Augmentation of host defense against bacterial infection pretreated intraperitoneally with an alpha-glucan RBS in mice

Protection against Listeria monocytogenes and Escherichia coli in mice was enhanced by an intraperitoneal (i.p.) administration of polysaccharide "RBS". Peritoneal macrophages from mice administered i.p. with 30 mg/kg doses of RBS 4 days earlier exhibited increased scavenger functions as assessed by in vivo phagocytosis, in vitro intracellular killing and generation of superoxide anion. When cytokine production of the macrophages was assessed by biological assay and Northern blotting analysis, interleukin (IL)-1 production and IL-1 alpha gene expression were significantly increased in macrophages from RBS-treated mice. On the other hand, tumor necrosis factor (TNF) alpha gene was expressed in macrophages from RBS-treated mice at a much reduced level as compared with those in mice treated i.p. with Corynebacterium parvum on 4 days earlier. In correlation with expression of TNF gene in the macrophages, RBS-treated mice were less susceptible to the lethal toxicity of LPS than C.parvum-treated mice. In RBS-treated mice, in vivo elimination of bacteria was enhanced at the early phase of infection with L.monocytogenes or E.coli, resulting in augmentation of host defense against these bacterial infection. These results suggest that adequately enhanced activities of macrophages acting as scavenger phagocytes play important roles in the enhanced resistance against bacteria in mice treated i.p. with RBS.     

Monocytes of individual human subjects display heterogeneous bacterial uptake and antilisterial activity.

Peripheral blood monocytes (Mo) of normal human donors simultaneously exhibit two subsets differing in their functional activity towards the facultative intracellular bacterium Listeria monocytogenes. One subset (on average, 25% of total Mo) was characteristically able to ingest a large number of L. monocytogenes bacteria and permitted intracellular growth of these bacteria. The other Mo subpopulation (on average, 75% of total Mo) was far less active in phagocytosing L. monocytogenes and restricted intracellular L. monocytogenes growth. Electron microscopy revealed that the Listeria-permissive Mo subset allowed the bacteria to escape to the cytosol, a mechanism by which these bacteria evade the lethal attack of phagocytes. The Listeria-restrictive Mo subset, on the other hand, confined the bacteria to the phagolysosomes, where they were exposed to the killing mechanisms of the Mo. Permissiveness for L. monocytogenes growth was further associated with differences in the capacity of the Mo subsets to synthesize tumor necrosis factor alpha TNF-alpha), an important mediator in the defense against intracellular bacteria. Following challenge with L. monocytogenes, the Listeria-restrictive Mo subset secreted two to six times more TNF-alpha than did the Listeria-permissive Mo subset. Enhanced TNF-alpha secretion was paralleled by increased accumulation of TNF-alpha mRNA as assessed by quantitative PCR. Despite these functional differences, the two Mo subsets were indistinguishable with respect to expression of cell surface markers known to be involved in adherence and phagocytosis of microbes. A speculative physiological role of the two Mo subsets may lie in the dual function of Mo as microbicidal effector cells and accessory cells for antigen-specific immune reactions.     

Immunostimulating properties of intragastrically administered Acetobacter-derived soluble branched (1,4)-beta-D-glucans decrease murine susceptibility to Listeria monocytogenes

We previously found that AC-1, an extracellular polysaccharide, produced by Acetobacter xylinum and composed of (1,4)-beta-D-glucan with branches of glucosyl residues, showed a strong activity to induce production of interleukin-12 (IL-12) p40 and tumor necrosis factor alpha by macrophages in vitro via Toll-like receptor 4 (TLR-4) signaling. In the present study, we examined the effect of oral administration of AC-1 on protective immunity against Listeria monocytogenes. Mice were given AC-1 or phosphate-buffered saline (PBS) intragastrically 2 days before, on the day of, and 2 days after an intraperitoneal inoculation of L. monocytogenes. The survival rate of AC-1-treated mice was significantly improved and bacterial growth in AC-1-treated mice was severely retarded compared to those of PBS-treated mice after infection with L. monocytogenes. IL-12 p40 levels in serum and magnitudes of CD4+ Th1 and CD8+ Tc1 responses against Listeria antigen were significantly higher in AC-1-treated mice than in PBS-treated mice. The effect of AC-1 on antilisterial activity was diminished in C3H/HeJ mice carrying mutated TLR-4. Thus, AC-1, a potent IL-12 inducer through TLR-4, enhanced protective immunity against L. monocytogenes via augmentation of Th1 responses. These results suggest that infectious processes driven by intracellular microorganisms could be prevented to develop by the (1,4)-beta-D-glucan.     

