the non-rosetted cells at the interphase were col-lected, washed twice, and readjusted to 5 x 106 cells/mlTC- 199. This procedure was repeated once. Theobtained cells were called 'B-cell enriched tonsil cells'.EU- I cell line cells, which lack all known immunolo-
gical markers except Ia-like antigen and are derived
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Xenoantiserum to human C3 receptors
from an immunoblastic lymphoma of the B-cell type,were a generous gift from Dr U. Schneider, Erlangen,Germany. A large quantity of cells from a lymphoblas-tic lymphoma of the mature thymocytic type (EACrosette-negative, strongly human T-lymphocyte anti-
gen-positive, and spontaneous E, rosette-positive at370) were obtained from the mediastinum of a 7 year
old child during thoracic surgery. Cells from a patientwith Fcy receptor-positive prolymphocytic leukaemiawere generously donated by Dr J. Muller, Hamburg,Germany.
Tissue homogenatesFor absorption of the antisera, liver, spleen, kidney,thymus, and brain autopsy tissue was homogenized insaline with an Ultra Turrax homogenizer (Janke &Kunkel, Staufen i.Br., Germany) and then washedseveral times.
Trypsinization of tonsil cells, human erythrocytes, andsheep erythrocytesFresh tonsil cells were incubated at 370 for 30 min and1 day old EhU and E, for 3 h in TC-199 supplementedwith 0 25% trypsin (Serva, Heidelberg, Germany) fol-lowed by three washings in GVB.
Erythrocyte-antibody-complement complexes (EA C)Trypsinized E, were sensitized with rabbit anti-eryth-rocyte antibodies of the IgM type isolated byammonium sulphate precipitation and passage over
an Ultrogel AcA-34 column (LKB, Grafelfing, Ger-many). For the preparation of EACrC6-def, EA com-
plexes (5 x 101 EA/ml GVB) were incubated with an
equal volume of diluted (1: 4 with GVB) fresh C6-defi-cient rabbit serum for 30 min at 370 (the C6-deficientrabbits were kindly donated by Professor Dr K. Rother,Heidelberg, Germany). The resulting EACra-C6defwerewashed three times in GVB and readjusted to a con-
centration of 1 x 108 cells/ml. EACra were prepared byincubating equal volumes ofEA (5 x 108 EA/ml GVB)and a sublytic dilution of fresh rabbit normal serum
with relatively low EA-lytic activity (EA-lytic titre of1: 16) for 30 min at 37°.EACl-3b were prepared by the procedure described
by Bokisch & Sobel (1974) as modified by Stein,Siemssen & Lennert (1978), using functionally pure
complement components purchased from Cordis(Miami, Florida, U.S.A.).
IgG-EoxAIgG-Eo0A were prepared by incubating ox erythro-
cytes (E0x) in a rabbit anti-E0x serum, which had a lowE., agglutination titre (1:4) and a high haemolysistitre (1:2000), at 370 for 30 min. The rabbit anti-E.,serum was raised by immunizing rabbits once a weekiv. with 1 x 1010 E., for 3 months. Among five anti-E.,sera, there was one with the desired properties.
EAC and IgG-E0xA rosette assays1-5 x 106 target cells were incubated with 2-5 x 107EAC or IgG-E0xA in a total volume of 450 yl TC- 199for 5 min at 37°. then centrifuged at 200 g for 5 min,and incubated again at 37° for 20 min. Afterwards, thecells were gently resuspended and kept on ice untilcounting. The proportion of rosette-forming cells(three or more bound indicator cells per target cell)was determined by viewing 200 viable target cells in ahaemocytometer with a Zeiss (Oberkochen, Germany)phase-contrast microscope.
Immune adherenceThe immune adherence receptor on Ehu was demon-strated by agglutinating Ehu with EAC3b as describedby Bokisch & Sobel (1974).
Sheep erythrocyte rosette assay1 5 x 106 tonsil cells were incubated with 3 x 107 E,treated with neuraminidase (Behringwerke, Marburg,Germany) (50 mU/5 x 108 E, at 370 for 20 min) in atotal volume of 450 p1 TC- 199 for 5 min at 37°, centri-fuged at 200 g for 5 min, and incubated again at 40 for60 min. The cells were very gently resuspended, andthe proportion of rosette-forming cells was evaluated.
