Gamma interferon mediates Propionibacterium acnes-induced hypersensitivity to lipopolysaccharide in mice.

Pretreatment of lipopolysaccharide (LPS)-responder C57BL/10ScSn mice with killed Propionibacterium acnes enhanced tumor necrosis factor alpha (TNF-alpha) production and lethality in response to a subsequent challenge with LPS. Sensitization to LPS increased with time of pretreatment and reached its maximum after 7 days. Sensitization was paralleled by gamma interferon (IFN-gamma) production that was detectable from day 3 onward. In contrast, a similar P. acnes pretreatment of LPS-nonresponder C57BL/10ScCr mice had no apparent effect on their high resistance to LPS. Challenge with LPS at any time during the 7-day period after P. acnes treatment led to no detectable TNF-alpha formation and caused no lethal effects. The absence of sensitization in C57BL/10ScCr mice was paralleled by an absence of IFN-gamma production. Administration of monoclonal IFN-gamma antibodies in C57BL/10ScSn mice up to day 3 of P. acnes treatment completely inhibited the overproduction of TNF-alpha by LPS. Anti-IFN-gamma administered later than day 3 had only a partial, although significant, inhibitory effect. Injection of appropriate amounts of anti-IFN-gamma also abolished the development of hypersensitivity to the lethal action of LPS. The effect of exogenously administered IFN-gamma on LPS sensitivity (e.g., TNF-alpha production, lethal effects) was studied in LPS-responder and nonresponder mice. Administration of murine recombinant IFN-gamma increased the sensitivity of C57BL/10ScSn mice to LPS and established LPS responsiveness in LPS-nonresponder C57BL/10ScCr and C3H/HeJ mice. The data provide evidence that IFN-gamma mediates the sensitization towards LPS induced by P. acnes.     

Effect of Corynebacterium acnes on interferon production in mouse peritoneal exudate cells.

Corynebacterium acnes, an organism closely related to C. parvum, has been recognized to have a striking effect on the reticuloendothelial system, as well as on both humoral and cellular immunity. In mice previously exposed to C. acnes, serum interferon levels induced by injection of Newcastle disease virus (NDV), Chikungunya virus (CV), and polyinosinic-polycytidylic acid are suppressed. When peritoneal macrophages and lymphocytes from animals exposed to C. acnes were cultivated in vitro, their capacity to produce interferon in response to NDV and CV was reduced. Furthermore, the interferon-producing capacity of these cells in tissue culture was inhibited after exposure to C. acnes to vitro. Exposure of separated populations of peritoneal macrophages and lymphocytes to C. acnes in vitro demonstrated that the interferon response to NDV by both cell types is inhibited. Peritoneal macrophages appear to be the major contributor to the interferon response in this system. Finally, this inhibitory effect was shown to occur after exposure to a purified cell wall preparation of C. acnes organisms, as well as a lipid extract of this preparation.     

Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells.

We examined the ability of interleukin-12 (IL-12) and IL-18 to induce the production of gamma interferon (IFN-gamma) and nitric oxide (NO) by murine peritoneal exudate cells (PEC) and to stimulate the growth-inhibitory activity of these cells against Cryptococcus neoformans. PEC produced IFN-gamma and NO when stimulated with a combination of IL-12 and IL-18 but little or no IFN-gamma or NO when either cytokine was used alone. PEC anticryptococcal activity was mediated by IFN-gamma and NO production, since it was completely inhibited by a neutralizing anti-IFN-gamma monoclonal antibody (MAb) and N(G)-monomethyl-L-arginine, a competitive inhibitor of NO synthesis, respectively. To identify the IFN-gamma-producing cells among PEC stimulated with IL-12 and IL-18, we depleted NK cells, gammadelta T cells, or CD4+ T cells by treating PEC with specific Abs and complement. NK cell depletion strongly suppressed IFN-gamma production and almost completely inhibited NO production and anticryptococcal activity, while depletion of other cells had no such influence. Alternatively, purified NK cells by two cycles of glass adherence and magnetic separation with anti-CD3, -CD4, -CD8, and -B220 MAbs produced a greater amount of IFN-gamma by stimulation with IL-12 and IL-18 than unseparated non-glass-adherent PEC. Our results demonstrated that IL-12 and IL-18 synergistically induced NO-dependent anticryptococcal activity of PEC by stimulating NK cells to produce IFN-gamma.     

Early gamma interferon production by natural killer cells is important in defense against murine listeriosis.

