Protection of mice against the intracellular bacterium Listeria monocytogenes by recombinant immune interferon

Immune interferon, available at high specific activity through recombinant DNA technology, is known to activate macrophages to intra- and extracellular cytotoxicity. We now report that murine recombinant IFN-gamma activates macrophages to cytotoxicity also when applied in vivo. Furthermore, recombinant IFN-gamma can protect mice in vivo against the intracellular bacterial pathogen Listeria monocytogenes in a local as well as in a systemic infection model. The role of T lymphocyte-produced lymphokines in acquired resistance to facultative intracellular pathogens and their possible involvement in novel immunotherapy are discussed.     

Hemolysin supports survival but not entry of the intracellular bacterium Listeria monocytogenes.

The gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen. The only known property of L. monocytogenes which has been shown to be involved in virulence is a hemolysin, listeriolysin (J. L. Gaillard, P. Berche, and P. Sansonetti, Infect. Immun. 52:50-55, 1986; S. Kathariou, P. Metz, H. Hof, and W. Goebel, J. Bacteriol. 169:1291-1297, 1987). Using our previously obtained transposon Tn916-induced hemolysin-negative mutants of L. monocytogenes Sv1/2a (Mackaness strain), we demonstrated that the loss of hemolysin reduced significantly the rate of survival of the bacteria in mouse peritoneal macrophages but did not reduce their uptake. It was further shown that virulent L. monocytogenes strains could invade the mouse embryo fibroblast 3T6 cell line, i.e., mammalian cells which are nonprofessional phagocytes. This uptake was inhibited by cytochalasin B and hence seems to be accomplished by parasite-induced endocytosis. Hemolysin was not essential for this step. Strains of other Listeria species could not efficiently penetrate the 3T6 cells.     

Treatment of mice with human recombinant interleukin-2 augments resistance to the facultative intracellular pathogen Listeria monocytogenes.

The effects of exogenously administered human recombinant IL-2 (hrIL-2) on resistance to Listeria monocytogenes infection were examined. Intravenous injection of hrIL-2 significantly enhanced antibacterial resistance in both BDF1 and C3H/HeJ mice. The beneficial effect of hrIL-2 was observed with as little as 0.6 micrograms per mouse, whereas optimum protection occurred at 6 micrograms per mouse, hrIL-2 was equally protective when administered concomitant with the listeriae or up to 24 h prior to infection; it had little effect if given after the bacterial challenge. Kinetic experiments indicated that both the peak bacterial burden and the time lag before L. monocytogenes began to be cleared from the spleen and liver were reduced in hrIL-2-treated mice as compared with control mice. Histopathological examination of spleens and livers confirmed that hrIL-2-treated Listeria-infected mice experienced considerably less damage to these organs than did control mice. Spleen cells from Listeria-infected mice exhibited depressed levels of mitogen-induced proliferation coincident with the peak bacterial burden in the spleen and liver and during the subsequent recovery from the infection. Administration of hrIL-2 to uninfected mice had no effect on spleen cell proliferation in response to mitogens in vitro, nor did hrIL-2 treatment restore normal levels of splenocyte proliferative responses to Listeria-infected mice. In addition, hrIL-2 treatment resulted in attenuated levels of serum colony-stimulating activity in infected mice as compared with control infected mice. Coadministration of both hrIL-2 and human recombinant interleukin-1 alpha at various dose and time combinations had no detectable additive or synergistic effect. Although these data do not suggest an obvious mechanism of action, they clearly demonstrate that hrIL-2 can augment host defense against the facultative intracellular pathogen L. monocytogenes.     

Activation of mouse peritoneal cells to kill Listeria monocytogenes by T-lymphocyte products.

An in vitro system has been used to demonstrate that glass-adherent mouse peritoneal cells can be activated to kill intracellular Listeria monocytogenes by antigen-stimulated T-lymphocytes derived from immunized mice. The soluble products of such stimulated lymphocyte cultures could only be shown to similarly activate peritoneal cells if the antigen used in both the immunization and lymphocyte stimulation was also present on the target intracellular organism.     

Fungicidal activation of murine macrophages by recombinant gamma interferon.

