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The Journal of Immunology, 2001, 167: 5558-5564.
Copyright © 2001 by The American Association of Immunologists

Fc{gamma}RIIB in IgG-Mediated Suppression of Antibody Responses: Different Impact In Vivo and In Vitro1

Mikael C. I. Karlsson2, Andrew Getahun and Birgitta Heyman3

Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The suppressive effect of IgG on Ab responses to particulate Ags such as erythrocytes is well documented. IgG-mediated suppression is used clinically in rhesus prophylaxis to prevent RhD-negative mothers from becoming immunized against their Rh D-positive fetuses. We have recently shown that IgG anti-SRBC, passively administered together with SRBC, can induce efficient suppression of primary Ab responses to SRBC in mice lacking the known FcRs for IgG (Fc{gamma}RI, Fc{gamma}III, and Fc{gamma}RIIB or the neonatal FcR). The lack of a demonstrable effect of the inhibitory Fc{gamma}RIIB was particularly surprising, and, in this study, the involvement of this receptor is further investigated during broader experimental conditions. The data show that SRBC-specific IgG administered up to 5 days after SRBC can induce suppression both in wild-type and Fc{gamma}RIIB-deficient mice. Suppression of secondary Ab responses to SRBC in vivo was similar in the two strains. In contrast, IgG-mediated suppression of Ab responses in vitro was impaired in cultures with primed Fc{gamma}RIIB-deficient spleen cells. In conclusion, inhibition of in vivo Ab responses to SRBC by passively administered IgG can take place via an Fc{gamma}RIIB-independent pathway. This pathway causes >99% suppression and operates during all experimental conditions studied so far. The nature of the mechanism can at present only be hypothesized. Masking of epitopes and/or rapid elimination of IgG-Ag complexes would both be compatible with the observations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The primary Ab response to particulate Ags such as erythrocytes can be completely suppressed by administration of specific Ab along with the Ag (1). The ability of IgG to suppress Ab responses has been applied clinically in rhesus prophylaxis (2). Several mechanisms have been proposed to explain the suppression by IgG. One possibility is that masking of antigenic epitopes by IgG could prevent Ag-specific B cells from recognizing and reacting with the Ag. This mechanism would act independently of the IgG(Fc) part. Alternatively, IgG-Ag complexes could be rapidly eliminated by FcR-mediated phagocytosis before they are able to induce an Ab response. The most widely accepted explanation is that suppression results from inhibition of B cell activation when the IgG-Ag complexes cocross-link the B cell receptor (BCR)4 Fc{gamma}RIIB. Fc{gamma}RIIB contains an immunoreceptor tyrosine-based inhibitory motif that inhibits signaling from receptors containing immunoreceptor tyrosine-based activation motifs, e.g., the BCR (3, 4, 5, 6).

Both in vitro and in vivo models have been used to study IgG feedback suppression. A central question is whether suppression requires the IgG(Fc) part, but an unequivocal answer has been difficult to obtain. In some in vivo experiments, F(ab')2 of IgG could induce efficient suppression (7, 8, 9), whereas in others, they were less potent than intact IgG (10, 11, 12, 13). Fc dependence was implicated by the finding that IgG-mediated suppression in many cases was nonepitope-specific (11, 12, 14, 15, 16), i.e., that IgG specific for one epitope suppresses the response to other epitopes on the Ag. In contrast, epitope-specific suppression has also been observed (17, 18). In a novel approach to study the Fc dependence of suppression, we recently showed that IgG can suppress the response to SRBC in mice lacking Fc{gamma}RIIB (Fc{gamma}RIIB-/-), Fc{gamma}RI and III (owing to the lack of the common {gamma}-chain (FcR{gamma}), Fc{gamma}RI, II, and III (Fc{gamma}RIIB-/- x FcR{gamma}-/- double knockouts) or the neonatal FcR, FcRn (owing to the lack of {beta}2-microglobulin, which constitutes part of the receptor). In addition, monoclonal IgE as well as F(ab')2 of monoclonal IgG2a were able to suppress (9). These findings strongly argued against a dominant role of the Fc part of IgG.