The contribution of both oxygen and nitrogen intermediates to the intracellular killing mechanisms of C1q-opsonized Listeria monocytogenes by the macrophage-like IC-21 cell line

Listeria monocytogenes is a facultative intracellular pathogen which is internalized by host mammalian cells upon binding to their surface. Further listerial growth occurs in the cytosol after escape from the phagosomal-endosomal compartment. We have previously reported that C1q is able to potentiate L. monocytogenes phagocytosis upon bacterial opsonization by ingestion through C1q-binding structures. In this report, we analysed the post-phagocytic events upon internalization of C1q-opsonized L. monocytogenes and found an induction of macrophage (Mphi)-like IC-21 cell bactericidal mechanisms displayed by the production of oxygen and nitrogen metabolites. Both types of molecules are effective in L. monocytogenes killing. Further analysis of the cellular responses promoted by interaction of C1q with its surface binding structures, leads us to consider C1q as a collaborative molecule involved in Mphi activation. Upon interaction with surface binding structures, C1q was able to trigger and/or amplify the production of reactive oxygen and nitrogen intermediates induced by stimuli such as interferon-gamma and L. monocytogenes phagocytosis.     

Effect of 6-Hydroxydopamine on Host Resistance against Listeria monocytogenes Infection

Recent studies have shown that immunocompetent cells bear receptors of neuropeptides and neurotransmitters and that these ligands play roles in the immune response. In this study, the role of the sympathetic nervous system in host resistance against Listeria monocytogenes infection was investigated in mice pretreated with 6-hydroxydopamine (6-OHDA), which destroys sympathetic nerve termini. The norepinephrine contents of the plasma and spleens were significantly lower in 6-OHDA-treated mice than in vehicle-treated mice. The 50% lethal dose of L. monocytogenes was about 20 times higher for 6-OHDA-treated mice than for vehicle-treated mice. Chemical sympathectomy by 6-OHDA upregulated interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) production in enriched dendritic cell cultures and gamma interferon (IFN-gamma) and TNF-alpha production in spleen cell cultures, whereas chemical sympathectomy had no apparent effect on phagocytic activities, listericidal activities, and nitric oxide production in peritoneal exudate cells and splenic macrophages. Augmentation of host resistance against L. monocytogenes infection by 6-OHDA was abrogated in IFN-gamma(-/-) or TNF-alpha(-/-) mice, suggesting that upregulation of IFN-gamma, IL-12, and TNF-alpha production may be involved in 6-OHDA-mediated augmentation of antilisterial resistance. Furthermore, adoptive transfer of spleen cells immune to L. monocytogenes from 6-OHDA-treated mice resulted in untreated naive recipients that had a high level of resistance against L. monocytogenes infection. These results suggest that the sympathetic nervous system may modulate host resistance against L. monocytogenes infection through regulation of production of IFN-gamma, IL-12, and TNF-alpha, which are critical in antilisterial resistance.     