Alveolar macrophages from BALB/c mice readily phagocytized endospores (2 to 5 micron) and arthroconidia of Coccidioides immitis in vitro. Within 24 to 30 h at 37 degrees C, the phagocytized endospores started developing into spherules, and the arthroconidia formed germ tubes and hyphae. However, these processes did not occur if the macrophages were incubated with murine recombinant gamma interferon (rIFN-gamma) during infection with C. immitis. Treatment with rIFN-gamma activated the fungicidal capabilities of the alveolar macrophages, as evidenced by the 50% reduction in the CFU which could be recovered from macrophages infected in the presence of gamma interferon compared with alveolar macrophages infected without gamma interferon (P less than 0.05). Similar results were seen with peritoneal macrophages incubated with rIFN-gamma and infected with C. immitis. As little as 10 U of rIFN-gamma per ml reduced by half the number of C. immitis CFU which could be recovered from the phagocytes 8 h after infection with arthroconidia, although interferon alone did not affect the viability of the fungi.     

Mechanism for candidacidal activity in macrophages activated by recombinant gamma interferon.

Candidacidal activity in macrophages activated by recombinant gamma interferon was examined kinetically in relation to acidification of phagolysosomes. In resident peritoneal macrophages (PMPs) of BALB/c mice, enhanced killing activity against Candida albicans was demonstrated after incubation with 100 U of gamma interferon per ml for 24 h but not after incubation for 48 to 72 h. Conversely, increased generation of H2O2 was exhibited in PMPs incubated from 48 to 72 h but not in PMPs incubated for 24 h. In normal PMPs, fusion of lysosomes to candida-containing phagosomes was readily accomplished and phagosome-lysosome fusion was not enhanced further by activation. The candidacidal substance was extracted from granule-rich fractions of either normal or activated PMPs by using citric acid (pH 2.7) in equal amounts; the substance showed a noncationic, heat-stable protein nature. In addition, when phagolysosomal pH was determined by flow cytometry of intraphagolysosomal fluorescein isothiocyanate-labeled C. albicans, phagolysosomes with low pH (less than 4.0) were detected in about 40% of PMPs activated for 24 h but not in those activated for 72 h or in normal PMPs. Moreover, increasing the intralysosomal pH with NH4Cl resulted in a significant reduction of candidacidal activity in activated PMPs. These results indicate that the candidacidal activity of gamma interferon-activated PMPs correlates well with enhanced acidification of their phagolysosomes and suggest that the candidacidal activity of activated PMPs is independent from reactive oxygen molecules and is mediated by proteinaceous substance(s) generated only in a strong acidic milieu of phagolysosomes by activation.     

Capacity of recombinant gamma interferon to activate macrophages for Salmonella-killing activity.

The ability of recombinant gamma interferon (rIFN-gamma) to activate macrophages for Salmonella-killing activity was kinetically examined in relation to phagosome-lysosome fusion and H2O2 generation. Resident peritoneal macrophages of BALB/c mice incubated with 10(2) to 10(3) U of rIFN-gamma per ml for 12 h exhibited enhanced bactericidal activity against Salmonella typhimurium, although H2O2 generation was unaltered. In contrast, macrophages incubated with equal doses of rIFN-gamma for 48 h showed both an enhanced Salmonella-killing activity and an increased generation of H2O2. To evaluate Salmonella-killing activities of macrophages, intracellular bacteria were assayed at 0, 2, and 8 h after infection. During the initial 2 h of infection, 12-h-activated macrophages, as well as the unstimulated control macrophages, showed a decline in bacterial population at the same rate. Over the next 6 h of infection, however, the number of viable bacteria in activated macrophages remained unchanged, whereas the number of bacteria in control macrophages significantly (P less than 0.05) increased. Similar results were obtained in 48-h-activated macrophages. On the other hand, macrophages incubated with 10 to 10(3) U of rIFN-gamma exhibited enhanced fusion of lysosomes to Salmonella-containing phagosomes in both the 12-h- and 48-h-stimulated stages. Moreover, when 48-h-activated macrophages were incubated concomitantly with superoxide dismutase and catalase, Salmonella-killing activity was not affected. These results indicate that rIFN-gamma per se is able to activate peritoneal macrophages to induce Salmonella-killing activity and suggest that increased phagosome-lysosome fusion followed by an oxygen-independent killing mechanism is primarily responsible for the enhanced Salmonella-killing activity in rIFN-gamma-activated macrophages.     

