Induction of interferon-gamma and tumor necrosis factor by Legionella pneumophila: augmentation of human neutrophil bactericidal activity

We have previously reported that Legionella pneumophila antigens can induce interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) in vitro and in vivo in mice. Furthermore, treatment of murine polymorphonuclear leukocyte (PMN) cultures with these cytokines resulted in augmented killing of the bacteria in vitro. The purpose of the present study was to determine if these findings could be extended to human responses. Here we report that Legionella antigens induced IFN-gamma and TNF in nonimmune human leukocytes cultures, and that these cytokines were able to stimulate the bactericidal activity of isolated PMN against L. pneumophila in vitro. Furthermore, optimal production of IFN-gamma was found in cultures which were enriched for large granular lymphocytes (LGL). The phenotype of IFN-producing cells was determined to be CD11+, CD16+, CD2+, and negative for CD4, CD8, CD14, and Leu 7. Additionally, Legionella-infected monocytes were found to produce TNF in a dose-dependent response to the number of infecting bacteria, and the addition of recombinant IFN-gamma to infected monocytes resulted in augmented production of TNF in a synergistic manner. Finally, treatment of PMN with recombinant IFN-gamma and recombinant TNF augmented their bactericidal activity against Legionella in a dose-dependent response. Thus, cytokines which can be induced by L. pneumophila antigens are able to stimulate PMN function in vitro, suggesting that resistance to infection results from a complex interaction of cytokines and cell responses.     

Regulation of Trypanosoma cruzi infection in mice by gamma interferon and interleukin 10: role of NK cells.

Gamma interferon (IFN-gamma) plays an important role in experimental Trypanosoma cruzi infections, presumably by controlling the early replication of parasites in host macrophages. In this work, we show that NK cells represent an important cell type responsible for the production of most of the IFN-gamma in the early stage of T. cruzi infection and that the in vivo treatment of mice with anti-NK1.1 monoclonal antibody made resistant animals susceptible to the infection. Through in vitro experiments, we demonstrate that normal splenocytes from euthymic or athymic nude mice cultivated for 48 h with live T. cruzi trypomastigotes produced elevated levels of IFN-gamma. In addition, NK-depleted splenocytes show a drastic reduction of IFN-gamma production in response to live T. cruzi trypomastigotes. We also demonstrated that IFN-gamma production is dependent on a factor secreted by adherent cells. Supernatants of spleen cells from athymic nude mice are able to induce IFN-gamma production by normal splenocytes when cultured with trypomastigotes. The addition of anti-interleukin-10 to these cultures resulted in a marked increase in IFN-gamma production. On the other hand, the absence of NK cells led to an increased secretion of interleukin-10 upon in vitro stimulation with T. cruzi. Taken together, these results suggest that NK cells are the major source of IFN-gamma that could be involved in limiting the replication of T. cruzi in host macrophages during the early acute phase of the infection.     

NK cytokine secretion and cytotoxicity occur independently of the SLP-76 adaptor protein.

The adapter protein SLP-76 is required for T cell development and TCR signal transduction. SLP-76 is also expressed in NK cells, yet splenic populations of NK cells develop normally in SLP-76-deficient mice. We examined the effects of SLP-76 deficiency upon cellular activation through studies of NK function in SLP-76(-/-) mice. This study presents evidence that NK populations in both spleen and liver of SLP-76(-/-) mice remain intact. Natural cytotoxic responses of SLP-76(-/-) splenocytes proceed in a manner comparable to those of wild-type control splenocytes. Similar to controls, SLP-76(-/-) splenocytes exhibit enhanced survival and augmented cytotoxic capacity after in vitro culture with IL-2. IL-2-stimulated SLP-76(-/-) splenocytes also retain normal antibody-dependent cellular cytotoxicity and the ability to secrete IFN-gamma in response to IL-12 stimulation. These results indicate that, unlike events stimulated by TCR engagement, signaling cascades engaged by IL-2 and IL-12 receptors, by Fc gammaRIIIA (which mediates antibody-dependent cellular cytotoxicity), and by natural cytotoxicity-associated receptors on murine NK cells can occur independently of SLP-76.     

