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The Journal of Immunology, 1998, 160: 5246-5252.
Copyright © 1998 by The American Association of Immunologists

In Vivo Inflammatory Response to a Prototypic B Cell Superantigen: Elicitation of an Arthus Reaction by Staphylococcal Protein A1

Lisa M. Kozlowski2,*, Weiping Li*, Michael Goldschmidt{dagger} and Arnold I. Levinson3,*

* Division of Allergy and Immunology, University of Pennsylvania School of Medicine and {dagger} Laboratory of Pathology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Staphylococcal protein A (SpA) is representative of a new class of Ags, the B cell superantigens (SAgs). These SAgs, unlike conventional Ags, bind to the Fab regions of Ig molecules outside their complementarity-determining regions. In addition, B cell SAgs can react with a substantial amount of a host’s serum Igs by virtue of their ability to interact with many members of an entire variable heavy chain (VH) or variable light chain gene family. For example, SpA reacts with the Fabs of most human Igs using heavy chains from the VH3 gene family (VH3+). Members of this gene family are expressed on 30 to 60% of human peripheral B cells. We sought to determine whether the interaction of a B cell SAg with its reactive Igs can elicit immune complex-mediated tissue injury. Using the Arthus reaction in rabbits as an in vivo model of immune complex-mediated tissue inflammation, we demonstrated that untreated rabbits, which were administered SpA intradermally (i.d.), do not develop a cutaneous inflammatory response. However, when rabbits were pretreated i.v. with human IgG (hIgG), i.d. injections of SpA induced an inflammatory response with the classical histologic features of an Arthus reaction. To determine whether this Arthus-like response occurred via a B cell superantigenic mechanism, the rabbits were pretreated with VH3-depleted hIgG and then were administered SpA i.d. We found that the induction of a prominent inflammatory response by SpA was dependent upon the presence of VH3+ molecules in the hIgG pretreatment. These results provide compelling evidence that an interaction of the B cell SAg, SpA, with its reactive (VH3+) IgGs leads to an immune complex-mediated inflammatory response in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Staphylococcal protein A (SpA)4, a cell wall component of Staphylococcus aureus, binds to the Fc fragment of IgG. In addition, an alternative site on SpA has been defined that binds to the Fab region of Igs independently of the heavy chain isotype (1, 2, 3, 4, 5, 6). Studies have mapped the Fab determinants to framework regions 1 and 3 in the variable heavy chain (VH) region (7, 8, 9), with a possible contribution of residues in complementarity-determining region 2 (8). The binding of this alternative site on SpA is restricted to human Igs using heavy chains from the VH3 gene family (VH3+) (9, 10). The VH3 gene family is the largest of the seven human VH gene families and is expressed by 30 to 60% of human peripheral B cells (11, 12, 13). The cross-linking of membrane IgM by the alternative binding site on SpA accounts for the ability of this bacterial cell wall protein to activate human B cells in a VH-selective manner (6). These properties are reminiscent of those of a T cell superantigen (SAg) and have led SpA to be characterized as a B cell SAg (9, 10, 14, 15, 16, 17, 18). Recently, several other proteins have also been defined as B cell SAgs, including HIV gp120, protein Fv (a human liver sialoprotein), protein L (a coat protein of Peptostreptococcus magnus), and staphylococcal enterotoxin D (19, 20, 21, 22).

Given its ability to react with a large amount of Ig molecules, a B cell SAg could inflict tissue damage through a number of inflammatory mechanisms. Its interaction with cytophilic IgG, IgE, or IgA molecules could lead to the cross-linking of the respective Ig FcRs on inflammatory cells, thereby resulting in the release of inflammatory mediators. Indeed, SpA, protein L, and protein Fv induce histamine release from human basophils by interacting with the Fab region of IgE molecules that are bound to Fc{epsilon}R on these cells (23, 24, 25); protein L and protein Fv also degranulate human mast cells (24, 25). The interaction of a B cell SAg with fluid-phase IgG could lead to immune complex-mediated tissue injury, a possibility that has not been formally investigated. However, based on our recent findings (26), it is now known that the immune complexes that are formed by a B cell SAg with reactive serum Igs cause activation of the classical complement cascade.

