Brucella abortus regulates bovine macrophage-T-cell interaction by major histocompatibility complex class II and interleukin-1 expression

T-cell activation is dependent on nominal antigen associated with major histocompatibility complex (MHC) class II molecules and interleukin-1 (IL-1), both provided by antigen-presenting cells. We have studied the effects of Brucella abortus and recombinant bovine gamma interferon (IFN-gamma) on bovine macrophage expression of MHC class II and IL-1 molecules and subsequent T-cell proliferation in response to B. abortus. When peripheral blood mononuclear cells were cocultured with B. abortus and IFN-gamma, increasing amounts of IFN-gamma, from 1 to 100 U/ml, down regulated T-cell proliferation. Expression of MHC class II molecules on macrophages was increased independently by IFN-gamma or B. abortus treatment. Thus, class II molecule expression was not the cause of down regulation of T-cell proliferation as observed in other systems. However, B. abortus-IFN-gamma-treated macrophages obtained from overnight cultures had minimal membrane IL-1 compared with macrophages treated with B. abortus alone. Loss of membrane IL-1 required IFN-gamma and the o-polysaccharide of the lipopolysaccharide. IFN-gamma at 1 U/ml in combination with B. abortus produced a 32% decrease in T-cell response, while IFN-gamma at 100 U/ml added to B. abortus-treated cultures produced an 82% reduction in T-cell response. Membrane IL-1 levels were not altered when recombinant bovine IFN-alpha or the rough strain 45/20 of B. abortus, which lacks the o-polysaccharide, was used. Secreted IL-1 levels were unaffected by IFN-gamma and B. abortus treatment. The addition of recombinant bovine IL-1 beta (0.001 to 0.1 ng/ml) to B. abortus- and IFN-gamma-treated cultures failed to provide a signal necessary for T-cell proliferation. These data suggest that membrane IL-1 has a key role in T-cell activation in response to B. abortus. When the o-polysaccharide of B. abortus lipopolysaccharide is combined with IFN-gamma at an inappropriate time during an immune response, T-cell proliferation is prevented and cannot be restored by the addition of exogenous IL-1.     

Differential secretion of interleukin-12 (IL-12) subunits and heterodimeric IL-12p70 protein by CD-1 mice and murine macrophages in response to intracellular infection by Brucella abortus

The secretion of interleukin-12 (IL-12) following intracellular infection with virulent Brucella abortus strain 2308 was investigated in CD-1 mice and in CD-1 cultured peritoneal macrophages. Bioactive IL-12p70 and free non-immunoactive p40 subunits (IL-12p40) were determined by enzyme-linked immunosorbent assays. In CD-1 mice, B. abortus 2308 was a potent inducer of IL-12p40 (maximum levels were 5.9 and 3.4 ng/ml in sera and spleen homogenates, respectively). Secretion of IL-12p70 was also demonstrated in vivo, although at much lower levels (216.6 and 198.9 pg/ml in sera and spleen homogenates, respectively). Production of IL-12 over the first 7 days after infection was accompanied by active multiplication of B. abortus in the spleens of infected mice. CD-1 cultured peritoneal macrophages secreted only IL-12p40 (878.4 pg/10(7) macrophages) in response to B. abortus infection and no production of IL-12p70 was observed. In contrast, CD-1 peritoneal macrophages secreted detectable amounts of IL-12p70 (16.2 pg/10(7) macrophages) in response to purified lipopolysaccharide (S-LPS) from B. abortus 2308. The macrophages also secreted significant amounts of interferon-gamma (IFN-gamma) (520.1 pg/10(7) macrophages) in response to intracellular B. abortus. These results indicate that B. abortus 2308 is not a potent inducer of IL-12p70 production, whereas purified S-LPS from B. abortus 2308 induces the secretion of this bioactive form of IL-12 in cultured peritoneal macrophages. CD-1 peritoneal macrophages were able to secrete IFN-gamma, as well as high amounts of IL-12p40, in response to intracellular infection by B. abortus.     

Interleukin-10 downregulates protective immunity to Brucella abortus.

In vivo neutralization of interleukin-10 (IL-10) with an anti-IL-10 monoclonal antibody resulted in up to 10-fold fewer bacteria in the spleens of BALB/c mice infected with the virulent Brucella abortus strain 2308. In vitro neutralization of endogenous IL-10 in brucella antigen-stimulated cultures of splenocytes from infected mice resulted in increased gamma interferon production in these cultures, whereas exogenous recombinant IL-10 inhibited the ability of peptone-elicited peritoneal macrophages to control intracellular brucellae. These data suggest that IL-10 may be downregulating the immune response to B. abortus by affecting both macrophage effector function and the production of the protective Th1 cytokine gamma interferon.     

