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.     

Effects of cytokines on intracellular growth of Brucella abortus.

Interleukin 1 alpha (IL-1 alpha), IL-2, IL-4, IL-6, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocyte macrophage colony-stimulating factor (GM-CSF) were tested for their abilities to alter the growth of Brucella abortus in BALB/c J774A.1 murine macrophages. IL-1 alpha, IL-4, IL-6, tumor necrosis factor alpha, and granulocyte macrophage-colony-stimulating factor had no consistent or significant effect on the growth of the avirulent B. abortus strain 19. In contrast, the addition of either IFN-gamma or IL-2 at 100 U/ml to the macrophage cultures resulted in a significant reduction in the number of intracellular bacteria that was not attributable to decreased infection rates. With IL-2, the reduction was most often apparent only during the first 24 h after infection, while inhibition with IFN-gamma was apparent throughout the culture period of 48 h. The addition of either IL-2 or IFN-gamma to macrophage cultures also resulted in reduced intracellular CFU of the virulent B. abortus strain 2308 and the attenuated rough mutant B. abortus strain RB51. Inhibition of intracellular growth was not augmented by combinations of cytokines. Additional studies with IFN-gamma and IL-2 indicated that they could mediate the inhibition of intracellular growth of B. abortus in resident and thioglycolate broth-induced BALB/c peritoneal macrophages and in splenic macrophages. IFN-gamma also inhibited bacterial growth when added after infection of the macrophages, although the magnitude of the antibrucellae effects was less than that when it was added before infection. Furthermore, the maximal inhibitory effect was sustained only when IFN-gamma remained in the cultures after infection of the macrophages.     

Lipopolysaccharide augments HLA-A,B,C molecule expression but inhibits interferon-gamma-induced HLA-DR molecule expression on cultured human endothelial cells.

The effect of bacterial lipopolysaccharide (LPS) on the expression of class I and II major histocompatibility complex (MHC) molecules on the surface of cultured human umbilical vein endothelial cells (HUVEC) was determined by indirect immunofluorescent staining followed by flow cytometric analysis. LPS at concentrations higher than 0.01 micrograms/ml augmented class I MHC (HLA-A,B,C) expression on HUVEC in a concentration-dependent manner. Optimal augmentation, approximately sixfold compared with control, was seen with 10 micrograms/ml of LPS. Time-course experiments indicated that the augmentation was maximal on Day 4. In contrast, LPS had no effect on the induction of class II MHC (HLA-DR) molecules and at concentrations higher than 0.01 micrograms/ml inhibited the interferon-gamma(IFN-gamma)-induced class II MHC expression. The inhibition was about 60% at the concentration of 100 micrograms/ml of LPS. Interleukin-1 (IL-1) had a similar effect as LPS on class I and II MHC expression. However, LPS appeared to affect MHC expression directly and not through production of IL-1 or cyclo-oxygenase pathway products, since anti-IL-1 antibodies or an inhibitor of cyclo-oxygenase pathway products, indomethacin, failed to reverse the effects of LPS. These data stress the role of LPS as a direct modulatory factor of class I and II MHC expression on endothelial cells during the development of immune and inflammatory response against Gram-negative bacteria.     

Vitamin D differentially regulates B7.1 and B7.2 expression on human peripheral blood monocytes.

The hormonal active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1, 25(OH)2D3), inhibits (through an unknown mechanism) the ability of monocytes/macrophages to induce T-cell activation. For T cells to be optimally activated, recognition of antigen/major histocompatibility complexes (MHC) by the T-cell receptor (TCR) must be accompanied by a second costimulatory signal. Considerable experimental data now suggest that this costimulatory signal is predominantly generated by B7.1 and/or B7.2 molecules, expressed on antigen-presenting cells (APC), when engaged to their counter-receptor, CD28, present on T cells. To determine whether the inhibitory effect of 1,25(OH)2D3 on monocytes/macrophages might involve modulation of the expression of B7.1 and B7.2 molecules, we analysed (by flow cytometry) the influence of 1,25(OH)2D3 and an analogue, KH 1060, on the expression of these two molecules at the surface of resting human peripheral blood monocytes. In parallel, we tested the effect of these two agents on human monocyte expression of cell-surface markers (CD14 and CD4) and antigen-presenting molecules (MHC class I and MHC class II). Our results showed that both 1,25(OH)2D3 and KH 1060 inhibited the basal expression of B7.2 in a dose- and time-dependent manner, without affecting B7.1. Moreover, these two compounds increased CD14 and reduced MHC class II and CD4 expression. Furthermore, the effect of 1,25(OH)2D3 on B7 molecule expression in combination with lipopolysaccharide (LPS) or cytokines, including interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), was studied. The 1,25(OH)2D3-induced B7.2 down-regulation was still detectable when monocytes were activated by IL-10, IFN-gamma and TNF-alpha but not with LPS. Moreover, the induction of B7.1 by TNF-alpha was inhibited by addition of 1, 25(OH)2D3. We conclude that the ability of 1,25(OH)2D3 to decrease B7.2 expression on human monocytes might contribute to its inhibitory effect on APC-dependent T-cell activation and to its immunosuppressive properties observed in autoimmune diseases and organ transplantation.     

