Immunology 1979 36 659

Antigenic modulation of the cytophilic binding of guinea-pig IgG and IgM antibodies to homologous macrophages

R.

0.

WEBSTER* & D. A. LAWRENCEt Department of Microbiology and Immunology, Albany Medical College, and the New York State Kidney Disease Institute,§ Albany, New York, U.S.A.

Received 18 April 1978; acceptedfor publication 11 August 1978

Summary. The cytophilic binding of immune complexes by peritoneal exudate cells (PEC) from adjuvant-stimulated guinea-pigs was studied using 125[-labelled guinea-pig IgGI, IgG2 and IgM antibodies to the dinitrophenyl (DNP) group. The influence of hapten density upon cytophilic activity was studied by the addition of DNP-conjugated antigens to antibody in 2-200 molar ratios of DNP: antibody. Only IgG2 binding was enhanced by immune complex formation, and the increased binding of IgG2 antiDNP was dependent on the number of DNP determinants per antigen molecule. Cytophilic activity with e-DNP-L-lysine (DNP-LYS), a,8-di-DNP-L-lysine (DNP-LYS-DNP), or DNP1 8-BSA was no greater than that seen in the absence of hapten. Increased cytophilic binding was noted only with DNP2041-BSA. The binding of IgG2 and IgG2 antiDNP: DNP-bovine serum albumin (BSA) complexes * Present address: Department of Pediatrics, National Jewish Hospital and Research Center, Denver, Colorado 80206, U.S.A. t A Sinsheimer Scholar Awardee. § The Kidney disease Institute is a unit of the New York State Department of Health, Division of Laboratories and Research. Correspondence: Dr David A. Lawrence, The Albany Medical College, Department of Microbiology and Immunology, The Neil Hellman Medical Research Building, Albany, New York 12208, U.S.A.

0019-2805/79/0400-0659$02.00 ©) 1979 Blackwell Scientific Publications

659

was inhibited by monomeric IgG2. The relative cyto-

philic capacities of guinea-pig immunoglobulins appeared as follows: IgG2 > IgG I > IgM. IgG I and IgM binding of DNP conjugates did not enhance their cytophilic activity; therefore, IgG I and IgM cytophilic binding to PEC was considered biologically insignificant. This investigation provides further evidence that cytophilic binding of immune complexes to macrophages is due to the co-operative action of multiple Fc sites rather than a conformational change in the IgG2 antibodies, and serum proteins, notably complement components, can alter the binding and/or phagocytosis of IgG2 anti-DNP: DNP-BSA complexes.

INTRODUCTION The role of antigen in the expression of biological activity of immunoglobulin (Ig) such as the ability to initiate the complement reaction sequence or the ability of Ig to bind cytophilically to macrophages has resulted in at least two major theories for its mode of action. The first theory suggests that through combination with antigen at the antibody-combining sites, a structural perturbation of the antibody molecule takes place which results in biological activation of the antibody molecule. The second theory postulates that the observed biologic activity is the result of the Ig complexes, that is, multiple antibody Fc sites are brought into close proximity due to antigen binding;

660

R. 0. Webster & D. A. Lawrence

thus, the biological activities of the antibody molecules are increased. These theories are not mutually exclusive. Boyden & Sorkin (1960, 1961) demonstrated the presence of cytophilic Ig in immune serum and the ability of the homologous antigen to enhance the cytophilic binding of the antibodies. Berken & Benacerraf (1966) confirmed and extended these initial studies by showing that guinea-pig IgG2 was more cytophilic than IgGI and the cytophilic site is in the Fc fragment. Attempts mnade to obtain physical evidence for conformational changes of antibodies in combination with antigen have failed to yield definitive results (Metzger, 1974; Brown & Koshland, 1975). Schlessinger, Steinberg, Givol, Hochman & Pecht (1975) recently have reported that antigen can induce a conformational change in antibodies and that a conformational change may be necessary, but not always sufficient, for the triggering of the functions of Fc. Evidence for increased binding of immune complexes to macrophages resulting from a summation of individual binding sites has appeared in several reports (Leslie, Alexander & Cohen, 1976; Leslie & Cohen, 1974; PhillipsQuagliata, Levine, Quagliata & Uhr, 1971). Although the mechanism(s) of enhanced cytophilic binding of IgG after complexing with antigen has been debated, all reports agree that IgG is cytophilic. The cytophilic binding of IgM is more controversial. Various forms of IgM molecule showed only weak or no binding to lymphocytes, neutrophils, and monocytes in comparison to IgG (Ramasamy, Richardson & Feinstein, 1976; Lawrence, Weigle & Spiegelberg, 1975; Inchley, Grey & Uhr, 1970; Berken & Benacerraf, 1968). On the other hand, IgM has been reported to bind to lymphocytes (Moretta, Ferrarini, Durante & Mingari, 1975; Ferrarini, Hoffman, Fu, Winchester & Kunkel, 1977). Lay & Nussenzweig (1969) have reported that IgM-sheep erythrocyte complexes would bind to macrophages but not monocytes, and this binding was Ca2 +dependent;.they suggested that a conformational change in the IgM antibody induced by antigen binding resulted in cytophilic IgM. Despite numerous investigations, the ability of antigen to induce the biologic activities of IgG and IgM antibodies has not been resolved. We proposed to examine antigenic enhancement of the cytophilic activity of anti-DNP antibodies and enhancement of the phagocytosis of the complexes by the PEC using conjugates with various DNP densities and guinea-pig IgG and IgM anti-DNP antibodies. In addition, the effect of Clq, a known cross-linking agent of Ig, and