Tumor necrosis factor-alpha: central regulatory cytokine in the induction of macrophage antimicrobial activities

Expression of activated macrophage resistance to infection requires the cooperative interaction of interferon-gamma (IFN-gamma) and either interleukin-2 (IL-2), interleukin-4 or granulocyte/macrophage-colony-stimulating factor: no single cytokine is effective. For IFN-gamma and IL-2, the effector activity can be suppressed by the presence of anti-tumor necrosis factor-alpha (TNF-alpha) antibodies in the reaction mixture. IFN-gamma and IL-2, only in combination, induce TNF-alpha-specific mRNA and secretion of this cytokine by macrophages. Development of intracellular killing activity by activated macrophages also requires the autocrine effects of TNF-alpha. IFN-gamma provides the first signal for the production of nitric oxide (NO), the effector molecule for intracellular destruction of parasites. When IFN-gamma-treated cells are infected with pathogens, they are stimulated to make TNF-alpha. Expression of intracellular killing, as well as production of NO, is inhibited by anti-TNF-alpha antibody.     

Growth of Brucella abortus in macrophages from resistant and susceptible mouse strains

C57Bl/10 mice have a superior ability to control chronic infections with virulent strains of the intracellular bacteria Brucella abortus compared with BALB/c mice. While a number of differences in the cytokines produced by lymphocytes following infection of these two strains of mice have been shown, macrophages have not been evaluated for their role in conveying relative resistance. The importance of macrophages in control of brucella infections is demonstrated by the observations that intracellular survival of various strains of B. abortus directly correlates with their virulence in vivo, and the ability of macrophages to control brucellae in vitro has been shown to correlate with a brucella-resistant phenotype in ruminants. While both BALB/c and C57Bl are Nramp-susceptible mouse strains, additional differences in macrophage function outside of the Nramp1 gene effects could influence susceptibility to brucellosis. The studies conducted here comparing the ability of macrophages from C57Bl/10 and BALB/c mice indicate that the macrophages from resistant mice did not control intracellular growth of B. abortus strain 2308 more efficiently than those from the susceptible mice, either in the absence of, or following, interferon-gamma activation or iron supplementation. A number of different conditions for culturing macrophages were evaluated to rule out the influence of antibiotics on the conclusions drawn from the results.     

The role of integrase/recombinase xerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo

Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.     

Differential chemokine response of murine macrophages stimulated with cytokines and infected with Listeria monocytogenes

During inflammatory processes the infected macrophage is a rich source of chemokines which induce infiltration of leukocytes to the site of infection. We investigated the regulation of chemokine production by murine macrophages in response to infection with the intracellular bacterial pathogen, Listeria monocytogenes. As a source of quiescent macrophages, murine bone marrow-derived macrophages (BMM) cultured under serum-free conditions were used. With RT-PCR, we detected induction of RNA message for the chemokines macrophage inflammatory protein (MIP)-2, KC, MIP-1alpha, MIP-1beta, IFN-gamma-inducible protein- 10 and RANTES in L. monocytogenes-infected macrophages. Accordingly, ELISA-detectable MIP-1alpha, MIP-2 and KC protein was induced by infection with L. monocytogenes. In contrast, L. monocytogenes infection of BMM alone failed to induce considerable expression of monocyte chemoattractant protein (MCP)-1 at the mRNA or protein level, but co-treatment with IFN-gamma was necessary. Release of infection- triggered MIP-2, MIP-1alpha and KC was negatively regulated by IFN- gamma. Similarly, IL-4 stimulated MCP-1 release by infected macrophages but reduced production of MIP-1alpha, MIP-2 and KC. IL-10 turned out to be a general deactivator in terms of macrophage chemokine production. IL-13 had no effect on MIP-1alpha, MIP-2 and KC production by infected BMM, but slightly reduced MCP-1 release. By using IFN-gamma and IL-4 gene deletion mutant mice, in vivo regulation of these chemokines by IL- 4 and IFN-gamma in listeriosis was studied. In summary, our results show that chemokines are produced by macrophages infected with L. monocytogenes, and that chemokine release is differentially regulated by the macrophage modulators IFN-gamma, IL-4, IL-10 and IL-13.      

