Search

The resistance to bacteria was studied at the site of delayed footpad reaction in mice immunized with Listeria monocytogenes. When a challenge injection of listeria was given into the footpad of immune mice, no enhancement of bacterial elimination was observed before the generation of delayed footpad reactivity. After the generation of delayed reactivity, an enhanced elimination of listeria or Salmonella typhimurium was observed only at the site of strongly positive delayed footpad reaction elicited with listerial antigen. Such an enhancement in bacterial elimination was also found at the site of delayed footpad reaction induced by immunization with heterologous erythrocytes. Both delayed footpad reaction and local resistance could be transferred locally by immune spleen cells in a dose-dependent fashion, but were completely abrogated when recipient mice were treated with carrageenan or whole-body X irradiation. Macrophage accumulation was an important factor in the expression of resistance at the reaction site. These results suggest that the delayed footpad reaction contributes to host defense by enhancing the local resistance to bacteria.

Age-related modification of protection to L. monocytogenes by macrophages was examined. Cumulative mortality rates of old (15-month-old) or very old (24 to 26-month-old) mice were lower than that of young (3-month-old) mice after an intravenous inoculation of L. monocytogenes. The numbers of bacteria in the livers and spleens of old or very old mice were smaller than that of young mice at an early stage of infection (1 day or 3 days). Adversely, the numbers of bacteria in old or very old mice were larger than in young mice at a late stage (7 days). Enhanced resistance to L. monocytogenes was shown not only in the case of systemic infection after an intravenous bacterial challenge, but also in the case of local infection in the thigh muscle or subcutaneous air cavity. The number of macrophages accumulating to the infected sites in old mice was not larger over a five day period than that in young mice in the case of local infection in a subcutaneous air cavity. Peritoneal macrophages derived from old mice suppressed more effectively the bacterial growth within macrophages in vitro than did those derived from young mice. These results suggest that the macrophage function of aged mice is not impaired and non-specifically activated macrophages contribute to the protection to L. monocytogenes in aged mice.

Monoclonal antibodies that bind to surface membranes of developing schistosomula and/or cercarial tails were generated from mice immunized with living schistosome eggs or soluble egg antigen. These monoclonal antibodies detected at least three different surface epitopes. One surface antigen detected by anti-egg monoclonal antibody EG1C4B1 (E.1) persisted on the surface of developing schistosomula for 96 h posttransformation . The same or a cross-reactive antigen was also detected on the surfaces of S. japonicum and S. haematobium schistosomula and cercarial tails. Monoclonal antibody E.1 killed schistosomula in vitro as well or better than infected mouse sera and transferred immunity to naive mice when administered in vivo. The monoclonal antibody reduced the number of lung worms recoverable on day 4 postchallenge by up to 85% and reduced the adult worm burden up to 41% as compared with controls. The data also show that the molecular weights of the egg antigens detected by monoclonal antibody E.1 were different from those detected on schistosomula.

The augmentation of resistance against Listeria monocytogenes after an intraperitoneal (ip) administration of shosaiko-to in mice was shown to depend on the time interval between the treatment and the infection. A maximal effect was expressed in mice treated 4 days before ip infection. The time dependent resistance correlated to the accumulation of macrophages in the peritoneal cavity just before the infection, but not to bactericidal activity as judged by the fact that peritoneal macrophages from untreated mice and those from mice treated with shosaiko-to 4 days before showed a high bactericidal activity of the same degree. Resistance to the infection in untreated mice may be attributable to newly accumulating macrophages with a low level of bactericidal activity, but not to resident macrophages with a high level of the activity. After intravenous infection, on the other hand, a maximal effect was expressed in mice treated with shosaiko-to 2 days before. The resistance correlated to accumulation of macrophages and bactericidal activity in the spleen just before the infection. Participation of cytokines in an augmenting effect of shosaiko-to on protection against the infection was examined. Shosaiko-to induced a transient elevation of serum CSF activity that was maximal at 3 hours after the administration in uninfected mice, though it did not augment the CSF activity induced by the infection. The elevation of CSF activity may induce accumulation of macrophages with a high level of bactericidal activity in the spleen 2 days after administration of shosaiko-to and then in the peritoneal cavity 4 days after administration. IFN-gamma and TNF-alpha did not participate in the effect because administration of anti-IFN-gamma or anti-TNF-alpha just before administration of shosaiko-to or just before infection did not abrogate the inhibitory effect of shosaiko-to on the bacterial growth in the early stage of infection. Shosaiko-to also induced an increase of CFUm number in the spleen. The effect may contribute to the augmentation of resistance in the late stage of infection by differentiating to mature macrophages.