Expression of functional IL-2 receptors on mature splenic dendritic cells

We report here the expression of functional IL-2 receptor (IL-2R) on mature splenic dendritic cells (DC) and synergistic effect of IL-2 on IFN-gamma production by DC. IL-2 augmented IL-12-dependent IFN-gamma production by DC purified from both splenocytes of wild-type and anti-asialoGM1 Ab-treated Rag-2(-/-) splenocytes devoid of T, B, NK and NKT cells. A neutralizing mAb against IL-2Ralpha blocked such enhancing effect of IL-2 on IFN-gamma production, indicating the presence of functional IL-2R on DC. Synergistic effects of IL-2 were also observed on IFN-gamma production by DC stimulated through CD40 or MHC class II, suggesting that T cell-derived IL-2 can act on DC during antigen presentation. Furthermore, we provide evidence that DC produce IFN-gamma during interaction with allogeneic CD4(+) T cells from IFN-gamma(-/-) mice. These results suggest that IL-2 produced by naive T cells upon antigen stimulation is an important factor during Th0 to Th1 differentiation by inducing IFN-gamma from DC.     

Differential induction of gamma interferon in Legionella pneumophila-infected macrophages from BALB/c and A/J mice

Gamma interferon (IFN-gamma), a pleiotropic cytokine, is now known to be produced by macrophages as well as by NK cells, gammadelta cells, and activated T cells. The autocrine biological functions of IFN-gamma on the macrophage include the upregulation of major histocompatibility complex MHC class II and the activation to an antiviral state. In this study, the production of IFN-gamma by macrophages was demonstrated to correspond to antibacterial activity. Legionella pneumophila replicates intracellularly in thioglycolate (TG)-elicited macrophages (TG-macrophages) from A/J mice, while TG-macrophages from BALB/c mice restrict bacterial growth after an initial period of growth. BALB/c TG-macrophages were shown to express IFN-gamma mRNA at 24 and 28 h, which corresponded to the initiation of anti-L. pneumophila activity. Moreover, IFN-gammaneutralization by antibody treatment of the cultures resulted in increased L. pneumophila growth in the macrophages. In contrast, no IFN-gamma mRNA was expressed in TG-macrophages from A/J mice, where L. pneumophila grew unrestricted. As would be expected, IFN-gamma treatment decreased bacterial growth. An IFN-gamma-mediated antibacterial activity was, however, inducible in A/J macrophages by the addition of interleukin-12 following L. pneumophila infection. Thus, autocrine IFN-gamma is involved in anti-L. pneumophila activity associated with different growth patterns and appears to be important during intracellular infection.     

Production of gamma interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha.

Previous studies of mice have implicated natural killer (NK) cells as mediators of protective activity against Toxoplasma gondii through their production of gamma interferon (IFN-gamma). In the present study, we have compared NK-cell activity in infected and uninfected SCID mice. Our data reveal that infection results in increased levels of IFN-gamma in serum and elevated NK-cell activity but that these NK cells were not cytotoxic for T. gondii-infected P815 cells. Treatment with anti-IFN-gamma antibody abrogated the increase in NK-cell activity and resulted in earlier mortality of infected mice. In vivo treatment with anti-asialo GM1 antiserum reduced NK cell activity and levels of IFN-gamma in serum but did not alter time to death. Spleen cells from infected mice produced higher levels of IFN-gamma than those from uninfected mice when stimulated in vitro with live T. gondii or parasite antigen preparations. Further analysis revealed that interleukin 10 (IL-10) inhibited, whereas tumor necrosis factor alpha (TNF-alpha) and IL-12 enhanced, IFN-gamma production by spleen cells from infected or uninfected mice. The combination of IL-12 and TNF-alpha induced higher levels of IFN-gamma from whole spleen cells of infected mice than from those of uninfected mice. Depletion of the adherent cell population from the spleen cells of infected mice led to a significant reduction in the levels of IFN-gamma produced after stimulation with IL-12 plus TNF-alpha. Similar results did not occur with cells from uninfected mice. These data indicate that other cytokines produced by the adherent cell population from infected mice may be involved in maximal production of IFN-gamma by NK cells stimulated with IL-12 and TNF-alpha. To assess the importance of endogenous IL-12, a polyclonal anti-IL-12 was administered to infected SCID mice. This treatment led to earlier mortality, indicating that endogenous IL-12 mediates resistance to T. gondii.     

