Inheritance of lipopolysaccharide-enhanced nonspecific resistance to infection and of susceptibility to endotoxic shock in lipopolysaccharide low-responder mice.

In a previous study, we demonstrated that lipopolysaccharide (LPS) and other bacterial immunostimulants, in contrast to their activity in a closely related high-responder subline, failed to elicit nonspecific resistance in LPS low-responder mice against Klebsiella pneumoniae infection. To investigate the type of inheritance controlling the LPS-induced nonspecific resistance to infection, the present study was performed in low- and high-responder C3H sublines and in F1 and F2 hybrids. In addition, F1 mice were backcrossed to each parental type. Inheritance of susceptibility to endotoxin was also tested in both sublines and their hybrids and backcross progeny. For these latter assays, mice were previously adrenalectomized because removal of this gland considerably enhances their sensitivity. Our present findings are consistent with the hypothesis that LPS enhances nonspecific resistance to infection and that susceptibility to endotoxin shock in the absence of corticoids may be determined by a single autosomal dominant gene.     

Failure of endotoxin to increase nonspecific resistance to infection of lipopolysaccharide low-responder mice.

In vitro and in vivo responses to lipopolysaccharide (LPS) and various other bacterial immunostimulants were compared in c3H/He low-responder mice. The principal findings were as follows. (i) Their splenic lymphocytes were stimulated by various gram-negative mitogens such as an Escherichia coli peptidoglycan, a detoxified derivative of LPS, and even endotoxins extracted by trichloroacetic acid that are known to contain protein; spleen cells of these mice were also transformed by two other B-cell mitogens extracted from acid-fast organisms. (ii) Their macrophages were refractory to LPS and weakly responsive to a mycobacterial prepartion. (iii) LPS failed to elicit nonspecific resistance in these mice against Klebsiella pneumoniae infection. (iv) Endotoxin extracted by trichloroacetic acid and a mycobacterial preparation that could increase nonspecific resistance to infection in other strains did not protect C3H/He mice against a challenge by K. pneumoniae, although both prepartions could evoke nonspecific responses of B cells in this low-responder subline.     

Genetic analysis of lymphocyte activation by lipopolysaccharide Endotoxin.

A genetic study of in vitro lymphocyte blastogenesis by the B-cell mitogen lipopolysaccharide endotoxin has been performed, using both low-responder C3H/HeJ mice and high-responder CBA/J mice. The crossbreeding of these strains produced F1 progeny that were intermediate in responsiveness. Responder types from the backcross of the F1 to the C3H/HeJ strain segregated into intermediate- and low-responder phenotypes, whereas intermediate- and high-responder phenotypes were produced from the backcross of the F1 to the CBA/J strain. The F2 generation consisted of all three responder phenotypes in frequencies that fit the classical Mendelian ratio of 1:2:1, indicating that the mitogenic response to lipopolysaccharide endotoxin in mice is most likely governed by a pair of autosomal co-dominant genes.     

Failure of bacterial lipopolysaccharide to elicit a cytostatic effect on Plasmodium vinckei petteri in C3H/HeJ mice.

Malarial parasites, Plasmodium vinckei petteri, taken from lipopolysaccharide (LPS) high-responder (C3H/HeJGiFWeHi) mice which had been injected 7 to 8 h previously with either Escherichia coli LPS B or LPS W incorporated the purine nucleotide precursor hypoxanthine more slowly in an in vitro assay than parasites taken from saline-injected controls. In contrast, malarial parasites taken from LPS low-responder C3H/HeJ mice after injection of either LPS B or LPS W did not show reduced levels of hypoxanthine incorporation. These differing results with LPS high- and low-responder mouse strains demonstrated that the cytostatic effect on the parasites seen in the high-responder strain was not due to the direct action of LPS and implied that the cytostasis was mediated via host lymphoreticular cells. Furthermore, the failure of LPS B, a lipid A-associated protein-containing LPS preparation, to elicit a cytostatic effect on P. vinckei petteri in C3H/HeJ mice suggested that the LPS-induced effector mechanisms acting against malarial parasites may be similar to those reported against bacteria and tumors.     

