Effects of interferon gamma on the antibody response to Pseudomonas aeruginosa lipopolysaccharide in mice.

Different strains of mice were examined for the capacity to produce an Ig subclass-specific antibody response to purified Pseudomonas aeruginosa lipopolysaccharide (PALPS). With the exception of the AKR strain, the predominant isotype for most of the strains tested was IgG3 whereas the least frequent isotype expressed was either IgG2b or IgG1. AKR mice were unique in that the predominant isotype produced was IgG2a, rather than IgG3; however, the administration of anti-interferon gamma antibody, at the time of immunization with PALPS caused a substantial decrease in the IgG2a antibody response. Selected B10 congenic strains were used to assess the relationship between the antibody responses and the major histocompatibility complex (MHC) genes. Here, the isotype-patterns for the antibody responses were essentially the same regardless of the MHC haplotype. Interestingly, an increase in IgG2a, with a concomitant decrease in IgM and IgG1 antibody was noted when C3H mice were given interferon gamma at the time of immunization. These studies indicate that, in general, the antibody response to PALPS consists of IgG3 antibody as the predominant isotype, and that the antibody response can be modified by interferon gamma.

Molecular structures that influence the immunomodulatory properties of the lipid A and inner core region oligosaccharides of bacterial lipopolysaccharides.

The relationship between chain length as well as the position of fatty acyl groups to the ability of lipid A to abolish the expression of suppressor T-cell (Ts) activity was examined. Fatty acyl chain lengths of C12 to C14, as in the lipid A of Escherichia coli and Salmonella minnesota, appear to be optimal for this bioactivity, since lipid A preparations with fatty acyl groups of relatively short chain length (C10 to C12 for Pseudomonas aeruginosa and Chromobacterium violaceum) or predominantly long chain length (C18 for Helicobacter pylori) are without effect. The presence of an acyloxyacyl group of appropriate chain length at the 3' position of the glucosamine disaccharide backbone of lipid A also plays a decisive role. By contrast, the lipid A proximal inner core region oligosaccharides of some bacterial lipopolysaccharides increase the expression of Ts activity; this is due mainly to the capacity of such oligosaccharides, which are relatively conserved in structure among gram-negative bacteria, to enlarge or expand upon the population of CD8+ Ts generated during the course of a normal antibody response to unrelated microbial antigens. The minimal structure required for the expression of the added immunosuppression observed appears to be a hexasaccharide containing one 2-keto-3-deoxyoctonate residue, two glucose residues, and three heptose residues to which are attached two pyrophosphorylethanolamine groups. The relevance of these findings to virulence and to the pathogenesis of gram-negative infections is discussed.

The influence of monophosphoryl lipid A (MPL) on erythrocyte autoantibody formation.

The onset and the amount of erythrocyte autoantibodies induced by the injection of C57BL/6N mice with rat red blood cells (RRBC) were hastened and increased, respectively, after the administration of monophosphoryl lipid A (MPL); this was not the case for similarly treated BALB/cAnN mice, which make a lower autoantibody response after immunization with RRBC. The transfer of spleen cells from donor C57BL/6N mice immunized with RRBC suppressed autoantibody formation in recipient mice subsequently immunized with RRBC; however, treatment with MPL prevented neither the induction nor the expression of such suppression. This suggests that the increased autoantibody response in RRBC-immunized C57BL/6N mice treated with MPL is not due to the inactivation of suppressor cell activity which, in other studies, was found to be extremely sensitive to MPL.

Effects of IL-4 depletion on the antibody response to Pseudomonas aeruginosa lipopolysaccharide in mice.

These studies were done to examine the role of interleukin-4 (IL-4) in the generation of isotype specific antibody responses of mice to Pseudomonas aeruginosa lipopolysaccharide (PALPS) by neutralization of IL-4 in vivo using anti-IL-4 antibody (11B11). We found that the administration of anti-IL-4 antibody (11B11) 24 h before immunization with PALPS resulted in a decreased PALPS-specific antibody response for all isotypes examined (IgM, IgG1, IgG2a, IgG2b, IgG3). By contrast, we observed that the non-antigen-specific (polyclonal) IgM response of mice following treatment with 11B11 antibody and PALPS was increased while the polyclonal responses for the other isotypes were unaffected. When mice were given recombinant IL-10 at the time of immunization with PALPS there was a decrease in the PALPS-specific antibody response but an increase in the polyclonal IgM, IgG2a, IgG2b, IgG3 response whereas the polyclonal IgG1 response was decreased by a five-fold margin. The results of these studies suggest that both the antigen-specific and the polyclonal response can be influenced in a different manner by IL-4 or by IL-10.

Immunosuppressive effects induced by the polysaccharide moiety of some bacterial lipopolysaccharides.

