Stimulation of the Synthesis of Histamine and Putrescine in Mice by a Peptidoglycan of Gram-Positive Bacteria

To clarify the base of in vivo biological activities of peptidoglycans of Gram-positive bacteria, the effects of a polysaccharide peptide of Staphylococcus epidermidis peptidoglycan (SEPS) on the synthesis of histamine and putrescine in BALB/c mice were examined and compared with those of a lipopolysaccharide (LPS or endotoxin) of Gram-negative bacteria. Within a few hours after its injection into BALB/c mice, SEPS induced histidine decarboxylase (HDC), the enzyme forming histamine, in the liver, lung, spleen and bone marrow, and ornithine decarboxylase (ODC), the enzyme forming putrescine, in the tissues except for the lung. SEPS induced HDC activity even in mast cell-deficient mice and in nude mice. These effects of SEPS were essentially the same as those of LPS. However, the dosage of SEPS capable of inducing HDC and ODC was much higher (100 to 1,000 times) than that of LPS. We have reported that C3H/HeN mice are resistant to SEPS in producing acute arthritis, and their productions of IL-1 and prostaglandin E2 are less than BALB/c mice sensitive to producing acute arthritis. In the present study, it was also found that C3H/HeN mice were markedly resistant to SEPS in inducing HDC activity.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

Experimental Dental Caries on Gnotobiotic Inbred Mice

The purpose of this study in mono-infected gnotobiotic BALB/cA and C3H/HeN mice was to evaluate the cariogenicity of Enterococcus faecalis. The caries incidence and mean caries score in the BALB/cA mice were significantly higher than those in the C3H/HeN. In both of the mouse strains, the mean number of E. faecalis isolated from the cecum content was almost the same, however, the mean number of E. faecalis from the maxilla of BALB/cA was significantly higher than that of C3H/HeN. These results indicate that C3H/HeN has some factors that prevent E. faecalis from attaching to the tooth surfaces.     

Levels of Endogenous lnterleukin-1, Interleukin-6, and Tumor Necrosis Factor in Congenic Mice Infected with Borrelia garinii

This study describes the levels of interleukin-1 alpha (IL-1α), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) in the sera and parenchymal organs of various congenic mouse strains infected with Borrelia garinii. A significant elevation of inflammatory cytokine levels was found in the organs of C3H/HeN (H-2^k) and B10.BR (H-2^k) mice but not in those of BALB/c mice (H-2^d). Focally produced cytokines can contribute to antimicrobial defense against these organisms. High levels of IL-1α were observed in the sera of C3H/HeN, B10.BR and B10 (H-2^b) mice infected with B. garinii and they were associated with the presence of spirochetes in the skin. Thus, susceptible mice demonstrated a stronger cytokine response than resistant mice. This study presents in vivo evidence that B. garinii infection affects the immunopathogenesis of Lyme disease.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.     

Lipopolysaccharide-induced suppression of antibody-directed cell-mediated cytotoxicity to chicken red blood cells

Prior treatment with commercially prepared and acetone-extracted lipopolysaccharide (LPS) was found to suppress the expression of antibody-directed, cell-mediated cytotoxicity (ADCC) to chicken red blood cells (CRBC) by spleen cells from C57/BL10, C3H, and BALB/c mice. The in vitro incubation with commercial LPS suppressed ADCC-CRBC activity of spleen cells from both C3H/HeJ and C3H/HeN mice. Only the C3H/HeN strain was suppressed when treated with purified LPS. ADCC-CRBC activity of neonatal spleen cells could be suppressed after a 3-hr in vivo incubation with LPS while adult spleen cells required a minimum of 15 hr preincubation.     

Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice

PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-gamma production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-gamma production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-gamma production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-gamma production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-gamma and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.     

Biological significance of elevated TNF levels: In vivo administration of monoclonal antibody against TNF following haemorrhage shock increases the capacity of macrophages to release TNF while restoring immuno-responsiveness

Haemorrhagic shock results in a severe depression of the cellular and humoral immunity, thus rendering the host increasingly susceptible to sepsis. To study the effect of elevated TNF release following haemorrhagic shock on depressed macrophage and splenocyte functions, C3H/HeN mice were pretreated intraperitoneally with either anti-murine TNF-Ab or saline. Twenty hrs later, mice were bled to and maintained at a mean BP of 35 mmHg for 60 min followed by adequate fluid resuscitation. Pretreatment with anti-TNF-Ab completely neutralized elevated TNF plasma levels following haemorrhage. This was associated with an increased (P < 0.05) capacity of pMø isolated 24 h after haemorrhagic shock to release TNF, while the ability to secrete IL-6 and PGE₂ was reduced. Haemorrhagic shock-induced suppression of pMø antigen presentation capacity and MHC class II antigen expression, as well as depression of splenocyte proliferation and lymphokine production was also attenuated (P < 0.05) by anti-TNF-Ab pretreatment. These data indicate that elevated circulating TNF levels play a pivotal role in the depression of essential macrophage and splenocyte functions following haemorrhagic shock.     

