Jeffry K. Russell

Staphylococcal enterotoxin microbial superantigens.

Staphylococcal enterotoxins are a family of structurally related proteins that are produced by Staphylococcus aureus. In addition to their role in the pathogenicity of food poisoning, these microbial superantigens have profound effects on the immune system, which makes them useful tools for understanding its mechanism of action. These molecules (24‐30 kDa) are highly hydrophilic and exhibit low α helix and high β pleated sheet content, suggesting a flexible, accessible structure. Staphylococcal enterotoxins are among the most potent activators of T lymphocytes known. The receptors for staphylococcal enterotoxins on antigen‐presenting cells are major histocompatibility complex (MHC) class II molecules. Further, the α‐helical regions of the class II molecule are essential for function and appear to interact directly with the NH2‐terminal region of staphylococcal enterotoxins such as SEA. Recent studies have shown that a complex of staphylococcal enterotoxin and MHC class II molecules is required for binding to the Vβ region of the T cell antigen receptor. Staphylococcal enterotoxin mitogenic activity is dependent on induction of interleukin 2, which may be intimately involved in the mechanism of toxicity. The mouse minor lymphocyte stimulating (Mls) “endogenous” self‐superantigen has been shown to be a retroviral gene product, so this too is apparently a microbial superantigen. An understanding of the mechanism of action of these microbial superantigens has implications for normal and pathological immune functions.—Johnson, H. M.; Russell, J. K.; Pontzer, C. H. Staphylococcal enterotoxin microbial superantigens. FASEB J. 5: 2706‐2712; 1991.

The I-Aβb region (65–85) is a binding site for the superantigen, staphylococcal enterotoxin A

Ia antigen is a receptor for the superantigen staphylococcal enterotoxin A (SEA). Peptides I-A beta b(30-60), I-A beta b(50-70), I-A beta b(65-85), and I-A beta b(80-100) of the MHC class II antigen beta chain on mouse (H-2b) accessory cells were synthesized. Only I-A beta b(65-85) inhibited SEA binding to the mouse B-cell lymphoma line, A20 (H-2d) and the human Burkitts lymphoma line, Raji (HLA-DR). The I-A beta b(65-85) sequence is a predicted alpha-helix along the hypothetical antigen binding cleft of the Ia molecule. I-A beta b(65-85) also directly and specifically bound both the intact SEA molecule and its Ia binding site, represented by the peptide SEA(1-45). The results suggest that I-A beta b region (65-85) is a necessary site for Ia molecular interaction with the superantigen SEA. Further, the data suggest that the same helical region of other Ia antigens binds SEA irrespective of haplotype and species.

Structural basis for arachidonic acid second messenger signal in gamma-interferon induction.

Gamma interferon (IFNy) is an extremely important immunoregulatory lymphokine. It plays a pivotal role in the regulation of a variety of essential immune functions (reviewed in reference 1). Among these are: (a) modulation of expression of products of the major histocompatibility complex on the cell membrane, particularly the enhancement of Ia antigen expression on macrophages; (b) priming/activation of macrophages for tumor cell killing; (c) enhancement of cytotoxicity of lymphocytes, including that of antigen-stimulated T lymphocytes and that of natural killer cells against tumor cells; and (d) maturation of B cells for immunoglobulin production and secretion that, depending on the stage of clonal expansion of the cells, is manifested as either enhancement or suppression of antibody production. IFNy production is regulated by a dynamic interaction between macrophages and various T cell subsets, involving helper cells, suppressor cells, and IFNy-producing cells. A positive regulatory loop for production of IFNy, as well as for other lymphokines and cytokines, is summarized in FIGURE 1. This loop is based on data generated in several laboratories that show that: (a) helper cells are required for IFNy production and provide the helper signal via interleukin-2 (IL-2);* (b) IFNy alone or with lipopolysaccharide induces macrophages to produce interleukin1 (ILand a cytolytic factor(s) for tumor cells;5 (c) macrophages are required for IFNy production6 and the macrophage requirement is thought to be mediated through IL-1; and (d) IL-1 stimulates helper cells to produce IL-2,’ thereby completing the loop. Interestingly, the IL-2 requirement for I F N y production can be replaced by the neuroendocrine hormone, arginine vasopressin (AVP), and structurally related n e u r o p e p t i d e ~ , ~ ~ ~ as well as by the growth factors, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF).’’ Therefore, IFNy production can involve the interaction of the immune system with several important hormonal or hormonal-like systems.