For many years, investigators have exploited the Arthus reaction as a model of in vivo immune complex-mediated tissue injury (27, 28, 29, 30). In the classical model of the Arthus reaction, animals are immunized with an Ag until they have appreciable levels of precipitating IgG Abs. Intradermal (i.d.) injection of the same Ag elicits a local inflammatory response (28). This response is characterized grossly by erythema, edema, and hemorrhage and microscopically by a prominent polymorphonuclear cell (PMN) infiltrate that peaks at 8 h after cutaneous challenge.

In the present study, we sought to determine whether a B cell SAg could elicit immune complex-mediated tissue injury. We now show that rabbits injected i.d. with the model B cell SAg, SpA, do not develop a cutaneous Arthus reaction, as previously reported (30). However, when rabbits are pretreated i.v. with human IgG (hIgG) from healthy donors, they do develop a cutaneous reaction with the histologic features of the Arthus reaction at the sites that were injected i.d. with SpA. This reaction is mediated by the Fab-binding site on SpA, since it was not induced in animals that were pretreated with (hIgG) depleted of VH3+ molecules (VH3-hIgG). These data provide the first evidence that the interaction of a B cell SAg, SpA, with its reactive (VH3+) Igs leads to an immune complex-mediated inflammatory reaction in vivo.


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

Male and female New Zealand white rabbits (2–4 kg) were obtained from Ace Animals (Boyertown, PA) and housed in the animal facility at the University of Pennsylvania (Philadelphia, PA).

Administered reagents

Lyophilized, pooled normal hIgG (kindly provided by Sandoz Pharmaceutical, East Hanover, NJ) was reconstituted to 60 mg/ml with sterile 0.9% saline. rSpA (Repligen, Needham, MA) and human serum albumin (HSA) (low endotoxin) (Calbiochem, La Jolla, CA) were sterile filtered through 0.2-µm filters (Millipore, Bedford, MA).

Preparation of hyperiodinated SpA (Mod SpA)

rSpA was hyperiodinated to abrogate its IgG Fc-binding activity, as previously described (6).

Conjugation of Mod SpA to Sepharose 4B

We suspended 300 µg of freeze-dried CNBr-activated Sepharose 4B (Seph) (Sigma, St. Louis, MO) in 12.5 ml of 1 mM HCl; the Seph was subsequently rotated for 15 min at room temperature to swell the beads (300 µg of Seph yields 1 ml of gel.) The beads were then centrifuged at 2500 rpm for 2 min at room temperature. The supernatant was aspirated, and the beads were resuspended in 10 ml of 1 mM HCl and centrifuged as described above. Following the repetition of these steps, the beads were washed in coupling buffer (0.1 M NaHCO3 and 0.5 M NaCl (pH 8.3)). The supernatant was aspirated, and 4 mg Mod SpA plus coupling buffer or coupling buffer alone (for a total volume of 1.5 ml) was added to 1 ml of swelled Seph. Following rotation for 2 h, the beads were centrifuged as described above and washed once in coupling buffer. Next, 2 ml of 0.2 M glycine was added to 1 ml of swelled Seph and rotated for 1 h at room temperature. The Seph beads were centrifuged, washed with an acetate buffer (0.1 M sodium acetate and 0.5 M NaCl (pH 4.0)), and then washed with a Tris buffer (0.1 M Tris-HCl and 0.5 M NaCl (pH 8.0)). This procedure was repeated two more times. The Mod SpA-conjugated and unconjugated Seph beads were stored in 0.1% BSA/PBS with 0.02% sodium azide at 4°C until needed. At the time of use, the Seph beads were washed extensively with the appropriate buffer.