A spot enzyme-linked immunosorbent assay was used to show that subcutaneous inoculation of a sublethal number of Listeria monocytogenes resulted in the early appearance of gamma interferon (IFN-gamma)-producing cells in the draining lymph nodes. In contrast, inoculation of UV-killed L. monocytogenes failed to cause the appearance of IFN-gamma-producing cells. The appearance of IFN-gamma-secreting cells in response to the living organisms peaked at 24 h of infection and then declined. The draining lymph node cells responsible for secreting IFN-gamma belonged to a cell population that was positive for the NK1.1, asialo-GM1, and Thy-1 markers but negative for the CD4 and CD8 T cell subset markers. Early elimination of natural killer (NK) cells by treatment with anti-NK cell antibodies resulted in severe exacerbation of infection, as did early neutralization of endogenous IFN-gamma by treatment with a rat anti-murine IFN-gamma monoclonal antibody. In contrast, depletion of CD4+ and CD8+ T cells failed to exacerbate infection. The results serve to show that the early production of IFN-gamma by NK cells, rather than by T cells, is an essential event in resistance to listeriosis.     

Induction of tumoricidal peritoneal exudate cells by administration of Lactobacillus casei

The lifespan of mice into which Meth A fibrosarcoma was transplanted intraperitoneally (i.p.) was prolonged by i.p. treatment with Lactobacillus casei YIT 9018 (LC 9018). Treatment with LC9018 before the tumor inoculation (pre-treatment), was more effective than after the tumor inoculation (post-treatment). When tumor cells were inoculated subcutaneously along with peritoneal exudate cells (PEC) harvested from LC 9018-treated mice in the Winn type test, the tumor growth was significantly inhibited, but it was not inhibited by spleen cells. Adherent and nonadherent cells of LC 9018-induced PEC suppressed tumor growth in the Winn type test. PEC induced with LC 9018 showed significant cytolytic activity for 51Cr-labeled Meth A from 3 days to at least 2 weeks after a single injection of LC 9018. The adherent cells lysed tumor cells in the cytolytic test, though nonadherent cells did not show cytolytic activity. These results show that LC 9018 can induce two cell populations possessing the ability to kill tumor cells in vivo. One may be activated macrophages which directly kill tumor cells and the other may be T-lymphocytes which can induce cytotoxic cells.     

Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes.

Mycobacterium bovis BCG was genetically engineered to express and secrete mouse interleukin-2 (IL-2) and rat IL-2. Genes encoding IL-2 were inserted into an Escherichia coli-BCG shuttle plasmid under the control of the BCG HSP60 promoter. To facilitate study of proteins produced in this system, the IL-2 gene product was expressed (i) alone, (ii) with the mycobacterial alpha-antigen secretion signal sequence at the amino terminus, (iii) with an influenza virus hemagglutinin epitope tag at the amino terminus, and (iv) with both the secretion signal sequence and the epitope tag. When expressed with the alpha-antigen signal sequence, biologically active IL-2 was secreted into the extracellular medium. Western blot (immunoblot) analysis of the intracellular IL-2 and extracellular IL-2 revealed that the secretion signal was appropriately cleaved from the recombinant lymphokine upon secretion. To assess the ability of recombinant BCG to stimulate cytokine production in a splenocyte population, mouse splenocytes were cultured together with wild-type or IL-2-producing BCG. IL-2-secreting BCG clones stimulated substantial increases in gamma interferon production, which could be reproduced by the addition of exogenous IL-2 to BCG. Levels of IL-6, IL-10, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor were not significantly changed, while IL-4 and IL-5 remained undetectable (less than 50 pg/ml). The enhanced production of gamma interferon in response to IL-2-secreting BCG was strain independent. Recombinant BCG expressing mammalian cytokines provides a novel means to deliver cytokines and may augment the immunostimulatory properties of BCG in immunization and cancer therapy.     

Functions of uterine natural killer cells are mediated by interferon gamma production during murine pregnancy.

The dominant lymphocytes in healthy human and murine implantation sites are pregnancy-associated uterine natural killer (uNK) cells. These cells produce 90% of pregnancy-induced, uterine interferon (IFN)- gamma, a cytokine that regulates expression of more than 0.5% of the mouse genome. Implantation sites in uNK cell-deficient and IFN- gamma -signal-disrupted mice display anomalies in decidua and its spiral arteries. Reconstitution of uNK cell-deficient females with bone marrow containing normal NK cell progenitors, establishes uNK cells and reverses the anomalies. Grafts from IFN- gamma(-/-)mice are restored uNK cells, but the uNK cells did not reverse the phenotypes. This review focuses on the functions of uNK cell-derived IFN- gamma and the genes that it may regulate in the pregnant uterus.     