the effect of complement (foetal calf serum) were tested. MATERIALS AND METHODS Animals

Albany strain guinea-pigs (250-350 g) were obtained from a closed colony maintained by the New York State Department of Health, Division of Laboratories and Research. Immunization Guinea pig IgGI and IgG2 anti-DNP antibodies were raised by intradermal and footpad injections of 100 pg DNP62-Human Gamma Globulin (DNP62-HGG) in Freund's incomplete and complete adjuvant (1: 1, v: v), respectively. Guinea-pig IgM anti-DNP antisera was raised by intradermal and intraperitoneal injection with 1 mg of DNP40-Ficoll followed by bleeding 7 days later. The antisera from each animal were kept separate until tested for antibody activity after which the high-titred sera were pooled and stored at - 20°.

Antigens s,aDNP-L-lysine and s-DNP-L-lysine (Sigma Chemical Co., St. Louis, Mo.) were purified by ion exchange column chromatography (Revoltella, Adler & Osler, 1971). Concentrations of these haptens were estimated spectrophotometrically on the basis of their molar extinction coefficient of 17,400 at 360nm. Dinitrophenylated bovine serum albumin (BSA) and HGG were prepared as described by Little & Eisen (1967). DNP141-BSA were prepared by varying the dinitrobenzene sulphonic acid: BSA ratio and the incubation times. Unreacted DNP was removed by exhaustive dialysis against distilled water containing potassium carbonate. The degree of DNP substitution was estimated spectroscopically. DNP40-Ficoll, used for immunization, was prepared according to the procedure of Inman (1975). The degree of DNP substitution was estimated spectroscopically. Preparation ofspecific immunoglobulins IgGl and IgG2 were isolated by 35% ammonium sulphate fractionation from the anti-DNP antisera, followed by DEAE cellulose chromatography using 0-015 M phosphate, pH 8-0. IgGI and IgG2 were separated and purified either by (a) passage over a QAESephadex (Pharmacia, Piscataway, N.J.) column according to the method described by Ma, Rudofsky,

Antigenic modulation ofcytophilic immunoglobulin binding Esposito, Dilwith, Pollara and Steblay (1974) or by (b) starch-block electrophoresis as described by Benacerraf, Ovary, Bloch & Franklin (1963). IgM from anti-DNP sources was obtained by passage of two-fold concentrated, pooled serum over Sephadex G-200 (Pharmacia, Piscataway, N.J.) eluted with 0-01 M Tris, 0-14 M NaCl, pH 7 5. Fractions of the first protein elution peak were pooled and concentrated while dialysing against 0-05 M sodium phosphate buffer, pH 8-0. In order to remove any trace IgG contaminants the IgM fraction was further purified by DEAE-cellulose column chromatography. Anti-DNP antibodies were isolated by affinity column chromatography on a CNBr-coupled DNP24BSA-Sepharose 4B column eluted with 3 5 M sodium thiocyanate. The specific immunoglobulin fraction was then dialysed against 015 M NaCl followed by dialysis against 001 M sodium phosphate, 0-14 M NaCl, pH 7S5. Characterization of immunoglobulins The isolated immunoglobulins were analysed by immunoelectrophoresis using both goat anti-guinea-pig IgG and goat anti-whole guinea-pig serum. Specificity for anti-DNP activity was measured by a double immunodiffusion assay with DNP24-BSA (0 5 and 10 mg/ml). Samples were incubated at 250 for 24-48 h, washed in saline, dried, stained with Amido black lOB and decolourized in tap water. Passive microhaemagglutination activity against DNP41-BSA-conjugated sheep red blood cells (DNP-SRBC) was done using two-fold dilutions of serum (heat inactivated at 560 for 30 min) made in 25 pl of saline containing 0-4% rabbit serum absorbed with DNP-SRBC. Fifty microlitres of 1% suspension of DNP-SRBC were added to each well. After shaking, the microplates were incubated at 40. The results are expressed as the specific activity of the isolated antibodies based on the protein concentration of the Ig at the highest dilution with which definite haemagglutination was evident. Radioiodination The various proteins were labelled in small volumes with 1251I (New England Nuclear Corp.) by a chloramine-T procedure (Unanue, 1971). The specific activity of the different preparations varied from 14 to 31 pCi/,ug. The proteins were 90-95% precipitable with 10% TCA at 00.