We established an in vitro system generating L. monocytogenes-specific T cells primarily from unprimed spleen cells of mice. Normal spleen cells were cultured for 5 days in the presence of L. monocytogenes in vitro. Viable cells were harvested and assessed for their capacity to confer acquired cellular resistance (ACR) and delayed footpad reaction (DFR) upon local passive transfer to naive syngeneic recipient mice. When normal spleen cells were stimulated with viable L. monocytogenes, the viable cells that were recovered after 5 days of culture conferred a high level of ACR and DFR. Negative selection revealed that the effector cells obtained in primary in vitro culture were Thy 1+, L3T4+, Lyt2- cells. T cells mediating ACR could not be generated in the culture of normal spleen cells with heat-killed bacteria; however, cells mediating only DFR were generated in the presence of a large number of killed L. monocytogenes. The expression of DFR and ACR by T cells generated in this primary culture system was Listeria-specific; reactions were not observed against unrelated bacterial antigens including S. typhimurium, S. aureus, E. coli and PPD. FACS analysis of the cells in culture showed that L3T4+ and Lyt2- T cells were being enriched during culture. The primary generation of antigen-specific T cells in vitro was also possible with spleen cells from NTx mice but not with cells from nude mice, suggesting the presence of Listeria-specific precursors in NTx mice.

Meth A tumor-bearing mice were examined for changes in the host defense mechanism against infection with Listeria monocytogenes. The resistance of tumor-bearing mice to systemic, i.e., intravenous, infection in an early phase of the infection was suppressed for 1-3 days after tumor implantation (5 x 10(5) cells/mouse, subcutaneously), but was augmented thereafter even on the 35th day as compared with normal mice. Suppression and enhancement of the resistance of tumor-bearing mice to primary infection with L. monocytogenes was also observed in tumor-bearing athymic nude mice. Splenic macrophages from tumor-bearing mice on the 14th day after tumor implantation exerted potent bactericidal activity against L. monocytogenes in vitro as compared with those from normal mice. The function of macrophages as nonimmune scavenger cells seemed to be activated in mice bearing a progressing tumor. However, some of the tumor-bearing mice challenged with a sublethal dose of L. monocytogenes showed diminished resistance in the late phase of the infection; moreover listeria-immunized tumor-bearing mice showed less resistance to a secondary challenge with the bacteria than did normal immunized mice. This suppression of the specific immune response of tumor-bearing mice to L. monocytogenes was shown also in the assay of the delayed-type footpad reaction. The bacterial growth-inhibiting function of listeria-immune T lymphocytes, determined by the effect of adoptive transfer of the cells on the growth of Listeria, was also reduced in tumor-bearing mice as compared with normal mice.

The role of gamma interferon (IFN-gamma) in an increase in Ia-bearing macrophages during Listeria monocytogenes infection was studied. The peritoneal macrophages from L. monocytogenes-infected mice contained a high proportion of Ia. Intraperitoneal injection of the supernatant from a culture of spleen cells from L. monocytogenes-infected mice induced Ia-rich exudates in normal mice. The Ia-inducing activity in the culture supernatant was abrogated by the pretreatment of spleen cells with anti-Thy-1.2 antibody plus complement. Immunoadsorption of the culture supernatant with anti-recombinant IFN-gamma antibody and protein A-Sepharose CL-4B completely abrogated its Ia-inducing activity. These results suggested that an increase in Ia-bearing macrophages during L. monocytogenes infection was attributable to T-cell-derived IFN-gamma.

The effects of macrophage chemotactic factor (MCF) and macrophage-activating factor (MAF) on protection to Listeria monocytogenes were analysed using 'MCF-rich fraction' and murine recombinant interferon-gamma (rIFN-gamma). Recombinant IFN-gamma showed no macrophage chemotactic activity in the assays performed in vitro and in vivo. Although a single injection of either MCF-rich fraction or rIFN-gamma into the footpads of mice led to a significant degree of anti-listerial activity, the highest degree of protection was observed when injected together. The i.v. administration of 20,000 U rIFN-gamma did not raise significant protective activity against Listeria; however, injection with rIFN-gamma prior to that with the MCF-rich fraction into the footpad produced a higher level of protective activity than the group treated with MCF alone. In addition to MAF(IFN-gamma), MCF seems to play another important role in the full expression of protection against L. monocytogenes.