In the absence of endogenous gamma interferon, mice acutely infected with Neospora caninum succumb to a lethal immune response characterized by inactivation of peritoneal macrophages.

Following infection with Neospora caninum, BALB/c mice were shown to be resistant to an acute infection but developed a latent chronic infection. However, BALB/c background gamma interferon (IFN-gamma)-deficient mice were sensitive to the acute infection. Since the immune response in IFN-gamma-deficient mice is scantly known, we examined the function of macrophages, major histocompatibility complex (MHC) class II expression, T-cell responses, and serum cytokine levels in the mice. All IFN-gamma-deficient mice died within 9 days of infection with N. caninum, whereas those treated with exogenous IFN-gamma lived longer. Although N. caninum invaded various organs in both types of mice at the early stage of infection, the parasite was not detected in the brains of resistant hosts until 21 days postinfection (dpi). Peritoneal macrophages from IFN-gamma-deficient mice were activated by exogenous IFN-gamma associated with inhibition of parasite growth and nitric oxide production as were those from BALB/c mice. IFN-gamma-deficient mice failed to increase MHC class II expression on macrophages. Moreover, BALB/c mice induced T-cell proliferation while IFN-gamma-deficient mice did not. However, in vivo treatment with exogenous IFN-gamma induced up-regulated MHC class II expression in IFN-gamma-deficient mice. BALB/c mice treated with an antibody to CD4 showed an increase in morbidity and mortality after parasite infection. In serum, significant levels of IFN-gamma and interleukin-4 (IL-4) were detected in resistant hosts, whereas IL-10 was detected in IFN-gamma-deficient mice. The levels of IL-12 in IFN-gamma-deficient mice were higher than those in BALB/c mice at 7 dpi. The present study indicates that early IFN-gamma production has a crucial role in the activation of peritoneal macrophages for the induction of protective immune responses against N. caninum.     

Distinct Effects of γ Interferon on Human B-Lymphocyte Precursor Cell Lines

The effect of gamma interferon (IFN-gamma) on proliferation and antigenic characteristics of cell lines belonging to the B-cell progenitor compartment was studied. We observed a selective effect of recombinant IFN-gamma but not IFN-alpha on proliferation of the B-precursor cell lines Reh and KM3. On day 4, after addition of 400 mu/ml IFN-gamma the [3H]thymidine uptake in these cells was reduced to 60% and 45% respectively, while no effect of IFN-gamma was evident on the proliferation of the more mature B-cell lines Raji, Ramos, B85, and Daudi. On the other hand, both Reh and Ramos showed induction of major histocompatibility complex (MHC) class I antigen expression in response to 400 mu/ml IFN-gamma. In contrast to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), IFN-gamma did not induce increased MHC class II antigen expression on Reh cells. Taken together, our results indicate that IFN-gamma fulfils distinct functions at different levels in the development of B cells.     

Differential expression of alternative H2-M isoforms in B cells, dendritic cells and macrophages by proinflammatory cytokines