Conversion of immune response patterns from high to low and low to high by an RNase-sensitive thymocyte extract.

In vitro immune responses to human serum albumin (HSA) and synthetic polypeptides (T,G)-A-L, and (H,G)-A-L, associated with the maturation IgG-forming plasma cells were studied in relation to thymic low molecular weight RNA. This RNA, from normal low-responder animals to these antigens, converted high-responder bone marrow cells to low responders, whereas RNA from high responders converted low-responder bone marrow cells to high responders. These changes of immune response patterns were observed not only in the combination of allogeneic mouse cells and RNA, but also in xenogeneic rat and mouse systems. Thymic RNA from low-responder animals had no suppressive activity to high-responder RNA, when both were added together to one dish with antigen. High doses (x 50) of low-responder thymic RNA stimulated the same immune response level as a regular amount of high-responder thymic RNA. Intact thymocytes derived from low-responder mice added together with high-responder thymic RNA did not suppress immune response of high-responder bone marrow cultures. Intact allogeneic thymocytes also induced the same response as allogeneic thymic RNA. These results indicate that humoral immune responses can be influenced by thymic RNA derived from normal animals.     

Age-dependent changes in tolerizability with rabbit gamma-globulin in the Biozzi high and low-responder lines of mice.

The antibody response to rabbit gamma-globulin (RGG) of high-responder, but not of low-responder Biozzi mice decreased with age. Injection with aggregate-freed RGG reduced the response of high-responder but not of low-responder mice to subsequent injections with aggregated RGG. This reduction in the antibody response, formed by high-responder mice, decreased with increasing age; aggregate-freed RGG appeared to sensitize 6-month-old low-responder mice to a subsequent injection with aggregated RGG. When animals, not pretreated with aggregate-freed RGG, were immunized with RGG and lipopolysaccharide (LPS), the immune response was greatly enhanced. The response of both pretreated low and high responders was substantially smaller than that of corresponding animals which were not given aggregate-freed RGG, prior to immunization. LPS revealed an inhibitory effect on low-responder mice of aggregate-freed RGG, which was not detected upon immunization with heat-aggregated RGG alone. The involvement of nonspecific suppressor cells and of B cell tolerance in low-responder mice is discussed.     

Bacterial Lipopolysaccharides Bind Selectively to Lymphocytes from Lipopolysaccharide High-responder Mouse Strains

Three different concentrations of horseradish peroxidase-labelled lipopolysaccharide (LPS-HRP) were added in vitro to spleen cells from the LPS high-responder strain C3H/Tif and to cells from the low-responder strain C3H/HeJ. After being washed and fixed the cells were exposed to the substrate and prepared for electron microscopy. After addition of 7 and 0.7 microgram/ml of labelled LPS only lymphocytes from the high-responder strain were labelled. About 5-10% of the cells from C3H/Tif bound LPS, which is in accordance with the known frequency of B cells possessing the genetically determined LPS receptor. At the highest dose of labelled LPS (70 microgram/ml) a large proportion of lymphocytes from the low-responder strain also bound LPS. Erythrocytes from both strains bound LPS at all concentrations. It is concluded that LPS-HRP allows the detection at the cellular level of LPS binding to the genetically controlled membrane receptor for LPS.     

Immunosuppression induced by attenuated Salmonella: effect of LPS responsiveness on development of suppression.