Gamma interferon production is critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO production nor NK cell activation is critical

We have examined the roles of gamma interferon (IFN-gamma), nitric oxide (NO), and natural killer (NK) cells in the host resistance to infection with the blood-stage malarial parasite Plasmodium berghei XAT, an irradiation-induced attenuated variant of the lethal strain P. berghei NK65. Although the infection with P. berghei XAT enhanced NK cell lytic activity of splenocytes, depletion of NK1.1(+) cells caused by the treatment of mice with anti-NK1.1 antibody affected neither parasitemia nor IFN-gamma production by their splenocytes. The P. berghei XAT infection induced a large amount of NO production by splenocytes during the first peak of parasitemia, while P. berghei NK65 infection induced a small amount. Unexpectedly, however, mice deficient in inducible nitric oxide synthase (iNOS-/-) cleared P. berghei XAT after two peaks of parasitemia were observed, as occurred for wild-type control mice. Although the infected iNOS-/- mouse splenocytes did not produce a detectable level of NO, they produced an amount of IFN-gamma comparable to that produced by wild-type control mouse splenocytes, and treatment of these mice with neutralizing anti-IFN-gamma antibody led to the progression of parasitemia and fatal outcome. CD4(-/-) mice infected with P. berghei XAT could not clear the parasite, and all these mice died with apparently reduced IFN-gamma production. Furthermore, treatment with carrageenan increased the susceptibility of mice to P. berghei XAT infection. These results suggest that neither NO production nor NK cell activation is critical for the resistance to P. berghei XAT infection and that IFN-gamma plays an important role in the elimination of malarial parasites, possibly by the enhancement of phagocytic activity of macrophages.     

The enhancement of avian NK cell cytotoxicity by thymulin is not mediated by the regulation of IFN-gamma production.

Preincubation with either thymulin or IFN-gamma can enhance NK activity. In addition, overnight in vitro pre-treatment with thymulin and IFN-gamma increases NK activity further than either treatment alone. It has been hypothesized that thymulin increases the responsiveness of immune cells to IFN-gamma by either increasing the expression of IFN-gammaR or by increasing the production and/or secretion of IFN-gamma. The effects of thymulin on IFN-gamma production and secretion were examined in this study. While an overnight incubation with the polyclonal activator Con A increased the number of cells positive for intracellular IFN-gamma, a similar incubation with thymulin produced no change in the percentages of cells labeling positive for intracellular IFN-gamma when compared to the media control cells. In addition, IFN-gamma was not secreted by splenocytes following an overnight incubation with thymulin, but increased secretion was induced by Con A stimulation. Taken together, these results suggest that thymulin does not increase IFN-gamma production or induce IFN-gamma secretion by avian splenocytes.     

Role of natural killer cells in iscador mediated inhibition of metastasis by adoptive immunotherapy

Iscador activated (in vivo and in vitro) splenocytes were found to inhibit metastatic tumour growth in C57BL/6 mice. In order to check whether NK cells are involved in the antimetastatic activity of Iscador activated splenocytes ,animals were depleted of NK cells using antiasialo GMI antibodies. When spleen cells activated in vivo with Iscador were injected into animals pretreated with Antiasialo GM I antibodies, there was an average of 44.6 tumour nodules on 21st day indicating that stimulation of NK cell activity produced by the Iscador compensate for the NK cell depletion by Antiasialo GM I antibody. Animals treated with Iscador activated splenocytes showed an average survival period of 68 days whereas that of control tumour bearing animals treated with Ab the average survival was 19.3 days. The lung collagen hydroxyproline content, serum sialic acid levels, markers of metastasis were also significantly (P<0.001) lowered in these animals compared to those of the untreated tumour bearing animals. gamma-glutamyl transpeptidase a marker of neoplastic growth, was also significantly reduced (P<0.001) in animals treated with activated splenocytes.     