Isolation ofperitoneal exudate cells (PEC) Guinea-pigs (350-500 g) were injected intraperi-

661

toneally with 10 ml of an equal mixture of sterile normal saline and Freund's complete adjuvant. Four to five days later, PEC were harvested from the peritoneal cavity using balanced salt solution (BSS) containing 5 u heparin/ml. The cells were centrifuged at 400 g for 10 min and then transferred to a clean centrifuge tube and washed twice at 4° with 50 ml of BSS-heparin solution. The cells were then resuspended to 50 ml with 0 87% ammonium chloride, allowed to incubate at 250 for 5 min, then centrifuged and washed three times with normal BSS. The cells were then washed twice with cytophilic buffer (BSS containing 0-25% BSA and 0 7 mm EDTA).

Cytophilic assay The assay employed was essentially that described by Lawrence et al. (1975) with minor modifications. Suspensions of 5 x 106 PEC in 0 1 ml cytophilic buffer were placed into plastic tubes in wet ice. Forty nanograms of 1251-labeled protein were added in a volume of 10 pl and the cells were gently mixed and incubated for 30 min at 40. Ten microlitres of antigen was then added to the respective tubes and the cells further incubated at 40 or 370 for 30 min. After incubation, the cells were washed twice through 100% foetal calf serum. The results were expressed as picograms of bound immunoglobulin per 106 cells. All analyses were performed in duplicate and usually did not vary by more than 10%; if a greater variation was observed, the results were discarded. As a control of non-specific sticking to or trapping by the cells, bovine serum albumin was similarly labelled and tested. In inhibition studies, various amounts of unlabelled monomeric IgG2 antibody in 10 pl volumes were added to the cells. The mixtures were preincubated for 30 min at 40 and washed with cytophilic buffer before addition of 1 251-IgG2. All of the 125II-labelled proteins, 4 pg/ml, were deaggregated by ultracentrifugation in a Beckman SW50. 1 rotor (Beckman Instruments, Inc., Spinco Division, Palo Alto, California) at 100,000 g for 90 min. All calculations were corrected for acid precipitable label. In some assays, 125I-labeled antibodies and antigens were preincubated (370, 30 min) prior to their addition to the PEC. 1251-labelled monomeric anti-DNP antibodies (4 pg/ml) were added to DNP-lysine, DNP-lysine-DNP or DNPn-BSA. DNP: antibody ratios were calculated as multiples of optimal binding assuming two DNP moieties per IgG. DNP: antibody molar ratios of 2-0-200:1 were assayed.

662

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Webster & D. A. Lawrence

Acid stripping of PEC As noted in certain experiments, the extrinsic proteins on guinea-pig PEC were eluted with a low pH glycine buffer (stripping buffer) according to a procedure described by Kulczycki & Metzger (1974). At the appropriate time 1 ml of acid stripping buffer was added to the peritoneal exudate cells and after 90 s incubation at 00 the cells were centrifuged. After the supernatant was decanted, the cells were washed and resuspended in 1 0 ml of cytophilic buffer. This method did not affect cell viability throughout the cytophilic assay as determined by trypan blue exclusion. The antigen-induced phagocytic index was calculated as follows: Antigen (c.p.m. per 106 acid-stripped cells c.p.m. per 106 non-acid-stripped cells) No Antigen (c.p.m. per 106 acid-stripped cells c.p.m. per 106 non-acid-stripped cells) Preparation ofhuman Clq Human Clq was isolated from fresh serum by serial precipitation in low ionic strength EDTA buffers at 00 by the method of Volanakis & Stroud (1972). The only change was the use of relative salt concentrations of 0 04 M NaCl and 0 078 M NaCl, respectively, for the first and second precipitation steps. The concentration of purified Clq was 209 pg/ml. Clq was judged pure by the criteria of a single line on immunoelectrophoresis and electrophoresis in 3-75% polyacrylamide gel containing 0-1% sodium dodecyl sulphate.

RESULTS Characterization of immunoglobulin reagents Purified anti-DNP IgGI, IgG2 and IgM antibodies, each developed as a single line on immunoelectrophoresis against goat anti-guinea-pig serum. Ouchterlony double diffusion analysis showed precipitation lines with IgGl, IgG2 and IgM anti-DNP when tested against DNP24-BSA. Haemagglutination specific activities for IgGl, IgG2 and IgM anti-DNP were 12, 4 and 32 Mg, respectively.