Major histocompatibility (MHC) class II heterodimers bind peptides generated by degradation of endocytosed antigens and display them on the surface of antigen presenting cells (APCs) for recognition by CD4+ T cells. Efficient loading of MHC class II molecules with peptides is catalyzed by the MHC class II-like molecule H2-M. The coordinate regulation of MHC class II and H2-M expression is a prerequisite for efficient MHC class II/peptide assembly in APCs determining both the generation of the T cell repertoire in the thymus and cellular immune responses in the periphery. Here we show that expression of H2-M and MHC class II genes is coordinately and cell type-specific regulated in splenic B cells, splenic dendritic cells (DCs) and peritoneal macrophages (Mphi) in response to proinflammatory and immunoregulatory cytokines, including GM-CSF, IFN-gamma, TGF-beta2, IL-4, IL-10 and viral IL-10. In addition, ratio-RT-PCR expression analysis of the duplicated H2-Mbeta-chain loci demonstrates for the first time that Mbl and Mb2 genes are differentially expressed in individual APC types. Mb2 is preferentially expressed in IL-4, GM-CSF, IL-10, vIL-10 and IFN-gamma stimulated splenic B cells, whereas splenic DCs express both Mb genes at almost equal levels. In contrast, peritoneal Mphi express predominantly Mb2 but stimulation with IFN-gamma induces a switch towards Mb1 expression. These data suggest a common mechanism that regulates coordinate expression of H2-M and MHC class II genes in professional APCs. Differential expression of Mb1 and Mb2, and by consequence alternative H2-M isoforms (Malphabeta1 or Malphabeta2), may influence the nature of the peptide repertoire presented by different APC types.     

Interleukin-10 differentially modulates MHC class II expression by mesangial cells and macrophages in vitro and in vivo.

Inhibition of major histocompatibility complex (MHC) class II expression by macrophages is the primary mechanism by which interleukin-10 (IL-10) exerts immune suppression. Little, however, is known of the effects of IL-10 on other types of cells which can be induced to express MHC class II during an inflammatory response. We therefore studied the effects of IL-10 treatment on the expression of MHC class II molecules in a rat model of immunologically induced glomerulonephritis. MHC class II mRNA levels in whole kidney were increased in saline-treated (control) animals with glomerulonephritis (2.6-fold increase versus normal, P = 0.028) and this was partially inhibited by treatment with IL-10 (P = NS). Double immunostaining of tissue sections was used to compare MHC class II expression by infiltrating macrophages and resident glomerular cells. IL-10 treatment reduced the proportion of glomerular macrophages which expressed detectable MHC class II (70% reduction, P = 0.03). In contrast, IL-10 treatment was associated with an increase in the number of resident glomerular cells expressing MHC class II, particularly within mesangial areas. Therefore, the effects of IL-10 on macrophages and mesangial cells were compared in vitro. IL-10 reduced constitutive MHC class II mRNA and cell surface expression by peritoneal macrophages. In contrast, IFN-gamma-stimulated mesangial cells (1097 cell line) cultured with IL-10 for 24 hr showed increased MHC class II mRNA (26% increase) and surface expression (72% increase in percentage MHC II+ by flow cytometry, P = 0.04) as compared with cells stimulated with IFN-gamma alone. IL-10 also directly up-regulated expression of ICAM-1 by 1997 cells. In conclusion, IL-10 was found to have contrasting effects on the production and cell surface expression of MHC class II molecules by mesengial cells and by macrophages, both in vitro and in vivo. The implications of these findings for IL-10-mediated immunosuppression are discussed.     

Effects of IFN-gamma and interleukin-1beta on major histocompatibility complex antigen and intercellular adhesion molecule-1 expression by 9L gliosarcoma: relevance to its cytolysis by alloreactive cytotoxic T lymphocytes.

To enhance the efficacy of cellular immunotherapy for gliomas, we tested the concept of using proinflammatory cytokine treatment with interferon-gamma (IFN-gamma) or interleukin-1beta (IL-1beta) or both to render glioma cells more susceptible to cytolysis by alloreactive cytotoxic T lymphocytes (aCTL). The cytokines, separately or in combination, were able to upregulate major histocompatibility complex (MHC) class I antigen or intercellular adhesion molecule-1 (ICAM-1) on Fischer rat 9L gliosarcoma cells. 9L cells were incubated in vitro for 24, 48, or 72 h with varying concentrations of rat IFN-gamma (0-2000 U/ml) or recombinant human IL-1 (rHUIL-1) (0-1000 U/ml) or both. By 48 h, IFN-gamma (500 U/ml) maximally induced the percentage of positive expressing cells and the relative antigen density of MHC class I and ICAM-1 on 9L cells, whereas IL-1 induced only ICAM-1 expression. Simultaneous incubation of IL-1 with IFN-gamma did not further affect the induction of class I on 9L cells more than that achieved with IFN-gamma alone. 9L cells with upregulated MHC class I and ICAM-1 expression were more sensitive to lysis by aCTL in in vitro cytotoxicity assays, regardless of whether the precursor aCTL came from naive or from 9L-immunized rats. Furthermore, inhibition of 9L cytotoxicity in assays that included blocking antibodies to MHC class I or to ICAM-1 revealed that T cell receptor (TCR) interactions with MHC class I and that ICAM-1 interactions with lymphocyte function-associated-1 (LFA-1) antigen account for a portion of the glioma lysis by aCTL.     

