Enhancement of Murine Immune Responses to Orally Administered Haptenated Streptococcus mutans

The induction of immune responses to orally administered trinitrophenyl (TNP)-haptenated Streptococcus mutans and its enhancement with muramyldipeptide (MDP), peptidoglycan (PG), and concanavalin A (Con A) were investigated in lipopolysaccharide (LPS)-non-responsive C3H/HeJ mice and the syngeneic, LPS-responsive C3H/HeN strain. Both mouse strains manifested similar immune responses, primarily of the IgM isotype, after a single gastric intubation (GI) with TNP-S. mutans. However, when groups of animals were first carrier-primed by GI with S. mutans for 2 consecutive days, followed by a single GI with TNP-S. mutans 1 week later, C3H/HeJ mice gave a significantly higher (P less than or equal to 0.01) splenic IgA anti-TNP plaque-forming cell (PFC) response than identically treated C3H/HeN mice. Furthermore, saliva, urine and serum from these C3H/HeJ mice possessed high levels of IgA anti-TNP antibodies as determined by the enzyme-linked immunosorbent assay, whereas C3H/HeN mice exhibited low antibody levels. Oral administration of Con A (either 250 micrograms or 500 micrograms/mouse) or purified PG (1 mg/mouse) at the time of TNP-S. mutans immunization resulted in significantly (P less than or equal to 0.01) enhanced splenic IgA anti-TNP PFC responses, especially in C3H/HeJ mice. On the other hand, MDP promoted IgA anti-TNP PFC responses in LPS-responsive C3H/HeN mice but did not augment responses in C3H/HeJ animals. A similar immune response pattern was seen when antibody levels were measured in serum, saliva, and urine of both mouse strains. These results demonstrate that haptenated S. mutans is a good antigen for the induction of high IgA responses in orally immunized C3H/HeJ mice and that this high response can be enhanced with the adjuvants Con A and PG. However, MDP is ineffective in C3H/HeJ mice but enhances IgA responses in normal LPS-responsive C3H/HeN animals.     

Mice Refractory to Lipopolysaccharide Manifest High Immunoglobulin A Responses to Orally Administered Antigen

Lipid A-nonresponding C3H/HeJ mice manifested high immune responses to orally administered (either by feeding or by intragastric immunization) heterologous erythrocytes when compared with syngeneic lipid A-responding C3H/HeN mice. Prolonged consumption of horse erythrocytes resulted in a significant secretory immune response in both C3H mouse strains as evidenced by high salivary agglutinin titers. Although salivary agglutinin titers were only slightly greater in C3H/HeJ mice than those of C3H/HeN mice, serum agglutinin titers and immunoglobulin A (IgA) levels were consistently higher (two- to fourfold) in C3H/HeJ mice. The appearance of anti-horse erythrocyte plaque-forming cell responses in spleens of immunized animals was followed by an increase in salivary anti-horse erythrocyte agglutinin activity. Peak levels of both responses were attained after approximately 3 weeks of immunization. Differences in immune responsiveness between C3H mouse strains were also evident at the cellular level since splenic IgA anti-horse erythrocyte plaque-forming cell responses in fed C3H/HeJ mice were twofold higher than those in similarly treated C3H/HeN mice. This higher response pattern was also observed when C3H/HeJ mice manifested threefold higher splenic IgM and IgA plaque-forming cell responses to intragastrically administered sheep erythrocytes. Thus, higher responsiveness was observed in the C3H/HeJ mice given heterologous erythrocytes by the oral route. Furthermore, levels of serum IgA in 10- to 12-month-old nonimmunized C3H/HeJ mice were higher than those of C3H/HeN mice. These findings suggest that lack of host responsiveness to lipopolysaccharide affects the manifestation of subsequent immune responses to orally administered antigens. The possible mechanisms and implications of this high responsiveness are discussed.     

Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants.

