Role of liver NK cells and peritoneal macrophages in gamma interferon and interleukin-10 production in experimental bacterial peritonitis in mice

Gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-10 (IL-10) production by liver, spleen, lung, peripheral blood mononuclear cells (MNC), and peritoneal exudate cells (PEC) in experimental bacterial peritonitis was examined by cecum ligation and puncture (CLP) (with an 18-gauge needle) of BALB/c mice. MNC of organs were cultured for 18 h, and cytokine levels in supernatants were examined. Cytokines contained in peritoneal lavage fluid were regarded as those produced by PEC. Only liver MNC and PEC produced substantial amounts of IFN-gamma, and PEC were the main source of IL-10, especially 12 h after CLP. As reflected by the cytokine production by liver MNC and PEC, serum IFN-gamma and IL-10 levels were elevated after CLP. C57BL/6 (B6) mice and BALB/c nude mice showed a similar pattern of cytokine production. TNF-alpha levels in culture supernatants, peritoneal lavage fluid, and sera were not significantly elevated compared to those of sham-operated mice. In vivo depletion of NK cells of B6 mice with anti-asialo GM1 or anti-NK1.1 antibody greatly decreased IFN-gamma levels in liver MNC culture supernatants and sera, suggesting that liver NK cells are IFN-gamma producers. On the other hand, plastic-adherent PEC macrophages are the major IL-10 producers. Mice subjected to a cecum ligation and cut procedure (which have a more severe peritonitis) showed much higher IFN-gamma and IL-10 levels than those subjected to CLP, while mice subjected to CLP with a smaller (22-gauge) needle showed low levels of these cytokines. These findings show that liver NK cells and PEC macrophages are important for the production of proinflammatory and anti-inflammatory cytokines in bacterial peritonitis.     

Regression of Ehrlich ascites carcinoma in BCG-sensitized mice

Intraperitoneal inoculation of CF1 mice with Bacillus Calmette-Guérin (BCG) protected many of them against the ascites form of Ehrlich carcinoma; and, for those that developed cancer, complete regression occurred in up to 50% of the cases at an advanced state of the neoplastic disease. In contrast, when a booster dose of BCG was administered in admixture with tumor cells, the incidence of the tumor was lower and tumor regressions were very rarely observed in mice that developed cancer. Trypan blue, an inhibitor of lysosomal enzymes of macrophages, was found to markedly suppress the natural (innate) antitumor resistance of control mice as well as the acquired resistance and tumor regressions of BCG-sensitized mice. Moreover, a comparison of the cytotoxic activity of the adherent (macrophages) and nonadherent (predominantly lymphocytes) cells isolated from the peritoneal cavity of BCG-sensitized mice, as measured by the inhibition of DNA synthesis, revealed that the effector cells were amongst the macrophages. In contrast, spleen macrophages were devoid of cytotoxicity. The spleen lymphocytes from both BCG-sensitized and control mice possessed about the same significant cytotoxic activity. These results indicate that the activated peritoneal macrophages, induced by a local injection of BCG, could play an important role in the antitumor immunity against Ehrlich carcinoma.     

Experimental cancer immunotherapy: comparison of tumor rejection if F344 rats given live Mycobacterium bovis (Strain BCG) and killed Corynebacterium parvum