Induction of eotaxin production by interleukin-4, interleukin-13 and lipopolysaccharide by nasal fibroblasts

BACKGROUND: There is growing evidence that eotaxin is a key mediator in the development of tissue eosinophilia. Fibroblasts are a major source of eotaxin. The severity of diseases with eosinophilic inflammation like nasal polyposis, atopic dermatitis and asthma, where Th2-type cytokines (IL-4 and IL-13) and TGF-beta are expressed locally, was shown to correlate with bacterial factors such as lipopolysaccharide (LPS) rather than allergen. OBJECTIVE: We examined eotaxin production by nasal fibroblasts stimulated with IL-4 or IL-13 alone or in combination with LPS, and the effect of TGF-beta(1) on it. Moreover, we compared the magnitude of eotaxin produced by nasal fibroblasts with that produced by lung or skin fibroblasts. METHODS: Fibroblast lines were established from human biopsy tissue. The expression of eotaxin mRNA was evaluated by RT-PCR. The amount of eotaxin in the supernatants was measured by ELISA. RESULTS: IL-4, but not IL-13, synergized with LPS to produce eotaxin in a dose- and time-dependent manner. Sequential treatment of nasal fibroblasts with IL-4 and LPS did not have any effect. But when IL-4 and LPS were added together, synergy for eotaxin production was observed. Moreover, this synergy was observed in nasal and skin fibroblasts, but not in lung fibroblasts. The production of eotaxin by IL-4 and LPS was modulated by TGF-beta(1). CONCLUSION: These results suggest that a co-stimulus like LPS is necessary for IL-4 to make a strong induction of eotaxin in eosinophilic inflammations such as nasal polyposis. Modulation by TGF-beta(1) may have important implications for the development of eosinophilic inflammation.     

Confirmation of destruction of salmonellae within murine peritoneal exudate cells by immunocytochemical technique.

A procedure was developed with which peritoneal exudate cell (PEC) preparations were fixed in a glutaraldehyde-picric acid mixture, post-fixed with osmium tetroxide, embedded in LR White resin and then stained with immunogold probe. It provided tissue sections showing both well-defined ultrastructures as well as specifically labelled Salmonella O antigens by electron microscopy. Inbred, male C57BL/6 mice were injected intraperitoneally with 2 x 10(7) virulent Salmonella typhimurium. Peritoneal exudate cells were harvested at 16 and 20 hr after infection. Disintegrating intracellular bacteria were identified as salmonellae by the immunogold markers. Deposition of gold particles in the cytoplasm of phagocytes also indicated that intracellular debris contained digested pathogen. This investigation therefore confirms previous findings of the destruction of salmonellae within inflammatory polymorphs and macrophages.     

In vitro activation of murine peritoneal exudate cells (PEC) and peritoneal macrophages by ST 789.

ST 789 is a new synthetic compound characterized by an amino acidic group joined to the N9 position of the hypoxanthine ring, which has been shown recently to have immunomodulating properties and minimal toxicity. The drug has been reported to protect immunosuppressed mice from microbial infections and tumour growth, and to restore the mitogen-induced proliferation of splenocytes from immunosuppressed young mice. In this study, we show that in vitro addition of ST 789 is able to markedly augment the sheep red blood cells (SRBC) phagocytosis by PEC, and to potentiate the cytotoxic activity of peritoneal exudate (PE) macrophages (M phi) vs the L-M tumour cell line. We also found that ST 789 enhanced the rIFN-gamma-induced NO2- release from cultured PE M phi. Similarly, in vitro addition of ST 789 to the latter cultures significantly increased the production of interleukin 1 (IL-1) and tumour necrosis factor (TNF) induced by lipopolysaccharide (LPS). These studies demonstrate that ST 789 is a potent phagocyte activator for the induction of cytokine release, phagocytosis and cytotoxic activity against tumour cells in vitro.     

Induction of interferon synthesis and cytotoxicity by murine peritoneal macrophages exposed to glycoprotein ligands.