Chemokine and cytokine production in susceptible C3H/HeN mice and resistant BALB/c mice during Orientia tsutsugamushi infection

To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production in susceptible (C3H/HeN) and resistant (BALB/c) mice after infection with O. tsutsugamushi Gilliam. C3H/HeN mice produced high levels of chemokines macrophage inflammatory proteins 1 alpha (MIP-1 alpha ), MIP-2, monocyte chemoattractant protein 1 (MCP-1), and cytokines gamma-interferon (IFN-gamma ), interleukin-12 (IL-12), IL-10, and tumor necrosis factor alpha (TNF-alpha ) in response to O. tsutsugamushi infection, compared to BALB/c mice. Chemokine profiles in infected mice correlated well with the kinetics of inflammatory cell infiltration. Hyperproduction of chemokines and cytokines was observed in another susceptible-infection model (BALB/c-Karp). These results suggest that hyperproduction of chemokines and cytokines are associated with susceptibility during O. tsutsugamushi infection.     

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.     

Comparative Analyses of Thymocyte and Thymic Low-Density Adherent Cell Functions

Thymocytes which have developed in the C3H thymus showed depressed proliferative responses to stimulation with anti-CD3 antibody as compared with those which have developed in the thymus of other strains of mice (i.e. AKR). The present study was conducted to analyze immunological functions of the thymic stromal cell population (low-density adherent cells, LDAC) in the C3H mice using allogeneic bone marrow (BM) chimeras established by BM transplantation in the reciprocal combination of AKR and C3H mice as donor or recipient. The thymic LDAC from C3H mice or the [AKR(donor)→C3H(recipient)] chimeras contained a high proportion of Mac-1⁺ cells as compared to AKR mice or the [C3H→AKR] chimeras. The proportion of Mac-1⁺ cells paralleled the IL-1- and PGE₂-secreting ability of the LDAC cultured either in the presence or absence of LPS and also paralleled the antigen-presenting cell functions of the LDAC. Furthermore, after anti-CD3 stimulation the PGE₂ inhibited more profoundly proliferative responses of [AKR→C3H] or normal C3H thymocytes than those of the [C3H→AKR] chimera or normal AKR thymocytes. A PGE₂ inhibitor, indomethacin, reversed the depressed responses of the thymocytes which had developed in the C3H thymus. These findings suggest that the lower responsiveness of thymocytes from [AKR→C3H] chimeras to anti-CD3 stimulation may be attributable to large amounts of PGE₂ secreted by LDAC and/or to increased sensitivity of thymocytes themselves to PGE₂.     

Pyridoxine deficiency and cytotoxicity of T lymphocytes in vitro.

The effect of pyridoxine deficiency on the proliferation and cytotoxicity of BALB/c mouse lymphocytes stimulated in vitro with irradiated spleen cells from C3H mice was studied. Cytotoxicity was measured by Na⁵¹CrO₄ release from L cells which have the same histocompatibility loci as C3H mouse cells. Pyridoxal-5′-phosphate (PLP) content in the spleen and liver of pyridoxine-deficient animals was determined with Escherichia coli B/1 t7A apotryptophanase. Maintenance of animals on a pyridoxine-deficient diet for 1 to 3 weeks affected neither proliferation of lymphocytes in vitro nor their cytotoxicity. Lymphocytes from mice fed a pyridoxine-deficient diet for 5 to 6 weeks had a reduced capacity to respond to foreign lymphoid cells in vitro. The Cytotoxicity of these lymphocytes was also significantly decreased. PLP, but not pyridoxal, added directly to the medium in vitro partially restored the impaired functions of T lymphocytes.     

