Proliferation of erythromycin-stimulated mouse peritoneal macrophages in the absence of exogenous growth factors.

Erythromycin (0.2-20 micrograms/ml) induced the proliferation of macrophages of mouse peritoneal exudate cells (PEC) in a liquid medium without exogenous growth factors. The proliferating macrophages formed giant colonies between days 22 and 26 of culture; these colonies continued to proliferate even after subculture. The erythromycin-induced cell proliferation was independent of fibroblasts, T cells, B cells, or endotoxins. This activity seemed to be specific to erythromycin since other antibiotics such as tetracycline, streptomycin, gentamicin, penicillin G, and josamycin did not induce the proliferation of macrophages. Any known cytokines, including IL-2, IL-3, IL-4, IL-6, and GM-CSF, were not detectable by ELISA tests in any of the culture supernatants sampled from day 7 through day 28. The culture supernatants, however, had the capability of inducing the growth of macrophages, only in the presence of bioactive erythromycin at concentrations higher than 1.6 micrograms/l. Moreover, the culture supernatants, sampled after giant colonies had been formed, were capable of inducing giant colonies in the culture of adherent PEC. Thus, the erythromycin-induced macrophage proliferation might be due to the direct effect of this antibiotic, whereas the formation of giant colonies might be due to the production of some unidentified soluble factor produced by the proliferating macrophages. These data indicate that mouse PEC contain a subset of peritoneal macrophages capable of responding to erythromycin by forming proliferating colonies without exogenous growth factors.

Mechanism of the protective immunity against murine typhoid: persistence of Salmonella L forms in the liver after immunization with live-cell vaccines.

Live-cell vaccines of Salmonella typhimurium, either a sub-lethal dose of a wild-type (strain LT2) or a high dose of its two-heptose Rd1 mutant (strain SL1004), induced acquired resistance to murine typhoid, which remained 180 days after immunization. Growth of S. typhimurium as a bacillary form ceased between days 30 and 60 of immunization, but L forms of this bacterium colonized the liver (the mean number of L forms in the liver: 600 L-forming units) even at 180 days post-immunization. In contrast, a high inoculum of either a Ra mutant (strain TV148) of strain LT2 or S. schottmülleri 8006 sharing the same O antigenic components with those of S. typhimurium induced only a short-lived protection in proportion to the number of L forms in the liver, and the protective immunity was lost before day 180. However, there was no significant difference in the salmonella-specific T-cell responses among groups of immunized mice on day 180 of immunization. A lethal infection with strain LT2 in mice which had been immunized 75 days previously with living cells of strain SL1004 resulted in a rapid clearance of the challenge inoculum, together with a rapid elevation of anti-S. typhimurium antibody responses. Thus, the present data suggest that the long-lived immunity conferred upon live S. typhimurium vaccines is attributable to the colonization of this bacterium in the liver as L forms and the ability to colonize the liver as L forms is independent of the chain length of salmonella O-antigens.

Contribution of interferon gamma and membrane-associated interleukin 1 to the resistance to murine typhoid of Ityr mice.

Resistance of mice to Salmonella typhimurium in the early phase of infection is known to be controlled by the expression of chromosome 1 locus Ity. To clarify the mechanism by which the genetically resistant (Ityr) mice can overcome the first phase of salmonellosis, the early response in DBA/2 (Ityr) and BALB/c (Itys) mice was compared after a subcutaneous injection of S. typhimurium. In both strains, the growth of S. typhimurium was controlled in livers and Kupffer cells until day 3, but thereafter the bacteria multiplied rapidly in BALB/c mice. Over the first 2 days nonspecific responses (changes in levels of blood leukocytes, plasma iron, and alpha 1-antitrypsin) were not significantly different between the strains, and the capacity of Kupffer cells isolated from infected mice of both strains to produce interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) was of the same degree. Thereafter, only DBA/2 Kupffer cells were able to produce membrane-associated IL-1 (ma IL-1) as well as TNF-alpha. Moreover, only DBA/2 splenocytes were able to produce interferon gamma (IFN-gamma) upon stimulation with Salmonella antigens, although concanavalin A-stimulated splenocytes of both strains produced the same level of interleukin 2. Furthermore, administration of recombinant murine IFN-gamma and DBA/2 Kupffer cells of day 6 to BALB/c mice 3 days after infection resulted in a significant level of protection, whereas neither of these materials alone induced protection. Injection of anti-TNF-alpha antibodies did not affect the resistance of DBA/2 mice. Thus, these findings suggest that the early resistance of Ityr mice is partly attributable to their capacity to produce IFN-gamma and ma IL-1 after infection.