In the present study, we compared the ability of the soluble adjuvants concanavalin A (ConA), muramyl dipeptide (MDP), and peptidoglycan (PG) to enhance immune responses to orally administered particulate antigens of Streptococcus mutans 6715 in gnotobiotic rats. The isotype and levels of antibody in saliva and in serum from experimental rats were determined by an enzyme-linked immunosorbent assay using S. mutans whole cells (WC) as the coating antigen. The specificities of salivary and serum immunoglobulin A (IgA) antibodies to particulate S. mutans antigens, lipoteichoic acid, S. mutans serotype g carbohydrate, and dextran were also determined. When 50 micrograms of ConA was used as the oral adjuvant with S. mutans 6715 WC immunogen, a slight enhancement of immune responses was obtained. A higher dose of ConA suppressed humoral responses to the immunogen. Enhanced immune responses, especially of the IgA isotype, in both serum and saliva were induced in gnotobiotic rats given MDP and either S. mutans 6715 WC or purified cell walls (CW) by gastric intubation. Elevated IgA antibody levels to CW, lipoteichoic acid, and carbohydrate were observed in rats given S. mutans WC and MDP by gastric intubation, whereas oral immunization with S. mutans CW and MDP resulted in higher antibody levels to CW and carbohydrate and lower levels to lipoteichoic acid when compared with the antibody levels in rats given antigen alone. Rats orally immunized with either S. mutans WC or CW and MDP and challenged with virulent S. mutans 6715 exhibited significantly (P less than or equal to 0.05) lower plaque scores, numbers of viable S. mutans in plaque, and caries scores than did rats immunized with antigen alone or in infected-only controls. In another series of experiments, a PG fraction derived from S. mutans 6715 CW was assessed for adjuvant properties. The oral administration of PG and either S. mutans WC or CW induced good salivary and serum IgA antibody responses. The specificity of the antibodies was similar to that obtained in rats given antigen and MDP. Rats receiving either S. mutans WC or CW and PG and challenged with virulent S. mutans 6715 had lower plaque scores, fewer numbers of viable S. mutans in plaque, and lower caries activity than did infected rats receiving S. mutans WC or CW immunogen alone. These results provide evidence that soluble adjuvants derived from the gram-positive bacterial CW, e.g., MDP and PG, are effective oral adjuvants and augment IgA immune responses to particulate S. mutans antigens which are protective against the mucosally associated disease, dental caries.     

Effective immunity to dental caries: gastric intubation of Streptococcus mutans whole cells or cell walls induces protective immunity in gnotobiotic rats.

Gnotobiotic rats were given Streptococcus mutans 6715 whole cells (WC), purified cell walls (CW), or cell wall lysate by gastric intubation (GI), and assessments were made of humoral immune responses in serum and saliva and of caries protection. Levels of secretory immunoglobulin A (IgA) and IgG antibodies to S. mutans WC in saliva samples from experimental rats were determined by an enzyme-linked immunosorbent assay. Serum antibody levels of the IgM, IgG, and IgA isotypes were also determined. Similar levels of salivary antibodies were induced in rats given S. mutans WC or CW by GI, whereas lower salivary antibody titers were observed in rats given cell wall lysate by the oral route. The level of serum antibodies in the various groups of rats also reflected the oral antigen used. The specificity of salivary IgA and serum IgG antibodies in the various groups of rats was determined by enzyme-linked immunosorbent assay with lipoteichoic acid, serotype g carbohydrate, dextran, CW, and WC as coating antigens. Salivary IgA and serum IgG antibodies in rats given S. mutans WC or CW by GI were primarily directed to lipoteichoic acid and serotype g carbohydrate. The presence of salivary IgA antibodies to S. mutans in rats given either S. mutans WC or CW by GI correlated with a significant reduction in the levels of plaque, numbers of viable S. mutans in plaque, and caries scores when compared with the control animals (infected only). These results demonstrate that particulate antigens of S. mutans induce salivary immune responses when given by GI to gnotobiotic rats and that the presence of these antibodies correlates with caries protection.     

Enhancement of Natural Resistance to Influenza Virus in Lipopolysaccharide-Responsive and -Nonresponsive Mice by Propionibacterium acnes

Lipopolysaccharide-responsive C3H/HeN mice were rendered resistant to a mouse-adapted strain of influenza (Aichi, H₃N₂) virus when Propionibacterium acnes was given either intranasally or intraperitoneally several days before virus infection. The time of P. acnes treatment was important since no protection was demonstrated when this agent was given either on the same day as or several days after virus challenge. In contrast, lipopolysaccharide-nonresponsive C3H/HeJ mice were not protected when P. acnes was administered intranasally at any time before infection; however, protection was demonstrated when P. acnes was given by the intraperitoneal route. Depending on the route of inoculation, P. acnes induced several distinctive immunological responses in the lungs of both C3H/HeN and C3H/HeJ mice. Intranasal inoculation was more effective in activating pulmonary macrophages in C3H/HeN than in C3H/HeJ mice. In contrast, intraperitoneal inoculation activated pulmonary natural killer cells in both mouse lines but did not activate pulmonary macrophages.     