F344 rats received grafts of syngeneic 13762 mammary adenocarcinoma cells previously admixed with either living BCG of killed Corynebacterium parvum administered sc or intradermally (id). Animals given id transplants of tumor cells admixed with either BCG or killed C. parvum exhibited tumor growth for an average of 10 days, then regression in size and rejection of the tumor nodules. Lesions were found in rats given sc transplants of tumor cells admixed with the killed microorganism for an average of 13 days with the same results. When live BCG was added to the sc transplants, accelerated rates of tumor growth and early death were noted, compared with the group receiving tumor cells alone sc. Suppressed rates of tumor growth and prolonged survival were observed in the groups receiving id inoculations of tumor cells followed by treatment with killed C. parvum administered weekly ip or id 1 cm away and around the growing tumor. On the other hand, weekly treatment of BCG injected either ip or id 1 cm away and around the growing tumor resulted in accelerated rates of tumor growth and early death. Animals exhibiting C. parvum of BCG-mediated tumor rejection displayed tumor-specific protection to sc challenge injections of the cell line initially used, but they died with growing tumors and metastases when challenged with tumor cells of an antigenically different line syngeneic to F344 RATS. Microscopic examination of histologic sections of tumors formed from id inoculations of tumor cells admixed with either BCG or killed C. parvum revealed a nonspecific infiltrate of macrophages and polymorphonuclear leukocytes in the tumor, whereas sections of tumors formed from sc grafts of cells admixed with killed C. parvum revealed a specific organized infiltrate of mostly macrophages around the tumor follicles.     

The cellular immune responses induced in the follow-up of interferon-alpha treated patients with chronic hepatitis C may determine the therapy outcome

To elucidate the mechanism of human cell elimination from severe combined immunodeficient (SCID) mice transplanted with human peripheral blood lymphocytes (hu-PBL-SCID mice), we explored the immunocytes in the peritoneal cavity in SCID mice where human PBL were transferred. When the phenotype of peritoneal exudate cells (PEC) was compared by flow cytometry among three congenic strains of SCID mice that differ in their acceptability for human PBL, the PEC in NOD-scid mice, which exhibit the highest acceptability, contained the smallest number of F4/80lo/-Mac-1(+)-activated macrophages. Moreover, the proportions of natural killer cells in PEC of the three strains of SCID mice were not always correlated with the acceptability. These findings suggest the possibility that peritoneal macrophages eliminate human cells in hu-PBL-SCID mice. To verify this hypothesis, we evaluated the engraftment of human PBL into SCID mice that were treated with liposome-encapsulated dichloromethylene diphosphonate, which selectively depletes macrophages by inducing apoptosis, or 8-aminoguanidine hemisulphate salt, an inhibitor of inducible nitric oxide synthase of macrophages. As a result, both of these regimens improved engraftment of human PBL, indicating that peritoneal macrophages take part in human cell elimination in the peritoneal cavity of hu-PBL-SCID mice and that it is mediated, at least in part, by direct macrophage cytotoxicity utilizing nitric oxide.     

The synthesis of transcobalamin II, a vitamin B12 transport protein, by stimulated mouse peritoneal macrophages

The concentration of transcobalamin II (TCII), the vitamin B12 binding protein which delivers vitamin B12 to the tissues, was determined in stimulated and non stimulated mouse peritoneal exudate cells (PEC). Following a single intraperitoneal thioglycollate injection there was a marked increase in TCII which was shown to be produced by the adherent cells of the PEC i.e. the macrophages. It has been concluded that the PEC macrophages synthesize TCII.     

Influence of twinline, an elemental diet, on the generation of nitric oxide and reactive-oxygen intermediates from mouse peritoneal macrophages and polymorphonuclear leukocytes

The influence of Twinline (SNN-6010), an elemental diet containing medium-chain triglycerides, on the generation of nitric oxide (NO) and superoxide (O2.-) has been examined in mouse peritoneal macrophages and polymorphonuclear leukocytes (PMN). When PMN and peritoneal macrophages obtained from untreated mice were cultured in medium containing 0.1% and 1% (v/v) Twinline for 48h and stimulated with phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine, their chemiluminescence and O2.- generation were strongly suppressed, as was NO generation from peritoneal macrophages. PMN and peritoneal macrophages obtained from mice fed Twinline for 30 days generated much smaller amounts of 02.- and NO compared with PMN and peritoneal macrophages from control mice. In conjunction with this suppressed NO generation, inducible NO synthase and its mRNA expression in peritoneal macrophages were suppressed by Twinline both in-vivo and ex-vivo. Although phagocytosis of PMN and peritoneal macrophages was not suppressed by Twinline; their candida-killing activity was markedly suppressed. These results indicate that Twinline suppresses the host-defence function of PMN and peritoneal macrophages by down-regulating their generation of reactive-oxygen intermediates and NO.     