A live, avirulent strain of Salmonella typhimurium, SL3235, was previously shown to afford protection against virulent Salmonella challenge in three mouse strains of the C3H lineage, C3H/HeJ, C3HeB/FeJ, and C3H/HeNCrlBR, which differ in their innate susceptibility to Salmonella infection, as well as in their responsiveness to lipopolysaccharide (LPS). Concurrent with protection, however, SL3235 was found to induce greater than 90% reduction in proliferative responses of splenocytes from immunized mice to a panel of B and T cell mitogens. Suppression appeared to be independent of susceptibility to Salmonella infection, since the mitogenic responses of hypersusceptible C3H/HeJ and C3HeB/FeJ, as well as resistant C3H/HeNCrlBR mice, were suppressed. The suppressor cell population in immunized C3HeB/FeJ mice was recently shown to be of monocytic lineage. Using transwell plates, co-culture studies indicated that suppression was mediated by soluble factors. In the present study, the effect of LPS responsiveness on susceptibility to SL3235-induced suppression was evaluated in C3H mice by studying their ability to mount plaque-forming cell (PFC) responses to sheep red blood cells (SRBC) and in vivo antibody responses to tetanus toxoid. Comparison of PFC responses as a function of SL3235 dose in C3HeB/FeJ and C3H/HeJ mice, revealed that the latter strain was markedly more resistant to the development of suppression, as evidenced by the significantly higher (10-35-fold) SL3235 doses needed to achieve comparable suppression to those seen in C3HeB/FeJ mice. In contrast to C3HeB/FeJ mice, suppression in C3H/HeJ mice required direct cell-cell contact. In both mouse strains, suppression was alleviated by pre-treatment of immune splenocytes with either mitomycin C or x-irradiation, indicating that actively proliferating cells are required for suppressor function. Resistance of C3H/HeJ mice to SL3235-induced suppression was not due to a lesser bacterial load in vivo, since a higher number of SL3235 organisms were seen in C3H/HeJ spleens compared to C3HeB/FeJ mice. Rather, resistance of C3H/HeJ mice correlated with their reduced ability to recruit macrophages and other inflammatory cells into the spleen, as evidenced by the significantly smaller degree of splenomegaly induced in these mice following immunization with SL3235.     

Microbicidal activity and morphological characteristics of lung macrophages in Mycobacterium bovis BCG cell wall-induced lung granuloma in mice.

Morphological and functional changes in lung macrophages from mice injected intravenously with Mycobacterium bovis BCG cell walls (CWs) were studied. In BCG CW high-responder mice (C57BL/6 [B6] strain), an increase in the size and the acid phosphatase activity of lung macrophages was observed. These lung macrophages showed greater microbicidal activity to M. bovis Ravenel and Listeria monocytogenes EGD, enhanced superoxide anion production index, and greater macrophage migration inhibition activity, as compared with lung macrophages from BCG CW low-responder mice (C3H/He strain), which were small in size and showed weak acid phosphatase activity, low antimicrobial activity, and low superoxide anion production index upon intravenous injection of the mice with BCG CW. These results indicated that lung macrophages from B6 mice injected with BCG CWs were morphologically and functionally activated, but not those from C3H mice.     

Functional role of interleukin 1 in periodontal disease: induction of interleukin 1 production by Bacteroides gingivalis lipopolysaccharide in peritoneal macrophages from C3H/HeN and C3H/HeJ mice.

Hot phenol-water-extracted lipopolysaccharide (LPS) from Bacteroides gingivalis 381 was purified by Sephadex G-100 chromatography with Tris buffer supplemented with sodium deoxycholate and EDTA (B-LPS). In the present study, B-LPS was examined for its ability to induce interleukin 1 (IL-1) production, a mitogenic response, and macrophage activation in LPS high-responder C3H/HeN and low-responder C3H/HeJ mice. A significant increase in IL-1 production was observed in C3H/HeN and C3H/HeJ peritoneal macrophages treated with various doses (1.0 to 50 micrograms/ml) of B-LPS. IL-1 production by C3H/HeN macrophages treated with B-LPS (10 micrograms/ml) was about seven times greater than that by C3H/HeJ macrophages. However, the IL-1 production induced by B-LPS (10 micrograms/ml) in C3H/HeN macrophages was four times lower compared with that induced by Escherichia coli O111 B4 LPS. Also, a significant increase in IL-1 production was found in human monocytes stimulated with B-LPS. That B-LPS-induced IL-1 exhibits some molecular weight heterogeneity was indicated from Sephadex G-75 gel filtration profiles. A significant, high mitogenic response by whole spleen cells with 1 X 10(5) to 5 X 10(4) cells of either mouse strain per well treated with B-LPS (10 to 50 micrograms/ml) was observed. However, the response of C3H/HeJ mice was less than that of the C3H/HeN strain. Also, glucose consumption assays indicated that enhanced macrophage activation occurred in C3H/HeN but not in C3H/HeJ mice treated with B-LPS. In light of recent studies showing that IL-1 stimulates bone resorption in a mouse calvaria system and collagenase production in fibroblasts, we suggest that B-LPS-induced IL-1 may play a significant role in the pathogenesis of adult periodontal disease.     