Vitamin E supplementation increases T helper 1 cytokine production in old mice infected with influenza virus

Compared with young mice, old mice infected with influenza virus have significantly higher pulmonary viral titres, although these can be reduced significantly with dietary vitamin E supplementation. T helper 1 (Th1) cytokines, especially interferon-gamma (IFN-gamma), play an important role in defending against influenza infection. However, there is an age-associated loss of Th1 cytokine production. Prostaglandin E2 (PGE2) production, which increases with age, can modulate the T helper cell function by suppressing Th1 cytokine production. To investigate the mechanism of vitamin E supplementation on reduction of influenza severity in old mice, we studied the cytokine production by splenocytes, and PGE2 production by macrophages (Mphi), in young and old C57BL mice fed semipurified diets containing 30 (control) or 500 parts per million (ppm) (supplemented) vitamin E for 8 weeks, and then infected with influenza A/PC/1/73 (H3N2). Old mice fed the control diet had significantly higher viral titres than young mice; old mice fed the vitamin E-supplemented diet had significantly lower pulmonary viral titres than those fed the control diet (P = 0.02 and 0.001 for overall age and diet effect, respectively). Following influenza infection, interleukin (IL)-2 and IFN-gamma production was significantly lower in old mice than in young mice. Vitamin E supplementation increased production of IL-2 and IFN-gamma in old mice; higher IFN-gamma production was associated with lower pulmonary viral titre. Old mice fed the control diet showed significantly higher lipopolysaccharide (LPS)-stimulated Mphi PGE2 production than old mice fed the vitamin E diet or young mice fed either diet. There was no significant age difference in IL-6, IL-1beta, or tumour necrosis factor-alpha (TNF-alpha) production by splenocytes. Young mice fed the vitamin E-supplemented diet had significantly lower IL-1beta (day 7) and TNF-alpha production (day 5) compared with those fed the control diet. Old mice fed the vitamin E-supplemented diet had significantly lower TNF-alpha production (day 2) than those fed the control diet. Our results indicate that the vitamin E-induced decrease in influenza viral titre is mediated through enhancement of Th1 cytokines, which may be the result of reduced PGE2 production caused by vitamin E.     

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.     

Ontogeny of macrophage-mediated protection against Listeria 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.     

Age-associated changes in immune and inflammatory responses: impact of vitamin E intervention

Aging is associated with dysregulated immune and inflammatory responses. Declining T cell function is the most significant and best-characterized feature of immunosenescence. Intrinsic changes within T cells and extrinsic factors contribute to the age-associated decline in T cell function. T cell defect seen in aging involves multiple stages from early receptor activation events to clonal expansion. Among extrinsic factors, increased production of T cell-suppressive factor PGE(2) by macrophages (Mphi) is most recognized. Vitamin E reverses an age-associated defect in T cells, particularly na?ve T cells. This effect of vitamin E is also reflected in a reduced rate of upper respiratory tract infection in the elderly and enhanced clearance of influenza infection in a rodent model. The T cell-enhancing effect of vitamin E is accomplished via its direct effect on T cells and indirectly by inhibiting PGE(2) production in Mphi. Up-regulated inflammation with aging has attracted increasing attention as a result of its implications in the pathogenesis of diseases. Increased PGE(2) production in old Mphi is a result of increased cyclooxygenase 2 (COX-2) expression, leading to higher COX enzyme activity, which in turn, is associated with the ceramide-induced up-regulation of NF-kappaB. Similar to Mphi, adipocytes from old mice have a higher expression of COX-2 as well as inflammatory cytokines IL-1beta, IL-6, and TNF-alpha, which might also be related to elevated levels of ceramide and NF-kappaB activation. This review will discuss the above age-related immune and inflammatory changes and the effect of vitamin E as nutritional intervention with a focus on the work conducted in our laboratory.     