Ia-bearing macrophages in athymic mice: antigen presentation and regulation

Ia-bearing macrophages from spleens and peritoneal cavities of athymic (nu/nu) and euthymic (nu/+) were compared. Macrophages from both strains of mice had equal antigen-presenting ability. The basal numbers of unstimulated Ia-bearing peritoneal macrophages were equal, but only euthymic mice recruited large numbers of Ia-bearing macrophages after Listeria infection. The i.p. injections of a "macrophage Ia-recruiting factor" induced exudates rich in Ia-bearing macrophages in both athymic and euthymic mice. These data suggest 2 levels of control of Ia-bearing macrophages: a basal T cell-independent level and a stimulated level dependent on mature T cells.     

Generation of nitric oxide and induction of major histocompatibility complex class II antigen in macrophages from mice lacking the interferon gamma receptor

Availability of mice with a targeted disruption of the interferon gamma (IFN-gamma) receptor gene (IFN-gamma R0/0 mice) made it possible to examine parameters of macrophage activation in the absence of a functional IFN-gamma receptor. We asked to what extent other cytokines could replace IFN-gamma in the induction of nitric oxide or major histocompatibility complex class II antigen (Ia) expression in peritoneal macrophages. In thioglycollate-elicited macrophages from wild-type mice, tumor necrosis factor (TNF) alone was virtually ineffective in inducing release of NO2- (the endproduct of nitric oxide generation), but TNF enhanced NO2- release in the presence of IFN-gamma. In macrophages from IFN-gamma R0/0 mice, which were unresponsive to IFN-gamma, TNF completely failed to stimulate NO2- release. The stimulatory actions of IFN-alpha/beta on NO2- release were indistinguishable in wild-type and IFN-gamma R0/0 macrophages: IFN-alpha/beta was ineffective on its own, showed marginal stimulation of NO2- release in combination with TNF, and was moderately effective in the presence of lipopolysaccharide. The level of constitutive Ia antigen expression was not significantly different in peritoneal macrophages from wild-type and IFN-gamma R0/0 mice. An increased Ia expression was induced by IL-4 and granulocyte-macrophage colony-stimulating factor in both wild-type and IFN-gamma R0/0 macrophages, but the magnitude of this induction was less than with optimal concentrations of IFN-gamma in macrophages from wild-type mice. IFN-alpha/beta showed only a minor stimulatory effect on Ia expression in both wild-type and IFN-gamma R0/0 macrophages. Simultaneous treatment of wild-type macrophages with IFN-alpha/beta and IFN-gamma reduced the IFN-gamma-induced Ia expression in wild-type macrophages, but IFN-alpha/beta did not show an inhibitory effect on IL-4- or granulocyte-macrophage-colony-stimulating factor-induced Ia expression in either wild-type or IFN-gamma R0/0 macrophages. The important role of IFN-gamma in the regulation of the induced expression of major histocompatibility complex class II antigen was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis resident peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from wild-type mice. The inability of other cytokines to substitute fully for IFN-gamma in macrophage activation helps to explain the earlier observed decreased resistance of IFN-gamma R0/0 mice to some infections.     

Macrophage colony-stimulating factor stimulates synthesis and secretion of a mouse homolog of a human IgE-dependent histamine-releasing factor by macrophages in vitro and in vivo

