Partial restoration of the lipopolysaccharide-induced proliferative response in splenic B cells from C3H/HeJ mice

C3H/HeJ mice are hyporesponsive to the biologic effects of bacterial lipopolysaccharide (LPS), and their splenic B cells do not proliferate after exposure to LPS. The molecular basis of this hyporesponsiveness is unknown but it may result from defective membrane signal transduction after LPS binding. To examine this possibility, we added bioactive compounds in combination with LPS to C3H/HeJ B cell cultures in order to bypass the putative defect. The addition of PMA, monensin, or ionomycin, either alone or in combination, had no effect on C3H/HeJ B cell responses to LPS. In contrast, the addition of trypsin together with LPS resulted in a partial restoration of the proliferative response in C3H/HeJ splenic B lymphocytes. The maximal C3H/HeJ B cell response varied from 25 to 60% of the C3Heb/FeJ (LPS responder) B cell response. The trypsin-mediated enhancement of the LPS response was abrogated by pretreatment of the trypsin with the trypsin inhibitors DFP or TLCK. Pretreatment of the LPS with polymyxin B, which blocks lipid A-dependent reactions, also abrogated the trypsin effect. Because the C3H/HeJ B cell responds to all other B cell mitogens, we suggest that the defect is in an LPS-specific step and that the action of trypsin results in the restoration of the missing signal. At the present time the identity of this signal is not known, but the experiments described in this report provide a unique model to elucidate the basis of LPS hyporesponsiveness in splenic B cells from C3H/HeJ mice.     

Calcium ionophore A23187 does not stimulate lipopolysaccharide nonresponsive C3H/HeJ peritoneal macrophages to produce interleukin 1

C3H/HeJ mice are hyporesponsive to the biologic effects of bacterial lipopolysaccharide (LPS). The defect in the strain of mice is believed to be due to the expression of a mutant allele designated Lpsd at the chromosome four locus. The molecular basis of this hyporesponsiveness is not known, but it may result from some defective membrane signal transductions. To examine this possibility, we compared the abilities of interleukin 1 (IL-1) production by C3H/HeJ macrophages with those by C3H/He macrophages (LPS responsive) after stimulation with the calcium ionophore A23187 or phorbol myristate acetate (PMA). A23187 induced IL-1 production by C3H/He macrophages, but it did not induce IL-1 production by C3H/HeJ macrophages and neither did LPS. However, it had the ability to increase intracellular free Ca2+ in C3H/HeJ macrophages as well as in C3H/He macrophages, this being examined by the changes in cytosolic Ca2+ in the macrophages by using Quin 2. In contrast, PMA was able to induce IL-1 production by both C3H/He and C3H/HeJ macrophages without increasing intracellular Ca2+. Since polymyxin B did not inhibit A23187- or PMA-induced IL-1 production by C3H/He macrophages, these results are not due to the little amount of LPS in culture medium, but due to their own characteristics. A calmodulin antagonist W-7 effectively inhibited A23187-induced IL-1 production by C3H/He macrophages. However, it hardly inhibited LPS-induced IL-1 production except at high concentration, and it caused no inhibition of the PMA-stimulated one. These results suggest that the blocking sites expressed phenotypically by the Lpsd are shared by LPS- and A23187-stimulated cellular processes, although the actions of LPS and A23187 are different from each other. In addition to the direct study with LPS or lipid A, A23187 should provide another useful approach to clarify the molecular mechanisms of Lpsd defect in C3H/HeJ macrophages.     

Defect of calmodulin-binding protein in expression of interleukin-1 beta gene by LPS-nonresponder C3H/HeJ mouse macrophages

Peritoneal macrophages of Lipopolysaccharide (LPS)-refractory C3H/HeJ mouse failed to express the mRNA coding interleukin 1 (IL-1) beta when stimulated by the Ca2+ ionophore A23187 or LPS, though macrophages of LPS-responsive C3H/He responded to these stimulants. These results suggest that the defect of the response in C3H/HeJ macrophages toward LPS stimulation may be related to the Ca2+-dependent signal pathway. The extracts from the C3H/HeJ macrophages showed normal activities of both protein kinase C (PKC) and calmodulin (CaM) in comparison with those from LPS-responsive C3H/He macrophages. However, one species of CaM-binding proteins could hardly be detected by the cross-linking assay with 125I-CaM in C3H/HeJ macrophages stimulated by LPS. These results suggest that the LPS-refractory site in C3H/HeJ macrophages is related to the lack of this CaM-binding protein, and the Ca2+-dependent CaM system may play an important role in the activation of cells by LPS.     