Depletion of VH3+ IgG from pooled hIgG

The pooled normal hIgG was passed over either a Mod SpA-conjugated Sepharose column (Mod SpA-Seph column) or an unconjugated Sepharose column (Seph column). Polyprep chromatography columns (Bio-Rad, Hercules, CA) were packed with 10 ml of either Mod SpA-Seph or Seph alone and washed with 100 ml of 1x running buffer (1:5 dilution of 5x running buffer (pH 7.0): 0.079 M KH2PO4, 0.48 M Na2HPO4, and 0.77 M NaCl). Next, 1 ml of pooled IgG (60 mg/ml) diluted with 250 µl of 5x running buffer was added to each column and allowed to pass into the Seph. The column was then closed, and the IgG was incubated in the column for 5 min. This procedure was repeated twice for each column. Afterward, 10 ml of 1x running buffer was added, and 1 ml fractions were collected. This process was repeated until the A280 of the fractions was <0.100. The column was subsequently flushed with 50 ml of 1x running buffer. The IgG concentrations of the collected fractions were determined by the following method: A280/extinction coefficient for IgG (1.43). Effluent fractions with a concentration of >1 mg/ml were pooled together and dialyzed two times against PBS at 4°C. The samples were concentrated in an Ultrafree-50 centrifugal filter (Millipore), and IgG concentrations were determined again as described above. The effluent fractions were then passed over their respective columns a second time, and the dialysis and concentration procedures were repeated.

ELISA for determining the binding of IgG fractions to Mod SpA or SpA

Half-area microtiter plate wells (Costar, Cambridge, MA) were coated with 100 µl Mod SpA, SpA, or BSA (Calbiochem) at 10 µg/ml in PBS overnight at 4°C. Each well was subsequently saturated with 100 µl 1% BSA/PBS for 2 h at room temperature. The wells were washed three times with 0.05% Tween-20 (Sigma) in PBS. We incubated 100 µl aliquots of varying concentrations of unfractionated hIgG or hIgG fractions from the Mod SpA-Seph column or the Seph column for 2 h at room temperature. The wells were washed as described above, and peroxidase-conjugated goat F(ab')2 anti-hIgG Fc Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) was added for 1 h at room temperature. Following this incubation, the plates were washed as described above, the bound Ab was detected by the addition of o-phenylenediamine substrate (Eastman Kodak, Rochester, NY) in 20 mM citrate buffer (pH 4.0) plus 0.05% hydrogen peroxide, and the OD was read spectrophotometrically at 450 nm.

Arthus reaction

We adapted a previously described model (30) to determine whether the Arthus reactivity induced by SpA was mediated by the interaction of the Fab-binding site on SpA with reactive IgG molecules. In this model, untreated rabbits injected i.d. with SpA failed to develop inflammatory reactions at injection sites. However, animals pretreated i.v. with hIgG developed Arthus reactions at cutaneous sites that had been injected with SpA. The rabbits in our experiments were sedated with 150 mg ketamine HCl (Ketaject, Phoenix Pharmaceuticals, St. Joseph, Missouri) and 10 mg xylazine (Xylaject, Phoenix Pharmaceuticals), and their backs were shaved. The animals were rested for 48 h before injections were performed to allow any inflammation that might have occurred from the shaving to subside. After 48 h, the animals were sedated with ketamine/xylazine as described above and then injected i.v. with either 170 mg hIgG, 170 mg IgG effluent from the Mod SpA-Seph column or the Seph column, or saline (5 ml), followed by 3 to 5 ml of 0.9% saline. At 10 to 15 minutes after the i.v. injection, the dorsal skin of the rabbits was prepared with alcohol. The i.d. injections of 200 µl of 900 µg of SpA or HSA (negative control) were performed at duplicate sites. The same volume of saline was injected i.d. at duplicate sites as an additional negative control. After 8 h, the dorsal skin was examined macroscopically for erythema, edema, and hemorrhage. The animals were then sacrificed, and skin biopsies were obtained from the i.d. injection sites. The specimens were fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. The histologic procedures were performed by the Laboratory of Pathology at the University of Pennsylvania School of Veterinary Medicine (Philadelphia, PA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of a cutaneous inflammatory response by SpA

To establish the Arthus model in our laboratory, three rabbits were injected i.v. with 170 mg of hIgG and then injected i.d. with 900 µg of SpA, HSA, or saline at duplicate sites 10 to 15 min later. Three additional rabbits were pretreated i.v. with saline and then injected i.d. with the above reagents as described. All rabbits were examined grossly and then sacrificed at 8 h after the i.d. injections were administered.