Treatment of murine resident peritoneal macrophages with macrophage-CSF (M-CSF) up-regulated the synthesis of a discrete set of proteins, including a 26-kDa protein (p26). The sequence of 20 NH2-terminal amino acids of the purified p26 was identical with the mouse homolog of a human IgE-dependent histamine-releasing factor (HRF). Among macrophage activators tested (M-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, IFN-gamma, and LPS), only M-CSF could up-regulate the p26 HRF synthesis by cultured macrophages. M-CSF not only increased the levels of p26 HRF mRNA and protein, but also stimulated the secretion of an N-glycosylated p26 HRF with a m.w. of 30 kDa. Repeated injections of M-CSF into mouse peritoneal cavity for 4 days elicited macrophages expressing abundant p26 HRF. A single i.p. injection of M-CSF failed to increase the p26 HRF level in peritoneal macrophages of thioglycollate-, LPS-, or adjuvant-treated mice, while M-CSF challenge to OVA-immunized mice caused macrophage infiltration and overproduction of p26 HRF, similarly as did OVA challenge. The Ag-specific priming for enhanced synthesis and secretion of p26 HRF by M-CSF was also demonstrated in cultured macrophages prepared from OVA-immunized mice. An i.p. injection of M-CSF or recombinant p26 HRF triggered eosinophil recruitment, even in the absence of the Ag, in the sensitized mice, but not in normal mice. Furthermore, recombinant p26 HRF could induce eosinophilia without marked macrophage and lymphocyte infiltrations. Our results suggest that p26 HRF secreted by M-CSF-stimulated macrophages may be an important mediator for the late phase allergic inflammation.     

Changes in intracellular localization of proteasomes in immortalized ovarian granulosa cells during mitosis associated with a role in cell cycle control

The antitumor effect of oenothein B, a macrocyclic ellagitannin from Oenothera erythrosepala Bordas, on rodent tumors was studied. Oenothein B exhibited a strong antitumor activity against MM2 ascites tumors upon intraperitoneal administration to the mice before or after the tumor inoculation. The tannin also inhibited the growth of Meth-A solid type tumor in mice. This antitumor effect of the tannin could not be attributed to its direct cytotoxic action on tumor cells, because the cytotoxicity was very weak in the presence of serum protein. When oenothein B was injected into the peritoneal cavity of mice, peritoneal exudate cells, including cytostatic macrophages, were induced. Furthermore, in the in vitro treatment of macrophages from mice and humans, the tannin stimulated release of an interleukin 1 (IL-1)-like activity and IL-1 beta from the cells. These results suggest that oenothein B exerts its antitumor effect through potentiation of the host-immune defense via activation of macrophages.     

A generalized consideration of myocardial preservation with cold crystalloid versus warm blood cardioplegia in heart valve replacement

The spontaneous as well as mitogen-induced in vitro production of interleukin-6 (IL-6) was studied in cultures of peripheral blood mononuclear cells (PBMC) from 14 children with marginal protein-energy malnutrition, 43 children with definite protein-energy malnutrition and 38 eutrophic controls of similar age, sex, race and socioeconomical condition. PBMC were cultured without added mitogen or stimulated with either lipopolysaccharide (LPS) or phytohemagglutinin (PHA). After 48 h incubation, cell-free culture supernatants were collected and stored at -70 degrees C. The amount of IL-6 in the supernatants was determined by a specific bioassay based on the proliferation of B9 hybridoma cells using human rIL-6 as standard. The mean level of IL-6 was significantly increased in supernatants from nonstimulated PBMC cultures from definitely malnourished children as compared with that observed in those of the controls. Stimulation with either LPS or PHA induced a rise in cytokine bioactivity in the supernatants of PBMC cultures from the different nutritional groups tested. Interestingly, IL-6 was significantly increased in the supernatants of PHA-stimulated cultures from malnourished children as compared with those of the controls.     

Comparison of the potentiation of specific tumor immunity in mice by Corynebacterium parvum or BCG

Two independent studies have compared killed C. parvum (CP) vaccine with viable BCG (Pasteur) and BCG (Glaxo) vaccines, respectively, for potentiation of antitumor immunity when injected with irradiated tumor cells into B6D2F1 AND CBA-T6T6 mice. Both studies concurred that, for a given number of irradiated tumor cells, larger amounts (dry weight equivalent) of BCG than CP were required to produce similar degrees of tumor immunity. Evidence also showed that stronger and more protracted immunity was mediated by CP than by BCG.     