IFNγ expression by an attenuated strain of Salmonella enterica serovar Typhimurium improves vaccine efficacy in susceptible TLR4-defective C3H/HeJ mice

C3H/HeJ mice carry a mutated allele of TLR4 gene (TLR4 ^d ) and thus are hyporesponsive to the lethal effects of lipopolysaccharide (LPS). Characteristically, however, the mice are also hypersusceptible to infections, particularly by Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) and are known to be difficult to vaccinate against virulent exposure. This is observed despite the expression of wild-type allele of Nramp1 gene, another important determinant of Salmonella susceptibility. In contrast, C3H/HeN mice (TLR4 ⁿ Nramp1 ⁿ ) express a functional TLR4 protein and are resistant to infection, even by virulent strains of S. typhimurium. In the present study, we describe the immune system-enhancing properties of an attenuated strain of S. typhimurium engineered to express murine IFN-γ. This strain (designated GIDIFN) was able to modulate immune responses following systemic inoculation by upregulating the production of inflammatory mediators (IL-6 and IL-12) and anti-bacterial effector molecules (nitric oxide; NO). Consequently, this led to a more effective control of bacterial proliferation in systemic target organs in both C3H/HeJ and C3H/HeN mice. Although evidence for the enhancement in immune responses could be observed as early as few hours post-inoculation, sustained improvements required 2–3 days to manifest. Vaccination of C3H/HeJ mice with GIDIFN strain, even at low doses, conferred a significantly higher degree of protection against challenge with virulent Salmonella in susceptible C3H/HeJ mice. Our data demonstrate that IFNγ-expressing Salmonella are immunogenic and confer excellent protection against virulent challenge in susceptible C3H/HeJ mice; in addition they may be used as an effective mucosal delivery vectors against virulent infection and for boosting immune responses in immunodeficient hosts.     

Enhancement of in Vitro Immune Responses of Murine Peyer's Patch Cultures by Concanavalin A, Muramyl Dipeptide and Lipopolysaccharide

Immunofluorcscent and esterase staining of Peyer's patch (PP) cell preparations from six mouse strains showed that each strain possessed approximately eqtial numbers of T and B cells but less than 1% esterase-positive cells (macrophages; Mψ). The addition of concanavalin A (Con A) to either lipopolysaccharide (LPS)-responsive C3H/HeN or LPS-non-responsive C3H/Hej PP cultures immunized with sheep erythrocytes (SRBC) resulted in good anti-SRBC plaque-forming cell responses, whereas the addition of Con A to PP cultures from SRBC orally primed C3H/HeJ mice resulted in significantly higher in vitro responses to trinitrophenyl-conjugated SRBC than similarly treated cultures from C3H/HeN mice. On the other hand, muramyl dipeptide (MDP) promoted higher responses in PP cultures derived from either normal or carrier-primed LPS-rcsponsive (C3H/HeN) mice than were observed with C3H/HeJ PP cultures. A similar pattern of responsiveness was seen when LPS prepared by a phenol-water extraction procedure (LPS(Ph)) was added to PP cultures. When LPS prepared by a bntanol-water extraction method was used, PP cultures from both C3H/HeN and C3H/HeJ mice were enhanced; however, the response of C3H/HeN PP cultures was significantly greater than that seen with C3H/HeJ PP cultures. The enhanced responsiveness of C3H/HeN PP cultures to MDP was probably due to an effect on the Mψ, since addition of MDP to either C3H/HeN or C3H/HeJ PP cultures containing C3H/HeN splenic adherent cells resulted in significantly enhanced immune responses. C3H/HeJ splenic Mψ did not promote adjuvant responses. LPS(Ph) augmentation of immune responses required that Mψ (spleen) and PP cultures be derived from LPS-re-sponsive C3H/HeN mice. These results demonstrated that Con A, MDP, and LPS promoted immune responses of PP cultures to the T-dependent antigen, SRBC. Evidence is presented that Con A enhanced T-helper-cell activity, whereas MDP and LPS required the presence of LPS-responsive Mψ for augmentation of immune responses.     