The lack of a demonstrable role for Fc{gamma}Rs, in particular Fc{gamma}RIIB, in feedback suppression was unexpected. Our previous report on IgG-mediated suppression in Fc{gamma}RIIB-/- mice was limited to the study of in vivo primary Ab responses where SRBC and IgG were administered either as preformed immune complexes or within 1 h of each other. It cannot be excluded that these conditions predispose for Fc-independent effector functions. It is known, for example, that expression of Fc{gamma}RIIB is up-regulated on activated B cells (19), and it may therefore be easier to detect involvement of this receptor in secondary Ab responses. Moreover, masking of epitopes by IgG would presumably be most efficient when IgG is administered in close temporal relation to SRBC. Therefore, in this study we investigate the role of Fc{gamma}RIIB in suppression of secondary Ab responses, both in vivo and in vitro, and when IgG is administered several days after the Ag.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Founders for the Fc{gamma}RIIB-/- (20) were a gift from Dr. J. V. Ravetch (Rockefeller University, New York, NY). These mice (H-2b) were backcrossed to CBA/J mice (H-2k; Bommice, Ry, Denmark) for 10 generations. The H-2 haplotype (H-2k or H-2b) was analyzed by two PCR using three different primers: {alpha}K1 (5'-TATCAGTCTCCTGGAGAGATTG-3'), {alpha}K2 (5'-TTCCAAGTTGTGTTTTCCTG-3'), and {alpha}B2 (5'-ACTCCCAAGTTGTGTTTTACTA-3'). {alpha}K1/{alpha}K2 was used to detect H-2Ak and {alpha}K1/{alpha}B2 was used to detect H-2b. Gene amplification was done in 50 µl of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 0.2 mM dNTPs, 0.22 µM primers {alpha}K2 and {alpha}B2, 0.28 µM primer {alpha}K1, and 2.5 U of AmpliTaq DNA polymerase (PerkinElmer/Cetus, Norwalk, CT) for 25 cycles (30 s, 95°C; 40 s, 55°C; 30 s, 72°C). Both {alpha}K1/{alpha}K2 and {alpha}K1/{alpha}B2 give fragments of 180 bp in size. The Fc{gamma}RIIB genotype was analyzed in a PCR using three different primers: Neo (5'-CTCGTGCTTTACGGTATCGCC-3'), 5'EC1 (5'-AAACTCGACCCCCCGTGGATC-3'), and 3'EC1 (5'-TTGACTGTGTTAAACGTGTAG-3'). Gene amplification was done in 20 µl of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.2 mM MgCl2, 0.2 mM dNTPs, 0.22 µM of primers, and 1 U of AmpliTaq DNA polymerase (PerkinElmer/Cetus) for 35 cycles (30 s, 94°C; 30 s, 63°C; 40 s, 72°C). Neo/5'EC1 gives a 232-bp fragment and Neo/3'EC1 gives a 161-bp fragment. Animals were maintained and bred at the Department of Genetics and Pathology, Uppsala University (Uppsala, Sweden). Mice were age and sex matched within each experiment.

Antibodies

Polyclonal IgG anti-SRBC was prepared from hyperimmune CBA/J mouse serum. IgG was purified on a protein A-Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) (21). Abs were dialyzed against PBS, sterile filtered, and stored at -20°C. The Ab concentration was determined by absorbance at 280 nm (OD of 1.5 was assumed to equal 1 mg/ml Ab).

Antigens

SRBC and horse red blood cells (HRBC) were purchased from The National Veterinary Institute (Uppsala, Sweden) and were stored in sterile Alsever’s solution at 4°C. The cells were washed three times in PBS before use.

Immunizations

Mice were usually immunized in one of their lateral tail veins with 0.2 ml of IgG anti-SRBC plus SRBC plus HRBC (HRBC served as a specificity control) in PBS, preincubated for 1 h at 37°C immediately before injection. Controls were immunized with SRBC plus HRBC, "preincubated" with PBS. Variations to this protocol are described in the figure legends.