Administration of silica sensitizes lipopolysaccharide responsiveness of murine macrophages but inhibits T and B cell priming by inhibition of antigen presenting function

Macrophages play a key role in natural host defense against infection by a variety of pathogens. In addition, macrophages initiate the development of acquired immunity via antigen processing and presentation. The role of macrophages in resistance to pathogens, the development of autoimmune diseases and the induction of acquired immunity has been studied by treatment of rodents with reagents which are cytotoxic. We have studied the effects of one such reagent, silica, on the function of spleen macrophages and peritoneal exudate cells (PEC). Intraperitoneal administration of silica caused the accumulation of spleen macrophages and neutrophils, reduction in the number of B cells and had a modest effect on T cell abundance. The percentage of CD11b+ PEC was not affected by silica treatment but total PEC recovery was diminished 5-8 fold. Silica treatment did not cause release of TNF-alpha or IL-1-beta but, when stimulated with lipopolysaccharide (LPS) in vitro after silica treatment, PEC or spleen macrophages produced elevated levels of both cytokines compared to controls. In contrast, release of IL-12 from non-LPS treated PEC was stimulated 4-5 fold by silica treatment. In addition, sensitivity to LPS toxicity in vivo was significantly enhanced by silica. The ability of macrophages to present antigen to a T cell clone in vitro was found to be dramatically inhibited by silica treatment, as was the ability to prime antigen-specific T cells and B cells by antigen injection. Collectively these data demonstrate that silica treatment enhances macrophage sensitivity to LPS exposure but inhibits antigen processing and presentation.     

Bacillus Calmette-Guérin potentiates monocyte responses to lipopolysaccharide-induced tumor necrosis factor and interleukin-1, but not interleukin-6 in bladder cancer patients

During the past decade, particular attention has been focused on treatment of bladder cancer patients with the bacterial agent bacillus Calmette-Guérin (BCG). In these studies, bladder cancer patients were instilled with BCG (75 mg/50 ml) once per week for 6 weeks, 1-2 weeks following trans-urethral resection of the bladder. Cystoscopy was performed after 6 weeks and, unless tumor progression was present, monthly treatments were given for 1 year. Blood was drawn 2 h after the last instillation, and monocytes were isolated (5 x 10(6) cells/ml) and treated, or not, with lipopolysaccharide (LPS) (20 microgram/ml) for tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6) release. The levels of monokines were determined by a monokine-specific enzyme-linked immunosorbent assay. Our results clearly show that, after 18 h incubation, macrophages from BCG-treated bladder cancer patients produced from 2.8- to 1.9-fold and from 2.0- to 1.3-fold greater amounts of TNF alpha and IL-1 alpha respectively, compared to macrophages from healthy controls, 5-fold higher than bladder cancer patients not treated with BCG. IL-6 was not affected. In another set of experiments macrophages (5 x 10(6) cells/ml) from healthy subjects were pretreated, or not, with BCG (100 micrograms/ml) overnight and treated, or not, with LPS 20 microgram/ml alone and in combination with interleukin-1 receptor antagonist (IL-1ra) 250 ng/ml. Macrophages treated with BCG had a strong stimulatory effect on IL-1 alpha release (9.45 ng/ml) while LPS was less effective (3.59 ng/ml). The combination of BCG plus LPS produced an additive effect on IL-1 alpha release (13.71 ng/ml) compared to the effect of the compound alone. The addition of IL-1ra (250 ng/ml) to BCG was not effective, while when IL-1ra was added to BCG plus LPS only a partial inhibition of IL-1 alpha release was found (9.83 ng/ml), compared to BCG plus LPS without IL-1ra (13.71 ng/ml). These effects seem to be related to the inhibition of IL-1 alpha stimulated with LPS, but not BCG. The priming effect of BCG exerted on LPS-stimulated monocyte production of TNF alpha and IL-1 alpha from bladder cancer patients led us to study the possible modulation of fibrinogen and C-reactive protein in the serum of BCG-treated cancer patients. The plasma levels of fibrinogen and C-reactive protein were higher (approximately twice) in BCG-treated patients compared to values obtained in untreated patients or healthy controls. We conclude that the beneficial immunotherapeutic effects of BCG in bladder cancer patients are related to its capacity to prime macrophages to enhance the release of TNF alpha and IL-1 alpha, but not IL-6 in response to physiological secondary stimuli, or through the direct stimulation of BCG on IL-1 alpha or TNF alpha, which are directly involved in the killing of cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of activated macrophage products on neurite growth in an organotypic culture of chick embryo sensory ganglia]