Modified Immunogenicity of a Mucosally Administered Antigen

Streptococcus mutans is present in the saliva of most individuals and is modified by salivary components bound to the cells. These saliva-bound S. mutans are swallowed, exposed to high levels of acidity in the stomach, and presented to the common mucosal immune system. Much effort has been directed to identifying the specific S. mutans antigens that the mucosal immune responses are directed against. However, little is known about the host-altered antigenic determinants that the mucosal immune system recognizes. The immunogenicity of gastrically intubated untreated S. mutans cells, cells coated with whole human saliva, cells treated with HCl (pH 2.0), and saliva-coated and acid-treated cells in mice was investigated. Saliva and serum samples were assayed by enzyme linked immunosorbent assay for immunoglobulin A (IgA) and IgG antibodies, respectively, against the untreated or treated S. mutans cells. In general, the levels of salivary IgA and serum IgG antibodies to the antigen against which the mice were immunized were significantly higher (P ≤ 0.05). In addition, human saliva and serum samples from 12 subjects were assayed for naturally occurring antibody against the untreated or treated S. mutans cells. In every case, significantly higher reactivity was directed against the saliva-coated and acid-treated cells followed by the saliva-coated S. mutans. These results provide evidence for the altered immunogenicity of swallowed S. mutans in humans by coating native S. mutans antigens with salivary components and/or denaturing surface S. mutans antigens in the acidic environment of the stomach, which would lead to an immune response to modified S. mutans determinants and not to native S. mutans antigens.     

Th1-type immune responses by Toll-like receptor 4 signaling are required for the development of myocarditis in mice with BCG-induced myocarditis

The immunological aspects of autoimmune myocarditis are difficult to understand because of the existence of many infectious agents and animal models suggesting different mechanisms in autoimmune myocarditis. To overcome these difficulties, two strains of mice, C3H/HeN and C3H/HeJ, showing different immune responses to mycobacteria, were immunized with myosin mixed with BCG. The C3H/HeN mice with a wild-type Toll-like receptor 4 (TLR4) showed severe myocarditis, whereas the C3H/HeJ mice with nonfunctional mutated TLR4 did not. CD4(+) cells from both strains of mice exhibited appreciable proliferative responses following myosin stimulation; however, the cytokines from these cells differed between these two strains. The C3H/HeN mice showed T helper (Th)1-type cytokine responses, whereas the expressions of mRNA in C3H/HeJ mice were Th2-type cytokine. When both of these strains of immunized mice were inoculated with a plasmid encoding cDNA of interleukin (IL)-4 or agonistic IL-4, the development of myocarditis was inhibited in C3H/HeN mice. Moreover, C3H/HeJ mice, in which development of myocarditis was not induced by immunization of myosin mixed with BCG, showed myocarditis after injection of IL-4 antagonistic mutant DNA for the induction of Th1-type immune responses. The results suggested that the induction of autoimmune myocarditis by myosin is affected by Th1-type immune responses.     

Immunogenicity and In Vitro and In Vivo Protective Effects of Antibodies Targeting a Recombinant Form of the Streptococcus mutans P1 Surface Protein

Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1_39–512), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the present study, we further investigated the immunological features of P1_39–512 in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1_39–512 preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1_39–512-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1_39–512 antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1_39–512 as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1.     

BCG-induced enhancement of endotoxin sensitivity in C3H/HeJ mice. II. T cell modulation of macrophage sensitivity to LPS in vitro

BCG infection induces a marked increase in LPS sensitivity in vivo and will render genetically defective, LPS hyporesponsive, C3H/HeJ mice almost as sensitive to LPS as normal mice. In this study, we have examined the endotoxin sensitivity of lymphocytes and macrophages from BCG infected mice in order to determine the cellular basis of this effect. We have found that the alteration in endotoxin sensitivity is mediated by a primary effect of BCG infection on T lymphocytes rather than on macrophages. Macrophages from «LPS sensitive», BCG-infected C3H/HeJ mice remain unresponsive to LPS when tested in vitro. However, when peritoneal T lymphocytes from these LPS »corrected» mice were cocultured with LPS unresponsive C3H/HeJ macrophages, a conversion to the LPS-responsive state was observed as manifested by the ability of the macrophages to produce LAF (IL 1) upon LPS stimulation. T cells from normally LPS-responsive or BCG-infected C3H/HeN mice, but not from control C3H/HeJ mice, were also able to render C3H/HeJ macrophages sensitive to LPS. This activity was not affected by treatment of the column-purified T cells with anti-macrophage serum plus complement, indicating that the response was not due to residual LPS-responsive macrophages contaminating the T cell preparations. The ability of the T cell suspension to render C3H/HeJ macrophages capable of producing LAF (IL 1) in response to LPS was abrogated by treatment of the T cell preparations with anti-Thy 1.2 plus complement. These findings establish the importance of T lymphocytes in regulating the LPS sensitivity of macrophages in BCG infected C3H/HeJ mice and support the concept that macrophage LPS responsiveness is dependent upon a certain state of macrophage activation which is regulated by lymphocytes.     

