Gila Arad

Superantigen antagonist protects against lethal shock and defines a newdomain for T-cell activation

Superantigens trigger an excessive cellular immune response, leading to toxic shock. We have designed a peptide antagonist that inhibits superantigen-induced expression of human genes for interleukin-2, gamma interferon and tumor necrosis factor-b, which are cytokines that mediate shock. The peptide shows homology to a b-strand–hinge–a-helix domain that is structurally conserved in superantigens, yet is remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. Superantigens depend on this domain for T-cell activation. The peptide protected mice against lethal challenge with staphylococcal and streptococcal superantigens. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed protective antibodies against superantigen that rendered them resistant to further lethal challenges, even with different superantigens. Thus, the lethal effect of superantigens can be blocked with a peptide antagonist that inhibits their action at the beginning of the toxicity cascade, before activation of T cells takes place.

Binding of superantigen toxins into the CD28 homodimer interface is essential for induction of cytokine genes that mediate lethal shock.

Bacterial superantigen toxins bind directly to the dimer interface of CD28, the principal co-stimulatory receptor, to induce a lethal cytokine storm, and peptides that prevent this binding can suppress superantigen lethality.

Superantigen antagonist blocks Th1 cytokine gene induction and lethal shock

Bacterial superantigens trigger an excessive, Th1‐cytokine response leading to toxic shock. We designed a peptide antagonist that inhibits SEB‐induced expression of human genes for IL‐2, IFN‐γ, and TNF‐β, cytokines that mediate shock. The peptide antagonist shows homology to a β‐strand‐hinge‐α‐helix domain that is conserved structurally in superantigens produced by Staphylococcus aureus andStreptococcus pyogenes yet remote from known binding sites for the major histocompatibility class II molecule and T‐cell receptor. For Th1‐cell activation, superantigens depend on this domain. The peptide protected mice against lethal challenge with SEB or SEA. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed broad‐spectrum, protective immunity, which rendered them resistant to further lethal challenges with different staphylococcal and streptococcal superantigens. Thus, the lethal effect of superantigens, mediated by Th1 cytokines, can be blocked with a peptide antagonist that inhibits their action at the top of the toxicity cascade, before activation of T cells takes place.

Glutamine-stimulated modification and degradation of glutamine synthetase in hepatoma tissue culture cells

Effects of glutamine on glutamine synthetase (GS) activity of hepatoma tissue culture (HTC) cells were studied with the aid of a specific goat anti-rat GS serum. Immunodiffusion and immunoelectrophoretic tests show that rat liver GS and HTC cell GS are immunologically similar but not identical. Immunotitrations of HTC cell extracts demonstrate that in cells incubated in high concentrations (5 mM) of glutamine, a cross-reacting form of GS with a decreased enzyme-specific activity accumulates. On prolonged incubation of cells in high glutamine, there is net degradation of GS to form immunologically inactive products. Radio-immunoprecipitation experiments show that glutamine acts by accelerating the degradation of preformed GS.

Hyperinducible expression of the interferon-gamma (IFN-γ) gene and its suppression in systemic lupus erythematosus (SLE)

Transient expression of IFN‐γ and IL‐2 mRNA and its control by post‐transcriptional and suppressive mechanisms were analysed in phytohaemagglutinin‐induced peripheral blood mononuclear cells (PBMC) from 47 patients with SLE and 31 age‐matched normal donors, using quantitative hybridization with antisense RNA probes. In SLE, basal levels of gene expression did not deviate from those of normal donors, but strongly aberrant patterns were obtained upon induction. The ratio of subjects exhibiting highly inducible IFN‐γ gene expression in their PBMC to those showing moderate or low inducibility was increased five‐fold in SLE (P = 0.003). High inducibility was observed for 43% of SLE patients and was equally pronounced in partial remission, mild or active disease. Inducibility of IL‐2 mRNA, by contrast, remained similar to that for normal donors. However, regulation of IFN‐γ gene expression differed for mild SLE. Patients with mild disease showing high inducibility of IFN‐γ mRNA in their PBMC not only had the highest frequency of responders, but also the highest extent of an individual response, defined by superinduction of mRNA, to agents that relieve suppression (γ‐irradiation) or post‐transcriptional down‐regulation (cycloheximide). By contrast, patients with active SLE showing high IFN‐γ mRNA inducibility had normal suppressive capacity as well as post‐transcriptional control. Hence, both high inducibility of the IFN‐γ gene and its suppression are relevant to disease. Hyperactivation of the IFN‐γ gene may be alleviated in mild SLE by a vigorous, concomitant activation of post‐transcriptional control and of cell‐mediated suppression.

Aberrant regulation of interleukin-2 but not of interferon-γ gene expression in Down syndrome (trisomy 21)

The regulated expression of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) genes was analyzed in peripheral blood mononuclear cells derived from 29 noninstitutionalized Down syndrome individuals and compared to that of 32 normal donors. Culture conditions were chosen that measure the transient, phytohemagglutinin-induced expression of IL-2 and IFN-gamma mRNA, as well as the intactness of post-transcriptional and suppressor T cell-dependent mechanisms that control this expression. The latter was achieved by analyzing, respectively, the superinduction of IL-2 and IFN-gamma mRNA occurring upon culture with cycloheximide or after low-dose gamma-irradiation. A convenient, sensitive, and quantitative assay for specific mRNA was devised, suitable for measuring mRNA levels expressed in cells from 1 ml of peripheral blood. Analysis of individuals with Down syndrome revealed a pronounced decrease in inducibility of the IL-2 gene. By contrast, induction of IFN-gamma mRNA was as vigorous as that observed for normal donors. In cells from trisomic subjects, superinduction of IFN-gamma mRNA by cycloheximide was at least as extensive as for normal donors, while in the case of IL-2 mRNA, it was weaker. These abnormal patterns of IL-2 gene expression were seen irrespective of age. Our findings demonstrate a selective impairment of IL-2 gene expression in Down syndrome, rather than a general deficiency in helper T cells.