No macroscopic evidence of an inflammatory response was observed at any i.d.-injected sites of saline-pretreated rabbits, as previously reported (30). By contrast, erythema and induration were both seen at the SpA-injected sites of animals pretreated i.v. with hIgG (data not shown). Such reactions were not observed at the HSA- or saline-injected sites of these hIgG-pretreated animals (data not shown).

Histologic examination of the skin biopsies from the hIgG-pretreated rabbits revealed a prominent inflammatory cell infiltrate only at the sites injected with SpA (Fig. 1Go, A and B). The venules were congested with PMNs. The PMNs were also observed within the walls of the venules and in a perivascular distribution. Several areas of hemorrhage and diapedesis of erythrocytes were also observed. The arterioles were not involved. Rare PMNs were observed in the lumen of the venules that were examined at the HSA-injected sites (Fig. 1GoC), whereas no inflammatory cells were observed at the saline-injected sites (data not shown). In the saline-pretreated animals, rare PMNs were observed in venule lumens at the SpA- (Fig. 2Go) and HSA-injected sites (data not shown), while no PMNs were seen at cutaneous sites injected with saline (data not shown).



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  		FIGURE 1. Arthus reaction as seen in histologic sections from the skin of an hIgG-pretreated rabbit. Rabbits were pretreated i.v. with 170 mg of hIgG, and 900 µg of SpA (A, x31.2; B, x62.5), HSA (C, x31.2), or saline (data not shown) was administered i.d 10 to 15 min later. Animals were sacrificed after 8 h. Skin biopsies were fixed in 10% buffered formalin and stained with hematoxylin and eosin. The arrows in A demonstrate a prominent PMN infiltration in the venules. B shows a x62.5 magnification of the boxed area in A, demonstrating intense hemorrhage without the involvement of the arteriole. The section in C shows a lack of cellular infiltration and hemorrhage in the HSA-injected sites. Results are representative of duplicate injections from six rabbits.

 


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  		FIGURE 2. Lack of an Arthus reaction in a saline-pretreated rabbit. Rabbits were pretreated i.v. with saline, and 900 µg of SpA (x31.2), HSA (data not shown), or saline (data not shown) was administered i.d 10 to 15 min later. The animals were sacrificed after 8 h. Skin biopsies were fixed in 10% buffered formalin and stained with hematoxylin and eosin. This figure demonstrates the rare PMNs observed in SpA-injected sites from rabbits pretreated with saline. Results are representative of duplicate injections from three rabbits.

 
These results demonstrate that SpA induces a prominent cutaneous inflammatory response that has the features of an Arthus reaction, and that this response is only seen in rabbits pretreated with hIgG. The rabbit’s own IgG alone is not sufficient to sustain an Arthus reaction as demonstrated in the saline-pretreated animals challenged i.d. with SpA. Thus, these results confirm that SpA elicits an Arthus-like reaction in vivo, presumably via binding to hIgGs.

Depletion of VH3+ IgG molecules from hIgG

To establish that the observed inflammatory response required both SpA and human VH3+ IgGs, the hIgG preparation was separated into a VH3-depleted (VH3-) fraction. To obtain this fraction (as described in Materials and Methods), hIgG was passed twice over a Mod SpA-Seph column, and the effluent fractions were collected. The control IgG, containing both VH3+ and non-VH3+ hIgGs (VH3+/non-VH3+ hIgG, consisted of hIgG that was passed twice over a Seph column. Following fractionation over the Mod SpA-Seph column or the Seph column, the binding of the effluent IgG fractions to Mod SpA and SpA was examined by ELISAs (see Materials and Methods). As shown in Figure 3Go, the effluent IgG from the Mod SpA-Seph column had a 100-fold reduction in Mod SpA-binding IgGs as compared with equivalent concentrations of unfractionated hIgG and the effluent IgG from the Seph column. By contrast, IgG fractions from both columns bound SpA to a similar degree (data not shown). This finding demonstrates that the two types of IgG fractions contain roughly equivalent amounts of SpA-binding IgG molecules, and that the fractionation procedure does not lead to nonspecific IgG degradation. Neither of the IgG fractions bound BSA-coated wells (data not shown). Therefore, fractionation of hIgG over a Mod SpA-Seph column removed a sufficient amount of VH3+ IgGs to use this fraction as a VH3- population of IgGs. In addition, passage of hIgG over a Seph column did not remove VH3+ IgGs nor did it nonspecifically degrade the IgG molecules.