Macrophage stimulation by bacterial lipopolysaccharides. III. Selective unresponsiveness of C3H/HeJ macrophages to the lipid A differentiation signal.

Peritoneal macrophages from the endotoxin-unresponsive C3H/HeJ substrain of mice were entirely refractory to activation in vitro by protein-free LPS, a defect that was not overcome by co-culture of spleen cells from the responder C3H/St substrain with LPS resistant C3H/HeJ macrophages. The defect in responsiveness appears confined to the lipid A activation signal since C3H/HeJ macrophages were fully activated after in vitro treatment by lipid A protein (LAP)--LPS complex, isolated LAP, and BCG. Moreover, after exposure to allogeneic tumor cells in vivo, C3H/HeJ macrophages were cytotoxic for tumor target cells in vitro. By contrast, macrophages from the responder C3H/St strain were fully activated by protein-free LPS to become cytolytic for tumor cells in vitro. C3H/HeJ macrophages, therefore, exhibit a highly selective defect characterized by unresponsiveness to the lipid A activation signal of protein-free LPS and resistance to the toxic effects of high concentrations of LPS that were lethal to the responder C3H/St strain.     

Differing signal requirements for the activation of macrophages from C3H/HeJ and C3H/OuJ mice

Abstract Endotoxin-associated protein (EP) from Salmonella typhi stimulated the release of prostaglandin E2 (PGE2), interleukin-1 (IL-1), and interferon (IFN) activity in macrophages from the lipopolysaccharide (LPS) responder C3H/OuJ mouse strain. However, only PGE2 and IL-1 were stimulated by EP in macrophages from the LPS nonresponder C3H/HeJ mouse strain. LPS stimulated the release of PGE2, IL-1 and IFN activity in C3H/OuJ macrophages, but not in C3H/HeJ macrophages. The protein kinase C (PKC) activator phorbol myristic acid (PMA) stimulated PGE2 production in both strains but not IL-1 production, suggesting that signalling pathways other than PKC may be involved in IL-1 production. The calcium ionophore ionomycin stimulated PGE2 production in C3H/OuJ but not C3H/HeJ macrophages, suggesting a defective calcium-related pathway in the C3H/HeJ macrophages as compared to the C3H/OuJ cells.     

Restoration of LPS responsiveness of C3H/HeJ mouse lymphocytes by microinjection of cytoplasmic factor(s) from LPS-stimulated normal lymphocytes

Cytosol fractions of lymphocytes from LPS-responder mice (C3H/HeN) were prepared and injected into B lymphocytes of LPS-nonresponder mice (C3H/HeJ) by a microinjection technique utilizing polyethyleneglycol-mediated cell fusion. The B lymphocytes of C3H/HeJ mice microinjected with cytosol prepared from LPS-stimulated C3H/HeN cells became normally responsive to LPS. Microinjection of cytosol itself did not stimulate C3H/HeJ cells to proliferate or differentiate into immunoglobulin-producing cells, and the cells injected with cytosol had to be restimulated with LPS in order to proliferate and differentiate. These data suggested that C3H/HeJ B cells acquired LPS responsiveness by microinjection of cytoplasmic factor(s) from LPS-stimulated C3H/HeN cells and that these factor(s) may be one of the components involved in normal signal transmission from cell surface to nucleus in the early stages of the LPS response. The apparent m.w. of the cytoplasmic factor(s) is 100,000 by gel filtration. Chromatofocusing analysis suggested that these factors may consist of two components with the same m.w.     