Interaction of Brucella abortus lipopolysaccharide with major histocompatibility complex class II molecules in B lymphocytes.

Lipopolysaccharide (LPS), a major amphiphilic molecule located at the outer membrane of gram-negative bacteria, is a potent antigen known to induce specific humoral immune responses in infected mammals. LPS has been described as a polyclonal activator of B lymphocytes, triggering the secretion of antibodies directed against distinct sugar epitopes of the LPS chain. But, how LPS is handled by B cells remains to be fully understood. This task appears to be essential for a better knowledge of the anti-LPS humoral immune response. In this study, we examine the internalization of LPS and its interaction with antigen-presenting major histocompatibility complex (MHC) class II molecules in murine and human B-cell lines. By use of immunofluorescence, we observe that structurally different LPSs from Brucella and Shigella strains accumulate in an intracellular compartment enriched in MHC class II molecules. By use of immunoprecipitation, we illustrate that only Brucella abortus LPS associates with MHC class II molecules in a haplotype-independent manner. Taken together, these results raise the possibility that B. abortus LPS may play a role in T-cell activation.     

Brucella abortus as a potential vaccine candidate: induction of interleukin-12 secretion and enhanced B7.1 and B7.2 and intercellular adhesion molecule 1 surface expression in elutriated human monocytes stimulated by heat-inactivated B. abortus.

Development of a vaccine which is capable of generating a strong cellular immune response associated with gamma interferon (IFN-gamma) production and cytotoxic T-cell development requires that the immunogen be capable of inducing the secretion of interleukin-12 (IL-12), which is a pivotal factor for the differentiation of Th1 or Tc1 cells. We have previously shown that the heat-inactivated gram-negative bacterium Brucella abortus can induce IFN-gamma secretion by T cells. In the present study, we demonstrate that B. abortus and lipopolysaccharide (LPS) from B. abortus can induce IL-12 p40 mRNA expression and protein secretion by human elutriated monocytes (99% pure). p40 mRNA was detected within 4 h, and p40 protein could be measured at 24 h. This induction was abrogated by anti-CD14 monoclonal antibody, suggesting that monocytes recognize B. abortus via their receptor for LPS. The biological activity of IL-12 secreted by B. abortus-stimulated monocytes was demonstrated by its ability to upregulate IFN-gamma mRNA expression in T cells separated from monocytes and B. abortus by a transwell membrane. The B. abortus-induced IL-12 also enhanced NK cytolytic activity against K562 target cells. B. abortus was shown to rapidly increase the expression of the costimulatory molecules B7.1 and B7.2 and intercellular adhesion molecule 1 on human monocytes. Together, these data indicate that B. abortus can directly activate human monocytes and provide the cytokine milieu which would direct the immune response towards Th1-Tc1 differentiation.     

Brucella abortus inhibits major histocompatibility complex class II expression and antigen processing through interleukin-6 secretion via Toll-like receptor 2

The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-gamma)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-gamma production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection.     

Human peripheral blood CD4+ and CD8+ T cells express Th1-like cytokine mRNA and proteins following in vitro stimulation with heat-inactivated Brucella abortus.

Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells.     

Drug-induced inhibition of insulin recognition by T-cells: the antirheumatic drug aurothiomalate inhibits MHC binding of insulin peptide.

Previous work has shown that the Au(I) moiety of the antirheumatic drug disodium aurothiomalate (Au(I)TM) can selectively inhibit the response of murine CD4+ T-cell hybridomas to antigenic peptides containing two or more cysteine (Cys) residues. Here, we investigated the mechanism that underlies the inhibitory effect of Au(I)TM on T-cell recognition of bovine insulin (BI). We found that low concentrations of Au(I)TM (10 microM) inhibited the BI-induced proliferation of bulk T-cells from BI-immunized BALB/c mice as well as the IL-2 release of Ab- and Ad-restricted T-cell hybridoma clones. Au(I)TM was found to inhibit binding of the immunodominant BI peptide A1-14 to isolated MHC class II molecules. We suggest that Au(I) forms stable chelate complexes with thiol groups of two Cys residues in the BI A1-14 peptide. Conceivably, formation of these metal-peptide complexes keeps the peptide in a sterical conformation that cannot undergo binding to MHC class II molecules, resulting in an inhibition of T-cell activation due to insufficient peptide presentation.     