Mediation of the immunomodulatory effect of beta-estradiol on inflammatory responses by inhibition of recruitment and activation of inflammatory cells and their gene expression of TNF-alpha and IFN-gamma

BACKGROUND: Estrogen has long been reported to show immunomodulatory effects on immune responses, yet, its specific anti-inflammatory mechanism is not clear. METHODS: In this study, we analyzed the effects of beta-estradiol (E2), at its contraceptive dose, on both T cell-independent and T cell-dependent inflammations, and the associated immune mechanism, in female mice. The T cell-independent inflammation was locally induced either with an intradermal injection of olive oil in the footpad, or by an intraperitoneal injection of proteose peptone (PP). The T cell-dependent inflammation was induced by an intraperitoneal injection of the purified protein derivatives (PPD). RESULTS: While E2 inhibited olive oil-induced inflammation as monitored by the decrease in footpad swelling, it did not affect the gene expression of monocyte chemoattractant protein-1 and IL-6 by cells at the inflammatory locus. E2 also inhibited PP-induced inflammation as monitored by the decrease in the number of inflammatory peritoneal exudate cells (PEC) coinciding with a marked decrease in the number of macrophages and granulocytes (Gr. 1+). While E2 did not affect the gene expression of monocyte chemoattractant protein-1 and IL-6 by PP-elicited PEC, it decreased both gene expression and production of TNF-alpha. E2 also decreased the number of cells expressing the lymphocyte function-activated protein-1 in PP-elicited PEC, but not for CD62L. In purified protein derivative-induced T cell-dependent inflammation, E2 decreased the total cellularity of PEC and the relative numbers of CD3+ and CD4+ T cells, and the number of cells expressing the lymphocyte activation markers CD40, CD44, CD69 and IL-2Ralpha in PEC. Furthermore, while E2 did not affect the gene expression of the early T lymphocyte activation protein-1 by PEC, it decreased the gene expression of INF-gamma. CONCLUSION: Collectively, the results suggest that E2-mediated inhibition of inflammatory responses may be due to a combination of suppression of homing and activation of inflammatory cells and their production of TNF-alpha and IFN-gamma.     

Scavenger receptor-mediated delivery of muramyl dipeptide activates antitumor efficacy of macrophages by enhanced secretion of tumor-suppressive cytokines

We showed that muramyl dipeptide (MDP) conjugated to maleylated bovine serum albumin (MBSA) was internalized by macrophages (Mphi) through scavenger receptor (SCR)-mediated endocytosis, which leads to 50-fold higher cytotoxic activity against non-Mphi tumor cells compared with that elicited by free MDP-treated Mphi. The enhanced cytotoxic effect of MBSA-MDP was found to be a result of higher secretion of interleukin (IL)-1, IL-6, tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO) because the addition of antibodies directed against IL-1, IL-6, or TNF-alpha in combination with Mphi cultures totally abrogated the tumoricidal activity of MBSA-MDP. It is interesting to note that MBSA-MDP triggers the secretion of IL-12, whereas IL-10, a Mphi suppressor cytokine, could be detected only on free MDP treatment. The cytotoxic activity of MBSA-MDP was inhibited by indomethacin, indicating a regulatory role for prostaglandin E2 (PGE2). Efficient SCR-mediated intracellular delivery of MDP leading to elimination of cancer cells suggests the immunotherapeutic potential of this approach for treatment of neoplasia.     

Effects of IL-13 on murine listeriosis

Acquired resistance against Listeria monocytogenes is a typical T helper (Th) 1 dominated immune response, whereas Th2 cytokines are thought to worsen listeriosis. We investigated effects of recombinant IL-13 (rIL-13) on the host response to L. monocytogenes in mice. Although IL-13 has been described as a Th2 cytokine with deactivating anti-inflammatory activities, it was found to enhance antilisterial resistance. In vitro, rIL-13 increased IL-12 p40 and p70 production by bone marrow macrophages infected with L. monocytogenes. In vivo, numbers of viable bacteria in spleens and livers were decreased after treatment of mice with rIL-13. In addition, granuloma formation was impaired and NK cell activity of spleen cells was enhanced. At the onset of infection, frequencies of IL-12-producing cells were increased and numbers of IL-4- and IFN-gamma-secreting cells were diminished in rIL-13-treated mice as compared to controls. In contrast, on day 6 after infection, IL-12, IL-4 and IFN-gamma levels in rIL-13-treated animals were equal to or even higher than those in controls. Although direct activation of host macrophages by IL-13 is possible, we consider it more likely that IL-13 acted indirectly through stimulation of IL-12 production and inhibition of IL-4 release early after infection. In contrast, our data argue against an apparent role of IFN-gamma in IL-13- induced antilisterial resistance.      