Nonspecific stimulation of host defense by Corynebacterium kutscheri. III. Enhanced cytokine induction by the active moiety of C. kutscheri.

The present study was carried out to ascertain whether the active component of Corynebacterium kutscheri (CK-M) could stimulate host cells of mice to produce several cytokines. CK-M stimulated thioglycollate-induced peritoneal macrophages to produce interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) at concentrations of 1-100 ng/ml, and it also induced IL-2 and interferon-gamma (IFN-gamma) as well as IL-6 production by splenocytes. Maximum production of each cytokine induced by CK-M was obtained at the following doses: IL-1 at 5 ng/ml, TNF-alpha at 50 ng/ml, IL-2 at 1 microgram/ml, IL-6 at 500 ng/ml and IFN-gamma at 750 ng/ml. In contrast, IL-4 was not produced to a significant extent by CK-M-stimulated splenocytes. Furthermore, when mice were intravenously injected with 20 micrograms of CK-M, IL-2 and IFN-gamma production by splenocytes, upon stimulation with either formalin-killed C. kutscheri or mitogens, was significantly higher on day 10 of treatment than on day 2. Additionally, the cytotoxicity to L929 cells of this serum from CK-M-treated mice increased with time, and the activity in the serum of day 10 was not abrogated by the antibody to TNF-alpha. Data obtained here indicate that CK-M may preferentially stimulate type-1 helper T cells to produce IL-2 and IFN-gamma, and that the enhanced cytokine production could contribute to the nonspecific resistance induced by C. kutscheri.

Suppressive effect of a mouse testicular extract on lymphocyte activation.

The effect of mouse testicular extract (TE) on lymphocyte activation was investigated. TE, in the dose range 75-600 micrograms ml-1, suppressed significantly the blastogenic response of splenocytes to concanavalin A (Con-A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA) and lipopolysaccharide (LPS). TE also suppressed the blastogenic response of B-cells to LPS and of T-cells to PHA in a dose-dependent manner as well as suppressing the mixed lymphocyte reaction (MLR). Pretreatment of splenocytes with TE did not however, completely suppress their blastogenic response to Con-A, when the treated cells were washed prior to culturing. Furthermore, TE did not inhibit the on-going blastogenesis of splenocytes that had been activated already with Con-A for 48 h. Splenocytes obtained from TE-treated mice remained capable of responding to Con-A stimulation, whereas they did not respond to listerial antigens when mice were immunized with Listeria monocytogenes together with TE. The effects of TE were enhanced significantly by heating to 100 degrees C, but were resistant to pronase, RNase and DNase. These results suggest that TE affects non-specifically the stage of lymphocyte sensitization to antigens or mitogens.

Requirement of the conformational stability of a Salmonella ribosomal vaccine for its mouse protection.