BCG-induced enhancement of endotoxin sensitivity in C3H/HeJ mice. I. In vivo studies

C3H/HeJ mice exhibit a marked insensitivity to bacterial lipopolysaccharide (LPS) in vivo. Pretreatment of these mice with viable BCG organisms 11 days before LPS administration renders them sensitive to the lethal effects of a highly purified, phenol-extracted LPS. Other in vivo responses to LPS are increased in BCG-infected C3H/HeJ mice in parallel with enhanced lethality. These include 1) the elevation of serum interferon, 2) the production of the acute phase reactant, serum amyloid A (SAA), and 3) hypoglycemia. However, BCG infection has only a minimal effect on anti-LPS antibody production. BCG-infected C3H/HeJ mice approach the LPS sensitivity of normal C3H/HeN mice, but the enhanced LPS sensitivity is transient and decreases over a 2-month period. The ability of BCG to induce LPS sensitivity in C3H/HeJ mice demonstrates that LPS unresponsiveness is not due to an absolute defect in this strain, but rather, a partially reversible state of hyporesponsiveness. In addition, these findings, in conjunction with other observations, suggest that the enhancement of LPS sensitivity induced by BCG infection is mediated primarily through an effect on T cells and/or macrophages rather than B lymphocytes.     

Defective tumoricidal capacity of macrophages from C3H/HeJ mice

Peritoneal macrophages from C3H/HeN mice treated i.p. with T cell mitogens or viable BCG organisms were cytotoxic to syngeneic tumor cells in vitro. Macrophages from endotoxin-unresponsive C3H/HeJ mice treated with BCG or T cell mitogens, however, were not tumoricidal. Furthermore, unlike cells from C3H/HeN mice, macrophages from C3H/HeJ mice could not be activated for tumor cytotoxicity after in vitro treatment with bacterial endotoxins or with lymphokine-rich supernatants. The subnormal induction of cytotoxic macrophages after in vitro or in vivo treatments in C3H/HeJ mice appears to be a highly selective defect. Macrophage responses (yield, phagocytosis, or peroxidase staining) in inflammatory exudates induced by BCG, T cell mitogens, or heterologous serum in C3H/HeJ or C3H/HeN mice were identical. C3H/HeJ macrophages also responded normally in vitor to chemotactic lymphokines. Thus, C3H/HeJ macrophages possess a profound and selective defect in tumoricidal capacity. This defect was not dependent upon exogenous endotoxins. Defective macrophage cytotoxic responses may reflect non-LPS related functions regulated by the LPS gene.     

Defective helper T-cell activity in LPS-unresponsive C3H/HeJ mice

C3H/HeJ mice, unresponsive to LPS, exhibit a defective ability to mount antibody responses to T-dependent immunogens. The anti-TNP antibody response to TNP-HRBC, a T-dependent immunogen, was found to be lower in these mice as compared to LPS-responsive C3H/HeN mice, whereas the anti-TNP antibody response to TNP-Ficoll, a T-independent immunogen, was of the same magnitude in C3H/HeJ and C3H/HeN mice. An impaired helper activity of C3H/HeJ HRBC-primed spleen cells was demonstrated in a titration assay in which graded numbers of C3H/HeJ or C3H/HeN HRBC-primed spleen cells were added to cultures containing a constant number of unprimed spleen cells from either C3H/HeJ or C3H/HeN mice and the immunogen TNP-HRBC. The reduced helper T-cell activity of C3H/HeJ HRBC-primed spleen cells appears to be independent of macrophage defects, since C3H/HeJ and C3H/HeN macrophages were found equally effective in antigen presentation as evaluated by an in vitro antigen-specific T-cell proliferation assay. The difference in helper T-cell activity between these two substrains probably reflects a lower number and/or proliferation rate of antigen-responsive T cells in C3H/HeJ mice.     

Correlation of the biologic responses of C3H/HEJ mice to endotoxin with the chemical and structural properties of the lipopolysaccharides from Pseudomonas aeruginosa and Escherichia coli