Immunomodulatory role of endogenous interleukin-18 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection

The in vivo role of endogenous interleukin-18 (IL-18) in modulating gamma interferon (IFN-gamma)-mediated resolution of replicative Legionella pneumophila lung infection was assessed using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with virulent bacteria (10(6) L. pneumophila organisms per mouse) resulted in induction of IL-18 protein in bronchoalveolar lavage fluid (BALF) and intrapulmonary expression of IL-18 mRNA. Real-time quantitative RT-PCR analysis of infected lung tissue demonstrated that induction of IL-18 in BALF preceded induction of IL-12 and IFN-gamma mRNAs in the lung. Blocking intrapulmonary IL-18 activity by administration of a monoclonal antibody (MAb) to the IL-18 receptor (anti-IL-18R MAb) prior to L. pneumophila infection inhibited induction of intrapulmonary IFN-gamma production but did not significantly alter resolution of replicative L. pneumophila lung infection. In contrast, blocking endogenous IL-12 activity by administration of anti-IL-12 MAb) alone or in combination with anti-IL-18R MAb inhibited induction of intrapulmonary IFN-gamma and resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection. Taken together, these results demonstrate that IL-18 plays a key role in modulating induction of IFN-gamma in the lung in response to L. pneumophila and that together with IL-12, IL-18 regulates intrapulmonary growth of the bacteria.     

Interleukin-12- and interferon-gamma-mediated natural killer cell activation by Agaricus blazei Murill

Dried fruiting bodies of Agaricus blazei Murill (A. blazei) and its extracts have generally used as complementary and alternative medicines (CAMs). Here, we report that the oral administration of A. blazei augmented cytotoxicity of natural killer (NK) cells in wild-type (WT) C57BL/6, C3H/HeJ, and BALB/c mice. Augmented cytotoxicity was demonstrated by purified NK cells from treated wild-type (WT) and RAG-2-deficient mice, but not from interferon-gamma (IFN-gamma) deficient mice. NK cell activation and IFN-gamma production was also observed in vitro when dendritic cell (DC)-rich splenocytes of WT mice were coincubation with an extract of A. blazei. Both parameters were largely inhibited by neutralizing anti-interleukin-12 (IL-12) monoclonal antibody (mAb) and completely inhibited when anti-IL-12 mAb and anti-IL-18 mAb were used in combination. An aqueous extract of the hemicellulase-digested compound of A. blazei particle; (ABPC) induced IFN-gamma production more effectively, and this was completely inhibited by anti-IL-12 mAb alone. NK cell cytotoxicty was augmented with the same extracts, again in an IL-12 and IFN-gamma-dependent manner. These results clearly demonstrated that A. blazei and ABPC augmented NK cell activation through IL-12-mediated IFN-gamma production.     

Immunostimulatory DNA mediates inhibition of eosinophilic inflammation and airway hyperreactivity independent of natural killer cells in vivo.

BACKGROUND: Immunostimulatory DNA sequences (ISS) inhibit eosinophilic inflammation and airway hyperreactivity in mouse models of asthma. In vitro ISS activate natural killer (NK) cells to secrete IFN-gamma, and this cytokine is hypothesized to contribute to the antiallergic effect of ISS in vivo. OBJECTIVE: We investigated whether ISS activation of NK cells is important in mediating the reduction in airway hyperreactivity and the antieosinophilic effect of ISS in vivo. METHODS: We assessed whether ISS modulated the development of eosinophilic airway inflammation and airway hyperreactivity to methacholine in ovalbumin (OVA)-sensitized and OVA allergen-challenged mice pretreated with an antibody to deplete NK cells. RESULTS: Mice sensitized and challenged with OVA had significant bronchoalveolar lavage and lung eosinophilia, as well as airway hyperresponsiveness. ISS induced significant inhibition of bronchoalveolar lavage and lung eosinophilia, as well as airway hyperresponsiveness, in OVA-sensitized mice pretreated before OVA challenge with an NK cell-depleting antibody (NK(-) mice), as well as in mice pretreated with a control non-NK cell-depleting antibody (NK(+) mice). The NK cell-depleting antibody inhibited ISS-induced IFN-gamma production by spleen cells. CONCLUSION: These studies demonstrate that depletion of NK cells has no significant effect on ISS-mediated inhibition of airway eosinophilia and airway hyperresponsiveness in vivo, suggesting that non-NK cells and cytokines other than IFN-gamma derived from NK cells mediate the majority of the ISS-inhibitory effect on eosinophilic inflammation and airway hyperresponsiveness in vivo.     

Effector mechanisms responsible for gamma interferon-mediated host resistance to Legionella pneumophila lung infection: the role of endogenous nitric oxide differs in susceptible and resistant murine hosts.