Adoptive transfers

Fc{gamma}RIIB-/- and wild-type mice were immunized with 4 x 106 SRBC and 8 x 105 HRBC. Seventy-nine days later, spleen cells from these mice and from unprimed controls were adoptively transferred i.v. to irradiated syngeneic recipients (600 rad 24 h before transfer). These were challenged with IgG anti-SRBC, SRBC, and HRBC as described in the figures and table.

In vitro cultures

Cultures were set up as previously described (22). Fc{gamma}RIIB-/- and wild-type mice were immunized i.p. with 0.1 ml of a 10% SRBC suspension in PBS. After 4 days, spleens were removed, spleen cell suspensions were prepared, and cultures containing 1 x 106 spleen cells, 1 x 106 SRBC, and 1 x 106 HRBC in a total of 200 µl of culture medium (DMEM supplemented as described elsewhere (22)) were set up in flat-bottom tissue culture plates (Falcon 3072, Labora, Stockholm, Sweden). Twenty microliters of a 100 or 10 µg/ml solution of polyclonal IgG anti-SRBC (giving final concentrations of 9 or 0.9 µg/ml in each well) or DMEM (to controls) was added immediately after the Ag. Cultures were incubated at 37°C in 5% CO2 and were harvested for plaque-forming cell (PFC) assays after 4 days.

PFC assay

A modified version of the Jerne hemolytic PFC assay was used (23). Briefly, 100 µl of an appropriately diluted spleen cell suspension, 25 µl of a 10% SRBC or HRBC suspension, and 25 µl of guinea pig serum (as a source of complement, diluted 1/6; The National Veterinary Institute) were added to 300 µl of 0.5% agarose (50% Seaplaque GTG (low melting point); Amersham Pharmacia Biotech) and 50% agarose (U.S. Biochemical, Cleveland, OH) kept at 45°C. The mixture was quickly spread on a microscope slide and incubated in a humid chamber for 3 h at 37°C. All dilutions were made in HBSS. Duplicate samples were counted blindly under a magnifying glass.

ELISPOT assay

The ELISPOT assay for measuring SRBC- and HRBC-specific single-cell responses has been described previously (24). Briefly, microtiter plates (Immunolon 2HB; Dynex Technologies, Chantilly, VA) were coated with SRBC or HRBC. Spleen cells were appropriately diluted in culture medium (DMEM with 0.5% FCS), applied to wells in duplicates, and incubated at 37°C for 3 h. Abs produced by the single cells were visualized as spots following the addition of goat anti-mouse IgG-alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) and the precipitating substrate 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich, St. Louis, MO). Duplicate samples were counted blindly under stereomicroscope.

Statistics

Values are expressed as log10 and/or anti-log10 (geometrical mean). Statistical analyses were usually performed with Student’s t test. For the comparison of suppression between cultures containing Fc{gamma}RIIB-/- and wild-type spleen cells (see Fig. 5Go), a Wilcoxon rank sum test was used. NS, p > 0.05; *, p < 0.05; **, p < 0.01; or **, p < 0.001.



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  		FIGURE 5. IgG-mediated suppression in vitro with Fc{gamma}RIIB-/- and wild-type spleen cells. Fc{gamma}RIIB-/- and wild-type mice were immunized with 10% SRBC and HRBC i.p. Four days later, spleen cells from these animals were prepared and five replica cultures with 1 x 106 primed Fc{gamma}RIIB-/- ({circ}) or wild-type (•) spleen cells, 1 x 106 SRBC, 1 x 106 HRBC, and IgG were set up. Direct PFC were assayed after 4 days. Results are presented as the percentage of the control (wells receiving Ag alone, represented by a broken line (100%)). Seven independent experiments are shown and lines connect results within the same experiment. The significance of the IgG-mediated suppression within each strain and experiment was calculated using Student’s t test: *, p < 0.05; **, p < 0.01; or ***, p < 0.001. The overall significance of the difference between suppression in Fc{gamma}RIIB and wild-type mice, comparing all seven experiments, was calculated using a Wilcoxon rank sum test: p < 0.001, both when 10 and 100 µg of IgG was used.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Suppression in Fc{gamma}RIIB-/- mice when IgG is administered after SRBC