The basis of the biologic responses of C3H/HeJ mice to endotoxin administration in relation to the structural linkages in the lipid A portion of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa and Escherichia coli were investigated. P. aeruginosa LPS was found to be immunogenic, mitogenic, and toxic, but not lethal, in C3H/HeJ mice. The observed mitogenicity in spleen cells was directed toward immunoglobulin- (Ig) bearing cells, was present in response to isolated and solubilized lipid A, and was inhibitable by polymixin B. The P. aeruginosa LPS was chemically analyzed in order to define its composition and exclude the presence of contaminating proteins being responsible for the biologic responses of C3H/HeJ mice that were observed. Structural analysis of the linkages of the fatty acids to the glucosamine backbone in the lipid A of P. aeruginosa and E. coli revealed similarities in terms of the ratio of hydroxy fatty acids to straight chain fatty acids and the way in which these 2 types of fatty acids were linked to the backbone. Differences were seen in the carbon chain length of the fatty acid substituents, and the substituent on the hydroxy fatty acid that is directly ester linked to the glucosamine backbone. These data indicate that the refractivity of C3H/HeJ mice to the biologic effects after the administration of Gram-negative endotoxins may be limited to enterobacterial LPS. Those differences we found in the chain length and/or linkages of the fatty acid substituents in the lipid A portion of the LPS between P. aeruginosa and E. coli may be sufficient to render C3H/HeJ mice responsive to the biologic effects of nonenterobacterial endotoxins.     

Cellular mechanism of endotoxin unresponsiveness in C3H/HeJ mice.

B cells from C3H/HeJ mice fail to respond to an endotoxin (LPS K235) which is mitogenic for normal mice including the closely related C3H/HeN strain. The cellular basis for this unresponsive state has been investigated. The C3H/HeJ mice have normal numbers of B cells, which are capable of normal responses to other B cell mitogens, such as polyinosinic acid (Poly I). Addition of normal macrophages or spleen cells fails to reconstitute the normal response. Furthermore, neither macrophages nor spleen cells from the C3H/HeJ strain suppress the normal C3H/HeN spleen cells. Finally, spleen cells enriched for B cells by the removal of macrophages or T cells demonstrate the same differences in responsiveness to LPS. These results indicate that LPS unresponsiveness is a defect of the B cell itself and not due to suppressor cells or the absence of helper cells. When LPS is added to Poly I-stimulated cultures, there is additional enhancement of the response of normal C3H/HeN spleen cells. However, LPS causes a dose-dependent suppression of the Poly I response of C3H/HeJ spleen cells. This suppression is dependent on the time of addition of LPS to the Poly I-stimulated cultures. These data are interpreted as indicating that the binding of LPS to the membrane of C3H/HeJ B cells results in their inactivation or suppression, and that this is the basis of LPS unresponsiveness in this mouse strain.     

The inheritance of a defective lipopolysaccharide response locus in C3H/HeJ mice

F₁ hybrid mice from crosses between the lipopolysaccharide (LPS) nonresponder strain C3H/HeJ and a variety of LPS responder strains show quantitative or codominant inheritance of mitogenic responsiveness to LPS. Backcross segregation ratios indicate that a defect at a single locus is responsible for the lack of responsiveness in C3H/HeJ mice. Autoradiographic studies show that the number of LPS-responsive cells in F₁ cultures is approximately half the number of responsive cells in parent cultures. Kinetic patterns of mitogenic responsiveness to LPS differ in F₁ hybrid and parent cultures. The kinetic patterns of responsiveness vary with the cell concentrations of the culture and appear to correlate with the number of LPS-responsive cells in F₁ and parent cultures.     

Induction of activated macrophages in C3H/HeJ mice by avirulent Salmonella

A single injection of viable Salmonella typhimurium SL3235, an avirulent organism blocked in the aromatic pathway, induced the generation of activated peritoneal macrophages in three different C3H mouse strains, including macrophage-defective C3H/HeJ mice. Macrophages obtained from immunized mice were cytotoxic for B16 melanoma cells, P815 mastocytoma cells, and TU-5 fibrosarcoma cells and microbicidal in vitro for the obligate, intracellular, protozoan parasite Leishmania major. The capacity of live SL3235 to activate C3H/HeJ macrophages contrasts with the failure of live Bacillus Calmette-Guérin to induce activated macrophages in this mouse strain. Although viable SL3235 were capable of fully activating cells of both normal and defective mice, a dose-dependent difference was observed in the number of organisms necessary for induction of tumoricidal macrophages in C3HeB/FeJ (normal) and C3H/HeJ (defective) animals. As few as 80 viable SL3235 were capable of activating C3HeB/FeJ macrophages whereas 5 X 10(4) organisms were required to activate C3H/HeJ macrophages. Maximal macrophage activation occurred 7 to 10 days after SL3235 inoculation in C3H/HeJ and C3HeB/FeJ mice. Acetone-killed cells of SL3235 had some but not all of the activity of the living Salmonella. A single in vivo injection of the nonviable preparation resulted in the induction of tumoricidal macrophages in C3HeB/FeJ but not in C3H/HeJ mice, even when tested over a wide dosage range. Injection of acetone-killed cells of SL3235 did, however, result in a population of primed macrophages in C3H/HeJ mice, as explanted cells could be induced to express activated macrophage effector activities after additional treatment in vitro with either LPS or IFN-gamma. Thus, in vivo administration of viable SL3235 is, by itself, capable of eliciting the full series of steps required for activation of C3H/HeJ macrophages, whereas killed SL3235 only provides signals sufficient to prime these defective macrophages for further activation in vitro. AI 15613     