Preparation of C3 receptor-active lysatesSolubilized C3 receptor-active membrane fragmentsof tonsil cells were prepared according to Dierich &Reisfeld (1975). Briefly, 20,000g pellets prepared from1 x 1010 tonsil cells were suspended in 8 ml KHB,homogenized in a Potter-Elvehjem glass homogenizer,incubated at 40 for 30 min, and centrifuged at 100,000g for 60 min at 4°. The supernatant, designated as 'C3receptor-active lysate', was able to agglutinate EAC,with a titre ranging from 1:4000 to 1: 100,000. Theprotein concentration was determined by the methodof Lowry, Rosebrough, Farr & Randall (1951).
Absorption ofsolubilized C3 receptors with EA Crac6def5 x 108 packed EACraC6def were added to 500 p1 of theC3 receptor-active lysate diluted to a molarity of 0-5 MKBr; this mixture was incubated at 370 for 30 min with
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J. Gerdes, Ute Klatt & H. Stein
vigorous shaking to prevent agglutination. The result-ing complexes consisting of EACraC6-ef and C3 recep-tors were passed through a glass sieve (pores with adiameter of 60-80 tim) to eliminate agglutinates andthen washed three times in TC- 199. The binding of C3receptors to the EACra-C6.def was tested by the rosetteinhibition assay described by Dierich & Reisfeld(1975).
Preparation ofanti-C3 receptor seraSix rabbits were immunized once a week i.v. with5 x 108 EACraC6-deftC3 receptor complexes showingrosette formation with tonsil cells reduced by at least80%. Blood was obtained weekly, starting 21 daysafter the first immunization. For control, rabbits wereimmunized with 'non-incubated' EACra-C6-def or withEs incubated in C3 receptor-active lysate. All antiserawere routinely heat-inactivated (for 30 min at 560) andexhaustively absorbed with Es. Aliquots ofthe antiserawere absorbed with glutaraldehyde-insolubilizedhuman serum proteins, human brain homogenate, un-treated tonsil cells, tonsil cells made C3 receptor-nega-tive by trypsinization, Ehu, cells from a lymphoblasticlymphoma of the mature thymocytic type, or salinetissue homogenates of human thymus, spleen, liver, orkidney.
Rosette inhibition testA pellet of 5 x 106 target cells was resuspended in 1 mlof an anti-C3 receptor serum (diluted to a just non-agglutinating titre, usually 1:40 in this study) andincubated at 40 for 40 min with gentle shaking. Theincubated cells were then washed three times andtested for their capacity to bind Es, IgG-Eo0A, andEAC intermediates. Control sera and rabbit normalsera were usually used at the same dilutions as theanti-C3 receptor sera. The degree of rosette inhibitionwas calculated with the formula of Dierich & Reisfeld(1975) in the following modified form:
were then tested for their capacity to agglutinate withEAC3b as described above for immune adherence.
Indirect agglutination oferythrocytesSerial dilutions of 200 yl antiserum or control serumwere prepared in small conical reaction tubes. Twohundred microlitres of an erythrocyte suspension(1 x 108 E/ml GVB) were added to each tube and themixture was incubated at 370 for 30 min. After threewashings, the concentration was readjusted to 1 x 108E/ml GVB in each tube. Twenty-five microlitres ofthese suspensions were incubated on microtitre plateswith 25 pl of a sheep anti-rabbit IgG serum, which hadbeen absorbed with glutaraldehyde-insolubilizedhuman serum proteins, human brain tissue, Ehu, andhuman tonsil cells. The plates were agitated for 5 minand incubated at 370; the agglutination pattern wasread after 60-90 min.
Inhibition ofsolubilized C3 receptorsSerial dilutions of anti-C3 receptor serum or controlserum were mixed with 25 p1 of a C3 receptor-activelysate with an EAC-agglutination titre of 1: 16 to 1:32in microtitre wells and incubated at 370 for 40 min.Then, 2-5 x 106 EAC suspended in 25 p1 GVB wereadded to each well. The plates were agitated for 5 minat 22° and then incubated at 37°. The sedimentationpattern was evaluated after 60-90 min.