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  		FIGURE 3. Binding of IgG preparations to Mod SpA. hIgG was fractionated over a Mod SpA-Seph column or a Seph column. The binding of these IgG fractions and unfractionated hIgG to Mod SpA was analyzed in an ELISA as described in Materials and Methods. Various concentrations of hIgG (•), the IgG fraction from the Seph column ({circ}), or the IgG fraction from the Mod SpA-Seph column ({blacksquare}) were added to Mod SpA-coated ELISA plates. Background absorbance was determined in Mod SpA-coated wells in which no IgG proteins were incubated, and this absorbance was subtracted from the raw data. OD values represent the means of duplicate determinations from a representative experiment; similar results were obtained for all fractionated IgG preparations.

 
Requirement of human VH3+ IgGs for the SpA-induced Arthus reaction

As shown in Figure 1Go, SpA induced an Arthus reaction in rabbits pretreated with hIgG. To determine whether this reaction occurred via a B cell superantigenic mechanism and consequently was dependent on human VH3+ IgGs, four groups of rabbits (two rabbits per group) were injected i.v. with 170 mg of 1) VH3- hIgG, 2) hIgG that had been passed over a Seph column (VH3+/non-VH3+ hIgG), 3) unfractionated hIgG, and 4) saline, respectively. At 10 to 15 min after the i.v. injections, all animals were challenged i.d. at duplicate sites with 900 µg of SpA, HSA, and saline, respectively. After 8 h, the skin was examined grossly, and biopsies were obtained for histologic analysis. The histologic results from representative skin biopsies from hIgG- and saline-pretreated rabbits are depicted in Figures 1Go and 2Go, as described above.

Animals pretreated with VH3+/non-VH3+ hIgG showed erythema, edema, and hemorrhage only at the SpA-injected, i.d. sites upon gross examination (Fig. 4Go, A and B). In contrast, animals pretreated with VH3- hIgG demonstrated no erythema or edema at any injected sites (Fig. 4GoC). Thus, the depletion of VH3+ IgGs from the hIgG preparation abrogated the macroscopic signs of an Arthus reaction at SpA-injected, i.d. sites.



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  		FIGURE 4. Hemorrhage in an 8-h Arthus reaction. A and B, Rabbits were pretreated i.v. with 170 mg of hIgG fractionated over a Seph column, and 900 µg of SpA (A), HSA (B), or saline (data not shown) was administered i.d. 10 to 15 min later. C, Rabbits were pretreated i.v. with 170 mg of hIgG fractionated over a Mod SpA-Seph column, and 900 µg of SpA (C), HSA (data not shown), or saline (data not shown) was administered i.d 10 to 15 later. The shaved dorsal skin of all rabbits was examined after 8 h. The small dark spots observed on the rabbit skin were used to mark the site of the i.d. injections. Results are representative of duplicate injections in two rabbits per each type of IgG administered.

 
As expected from the gross examination, the SpA-injected sites in the VH3+/non-VH3+ hIgG pretreated rabbits demonstrated a prominent PMN inflammatory response (Fig. 5Go, A and B). Dense infiltrates of PMNs were observed in a perivascular distribution surrounded by areas of hemorrhage. Again, the arterioles were not involved. The HSA-injected sites contained few PMNs in the lumens of the venules (Fig. 5GoC), while no PMNs were seen at the saline-injected sites (data not shown).



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  		FIGURE 5. Arthus reaction as seen in histologic sections. Rabbits were pretreated i.v. with 170 mg of hIgG fractionated over a Seph column, and 900 µg of SpA (A, x39; B, x93.7), HSA (C, x31.2), or saline (data not shown) were administered i.d 10 to 15 min later. The animals were sacrificed after 8 h. Skin biopsies were fixed in 10% buffered formalin and stained with hematoxylin and eosin. A shows a prominent PMN infiltration in the venules and hemorrhage. B is a x125 magnification of the boxed area in A, demonstrating an intense PMN infiltration with diapedesis of erythrocytes. C shows rare PMNs in the venules. Results are representative of duplicate injections from two rabbits.