The immunomodulatory properties of several lipopolysaccharides (LPS) derived from clinical isolates of Pseudomonas aeruginosa, Branhamella catarrhalis, and Bordetella pertussis were evaluated for their capacity to influence the magnitude of the antibody response to type III pneumococcal polysaccharide (SSS-III), which is known to be regulated by suppressor and amplifier T cells (Ts and Ta, respectively). The administration of LPS, two days after immunization resulted in a significant increase in the antibody response. Such enhancement may be due mainly to the ability of the lipid A moiety of LPS to abolish the negative effects of activated Ts, thereby enabling Ta function to be more fully expressed; however, B cell mitogenicity of the LPS molecule also may be involved. By contrast, treatment with LPS at the time of immunization with SSS-III induces significant suppression of the SSS-III-specific antibody response; such suppression is not induced by LPS or lipid A derived from Escherichia coli and Salmonella minnesota, and is independent of the capacity of LPS to activate B cells polyclonally, an activity generally attributed to the lipid A fraction of LPS. Studies conducted with the LPS of P. aeruginosa indicated that the suppression induced is T cell dependent and mediated by the polysaccharide (PS) fraction of LPS; it appears to be due-at least in part-to the capacity of PS to expand or increase the size of the precursor pool of Ts, activated in response to SSS-III. The significance of these findings to the pathogenesis of certain gram-negative infections is discussed.

Differential effects of monophosphoryl lipid A on expression of suppressor T cell activity in lipopolysaccharide-responsive and lipopolysaccharide-defective strains of C3H mice.

Lipopolysaccharide (LPS)-responsive and LPS-defective strains of C3H mice did not differ in the capacity to make an antibody response to type III pneumococcal polysaccharide or in the degree of thymus-derived suppressor cell (Ts) activity generated following exposure to type III pneumococcal polysaccharide. However, treatment with monophosphoryl lipid A (MPL) abolished the expression of Ts function in LPS-responsive but not LPS-defective mice. Since this effect was elicited by different preparations of MPL, it appears to be a general property of MPL mediated by direct action of MPL on activated Ts.

Antigen-specific suppressor T cells respond to recombinant interleukin-2 and other lymphokines.

Previous studies have shown that transfer of whole spleen cell populations obtained from primed donors or transfer of purified T cells enriched for suppressor activity (Ts) to recipient mice decreased the antibody response to pneumococcal polysaccharide type III (SSS-III) when the animals were simultaneously immunized with SSS-III. In the present studies, such suppression of the antibody response was transferred with 10- to 100-fold fewer primed spleen cells when the cells were treated in vitro with recombinant interleukin-2 (rIL-2) before transfer; spleen cells from naive mice or mice primed with an unrelated antigen (dextran) and then treated with rIL-2 did not cause suppression of the antibody response to SSS-III, thereby eliminating the possibility of nonspecific carryover effects induced by rIL-2. In vivo administration of rIL-2 at the time of immunization with an optimally immunogenic dose of SSS-III resulted in significant (P less than 0.05) suppression of the antibody response relative to that of control animals, suggesting that IL-2 augments the clonal expansion of Ts cells in vivo. Further, the ability of passively administered anti-IL-2 receptor antibody to inhibit generation of Ts cells in vivo is consistent with such a view. Spleen cells from primed animals treated with rIL-4, rIL-5, or gamma interferon--but not those from primed animals treated with rIL-6--likewise were able to transfer suppression of the antibody response with fewer cells than those required when primed cells not treated with lymphokines were used. Thus, these studies indicate that Ts cell activity is greatly influenced by lymphokines produced by helper T cells. The studies also suggest that these lymphokines are required during activation and/or clonal expansion of Ts cells.

T cells regulate the IgM antibody response of BALB/c mice to dextran B1355.

The IgM antibody response of BALB/c mice to bacterial (Leuconostoc) dextran B1355 is influenced in a positive and negative manner by regulatory CD4+ and CD8+ T cells, respectively. Treatment with concanavalin A (ConA) at the time of immunization or 2 days later caused suppression and enhancement of the antibody response, respectively. Priming of mice with a sub-immunogenic dose of dextran resulted in profound suppression upon subsequent immunization 3 days later. None of these effects were demonstrable in athymic mice. Transfer of T cells from mice primed 18 h previously with a subimmunogenic dose of dextran suppressed the antibody response in immunized recipients; such suppression was abolished by the treatment of transferred cells with anti Thy 1.2 or anti Lyt 2.2 (CD8) antibody in the presence of complement. By contrast, the transfer of T cells from mice, which had been given an immunogenic dose of dextran 4 days previously, increased the antibody response in immunized recipients; such enhancement was abolished by treating transferred cells with anti Thy 1.2 or anti L3T4 (CD4) antibody in the presence of complement. These findings indicate that the immune response to dextran B1355 is regulated by CD4+ T-amplifier cells (Ta cells) and by CD8+ T-suppressor cells (Ts cells) which are activated during the course of a normal antibody response.