Cytophilic binding of monomeric IgG1, IgG2 and IgM The cytophilic binding of monomeric guinea-pig IgGI, IgG2 and IgM anti-DNP antibodies to guineapig PEC were compared in the absence of any antigen. A normal guinea-pig IgG fraction (N-IgG) and bovine serum albumin (BSA) were included in the assay as

Table 1. Cytophilic binding of

125I-labelled immunoglobulin (Ig) and bovine serum albumin (BSA) to peritoneal exudate cells (PEC)

Protein bound

1251-protein

(pg/106 PEC*)

BSA N-IgGt IgM anti-DNP IgGl anti-DNP IgG2 anti-DNP

18+10 98 + 21 59+ 10 137 + 56

473 + 108

* PEC (5 x 106/0.1 ml) were incubated with 40 ng of 1251-labelled protein for 60 min at 0-4' followed by the isolation of the cells and assessment of the amount of protein bound. The numbers represent the mean amount of protein bound to 106 cells + standard deviation. t N-IgG, normal guinea-pig IgG

immunoglobulin. positive and negative controls, respectively. As shown in Table 1, IgG2 was the most cytophilic, and it bound significantly better to PEC than the IgG 1, N-IgG, IgM or BSA. Surprisingly, IgGI did show significant binding; a previous study indicated that IgGI was not cytophilic (Berken & Benacerraf, 1966). The IgGI binding observed in this study could be due to the increased sensitivity of our assay or to a slight denaturation of IgGI, which may have resulted from the extensive purification procedures, leading to spontaneous aggregation after the ultracentrifugation. Aggregation of IgG is known to increase cytophilic potential (Berken & Benacerraf, 1966; Lawrence et al., 1975). IgM had the least cytophilic capacity of the Ig preparations. There was no significant difference between the binding of N-IgG and IgG1-anti-DNP; and IgG2-anti-DNP bound significantly better and IgM-anti-DNP bound significantly less than N-IgG. Effect of hapten density on IgG2 cytophilia Since guinea-pig IgG2 was the most cytophilic preparation, investigation of the influence of DNP conjugates on the cytophilia of IgG2 anti-DNP initiated this study (Fig. 1). 1251-labelled IgG2 anti-DNP was incubated with 5 x 106 PEC at 40 for 30 min; 10 Ml of various concentrations of DNP conjugates then were added, and the cells were incubated at 40 for an addi-

Antigenic modulation ofcytophilic immunoglobulin binding

663

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Figure 1. Effect of hapten density and its molar excess on IgG2 cytophilic binding. Forty nanograms of 1251-labelled IgG2 anti-DNP was incubated with 5 x 106 PEC at 40 for 30 min, followed by addition of 10 ul of various concentrations of DNP conjugates (2-200 molar ratio of DNP to antibody, open columns, 2; hatched columns, 20; cross-hatched columns, 200). The cells were then incubated at 40 for an additional 30 min. The bar height represents the average of ten experiments + standard deviation.

tional 30 min. The cytophilic activity of IgG2 antiDNP was altered only with DNP20-BSA and DNP41-BSA. Both conjugates with high DNP ratios produced significant increases in IgG2 binding, and enhanced cytophilia was most effective in antigen excess (200: 1, DNP: IgG2).

Inhibition of monomeric IgG2 and antigen aggregated IgG2 cytophilic activity Cytophilic binding of 125I-labelled IgG2 and 125I-labelled IgG2: DNP20-BSA (1:20) was inhibited by prior incubation of PEC with unlabelled IgG2. Table 2 shows the quantitative inhibition of monomeric and antigen-aggregated IgG2 by monomeric IgG2. Upwards of 85% inhibition was demonstrated in these experiments. This illustrated that the binding of IgG2 to PEC was specific, not simply absorption to the cell surface, and that monomeric IgG2 and antigen complexed IgG2 appear to bind similarly since the binding of both was inhibited by monomeric IgG2.

Influence of DNP conjugates on IgG and IgM cytophilia As shown in Table 3, only IgG2 anti-DNP binding was substantially enhanced by the presence of DNP20-BSA or DNP41-BSA. Only DNP conjugates with high DNP ratios are shown because earlier studies (Fig. 1) indicated that they were more efficient in enhancing IgG2 binding. DNP1 8-BSA were equally unable to enhance IgGl anti-DNP, IgM anti-DNP, and normal IgG (N-IgG) binding (date not shown). If the DNP conjugates had enhanced BSA or N-IgG binding, one could assume that DNP conjugates nonspecifically complex proteins with cells; this did not occur, however. Therefore, the preferential enhancement of IgG2 anti-DNP cytophilia by DNP2041-BSA suggests that these DNP conjugates function as specific antigens in complexing the DNP-specific antibodies and biologically affecting IgG2 anti-DNP cytophilia. IgGI and IgM anti-DNP were unaffected by antigen, and they probably play little or no role in cytophilic binding to macrophages in the guinea-pig. These data

R. 0. Webster & D. A. Lawrence

664

Table 2. Monomeric IgG2 inhibition of binding of '251-labelled IgG2 and IgG2: DNP41-BSA complexes to PEC*

Inhibition (%)

1251-Igt

IgG2 inhibitor (pg)

Exp I

Exp 2

Exp 3

IgG2:DNP41-BSA

0.1 1-0 50 10-0 50

ND ND 59 ND 61

ND ND 62 ND 53

32 73 ND 86 ND

IgG2

* PEC (5 x 106/0.1 ml) were preincubated with various amounts of monomeric IgG2 (inhibitor), washed, and added to the cytophilic Ig assay. t The binding of 40 ng monomeric 1251-labelled IgG2 or '251-labelled IgG2: DNP4 1-BSA (40 ng:9 4 ng) complexes to PEC were inhibited. ND, Not done.