Immunofluorescent stainingVital immunofluorescent staining of cell suspensionswas performed according to the principles originallydescribed by Moller (1961). Surface immunoglobulinwas stained by direct immunofluorescence, using goat-lgG rhodamine isothiocyanate (RHITC) conjugatedirected against human IgM, IgA, and IgG (Nordic,Tilburg, The Netherlands) at a dilution of 1:8. Theindirect procedure was used for the immunofluores-cent staining of the other cell surface structures. The
/0% rosette formation in the presence of anti-C3 receptor serum \
% rosette formation in the presence of rabbit normal serum J
Inhibition of agglutination ofhuman erythrocytes with following antisera were used as primary antisera,EAC3b diluted 1:4 to 1:40: rabbit anti-C3 receptor sera,1 x 108 packed Ehu (blood group 0) were incubated at rabbit anti-HL-B serum (Alpha Gamma Labs, Sierra370 for 30 min in 1 ml of anti-C3 receptor serum or Madre, California, U.S.A.), rabbit anti-a1-foetopro-control serum at various dilutions (1: 32-1: 512), fol- tein serum (Dakopatts, Copenhagen, Denmark), andlowed by three washings in GVB. The incubated Ehu rabbit normal serum absorbed with human brain,
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Xenoantiserum to human C3 receptors
human serum polymer, untreated or trypsinized tonsilcells, Es, or Ehu. As second antiserum, we used a goatIgG-RHITC conjugate directed against rabbit IgG(Nordic, Tilburg, The Netherlands). The goat anti-rabbit IgG-RHITC conjugate was additionallyabsorbed with human tonsil cells and applied at adilution of 1:40.
RESU LTS
Preparation of the anti-C3 receptor seraAs source of complement receptors, we used humantonsil cells, 32-68% of which showed rosette forma-tion with EACra, 39-65% with EACra-C6def, 28-67%with EAC3b, and 37-71% with EAC3d. C3 receptor-active lysates were prepared by solubilizing 20,000 gpellets of I x 1010 tonsil cells with 8 ml KHB. Aftercentrifugation at 100,000 g for 60 min at 4°, the resus-pended pellets contained 5988% of the total protein ofthe 20,000 g pellets and showed an EAC-agglutinationtitre ranging from 1:64 to 1:256. The supernatants,called 'C3 receptor-active lysates', contained 4022% ofthe total protein and showed an EAC-agglutinationtitre that varied from preparation to preparation(1 :4000-1: 100,000). For preparation of anti-C3receptor sera, Es were heavily coated with rabbit anti-bodies and rabbit complement components 1-5 using1:4 diluted C6-deficient rabbit serum (EACraC6def)and then incubated in receptor-active lysate. As shownin Table 1, EACr.C6-def rosette formation was mar-kedly inhibited by the pre-incubation in C3 receptor-active lysate, indicating a dense coating of the EACraC6-def with C3-binding protein. Rabbits were then im-munized with the EACraC6 detC3 receptor complexes.
Table 1. Inhibition of binding of EACra.C6.defto tonsil cells by C3 receptor-active lysates oftonsil cells
*5 x 108 packed EACraC6-def were pre-incu-bated in 500 p1 of C3 receptor-active lysate oftonsil cells, diluted to a molarity of 0 5 M KBr.
As a control of specificity, rabbits were immunizedwith non-incubated EACaC6-ef or with Es pre-incu-bated in C3 receptor-active lysate.
Reactivity of the anti-C3 receptor sera to sera, erythro-cytes, and complement components of various speciesThe anti-C3 receptor sera absorbed with E, showed nodetectable precipitation lines in the double diffusionassay (Ouchterlony, 1949) with sheep normal serum,rabbit normal serum, goat normal serum, guinea-pignormal serum, or human normal serum. The anti-C3receptor sera agglutinated Eh., but not rabbit erythro-cytes, mouse erythrocytes, chicken erythrocytes, orEo. The control sera did not agglutinate any of theerythrocytes tested, including Ehu (see Fig. 1). Todetermine whether serum complement components ofvarious species and rabbit IgG were recognized by the
1:4 1:16 1:64 1:256
.:
i:f
Figure 1. Direct and indirect agglutination ofhuman erythro-cytes (Ehu), trypsinized Ehu, ox erythrocytes (E,), mouseerythrocytes, and rabbit erythrocytes by an anti-C3 receptorserum. Row A: serial dilutions ofanti-C3 receptor serum plusEhu. Row B: serial dilutions of anti-C3 receptor serum plustrypsinized immune adherence-negative Ehu. Row C: serialdilutions of an antiserum raised against sheep erythrocytes(Es) pre-incubated in C3 receptor-active lysate plus Ehu. RowD: Ehu pre-treated with a serially diluted anti-C3 receptorserum plus a sheep anti-rabbit IgG serum. Row E: tryp-sinized immune adherence-negative Ehu pre-treated with ser-ially diluted anti-C3 receptor serum plus a sheep anti-rabbitIgG serum. Row F: E., pre-treated with serially dilutedanti-C3 receptor serum plus sheep anti-rabbit IgG serum.Row G: mouse erythrocytes pre-treated with serially dilutedanti-C3 receptor serum plus sheep anti-rabbit IgG serum.Row H: rabbit erythrocytes pre-treated with serially dilutedanti-C3 receptor serum plus a sheep anti-rabbit IgG serum.