Modulation of Mycobacterium avium growth in murine macrophages: reversal of unresponsiveness to interferon-gamma by indomethacin or interleukin-4

The ability of soluble factors to modulate the growth of a virulent strain of Mycobacterium avium in murine peritoneal macrophages was studied. The virulent strain, TMC 702, grew progressively in the organs of susceptible BALB/C mice. In addition, this strain of M. avium grew progressively in untreated peritoneal macrophages. Treatment of macrophage monolayers with interferon-gamma (IFN-gamma) did not change significantly the intracellular growth of M. avium. Addition of indomethacin to IFN-gamma-treated macrophage monolayers rendered them significantly more bacteriostatic than macrophages treated with interferon alone, suggesting a role for prostaglandins in inducing unresponsiveness to IFN-gamma in infected cells. Additionally, treatment with tumour necrosis factor-alpha led to a modest increase in bacteriostasis, as compared to untreated monolayers. Further experiments with recombinant interleukins showed that interleukin-4 (IL-4), on its own, could increase bacteriostatic activity against M. avium in a reproducible fashion. Experiments with interleukin combinations showed that IFN-gamma and IL-4 treatment of macrophages rendered these cells almost fully bacteriostatic against M. avium, inclusion of scavengers of reactive oxygen species did not modify the beneficial effect of IFN-gamma and IL-4. Overall, our results suggest an important role for interleukins in modulating the interaction between virulent mycobacteria and murine macrophages.     

Toxoplasma gondii Infection of Neurons Induces Neuronal Cytokine and Chemokine Production, but Gamma Interferon- and Tumor Necrosis Factor-Stimulated Neurons Fail To Inhibit the Invasion and Growth of T. gondii

The intracellular parasite Toxoplasma gondii has the capacity to persist in the brain within neurons. In this study we demonstrated that T. gondii infected murine cerebellar neurons in vitro and replicated within these cells. Stimulation with gamma interferon (IFN-gamma) and/or tumor necrosis factor (TNF) did not enable neurons to inhibit parasite invasion and replication. Cultured neurons constitutively produced interleukin 1 (IL-1), IL-6, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta but not transforming growth factor beta1 (TGF-beta1), IL-10, and granulocyte-macrophage colony-stimulating factor. Neuronal expression of some cytokines (IL-6, TGF-beta1) and chemokines (MIP-1beta) was regulated by infection and/or by IFN-gamma and TNF.     

Response to stimulation with recombinant cytokines and synthesis of cytokines by murine intestinal macrophages infected with the Mycobacterium avium complex.

Current evidence suggests that the gut is the chief portal of entry for organisms of the Mycobacterium avium complex (MAC) in AIDS patients. Bacterial invasion of intestinal mucosa presumably occurs through epithelial cells, and M cells in the Peyer's patches, where the bacteria have contact with immunocompetent cells such as macrophages and T and B lymphocytes. As mucosal macrophages are probably the first line of defense against MAC, we examined their ability to inhibit intracellular growth of MAC when properly stimulated. Mouse intestinal macrophages were purified, infected with MAC 101, serovar 1, and MAC 86-2686, serovar 16, and subsequently stimulated with recombinant tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage colony-stimulating factor (M-CSF). Viable intracellular bacteria were quantitated at 24 h after infection and again after 4 days of infection. Stimulation with TNF-alpha, IFN-gamma, and GM-CSF, but not M-CSF, was associated with mycobacteriostatic and/or mycobactericidal activity in macrophages. Treatment with 10(3) U of TNF-alpha, GM-CSF, and IFN-gamma per ml at 24 h prior to infection with MAC resulted in a significant enhancement in killing of MAC at 4 days after infection, compared with that observed for macrophages exposed to cytokines after infection. When stimulated with lipopolysaccharide or live MAC, intestinal macrophages had produced significantly less TNF-alpha and transforming growth factor beta than had splenic and peritoneal macrophages, although the levels of production of interleukin 6 and interleukin 10 among the three populations of cells were similar. Intestinal macrophages can be stimulated with cytokines to inhibit the intracellular growth of MAC, but they have differentiated abilities to produce cytokines which can modulate the anti-MAC immune response.     

Mycobacterium tuberculosis H37Ra and H37Rv differential growth and cytokine/chemokine induction in murine macrophages in vitro.

The role of tumor necrosis factor-alpha (TNF-alpha) in controlling growth of Mycobacterium tuberculosis in murine peritoneal macrophages infected in vitro was studied. TNF-alpha was shown to be required but not sufficient, and the amount of TNF-alpha produced by the infected cells did not correlate with the extent of growth control. In this system, TNF-alpha-dependent control of growth of the avirulent strain H37Ra was independent of inducible nitric oxide synthase (iNOS) and interferon-gamma (IFN-gamma), as shown by the infection of macrophages from selected gene-disrupted mice. TNF-alpha-mediated bacteriostasis of H37Ra in the infected macrophages was associated with increased expression of selected Th1-type cytokines and chemokines. In contrast, growth of the virulent strain H37Rv in macrophages involved upregulation by infected cells of Th2-type cytokines, including interleukin-5 (IL-5), IL-10, and IL-13. Taken together, these results suggest that the particular nature of macrophage activation and the cytokine and chemokine response to infection with different M. tuberculosis strains determine the ability of the cells to control the growth of the intracellular bacilli.     