Interleukin-22-Induced Antimicrobial Phospholipase A2 Group IIA Mediates Protective Innate Immunity of Nonhematopoietic Cells against Listeria monocytogenes

Listeria monocytogenes is a bacterial pathogen which establishes intracellular parasitism in various cells, including macrophages and nonhematopoietic cells, such as hepatocytes. It has been reported that several proinflammatory cytokines have pivotal roles in innate protection against L. monocytogenes infection. We found that a proinflammatory cytokine, interleukin 22 (IL-22), was expressed by CD3⁺ CD4⁺ T cells at an early stage of L. monocytogenes infection in mice. To assess the influence of IL-22 on L. monocytogenes infection in hepatocytes, cells of a human hepatocellular carcinoma line, HepG2, were treated with IL-22 before L. monocytogenes infection in vitro. Gene expression analysis of the IL-22-treated HepG2 cells identified phospholipase A2 group IIA (PLA2G2A) as an upregulated antimicrobial molecule. Addition of recombinant PLA2G2A to the HepG2 culture significantly suppressed L. monocytogenes infection. Culture supernatant of the IL-22-treated HepG2 cells contained bactericidal activity against L. monocytogenes, and the activity was abrogated by a specific PLA2G2A inhibitor, demonstrating that HepG2 cells secreted PLA2G2A, which killed extracellular L. monocytogenes. Furthermore, colocalization of PLA2G2A and L. monocytogenes was detected in the IL-22-treated infected HepG2 cells, which suggests involvement of PLA2G2A in the mechanism of intracellular killing of L. monocytogenes by HepG2 cells. These results suggest that IL-22 induced at an early stage of L. monocytogenes infection enhances innate immunity against L. monocytogenes in the liver by stimulating hepatocytes to produce an antimicrobial molecule, PLA2G2A.     

Endogenous Interleukin-4 Does Not Suppress the Resistance Against a Primary or a Secondary Listeria monocytogenes Infection in Mice

Interleukin-4 (IL-4), a cytokine produced by T-helper 2 (Th2) cells, can inhibit the development of T-helper 1 (Th1) cells, which results in a decreased release of cytokines by the latter. As interferon-γ (IFN-γ), produced by Th1 cells, is involved in the resistance against a Listeria monocytogenes infection, the role of endogenously formed IL-4 during a Listeria infection in mice was investigated. Neutralization of endogenously formed cytokines by subcutaneously injected alginate-encapsulated monoclonal antibody (MoAb)-forming cells results in high antibody titres in the circulation over a long time period. The aim of the present study was to re-evaluate the effect of neutralization of IL-4 during a primary Listeria infection and to investigate the role of IL-4 during a secondary infection in mice using encapsulated MoAb-forming cells. During the course of a primary infection in mice given anti-IL-4 antibody-forming cells (anti-IL-4-FC), the number of Listeria found in the liver and spleen was comparable to that found in control mice given anti-β-galactosidase antibody-forming cells (anti-β-gal-FC). Activation of macrophages measured by inhibition of Toxoplasma gondii proliferation and the release of reactive nitrogen intermediates (RNI) was not affected by anti-IL-4-FC treatment during infection. Furthermore, during a secondary L. monocytogenes infection the number of bacteria in the liver and spleen of anti-IL-4-treated immune mice was comparable to anti-β-gal-FC-treated, control, immune mice. The concentration of IFN-γ in plasma of anti-IL-4-treated immune mice was similar to that of control immune mice. Taken together, these findings demonstrate that neutralization of endogenously formed IL-4 does not affect resistance to a primary or a secondary L. monocytogenes infection in mice.     