Synthesis and secretion of interferon by murine fibroblasts in response to intracellular Listeria monocytogenes.

Listeria monocytogenes, a gram-positive facultative intracellular bacterium, was shown to be capable of infecting and proliferating in murine embryo fibroblasts. During exponential proliferation, the doubling time of the bacterium was determined to be 2.5 h intracellularly, compared with 25 min extracellularly. Progressive intracellular growth of listeriae ultimately resulted in the destruction of initially infected cells and the spread of infection to neighboring cells. Listeria infection induced fibroblasts to synthesize considerable quantities of an acid-stable interferon that proved to be antigenically indistinguishable from both polyinosinic-polycytidylic acid-induced and virus-induced interferon.     

Mycobacterium leprae-burdened macrophages are refractory to activation by gamma interferon

Mycobacterium leprae grows to enormous numbers in the nu/nu mouse footpad, producing granulomas resembling those of lepromatous leprosy in humans. Footpad granuloma cells gorged with M. leprae were established in primary cell culture to examine their functional capabilities. These cells were classified as macrophages by the following criteria: positive staining for nonspecific esterase, reduction of Nitro Blue Tetrazolium during phagocytosis of Candida albicans, possession of Fc receptors, and possession of Mac-1 antigen. Footpad macrophages also phagocytized and supported the intracellular growth of Toxoplasma gondii. However, unlike peritoneal macrophages, footpad macrophages could not be activated to kill or inhibit T. gondii by macrophage-activating factor produced by mitogen-stimulated spleen cells or by recombinant gamma interferon. Thus, although the lepromatous macrophages appeared to be normal in many of their functions, they were defective in response to macrophage-activating signals.     

Human monocyte-derived macrophages are lysed by schistosomula of Schistosoma mansoni and fail to kill the parasite after activation with interferon gamma.

In this study was examined the interaction between schistosomula of Schistosoma mansoni and human monocyte-derived macrophages activated with interferon gamma (IFN-gamma). Peripheral blood monocytes were matured for 6 days and activated by further culture with IFN-gamma (600 U/ml). These IFN-gamma-treated monocyte-derived macrophages are cytotoxic for the tumor cell line K562, which is not killed by nonactivated monocyte-derived macrophages. Activated monocyte-derived macrophages were incubated with schistosomula at ratios of 10(3):1 and 10(4):1 in the presence of serum pooled from patients with schistosomiasis. This antiserum promoted an increased adherence of cells to the parasite. However, the activated monocyte-derived macrophages failed to kill the schistosomula under all conditions tested. On the contrary, the monocyte-derived macrophages were killed by schistosomula in a time-dependent and antibody-dependent manner, which was most evident at a lower effector/target ratio, 200:1. Electron microscopy showed that monocyte-derived macrophages were lysed on the surface of schistosomula. Further, both monocyte-derived macrophages and contaminating blood platelets fused with the parasite surface membrane, so that the cell plasma membrane and the outer tegumental membrane formed a hybrid membrane. The results indicate that matured human monocyte-derived macrophages activated by IFN-gamma are unable to kill schistosomula. Instead, the effector cells fuse with the parasites and are lysed by them.     

L-arginine-dependent killing of intracellular Ehrlichia risticii by macrophages treated with gamma interferon.

Thioglycolate-induced murine peritoneal macrophages infected with Ehrlichia risticii and treated in vitro with gamma interferon (IFN-gamma) developed antiehrlichial activity that eliminated the intracellular bacteria. This antiehrlichial activity was suppressed by NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthesis from L-arginine, but not by L-tryptophan. Increased levels of nitrite, an oxidative product of nitric oxide, were measured in cultures of infected macrophages treated with IFN-gamma. Sodium nitroprusside, which spontaneously releases nitric oxide, also showed the antiehrlichial activity. The antiehrlichial activity by reactive nitrogen intermediates was not mediated by elevation of the cellular concentration of cyclic GMP since the addition of 8-bromo-cyclic GMP itself had no influence on ehrlichial infection of macrophages. Addition of the intracellular iron chelator deferoxamine also inhibited E. risticii infection in vitro. These results suggest that intracellular E. risticii survival is iron dependent and that production of reactive nitrogen intermediates triggers iron loss from critical target enzymes of E. risticii, leading to lethal metabolic inhibition. However, addition of excess FeSO4, ferric citrate, or iron-saturated transferrin did not counteract the antiehrlichial effect induced by IFN-gamma.     