Inactivation of suppressor T cell activity by the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides.

Antibody responses of mice immunized with type III pneumococcal polysaccharide were examined with and without treatment with nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides (Rs-LPS). The results obtained were similar to those described previously for mice treated with monophosphoryl lipid A (MPL) except that lower amounts of Rs-LPS were needed. Both were without effect when given at the time of immunization with type III pneumococcal polysaccharide but elicited significant enhancement when given 2 to 3 days later. Such enhancement was T cell dependent and not due to polyclonal activation of immunoglobulin M synthesis by B cells. Treatment with either Rs-LPS or MPL abolished the expression but not induction of low-dose paralysis, a form of immunological unresponsiveness known to be mediated by suppressor T cells (Ts). The in vitro treatment of cell suspensions containing Ts with extremely small amounts of Rs-LPS or MPI completely eliminated the capacity of such cells to transfer suppression to other mice. These findings indicate that the immunomodulatory effects of both MPL and Rs-LPS are mainly the result of eliminating the inhibitors effects of Ts; this permits the positive effects of amplifier T cells to be more fully expressed, thereby resulting in an increased antibody response. The significance of these and other findings to the use of Rs-LPS as a pharmacotherapeutic agent for gram-negative bacterial sepsis is discussed.

Characterization of the immunodeficiency of RIIIS/J mice: immune response to polysaccharide antigens.

RIIIS/J mice lack an autosomal dominant gene(s) that influences the magnitude of the antibody response to several polysaccharide antigens of bacterial origin. Low responsiveness is demonstrable whether polysaccharide is administered as a T-helper-cell-independent or -dependent antigen conjugated to an immunogenic carrier; however, RIIIS/J mice make good anti-hapten antibody responses to haptenated polysaccharides. The low antibody responses of RIIIS/J mice to type III pneumococcal polysaccharide do not appear to be the results of an imbalance in the activity of regulatory T lymphocytes. Compared with other strains of mice, RIIIS/J mice elicit low antibody responses to lipopolysaccharide (LPS). They do not develop a cyclic primary or secondary antibody response to Escherichia coli O113 LPS; the latter is not due to a lack of mitogenic response to E. coli O113 LPS. They also produce auto-anti-idiotypic antibody after being immunized with trinitrophenyl-Ficoll.

Enrichment of suppressor T cells by means of binding to monophosphoryl lipid A.

The binding and elution of spleen cells from plastic dishes coated with monophosphoryl lipid A (MPL) resulted in a greater than 1,000-fold enrichment of antigen-specific suppressor T-cell (TS) activity when spleen cells from mice 18 to 24 h after exposure to a low dose of type III pneumonococcal polysaccharide (SSS-III) were used. The removal of MPL-adherent TS cells resulted in an increase in the degree of amplifier T-cell (TA) activity present in the remaining MPL-nonadherent cell fraction; however, both TS and TA activities were found in the MPL-adherent cell fraction when spleen cells from mice 4 days after immunization with an optimal dose of SSS-III were examined. These findings, as well as others, suggest that both TS and TA, once activated, acquire a cell surface receptor that enables them to bind to MPL. Because of differences in the kinetics for the activation of TS and TA during the course of the antibody response and the fact that TS, but not TA, activity appears as early as 18 to 24 h after exposure to SSS-III, it is possible to use this experimental approach to obtain cell suspensions greatly enriched in TS activity.

Adjuvant effects of trehalose dimycolate on the antibody response to type III pneumococcal polysaccharide.

Treatment with trehalose dimycolate (TDM) increases the magnitude of the immunoglobulin M (IgM) antibody response of mice to type III pneumococcal polysaccharide (SSS-III). Such enhancement is demonstrable over a wide range of immunizing doses and does not require thymus-derived (T) cells to be elicited. Although young adult mice immunized with SSS-III do not usually make anti-SSS-III antibodies of the IgG1 and IgG3 classes, antibodies of one or both isotypes were produced after immunization and treatment with TDM and/or monophosphoryl lipid A (MPL); the additive nature of the effect produced by both TDM and MPL suggests that the two immunomodulators act by different mechanisms. TDM and MPL have different effects on the induction and expression of low-dose immunological paralysis, a form of unresponsiveness known to be mediated by suppressor T cells. The relevance of these findings to the modes of action of TDM and MPL is discussed.

Ability of monophosphoryl lipid A to augment the antibody response of young mice.