Murine peritoneal macrophages, activated by BCG vaccine, and human peripheral blood monocytes, activated by lipopolysaccharides, exerted neurite stimulating or neurite inhibiting effects in various periods of activation. The supernatants of these preparations were active in organotypic culture of chick embryo dorsal root ganglia. The inhibition of neurite growth on the 1st day of cultivation was followed by the neurite-stimulating effect. The fluctuation of neurite-inhibition and neurite-stimulation effect of macrophage supernatants suggest the availability of certain changes in cytokine composition in different periods of macrophage activation.     

Immunomodulatory activities of oat beta-glucan in vitro and in vivo

Previous studies have shown that beta-glucans extracted from yeast or fungi potentiate immune responses. In the present study, the immunomodulatory activities of beta-(1-->3,1-->4)-glucan, derived from oats, were investigated. The ability of oat beta-glucan (ObetaG) to stimulate IL-1 and TNF-alpha release from murine peritoneal macrophages and the murine macrophage cell line P338D1, was assessed. In vitro stimulation of macrophages with ObetaG resulted in the production of IL-1 in a dose and time-dependent manner, whereas only small amounts of TNF-alpha could be detected in the culture supernatants. ObetaG also induced the production of IL-2, IFN-gamma and IL-4 secretion in a dose-dependent manner in cultured spleen cells. The intraperitoneal administration of ObetaG in mice resulted in the accumulation of leucocytes, predominantly macrophages, in the peritoneal cavity. Furthermore, ObetaG was tested for its ability to enhance non-specific resistance to a bacterial challenge in mice. Survival of mice challenged with Staphylococcus aureus was enhanced by a single intraperitoneal administration of 500 microg of ObetaG 3 days prior to bacterial challenge. In conclusion, these studies demonstrated that ObetaG possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.     

Release of superoxide anion from activated mouse peritoneal macrophages during Mycobacterium tuberculosis infection

Mouse peritoneal macrophage monolayers infected with M. tuberculosis were cultured in RPMI up to 7 days. Release of superoxide was assayed on different days in presence or absence of Phorbol myristate acetate (PMA), a known stimulator of NADPH oxidase which is involved superoxide production. Basal level of superoxide release was significantly higher in M. tuberculosis infected peritoneal mouse macrophages (P < 0.01) as compared to normal mouse macrophages. When normal and tuberculoid macrophage cultures were stimulated with PMA, increased superoxide anion release was observed in both the cultures but the increase of superoxide was significantly higher in normal macrophages as compared to tuberculoid stimulated macrophages. Superoxide release was maximum in 4 day old cultured macrophages and gradually it declined in older cultures by day 7, both in vitro and in vivo. A defective macrophage function in killing of M. tuberculosis bacilli was observed after 4 days of in vitro and in vivo cultures.     

Production of tumor necrosis factor alpha and interleukin-6 by alveolar macrophages from patients with rheumatoid arthritis and interstitial pulmonary disease