Table 3. Influence of DNP20-BSA and DNP41-BSA on cytophilic binding of various 125I-labelled protein preparations

Binding index*

12II-protein

Antigen

2:1

IgG2anti-DNP IgGI anti-DNP N-IgG BSA IgG2 anti-DNP IgGl anti-DNP IgM anti-DNP N-IgG

DNP20-BSA

DNP20-BSA DNP20-BSA DNP20-BSA DNP41-BSA DNP41-BSA DNP41-BSA DNP41-BSA

20:1

200:1

1-53+04t

1-81 +05

1-13+0 2 101 +02

1-30+0-5

309+ 15 1 02+0 1 096+0 1

1 87+0 5 1-28 +01 1 09+0 1 1 05+0 2

124+0 1 108+0 1 3 40+ 12 101 +0-1 1.05 +01 1-10+01

4-38+ 1 9 1 01 +02 100+0 1 1 17+0 2

* Binding index was calculated as follows: amount 125I-labelled protein + DNP conjugate bound to 5 x 106 PEC + amount of 125I-labelled protein bound to 5 x 106 PEC in the absence of DNP conjugates. t The numbers represent the mean binding index + standard deviation of the various 1251-labelled proteins mixed with DNP20-BSA or DNP41-BSA. These values were calculated from two to five separate experiments.

(Fig. 1, Table 3) were interpreted as showing that IgG2 shows increased cytophilic activity due to the crosslinking of the membrane adsorbed IgG2 anti-DNP molecules by antigens with enough DNP per molecule to bridge the distance between antigen binding sites of adjacent anti-DNP antibody molecules.

Quantitative binding of IgGl and IgG2 antiDNP: DNP conjugate complexes In order to quantitatively assess the effects of DNP conjugates on the binding of IgGI and IgG2 antiDNP antibodies, DNP conjugates were pre-incubated (30 min, 370) with the antibody preparations in a 20: 1

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Antigenic modulation ofcytophilic immunoglobulin binding molar ratio, and the mixtures were added to PEC, incubated I h at 40, and processed as previously described. The DNP conjugates with less than six DNP moieties had no significant effect on IgGI or IgG2 anti-DNP binding, but DNP24-BSA significantly enhanced IgG2 anti-DNP binding and produced slight enhancement of IgGI anti-DNP binding (Fig. 2). Interestingly, the DNP conjugates with less than six DNP moieties per antigen molecule did not enhance anti-DNP binding or change the slope of the binding curve; whereas DNP24-BSA did both for IgG2. An increase in the binding slope suggests a higher binding avidity with immune aggregates of IgG2 antiDNP: DNP24-BSA. The IgGl anti-DNP: DNP24BSA binding also was increased; however, the slope of 50 F

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the binding curve was not altered. This suggests that this increase may be non-specific, but this evaluation is being further investigated.

Cytophilic binding and phagocytosis of IgG2 and IgG2: DNP complexes To distinguish between the cytophilic activity of the immunoglobulins and their phagocytosis in the presence ofvarious antigen doses, cells were treated with a low pH stripping buffer. Two groups were set up with 40 ng of IgG2 to which 5 x 106 PEC were added and incubated at 40 for 30 min; 10 p1 ofvarious doses of the DNP conjugates were added, and after an additional incubation at 40 for 30 min, one group was treated with stripping buffer while the other group was kept at 4°. After acid stripping, both groups were washed in the usual fashion. The residual '25I-label remaining with the acid-stripped group was used for determining the phagocytic index since any phagocytosed 1251-IgG2 would not be accessible to acid stripping. Again, cytophilic activity was enhanced only with DNP24-BSA and DNP41-BSA, and increased DNP: antibody ratios were more effective (Fig. 3). In addition to enhancing cytophilic binding, the 200:1 molar ratio of DNP24-BSA and DNP41-BSA:IgG2 anti-DNP increased the percentage of IgG2 phagocytosed to 31% and 47%, respectively. At 40, the degree of phagocytosis in the other samples was only 22 + 2%. Temperature effects on cytophilic binding and phagocytosis also were investigated (Fig. 4 a and b). As seen in the previous studies, only those antigens with a high density of haptenic groups showed an enhancement of cytophilic binding and phagocytosis. Incubation at 370 enhanced these differences. In addition, the 370 incubation with no DNP or with conjugates with low DNP densities increased the degree of phagocytosis from 21 + 2% at 40 to 72 + 8%, and DNP24-BSA and DNP41-BSA (200: 1) increased phagocytosis from 33 to 88% and 45 to 95%, respectively. Effect of Clq and foetal calf serum on phagocytosis The ability of Clq (a subcomponent of the first component of complement) to bind antigen-activated immunoglobulins is well documented (Muller-Eberhard, 1969). In order to determine if Cl q can further crosslink immune complexes and enhance the phagocytic process, human CIq was added in l-5, 15, 150, or 1500 ' ' ' molar excess amounts to either DNP3-BSA or DNP24-BSA: IgG2 complexes formed on the surface t