Intrinsic B lymphocyte and macrophage defects in C3H/HeJ mice

C3H/HeJ mice are genetically defective in their responsiveness to lipopolysaccharide (LPS). Their B cells also have a characteristically low cloning efficiency in semisolid agar cultures, where LPS does not seem to be required. Adherent macrophages facilitate clonal proliferation in such cultures via diffusible substances. C3H/HeJ macrophages functioned poorly in this respect, and addition of normal C3HeB/FeJ macrophages to cultures of C3H/HeJ B cells did not lead to normal colony numbers. Although immune interferon can stimulate normal resident peritoneal macrophages to function well in semisolid agar cultures, it did not improve the cloning efficiency of C3H/HeJ cells. Similarly, addition of indomethacin or interleukin 1 to the cultures did not reveal that abnormally elevated production of prostaglandins or a deficiency in interleukin 1 are responsible for poor C3H/HeJ colony formation. These results indicate that C3H/HeJ mice have defects intrinsic to both B cells and macrophages that are not overcome by interferon. Purified B cells from these mice cloned poorly and did not respond to stimulation in liquid cultures with anti-mu-coated beads plus factors. There was a tendency for the poor cloning of C3H/HeJ B cells to improve with age.     

Dysfunction of thymocytes of C3H/HeJ mice in blastogenic response to anti-thymocyte serum in vitro and its repair with lipopolysaccharide induced mediator.

In vitro mitogenic responses of thymocytes to rabbit anti-mouse thymocyte serum (ATS) have been compared in several mouse strains. The response of thymocytes of C3H/HeJ mice is about one-third of those of thymocytes of C3H/He, ATL or ATH strains. Phenol-extracted bacterial lipopolysaccharide (LPS) does not induce mitogenic response in cultured C3H/HeJ spleen cells, but the spleen cells of all other strains used are capable of responding to LPS. The low response of C3H/HeJ thymocytes to ATS is restored by adding “endotoxin soup” prepared from spleen cell cultures of LPS-responder mice in the presence of LPS. Neither soup prepared from C3H/HeJ spleen cell cultures without the addition of LPS nor soup prepared from cell cultures with LPS show such restoration of the response of C3H/HeJ thymocytes to ATS. The molecular size of the active factor in “endotoxin soup” was estimated on a Sepharose CL-4B column and determined to be about 20,000 daltons. The activity of “endotoxin soup” is destroyed by heating at 70 C for 30 min or 80 C for 10 min and diminished by digestion with trypsin. The mechanisms of restoration of low response of C3H/HeJ thymocytes to ATS by “endotoxin soup” are discussed.     

Immunologic properties of bacterial lipopolysaccharide (LPS). IV. Cellular basis of the unresponsiveness of C3H/HeJ mouse spleen cells to LPS-induced mitogenesis.