Thioglycollate-induced murine C57BL/6 and C3H/HeN peritoneal macrophages synthesized interferon-beta (IFN-beta) in response to exposure to glycoproteins such as horseradish peroxidase (HRP) or mannosyl or fucosyl bovine serum albumin (BSAman of BSAfuc, respectively), but not glucosylated or galactosylated BSA (BSAglu or BSAgal, respectively). These results suggest participation of the mannosyl-fucosyl receptor (MFR) in this response. IFN synthesis was augmented by culturing macrophages in L cell-conditioned medium prior to exposure to these substances. Macrophages obtained from lipopolysaccharide (LPS)-resistant C3H/HeJ mice did not produce IFN in response to HRP. Furthermore, IFN-induction by HRP was blocked by polymyxin B. In addition, exposure of macrophages to HRP or BSAman induced cytotoxicity against NIH 3T12 cells. Cytotoxicity was not inhibited by the presence of anti-IFN-alpha/beta. In contrast to IFN induction, however, macrophages activation was LPS-independent, since this activity was demonstrated in macrophages from C3H/Hej mice. The carbohydrate specificity of these responses suggests that the MFR or an another scavenger receptor may be involved in the responses to these substances, and that cytotoxicity and IFN-induction by glycoproteins follow unique pathways.     

Lipopolysaccharide and monophosphoryl lipid A differentially regulate interleukin-12, gamma interferon, and interleukin-10 mRNA production in murine macrophages.

Monophosphoryl lipid A (MPL) is a nontoxic derivative of the lipid A region of lipopolysaccharide (LPS) that is being developed as both an adjuvant and prophylactic drug for septic shock. We compared the ability of LPS and MPL to induce interleukin-10 (IL-10), IL-12 p35, IL-12 p40, gamma interferon (IFN-gamma), glucocorticoid receptor (GR), IL-1 receptor antagonist (IL-1ra), and inducible nitric oxide synthase mRNA expression in murine peritoneal macrophages. These genes were chosen for their ability to positively or negatively regulate the host immune response and thus for their potential involvement in MPL-induced adjuvanticity or in its ability to protect against sepsis. LPS was a more potent inducer of IL-12 p35, IL-12 p40, and IFN-gamma mRNA, as well as of IL-12 protein, than MPL. In contrast, MPL induced higher levels of IL-10 mRNA than did LPS from 1 to 1,000 ng/ml. In general, MPL was not a more potent inducer of negative regulatory genes, since MPL and LPS induced similar levels of GR and IL-1ra mRNA. Addition of anti-IL-10 antibody to cultures increased the induction of MPL-induced IL-12 p35, IL-12 p40, and IFN-gamma mRNA, suggesting that the enhanced production of IL-10 by MPL-stimulated macrophages contributes to decreased production of mRNA for IL-12 (p35 and p40) and IFN-gamma. Conversely, the addition of exogenous IL-10 to LPS-treated macrophages reduced the mRNA expression of these cytokine genes. These studies suggest that enhanced production of IL-10 by MPL-stimulated macrophages may contribute to the reduced toxicity of MPL through its negative action on induction of cytokines shown to enhance endotoxicity.     

Regulation of insulin and interleukin-1 release after Propionibacterium acnes-induced macrophage activation in mice

The administration of a potent activator of macrophages (M phi), Propionibacterium acnes, in nondiabetic mice was associated with the release of significant amounts of interleukin-1 (IL-1) in the peritoneal cavity and plasma within 4 hours after treatment. Shortly before IL-1 peaks were observed, the levels of pancreatic insulin, [3H]leucine-proinsulin, and insulin/total protein ratio were elevated, and followed by a transient but marked hyperinsulinemia at 4 hours after treatment. A single dose of recombinant murine IL-1 in mice was also associated with a 2- to 9-fold increase in the levels of insulin in the pancreas and plasma at 4 hours after treatment. During the period of observation after the administration of P. acnes, plasma glucose levels in treated mice were significantly less than in parallel controls. Mild hypoglycemia was observed at 7 to 10 days posttreatment. Although circulating IL-1-like activity could not be detected in plasma 1 to 10 days after P. acnes treatment, this activity was measured in activated peritoneal and liver M phi. IL-1-like activity (specific activity: 276 units/mg protein) was detected in plasma, after it was chromatographed on a Sephadex G-150 column to remove proteins with higher molecular weight. Peritoneal and liver M phi from P. acnes mice were also able to elaborate significant amounts of IL-1-like activity in their supernatants with or without Escherichia coli lipopolysaccharide. At the same time, total protein synthesis and insulin content in the pancreas in P. acnes mice were significantly lower than the parallel control (p less than 0.01). These results suggest that P. acnes-induced M phi activation in mice was associated with the modulation of insulin release and glucose homeostasis which may be attributed to the accumulation and release of IL-1 by activated M phi.     

Induction of interleukin-1 from murine peritoneal macrophages by Pseudomonas aeruginosa exotoxin A.