It has previously been reported that intact IgG, administered as late as 6 days after 1 x 108 SRBC, can suppress an already established Ab response (assayed as direct PFC 7–11 days after priming; 13). This effect was Fc-dependent because F(ab')2 were not suppressive (13). We wanted to test whether IgG, in a similar experimental set-up, could induce suppression in Fc{gamma}RIIB-/- animals. Fc{gamma}RIIB-/- and wild-type mice were immunized day 0 with 1 x 108 SRBC and 4 x 106 HRBC (as a specificity control) followed by IgG 2, 3, 4, or 5 days later. The direct PFC response 7 days after administration of Ag was assayed (Fig. 1Go). IgG administered 2, 3, or 4 days after Ag (i.e., 3, 4, or 5 days before the PFC assay) suppressed 75–89% of the SRBC response. The degree of suppression was similar in wild-type and Fc{gamma}RIIB-/- mice. IgG administered 5 days after SRBC (i.e., only 2 days before the PFC assay) had no significant suppressive effect on the response in either strain. The response against HRBC was not suppressed by IgG in these experiments, nor in any other experiments described in this study (data not shown).



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  		FIGURE 1. IgG-mediated suppression in Fc{gamma}RIIB-/- and wild-type mice when IgG is administered after high doses of SRBC. Fc{gamma}RIIB-/- ({circ}) or wild-type (•) mice were immunized with 108 SRBC and 4 x 106 HRBC at day 0. Two to 5 days later, groups of five Fc{gamma}RIIB-/- and wild-type mice received 10 µg of polyclonal IgG anti-SRBC. The direct SRBC- and HRBC-specific PFC/spleen were assayed 7 days after immunization with Ag. The response is shown as the percentage of the response in control groups receiving Ag alone. The broken line represents the control value (100%). SRBC-specific PFC/spleen in the control groups were 976 (Fc{gamma}RIIB-/-) and 649 (wild type). No significant suppression of the HRBC response was seen (data not shown). The p values vs the control group: *, p < 0.05; **, p < 0.01; or ***, p < 0.001.

 
The number of direct PFC as late as 7 days after immunization with SRBC is <10% of the number at the peak of the response, which occurs around day 4 (data not shown). Our reason for assaying PFC at day 7 was to match the previous study (13). However, data on suppression of a response that has already declined to near-background levels are prone to large relative variations. Because of this and because demonstration of efficient suppression in the absence of Fc{gamma}Rs (9) was performed in a system where IgG was administered along with 1 x 107 or 4 x 106 SRBC and PFC responses were assayed 5 days later, the experiments in Fig. 1Go were repeated using the latter protocol. Mice were immunized with 4 x 106 SRBC and 1.6 x 106 HRBC, followed after 1 h (day 0), or 1 or 2 days by 10 µg of polyclonal IgG anti-SRBC. Three to 5 days after administration of Ag, the number of spleen cells producing IgM anti-SRBC and -HRBC was measured. Three days after immunization with Ag (Fig. 2Goa), IgG given the same day as Ag (day 0) caused >90% suppression in both strains. IgG given 1 day after Ag suppressed significantly only in Fc{gamma}RIIB-/- mice, whereas IgG given 2 days after Ag had no effect in either strain (Fig. 2Goa). Four days after immunization with Ag (Fig. 2Gob), efficient suppression could be detected in wild-type and Fc{gamma}RIIB-/- mice given IgG day 0 and day 1. Significant suppression was caused in wild-type but not Fc{gamma}RIIB-/- mice by IgG administered at day 2 (Fig. 2Gob). Five days after immunization with Ag (Fig. 2Goc), IgG administered at days 0, 1, and 2 were equally suppressive in both strains.