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Figure 3. Influence of hapten density on cytophilic binding and phagocytosis of IgG2: DNP conjugates. A 2, 20, or 200 molar excess of DNP was added to 40 ng of IgG2 anti-DNP preincubated with 5 x 106 PEC at 4°. Hatched bars indicate acid-stripped cells. In this representative experiment the bar height is the mean of duplicate samples.

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Figure 4. Influence of temperature on cytophilic binding and phagocytosis. 200-fold molar excess of DNP-antigen was added to 40 ng of IgG2 preincubated at 4°. Reaction mixtures in panel A were kept at 40 for an additional 60 min, while the reaction mixtures in panel B were incubated at 370 for 60 min. In this representative experiment the bar height is the mean of duplicate samples. Hatched bars indicate acid stripped cells.

667

Antigenic modulation ofcytophilic immunoglobulin binding

of PEC at 40. The reaction tubes were then incubated at 370 for an hour, after which one group was acid stripped while the other group was kept at 40 until final processing. Figure 5 is representative of the data 6

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anti-DNP for 30 min at 4° with 5 x 106 PEC, 10 p1 of antigen (200 molar excess) was added for an additional 30 min at 40 followed by addition of 10 p1 of foetal calf serum (FCS) in ten-fold dilutions (equivalent to 10 p1, 1 pl, 0 I Ml, and 0-01 p1 of FCS) for 60 min at 370 before processing. Addition of foetal calf serum led to a marked decrease in both cytophilia and phagocytosis (Fig. 6). A 33% reduction in cytophilic activity and 37% reduction in phagocytosis was observed upon addition of undiluted foetal calf serum to the immune complex reaction.

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Amount of Clq added (,.Lg) Figure 5. Effect of Clq on cytophilic binding and phagocytosis. Human C I q was added to either DNP3-BSA (Panel A) or DNP24-BSA (Panel B): IgG2 immune complexes (200:1 ratio) preincubated with 5 x 106 PEC at 4° and incubated for an additional 60 min at 37°. In this representative experiment the bar height is the mean of duplicate samples. Hatched bars indicate acid stripped cells.

obtained in such an experimental system. C l q slightly enhanced the cytophilic binding of IgG2 anti-DNP antibodies complexes with DNP3-BSA but not DNP24-BSA. In addition, Clq produced moderate enhancement of the phagocytosis of IgG2 with DNP3-BSA and DNP24-BSA. Upwards of 35% more IgG2 was non-acid strippable (presumably phagocytosed) with 16 pg or 16-160 pg C l q for DNP3-BSA or DNP24-BSA complexes, respectively. Apparently, Clq can affect the cytophilic binding of DNP3-BSA complexes which are probably smaller than the DNP24-BSA complexes; whereas, the DNP24-BSA complexes were already bound efficiently and C l q was not able to enhance the binding. Clq, however, did enhance phagocytosis of the complexes most efficiently with the DNP24-BSA complexes. Since foetal calf serum appears to have an intact alternate complement reaction pathway (R. 0. Webster, unpublished observation), it was decided to see if addition of foetal calf serum in various dilutions would enhance cytophilic binding to PEC and phagocytosis by the PEC. After incubation of the IgG2

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Figure 6. Effect of foetal calf serum on cytophilic binding and phagocytosis. 200-fold excess DNP24-BSA was added to 40 ng IgG2 preincubated with 5 x 106 PEC at 4°. Ten-fold dilutions of FCS were added to the respective tubes and the reaction mixtures incubated at 370 for an additional 60 min. Panel A, no antigen; Panel B, DNP24-BSA. In this representative experiment the bar height is the mean of duplicate samples. Hatched bars indicate acid-stripped cells.