Pseudomonas exotoxin A, an ADP-ribosylating toxin produced by Pseudomonas aeruginosa, has been shown to stimulate the proliferation of murine thymocytes, which requires the participation of accessory cells. This requirement for accessory cells can be replaced by supernatant from adherent peritoneal exudate cells that have been stimulated with exotoxin A. Antibody to exotoxin A inhibits the induction of the thymocyte mitogenic activity from adherent peritoneal macrophages. However, antibody to exotoxin A had no effect on the thymocyte proliferation if the antibody was added to supernatant which contained thymocyte mitogenic activity. The thymocyte mitogenic activity was associated with a protein or protein complex with a molecular mass of greater than 10,000 daltons. D10 bioassays indicated the presence of interleukin-1 (IL-1) in the supernatant. Antibody to IL-1 inhibited the ability of supernatant to induce thymocytes to proliferate. Therefore, these data suggest that Pseudomonas exotoxin A can stimulate the production of IL-1 from adherent peritoneal cells, which induces murine thymocytes to proliferate.     

Effect of gamma interferon on hydrogen peroxide production by cultured mouse peritoneal macrophages.

Macrophage activation is thought to be mediated via a number of T-lymphocyte products, including gamma interferon (IFN-gamma). However, our studies indicate that IFN-gamma acts as a regulator molecule rather than solely as an activator. This depends upon the status of the macrophage. IFN-gamma treatment of resting macrophages and those activated or elicited by sodium caseinate, lipopolysaccharide, or Mycobacterium bovis BCG did not result in activation, as measured by hydrogen peroxide production; however, when thioglycolate was used as an eliciting agent, incubation with IFN-gamma resulted in a dramatic increase in hydrogen peroxide production compared with that by untreated controls.     

Roles of interleukin-12 and gamma interferon in murine Chlamydia pneumoniae infection

BALB/c and strain 129 mice infected intranasally with Chlamydia pneumoniae displayed a moderate-to-severe inflammation in the lungs and produced interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10, with peak levels on days 1 to 3 postinfection (p.i.), returning to basal levels by day 16 p.i. Anti-IL-12 treatment resulted in less-severe pathological changes but higher bacterial titers on days 3 and 7 p.i. By day 16 p.i., the inflammatory responses of control antibody-treated mice subsided. The bacterial titers of both anti-IL-12- and control antibody-treated mice decreased within 3 weeks to marginally detectable levels. Anti-IL-12 treatment significantly reduced lung IFN-gamma production and in vitro spleen cell IFN-gamma production in response to either C. pneumoniae or concanavalin A. In gamma-irradiated infected mice, cytokine production was delayed, and this delay correlated with high bacterial titers in the lungs. Following C. pneumoniae infection, 129 mice lacking the IFN-gamma receptor alpha chain gene (G129 mice) produced similar IL-12 levels and exhibited similarly severe pathological changes but had higher bacterial titers than 129 mice. However, by day 45 p.i., bacterial titers became undetectable in both wild-type 129 and G129 mice. Thus, during C. pneumoniae lung infection, IL-12, more than IFN-gamma, plays a role in pulmonary-cell infiltration. IFN-gamma and IL-12, acting mostly through its induction of IFN-gamma and Th1 responses, play an important role in controlling acute C. pneumoniae infection in the lungs, but eventually all mice control the infection to undetectable levels by IL-12- and IFN-gamma-independent mechanisms.     

Dissociated production of interleukin-2 and immune (gamma) interferon by phytohaemagglutinin stimulated lymphocytes in healthy infants.

Cord blood lymphocytes were stimulated with phytohaemagglutinin (PHA) to produce interleukin-2 (IL-2) and immune interferon (IFN-gamma). On PHA stimulation, cord blood lymphocytes produced efficiently IL-2 as much as adult ones. Antiviral activity generated on PHA stimulation was shown to consist mainly of IFN-gamma as assessed by the sensitivity to pH 2.0 treatment and neutralization with anti-human IFN-gamma antibody. In contrast to IL-2 production, cord blood lymphocytes released extremely low levels of IFN-gamma following PHA stimulation. The producing ability of IFN-gamma by lymphocytes on PHA stimulation gradually increased with child growth, but was significantly low at 1-2 years of age as compared with adult controls. Around 3 years of age or later, the producing ability of IFN-gamma by lymphocytes on PHA stimulation attained levels comparable to those of adult cells. These results suggested that IL-2 producing ability of lymphocytes appeared to be at a mature stage at birth, whereas lymphocytes in the early human life might be relatively deficient in their ability to produce IFN-gamma.