Characterization of a thyroiditis-inducing thyroglobulin-specific T-cell clone restricted by the H-2 molecule of a low responder mouse strain.

We established a thyroglobulin (Tg)-specific, thyroiditis-inducing T-cell clone, B12G, from B6C3F1 mice by the immunization of mouse Tg with lipopolysaccharide (LPS) from Klebsiella strain LEN (O3:K1). B12G was Thy-1.2+, CD3+, CD4+, CD18+, and CD8-, and could transfer thyroiditis to recipient mice after in vitro stimulation with mouse or bovine Tg. Histological examination showed severe thyroiditis with predominant infiltrations of polymorphonuclear cells; few mononuclear cells were observed. B12G proliferated in response to bovine, mouse, porcine, and rat Tg in the presence of irradiated spleen cells, but did not respond to chicken or human Tg. H-2b, a low-responder haplotype of experimental autoimmune thyroiditis, governed the response of the clone to Tg. B12G produced interleukin-4 (IL-4) and IL-6, but not IL-2 or interferon-gamma (IFN-gamma), on stimulation with mouse Tg. These findings were different from characteristics of previously reported Tg-specific T-cell clones from high-responder mice in terms of epitope specificity and cytokine production pattern, raising the possibility that the specificities and functions of T cells involved in the development of autoimmune thyroiditis in low-responder mice differ from those in high responders.     

Experimental Pneumocystis carinii pneumonia in C3H/HeJ and C3HeB/FeJ mice

C3H mice develop heavier degrees of Pneumocystis carinii pneumonia than other mouse strains tested. We have compared P. carinii pneumonia in two strains of C3H mice: C3H/HeJ mice, which are unresponsive to the effects of bacterial lipopolysaccharide (LPS), have defects in macrophage function, and have increased antibody responses to orally administered T-dependent antigens; and C3HeB/FeJ mice, which are immunologically normal. P. carinii pneumonia was induced by corticosteroids, and the intensity of the infection was judged by a semiquantitative histopathologic scoring system. Heavier degrees of infection were found in C3H/HeJ mice than in C3HeB/FeJ mice. Serum antibodies to P. carinii, measured by an indirect fluorescent antibody technique, were mainly of the IgG class in both strains of mice and varied inversely with the intensity of P. carinii infection in the lungs. Antibody levels were significantly higher in C3H/HeJ mice than in C3HeB/FeJ mice. These data suggest that C3H/HeJ have increased susceptibility to the effects of steroids of host defenses against P. carinii, and heightened serum antibody responses to the organism.     

Efficacy of oral vaccination against the murine intestinal parasite Trichuris muris is dependent upon host genetics.

Oral vaccinations with Trichuris muris adult worm homogenate antigen with cholera toxin as the adjuvant were successful in both high-responder BALB/c and low-responder C57BL/10 mice, resulting in high levels of protection against subsequent infection, but were ineffective in the low-responder B10.BR mice. Subcutaneous vaccination with antigen in Freund's complete adjuvant resulted in protection of all of these strains but was most effective in high-responder BALB/c and least effective in B10.BR mice. Oral vaccination resulted in a T. muris-specific intestinal immunoglobulin A response only in the two protected strains. High levels of serum immunoglobulin G1 antibody were induced by Freund's complete adjuvant vaccination in all cases. A relationship between vaccine efficacy, expulsion phenotype, and induced T-helper subset-associated cytokines (interleukin-5 and gamma interferon) was noted. It was concluded that effective vaccination against T. muris requires the induction of Th2 responses and that this can be achieved by both oral and parenteral administration of antigens.     