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J. Gerdes, Ute Klatt & H. Stein
antisera, EAC prepared with purified human comple-ment components, EAC prepared with rabbit normalserum, EAC prepared with mouse normal serum, andIgG-E.,A were mixed with serial dilutions of theanti-C3 receptor sera and the control sera. None of thesera agglutinated any of the EAC preparations orIgG-EA, indicating that none of the antisera con-tained detectable amounts of antibodies againsthuman, rabbit, or murine complement components orrabbit IgG.
Reactivity of the anti-C3 receptor sera to C3 receptorsof tonsil cellsAll anti-C3 receptor sera showed strong inhibition ofbinding of EACra, EAC3b, and EAC3d to tonsil cellsat a dilution of 1:40 (Fig. 2), whereas the control seradid not exhibit significant inhibition of rosette forma-
80 -
cn EAC Ra EAC 3b EAC 3d
E 60-
Ui0_ 40 -
1201
Figure 2. Inhibition of rosette formation of tonsil cells withEACra, EAC3b, and EAC3d by a diluted (1:40) anti-C3receptor serum before absorption (cross-hatched columns),after absorption with human erythrocytes (hatched col-umns), and after absorption with native human tonsil cells(filled columns), and by an antiserum raised against EACraC6-def (open columns).
tion at dilutions of less than 1: 5, with one exception.One out of the three antisera raised with Es pre-incu-bated in C3 receptor-active lysate reduced EACrosette formation by up to 15% at a dilution of 1:5.The anti-C3 receptor sera were also capable of inhibit-ing the agglutination of EAC3b by solubilized C3receptors (Fig. 3). The control sera did not have anyeffect on solubilized C3 receptors.
Reactivity of the anti-C3 receptor sera to the immuneadherence (C3b) receptor of human erythrocytesThe reactivity of the anti-C3 receptor sera to Ehu andtheir immune adherence receptors was investigated bythree different means. First, Ehu were titred withanti-C3 receptor sera (Fig. 1, row A). The agglutina-tion titre ranged from 1: 8 to 1: 32. Second, Ehu werepre-incubated with serially diluted anti-C3 receptorsera and washed. The pre-incubated Ehu were mixedwith an anti-rabbit IgG serum exhaustively absorbedwith Ehu (Fig. 1, row D). The resulting indirect agglu-tination titre varied from 1:32 to 1: 128. Trypsinized,immune adherence-negative Ehu were not agglutinatedby the anti-C3 receptor sera (Fig. 1, rows B and E).Third, Ehu were pre-incubated in increasing dilutions(1: 32-1: 512) of anti-C3 receptor sera and mixed withEAC3b. The agglutination of Ehu with EAC3b wascompletely inhibited by pre-incubation of Ehu inanti-C3 receptor sera at dilutions of 1: 32-1 :64.Anti-C3 receptor sera diluted more than 1:128 andcontrol sera and anti-C3 receptor sera absorbed fivetimes with packed Ehu amounting to one-third theantiserum volume did not inhibit agglutination (Fig.4).
Reactivity of the anti-C3 receptor sera to C3b receptorsof human granulocytesRosette formation by human granulocytes with
Dilutions of rabbitnormal serum
Dilutions of Fdifferent anti- . . . . * 0 GC3 receptor sera
Dilution: 1:2 1:8 1:32 1:128 1:512Figure 3. Inhibition of EAC agglutination by solubilized C3 receptors by means ofpre-treatment ofthe solubilized C3 receptorswith various anti-C3 receptor sera. First row: C3 receptor-active lysates diluted to an EAC-agglutination titre of 1: 32 andpre-treated with serially diluted rabbit normal serum plus EAC. Second, third, and fourth rows: C3 receptor-active lysatespre-treated with three different serially diluted anti-C3 receptor sera plus EAC.