DISCUSSION The cytophilic binding and phagocytosis of IgG2 but not IgGl or IgM antibodies was significantly enhanced by the presence ofantigens with high epitope densities. This data supports earlier findings with IgGl and IgG2 (Berken & Benacerraf, 1966) and extends these studies by evaluating IgG 1, IgG2, and IgM activities, binding v. phagocytosis, and the role of complement components. In addition, the data indicates that antigen enhances antibody cytophilia and phagocytosis via the formation of immune complexes. The cross-linking by antigen of cytophilic immunoglobulins bound to Fc receptors is proposed as the mechanism of this enhancement, which suggests that

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this mechanism is similar to antigenic cross-linkage of IgE which triggers histamine release from mast cells and basophils (Ishizaka & Ishizaka, 1975). The formation of immune complexes with DNP2041-BSA increased the cytophilic binding of IgG2 anti-DNP although the antibody alone was capable of binding to macrophages as previously had been reported (Berken & Benacerraf, 1966). The standard assay performed in this investigation was similar to that originally described by Boyden & Sorkin (1960, 1961), in that antibody was incubated with PEC before the addition of antigen (or hapten). It had been suggested that there are different receptors for monomeric Ig and aggregated Ig (Walker, 1976; Grey, Anderson, Heusser, Borthistle, von Eschen & Chiller, 1976); the ability of monomeric IgG2 to inhibit the binding of monomeric IgG2 and antigen-aggregated IgG2 (Table 2) suggests that there are cytophilic sites on native IgG2 molecules and that the same macrophage Fc receptors recognize these sites on the native or aggregated IgG2. Monomeric and antigen-aggregated IgG2 did show differential binding characteristics (Fig. 2), but these differences were probably due to the enhanced affinity of antigen cross-linked IgG2 and not to different receptors. Since only antigens with high epitope densities enhanced, we believe that there must be a certain minimal density of DNP groups per antigen molecule and a minimal number of antigen molecules for crosslinkage of the IgG2 molecules on the cell surface. Most likely, cross-linkage of the IgG2 and not a conformational change of the IgG2 is the mechanism for antigenic enhancement of antibody cytophilia since antigen molecules with ten DNP groups produced no enhancement. This is in agreement with PhillipsQuagliata et al. (1971) who have shown that the amount of antibody bound to macrophages and the affinity of binding are increased by cross-linking with polyvalent antigen at equivalence, but are unaffected by monovalent or excess polyvalent antigen. This differs from our study, in that enhancement was observed with a 200-fold molar excess of DNP: antibody; in our study, however, the antibody was usually already present on the cell surface, thus antigen excess probably enhanced cytophilia by enhancing the crosslinkage of the IgG2 on the cell surface. The ability of antigen excess to reduce the size of the immune complex presented to the macrophage would not be a factor since the antibody should be bound already to the cell. In addition, the present study evaluated the effect of temperature. Because the studies were per-

formed at 40 and 370 and included removal of extrinsic surface constituents, we could distinguish between the cytophilic binding and phagocytosis of the immune complexes. There was a marked difference between the binding and phagocytosis of IgG2 and antigen-aggregated IgG2 at 40 v. 370 (Fig. 4). The ability of the cytophilically bound immune complexes to be phagocytosed or pinocytosed at 40 increased with higher epitope densities. In fact, the differential effect of epitope densities on antigen-induced uptake (phagocytic index) was most apparent when the studies were done at 4°. These indices were the following: DNP-LYS, 0-87; DNP3-BSA, 1-04; DNP8-BSA, 0-95; DNA24-BSA, 1-59; and DNP41-BSA, 2-05 (Figs. 3 and 4). At 370, there was increased uptake of the IgG2 without antigen (Fig. 4). This was probably due to increased pinocytosis and/or increased spontaneous aggregation of the purified antibodies which lead to enhanced phagocytosis. The data obtained at 40 suggests that cross-linking of the IgG2 by antigen was required for the induction of phagocytosis and/or pinocytosis. The cytophilic binding of IgGI and IgM anti-DNP antibodies to macrophages was low in comparison to IgG2 binding, and antigen-aggregation did not enhance IgGI and IgM cytophilia or phagocytosis. The quantitative binding curves of IgG I with antigens possessing different DNP densities did not substantially differ (Fig. 2). Human IgG2 and IgG4, which in the monomeric form are not cytophilic, showed significant cytophilia after aggregation (Lawrence et al., 1975). It was suggested that aggregation increased their binding affinity so that binding could be detected. Since this does not appear to be the case with guineapig IgGI, the affinity of IgGI may be so low that it could not be appreciably enhanced by aggregation. In the absence of serum proteins, IgM also appeared to have no significant cytophilic activity. These results are in close agreement with earlier published reports (Ramasamy et al., 1976; Lawrence et al., 1975; Inchley et al., 1970; Berken & Benacerraf, 1968). Lawrence et al. (1975) could not demonstrate cytophilic binding of human IgM to human monocytes, neutrophils, or lymphocytes. Berken & Benecerraf (1968) could not demonstrate cytophilic activity of rabbit or mouse 19S antisera. The discrepancy between our findings and those of Lay & Nussenzweig (1969) who found that murine IgM: sheep erythrocytes could bind to homologous PEC may be that there are species differences between mouse and guinea-pig IgM and/or macrophages or that complement components, especially