Genetic Control of B-Cell Responses

The responsiveness of spleen cells from C3H/HeJ and C3H/Tif mice to lipopolysaccharide (LPS) was compared. Around 1,000-fold higher concentration of LPS were required to minimally activate HeJ cells, as compared with Tif high-responder cells, to both proliferation and polyclonal antibody secretion. However, HeJ cells did respond to higher LPS concentrations (100 and 1000 mug/ml). This selective pattern of responsiveness to LPS was also observed in the polyclonal responses of strains to LPS in vivo. Furthermore, the unresponsiveness of HeJ spleen cells was found to depend on a pure B-cell defect in the capacity to interact and/or generate triggering signals on interaction with LPS. Thus, adherent cells, thymus-derived lymphocytes, serum factors, and other non-specific conditions inherent in spleen cell suspensions of low-responder mice were not responsible for suppressing a putative B-cell response to LPS. The present findings are compatible with the possibility that the defect in C3H/HeJ B cells reflects the absence of a structure on the cell surface membrane that is functionally responsible for mediating LPS triggering.     

Enhancement of resistance to infections by endotoxin-induced serum factor from Mycobacterium bovis BCG-infected mice.

Necrosis of a variety of transplanted murine tumors can be induced by serum from Mycobacterium bovis BCG-treated mice challenged with a lethal dose of endotoxin. Results reported here show that the tumor necrosis serum (TNS) enhances resistance to infections, protecting mice against two types of challenges, either with Klebsiella pneumoniae or with the intracellular parasite Listeria monocytogenes. Moreover, TNS activity was demonstrated in animals which are refractory to lipopolysaccharide and very susceptible to infections, such as 8-day-old mice and adult C3H/He mice. Protection passively transferred by TNS was not related to antibodies, since it was not decreased by absorption with homologous organisms.     

Genetic Analysis of the Low Responsiveness to Dextran B512 in CB A/N and C57BL/10ScCr Mice

CBA/N and C57BL/10ScCr mice are low responders to the antigen dextran B512. This is due to the Xid gene in CBA/N mice and to unknown genes in C57BL/10ScCr mice, although this strain is unresponsive to lipopolysaccharide (LPS) due to a defective gene in the fourth chromosome. The female F1 hybrids (C57BL/10ScCr X CBA/N) and (CBA/N X C57BL/10ScCr) were low responders to dextran, although the Xid gene is not expressed in these hybrids, indicating lack of genetic complementation. In contrast, female F1 hybrids between the dextran high-responder strains CBA or C57BL/10 as one parental strain and the low-responder strains CBA/N or C57BL/10ScCr as the other parental strain, respectively, were responders to dextran. The C57BL/10ScCr mice did not appear to have an X-linked gene determining low responsiveness to dextran. The findings suggest that the only defect in CBA/N mice cannot be the Xid gene and the only defect in C57BL/10ScCr mice cannot be the gene determining unresponsiveness to LPS.     

Failure of C3H mice to develop lung granuloma after intravenous injection of BCG cell wall vaccine. Demonstration of a defect in lymphoid cells.

C57Bl/6 (B6) mice produced highly developed granulomata in the lung 3 or 4 weeks after intravenous injection with oil-associated BCG cell walls (CW), but C3H/He (C3H) mice did not. The potential to develop granulomata is genetically controlled (Yamamoto & Kakinuma, 1978). It is probable that the genes control only T-cell mediated granuloma and not non-specific inflammation prior to onset of immunopathological response. The retention of an FITC-labelled BCG CW preparation in the lungs of B6 and C3H mice did not differ until 10 days after injection, although 24 hr uptake of 125 I-labelled BCG CW in B6 lung was twice as much as that in C3H lung. Furthermore, C3H mice did not show a secondary type granuloma response in the lungs after subsequent injections of BCG CW. In experiments using radiation chimaeras, B6 mice reconstituted with C3H bone marrow cells were unable to produce a granulomatous response to BCG CW. In contrast, C3H mice reconstituted with B6 bone marrow cells showed a good granulomatous response. These results suggest that the C3H mice possessed a deficiency within their lympho-haematopoietic cells.     