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  		FIGURE 2. IgG-mediated suppression in Fc{gamma}RIIB-/- and wild-type mice when IgG is administered after low doses of SRBC. Fc{gamma}RIIB-/- ({circ}) or wild-type (•) mice were immunized with 4 x 106 SRBC and 1.6 x 106 HRBC at day 0. One hour (day 0) or 1 or 2 days later, groups of five Fc{gamma}RIIB-/- and wild-type mice received 10 µg of polyclonal IgG anti-SRBC. The direct SRBC- and HRBC-specific PFC/spleen were assayed 3 (a), 4 (b), and 5 (c) days after immunization with Ag. The response is shown as the percentage of the response in control groups receiving Ag alone. The broken line represents the control value (100%). SRBC-specific PFC/spleen in the respective control groups were day 3: 6,483 (Fc{gamma}RIIB-/-) and 4,117 (wild type) (a); day 4: 30,360 (Fc{gamma}RIIB-/-) and 18,108 (wild type) (b); and day 5: 17,525 (Fc{gamma}RIIB-/-) and 10,958 (wild type) (c). No significant suppression of the HRBC response was seen (data not shown). The p values vs the control group: *, p < 0.05; **, p < 0.01; or ***, p < 0.001.

 
Clearly, SRBC-specific IgG given after either high or low doses of SRBC can suppress responses efficiently in the absence of Fc{gamma}RIIB when IgG was allowed to interact with the immune system for 3 days or more. When PFC responses are assayed already 2 days after administration of IgG, wild-type mice sometimes had lower PFC levels (Figs. 1Go and 2Gob) and sometimes had higher PFC levels (Fig. 2Goa) than Fc{gamma}RIIB-/- mice. Therefore, we cannot state whether suppression was more prominent in wild-type than in Fc{gamma}RIIB-/- mice at this early time point.

Suppression of secondary Ab responses in Fc{gamma}RIIB-/- mice in vivo

Next, we wanted to analyze whether IgG could suppress a secondary Ab response in the absence of Fc{gamma}RIIB. Wild-type and Fc{gamma}RIIB-/- mice were primed with SRBC and HRBC or left unprimed. After 89 days, primed and unprimed mice were immunized with SRBC and HRBC alone, with SRBC plus HRBC plus IgG anti-SRBC, or were left without booster injection. Five days later, PFC and ELISPOT assays were performed (Fig. 3Go). Both the primary (wild type, 1% of control and Fc{gamma}RIIB-/-, 2% of control) and secondary (wild type, 14% of control, and Fc{gamma}RIIB-/-, 10% of control) IgM responses were significantly suppressed in the two strains. As expected, there was no detectable primary IgG response at this early time point. The secondary IgG response was suppressed to a similar degree in wild-type and Fc{gamma}RIIB-/- mice (23 and 12% of control, respectively).



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  		FIGURE 3. Suppression of the secondary Ab response in Fc{gamma}RIIB-/- and wild-type mice boosted in situ. Fc{gamma}RIIB-/- and wild-type mice were immunized i.v. with 4 x 106 SRBC and 8 x 105 HRBC or were left unprimed. After 89 days, primed and unprimed Fc{gamma}RIIB-/- and wild-type mice were immunized i.v. with 4 x 106 SRBC and 8 x 105 HRBC alone or in combination with 10 µg of polyclonal IgG anti-SRBC (n = 4–6). As a control for background IgG/IgM production, some mice were not boosted. Five days later, the number of B cells producing IgG and IgM anti-SRBC and HRBC were measured in ELISPOT (IgG) or direct PFC assay (IgM). The IgM and IgG responses are shown as the number of PFC or spot-forming cells (SFC) per spleen, respectively. Suppression is also presented as the percentage of the response in the control groups given Ag alone (100% response). The suppression was Ag specific because no suppression of the HRBC response could be detected (data not shown). The p values vs the control group: *, p < 0.05; **, p < 0.01; or ***, p < 0.001. nd, Not done. a, For the six groups at the bottom of the figure, injections described in this column were primary immunizations.