 
In contrast to the results observed in VH3+/non-VH3+ hIgG pretreated rabbits, only rare inflammatory cells were seen at the SpA-injected sites of animals pretreated i.v. with VH3- hIgG (Fig. 6Go). The rare PMNs were confined to the lumens of the venules. The HSA-injected sites showed rare PMNs in and around the venules, while no PMNs were seen at the saline-injected sites (data not shown). These results indicate that SpA elicits a prominent Arthus-like reaction in rabbits only when they are pretreated with hIgG. Moreover, this inflammatory response is dependent upon the presence of VH3+ IgGs in the hIgG preparation.



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  		FIGURE 6. Lack of an Arthus reaction at the SpA-injected site in a rabbit pretreated with VH3- hIgG and SpA. Rabbits were pretreated i.v. with 170 mg of hIgG fractionated over a Mod SpA-Seph column, and 900 µg of SpA (x31.2), HSA (data not shown), or saline (data not shown) was administered i.d 10 to 15 min later. The animals were sacrificed after 8 h. Skin biopsies were fixed in 10% buffered formalin and stained with hematoxylin and eosin. This figure demonstrates the rare PMNs observed in SpA-injected sites from rabbits that were pretreated with VH3- hIgG. Results are representative of duplicate injections from two rabbits.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies reported herein were undertaken to determine whether the interaction of a B cell SAg with its reactive IgG molecules leads to immune complex-mediated tissue injury. Although the B cell SAgs interact with variable region determinants on Ig molecules outside the complementarity-determining regions (7, 8, 9), the complexes that are formed can activate the classical complement cascade (26) and should be able to give rise to tissue inflammation. Unlike a conventional Ag, a B cell SAg can react with sizable amounts of Ig molecules. Immunization is not a prerequisite for this reaction to occur, since the reactive VH or variable light chain Abs are found in the standing pool of serum Igs in the host. Accordingly, there is great potential for a B cell SAg to engender immune complex-mediated tissue injury.

We chose to examine the Arthus reaction as an in vivo model of immune complex-mediated tissue inflammation. Although this model of inflammation was formerly thought to be mediated by complement products liberated by the action of immune complexes (28, 29, 31), recent studies have underscored the importance of Fc{gamma}RIII-bearing cells (32, 33, 34). In particular, mast cells, neutrophils, macrophages, and Langerhans cells were incriminated as critical Fc{gamma}RIII-bearing effector cells (33). These studies also suggested that complement-activation products likely augment the Arthus reaction. We decided to investigate the Arthus-inducing potential of SpA. Unlike other B cell SAgs, SpA has two Ig-binding sites. One site binds to the Fc region of IgG, while the second site binds to VH region residues on VH3+ human Igs independently of their heavy chain isotype. It is this second site that endows SpA with its B cell superantigenic properties.

Before the characterization of this Ig Fab-binding site on SpA, several groups investigated the similarities between conventional Ag/Ab complexes and SpA/IgG complexes (35, 36, 37, 38, 39). Indeed, one group hypothesized that SpA/IgG complexes could elicit an Arthus reaction based on the observation that Ag/Ab complexes and aggregated IgG elicit such an inflammatory reaction (30). These investigators found that i.d. injections of SpA into a rabbit did not cause an Arthus reaction. However, when the rabbits were pretreated i.v. with hIgG, an Arthus reaction occurred at SpA-injected, i.d. sites. The investigators concluded that the Arthus reaction was induced via an interaction between SpA and the Fc region of hIgG molecules. However, the authors failed to explain why an interaction with hIgG Fc, but not rabbit IgG Fc, would lead to an Arthus reaction.

One explanation for the inability of SpA to elicit an Arthus reaction in an untreated rabbit is that the binding of SpA to residues in the rabbit IgG Fc region blocks the binding sites for C1q and Fc{gamma}R. The C1q-binding site includes residues 285 to 340 in the constant heavy chain 2 domain of IgG Fc (40, 41, 42, 43). This region overlaps the SpA-binding site on IgG Fc (44), although it does not bind directly to the same residues. The Fc{gamma}R-binding site on IgG Fc includes residues 234 to 237 in the lower hinge region and residue 331 in the constant heavy chain 2 domain (45, 46). These residues do not overlap the SpA-binding site, but the binding of SpA to IgG Fc may still sterically hinder the binding of the IgG molecule to Fc{gamma}R. If this explanation is correct for rabbit IgGs, then it should also apply to hIgGs. However, SpA does elicit an Arthus reaction when rabbits are pretreated with hIgGs. Thus, it is unlikely that SpA blocks the binding site on IgG Fc for Fc{gamma}R or C1q, at least not on hIgG molecules.