Enhanced resistance against Listeria monocytogenes at an early phase of primary infection in pregnant mice: activation of macrophages during pregnancy.

We investigated the pregnancy-induced changes in macrophage activity which are important in the expression of host defense against infections. Several macrophage functions were examined by using Listeria monocytogenes. In pregnant mice, prolonged survival and enhanced in vivo elimination of bacteria were observed in the early phase of primary infection. Functions of peritoneal macrophages, including in vitro phagocytosis intracellular killing, glucose consumption, generation of superoxide anion, and intracellular beta-glucuronidase activity were shown to be enhanced in pregnant mice. These findings indicate that pregnancy enhances macrophage functions qualitatively. Possible mechanisms for this enhancement and the significance of macrophage activation for pregnant hosts are discussed.     

Mechanism of murine Vgamma1+ gamma delta T cell-mediated innate immune response against Listeria monocytogenes infection

Murine gamma delta T cells participate in innate immune response against infection of the intracellular bacterium Listeria monocytogenes. In the present report, we analyzed the mechanism of the gamma delta T cell-mediated response against L. monocytogenes infection. gamma delta T cell-enriched spleen cells of L. monocytogenes-infected mice produced IFN-gamma in vitro in response to L. monocytogenes-infected spleen cells. The IFN-gamma production was abrogated by depletion of Vgamma1+ gamma delta T cells. IFN-gamma production of the Vgamma1+ gamma delta T cells in response to L. monocytogenes-infected spleen cells required IL-12. However, addition of Fab fragment of anti-TCR gamma delta monoclonal antibodies (mAb) failed to block the response, suggesting that the response requires no TCR-mediated antigen recognition. Interestingly, Vgamma1+ gamma delta T cells of naive mice also produced IFN-gamma in response to L. monocytogenes-infected spleen cells in an IL-12-dependent manner. Furthermore, the IL-12 receptor (IL-12R) gene was expressed on the Vgamma1+ gamma delta T cells of naive mice as well as those of L. monocytogenes-infected mice although naive alpha beta T cells lack IL-12R expression. All the results suggest that the Vgamma1+ gamma delta T cells participate in immune surveillance against intracellular bacterial infection through quick production of IFN-gamma in response to infection-induced IL-12 without antigen-driven clonal expansion and maturation.     

Reduced antilisterial activity of TNF-deficient bone marrow-derived macrophages is due to impaired superoxide production.

Mice deficient for TNF ligand and receptor type 1 have demonstrated the importance of TNF in the host defense against Listeria monocytogenes. To investigate the particular deficiency of macrophages derived from TNF/lymphotoxin (LT)-alpha(-/-) mice in antilisterial growth control, bone marrow-derived macrophages (BMDM) were used for in vitro infection experiments. After the combined treatment with IFN-gamma and lipopolysaccharide (LPS), production of NO by wild-type (wt) and TNF/LT-alpha(-/-) BMDM was induced to comparable levels, but only wt BMDM controlled L. monocytogenes growth efficiently. Nevertheless, inhibition of NO production led to a remarkable loss of antilisterial activity. This suggests that presence of NO is necessary but not sufficient for L. monocytogenes killing and that elimination of L. monocytogenes requires additional effector molecules. The LPS-inducible superoxide production of TNF/LT-alpha(-/-) BMDM was impaired. Accordingly both scavenging of superoxide and peroxynitrite led to reduced L. monocytogenes killing by wt BMDM. In addition, peroxynitrite was able to kill L. monocytogenes in vitro. Together these findings suggest that the defective host defense of TNF/LT-alpha-deficient mice against L. monocytogenes partially stems from reduced superoxide production of macrophages due to the absence of TNF and imply a function for peroxynitrite, the reaction product of NO and superoxide, in the intracellular killing of L. monocytogenes.     