 
Previous work has shown that endogenous IgG, produced during a primary response, will act to feedback-suppress the response when these mice are boosted with the same Ag (25). The primary IgG anti-SRBC response in Fc{gamma}RIIB-/- mice is higher and declines slower than in wild-type mice (20). To avoid suppressive effects caused by the varying levels of endogenously produced IgG, similar experiments as those described in Fig. 3Go were performed in an adoptive transfer system. Fc{gamma}RIIB-/- and wild-type mice were primed with SRBC and HRBC or left unprimed. After 79 days, spleen cells were transferred to syngeneic irradiated recipients. The recipients were immunized with SRBC and HRBC alone, with SRBC plus HRBC plus different doses of IgG anti-SRBC, or were left unimmunized. Five days later, PFC and ELISPOT assays were performed (Fig. 4Go). The secondary SRBC-specific IgM and IgG responses were significantly suppressed in Fc{gamma}RIIB-/- and wild-type mice. No major difference in magnitude of suppression between the two groups was seen. Importantly, suppression was equally efficient in Fc{gamma}RIIB-/- mice when suboptimal concentrations of IgG (0.4 µg) were used. In this situation,complete epitope masking does not take place. Therefore, free epitopes should be available for the BCR, and in wild-type mice, cocross-linking of Fc{gamma}RIIB and BCR by the IgG-SRBC complexes would be possible. The lack of a visible effect of Fc{gamma}RIIB during conditions of suboptimal suppression was also noted in the primary Ab response (9). Recipients of spleen cells from unprimed mice had undetectable IgG responses, confirming that a secondary type of response was indeed induced in mice given booster injections.



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  		FIGURE 4. Suppression of the secondary Ab response in Fc{gamma}RIIB-/- and wild-type mice boosted after adoptive transfer. Fc{gamma}RIIB-/- and wild-type mice were immunized i.v. with 4 x 106 SRBC and 8 x 105 HRBC or were left unimmunized. Animals were sacrificed 79 days after immunization, spleen cells were prepared, and 4 x 107 cells were adoptively transferred i.v. to irradiated (600 rad 24 h before transfer) syngeneic recipients. The following day, groups of four to five recipient mice were boosted with 4 x 106 SRBC and 8 x 105 HRBC alone or along with 0.4 or 10 µg of polyclonal IgG anti-SRBC. As a control for background IgG/IgM production, some mice (n = 3/group) were not boosted. Five days later, the number of B cells producing IgG- and IgM-anti-SRBC and HRBC were measured in ELISPOT (IgG) or direct PFC assay (IgM). The IgM and IgG responses are shown as the number of PFC or spot-forming cells per spleen, respectively. Suppression is also presented as the percentage of the response in the control groups given Ag alone (100% response). Suppression was Ag specific because no suppression of the HRBC response could be detected (data not shown). The p values vs the control group: *, p < 0.05; **, p < 0.01; or ***, p < 0.001. nd, not done.

 
In conclusion, IgG can induce efficient suppression of primary IgM as well as secondary IgM and IgG responses in the absence of Fc{gamma}RIIB both after secondary immunization in situ (Fig. 3Go) and in adoptive transfer systems (Fig. 4Go).

Impaired suppression of secondary Ab responses in vitro without Fc{gamma}RIIB

To investigate whether Fc{gamma}RIIB was involved in IgG-mediated suppression in vitro, spleen cells from primed Fc{gamma}RIIB-/- and wild-type mice were incubated with either SRBC and HRBC alone or along with 10 or 100 µg of IgG anti-SRBC. Four days later, the IgM anti-SRBC and anti-HRBC responses were measured. Seven independent experiments are shown in Fig. 5Go. One hundred micrograms of IgG induced significant suppression of the SRBC-specific response in all experiments with wild-type cells, but only in four experiments with Fc{gamma}RIIB-/- cells. Ten micrograms of IgG induced significant suppression in all experiments with wild-type cells, but in none of the experiments with Fc{gamma}RIIB-/- cells. Moreover, the magnitude of suppression was always lower in cultures containing Fc{gamma}RIIB-/- cells than in cultures containing wild-type cells.