To facilitate identification of the effector mechanism(s) responsible for gamma interferon (IFN-gamma)-mediated host resistance to Legionella pneumophila, a murine model of legionellosis in BALB/c mice with a targeted disruption in the IFN-gamma gene (gamma knockout [GKO] mice) was developed. Immunocompetent BALB/c mice and GKO mice were inoculated intratracheally with virulent L. pneumophila (10(6) bacteria per mouse), and bacterial clearance and the pulmonary inflammatory response were assessed. L. pneumophila did not replicate in, and was rapidly cleared from, the lungs of immunocompetent BALB/c mice, demonstrating that immunocompetent BALB/c mice are resistant to replicative L. pneumophila pulmonary infections. In contrast, similarly infected GKO mice developed persistent, replicative intrapulmonary L. pneumophila infections with extrapulmonary dissemination of the bacteria to the spleen. Histopathologic and flow cytometric analysis of L. pneumophila-infected lung tissue demonstrated that while immunocompetent BALB/c mice develop multifocal pneumonitis which resolves, similarly infected GKO mice develop diffuse pneumonitis with persistent neutrophil recruitment into the lung. Intratracheal administration of exogenous IFN-gamma to L. pneumophila-infected GKO mice facilitated intrapulmonary clearance of the bacteria, confirming the pivotal role of IFN-gamma in innate host defenses to L. pneumophila lung infection in this murine host. The potential role of endogenous reactive nitrogen intermediates, including nitric oxide (NO), in IFN-gamma-mediated resistance to L. pneumophila pulmonary infections in immunocompetent BALB/c mice was subsequently assessed. Macrophage inducible nitric oxide synthetase (an enzyme responsible for the production of NO) was induced in alveolar cells from L. pneumophila-infected immunocompetent BALB/c mice (with maximal expression at 48 h postinfection) but was not induced in similarly infected GKO mice. However, administration of the NO synthetase inhibitor N-monomethyl-L-arginine did not significantly inhibit clearance of L. pneumophila from the lung of immunocompetent BALB/c mice (compared with that in similarly infected mice not administered N-monomethyl-L-arginine). In contrast, we have previously demonstrated that IFN-gamma-induced host resistance to replicative L. pneumophila lung infections in a susceptible murine host (A/J mice) is mediated, in part, by endogenous NO. Taken together, these studies identify a differing role of endogenous NO in IFN-gamma-mediated resistance to L. pneumophila pulmonary infection in susceptible and resistant murine hosts.     

Human NK1 and NK2 subsets determined by purification of IFN-gamma-secreting and IFN-gamma-nonsecreting NK cells

The in vivo existence of human NK cell subsets similar to Th1 and Th2 cells was demonstrated in freshly isolated IFN-gamma-secreting and IFN-gamma-nonsecreting NK cells. The IFN-gamma-secreting NK subset showed a typical cytokine pattern with predominant expression of IFN-gamma, but almost no IL-4, IL-5 and IL-13. In contrast, the IFN-gamma-nonsecreting NK subset was composed of IL-4, IL-5 and IL-13-producing NK cells. Short-time stimulation or 2 weeks of in vitro differentiation of NK cells led to distinct patterns of cytokine production similar to freshly-purified IFN-gamma (+) or IFN-gamma (-) NK cell subsets. NK cells stimulated with IL-12 produced increased levels of IFN-gamma and decreased levels of IL-4. In contrast, stimulation of NK cells with IL-4 inhibited IFN-gamma, but increased IL-13 production. Freshly-purified IFN-gamma (+) and IFN-gamma (-) or in vitro differentiated NK1 and NK2 subsets showed similar cytotoxicity to K562 cells. These results demonstrate that circulating NK cells retain effector subsets in humans with distinct cytokine profiles and may display different inflammatory properties.     