Neopterin release from human endothelial cells is triggered by interferon-gamma.

Human umbilical vein endothelial cells (HUVEC) were investigated for their ability to produce neopterin, a biochemical marker for an activated immune system. Interferon-gamma (IFN-gamma), IL-1 alpha, IL-2, IL-6, tumour necrosis factor-alpha, granulocyte/macrophage colony stimulating factor, phytohaemagglutinin and concanavalin A were used to stimulate HUVEC. While IFN-gamma induced neopterin release from HUVEC in a time- and dose-dependent manner, all the other cytokines used had no effect on neopterin production. High neopterin levels are found in patients with rejection episodes or infections. Our results suggest that not only monocytes and macrophages, which are known to synthesize neopterin, but also endothelial cells are responsible for these high serum neopterin levels.     

High levels of nitric oxide production decrease early but increase late survival of Brucella abortus in macrophages.

Nitric oxide (NO), produced by the iNOS protein, is known as a defense mechanism against various pathogens and an apoptotic inducer of cells. Apoptosis can also be a host protective mechanism against intracellular bacteria. The intracellular survival of Brucella abortus in RAW264.7 macrophages was examined under conditions of the apoptotic inducer, NO. Since B. abortus does not induce high output of NO, Escherichia coli LPS and IFN-gamma, as potential therapeutic modalities, were added to increase the expression of iNOS, and thus NO. Using 10 ng/ml E. coli LPS and 25 U/ml IFN-gamma, nitrite production was as high as 140 microM by 72 h. However, when macrophages were infected with B. abortus, the nitrite concentration was 60 microM after 72 h post infection, greater than a two-fold decrease. The number of surviving bacteria decreased, from 6 to 24 h, in the presence of nitrite accumulation. In the absence of B. abortus there was an increase in apoptotic cells at 72 h with high nitrite accumulation. In contrast, the number of macrophage apoptotic bodies decreased in the presence of B. abortus. The data suggest that: (i) NO accelerates the killing of intracellular B. abortus, but not to completion during the first 24 h of infection; (ii) B. abortus can prevent apoptosis as an advantage for bacterial survival inside macrophages and (iii) surviving intracellular bacteria then replicate steadily after 24 h. B. abortus probably expresses genes that counteract the effect of a high NO environment or activates genes to utilize NO as a nitrogen source, as the Brucella genome codes for nitric and nitrous oxide reductase genes.     

Legionella pneumophila replication in macrophages inhibited by selective immunomodulatory effects on cytokine formation by epigallocatechin gallate, a major form of tea catechins

Epigallocatechin gallate (EGCg) is a major form of tea catechin and has a variety of biological activities, including antitumor as well as antimicrobial activity against some pathogens. Although the biological activities of EGCg have been extensively studied, its immunological effects are not well known. In the present study, the ability of EGCg to modulate macrophage immune functions in an in vitro Legionella pneumophila infection model of macrophages was examined. The study showed that EGCg inhibited the growth of L. pneumophila in macrophages at a concentration as low as 0.5 microg/ml without any direct antibacterial effect on the organisms. The EGCg selectively upregulated the production of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) and downregulated IL-10 production of macrophages induced by L. pneumophila infection in a dose-dependent manner, but did not alter IL-6 production even at a high dose. The upregulation of the levels of macrophage gamma interferon (IFN-gamma) mRNA by EGCg was also demonstrated. Treatment of macrophage cultures with anti-TNF-alpha and anti-IFN-gamma monoclonal antibodies markedly abolished the anti-L. pneumophila activity of macrophages induced by the EGCg treatment. These results indicate that EGCg selectively alters the immune responses of macrophages to L. pneumophila and leads to an enhanced anti-L. pneumophila activity of macrophages mediated by enhanced production of both TNF-alpha and IFN-gamma. However, the enhancement of in vitro anti-L. pneumophila activity by EGCg may not be directly mediated by IL-10 and IL-12 production modulation. Thus, the results of this study revealed the immunomodulatory effect of EGCg on macrophages, which have a critical role in infections.     

