Intravenous gamma immunoglobulin (IVIG) is used as therapy in superantigen-mediated disease, yet its mode of action is not clear. Pooled immunoglobulin G contains high concentrations of staphylococcal exotoxin (SE)-specific antibodies which inhibit the in vitro activation of T cells. However, SE and streptococcal exotoxins are potent stimulators of monocytes as well. Monocytes exposed to SE in vitro release large amounts of tumor necrosis factor alpha (TNF-␣). The purpose of the present study

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... s to determine if SE-specific antibodies in IVIG can inhibit the activation of monocytes by SE. We examined the in vitro effect of IVIG on the ability of staphylococcal exotoxin A (SEA) and staphylococcal exotoxin B (SEB) to stimulate release of TNF-␣ from human mononuclear phagocytes (MO). Pretreatment of SEA with 0.1 mg of IVIG per ml resulted in a slight decrease of SEA-induced TNF-␣ secretion by MO. In contrast, pretreatment of SEB with 0.1 mg of IVIG per ml resulted in significant (greater than 50%) inhibition of SEB-induced TNF-␣ secretion at 24, 48, 72, and 96 h (P < 0.05 for TNF-␣ levels induced by SEB alone versus SEB pretreated with IVIG at all time points). Enzyme-linked immunosorbent assay and Western immunoblotting assays of the IVIG revealed high concentrations of antibodies against SEB and lower concentrations of antibodies to SEA. These data indicate that IVIG can act in a toxin-specific manner to decrease the MO TNF-␣ response to superantigens. Such inhibition may be one mechanism by which IVIG exerts an immunoregulatory role in superantigen-mediated disease. MATERIALS AND METHODS Reagents. Polymyxin B, SEB, SEA, and bacterial lipopolysaccharide (LPS) from Escherichia coli were purchased from Sigma Chemical Co. (St. Louis, Mo.). The toxins were Ͼ95% pure as determined by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. Lyophilized toxins were reconstituted in deionized water and stored in aliquots at Ϫ20ЊC until used. Human gamma interferon (IFN-␥) was purchased from Genzyme Corp., Cambridge, Mass. Two commercially available preparations of IVIG were used in the experiments. The majority of the experiments were conducted with Gammagard (batch 3312 M123 AA), purchased from Baxter-Healthcare Corp., Glendale, Calif. For comparison, select experiments were conducted with Polygam S/D (batch 26206030AA), manufactured by Baxter-Healthcare Corp. and distributed by the American Red Cross Blood Services, Washington, D.C. Both products are manufactured by cold ethanol fractionation followed by ultrafiltration and ion-exchange chromatography. Polygam was further treated with an organic solvent-detergent mixture composed of tri(n-butyl)phosphate, octoxynol 9, and polysorbate 80. Cell isolation and culture. Blood samples were collected from healthy adult volunteers by using sterile heparinized syringes (10 3 U of heparin/liter of blood). After collection, the blood was diluted 1:1 with sterile phosphate-buffered saline (PBS). The cell suspension was overlaid on Histopaque 1077 (Sigma Chemical Co.) and the mononuclear cells were separated by density gradient centrifugation. These cells were washed once with sterile PBS and then incubated for 10 min at 37ЊC in 0.01 M Tris-0.83% NH 4 Cl (pH 7.2 to 7.4) to lyse contaminating