Administration of monoclonal anti-IFN-gamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major

C3H/HeN mice that are naturally resistant to cutaneous disease and systemic infections with the protozoan parasite, Leishmania major, were treated at the time of infection, and weekly thereafter, with mouse anti-rat IFN-gamma mAb or an irrelevant antibody of similar isotype. Anti-IFN-gamma-treated mice developed cutaneous lesions; parasites spread to the regional lymph nodes and then metastasized to spleens and livers. The course of disease in these animals was similar to that of genetically susceptible BALB/c mice. Two exceptions in the pathology of L. major infections were noted between BALB/c and anti-IFN-gamma-treated C3H/HeN mice: 1) BALB/c mice died of systemic complications, whereas C3H/HeN mice did not, and 2) multinucleated giant cells were observed in lymph nodes and spleens of infected BALB/c mice, whereas these cells were not observed in infected C3H/HeN mice. Control mice, those treated with either saline or irrelevant antibody of the same isotype as the anti-IFN-gamma monoclonal, showed no evidence of cutaneous disease (development of footpad lesions) or systemic infection (by histopathology). Total abrogation of the natural resistance of C3H/HeN mice could be achieved by treatment with as little as 0.5 mg/mouse/wk of anti-IFN-gamma antibody, or by a single dose of 1 mg/mouse anti-IFN-gamma antibody administered at the time of parasite inoculation. If antibody treatment was delayed for as little as 1 wk after parasite inoculation, the infections in treated animals resembled that of untreated or control antibody-treated mice: no cutaneous lesions (by footpad swelling or viable counts of leishmania in footpad tissue) or systemic disease (by microscopic analysis of touch preparations of internal organs, and histopathology of same). The production of IFN-gamma during the initial interaction of the parasite and host cells appears to be a major component of genetic control of natural resistance to infection with L. major in C3H/HeN mice.     

Mechanisms of killing of measles virus-infected cells by human lymphocytes: interferon associated and unassociated cell-mediated cytotoxicity

The recent interest in natural killer (NK) cells in immunosurveillance and the ability of infection with certain organisms to modulate NK activity led us to examine the influence of Toxoplasma gondii infection on mouse NK cells. Infection of BALB/c mice with 5 × 10³ virulent Toxoplasma intraperitoneally (ip) resulted in significantly enhanced NK activity in peritoneal exudate cells (PC) and in spleen cells (SC). Intravenous (iv) and subcutaneous (sc) challenge of BALB/c mice with Toxoplasma also resulted in enhanced natural killer (NK) activity in PC and SC. In BALB/c mice, as well as in other strains (A/J, C57BL/6, C3H/HeJ, CeH/HeN, [A/J × C3H]F₁), peak augmentation of PC and SC NK activity was observed 3 days following ip Toxoplasma challenge. Administration of silica to mice abolished Toxoplasma-induced NK cytotoxicity. BALB/c mice chronically infected with Toxoplasma had significantly higher endogenous NK activity than did controls in PC but not in SC. Chronically infected BALB/c mice boosted with virulent Toxoplasma ip exhibited significantly enhanced NK activity in PC but not in SC. Thus, acute and chronic infection with Toxoplasma modulates NK activity in addition to macrophage activation and thereby provides a system that should facilitate study of the relative contribution of NK cells and activated macrophages in resistance to tumor growth and spread.     

Leishmania major: Secreted antigens of Leishmania major promastigotes shift the immune response of the C57BL/6 mice toward Th2 in vitro

BALB/c mice are sensitive to Leishmaniamajor infection, while C57BL/6 mice are resistant and able to mount an effective immune response against the parasite. Since the secreted antigens of L. major suppress the proliferation of BALB/c mice lymphocytes in vitro, we analyzed their effects on the immune system of resistant C57BL/6 mice. Secreted antigens were semi-purified and two fractions with immunosuppressive activity were isolated. 15 μg/ml of fraction could suppress 60% of lymphocyte proliferation and prevent the stimulated lymphocytes entering from G1 phase into the S phase of the cell cycle. These fractions decreased the production of IFN-γ, increased IL-4 level in the lymphocyte culture and down-regulated the nitric oxide production by activated macrophages. These results may suggest that L. major parasite by secreting immunosuppressive factors could down-regulate the immune system of both sensitive and resistant mice for own survival advantage.     

Susceptibility of several strains of mice to Echinostoma hortense infection

Susceptibilities of 5 different mice strains, including C3H/HeN, BALB/c, C57BL6, FvB and ICR, to Echinostoma hortense infection, was evaluated. The worm expulsion rate, worm size and egg production were observed from 1 to 8 weeks after infection with 30 metacercariae. C3H/HeN and ICR mice showed the highest worm maturation rates. The worm recovery rate and the number of eggs per gram (EPG) of feces was also higher in C3H/HeN and ICR mice than in BALB/c, C57BL6, and FvB mice. It is suggested that E. hortense is highly infectious to ICR and C3H/HeN mice, but not to the other strains of mice. Based on the results obtained, we believe that the susceptibility of different mouse strains to E. hortense infection is dependent on the genetic and immunologic background of mice.     