Lipopolysaccharides (LPS) of oral black-pigmented bacteria induce tumor necrosis factor production by LPS-refractory C3H/HeJ macrophages in a way different from that of Salmonella LPS

Some lipopolysaccharide (LPS) preparations from S- or R-form members of the family Enterobacteriaceae and oral black-pigmented bacteria (Porphyromonas gingivalis and Prevotella intermedia) are known to activate LPS-refractory C3H/HeJ macrophages. When contaminating proteins are removed from R-form LPS of Enterobacteriaceae by repurification, however, this ability is lost. In the present study, we investigated the capacity of LPS from P. gingivalis, P. intermedia, Salmonella minnesota, and Salmonella abortusequi to induce production of tumor necrosis factor (TNF) in gamma interferon-primed C3H/HeJ macrophages before and after repurification. P. abortusequi S-LPS was fractionated by centrifugal partition chromatography into two LPS forms: SL-LPS, having homologous long O-polysaccharide chains, and SS-LPS having short oligosaccharide chains. Prior to repurification, all LPS forms except SL-LPS induced TNF production in both C3H/HeJ and C3H/HeN macrophages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that repurification removed contaminating protein from the preparations, and repurified SS-LPS and S. minnesota Ra-LPS no longer stimulated TNF production in C3H/HeJ macrophages, although C3H/HeN macrophages remained responsive. In contrast, repurified oral bacterial LPS retained the capacity to induce TNF production in C3H/HeJ macrophages. Oral bacterial LPS preparations also were not antagonized by excess inactive, repurified SL-LPS; Ra-LPS; Rhodobacter sphaeroides lipid A, a competitive LPS antagonist, or paclitaxel, an LPS agonist, and they were comparatively resistant to polymyxin B treatment. Nevertheless, oral bacterial LPS was less toxic to D-galactosamine-treated C3H/HeN mice than was LPS from Salmonella. These findings indicate that the active molecule(s) and mode of action of LPS from P. gingivalis and P. intermedia are quite different from those of LPS from Salmonella.     

Intranasal Immunization against Dental Caries with a Streptococcus mutans-Enriched Fimbrial Preparation

Streptococcus mutans has been identified as the major etiological agent of human dental caries. The first step in the initiation of infection by this pathogenic bacterium is its attachment (i.e., through bacterial surface proteins such as glucosyltransferases, P1, glucan-binding proteins, and fimbriae) to a suitable receptor. It is hypothesized that a mucosal vaccine against a combination of S. mutans surface proteins would protect against dental caries by inducing specific salivary immunoglobulin A (IgA) antibodies which may reduce bacterial pathogenesis and adhesion to the tooth surface by affecting several adhesins simultaneously. Conventional Sprague-Dawley rats, infected with S. mutans at 18 to 20 days of age, were intranasally immunized with a mixture of S. mutans surface proteins, enriched for fimbriae and conjugated with cholera toxin B subunit (CTB) plus free cholera toxin (CT) at 13, 15, 22, 29, and 36 days of age (group A). Control rats were either not immunized (group B) or immunized with adjuvant alone (CTB and CT [group C]). At the termination of the study (when rats were 46 days of age), immunized animals (group A) had significantly (P < 0.05) higher salivary IgA and serum IgG antibody responses to the mixture of surface proteins and to whole bacterial cells than did the other two groups (B and C). No significant differences were found in the average numbers of recovered S. mutans cells among groups. However, statistically fewer smooth-surface enamel lesions (buccal and lingual) were detected in the immunized group than in the two other groups. Therefore, a mixture of S. mutans surface proteins, enriched with fimbria components, appears to be a promising immunogen candidate for a mucosal vaccine against dental caries.     