The 43-kDa non-O antigenic component isolated from the crude ribosomal fraction of Salmonella typhimurium [9] was further purified by affinity chromatography (43-kDa protein: 43-kDp). Immunization with 43-kDp did not induce complete mouse protection in CF1 mice to 500 LD50 of S. typhimurium, although it elicited a substantial IgG antibody response. The 43-kDp exhibited the mitogenicity to splenocytes (CF1 and C3H/HeJ) and B cell-rich populations (CF1). Complexing 43-kDp with the compact ribosomes of Streptococcus pyogenes by formaldehyde (complex vaccine: CV) elicited both IgM and IgG antibodies to 43-kDp. CV induced a boosting effect to enhance IgG antibody response. Moreover, CV generated delayed-type hypersensitivity to salmonella antigens and also conferred complete protection against 500 LD50 challenge of S. typhimurium to CF1 mice. These abilities of CV were reduced or impaired by RNase digestion. CV was able to induce partial or complete protection in inbred mouse strains (C3H/HeN, C3H/HeJ, DBA/2 and A/J). These data, in addition to other reports, suggest that conformational stability between ribosomes and contaminating substances such as 43-kDp or O-antigens might be required for the overall effects of the ribosomal vaccine.

Virulence of transparent and opaque colony types of Neisseria gonorrhoeae for the genital tract of mice.

The virulence of transparent (Tr) and opaque (Op) colony types of Neisseria gonorrhoeae in the genital tract of female mice was evaluated at two stages of oestrous. Isogenic pairs of Tr and Op variants were isolated from N. gonorrhoeae strain 57-120. Both variants exhibited a T2 morphology, but only the Op variant possessed protein II (P.II) in outer-membrane fractions. When administered by intravaginal inoculation Op gonococci were highly infective only for mice in late pro-oestrous, whereas Tr gonococci were virulent for mice at both late pro-oestrous and dioestrous. Gonococci recovered from the uterus were of both Tr and Op phenotypes in equal proportions when mice were infected at dioestrous with Tr cells. In contrast, greater than 90% of recovered colonies were of Op phenotype when mice were infected at late pro-oestrous with either Op or Tr cells. These results indicate that the virulence of gonococci for the genital tract of female mice differs from that for the chicken embryo. Furthermore, gonococcal survival in the female genital tract might be attributable to phase variation from Tr to Op phenotypes.

Biological functions of the water-insoluble fraction of mouse seminal vesicle fluid. I. Suppression of the blastogenic response of lymphocytes.

The effects of the water-insoluble fraction of mouse seminal vesicle fluid (WIF-SVF) on lymphocytes was investigated to clarify its role in reproductive immunity. WIF-SVF inhibited the blastogenic response of T-cells to concanavalin-A (Con-A), but it did not inhibit the blastogenic response of B-cells to lipopolysaccharide (LPS). Pretreatment of splenocytes with WIF-SVF did not suppress the blastogenic response of splenocytes to Con-A when treated cells were washed prior to culture. WIF-SVF did not inhibit the proliferation of Con-A activated splenocytes, the response of listeria-immune splenocytes to listerial antigen, or the proliferation of IL 2-dependent HT-2 cells, or the growth of tumour cells (Yac 1 cells, Ehrlich ascites carcinoma cells, EL-4 cells). A listerial antigen-specific immune response was not induced after mice were immunized with both listerial antigen and WIF-SVF. WIF-SVF is mainly composed of protein and its suppressive activity was enhanced by heating at 100 degrees C. These results suggest that WIF-SVF inhibits the responsiveness of T-cells to antigens or mitogens non-specifically at the initial stage.

Suppression of virulence factors of Pseudomonas aeruginosa by erythromycin.