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Xenoantiserum to human C3 receptors
EAC 3b EAC 3b
Ehu untreated
Ehu pretreated withrabbit normal serum
Ehu pretreated with different Janti-C3- receptor- sera
Ehu pretreated withrabbit normal serum
Ehu pretreated with differentanti- C3-receptor-seraabsorbed with Ehu
Figure 4. Inhibition of immune adherence by pre-treatment of human erythrocytes (Ehu) with three different anti-C3 receptorsera. The antisera were diluted 1: 40 with GVB.
EAC3b was strongly inhibited by the anti-C3 receptorsera at a dilution of 1:40 (Table 2). Absorption of theanti-C3 receptor sera five times with Ehb removed theinhibitory effect of the antisera on the C3b receptors ofgranulocytes. Control sera did not influence theEAC3b rosette formation of granulocytes.
Reactivity of the anti-C3 receptor sera to Fcy receptorsand sheep erythrocyte receptorsThe anti-C3 receptor sera absorbed with Es and insolu-bilized human serum protein were investigated fortheir effect on the binding of Es and IgG-E0xA to tonsilcells and cells from a case of Fcy receptor-positiveprolymphocytic leukaemia. Es rosette formation wasnot affected at all by the anti-C3 receptor sera. IgG-
Table 2. Inhibition of C3b receptors on granulocytes by anti-C3receptor sera and effect of absorption of the antisera withhuman erythrocytes (Ehu)
Inhibition of EAC3bAntiserum Absorption rosette formation (%)
Control sera* Est 0Anti-C3 receptor serum I Es 51
Es + Ehu 3Anti-C3 receptor serum 2 Es 59
Es+ Ehu 3Anti-C3 receptor serum 3 Es 62
Es + Ehu 5
*Sera obtained from three rabbits immunized with sheeperythrocytes pre-incubated in C3 receptor-active lysate.
tEs= sheep erythrocytes.
E0,A rosette formation, on the other hand, wasslightly (up to 19%), but significantly reduced. Con-trols with rabbit normal serum showed a similardegree of IgG-E0xA rosette inhibition; the inhibitoryeffect of the other control sera was not as great. Theinhibitory effect of the anti-C3 receptor sera and allcontrol sera at a dilution of 1:30 on IgG-E0xA rosetteformation could be completely abolished by absorp-tion with trypsinized tonsil cells.
Immunofluorescent staining of various types of cellswith anti-C3 receptor seraIn indirect immunofluorescence, the various anti-C3receptor sera absorbed with Es did not stain, or onlyfaintly stained peripheral blood lymphocytes and thy-mocytes. In contrast, EAC-binding B cells from CLL,granulocytes, and Ehu exhibited brilliant staining withthe anti-C3 receptor sera at dilutions of up to 1:40.The faint staining of T cells found with some of theanti-C3 receptor sera could be completely abolished,without affecting the bright staining of EAC-reactivecells, by absorption with C3 receptor-negative cellsfrom a lymphoblastic lymphoma ofthe mature thymo-cytic type. Table 3 shows the proportions of cells ofvarious types that stained with the absorbed anti-C3receptor sera. The percentage of stained cells coin-cided with the percentage of EAC rosette-formingcells. Control stainings with antiserum raised againstEs pre-incubated in C3 receptor-active lysate, rabbitnormal serum, or anti-a,1-foetoprotein serum diluted1:4 were negative, with the exception of a few veryfaintly stained cells.
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J. Gerdes, Ute Klatt & H. Stein
Table 3. Correlation between cells that stained with anti-C3 receptor serum, absorbed with T-type lymphoblastic lymphomacells, and cells that reacted with EAC or other cell surface markers
* Antiserum raised against sheep erythrocytes pre-incubated in C3 receptor-active lysate.t EN, sheep erythrocytes treated with neuraminidase.t NT, not tested.§ Very weak staining."Only small rosettes usually consisting of three to five EAC3d bound to each granulocyte.