In our opinion, a more appealing explanation is that SpA only induces an Arthus reaction when it interacts with the Fab region of reactive IgGs, such as human VH3+ IgGs. SpA binds to IgG molecules from most species via their Fc and Fab regions. However, the rabbit is one of the few species in which SpA binds only to the Fc region of its IgG molecules (47, 48). Thus, it is possible that administrating SpA to a rabbit does not induce an Arthus reaction, because SpA cannot bind to the Fab region of rabbit IgGs. On the other hand, SpA elicits an Arthus reaction in rabbits pretreated with hIgGs, presumably because it can bind to the Fab region of a large proportion of hIgGs, namely VH3+ IgGs. Proof of this hypothesis would strongly suggest that a B cell SAg can trigger an in vivo immune complex-mediated inflammatory response.

Accordingly, our initial experiments were performed to confirm that SpA elicits an Arthus reaction in rabbits pretreated with hIgG but does not cause an Arthus reaction in untreated rabbits (30). We injected hIgG or saline i.v. into several rabbits and then injected the rabbits i.d. with SpA, HSA, or saline. Only animals pretreated with hIgG and subsequently injected i.d. with SpA developed erythema and edema at injected sites. Histologic analysis of the grossly inflamed, SpA-injected sites revealed a prominent inflammatory response characterized by infiltrating PMNs (Fig. 1Go, A–C) and dermal hemorrhage. Animals pretreated with saline had rare luminal PMNs only at SpA- (Fig. 2Go) and HSA-injected sites. Thus, these results demonstrate that SpA that is i.d.-administered to a rabbit pretreated with hIgG induces an inflammatory response that has the classical histologic features of an Arthus reaction (30). In addition, our results confirm that i.d. administration of SpA to untreated rabbits does not lead to the development of such an inflammatory reaction.

To prove that this Arthus reaction reflected a B cell superantigenic property of SpA, it was necessary to show that it was mediated by VH3+ IgGs in the hIgG preparation. Therefore, we pretreated rabbits with VH3- hIgG. The VH3- hIgG fraction had a 100-fold reduction in Mod SpA-binding (VH3+) IgG when compared with unfractionated hIgG and a VH3+/non-VH3+ hIgG fraction which was passed over a Seph column (Fig. 3Go). Moreover, we demonstrated that the VH3- hIgG fraction had an equivalent amount of SpA-binding IgG molecules as compared with unfractionated hIgG and the VH3+/non-VH3+ hIgG fraction (data not shown). The marked reduction in Mod SpA binding of this fraction attested to the completeness of the VH3+ IgG depletion. Following this pretreatment, we injected the rabbits i.d. with SpA, HSA, and saline. The cutaneous responses were compared with the reactions in animals pretreated with VH3+/VH3- IgG, hIgG, or saline.

Inspection of the skin of the animals pretreated with VH3+/non-VH3+ hIgG revealed erythema, edema, and hemorrhage only at the SpA-injected, i.d. sites (Fig. 4Go, A and B). In contrast, no erythema or edema was observed at any injected sites from the VH3- hIgG-pretreated animals (Fig. 4GoC). These results demonstrated that SpA required the presence of human VH3+ IgGs to elicit the macroscopic appearance of an Arthus reaction. Histologic examination of skin biopsies from rabbits pretreated with VH3+/non-VH3+ hIgG demonstrated a prominent inflammatory cell infiltrate and dermal hemorrhage only at SpA-injected sites (Fig. 5Go, A–C). In striking contrast, the VH3- hIgG-pretreated rabbits contained only minimal PMN infiltration at the SpA-injected sites (Fig. 6Go). These results demonstrate that SpA is only able to elicit a prominent inflammatory response in rabbits that are pretreated with hIgG containing VH3+ IgGs. Even though SpA can bind with high affinity to rabbit IgG Fc, the Arthus reaction does not occur when human VH3+ IgGs are not present. Therefore, an interaction between SpA, as a B cell SAg, and its reactive hIgGs is necessary to cause tissue inflammation in vivo.