The aim of this study was to measure the production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by alveolar macrophages in patients with rheumatoid arthritis and interstitial lung disease (ILD). Rheumatoid arthritis patients diagnosed by ACR criteria (n = 8) with associated ILD documented by pulmonary function tests and high resolution computerized tomography scanning, and 12 healthy volunteers (6 smokers and 6 nonsmokers). We determined the spontaneous and induced production of bacterial lipopolysaccharides (LSP), TNF-alpha and IL-6 by alveolar macrophages obtained by bronchoalveolar lavage. The macrophages were isolated by Ficoll-Hypaque gradient centrifugation and plastic adherence and cultured in serum-containing medium (low endotoxin) in the presence and absence of LPS (100 ng/ml). TNF-alpha and IL-6 levels contents were determined in supernatants by ELISA. In the patient group both spontaneous and induced production of TNF-alpha were significantly higher than in controls (p < 0.01). Macrophages stimulated with LPS in patients with rheumatoid arthritis and ILD also released greater amounts of IL-6 than did those of the healthy controls. IL-6 spontaneous and induced production was significantly lower in smokers than in nonsmokers. TNF-alpha and IL-6 production in patients with rheumatoid arthritis and ILD, studied in bronchoalveolar lavage specimens reveals that alveolar macrophages are hyperreactive in these patients, who are possibly sensitized as a consequence of the inflammatory lung process inherent to the disease. Further study is needed to define the pathogenic role of these mediators.     

Murine peritoneal macrophages activated by the mycobacterial 65-kilodalton heat shock protein express enhanced microbicidal activity in vitro

After an intraperitoneal (i.p.) injection of purified protein derivative, peritoneal macrophages from mice infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) show an enhanced respiratory burst, inhibit the intracellular proliferation of Toxoplasma gondii, and kill Listeria monocytogenes more efficiently than peritoneal macrophages from normal mice. One of the immunodominant antigens of Mycobacterium spp. is the 65-kDa heat shock protein (Hsp 65), and in the present study, we determined whether injection of this protein into mice leads to activation of their peritoneal macrophages. After an i.p. injection of Hsp 65, peritoneal macrophages from BCG-infected CBA/J mice also released more H2O2, inhibited the proliferation of T. gondii, and killed L. monocytogenes faster than peritoneal macrophages from normal mice, although Hsp 65 was less effective than purified protein derivative. When normal mice were injected with Hsp 65 suspended in saline after a booster injection with Hsp 65, their macrophages did not display enhanced antimicrobial activity, indicating that an adjuvant was required for a cellular immune response against Hsp 65. In the present study, the adjuvant dimethyl dioctadecylammonium bromide (DDA) was preferred because it contains no endotoxin or mycobacterial antigens and because it has been reported that DDA does not induce the production of gamma interferon. Peritoneal macrophages from C57BL/6 and CBA/J mice that had received a subcutaneous injection of Hsp 65 suspended in DDA followed by an i.p. booster injection of Hsp 65 suspended in saline were activated, as indicated by the enhanced production of H2O2, inhibition of the intracellular proliferation of T. gondii, and increased rate of intracellular killing of L. monocytogenes in vitro relative to that by resident peritoneal macrophages and peritoneal macrophages obtained from mice that had received ovalbumin instead of Hsp 65. The rate of phagocytosis of L. monocytogenes was not affected by Hsp 65 treatment. Despite the in vitro expression of enhanced microbicidal activity of peritoneal macrophages, no difference in the growth of L. monocytogenes in the liver and spleen between Hsp 65-treated and control mice was found.     

Amphotericin B-induced resistance to Pseudomonas aeruginosa infection in mice

We evaluated the effects of amphotericin B (AmB) against Pseudomonas aeruginosa (P. aeruginosa) infection in mice. Pretreatment with 2 mg/kg of AmB 24 hours before infection significantly increased the survival rates of mice intraperitoneally infected with either P. aeruginosa or Escherichia coli. To evaluate the mechanism of this AmB-induced resistance to infection, we conducted a number of experiments. Peritoneal macrophages exposed in vitro to AmB showed superior bactericidal activity compared to that of control macrophages. Interleukin-1 production by peritoneal macrophages from mice pretreated with 2 mg/kg of AmB was significantly higher than that in control mice. Serum tumor necrosis factor level after intravenous injection of P. aeruginosa was also higher in mice pretreated with 2 mg/kg of AmB than in control mice. These data indicate that AmB induces resistance to P. aeruginosa in mice. Furthermore AmB-induced activation of peritoneal macrophages and their production of interleukin-1 and tumor necrosis factor appeared to play important roles in this phenomenon.