Unimpaired suppression of secondary responses in Fc{gamma}RIIB-/- mice adoptively transferred with SRBC-primed spleen cells using a similar protocol as for in vitro studies

LPS-activated B cells express 10-fold higher numbers of Fc{gamma}RIIB than resting B cells (19). The significant role of Fc{gamma}RIIB for IgG-mediated suppression in vitro (Fig. 5Go) raised the question of whether this was due to the presence of primed B cells, with increased expression of Fc{gamma}RIIB, or whether it was an effect of the in vitro culture conditions per se. To test this, we used the same protocol for priming and boosting Fc{gamma}RIIB-/- and wild-type mice in vivo as were used for the in vitro studies. Animals were primed with 10% SRBC i.p. Four days later, their spleen cells were adoptively transferred to syngeneic irradiated recipients followed by immunization with SRBC and HRBC alone or in combination with IgG anti-SRBC. Four days later, the IgM anti-SRBC and anti-HRBC responses were measured. IgG induced the same degree of suppression in Fc{gamma}RIIB-/- as in wild-type mice (Table IGo). Thus, the use of primed B cells cannot explain why IgG-mediated suppression in vitro is dependent on Fc{gamma}RIIB.


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  		Table I. IgG-mediated suppression in Fc{gamma}RIIB-/- and wild-type mice with adoptively transferred primed spleen cells1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we confirm and extend the previous observation that IgG-mediated suppression of Ab responses to SRBC in vivo takes place efficiently in the absence of Fc{gamma}RIIB. Fc{gamma}RIIB-independent suppression operates in secondary Ab responses both when mice are boosted in situ and after adoptive transfer. It also operates when IgG is administered up to 5 days after SRBC. In the vast majority of the experiments, suppression was equally efficient in Fc{gamma}RIIB-/- as in wild-type mice. Earlier studies of mice lacking Fc{gamma}RIIB have shown that this receptor is able to negatively regulate inflammation and autoimmunity in a number of in vivo models (26). Only a few investigators have specifically studied the effect of Fc{gamma}RIIB on Ab responses in vivo. These studies show that Fc{gamma}RIIB-/- mice immunized with SRBC or keyhole limpet hemocyanin in physiological solutions or with complexes of IgG/BSA-2,4,6-trinitrophenyl had 5- to 189-fold higher Ab responses than wild-type mice (20, 27). Our failure to demonstrate an influence of Fc{gamma}RIIB in IgG-mediated suppression clearly shows that other efficient mechanisms for suppression are operative in vivo.

We also confirm previous findings that IgG administered several days after SRBC can suppress an already established Ab response (13). These observations suggest that maturing Ag-specific B cells need a continuous stimulation by Ag via their BCR to survive and/or produce Abs. Passively administered hyperimmune, high-affinity SRBC-specific IgG will presumably compete successfully for SRBC with early SRBC-specific B cells with low affinity. The correlation between affinity and the ability of IgG to suppress is compatible with this idea (11, 28). The competition for Ag between IgG and B cells may operate in a similar fashion as the competition proposed to take place between B cells of different affinity within the germinal center in the affinity maturation process (29).

Inhibition of B cell activation following cocross-linking of Fc{gamma}RIIB and the BCR has mostly been observed in in vitro experimental systems (3, 4, 5, 6). Therefore, it is interesting that IgG-mediated suppression in vitro, unlike suppression in vivo, to a large extent is dependent on Fc{gamma}RIIB. This agrees with previous in vitro studies in the same system showing that aglycosylated monoclonal IgG (that did not activate C nor bind to Fc{gamma}Rs) lost its ability to suppress (22), whereas monoclonal point-mutated IgG (that bound to Fc{gamma}Rs but did not activate C) retained its suppressive ability (30). The present finding also agrees with the observation that IgG-mediated suppression in vitro was counteracted by a mAb that blocked Fc{gamma}RIIB and Fc{gamma}RIII (31).