Use of reconstituted Sendai virus envelopes for fusion-mediated microinjection of double-stranded RNA: inhibition of protein synthesis in interferon-treated cells

Poly(I).poly(C) molecules were trapped with reconstituted Sendai virus envelopes when added to the reconstitution system. A quantitative estimation indicated that about 10% of the added poly(I).poly(C) remained associated with the fusogenic viral envelopes. About 50% of the associated poly(I).poly(C) were found to be RNAase A resistant, enclosed within the viral envelopes. Incubation of loaded viral envelopes with HeLa or L-cells resulted in strong inhibition of protein synthesis, indicating fusion-mediated microinjection of the enclosed poly(I).poly(C). Introduction of poly(I).poly(C) into cultured cells by the use of reconstituted Sendai virus envelopes was as efficient as the introduction of these polynucleotides using the calcium phosphate coprecipitation technique. The inhibition of protein synthesis in L-cells but not in HeLa cells was dependent upon pretreatment with interferon. Incubation of poly(I).poly(C)-loaded viral envelopes with interferon-treated variant cells of the NIH 3T3 line, which possess a very low amount of RNAase L, resulted in only 25% inhibition of protein synthesis, compared to 85% inhibition observed in L-cells.

Superantigen concomitantly induces Th1 cytokine genes and the ability to shut off their expression on re-exposure to superantigen.

Superantigens, exemplified by staphylococcal enterotoxin B (SEB), are the strongest known inducers of a cellular immune response; they elicit the production of excessive amounts of Th1 cytokines, IL-2, IFN-gamma and TNF, leading to toxic shock. We show that increasing doses of SEB cause not only a greater induction but also a more rapid cessation of IL-2 gene expression. Remarkably, exposure of human PBMC to a second dose of SEB, even at concentrations 10- or 100-fold lower than the initial inducing dose and even within 2 h after the first exposure to SEB, resulted in an immediate and essentially complete shutoff of the induced IL-2 and IFN-gamma mRNA expression. The shutoff response was observed when primary induction of IL-2 and IFN-gamma gene expression was by SEB but not when it was by phytohemaggutinin-P. Signaling by a superantigen thus results not only in a vigorous induction of Th1 cytokine genes but concomitantly induces the ability to shut off their expression upon re-exposure to superantigen. Without induction of this negative control mechanism, the cellular immune response to a superantigen would be even more pronounced.

Effects of glutamine, methionine sulfone and dexamethasone on rates of synthesis of glutamine synthetase in cultured hepatoma cells

Glutamine synthetase (EC 6.3.1.2) activity of hepatoma tissue culture cells is elevated by corticosteroids and depressed by glutamine (Kulka, R.G., Tomkins, G.M. and Crook, R.B. (1972) J. Cell Biol., 54, 175--179). The transfer of cells from high (1--5 mM) to low (0.2--0.4 mM) concentrations of glutamine causes a marked increase in glutamine synthetase activity. The addition of a glutamine antagonist, methionine sulfone (1 mM) to cells suspended in high (1 mM) concentrations of glutamine also causes an increase of glutamine synthetase activity which is greater than that elicited by the transfer of cells to low concentrations of glutamine. Rates of synthesis of glutamine synthetase have been measured by radioimmunoprecipitation in hepatoma tissue culture cells incubated under various conditions. Incubation of cells with the synthetic corticosteroid hormone, dexamethasone, markedly stimulates the relative rate of glutamine synthetase biosynthesis. Glutamine, or its analogue, methionine sulfone, have no effect on the relative rate of synthesis of the enzyme. However, total protein and RNA synthesis increase markedly with increasing external glutamine concentration in the range 0--1 mM. Methionine sulfone (1 mM) inhibits the degradation of glutamine synthetase in the presence of 1 mM glutamine. The data are consistent with the conclusion that the corticosteroid, dexamethasone, elevates glutamine synthetase activity by stimulating its rate of synthesis, whereas methionine sulfone elevates glutamine synthetase activity by inhibiting the glutamine-stimulated degradation of preformed enzyme.

CD28: Direct and Critical Receptor for Superantigen Toxins

Every adaptive immune response requires costimulation through the B7/CD28 axis, with CD28 on T-cells functioning as principal costimulatory receptor. Staphylococcal and streptococcal superantigen toxins hyperstimulate the T-cell-mediated immune response by orders of magnitude, inducing a lethal cytokine storm. We show that to elicit an inflammatory cytokine storm and lethality, superantigens must bind directly to CD28. Blocking access of the superantigen to its CD28 receptor with peptides mimicking the contact domains in either toxin or CD28 suffices to protect mice effectively from lethal shock. Our finding that CD28 is a direct receptor of superantigen toxins broadens the scope of microbial pathogen recognition mechanisms.