Site of nonrestrictive binding of SEA to class II MHC antigens

We have used the synthetic peptide approach to show that the N-terminal 45-amino acids of staphylococcal enterotoxin A (SEA), SEA(1-45), constitute an important part of its binding site on class II major histocompatibility complex (MHC) molecules. SEA(1-45) and to a lesser extent SEA(1-27) were able to displace SEA from HLA-DR on Raji cells as assessed by flow cytometry and to compete with radiolabeled SEA for interaction with HLA-DR in a direct binding assay. Specific binding of SEA to Ia on murine A-20 cells could be inhibited by the same peptides [i.e. SEA(1-45) greater than SEA(1-27)] that blocked binding to HLA-DR. Therefore, different class II MHC molecules associate with the same functional site on SEA. Further, an ELISA system was used to demonstrate that SEA(1-45) is able to directly bind to a mouse synthetic I-A beta b peptide, I-A beta b (65-85), which contains a binding site of the class II MHC molecule involved in SEA presentation to T cells. Thus, we have localized a site on SEA that is involved in selective surface association with class II MHC antigens and identified the region on the class II MHC antigen to which that site binds.

A Positive Feedback Loop for Staphylococcal Enterotoxin-A-Stimulated IFN-Gamma Production Requires Macrophage Immune-Associated Antigen Upregulation

The C57Bl/6-derived T cell line, L12-R4, produced murine interferon-gamma (IFN gamma) in response to mitogenic stimulation by phorbol myristate acetate (PMA) or concanavalin A (Con A), but not by staphylococcal enterotoxin A (SEA). Low levels of IFN gamma were produced by SEA stimulation of L12-R4 cells cocultured with C57Bl/6 bone marrow macrophages (BMM). Significantly increased yields of IFN gamma resulted from 48-hour pretreatment of the BMM with recombinant IFN gamma (100 U/ml) prior to coculture. Polyclonal anti-IFN gamma and anti-IFN alpha/beta were used to characterize the interferon as IFN gamma. Paraformaldehyde (0.1%) treatment of IFN gamma-pretreated BMM did not affect IFN gamma production, suggesting that processing of SEA was not required. IFN gamma treatment of BMM resulted in significantly increased expression of immune-associated (Ia) antigen as determined by flow cytometric analysis, suggesting that the accessory cell role of BMM involved Ia antigen. Polyclonal anti-Ia antibody selectively inhibited the production of IFN gamma by SEA-stimulated whole spleen cell cultures, consistent with the necessity of Ia antigen for BMM help in SEA induction of IFN gamma. More interestingly, induction of IFN gamma. These findings suggest that Ia antigen is necessary for BMM accessory function in SEA induction of IFN gamma. More interestingly, the results implicate class II molecules in a positive feedback loop for IFN gamma production by SEA.

Induction of interferon by transformed cells: Inhibition by retinoic acid

Retinoic acid (RA) inhibited transformed mouse L-929 and human WISH cell induction of interferon alpha/beta production by nonsensitized mouse spleen cells. The RA effect was both time and dose dependent and acted in near physiologic concentrations. The results suggest that the effect is due to a modulation of a previously described transformed cell surface associated glycoprotein IFN inducer.

Evidence for the α-helicity of class II MHC molecular binding sites for the superantigen, staphylococcal enterotoxin A

Circular dichroism (CD) spectra of class II MHC peptides revealed the alpha-helical conformation of superantigen-binding peptides I-A beta b(60-90), I-A beta b(65-85), and I-A alpha b(51-80), but not the nonbinding peptide I-A beta b(80-100). These CD spectra provide biophysical evidence for the alpha-helicity of class II MHC molecular binding sites for the superantigen, staphylococcal enterotoxin A (SEA). Alanine-substituted analogs of the SEA binding-site peptide, I-A beta b(65-85), were used to implicate beta-chain residues 72 and 80 in class II MHC-SEA binding. The data support SEA binding away from the class II antigen binding cleft along the faces of the alpha-helices.