Activation of murine dendritic cells and macrophages induced by Salmonella enterica serovar Typhimurium

Macrophages and dendritic cells (DCs) are antigen-presenting cells (APCs), and the direct involvement of both cell types in the immune response to Salmonella has been identified. In this study we analysed the phenotypic and functional changes that take place in murine macrophages and DCs in response to live and heat-killed Salmonella enterica serovar Typhimurium. Both types of cell secreted proinflammatory cytokines and nitric oxide (NO) in response to live and heat-killed salmonellae. Bacterial stimulation also resulted in up-regulation of costimulatory molecules on macrophages and DCs. The expression of major histocompatibility complex (MHC) class II molecules by macrophages and DCs was differentially regulated by interferon (IFN)-gamma and salmonellae. Live and heat-killed salmonellae as well as lipopolysaccharide (LPS) inhibited the up-regulation of MHC class II expression induced by IFN-gamma on macrophages but not on DCs. Macrophages as well as DCs presented Salmonella-derived antigen to CD4 T cells, although DCs were much more efficient than macrophages at stimulating CD4 T-cell cytokine release. Macrophages are effective in the uptake and killing of bacteria whilst DCs specialize in antigen presentation. This study showed that the viability of salmonellae was not essential for activation of APCs but, unlike live bacteria, prolonged contact with heat-killed bacteria was necessary to obtain maximal expression of the activation markers studied.     

Dual roles for class II major histocompatibility complex molecules in staphylococcal enterotoxin-induced cytokine production and in vivo toxicity.

The staphylococcal enterotoxins (SE) specifically bind to class II major histocompatibility complex (MHC) proteins, resulting in activation of monocytes and T cells. The SE cause weight loss in mice, which is dependent on T-cell stimulation and tumor necrosis factor alpha (TNF-alpha) production. Here we use a mutant of staphylococcal enterotoxin A that binds class II MHC molecules and activates monocytes but not T cells to evaluate the relative contributions of monocyte- and T-cell-stimulatory activities to in vivo toxicity. The mutant toxin did not cause weight loss in B10. BR mice but did stimulate monocyte TNF-alpha production in vitro, as did the wild-type toxin. Addition of a supernatant from toxin-activated T cells enhanced monocyte-stimulatory activity of both mutant and wild-type toxins fivefold. The effect of the supernatant could be mimicked by recombinant gamma interferon (IFN-gamma) and was inhibited by antibody to IFN-gamma. These results suggest that toxin-induced monocyte TNF-alpha production is upregulated by IFN-gamma, which likely represents the T-cell requirement in SE-mediated weight loss. Our studies thus implicate two distinct class II MHC-dependent signaling pathways for SE, the first involving direct signal transduction through class II MHC molecules mediated by either mutant or wild-type toxin and the second requiring T-cell stimulation by toxin-class II MHC complexes with consequent production of IFN-gamma. We suggest that both pathways are required for optimal monocyte TNF-alpha production in vitro and SE-induced toxicity in vivo.     

Systematic evaluation of the conditions required for the generation of immature rat bone marrow-derived dendritic cells and their phenotypic and functional characterization

This study systematically evaluated the conditions required for generating immature rat bone marrow-derived dendritic cells (BMDCs) and characterized their phenotype. The culture of Wistar rat bone marrow cells for 7 days in an optimal cytokine environment (granulocyte macrophage-colony stimulating factor (GM-CSF), 10 ng/ml; IL-4, 5 ng/ml) resulted in adherent and non-adherent cell populations, but only the adherent population predominantly expressed the rat DC marker OX62. Adherent OX62+ cells were immature, in that they expressed lower levels of CD86 and MHC class II and were more phagocytic than their non-adherent OX62+ counterparts. Adherent BMDCs constitutively produced low levels of IL-12 and nitric oxide (NO), levels of both of which were markedly increased following lipopolysaccharide (LPS) activation. Activation also increased the proportion of OX62+ cells expressing CD40, CD54 and CD86 and their intensity of expression, however, unlike murine BMDCs, it had no effect on CD80 and MHC class II expression. Although the proliferation of allogeneic Lewis splenocytes in response to immature resting and LPS-activated (mature) Wistar BMDCs was of a similar magnitude, levels of IL-12 after 5 days were significantly higher in cultures containing LPS-activated BMDCs and the IFN-gamma/IL-4 cytokine ratio differed markedly (2.35 vs. 6.66, respectively). This study systematically defines conditions for generating immature rat BMDC populations and demonstrates qualitative differences in the phenotype of immune responses induced by resting and LPS-activated BMDC populations.     

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.