The effects of erythromycin stearate over a concentration range of 0.1-10 mg/l on production of elastase, protease and leucocidin by clinical isolates of Pseudomonas aeruginosa were investigated. Growth of P. aeruginosa N42 in broth was not affected significantly during 24 h culture with erythromycin (0.1-10 mg/l), although extracellular protein contents were reduced by erythromycin at concentrations of 0.1-1.0 mg/l. Production of elastase and protease by strain N42 was significantly suppressed by erythromycin with a maximum inhibition at 0.5 mg/l, but the complete inhibition of enzyme production was not achieved. In contrast, leucocidin production by strain N42 was completely impaired by erythromycin at concentrations of 0.1-5.0 mg/l. Although the leucotoxic activity, as determined by vital staining, was not detected, the leucocidin fraction prepared from the autolysate of strain N42 cultured with 10 mg/l of erythromycin induced morphological changes in human leucocytes, resulting in release of elastase. Erythromycin exerted similar effects on other clinical isolates of P. aeruginosa. These findings indicate that erythromycin might have a role in P. aeruginosa infection, although it has no direct antibacterial activity.

Hepatotoxic activity of Campylobacter jejuni.

Hepatotoxic factor(s) were isolated from whole-cell lysates of Campylobacter jejuni GIFU 8734 and purified by chromatography. A single intravenous injection of 10 micrograms of this factor reproducibly produced hepatitis in mice, as determined by histology and liver function tests. The hepatic lesions were very similar to those evoked by C. jejuni infection. Tissue-culture studies with mouse hepatocytes demonstrated that low concentrations of the factor caused release of hepatic enzymes into the medium without appreciable cytolysis. High concentrations of the factor induced cytolysis. These effects were neutralised by antiserum to the factor, but not by antisera to the lipopolysaccharide of C. jejuni or to the heat-labile enterotoxin of Escherichia coli. Among 20 clinical isolates of C. jejuni, only four evoked hepatitis in mice and produced the hepatotoxic factor.

Hormonal regulation of soluble immune response suppressor (SIRS): a possible role of SIRS in the maintenance of pregnancy.

This study was conducted to investigate the effects of sex hormones upon the nature of soluble immune response suppressor (SIRS) produced by concanavalin A-stimulated Lyt-2+ T cells. Conventional SIRS affected IgM PFC only. However, SIRS made with progesterone (20-400 ng/ml or Prog-SIRS) suppressed IgM PFC, one-way MLR, and generation CTL; and SIRS made with estrogen (0.2-50 ng/ml or Est-SIRS) enhanced these responses. The factor(s) (MW 40,000-55,000) to stimulate macrophages to produce the second soluble factor (M phi-SF) was isolated from all preparations by gel filtration. Furthermore, Est-SIRS contained a factor(s) (MW 10,000-30,000) to enhance IgM PFC, MLR, and mitogen-induced blastogenesis of both T and B cells; and Prog-SIRS possessed the suppressive factor(s) to IgM PFC, MLR, and mitogen-induced T-cell proliferation. These activities were not impaired by 2-mercaptoethanol. Moreover, the suppressive activity of Prog-SIRS was completely absorbed by T cells only, but the enhancing activity of Est-SIRS was not completely absorbed by a single-cell population. These data suggest that progesterone can contribute to the suppression of allograft rejection through soluble factors, and estrogen can enhance host responses which may be affected by several soluble factors during pregnancy.

Nonspecific stimulation of host defense by Corynebacterium kutscheri. II. Isolation of the active moiety.

The isolation and determination of biological activities of the active component of Corynebacterium kutscheri were attempted in the present investigation. The antitumor effect was confined to the subcellular particle fraction of this bacterium and was associated with a molecule of glycoprotein nature (40,000-38,000 Daltons) isolated from this fraction by affinity chromatography with concanavalin A-Sepharose 4B. This substance exerted mitogenic activity on C3H/HeJ splenocytes and T cells, stimulatory activity on macrophages, and further exhibited antitumor effect on P388 leukemia in CDF1 mice. The Winn assay disclosed that the antitumor effect induced by this substance was dependent on L3T4+ T cells. Furthermore, both the mitogenic and antitumor activity of this moiety were resistant to heating at 100 degrees C for 30 min or RNase digestion, but sensitive to trypsin digestion, or low or high pH. These results indicate that the antitumor effect of C. kutscheri is attributable to the heat-stable glycoprotein moiety which can directly stimulate T cells and macrophages.