Formation of interferon-gamma and tumor necrosis factor in mice during Salmonella typhimurium infection

Formation of interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) during Salmonella typhimurium infection was investigated in lipopolysaccharide (LPS)-sensitive C3H/HeN and C57BL/10ScSn(B10ScSn), and LPS-resistant (lpsd mutant) C3H/HeJ and C57BL/10ScCr(B10ScCr) mice. When infected with 50 colony-forming units (CFU) of S. typhimurium C5, C3H/HeN and B10ScSn mice became hypersensitive to the lethal effect of LPS. In the case of lpsd mutants, only C3H/HeJ mice became hypersensitive to LPS, while B10ScCr mice remained resistant. C3H/HeJ as well as B10ScSn mice produced significant amounts of plasma IFN-gamma on day 3 after infection. By this time bacterial CFU in the liver of B10ScSn and C3H/HeJ mice were 10(6.7) and 10(7.1), respectively. In B10ScCr mice, however, IFN-gamma was not detectable although bacteria present in the liver exceeded 10(8) CFU. On the other hand, plasma TNF was not detectable in any of the mouse strains during S. typhimurium infection. When S. typhimurium-infected mice were challenged with LPS on day 3, significant amounts of plasma TNF were measured in C3H/HeN and B10ScSn mice, while in the lpsd mutant C3H/HeJ and B10ScCr mice plasma TNF was undetectable.     

Comparative effects of particulate and soluble glucan on macrophages of C3H/HeN and C3H/HeJ mice.

In order to compare both the actions of soluble glucan (glucan-F) and particulate glucan (glucan-P) on macrophages and the responsiveness of macrophages from C3H/HeJ and C3H/HeN mice to these immunomodulators, interleukin-1 (IL-1) levels, phagocytosis and superoxide production were monitored after an in vitro exposure to glucan-F or glucan-P. A 2 or 20 h exposure to either glucan preparation decreased the ability of both C3H/HeJ and C3H/HeN macrophages to ingest zymosan. In contrast, glucan-P, but not glucan-F, decreased (after a 20 h exposure) the uptake of both IgG opsonized erythrocytes and latex beads. Furthermore, glucan-P, but not glucan-F was as effective as zymosan (after a 1 h exposure) in inducing superoxide release by macrophages isolated from both C3H/HeN and C3H/HeJ mice. While the effects of glucan-P on PMA-induced superoxide release and IL-1 levels were similar in macrophages from C3H/HeJ and C3H/HeN mice, glucan-F was ineffective at enhancing PMA-induced superoxide release or increasing IL-1 levels in C3H/HeJ mice. Thus (1) the effects of glucan-P on phagocytosis of opsonized erythrocytes and latex beads are not mimicked by glucan-F and (2) while macrophages from C3H/HeJ mice respond normally (as compared with C3H/HeN macrophages) to glucan-P, they are hyporesponders to glucan-F. These findings indicate that the activation of macrophages by glucan-P involves different (or additional) pathways from those activated by glucan-F.     

Mechanism of lipopolysaccharide-induced tumor necrosis: requirement for lipopolysaccharide-sensitive lymphoreticular cells.

Lipopolysaccharide (LPS) induces rapid necrosis of intradermal fibrosarcomas in mice. The mechanism(s) by which LPS produces tumor necrosis has been investigated using histocompatible LPS-sensitive (C3H/HeN) and LPS-resistant (C3H/HeJ) mouse strains. C3H/HeN- or C3H/HeJ-derived fibrosarcomas were necrotized by LPS when they were grafted onto C3H/HeN mice but were not affected when growing on C3H/HeJ mice, indicating that LPS does not act directly on the tumor itself. In contrast, lethally X-irradiated C3H/HeJ mice exhibit necrosis of their tumors when reconstituted with C3H/HeN bone marrow cells, whereas C3H/HeN mice no longer exert LPS-induced tumor necrosis after the adoptive transfer of C3H/HeJ bone marrow cells. These findings clearly indicate that LPS produces necrosis of tumors by activating host lymphoreticular cells.     