Central role of endogenous gamma interferon in protective immunity against blood-stage Plasmodium chabaudi AS infection

The role of endogenous gamma interferon (IFN-gamma) in protective immunity against blood-stage Plasmodium chabaudi AS malaria was studied using IFN-gamma gene knockout (GKO) and wild-type (WT) C57BL/6 mice. Following infection with 10(6) parasitized erythrocytes, GKO mice developed significantly higher parasitemia during acute infection than WT mice and had severe mortality. In infected GKO mice, production of interleukin 12 (IL-12) p70 and tumor necrosis factor alpha in vivo and IL-12 p70 in vitro by splenic macrophages was significantly reduced compared to that in WT mice and the enhanced nitric oxide (NO) production observed in infected WT mice was completely absent. WT and GKO mice had comparable numbers of total nucleated spleen cells and B220(+) and Mac-1(+) spleen cells both before and after infection. Infected WT mice, however, had significantly more F4/80(+), NK1.1(+), and F4/80(+)Ia(+) spleen cells than infected GKO mice; male WT had more CD3(+) cells than male GKO mice. In comparison with those from WT mice, splenocytes from infected GKO mice had significantly higher proliferation in vitro in response to parasite antigen or concanavalin A stimulation and produced significantly higher levels of IL-10 in response to parasite antigen. Infected WT mice produced more parasite-specific immunoglobulin M (IgM), IgG2a, and IgG3 and less IgG1 than GKO mice. Significant gender differences in both GKO and WT mice in peak parasitemia levels, mortality, phenotypes of spleen cells, and proliferation of and cytokine production by splenocytes in vitro were apparent during infection. These results thus provide unequivocal evidence for the central role of endogenous IFN-gamma in the development of protective immunity against blood-stage P. chabaudi AS.     

Modulation of IL-2, IFN-gamma, TNF-alpha and IL-4 production in mice of different ages by thymopentin.

The effect of an immunomodulator drug thymopentin (TP5) on the production of various cytokines (IFN-gamma, IL-2, IL-4, TNF-alpha) in mice of different ages has been studied. TP5 enhanced IL-2, TNF-alpha and IFN-gamma production but reduced the IL-4 secretion by splenocytes from aged mice (greater than 120 week old) in vitro. However, it had no effect on the IL-2, IFN-gamma, TNF-alpha or IL-4 production by splenocytes from young and adult mice. TP5 injected subcutaneously was able to induce high levels of IL-2 production by splenocytes from all groups of mice. The TP5 effect on TNF-alpha and IFN-gamma was similar, even though it was significant only in old mice. Furthermore, TP5 was able to significantly reduce IL-4 production in old mice, which normally produced high levels of this cytokine after mitogen stimulation. Since it has been observed in the mouse that the Th1 cells secrete IFN-gamma and IL-2, whereas the Th2 cells preferentially produce IL-3, IL-4 and IL-5, these results indicate that the immunopotentiatory activity of TP5 is due to the preferential up-regulation of Th1 cells.     

Gamma interferon levels during Chlamydia trachomatis pneumonia in mice.

Host defense against murine Chlamydia trachomatis (mouse pneumonitis agent [MoPn]) in a murine model was investigated. Gamma interferon (IFN-gamma) was produced in the lungs by both MoPn-susceptible nude athymic (nu/nu) and MoPn-resistant heterozygous (nu/+) mice. In vivo depletion of IFN-gamma in nu/nu mice led to exacerbation of infection. Fluorescence-activated cell sorter analysis disclosed induction of GL3 antibody-positive cells (putatively gamma/delta+ T cells) in nu/nu mouse lung during infection with MoPn. Treatment of nu/nu mice in vivo with antibody to NK cells (anti-asialo GM1 antibody) or to gamma/delta cells (UC7-13D5) did not significantly decrease IFN-gamma production in the lung. However, treatment of severe combined immunodeficiency mice (which lack gamma/delta cells) with antibody to NK cells significantly reduced lung IFN-gamma levels.     

Lactic acid bacterium potently induces the production of interleukin-12 and interferon-gamma by mouse splenocytes