Recombinant murine gamma interferon induces enhanced resistance to Listeria monocytogenes infection in neonatal mice.

Neonatal mice within 24 h of birth were highly susceptible to intraperitoneal infection with Listeria monocytogenes. The 50% lethal dose of bacterial cells was 6.3 X 10(1) CFU for neonates and 3.2 X 10(6) CFU for adult mice. A single injection of recombinant murine gamma interferon (rMuIFN-gamma) protected neonatal mice from simultaneous challenge with a lethal dose of L. monocytogenes cells. The rMuIFN-gamma effect was dose dependent: protection was consistently observed in mice treated with rMuIFN-gamma at doses of more than 4 X 10(2) U (0.1 microgram of protein) per mouse. Bacterial growth in the spleens and livers of rMuIFN-gamma-treated neonates was significantly suppressed in comparison with that in the nontreated controls. The infected neonatal mice showed acquired antilisterial resistance against secondary intravenous infection after 4 weeks of age, and this resistance was significantly augmented in mice that had been treated with rMuIFN-gamma.     

Inhibition of the intracellular growth of Histoplasma capsulatum by recombinant murine gamma interferon.

Recombinant murine gamma interferon as well as lymphokines prepared from immune splenocytes and concanavalin A-stimulated T-cell hybridoma activated normal mouse peritoneal macrophages to inhibit the intracellular growth of Histoplasma capsulatum. The activities of the lymphokine from immune splenocytes and of recombinant murine gamma interferon were neutralized by rabbit anti-murine gamma interferon antibody. The intracellular yeasts were not killed by the interaction even though growth was completely inhibited.     

Endogenous tumor necrosis factor, interleukin-6, and gamma interferon levels during Listeria monocytogenes infection in mice.

Mice were infected intravenously with a sublethal dose of Listeria monocytogenes cells and then levels of tumor necrosis factor (TNF), interleukin-6 (IL-6), and gamma interferon (IFN-gamma) in the bloodstreams, spleens, and livers were monitored. The maximum level of TNF was detected at 72 h in the spleens and livers, but TNF was never detected in the bloodstreams. IL-6 appeared in the bloodstreams and spleens and peaked at 48 h. The maximum level of IFN-gamma could be detected in all three specimens, and the highest titer was shown in the spleens. Endogenous TNF production was suppressed by in vivo administration of anti-CD4 monoclonal antibody (MAb) or anti-asialo GM1 antibody but not by anti-CD8 MAb, whereas none of these antibodies suppressed endogenous IL-6 production. Endogenous production of neither IL-6 nor IFN-gamma was inhibited in rabbit anti-recombinant mouse TNF-alpha antibody-treated mice. Similarly, production of TNF and IL-6 did not decrease in anti-mouse IFN-gamma MAb-treated animals, but TNF production was augmented in these animals. These results suggest that the these endogenous cytokines are produced by different mechanisms in L. monocytogenes infection.     

The Listeria monocytogenes lemA gene product is not required for intracellular infection or to activate fMIGWII-specific T cells

Clearance of the intracellular bacterial pathogen Listeria monocytogenes requires antigen-specific CD8(+) T cells. Recently it was shown that activation of class Ib major histocompatibility complex (MHC)-restricted CD8(+) T cells alone is sufficient for immune protection against listeriae. A major component of the class Ib MHC-restricted T-cell response is T cells that recognize formylated peptide antigens presented by M3 molecules. Although three N-formylated peptides derived from L. monocytogenes are known to bind to M3 molecules, fMIGWII is the immunodominant epitope presented by M3 during infection of mice. The source of fMIGWII peptide is the L. monocytogenes lemA gene, which encodes a 30-kDa protein of unknown function. In this report, we describe the generation of two L. monocytogenes lemA deletion mutants. We show that lemA is not required for growth of listeriae in tissue culture cells or for virulence during infection of mice. Surprisingly, we found that fMIGWII-specific T cells were still primed following infection with lemA mutant listeriae, suggesting that L. monocytogenes contains at least one additional antigen that is cross-reactive with the fMIGWII epitope. This cross-reactive antigen appears to be a small protease-resistant molecule that is secreted by L. monocytogenes.     