Mphi1 and Mphi2 can be re-polarized by Th2 or Th1 cytokines, respectively, and respond to exogenous danger signals

Macrophages (Mphi) represent a dynamic cell population that develops and operates within a changing microenvironment. In parallel to Th1/Th2 cells, primary Mphi may undergo classical (Mphi1) or alternative (Mphi2) activation. Here, we investigated whether Mphi1/Mphi2 may be re-polarized by a secondary stimulation by Th1 or Th2 cytokines or by exogenous danger signals. We established that Mphi1IFNgamma respond to alternative activation by IL-4 and IL-10 by de novo secretion of Th2 cytokines AMAC-1 and IL-1ra, and by an increase in phagocytic capacity and a decrease in bactericidal activity. Vice versa, Mphi2 responded to classical activation by IFNgamma exhibiting reduced phagocytosis and significantly increased bacterial killing while being refractory regarding secretion of TNFalpha, IL-1beta and IL-12. In response to the bacterial danger signals LPS and MDP, both Mphi1 and Mphi2 produced IL-1beta and TNFalpha; in addition Mphi2 expressed the Th1-inducing cytokine IL-12. The ability of Mphi to be re-polarized and to react to exogenous danger signals is a precondition to down-regulate an outdated immune reaction and to retain the capacity to mount an adequate anti-bacterial response. Selective refractoriness of Mphi1 and Mphi2 to IFNgamma- and LPS-induced cytokine secretion may contribute to prevent autoimmunity.     

Murine T-cell clones against Entamoeba histolytica: in vivo and in vitro characterization

Eleven T-cell clones were raised from the spleens of BALB/c mice hyperimmunized against a crude soluble extract of Entamoeba histolytica trophozoites. Seven clones were of the Lyt-1+, and four of the Lyt-23+ phenotype. All clones proliferated in the presence of E. histolytica antigens but not to a purified protein derivative; five clones proliferated to a crude extract of the E. histolytica-like Laredo amoebae. Ten clones secreted T-cell growth factors in response to E. histolytica antigens. Two clones (Lyt-23+) mediated direct lymphocytotoxicity (73% and 86%) against amoebic trophozoites that was inhibited with rabbit anti-mouse TNF-alpha. Supernatants of five of the clones (all Lyt-1+) activated mouse peritoneal macrophages (Mphi) to kill E. histolytica trophozoites in vitro, seemingly independent of secreted reactive oxygen intermediates (O2- and H2O2) in the case of three clones supernatants. All of the clones that were activating Mphi to kill amoeba in vitro also mediated a local DTH reaction in mouse footpad. Our results demonstrate direct lymphocyte cytotoxicity via a cytolytic molecule antigenically related to TNF-alpha and lymphokines activating Mphi for amoebic killing by oxidative and non-oxidative mechanisms, the latter process mediated by a macrophage-activating factor (MAF) distinct from interferon-gamma (IFN-gamma).     

Interference between host resistance to Listeria monocytogenes infection and ovalbumin-induced allergic responses in mice

Listeria monocytogenes promotes the induction of the T-helper 1 (Th1) cell response, while ovalbumin (OVA) induces a Th2 cell response and allergic reactions, such as airway hyperreactivity and immunoglobulin E (IgE) production. When mice were immunized with OVA on day 7 after L. monocytogenes infection, eosinophilia in bronchoalveolar lavage and the production of total IgE, OVA-specific IgE, interleukin-4 (IL-4), and IL-5 in the circulation were markedly suppressed. Cytokine responses, including IL-4, IL-5, IL-10, IL-13, and gamma interferon, to OVA were decreased in the spleen cell cultures obtained from OVA-immunized mice that had been infected with L. monocytogenes. Conversely, when OVA-immunized mice were infected with L. monocytogenes, conversion from the nonlethal infection to the lethal infection occurred. Host resistance to L. monocytogenes infection in OVA-immunized mice was enhanced by the administration of anti-IL-10 monoclonal antibody. The present study indicates that striking interference is observed between Th1-inducing L. monocytogenes infection and Th2-driven OVA-induced airway hyperreactivity.