We investigated the ontogenic development of macrophage functions which are important in the expression of host defense against infection by Listeria monocytogenes. Macrophage functions, including accumulation in response to local stimuli, chemotaxis in vitro, and intracellular killing, as well as number of macrophages, were examined by using mice 1, 2, 3, 4, and 8 weeks old. The number of peritoneal macrophages was extremely low in younger mice even when their body weights were taken into consideration. Macrophage accumulation in response to infectious stimulus with viable listeria was poor in younger mice and showed an age-dependent development. In younger groups, chemotaxis in vitro was as immature as chemotaxis in vivo. In 1- and 2-week-old mice, macrophages did not show any intracellular killing activity against L. monocytogenes, but killing was observed in mice over 3 weeks of age. These functions developed in an age-dependent manner and reached the 8-week-old adult level after the mice were 4 weeks of age. In adult mice, these macrophage functions were shown to be enhanced after immunization with viable listeria; however, such an immunization-induced enhancement was very poor in the younger groups of mice. Protection judged by mortality and in vivo bacterial growth was weaker in the younger groups against both primary and secondary challenges. In vivo protection against L. monocytogenes seemed to develop in the same age-dependent manner as the development of macrophage functions. These results indicate that age-dependent immaturity of macrophage functions mainly comprises the age-dependent immaturity of protection against L. monocytogenes.

Monoclonal antibodies (MAbs) were raised against the purified fimbriae of Serratia marcescens US46, a strain expressing three morphologically distinct fimbriae. The widths of these fimbriae were 7, 4.5, and 3 nm, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fimbriae showed three bands with molecular weights of 21,000, 20,000, and 19,000, respectively. This strain had mannose-resistant (MR) hemagglutinating activity and was agglutinated by yeast cells. Therefore, strain US46 appeared to have both MR and mannose-sensitive fimbriae. In the immunoblot analysis, all MAbs reacted with the 20,000-molecular-weight subunit when given a choice of three differently sized subunits. Immunoelectron microscopy showed these MAbs attached to the MR fimbriae with the largest width (7 nm). The antigenic cross-reactivity of fimbriae was examined by an MAb-mediated agglutination test. All MR strains of S. marcescens and some mannose-sensitive strains were agglutinated by the MAbs. The serological homogeneity of MR fimbriae was confirmed by a spot test, using the crude purified fimbriae from several MR strains of S. marcescens. In other gram-negative rods, clinical isolates of Klebsiella spp. with hemagglutinating activity were agglutinated, but clinical isolates of Escherichia coli and Enterobacter spp. were not.

Bacterial growth and lethality of Listeria monocytogenes in mice were augmented by carrageenan-treatment and X-irradiation (8 J kg-1), whereas growth and lethality of Pseudomonas aeruginosa were augmented by X-irradiation but not by carrageenan-treatment. Protection against L. monocytogenes, at least in the early phases, appears to depend mainly on macrophages, since carrageenan depletes macrophages but not polymorphonuclear cells (PMN), whereas protection against P. aeruginosa appears to depend mainly on PMN. Ineffectiveness of PMN in elimination of L. monocytogenes is supported by histological examination and observation of intracellular killing in vitro.

Protective mechanisms against Listeria monocytogenes were studied in young (3-month-old) and old (15-month-old) mice of C3H/He strain. Cumulative mortality rates of old mice were lower than those of young mice after intravenous inoculation of the same doses of bacteria. The numbers of bacteria in the liver and spleen on days 1 and 3 were larger in young than old mice. Bacterial growth at this stage of infection is suppressed by accumulation of non-immune macrophages. On day 7, however, the numbers of bacteria in the liver were smaller in young than old mice. Bacterial elimination at this stage depends upon immune macrophages. These results suggest that the enhanced resistance to lethal effects of bacteria in old mice may be ascribed to activation of non-immune macrophages in the presence of depressed capacities to raise cell-mediated immunity.

Monoclonal antibody M.1 was generated from mice immunized with membrane enriched extracts of mechanically transformed schistosomula. M.1 bound to the surface membranes of cercariae and young (0-24 h post-transformation) schistosomula but did not bind to older schistosomula or cultured worms. M.1 immunoprecipitated an antigen of approximate molecular weight 27-28 kDa from schistosomula. Experiments using metabolic labeling showed that the antigen was actively synthesized by developing schistosomula. Further M.1 immunoprecipitated a similar 27-28 kDa antigen from membrane-enriched extracts of miracidia, lung and adult worms as well as from schistosomula.