Table 4. Inhibition of C3 receptors of tonsil cells by anti-C3 receptorsera absorbed with sheep erythrocytes, after second absorption withvarious cells and tissue homogenates
* Mean value calculated from the results obtained with three differ-ent anti-C3 receptor sera. The antisera were diluted 1:40 with GVB.
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Xenoantiserum to human C3 receptors
Effect of absorption of the anti-C3 receptor sera withvarious types of cells and tissue on C3 receptor-blockingactivityThe anti-C3 receptor sera were absorbed with tissue orcells from various organs. The results are shown inTable 4. Absorption with glutaraldehyde-insolubi-lized human serum proteins or with human brain,human thymus, human liver, or the cells of a C3receptor-negative cell line (EU-1) did not alter theEAC rosette inhibitory capacity of the anti-C3 recep-tor sera. As expected, absorption with splenic tissue ortonsil cells completely removed the C3 receptor-block-ing activity, whereas absorption with trypsinized ton-sil cells did not. Absorption with Ehu or kidneyremoved the capacity of the anti-C3 receptor sera toinhibit EAC3b, but not EAC3d rosette formation(Fig. 2, Table 4).
DISCUSSION
The data presented here show that antisera highlycapable of blocking C3 receptors can be prepared byimmunizing rabbits with Es coated with rabbit anti-bodies, rabbit complement, and human C3 receptorprotein. The anti-C3 receptor sera obtained in thismanner had a marked blocking effect on both comple-ment receptor subtypes (C3b and C3d) of human ton-sil cells, irrespective of whether the complement recep-tors were attached to the cell membrane or were solu-bilized. Control sera raised with non-incubatedEACraC6-def did not inhibit C3 receptors at all, andonly one out of three antisera raised with Es incubatedin C3 receptor-active lysates showed a slight inhibitoryeffect on the binding ofEAC to tonsil cells. The block-ing activity of the anti-C3 receptor sera could beremoved only with tissue or cells that displayed C3receptors. Absorption with brain tissue, human serumpolymer, or liver did not abolish or reduce the anti-C3receptor reactivity. This finding suggests that humanserum, brain, and liver are either devoid of, or mar-kedly deficient in C3 receptors, or they have C3 recep-tors that are antigenically different from those of tonsilcells. The latter possibility might apply to liver, sinceHopf et al. (1976) demonstrated that isolated liver cellsare capable of binding fluid phase C3.To obtain information about the specificity of the
anti-C3 receptor sera, the reactivity of the antisera tosera and erythrocytes of various species, to human,murine, and rabbit complement components, and torabbit IgG was investigated. With the exception ofC3b receptor-positive Ehu, the anti-C3 receptor sera
did not react with any of the tested substrates. Re-markably, Ehu made C3b receptor-negative by pre-treatment with trypsin were not agglutinated byanti-C3 receptor sera. In contrast, trypsinized Ehu areknown to be agglutinated by rabbit antisera raisedagainst uncoated Ehu at up to the same, or even higherdilutions than those for untreated Ehu. This pheno-menon enables the coating of trypsinized Ehu anderythrocytes of other species with anti-E antibodies.The effect of the anti-C3 receptor sera on rosette
formation with Es and IgG-E0xA was also tested. Esrosette formation was not influenced at all by theanti-C3 receptor sera. IgG-E.,A rosette formation, onthe other hand, was slightly, but significantly reducedby the anti-C3 receptor sera. To remove this non-anti-C3 receptor activity, we absorbed the anti-C3receptor sera with trypsinized tonsil cells, since trypsindestroys complement receptors, but does not affectFcy receptor activity (Fr0land, Natvig & Wisl0ff,1973). Absorption with trypsinized tonsil cells causedtotal depletion of IgG-E0xA rosette inhibition,whereas it did not change the inhibitory effect on C3receptors.
