Reversal of lipopolysaccharide-analog-induced antibody suppression by anti-transforming growth factor beta and indomethacin.

Monophosphoryl lipid A and a synthetic, nontoxic monosaccharide analog of lipid A, termed GLA 60, both strongly suppressed antibody production when administered 1 to 2 days prior to antigen. Evidence is presented that this suppression was mediated by two cytokines, prostaglandin E and transforming growth factor beta, because it was reversed by multiple injections of the cyclooxygenase inhibitor indomethacin and by in vitro addition of antibody to transforming growth factor beta.

Polyadenylic: polyuridylic acid-induced determinants of host resistance to cytomegalovirus and their potentiation by hyperthermia.

The ability of spleen cells from poly A:poly U-treated mice to inhibit murine cytomegalovirus (MCMV) replication in confluent monolayer cells of secondary mouse embryo fibroblasts (MEFs) cultured at 37 and 40 degrees C was investigated. When spleen cells from BALB/c mice injected 48 h earlier with poly A:poly U were added to MEFs infected 2 h previously with MCMV, 37% less plaques were observed than in cultures containing control cells. Of interest, the poly A:poly U-induced antiviral activity at the elevated temperature (40 degrees C) resulted in a further drop to 61% in MCMV-induced plaques compared to those of the normothermic (37 degrees C) cultures. The antiviral function of spleen cells induced by poly A:poly U was evident in the supernatant fluid when cultured for 48 h at 37 degrees C. MCMV-induced plaques were reduced to 52 and 5% of controls in the plaque assays performed at 37 and 40 degrees C, respectively. Supernatant fluids generated at 40 degrees C, however, inhibited MCMV replication only when incubated at 40 degrees C. No direct inhibitory effect of the supernatant fluids on MCMV was evident; rather, inhibition was effected directly on the MEFs. The NK cell fraction of spleen cells from poly A:poly U-treated mice alone showed only a slight inhibitory effect at 40 degrees C. However, in the presence of the supernatant fluid from poly A:poly U-exposed spleen cells, the antiviral activity of NK cells was significantly increased both at 37 and 40 degrees C. The cellular source of the culture fluid showing poly A:poly U-induced antiviral activity appeared to be in the T-cell population. It was completely neutralized by monoclonal anti-IFN gamma antibody but not by anti-IFN beta, anti-IL4, anti-transforming growth factor, or anti-prostaglandin E2. In conclusion, these data document the ability of spleen cells from poly A:poly U-treated mice to inhibit MCMV replication and this activity is potentiated by hyperthermic conditions. The antiviral function of poly A:poly U-treated spleen cells appeared to be due mainly to the action of IFN gamma produced by T cells. The enhanced antiviral activity by hyperthermia appeared to be related to the action of IFN gamma rather than its production.

Immunosuppression induced by non-reducing acylated monosaccharide subunits of lipid A.

Synthetic acylated glucosamine monosaccharides, representative of the non-reducing subunit of lipid A, were compared for their ability to induce non-specific suppression of antibody forming cells. Five of nine analogs were found to be functional in this respect, indicating that these compounds, carrying a phosphate at C4 and acyl substituents at C2 and C3 are the smallest synthetic analogs of lipid A capable of eliciting non-specific immunosuppression. A comparison of the analogs inducing suppression with those testing negative revealed that (i) a single 3-hydroxymyristoyl group at C2 is common to 4/5 analogs inducing suppression; (ii) addition of an oxytetradecanoyl group to the 3-hyroxymyristoyl group at either C2 or C3 negated suppression; and (iii) extreme specificity was exhibited in suppression induction such that substitution of either lauric (C12) or palmitic (C16) for myristic acid (C14) at C2 voided transmission of the suppressive signal.

Characterization of immune suppression induced by polyribonucleotides.

Synthetic polyribonucleotide complexes, which have been shown to be potent adjuvants to the immune response of animals and humans were tested for their capacity to activate cells involved in suppressing antibody synthesis. Poly A:poly U and poly I:poly C inhibited murine antibody forming spleen cells when given 1-6 days before antigenic stimulus. To determine the cellular and molecular mediators of this suppression, individual cell populations were isolated or deleted and the resulting cell populations tested for induction of suppression. When the natural killer (NK) cell population was rendered non-functional with anti-asialo GM1 antiserum no diminution in suppressive activity was observed. Further experiments implicated adherent cells as the population responsible for mediating suppression. Supernatants from poly A:poly U-treated adherent cells were found both to contain increased levels of prostaglandin E (PGE) and to induce a significant decrease in antibody production when added to in vitro spleen cell cultures. In addition, indomethacin, an inhibitor of the cyclo-oxygenase pathway of the arachidonic acid cascade was found to reverse the suppression of antibody induced by poly A:poly U. Thus, the polyribonucleotide complexes appear to suppress antibody synthesis by inducing macrophages to secrete PGE, a known immune suppressant.

Involvement of gamma interferon in antibody enhancement by adjuvants.

In a previous study the adjuvant action of a monophosphoryl lipid A, a nontoxic derivative of endotoxic lipopolysaccharide (LPS), was found to be negated by a monoclonal anti-gamma interferon (anti-IFN-gamma) antibody. The present investigation centered on three other adjuvants of diverse microbial origins, testing for their capacity to affect the release of IFN-gamma as an explanation for their antibody-enhancing action. The adjuvant action of each of the three, a wild-type LPS, synthetic poly(A)-poly(U) complexes, and a synthetic muramyl dipeptide, n-acetylmuramyl-L-alanyl-D-glutaminyl-n-butyl ester (murabutide), was transferable by adjuvant-stimulated T cells to normal spleen cells on coculture. Supernatant fluids from these T cells contained increased levels of IFN-gamma. Addition of a monoclonal anti-IFN-gamma antibody to adjuvant-stimulated spleen cell cultures reduced the adjuvant action by approximately one-half. Removal of natural killer cells from spleen cell populations prior to culture with antigen had no effect on the enhancement induced by LPS and monophosphoryl lipid A. It was concluded that the enhancement induced by the adjuvants LPS, poly(A)-poly(U), and murabutide is mediated in part by their action on T cells resulting in release of IFN-gamma suggesting activation of a common transmembrane signal.

Suppression of the mixed leukocyte reaction by serum from polynucleotide-injected mice.

Sera from mice which have been injected iv with either poly(A) X poly(U) or poly(I) X poly(C) 1 1/2 hr prior to bleeding were found to suppress the mixed lymphocyte reaction. This effect was reduced considerably by 18 hr. Characterization of the suppressive sera revealed it (a) was stable to heating at 56 degrees C for 1 hr and freezing at -20 degrees C for 1 month; (b) had a molecular weight greater than 30,000; (c) could be induced in sera from athymic nude mice; and (d) was present to a lower degree in sera from aging mice.