A 38-kDa Antigen of Mycobacterium tuberculosis Predominantly Induces the Secretion of Interleukin-2, Interferon-gamma and IgG2a Antibodies

In the present study, mice of 3 different haplotypes (H-2d, H-2k and H-2b) were sensitized subcutaneously with heat-killed H37Ra or 38-kDa antigen of Mycobacterium tuberculosis. Lymphocytes obtained from immunized animals were challenged in vitro with 38-kDa antigen in both cases. The dominant pattern of Th1-like lymphokines (IL-2 and IFN-gamma) and preferential production of 38-kDa specific IgG2a-type antibody were observed. It was noted that 38-kDa antigen was recognized permissively by all 3 strains of mice used in the present study. It was interesting to note that C3H/HeJ mice, which express BCG-resistant alleles showed a higher level of proliferative as well as cytokine response as compared to BALB/c and C57BL/6 mice, which bear BCG-susceptible alleles. These results suggest that not only in recall responses but also during the induction as well as expression phase of the immune response mediated by 38-kDa antigen of M. tuberculosis the Th1-like immune response predominates.     

Regulatory effects of Spirulina complex polysaccharides on growth of murine RSV‐M glioma cells through Toll‐like receptor 4

This study is the first to report that Spirulina complex polysaccharides (CPS) suppress glioma growth by down‐regulating angiogenesis via a Toll‐like receptor 4 signal. Murine RSV‐M glioma cells were implanted s.c. into C3H/HeN mice and TLR4 mutant C3H/HeJ mice. Treatment with either Spirulina CPS or Escherichia coli (E. coli) lipopolysaccharides (LPS) strongly suppressed RSV‐M glioma cell growth in C3H/HeN, but not C3H/HeJ, mice. Glioma cells stimulated production of interleukin (IL)‐17 in both C3H/HeN and C3H/HeJ tumor‐bearing mice. Treatment with E. coli LPS induced much greater IL‐17 production in tumor‐bearing C3H/HeN mice than in tumor‐bearing C3H/HeJ mice. In C3H/HeN mice, treatment with Spirulina CPS suppressed growth of re‐transplanted glioma; however, treatment with E. coli LPS did not, suggesting that Spirulina CPS enhance the immune response. Administration of anti‐cluster of differentiation (CD)8, anti‐CD4, anti‐CD8 antibodies, and anti‐asialo GM1 antibodies enhanced tumor growth, suggesting that T cells and natural killer cells or macrophages are involved in suppression of tumor growth by Spirulina CPS. Although anti‐interferon‐γ antibodies had no effect on glioma cell growth, anti‐IL‐17 antibodies administered four days after tumor transplantation suppressed growth similarly to treatment with Spirulina CPS. Less angiogenesis was observed in gliomas from Spirulina CPS‐treated mice than in those from saline‐ or E. coli LPS‐treated mice. These findings suggest that, in C3H/HeN mice, Spirulina CPS antagonize glioma cell growth by down‐regulating angiogenesis, and that this down‐regulation is mediated in part by regulating IL‐17 production.     

Inflammatory and Phagocytic Response to Experimental Campylobacter jejuni Infection in Mice

After intraperitoneal inoculation with Campylobacter jejuni BALB/c, Swiss and DBA mice show a peritoneal inflammatory response of different intensity. Only BALB/c mice have a strong peritoneal response. Simultaneous intraperitoneal inoculation of C. jejuni plus FeCl₃ increase both inflammatory response and phagocytic activity in Swiss mice, without production of diarrhea. Some thermostable compounds of C. jejuni have a very strong chemotactic activity against peritoneal cells of mice, whereas a diffusible, thermolabile and glutaraldehyde-resistant factor has an inhibitory effect over murine peritoneal cell phagocytosis. Bactericidal activity of peritoneal cells increased after in vitro re-challenge with C. jejuni. Bacteremia is present in all the mice strains tested, but the clearance is quick in DBA and slow in BALB/c and Swiss mice. These experiments confirm that in mice, peritoneal non-specific mechanisms of defense, such as macrophages, play an important role in order to control C. jejuni infection.