Lipopolysaccharide-Mediated Induction of Calprotectin in the Submandibular and Parotid Glands of Mice

S100A8 and S100A9 constitute a heterodimeric protein, calprotectin. The mRNAs of S100A8 and S100A9, being expressed at minimal levels in the submandibular and parotid glands (SMG and PG, respectively) of C3H/HeN mice, were induced strongly and transiently by lipopolysaccharide (LPS). Among the mRNAs of members of the S100 protein family examined, those of S100A8 and S100A9 were specifically induced by LPS in the salivary glands. The induction was assumed to be mediated via toll-like receptor 4 (TLR4), since their elevation was limited in C3H/HeJ mice, a TLR4-mutant strain. These proteins became expressed in the granular convoluted tubular cells and striated duct cells in the SMG, and in both acinar and duct cells in the PG (all in the cytoplasm). The salivary calprotectin level was not increased by LPS treatment, implying that elevated calprotectin was not secreted into the saliva and that they may function in microcellular environment of the salivary gland.     

Monoclonal antibodies to salmonella lipopolysaccharide: functional analysis of anti-lipid A antibodies.

We have produced monoclonal antibodies (MoAb) to the Rb core and lipid A regions of Salmonella lipopolysaccharide (LPS) and have assessed their ability to inhibit LPS-mediated mitogenic responses in vitro, and to protect against LPS toxicity and lethal Salmonella infection in vivo. Monoclonal antibodies RC-8 and RC-16 were specific for LPS Rb core determinants, and MoAb LA-1, LA-2, LA-3, LA-4 and LA-5 were specific for lipid A. Anti-lipid A MoAb LA-2, LA-3 and LA-5 were found to abrogate mitogenic responses of C3H/HeN spleen cells to smooth S. typhimurium LPS (S LPS) and to rough S. minnesota R595 LPS (Re LPS). Monoclonal antibody LA-5 was effective in extending the median length of survival of C3H/HeN mice challenged with a lethal dose of either S LPS or Re LPS. Antibody LA-2 could extend the median length of survival of C3H/HeJ mice challenged with Re LPS but not with S LPS, and failed to extend significantly the length of survival of S LPS-challenged C3H/HeN and DBA/2 mice. Neither 20 micrograms of anti-Rb core or anti-lipid A MoAb nor 200 micrograms of anti-lipid A MoAb were able to protect C3H/HeN or BALB/c mice, respectively, against lethal infection with S. typhimurium SR-11. These results suggest that the importance of anti-lipid A antibodies in host defence may lie more in their ability to neutralize pathological effects of LPS, than in their ability to protect against bacterial infection.     

Defective prostaglandin synthesis by C3H/HeJ mouse macrophages stimulated with endotoxin preparations.

Macrophages obtained from C3H/HeN mice produced significant amounts of prostaglandin E when exposed to phenol-extracted lipopolysaccharides (LPS), whereas macrophages from C3H/HeJ mice were unresponsive. The lipid A fraction from phenol-extracted LPS was an effective inducer or prostaglandin synthesis by macrophages from C3H/HeN mice. The polysaccharide portion of the LPS molecule had no effect. In contrast, the C3H/HeJ macrophages did not produce prostaglandin E in response to the lipid A moiety of phenol-extracted LPS. LPS prepared by butanol extraction stimulated the production of prostaglandin E by macrophages from both C3H/HeN and C3H/HeJ mice. The component of butanol-extracted LPS that stimulated the C3H/HeJ macrophages was shown to be a lipid A-associated protein. Further studies demonstrated a correlation between prostaglandin production by the macrophages of these two strains of mice in response to butanol- and phenol-extracted LPS and the lethal effects of the endotoxin preparations.     

Protective Roles of γδ T Cells and Interleukin-15 in Escherichia coli Infection in Mice

The number of γδ T cells in the peritoneal cavity was increased after an intraperitoneal (i.p.) infection with Escherichia coli in lipopolysaccharide (LPS)-responsive C3H/HeN mice but not in LPS-hyporesponsive C3H/HeJ mice. The γδ T cells preferentially expressed invariant Vγ6 and Vδ1 chains and proliferated to produce a large amount of gamma interferon in the presence of LPS. Mice depleted of γδ T cells by T-cell receptor δ gene mutation showed impaired resistance against E. coli as assessed by bacterial growth. Macrophages from C3H/HeN mice infected with E. coli expressed higher levels of interleukin-15 (IL-15) mRNA than those from the infected C3H/HeJ mice. Administration of anti-IL-15 monoclonal antibody inhibited, albeit partially, the appearance of γδ T cells in C3H/HeN mice after E. coli infection and diminished the host defense against the infection. These results suggest that LPS-stimulated γδ T cells play an important role in the host defense against E. coli infection and that IL-15 may be partly involved in the protection via an increase in the γδ T cells.