We have analyzed the priming process of Listeria-specific T cells using an in vitro primary culture system. Listeria-specific T cells mediating delayed footpad reaction (DFR) and acquired cellular resistance (ACR) upon passive local transfer into naive recipients were generated from non-immune mouse spleen cells when stimulated with viable Listeria monocytogenes primarily in vitro. The effectors were detected on the third day of culture, and culturing for 5 days was sufficient for the generation of effectors mediating the peak level of DFR and ACR. The requirement of T cell subsets, Ia antigen and interleukin 2 (IL 2) for inducing effectors was studied. Presence of macrophages (M phi) and their contact to T cells were required for priming of Listeria-specific T cells in vitro. The presence of Ia antigens on macrophages was absolutely required for priming, but this requirement was lower than that in secondary immune response of Listeria-specific T cells. Effectors could not be generated when L3T4+ cells were depleted, but effectors capable of conferring a full level of DFR and ACR were induced even after the depletion of Lyt2+ cells. Contribution of IL 2 to the generation of effectors during early phase of priming was not observed. IL 2 was not produced in the supernatant of the in vitro primary culture. Precursor cells of the effectors did not respond to exogenously added recombinant IL 2 (rIL 2). Some mechanisms operating in the induction phase of Listeria-specific T cells were clarified in this study.

In the assessment of immunity to the encapsulated virulent strain of Klebsiella pneumoniae and its avirulent mutant defective for capsular polysaccharide (CPS), killed bacterial vaccine of both strains could protect mice equally against challenge with 100 x LD50 of encapsulated wild strain. Antisera to each strain conferred the same level of protection on naive mice upon transfer; the protective anti-mutant serum was highly capable of opsonizing the encapsulated bacteria. In addition to the common antigenic components shared by both strains, the wild strain had antigen(s) unrelated to the mutant since the protective capacity of the anti-wild serum was not affected by preabsorption with the mutant strain; the protection conferred by the anti-mutant serum was mediated by antibodies against non-capsular antigens since the antiserum did not contain antibodies against purified CPS detectable by ELISA. As possible candidates among the non-capsular antigens, outer-membrane proteins (OMPs) extracted from the mutant strain were examined for their immunogenicity. Immunoblotting of the protein-containing fraction and ELISA using LPS-free OMP suggested that a number of proteins were involved in the immune response evoked by K. pneumoniae. Furthermore, mice immunized with OMP or anti-OMP serum could overcome a lethal challenge with the wild strain. These results indicated that OMPs of K. pneumoniae are implicated as the protective antigens and may pave the way for the development of non-capsular, proteinaceous vaccines.

To determine whether the presence of bacterial flora contributes to the ontogenic development of macrophage function, the ability of macrophages to release superoxide anion (O2-) in response to stimulation with phorbol myristate acetate was compared in conventional and germfree mice of various ages after birth. One-week-old conventional mice showed a very low level of O2- release by their macrophages, and gradual increases were observed in 2-, 3-, and 4-week-old mice in an age-dependent manner. Macrophages from germfree mice always showed a significantly lower level of O2- release compared with conventional mice of the same age; however, age-dependent functional development was seen also in the germfree group. The poor level of O2- release by macrophages from adult germfree mice could be restored to more than the level by conventional mice when the mice were conventionalized for 3 weeks. These results suggested that the ontogenic development of macrophage function is not controlled by the presence of bacterial flora but that the full-scale expression of function at each age is under the influence of microflora.

Lethal effect of Listeria monocytogenes (L. monocytogenes) in mice was prevented by an intraperitoneal (ip) injection of a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), 4 days before ip bacterial infection. The numbers of bacteria in the peritoneal cavity and liver were smaller in shosaiko-to-treated mice from one day after the infection. Macrophage accumulation in the peritoneal cavity after ip inoculation of L. monocytogenes was observed in both untreated and shosaiko-to-treated mice. Although rates of such increases were almost the same between both groups, the absolute number of macrophages was larger in shosaiko-to-treated than in untreated mice because of a higher level of the macrophage number at 4 days after ip injection of shosaiko-to. In untreated mice, bactericidal activity of peritoneal macrophages decreased from one day to 3 days after ip injection of killed L. monocytogenes. Such an activity was maintained at the same level from 1 to 3 days in shosaiko-to-treated mice. Augmented accumulation of macrophages and maintenance of their bactericidal activity may be main mechanisms of the augmented resistance in shosaiko-to-treated mice. Augmented resistance against bacterial growth in the thigh muscle in ip shosaiko-to-treated mice may be caused by such mechanisms. The effect of shosaiko-to observed at an early stage of infection may be T cell-independent, since such an effect was observed in athymic nude mice and delayed footpad reaction could not be detected at such a timing in euthymic normal mice.