Antigenic modulation ofcytophilic immunoglobulin binding Clq, may be involved since Cl is a potential contaminant of IgM preparations and the IgM binding to macrophages was Ca2+ dependent. Cl activation is Ca2 + dependent (Muller-Eberhard, 1969), cells have been shown to have Clq receptors (Sobel & Bokishch, 1975), and as discussed below, Clq can influence cytophilic binding and phagocytosis. The role of Clq in IgM binding to macrophages, monocytes and lymphocytes presently is under investigation. To a limited degree, C lq enhanced the binding and phagocytosis of IgG2 antigen complexes. Little or no effect, however, was seen in the cytophilic binding of IgG2 antibodies in the presence of DNP24-BSA, an antigen with a DNP density with good enhancing capabilities. In the presence of DNP3-BSA, an antigen which by itself did not enhance, C lq (1 6pg) increased binding and phagocytosis. Although Clq did not enhance the binding of DNP24-BSA:IgG2, Clq (1 -6-16 jg) did produce moderate enhancement of its phagocytosis (Fig. 5). Clq appeared to influence the binding of smaller complexes (DNP3-BSA: IgG2) but not larger complexes which already were bound well. In addition, Clq increased the efficiency of phagocytosis possibly by including an additional membrane receptor, the Clq receptor (Sobel & Bokisch, 1975). The participation of C3b in phagocytosis has been described by several workers (Henson, 1976). It was, therefore, of interest to see if foetal calf serum, which appears to contain an intact alternate pathway of complement activation (R. 0. Webster, unpublished observation) could further enhance cytophilic binding and phagocytosis of the IgG2 anti-DNP: DNP-BSA complexes. Instead of enhancement, a marked decrease in both cytophilia and phagocytosis was noted (Fig. 6). The inhibition was reduced as the foetal calf serum was diluted. One possible explanation for this effect could be that solubilization of the immune complexes takes place in the presence of excess complement components. Suggestive evidence for this idea comes from the work of Nussenzweig and colleagues who have shown that immune complexes can be solubilized by complement (Czop & Nussenzweig, 1976; Takahaski, Tack & Nussenzweig, 1977). Our data suggests that such a phenomenon may take place on the cell surface before ingestion of the complex by the macrophage. An alternate possibility for the reduction in cytophilia and phagocytosis may have been due to trace amounts of bovine IgG in the FCS. This study supports the theory that antigen enhances cytophilic immunoglobulin binding by cross-linkage of the cell-bound immunoglobulin

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which results in an increased affinity of the complex. Although an antigen-induced allosteric change in the immunoglobulin which results in increased affinity can not be completely ruled out, the evidence favours the former hypothesis. Although antigen appears to be able to induce a conformational change of the antibody, antigen alone seems to be insufficient in the enhancement of cytophilia. It also has been shown to be insufficient in the activation of IgG for optimal complement binding; aggregation of IgG molecules was required (Cohen, 1968). In a recent publication, Barisas, Singer & Sturtevant (1977) presented kinetic evidence for conformational antibody changes upon binding of antigens with large epitope density. No attempt was made, however, to see what effect this change had on biological activity of the antibody molecule. On the other hand, a recent report by Segal & Hurwitz (1977) on the binding of rabbit IgG oligomers to Fc receptor-bearing tumour cells showed that trimeric IgG bound with greater affinity than did dimer which, in turn, bound better than monomer. This suggests that aggregation plays a major role in cytophilic binding; their study, however, did not look at the binding of monomeric IgG complexed with antigen. The current report shows that there is no difference in cytophilic binding of monomeric Ig in the absence or presence of monovalent antigen (DNP-LYS, DNP1.0-BSA), as well as DNP3-BSA to DNP,o-BSA. Goers, Schumaker, Glovsky, Rebek & Muller-Eberhard (1975) have shown that a univalent hapten-antibody complex (DNPI-LYS20:IgG antiDNP) was capable of complement activation. In our study, increasing the size of the carrier (LYS to BSA) was not able to alter the cytophilic activity of IgG2 anti-DNP as long as the DNP:carrier ratio was low. This suggests that the ability of monovalent antigen to induce the activation of the Fc complement site vs. the Fc cytophilic site may differ. The binding indices of IgG2 anti-DNP complexed with monovalent to polyvalent antigens (Fig. 1) and the binding curves (Fig. 2) strongly suggest that the major factor influencing IgG cytophilia is the aggregation of Fc sites. Antigens with higher epitope densities allow greater cross-linkage and, therefore, produce better cytophilic binding and subsequently better phagocytosis. ACKNOWLEDGMENTS We wish to thank Mr Walter Edge and Mrs Chris Schumaker for their technical assistance. We would

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also like to thank Mrs Marjorie McCormick and Mrs Kathy Benedetto for secretarial assistance. This work was supported in part by U.S. Public Health Service Grant Al 12527.

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