Lymphoid cells obtained from the C3H/HeJ mouse strain respond abnormally to LPS in vitro, as shown by the fact that they are unable to make a mitogenic response to some LPS preparations and make only a low mitogenic response to other LPS preparations. In contrast, cells from a closely related C3H substrain, the C3H/St, are highly responsive to both types of LPS preparations. Experiments were carried out to determine the cellular basis of these genetically determined LPS response differences. This question was approached by studying the mitogenic response to LPS in cultures containing mixtures of various combinations of B cells, T cells, and macrophages from C3H/HeJ and C3H/St mice. Experiments utilizing an LPS preparation to which the C3H/HeJ is totally unresponsive (negative LPS) revealed, first, that either spleen cells, or partially purified T cells and/or macrophages obtained from C3H/St, could not restore the ability of C3H/HeJ spleen cells to respond to LPS, indicating that the C3H/HeJ is not deficient in an LPS-specific helper cell population which may be required for mitogenesis. Secondly, the addition of either spleen cells or partially purified T cells or macrophages from the C3H/HeJ to spleen cells from the C3H/St did not inhibit the mitogenic response to LPS, suggesting that the presence of suppressor cell activity is also not involved. Experiments analogous to those described, except utilizing another LPS preparation to which the C3H/HeJ is partially responsive (positive LPS), also failed to demonstrate reconstitutive or suppressive effects when C3H/HeJ and C3H/St spleen cells were admixed. The results obtained indicate that the defect in the C3H/HeJ mouse strain that limits its responsiveness to positive LPS and which renders it totally unresponsive to negative LPS appears to be an intrinsic defect in the capacity of B cells to react to the mitogenic stimulus of LPS.     

Lipopolysaccharide-Induced Mediators Assisting the Proliferative Response of C3H/HeJ Thymocytes to Concanavalin A

³H-Thymidine uptake of thymocytes from LPS-responder Balb/c mice in the presence of a submitogenic dose (0.5 μg/ml) of con A in vitro was significantly enhanced by adding LPS (0.1 to 2.5 μg/ml), while the uptake of thymocytes from LPS-nonresponder C3H/HeJ was not enhanced by LPS. However, “endotoxin soups,” which were prepared from the supernatants of LPS-responder murine spleen cell cultures in the presence of LPS, clearly increased the incorporation of ³H-thymidine into C3H/HeJ thymocytes in the presence of this small amount of con A. The soup prepared from C3H/HeJ spleen cell cultures did not show any synergistic effect with con A. Even if the major histocompatibility between soup-producer cells and responder cells to con A was different, the soups were still effective. The active substance in the “endotoxin soups” was eluted through a Sepharose CL-4B column, and its molecular size was estimated to be about 20,000 daltons. The activity of the soups was destroyed by heating at 70 C for 30 min or at 80 C for 10 min. Digestion with trypsin destroyed the activity of the soups, but digestion with DNase or RNase did not. The role of the active substance in the soups in synergy with con A and its relation to the synergistic effect of con A and LPS are discussed.     

Lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) in mice is principally due to maternal cause but not fetal sensitivity to LPS

The present study deals with whether lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) is related to LPS-susceptibility of either mother or fetus and how LPS or LPS-induced TNF causes IUFD. LPS-susceptible C3H/HeN or -hypo-susceptible C3H/HeJ pregnant mice and the mice mated reciprocally with these mice were used on days 14 to 16 of gestation for experiments. All of fetuses in pregnant C3H/HeN mice mated with either C3H/HeN males [HeN(HeN)] or C3H/HeJ males [HeN(HeJ)] were killed within 24 hr when injected intravenously (i.v.) with 50 or 100 microg of LPS. On the other hand, the majority of fetuses in C3H/HeJ females mated with either C3H/HeJ males [HeJ(HeJ)] or C3H/HeN males [HeJ(HeN)] survived when injected i.v. with even 400 microg of LPS. These findings indicate that LPS-induced IUFD depends on the maternal LPS-responsiveness. LPS injected into mothers could pass through placenta to fetuses, since an injection with 125I-labeled LPS or IgG into pregnant mice resulted in considerable levels of radioactivity in fetuses as well as placenta. Cultured peritoneal macrophages derived from F1 mice of HeJ(HeN) or HeN(HeJ) mice, produced nitric oxide (NO) and tumor necrosis factor (TNF) in response to LPS, although the levels of NO and TNF were lower in comparison with those of C3H/HeN macrophage cultures, suggesting a possibility that the fetus as well as F1 cells might be responsible to LPS. LPS-induced IUFD was not blocked by treatment with anti-TNF antibody which inhibited LPS-induced TNF production in pregnant females, although an injection of recombinant TNFalpha instead of LPS could induce IUFD, suggesting that the cause of IUFD cannot be attributed to mother-derived TNF alone. The roles of LPS passed through placenta and LPS-induced mediators on IUFD were discussed.     

Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice

We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21-28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.     

Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice

Abstract We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21–28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.