By the use of a culture supernatant containing a lymphokine capable of inducing Ia-rich peritoneal macrophages in vivo, we investigated the effect of increased macrophage Ia expression on the induction of antibody response to sheep erythrocytes (SRBC) in mice. Peritoneal macrophages from mice injected intraperitoneally (i.p.) with lymphokine 3 and 4 days earlier contained a high proportion of Ia-bearing macrophages without any significant increase in their phagocytic activity for SRBC. In lymphokine-treated mice, slight enhancement was observed in the primary IgG plaque-forming cell (PFC) response only at an early stage after i.p. immunization with SRBC. When primed mice were injected with lymphokine before secondary i.p. immunization with SRBC, the secondary IgG PFC response was greatly enhanced. On the other hand, lymphokine treatment did not affect either the primary or the secondary IgM PFC response. This effect was dependent on both timing of lymphokine injection and route of secondary immunization with SRBC. Secondary immunization via several routes other than the i.p. route never resulted in such an enhanced secondary IgG PFC response. These results suggest that lymphokine-induced Ia-rich peritoneal macrophages, which first encounter the antigen in the peritoneal cavity, play an important role in the modification of the subsequent antibody response.

The protective roles of alveolar macrophages and polymorphonuclear cells were analyzed against intratracheal challenge with Candida albicans in mice. When mice were treated with carrageenan, a known cytotoxic agent for macrophages, there was no change in susceptibilities to the challenge in terms of the survival and the progressive elimination of fungi from the lung and kidney, in spite of a decreased in vitro phagocytosis of Candida albicans by their alveolar macrophages. On the other hand, irradiated mice (whole body irradiation with 800 rads) showed an enhanced mortality and a progressive growth of Candida albicans in their lungs and kidneys, although no change was observed in the in vitro phagocytic activity of alveolar macrophages until day 6 after irradiation. In normal and carrageenan treated mice, there was a progressive increase in the recruitment of polymorphonuclear cells into the lung after the challenge as shown by bronchoalveolar lavage and histological examination. In irradiated mice, on the other hand, there was a decreased recruitment of polymorphonuclear cells at 24 hr after the challenge, and a complete impairment at a late stage. When phagocytes were obtained from normal mice and examined for in vitro phagocytic activity to Candida albicans, polymorphonuclear cells showed higher activity than that of alveolar macrophages. These results suggest that polymorphonuclear cells play a very important role in the protection against intratracheal infection with Candida albicans.

A galactosaminoglycan moiety was obtained from an antitumor polysaccharide fraction (SN-C) isolated from Cordyceps ophioglossoides culture. SN-C was subjected to sonication, then a protein-bound galactosaminoglycan (CO-N) was isolated specifically by precipitation with 10% ammonium hydroxide. When given intraperitoneally to mice, CO-N inhibited the proliferation of sarcoma 180 cells inoculated into the peritoneal cavity and exhibited a marked life-prolonging effect against ascitic tumors such as Ehrlich carcinoma and IMC carcinoma. CO-N also showed an inhibitory effect against solid Ehrlich carcinoma when given intratumorally and significantly inhibited the growth of a syngeneic solid tumor (MM46 mammary carcinoma) upon intravenous administration at a low dose. CO-N showed a cytocidal effect against cultured cells of IMC and P388D1 in vitro. Flow cytometric analysis demonstrated that fluorescein isothiocyanate-CO-N binds to the surface of Ehrlich cells.

Analysis was done on macrophage chemotactic factor (MCF) produced in the culture supernatant of spleen cells from mice immunized with Listeria monocytogenes. MCF was produced by Thy-1+, Lyt-1+ lymphocytes. MCF activity was resistant against pH 2 treatment and heating at 56 degrees C for 30 min, but was abolished by digestion with trypsin. G-100 gel filtration chromatography revealed that the approximate molecular weight of MCF was 15,000. MCF-rich fraction obtained by gel filtration chromatography showed neither MAF activity nor interferon activity. MCF activity in MCF-rich fraction was not affected by treatment with anti-rIFN-gamma antibody. An injection of MCF-rich fraction into the peritoneal cavity of mice induced a significant degree of accumulation of polymorphonuclear leukocytes (PMN) in a very short time after injection and macrophages thereafter. Resistance against listerial infection was augmented at the site where macrophage accumulation was provoked by the injection with MCF-rich fraction. It was shown that MCF plays an important role by itself in the protection against listerial infection by the accelerated accumulation of macrophages.

L18-MDP(Ala), a synthetic derivative of muramyl dipeptide, enhanced resistance against Escherichia coli and Candida albicans infections in aged mice and younger adult mice whose defense mechanism(s) had been depressed by X-ray irradiation or by treatment with cyclophosphamide.