Biological functions of mouse seminal vesicle fluid. II. Role of water-soluble fraction of seminal vesicle fluid as a nonspecific immunomodulator.

The suppressive mechanisms of T cells induced by water-soluble fraction of mouse seminal vesicle fluid (WSF-SVF) were investigated to clarify its immunological roles in the reproductive immunity. WSF-SVF inhibited the blastogenic responses to concanavalin A (Con A) or phytohemagglutinin (PHA) of T cells. Pretreatment of splenocytes with WSF-SVF did not suppress the blastogenesis of splenocytes to Con A when treated cells were washed before cultures. WSF-SVF did not inhibit the proliferation of Con A-activated splenocytes, that of listeria-immune splenocytes to listeral antigen and growth of tumor cells (Yac 1 cells, Ehrlich ascites carcinoma cells, EL 4 cells). Listerial antigen-specific immune response was not observed when mice were immunized with both listerial antigen and WSF-SVF, whereas it was observed when mice were immunized with only listerial antigen. WSF-SVF also significantly inhibited allogenic MLR. WSF-SVF did not adsorb Con A, and its suppressive activity was rather enhanced by heating at 56 degrees C for 30 min. These results suggest that WSF-SVF inhibits the stage of sensitization of T cells with antigen or stimulant, such as mitogen nonspecifically, without adsorption to antigen or mitogen, and its substance is stable.

Alterations of host resistance to mouse typhoid infection by sex hormones.

The effect on mouse typhoid infection of a 3-day treatment of female virgin mice with 1 mg/day of female sex hormones (estrogen or progesterone), maintaining the same hormonal levels observed in pregnant mice for 30 days, was investigated in order to clarify the mechanisms of altered resistance during pregnancy. Estrogen-exposed mice were more susceptible to the intraperitoneal challenge with Salmonella typhimurium as compared with the vehicle control mice, while progesterone treatment increased the survival times of mice. Estrogen exposure increased the number of peritoneal cells after treatment, but the inflammatory cellular response after infection was significantly suppressed. Although the estrogen-treated and vehicle control mice had the same degrees of peritoneal cellular responses after infection, the death rates in the estrogen-treated mice were higher than those in the vehicle control mice against challenge with 1 LD50 of S. typhimurium. On the other hand, progesterone treatment resulted in the marked influx of peritoneal cells after treatment was terminated, and also it induced a significant increase in the number of peritoneal cells after infection. Although survival times in the progesterone group were higher than those in other groups, all progesterone-treated mice died after a challenge with 1,000 LD50 of S. typhimurium. These results suggest that progesterone enhances nonspecific resistance by increasing the influx of peritoneal cells after infection, while estrogen affects the acute inflammatory responses.

Nonspecific stimulation of host defense by Corynebacterium kutscheri. I. Antitumor effect.

The effect of local injection of formalin-killed Corynebacterium kutscheri (FK.CK) on mouse survival after the intraperitoneal inoculation of Ehrlich ascites carcinoma in outbred ddY mice or P388 leukemia cells in inbred CDF1 mice was investigated. Treatment of mice in the dose range of greater than 10(6) organisms per mouse conferred the substantial protection on both mice. The initial phase of antitumor effect consisted of the marked increase in the number of peritoneal exudate cells and the enhanced cytotoxicity of peritoneal exudate cells. The Winn assay disclosed that antitumor effect by which tumor-burden mice could survive was attributable to nonadherent splenocytes whose activity was impaired by treatment with anti-T cell serum and complement. A single injection of FK.CK induced the cytotoxicity to three different murine tumor cells in serum of treated mice without a boosting injection of endotoxin. Furthermore, the generation of effector cells and serum cytotoxicity seemed to be paralleled by that of the delayed-type hypersensitivity to this organism. Thus, the antitumor resistance induced by C. kutscheri is considered to be in part T cell mediated.