What then is the mechanism behind IgG-mediated feedback suppression? At present, no experimental data positively identifying a specific pathway is available. The major finding in the present report is that efficient suppression in vivo can take place without Fc{gamma}RIIB. We have previously discussed how the majority of the apparently conflicting in vivo data on Fc dependence of IgG-mediated suppression can be reconciled and we suggested that epitope masking plays a major role (1, 9, 32). Assuming that epitope masking is indeed involved, both reports on epitope specificity and nonepitope specificity of suppression can be correct. When the epitope density is high, nonepitope-specific suppression may result from steric hindrance. When the epitope density is low, epitope-specific suppression would be expected. Owing to the difficulties in obtaining pure F(ab')2 preparations and to their more rapid elimination than intact IgG, conclusions based on experiments with F(ab')2 will always be uncertain. The epitope masking hypothesis is compatible with the observations that monoclonal IgM (11), IgG3 (11, 15), and IgE (33) can suppress SRBC responses. IgM does not bind any Fc{gamma}R, and IgG3 does not bind Fc{gamma}RIIB. Although IgE has been reported to bind Fc{gamma}RIIB (34), it is suppressive in the absence of this receptor (33). Moreover, the fact that the ability of IgG to suppress correlates with its affinity (11, 28) is compatible with the epitope masking hypothesis.

However, epitope masking may not be the only way for IgG to cause suppression. Presumably, epitope masking would also be fully operative in vitro, and an argument against its exclusive role is our finding that suppression in vitro is less efficient without Fc{gamma}RIIB. In addition, some observations in the rhesus prophylaxis system are not easily explained by mere epitope masking (35). Doses of IgG anti-rhesus D, too low to cover all D Ags on human erythrocytes, suppressed the anti-D response (36). IgG, specific for the blood group Kell, suppressed the response against D Ags during conditions where steric hindrance was unlikely (37). It cannot be excluded that masking of <100% of D epitopes may be sufficient to decrease the immunogenicity of the erythrocytes sufficiently for a suppressive effect to be seen. However, an alternative resolution of the apparent paradox would be that IgG-mediated suppression operates via removal of IgG-erythrocyte complexes. This could take place in addition to epitope masking, or it could be the sole mechanism. Notably, if elimination of immune complexes were the only mechanism, and we also accept that suppression of SRBC responses in mice reflects suppression of anti-D responses in humans, it remains to be explained how the Ab-erythrocyte complexes are removed. Fc{gamma}R-mediated mechanisms seem unlikely because suppression of SRBC responses takes place in mice lacking not only Fc{gamma}RIIB, but also Fc{gamma}RI plus IIB plus III (9), and because both IgM and IgE can suppress (9, 11, 33). Complement-mediated effects also appear unlikely because IgG, unable to activate C, is still able to suppress (30). A thorough discussion about possible explanations for the apparently paradoxical findings in the mouse vs the human system has recently been published (35).

In conclusion, passively administered IgG can suppress in vivo Ab responses via Fc{gamma}RIIB-independent mechanisms, but the nature of these remain elusive. Epitope masking and/or rapid elimination of IgG-Ag complexes from the circulation are two possibilities. Should Fc{gamma}RIIB also be involved in suppression of erythrocyte responses by passively administered IgG, its effects are completely hidden by redundant suppressive pathways during all experimental conditions studied in vivo so far.


   Acknowledgments
 
We thank I. Brogren for skillful technical assistance and Dr. J. V. Ravetch for the gift of the Fc{gamma}RIIB-/- founder animals.


   Footnotes
 
1 This work was supported by Agnes och Mac Rudberg’s Foundation, by Ankarstrand’s Foundation, by Ellen, Walter and Lennart Hesselman’s Foundation, by Hans von Kantzow’s Foundation, by King Gustaf V’s 80 Year Foundation, by Lilly och Ragnar Åkerham’s Foundation, by The Swedish Association against Rheumatism, by The Swedish Foundation for Health Care Sciences and Allergy Research, by the Swedish Medical Research Council, by the Swedish Foundation for Strategic Research (Network for Inflammation Research), and by Uppsala University. Back

2 Current address: Laboratory of Molecular Genetics and Immunology, The Rockefeller University, 1230 York Avenue, Box 98, New York, NY 10021. Back

3 Address correspondence and reprint requests to Dr. Birgitta Heyman, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden. E-mail address: birgitta.heyman{at}genpat.uu.se Back

4 Abbreviations used in this paper: BCR, B cell receptor; HRBC, horse red blood cell; PFC, plaque-forming cell. Back

Received for publication April 16, 2001. Accepted for publication September 4, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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