Macrophage production during murine listeriosis: colony-stimulating factor 1 (CSF-1) and CSF-1-binding cells in genetically resistant and susceptible mice.

The concentration of the macrophage-specific colony-stimulating factor (CSF-1) and the numbers of bone marrow and spleen cells with specific receptors for that factor have been investigated in a number of mouse strains under normal conditions and after infection with the facultative intracellular bacterium Listeria monocytogenes. The CSF-1 concentration in serum and tissue was markedly elevated in infected mice, the degree of stimulation reflecting the dose of L. monocytogenes. The CSF-1 titer did not correlate with genetic resistance or susceptibility of the mice to L. monocytogenes. In contrast to the effect of lipopolysaccharide, Listeria infection was able to increase the level of CSF-1 in the lipopolysaccharide nonresponder strain C3H/HeJ. In line with earlier findings on colony-forming cells, cells bearing receptors for CSF-1 in uninfected susceptible BALB/cJ mice were only half those in resistant C57BL/6J mice. After infection the majority of these cells disappeared from the bone marrow and spleen cells of both resistant and susceptible mice. The number of CSF-1 receptor-bearing cells in the normal bone marrow may determine the degree of resistance to L. monocytogenes.     

Enhancement of antibacterial resistance of neutropenic, bone marrow-suppressed mice by interleukin-1 alpha.

The effect of recombinant human interleukin-1 alpha (IL-1) on the resistance of normal and bone marrow-suppressed mice against bacterial infection was evaluated. IL-1 induced neutrophilia and enhanced the resistance of normal mice against acute, systemic intraperitoneal infection with Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus. Mice with cyclophosphamide-induced bone marrow suppression were neutropenic and exhibited increased susceptibility to infection. Treatment of neutropenic C57BL/6 and C3H/HeJ mice with IL-1 before infection accelerated recovery of peripheral neutrophil counts and stimulated resistance against infection. Increases in neutrophils and enhancement of resistance induced by IL-1 were both dose and time dependent. Both neutrophilia and augmented resistance to infection were eliminated by a second dose of cyclophosphamide administered during the IL-1 treatments. Bone marrow-suppressed mice treated with IL-1 showed, at 4 h postinfection, greater increases in peripheral blood neutrophils and in numbers of peritoneal exudate neutrophils than suppressed mice treated with vehicle. The data suggest that the IL-1-stimulated recovery of myelopoiesis is an important factor in the enhancement of antibacterial resistance in bone marrow-suppressed, neutropenic mice. These findings indicate that IL-1 may be efficacious in limiting the duration of the neutropenia and of the increased risk for the development of bacterial infection associated with bone marrow suppression.     

Formation of intraperitoneal abscesses by Staphylococcus aureus.

Serum from Mycobacterium bovis BCG-infected mice treated with lipopolysaccharide was cytotoxic to tumor cells in vitro. Serum-induced cytotoxicity was estimated by measuring release of [3H]thymidine into culture supernatants of prelabeled tumor target cells. Serum from BCG-infected mice not treated with lipopolysaccharide or from uninfected mice treated with lipopolysaccharide was inactive. Moreover, although serum cytotoxic activity was evident with 10 syngeneic or allogeneic tumor cell lines, little or no effect was observed with normal embryonic fibroblast target cells. Maximal titers of serum cytotoxic activity were detected 14 days after BCG infection and 2 h after LPS treatment. Serum of BCT-infected, T-cell-deficient nude mice developed strong cytotoxic activity after LPS treatment; however, lipopolysaccharide-insensitive C3H/HeJ mice could produce this cytotoxic activity only after adoptive transfer with lipopolysaccharide-responsive C3H/HeN bone marrow. Physicochemical characterization of the serum cytotoxic activity revealed a heat-stable (56 degrees C, 30 min) entity with a molecular weight of about 60,000 and an isoelectric point at pH 4.8. Biological and physicochemical characteristics of this serum cytotoxic activity as defined by an in vitro assay were very similar to characteristics of tumor necrosis factor and suggest that this molecule may be a major effector mechanism for the antitumor actions of lipopolysaccharide.