Although the rabbit IgG does not appear to play a role by itself, it is possible that it might affect the complexes formed between SpA and hIgG. SpA could form complexes with IgG molecules by binding to the Fab and Fc regions of hIgGs, in addition to the Fc region of rabbit IgG. Such complexes would be large and possibly very stable, since SpA binds to rabbit IgG Fc with high affinity (Ka = 109-1010 M-1) (49). The presence of rabbit IgG might enhance the binding of these complexes to both rabbit Fc{gamma}R and rabbit complement components. However, this explanation also suggests that SpA may need to bind to both the Fab and Fc regions of IgG molecules to cause an inflammatory response, or that the additional binding of SpA to the rabbit IgG Fc region may not be required but may enhance the response. Additional studies are required to determine whether the Fc region of reactive or nonreactive IgG molecules is needed, in addition to the Fab region of reactive IgGs, to interact with SpA and subsequently lead to tissue inflammation in vivo.

Nevertheless, the results described in this paper demonstrate that SpA requires the presence of human VH3+ IgGs to induce an Arthus reaction. It is likely that this reaction occurs via an interaction of SpA and the Fabs of human VH3+ IgGs. Thus, these data provide the first evidence that the interaction of a B cell SAg, SpA, with its reactive (VH3+) Igs leads to an inflammatory reaction in vivo. Such an in vivo response to a B cell SAg could have profound clinical significance. For example, SpA immunoadsorption has been used as a novel therapy in a variety of diseases that are mediated by pathogenic autoantibodies (50, 51). In addition, the therapy is being investigated as a treatment for cancer, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, myasthenia gravis, and HIV (52, 53, 54). However, a significant number of these patients have developed severe adverse side effects, most notably leukocytoclastic vasculitis (52, 55). Histologically, the dermal lesions are characterized by superficial and deep perivascular infiltrates containing mostly neutrophils (52). In addition, diapedesis of erythrocytes was observed. These effects could be due to an interaction between SpA/VH3+ Igs and the formation of immune complexes that bind to and activate Fc{gamma}R-bearing cells, such as macrophages and neutrophils. Further studies will be necessary to determine whether this type of inflammatory reaction is associated with the in vivo response to other B cell SAgs.


   Acknowledgments
 
We thank Julie Burns of the Laboratory of Pathology, University of Pennsylvania School of Veterinary Medicine for expert technical assistance with the histologic procedures. We also thank the veterinary technicians of the University Laboratory Animal Resources Department for assistance with the i.v. and i.d. injections into the rabbits, Jeanette Tasey and Paul Barrow of the University of Pennsylvania Biomedical Communications Department for their excellent photography, and Dr. John Monroe for his critical review of this manuscript.


   Footnotes
 
1 This work was supported by a Biomedical Sciences Research grant from the National Arthritis Foundation and by National Institutes of Health Grant AI 22913. Back

2 Current address: Johns Hopkins University, 755 Ross Building, 1721 E. Monument Street, Baltimore, MD 21205. Back

3 Address correspondence and reprint requests to Dr. Arnold Levinson, 726 Clinical Research Building, 415 Curie Boulevard, Division of Allergy and Immunology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. Back

4 Abbreviations used in this paper: SpA, staphylococcal protein A; VH, variable heavy chain; SAg, superantigen; HSA, human serum albumin; Mod SpA, staphylococcal protein A hyperiodinated to abrogate its IgG Fc-binding ability; Seph, CNBr-activated Sepharose 4B; hIgG, human IgG; VH3- hIgG, VH3-depleted human IgG; VH3+ using heavy chains from the VH3 gene family; VH3+/non-VH3+ hIgG, human IgG containing both VH3+ and non-VH3+ IgGs; i.d., intradermal(ly); PMN, polymorphonuclear cell. Back

Received for publication October 10, 1997. Accepted for publication January 29, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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