Sep 15, 1987 - cells. J. Immunol. 128:1709-1711. 31. Morrison, D. C., and J. L. Ryan. 1979. Bacterial endotoxins and host immune responses. Adv. Immunol.
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Vol. 55, No. 12
IMMUNITY, Dec. 1987, P. 3093-3102
0019-9567/87/123093-10$02.00/0 Copyright C) 1987, American Society for Microbiology
Mechanisms of Specific Immunological Unresponsiveness Bacterial Lipopolysaccharides
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KAREN L. ELKINS,* PHILIP W. STASHAK, AND PHILLIP J. BAKER
Laboratory of Microbial Immunity, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892 Received 20 July 1987/Accepted 15 September 1987
Low-dose priming of mice with Escherichia coli 0113 lipopolysaccharide (LPS) results in the development of immunological memory, whereas low-dose priming with E. coli 055 LPS or Serratia marcescens LPS induces significant antigen-specific unresponsiveness. All three preparations of LPS induced proliferation of mouse splenocytes with similar time course and [3H]thymidine uptake. There was no correlation between the small amounts of serum antibody detected by enzyme-linked immunosorbent assay after low-dose priming and the subsequent generation of either memory or unresponsiveness. Further, the passive transfer of small amounts of LPS-specific antibody had no significant effect on the magnitude of the plaque-forming cell (PFC) response elicited after subsequent immunization. Reduction of the PFC response to E. coli 055 LPS occurred after low-dose priming of nulnu (as well as nul+) mice; however, unresponsiveness could not be generated in nu/nu mice by low-dose priming with S. marcescens LPS. Thus, although the development of low-dose unresponsiveness to S. marcescens LPS appears to involve T cells, the response to E. coli 055 LPS does not. Enhancement of the primary PFC response to S. marcescens LPS could be transferred with low-dose-primed spleen cells depleted of Lyt-2+ T cells; this suggests that the magnitude of the PFC response to this preparation of LPS is negatively influenced by Lyt-2+ T cells and positively influenced by Lyt-2- spleen cells (i.e., L3T4+ T cells). These findings indicate that T cells appear to be involved in regulating the magnitude of the antibody response to some types of bacterial LPS.
decrease the magnitude of the antibody response upon immunization with antigen (reviewed in reference 47), particularly in the case of E. coli 055 LPS (7), we examined the relationship between any LPS-specific serum antibody produced in response to low-dose priming and the expression of unresponsiveness or memory. We further evaluated the contributions of T-cell populations. The results indicate that specific antibody-mediated feedback does not contribute to the generation of memory or unresponsiveness. Antigenspecific unresponsiveness to S. marcescens LPS may be regulated by T cells, whereas regulation of the magnitude of the PFC response to E. coli 0113 or E. coli 055 LPS is not and appears to be more complex.
Previous studies have shown that the magnitude of the specific antibody response to some types of bacterial lipopolysaccharide (LPS) can be increased or decreased by prior exposure (priming) with a single injection of a subimmunogenic dose of LPS (4, 14, 37, 39, 50, 51). These effects are not due to changes in the isotype of the antibody produced after immunization or to alterations in the time course of the plaque-forming cell (PFC) response (14). The generation of memory or unresponsiveness is specific for the serotype of the LPS used for priming (14), a finding that is consistent with earlier work showing that immunological memory could be induced by priming with the nonmitogenic, 0-polysaccharide portion of the Escherichia coli 0113 LPS molecule (native protoplasmic polysaccharide or NPP; 50). In experiments using mice that were low-dose primed and then immunized with suboptimal, optimal, or supraoptimal amounts of E. coli 055 or Serratia marcescens LPS, the magnitude of the specific PFC response to all immunizing doses of homologous LPS tested was reduced by low-dose priming (14). This suggested that unresponsiveness is not due merely to the removal of injected LPS by circulating antibody, since it could thus not be abrogated by immunization with large amounts of LPS. We further considered it unlikely that unresponsiveness was due to antibodymediated feedback, because (i) few or no specific PFC could be detected in the spleen after low-dose priming with any LPS used, suggesting that little circulating LPS-specific serum antibody is produced upon priming, whereas relatively large amounts are involved in antibody-mediated feedback (7, 47); and (ii) unresponsiveness could be elicited by immunization only 2 days after priming with a subimmunogenic dose of LPS, i.e., at a time when little serum antibody could be present. Because preexisting specific serum antibody has been reported to both increase and *
MATERIALS AND METHODS Mice. Female BALB/cByJ mice were purchased from Jackson Laboratories (Bar Harbor, Maine); they were 8 to 12 weeks of age at the time of use. Female BALB/c nulnu and nul+ mice (matched littermates) were obtained from the National Institutes of Health Small Animal Section and were 8 to 10 weeks of age at the time of use. Antigens. All preparations of LPS used were extracted from cell walls of bacteria by the phenol-water procedure (27, 38). A preparation of E. coli 0113 LPS, used throughout, was a gift of J. A. Rudbach, Ribi ImmunoChem (Hamilton, Mont.); its immunological properties have been previously described (4, 22, 23, 38, 39, 50, 51). E. coli 055 LPS (lots RIR-326, RIR-342, and 026101) and S. marcescens LPS (lots RIR-322 and RIR-332) were purchased from Ribi ImmunoChem. These preparations of LPS appeared to be devoid of lipid A-associated protein, since they were not mitogenic in vitro for C3H/HeJ lymphocytes and were not immunogenic in vivo for C3H/HeJ mice; no protein could be detected by UV spectroscopy or by chemical (Lowry) analysis (data not shown). All solutions of LPS and other reagents used in vivo in this study were prepared in sterile,
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pyrogen-free saline (Abbott Laboratories, Chicago, Ill.). Mice were immunized intraperitoneally (i.p.) with the doses of LPS indicated in the text. Detection of antibody-producing cells. Antibody-producing PFC secreting immunoglobulin M (IgM) antibody specific for LPS were detected by a slide version of the technique of localized hemolysis-in-gel (5, 6). Sheep erythrocytes were sensitized with the appropriate LPS by the chromic chloride coupling method (6), using 1 mg of LPS to sensitize 0.5 ml of packed erythrocytes. The results obtained for individual mice (PFC per spleen) were corrected by subtraction for small numbers of sheep erythrocyte-specific PFC detected, so that only values for LPS-specific PFC are considered in this report. Since the values for PFC per spleen are log normally distributed (18), geometric means of the values for groups of similarly treated mice were calculated. The data are expressed as the mean log1o LPS-specific PFC per spleen ± standard error of the mean (SEM) for groups of 5 to 10 mice or as the antilog (actual PFC) of the mean. Student's t test was used to evaluate the significance of the differences observed (44). Differences were considered to be significant when probability (P) values of