Prevention by gamma interferon of fatal infection with Listeria monocytogenes in mice treated with cyclosporin A.

The significance of interferons (IFNs) induced by Listeria monocytogenes in the antilisterial defense mechanism was studied in mice. Cyclosporin A (CsA) had no effect on IFN-alpha production that was induced in the bloodstream after intravenous infection of mice with L. monocytogenes, whereas IFN-gamma that was induced in the bloodstreams of control mice 6 h after stimulation with specific antigen in the late phase of infection was suppressed in CsA-treated mice, depending on the dose of the drug injected. The decrease in IFN-gamma production caused an increase in bacterial growth in the spleens and livers of CsA-treated mice. Furthermore, administration of a daily dose of CsA at 80 or 100 mg/kg of body weight resulted in fatal listeriosis, even though the dose was nonlethal for normal mice. The administration of recombinant murine IFN-gamma on day 0 of L. monocytogenes infection prevented CsA-treated mice from developing fatal listeriosis and restored their ability to produce IFN-gamma in the bloodstream, in response to specific antigen in the late phase of infection.     

Effects of stimulated or immunologically activated macrophages on the induction of immune responses to Listeria monocytogenes

The influences of peritoneal macrophages induced by proteose peptone, Corynebacterium parvum (C. parvum) or Bacillus Calmette Guérin (BCG) on the initiation and development of immune responses and protection against Listeria monocytogenes infection were studied in mice. Mice treated intraperitoneally (i.p.) with proteose peptone 4 days previously showed much the same level of protection against an intraperitoneal infection with Listeria as untreated mice. Mice treated i.p. with C. parvum 4 days previously, of which peritoneal macrophages had increased abilities for intracellular killing of Listeria and O2- generation as compared with peptone-elicited macrophages, exhibited an enhanced resistance against the listerial infection. The degree of immune responses, as assessed by delayed footpad reaction (DFR), was rather depressed in these mice because C. parvum-activated macrophages acting as scavenger cells reduced the amount of effective antigenic stimulation. BCG-activated peritoneal macrophages from mice treated i.p. with BCG 14 days previously showed a strong ability for antigen presentation in correlation with increases in the number of Ia-bearing macrophages and in the level of interleukin 1 (IL 1) production. These mice showed an early appearance of DFR response and a markedly enhanced resistance against the listerial infection. These results suggested that the differences in macrophage activities as scavenger cells, cytokine-secreting cells and antigen presenting cells may account for the differences in the responsiveness against listerial infection in peptone-, C. parvum- and BCG-treated mice.     

Interactions between endogenous gamma interferon and tumor necrosis factor in host resistance against primary and secondary Listeria monocytogenes infections.

Intravenous injection of rat anti-mouse gamma interferon (IFN-gamma) monoclonal antibody as well as rabbit anti-mouse tumor necrosis factor (TNF) antibody into mice which had received a sublethal infection with Listeria monocytogenes cells resulted in acceleration of listeriosis. Endogenous IFN-gamma seemed to be produced early in infection, because suppression of antilisterial resistance was significant when a single injection of anti-IFN-gamma monoclonal antibody was given on day 0 or day 1 of infection. Production of TNF but not of IFN-gamma in the bloodstream early in infection was inhibited by administration of anti-IFN-gamma monoclonal antibody. The suppressive effect of anti-IFN-gamma and anti-TNF antibodies on antilisterial resistance was not augmented by simultaneous administration of these antibodies. On the other hand, in the secondary infection, simultaneous administration of anti-IFN-gamma and anti-TNF antibodies, but not of either of these antibodies alone, into L. monocytogenes-immune mice resulted in high mortality and explosive multiplication of bacterial cells in the spleens and livers. These results suggest that endogenously produced IFN-gamma and TNF are both essential to the host defense against L. monocytogenes infection and that these cytokines might act by different modes between the primary infection and the secondary infection.     

Protection of mice against Listeria monocytogenes infection by recombinant human tumor necrosis factor alpha.

Recombinant human tumor necrosis factor alpha (rHuTNF-alpha) administered intravenously to mice resulted in enhanced resistance to a lethal challenge infection of Listeria monocytogenes given 24 h later. The observed protection was lost following treatment of the rHuTNF-alpha preparations with rabbit polyclonal antibody rHuTNF-alpha but not with normal rabbit immunoglobulin G.