In indirect immunofluorescence, the anti-C3 recep-tor sera absorbed with Es intensely stained non-neo-plastic and leukaemic C3 receptor-positive B cells,neutrophils, and Ehu, but they did not stain, or onlyvery faintly stained T cells. After absorption with cellsfrom a lymphoblastic lymphoma ofthe mature thymo-cytic type, the anti-C3 receptor sera still showed adistinctly positive reaction with EAC-reactive cells,but constantly gave negative staining results with nor-mal and neoplastic T cells and EAC rosette-negativeEU-1 cell line cells. Control stainings with absorbedrabbit normal serum, absorbed control serum, anti-aI-foetoprotein serum, and anti-HL-B serum ensured thespecificity of the immunofluorescent staining results.All these findings suggest that the anti-C3 receptorsera that we raised contained predominantly anti-bodies against antigenic determinants ofC3 receptors.The anti-C3 receptor sera also inhibited the C3b
receptors of erythrocytes. Absorption with Ehuremoved the capcity of the anti-C3 receptor sera toblock the C3b receptors of both Ehu and tonsil cells.Absorption with Ehu had little or no effect, however,on the ability of the antisera to inhibit C3d receptors.These findings confirm those of Ross et al. (1973) andshow that (a) receptors for C3b and C3d are antigeni-cally distinct and (b) the C3b receptors of Ehu andtonsil cells might be identical, or at least have commonantigenic sites.
83
84 J. Gerdes, Ute Klatt & H. Stein
The capacity of the anti-C3 receptor sera to inhibitC3b receptors of neutrophils and the removability ofthis inhibitory effect from the anti-C3 receptor sera byabsorption with Ehu indicate that the C3b receptors ofneutrophils are also closely antigenically related tothose of lymphoid cells and erythrocytes. The C3breceptors of glomeruli also appear to be related, sinceabsorption with renal cortex tissue removed the abilityof the anti-C3 receptor sera to block the C3b receptorsof tonsil cells and Ehu.The results of this study show that by immunizing
rabbits with EAC coated with C3 receptor protein, onecan obtain anti-C3 receptor sera having strong C3receptor-blocking activity. These antisera could bemade at least operationally specific for C3d receptorsby absorbing the antisera with Ehu.
ACKNOWLEDGMENTS
The authors thank Petra Micheels and Ursula Oest forexcellent technical assistance and Martha Soehring forpreparation of the manuscript.
This work was supported by the Deutsche For-schungsgemeinschaft, SFB 111, Project D8.
REFERENCES
BIANCO C., PATRICK R. & NUSSENZWEIG V. (1970) A popula-tion of lymphocytes bearing a membrane receptor forantigen-antibody-complement complexes. J. exp. Med.132, 702.
BOKISCH V.A. & SOBEL A.T. (1974) Receptor for the fourthcomponent of complement on human B lymphocytes andcultured human lymphoblastoid cells. J. exp. Med. 140,1336.
BoYUM A. (1968) Isolation ofmononuclear cells and granulo-
cytes from human blood. Scand. J. clin. Lab. Invest.Suppl. 97, 77.
DIERICH M.P. & REISFELD R.A. (1975) C3 receptors on lym-phoid cells: isolation of active membrane fragments andsolubilisation of receptor complexes. J. Immunol. 114,1676.
FROLAND S..S., NATVIG J.B. & WISLOFF F. (1973) Humanlymphocytes with receptors for IgG. Scand. J. Immunol.2,83.
GELFAND M.C., FRANK M.M. & GREEN I. (1975) A receptorfor the third component of complement in the humanrenal glomerulus. J. exp. Med. 142, 1029.
HOPF U., MEYER ZUM BUSCHENFELDE K.H. & DIERICH M.P.(1976) Demonstration of the binding sites for IgG-Fc andthe third complement component (C3) on isolated hepa-tocytes. J. Immunol. 117,639.
HUBER H., POLLEY M.J., LINSCOTT W.D., FUDENBERG H.H.& MULLER-EBERHARD H.J. (1968) Human monocytes:distinct receptor sites for the third component of comple-ment and for immunoglobulin G. Science, 162, 1281.
KAPLAN M.E. & CLARK C. (1974) An improved rosettingassay for detection of human T lymphocytes. J. immunol.Meth. 5, 131.
LAY W.H. & NUSSENZWEIG V. (1968) Receptors for comple-ment on leucocytes. J. exp. Med. 128, 991.
LOWRY O.H., ROSEBROUGH N.J., FARR A.L. & RANDALL R.J.(1951) Protein measurement with the folin phenol re-agent. J. biol. Chem. 193, 265.
MOLLER G. (1961) Demonstration of mouse isoantigens atthe cellular level by the fluorescent antibody technique. J.exp. Med. 114,415.
NISHIOKA K. & LINSCOTT W.D. (1963) Components of guineapig complement. Separation of a serum fraction essentialfor immune hemolysis and immune adherence. J. exp.Med. 118, 767.