Interleukin-1 receptor-associated kinase 4 is essential for initial host control of Brucella abortus infection

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Recent studies have revealed that Toll-like receptor (TLR)-initiated immune response to Brucella spp. depends on myeloid differentiation factor 88 (MyD88) signaling. Therefore, we decided to study the role of the interleukin-1 receptor-associated kinase 4 (IRAK-4) in host innate immune response against B. abortus. After Brucella infection, it was shown that the number of CFU in IRAK-4(-/-) mice was high compared to that in IRAK-4(+/-) animals only at 1 week postinfection. At 3 and 6 weeks postinfection, IRAK-4(-/-) mice were able to control the infection similarly to heterozygous animals. Furthermore, the type 1 cytokine profile was evaluated. IRAK-4(-/-) mice showed lower production of systemic interleukin-12 (IL-12) and gamma interferon (IFN-γ). Additionally, a reduced percentage of CD4(+) and CD8(+) T cells expressing IFN-γ was observed compared to IRAK-4(+/-). Further, the production of IL-12 and tumor necrosis factor alpha (TNF-α) by macrophages and dendritic cells from IRAK-4(-/-) mice was abolished at 24 h after stimulation with B. abortus. To investigate the role of IRAK-4 in mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, macrophages were stimulated with B. abortus, and the signaling components were analyzed by protein phosphorylation. Extracellular signal-regulated kinase 1 (ERK1) and ERK2 and p38 as well as p65 NF-κB phosphorylation was profoundly impaired in IRAK-4(-/-) and MyD88(-/-) macrophages activated by Brucella. In summary, the results shown in this study demonstrated that IRAK-4 is critical to trigger the initial immune response against B. abortus but not at later phases of infection.     

Early in vitro priming of distinct T(h) cell subsets determines polarized growth of visceralizing Leishmania in macrophages

An in vitro priming system of murine naive splenocytes was established to investigate early immune responses to LEISHMANIA: chagasi, the agent of visceral leishmaniasis in the New World. Priming of splenocytes from resistant C3H and CBA or susceptible BALB and B10 mice with L. chagasi resulted in blast transformation and in proliferating parasite-specific CD4(+) T cells secreting a differential complement of cytokines (IFN-gamma and low IL-10 levels for resistant T cells; IFN-gamma, IL-4 and high IL-10 levels for susceptible T cells). After priming, intracellular parasite load was much higher in susceptible than in resistant-type splenocyte cultures. On the other hand, infection of purified splenic macrophages from either resistant or susceptible mice with live L. chagasi promastigotes, resulted in comparable parasite loads. Moreover, when early CD4(+) T cell priming in splenocyte cultures was disrupted with anti-CD4 mAb, polarized parasite growth was abolished, becoming comparable in resistant and susceptible cultures. Neutralizing IL-4 activity during splenocyte priming did not affect the final parasite load in susceptible cultures. However, neutralizing IL-10 activity markedly decreased parasite load in susceptible, but not in resistant splenic macrophages. These results suggest that IL-10 plays an important role in L. chagasi infection in susceptible hosts. The results also indicate that innate control of growth of a visceralizing LEISHMANIA: in splenic macrophages results from the ability to activate different CD4(+) T cell subsets.     

Role of IL-12 in macrophage activation during intracellular infection: IL-12 and mycobacteria synergistically release TNF-alpha and nitric oxide from macrophages via IFN-gamma induction

IL-12 is believed to play an important role in cell-mediated immunity against intracellular infection primarily by acting on T and NK cells. Recent evidence has suggested, however, that IL-12 has broader cellular targets than previously thought. In this study, we examined the role of IL-12 in macrophage TNF-alpha and nitric oxide (NO) release by using an in vitro model of intracellular infection. IL-12 alone released relatively little TNF-alpha and NO, whereas live mycobacteria alone released TNF-alpha markedly but little NO from murine alveolar macrophages. However, IL-12 and mycobacteria together enhanced TNF-alpha and NO release synergistically. Because IL-12 and mycobacteria also released IFN-gamma from macrophages synergistically, and exogenous IFN-gamma with mycobacteria enhanced TNF-alpha and NO release synergistically, we examined the role of endogenous IFN-gamma in IL-12/mycobacteria-stimulated macrophage activation. Using macrophages from mice deficient in IFN-gamma, we found that IL-12/mycobacteria-enhanced macrophage TNF-alpha and NO release was mediated through endogenous IFN-gamma. We further demonstrated that IFN-gamma and mycobacteria together had a selective effect on macrophage cytokine release because they released TNF-alpha synergistically but not macrophage chemotactic protein-1 (MCP-1). These findings reveal that IL-12 can activate macrophages potently during intracellular infection, and this activating effect is mediated primarily through its effect on macrophage IFN-gamma release.