We previously have reported that the lactic acid bacterium, Lactobacillus casei strain Shirota (LcS), shows marked antitumor activities and an ability to modify immune responses. In this study, we examined whether LcS can induce the production of interleukin (IL)-12 and interferon-gamma (IFN-gamma), which are important cytokines for antitumor and antimicrobial immunity, from murine splenocytes in vitro in order to clarify the mechanisms of its immune modification. Stimulation by LcS induced a marked production of IL-12 by X-ray-irradiated splenocytes (X-irr-Spl). The production of IL-12 by X-irr-Spl was independent of the presence of nylon wool column-passed splenocytes (NW-Spl). IFN-gamma was produced by splenocytes by the stimulation with concanavalin A (Con A). LcS showed a synergistic stimulatory effect on the ConA-induced production of IFN-gamma. In addition, X-irr-Spl were required for the IFN-gamma; production by NW-Spl treated with LcS. The IFN-gamma production was reduced by anti-IL-12 antibody treatment. NW-Spl produced IFN-gamma following treatment with recombinant IL-12. Thus, we confirmed that IFN-gamma production by splenocytes was the result of the production of IL-12 from X-irr-Spl stimulated by LcS. Furthermore, in BALB/c mice, the oral administration of viable LcS augmented the production of IFN-gamma but not that of IL-4 or IL-5 by splenocytes. Thus, we suggested that LcS primarily activated X-irr-Spl, probably macrophages, and these cells secreted IL-12. The IL-12 induced by LcS stimulated the production of IFN-gamma.     

Essential role of Toll-like receptors for dendritic cell and NK1.1(+) cell-dependent activation of type 1 immunity by Lactobacillus pentosus strain S-PT84

Activation of type 1 immunity plays a critical role in host defense mechanisms against infectious disease and tumor. Lactic acid bacteria, existing in the gastrointestinal tract, are one of the powerful tools to induce a type-1-dominant immunity, which may improve Th2-dependent allergic diseases. In the present work, we found that an oral intake of Lactobacillus pentosus strain, S-PT84 into mice significantly enhanced NK activity of spleen cells in vivo. We further revealed that NK1.1 positive NK cells and NKT cells are responsible cells for producing IFN-gamma after stimulation with S-PT84 in vitro. S-PT84 induced IFN-gamma-producing cells through activation of IL-12 production by CD11c(+)DCs in Toll-like receptor (TLR) 2- and/or TLR4-dependent manner. Interestingly, direct interaction between DCs and NK1.1(+) cells was also essential for the IFN-gamma production in response to the S-PT84 stimulation. Therefore, we concluded that S-PT84 effectively promoted type 1 immunity through IL-12 and IFN-gamma which were produced by DCs and NK1.1(+) cells, respectively. Thus, S-PT84 would be a nice immune modulator for improving immunobalance, which plays a pivotal role for controlling allergy, infectious diseases and tumor.     

Effects of macrophage colony-stimulating factor and interleukin-2 administration on NK1.1(+) cells in mice

We studied the effects of M-CSF and IL-2 on NK1.1(+) cell activity in vivo and in vitro. Administration of M-CSF increased the number of splenic NK1.1(+) cells (vs. saline: P<0.01). Moreover, the combination of M-CSF and IL-2 (M-CSF+IL-2) produced a synergistic expansion of the number of NK1.1(+) cells compared with each single treatment (vs. saline: P<0.001). The NK1.1(+) cells were isolated from the spleen of each treated mouse (four treatment groups: saline, IL-2 alone, M-CSF alone, M-CSF+IL-2) and their functions (IL-2-induced proliferation, IFN-gamma production and cytostatic activity) were evaluated in vitro. The NK1.1(+) cells from M-CSF alone and M-CSF+IL-2 treated mice showed greater responsiveness in terms of IL-2-induced proliferation, production of IFN-gamma and cytostatic activity than the cells from saline and IL-2 alone treated mice. The NK activity in vivo was enhanced by the administration of M-CSF and IL-2, as assessed by the 'Lung clearance assay' (clearance of Yac-1 cells in lung). And the M-CSF+IL-2 treatment induced the highest NK activity of the four treatments. To show a practical effect of upregulation of NK activity in vivo by M-CSF and IL-2 administration, the effect of the four treatments on an experimental tumor metastasis model was examined. The IL-2 alone, M-CSF alone and M-CSF+IL-2 treatment reduced the metastasis of B16 melanoma. And the M-CSF+IL-2 treatment proved of greater benefit to the antimetastatic activity than each single treatment. Our results demonstrated that the administration of M-CSF increases the number of NK1.1(+) cells, which have good responsiveness to IL-2. Furthermore, the combination treatment of M-CSF and IL-2 in vivo augments the increase of NK1.1(+) cells. And these effects can contribute to the antimetastatic activity in vivo.