Treatment with nontoxic monophosphoryl lipid A increased the magnitude of the immunoglobulin M (IgM) antibody response to type III pneumococcal polysaccharide in young (2- to 4-week-old) mice. This was accompanied by the appearance of significant numbers of IgG1- and IgG3- secreting antibody-forming cells in 4-week-old mice. These findings indicate that monophosphoryl lipid A can be used as an adjuvant to improve the immunogenicity of poorly immunogenic antigens in young, immunologically immature animals.

Examination of the differential characteristics of amplifier and contrasuppressor T cells.

Vicia villosa lectin-adherent Lyt-1+ spleen cells, obtained 4 days after immunization with an optimally immunogenic dose (0.5 micrograms) of Type III pneumococcal polysaccharide (SSS-III), increased the magnitude of the antibody response of mice to SSS-III upon transfer to recipients also immunized with the same antigen; however, the ability to demonstrate such enhancement depended greatly upon when such cells were transferred relative to immunization of recipients. Lectin-adherent cells augmented the antibody response of athymic nude (nu/nu) mice to SSS-III, and abrogated the expression - but not the induction - of low-dose immunological paralysis, a form of unresponsiveness mediated by suppressor T cells. These findings are consistent with effects usually attributed to the action of amplifier, rather than contrasuppressor, T cells.

Inactivation of suppressor T-cell activity by nontoxic monophosphoryl lipid A.

Treatment with nontoxic monophosphoryl lipid A (MPL), which was derived from a polysaccharide-deficient, heptoseless Re mutant of Salmonella typhimurium, was found to inactivate suppressor T-cell activity, as evidenced by a decrease in the degree of low-dose immunological paralysis expressed and an increase in the magnitude of the antibody response to type III pneumococcal polysaccharide. The effects produced, which could not be attributed to the polyclonal activation of immune B cells by MPL, were dependent upon the dose of MPL used, as well as the time when MPL was given relative to low-dose priming or immunization with type III pneumococcal polysaccharide. Neither amplifier nor helper T-cell activity was decreased by treatment with the same, or larger, doses of MPL. The significance of these findings to the use of MPL as an immunological adjuvant or an immunomodulating agent is discussed.

Increased activation of antigen-primed or memory B cells by bacterial lipopolysaccharide.

Treatment with bacterial lipopolysaccharide elicits the appearance of greater numbers of background antigen-specific plaque-forming cells (PFC) in the spleens of mice previously exposed or primed to subimmunogenic amounts of various non-cross-reacting antigens so as to generate detectable immunological memory. These findings suggest that treatment with lipopolysaccharide results in the activation of increased numbers of antigen-primed or memory B cells in mice previously exposed to antigen.

Increased amplifier T cell activity in autoimmune NZB mice and its possible significance in the expression of autoimmune disease.

The expression of amplifier- and helper-T cell activity was examined in NZB/N mice of different ages. Amplifier T cell activity develops in a cyclic manner; it decreases between 16 and 35 wk of age and then increases to maximal levels in mice older than 50 wk of age. The loss of suppressor T cell activity and increased amplifier T cell activity coincide with the development of autoimmune diseases in aging NZB/N mice. Although helper T cell activity is evident in young NZB/N mice, it is absent in old NZB/N mice expressing maximal amplifier T cell activity. This provides additional support for the fact that amplifier and helper functions are mediated by different subpopulations of T cells.

The role of antigen in the activation of regulatory T cells by immune B cells.

The transfer of B cells from mice immunized with Type III pneumococcal polysaccharide (SSS-III) results in the activation of suppressor and amplifier T cells that control the magnitude of the antibody response in recipient mice, immunized subsequently with SSS-III. Prior treatment of transferred B cells with an excess of enzyme (polysaccharide depolymerase) capable of hydrolyzing SSS-III, does not alter the capacity of these cells to activate regulatory T cells. These findings indicate that the activation of regulatory T cells by immune B cells is not mediated by residual antigen on the surface of transferred cells.

Genes on different chromosomes influence the antibody response to bacterial antigens.

B6.C congenic strains of mice, possessing histocompatibility (H) alleles from high responding BALB/cBy (C) mice on the genetic background of low responding C57BL/6By (B6) mice, were assayed for their ability to make an antibody response to Type III pneumococcal polysaccharide (SSS-III) and the alpha(1----3) epitope of bacterial (Leuconostoc) dextran B-1355. The results affirmed that the antibody response to SSS-III is multigenic and that genes making a positive contribution to responsiveness are located on different chromosomes, i.e., chromosomes 1, 3, 4, 5, and 9. At least one other gene also influences responsiveness to SSS-III; it is linked to the H-17 locus, which has not yet been assigned to a specific chromosome. Genes on chromosomes 1, 4, and 5 influence the magnitude of the antibody response to dextran B-1355. Some of these genes may be antigen-specific in their mode of action; however, others may not since they appear to exert a positive influence on the antibody response to both SSS-III and dextran B-1355.