Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus

Inflammation is a hallmark of brucellosis. Although Brucella abortus, one of the disease's etiologic agents, possesses cytokine-stimulatory properties, the mechanism by which this bacterium triggers a proinflammatory response is not known. We examined the mechanism whereby heat-killed B. abortus (HKBA), as well as its LPS, induces production of inflammatory cytokines in monocytes/macrophages. Polymyxin B, a specific inhibitor of LPS activity, did not inhibit the production of TNF-alpha- and IL-6-induced HKBA in the human monocytic cell line THP-1. HKBA induced the production of these cytokines in peritoneal macrophages of both C3H/HeJ and C3H/HeN mice, whereas B. abortus LPS only stimulated cells from C3H/HeN mice. Anti-TLR2 Ab, but not anti-TLR4 Ab, blocked HKBA-mediated TNF-alpha and IL-6 production in THP-1 cells. Because bacterial lipoproteins, a TLR2 ligand, have potent inherent stimulatory properties, we investigated the capacity of two B. abortus lipoproteins, outer membrane protein 19 (Omp19) and Omp16, to elicit a proinflammatory response. Lipidated (L)-Omp16 and L-Omp19, but not their unlipidated forms, induced the secretion of TNF-alpha, IL-6, IL-10, and IL-12 in a time- and dose-dependent fashion. Preincubation of THP-1 cells with anti-TLR2 Ab blocked L-Omp19-mediated TNF-alpha and IL-6 production. Together, these results entail a mechanism whereby B. abortus can stimulate cells from the innate immune system and induce cytokine-mediated inflammation in brucellosis. We submit that LPS is not the cause of inflammation in brucellosis; rather, lipoproteins of this organism trigger the production of proinflammatory cytokines, and TLR2 is involved in this process.     

Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection

Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c+ cells. In addition, IL-12 and TNF-alpha production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-alpha administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-alpha, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-alpha production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-gamma.     

Neisseria gonorrhoeae triggers the PGE2/IL-23 pathway and promotes IL-17 production by human memory T cells

PGE2 is a potent modulator of the T helper (Th)17 immune response that plays a critical role in the host defense against bacterial, fungal and viral infections. We recently showed high serum levels of interleukin (IL)-17 in patients with gonococcal infection and we hypothesized that Neisseria gonorrhoeae could exploit a PGE2 mediated mechanism to promote IL-17 production. Here we show that N. gonorrhoeae induces human dendritic cell (DC) maturation, secretion of prostaglandin E2 and proinflammatory cytokines, including the pro-Th17 cytokine IL-23. Blocking PGE2 endogenous synthesis selectively reduces IL-23 production by DC in response to gonococcal stimulation, confirming recent data on PGE2/IL-23 crosstalk. N. gonorrhoeae stimulated DC induce a robust IL-17 production by memory CD4(+) T cells and this function correlates with PGE2 production. Our findings delineate a previously unknown role for PGE2 in the immune response to N. gonorrhoeae, suggesting its contribute via Th17 cell expansion.     

Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.     

IL-6 production by pulmonary dendritic cells impedes Th1 immune responses

Mucosal tissues, such as the lung, are continually exposed to both foreign and environmental Ags. To counter the potential inflammatory tissue injury of chronic Th1-mediated responses against these Ags, mucosal sites may skew toward Th2 immune responses. However, the mechanism by which this occurs is unknown. Dendritic cells (DC), as orchestrators of the immune response, skew Th1/Th2 differentiation by cytokine secretion and expression of specific cell surface markers. We compared DC from mucosal and systemic locations. In this study, we show that the lung lacks a CD8alpha(+) DC subpopulation and contains DC that appear less mature than splenic DC. Furthermore, we demonstrate that pulmonary DC produce significant levels of IL-6 and fail to produce the Th1-polarizing cytokine IL-12. Importantly, we demonstrate that IL-6 negatively regulates IL-12 production, as pulmonary DC from IL-6(-/-) mice produce significant levels of IL-12 and induce Th1 polarization of naive CD4(+) T cells. Furthermore, we demonstrate that IL-6 is sufficient to explain the differential polarizing abilities of pulmonary and splenic DC, as splenic DC cocultures supplemented with IL-6 polarize naive T cells toward Th2, and pulmonary DC cultures in which IL-6 was removed with neutralizing Ab resulted in more Th1 polarization, pointing to IL-6 as the mechanism of Th2 polarization in the lung. We propose that the Th2 response seen in the lung is due to DC-mediated inhibition of Th1 responses via IL-6 production, rather than enhanced Th2 responses, and that this regulation decreases the likelihood of chronic inflammatory pathology in the lung.     

A novel role for IL-3: human monocytes cultured in the presence of IL-3 and IL-4 differentiate into dendritic cells that produce less IL-12 and shift Th cell responses toward a Th2 cytokine pattern

Dendritic cells (DC) derived from plasmacytoid precursors depend on IL-3 for survival and proliferation in culture, and they induce preferentially Th2 responses. Monocytes express not only GM-CSF receptors, but also IL-3Rs. Therefore, we examined whether IL-3 had an effect on the functional plasticity of human monocyte-derived DC generated in a cell culture system that is widely used in immunotherapy. DC were generated with IL-3 (instead of GM-CSF) and IL-4. Yields, maturation, phenotype (surface markers and Toll-like receptors), morphology, and immunostimulatory capacity were similar. Only CD1a was differentially expressed, being absent on IL-3-treated DC. In response to CD40 ligation DC generated in the presence of IL-3 secreted significantly less IL-12 p70 and more IL-10 compared with DC grown with GM-CSF. Coculture of naive allogeneic CD4(+) T cells with DC generated in the presence of IL-3 induced T cells to produce significantly more IL-5 and IL-4 and less IFN-gamma compared with stimulation with DC generated with GM-CSF. These data extend the evidence that different cytokine environments during differentiation of monocyte-derived DC can modify their Th cell-inducing properties. A hitherto unrecognized effect of IL-3 on DC was defined, namely suppression of IL-12 secretion and a resulting shift from Th1 toward Th2.     

Caspase-2-Dependent Dendritic Cell Death, Maturation, and Priming of T Cells in Response to Brucella abortus Infection

Smooth virulent Brucella abortus strain 2308 (S2308) causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs) are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs) than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs). More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+) and CD8(+) T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control). S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen presentation, and T cell priming.     

Mycobacterium leprae inhibits dendritic cell activation and maturation

Leprosy presents with a clinical spectrum of skin lesions that span from strong Th1-mediated cellular immunity and control of bacillary growth at one pole to poor Ag-specific T cell immunity with extensive bacillary load and Th2 cytokine-expressing lesions at the other. To understand how the immune response to Mycobacterium leprae is regulated, human dendritic cells (DC), potent inducers of adaptive immune responses, exposed to M. leprae, Mycobacterium tuberculosis (Mtb), and Mycobacterium bovis bacillus Calmette-Guerin (BCG) were studied for their ability to be activated and to prime T cell proliferation. In contrast with Mtb and BCG, M. leprae did not induce DC activation/maturation as measured by the expression of selected surface markers and proinflammatory cytokine production. In MLR, T cells did not proliferate in response to M. leprae-stimulated DC. Interestingly, M. leprae-exposed MLR cells secreted increased Th2 cytokines as well as similar Th1 cytokine levels as compared with Mtb- and BCG-exposed cells. Gene expression analysis revealed a reduction in levels of mRNA of DC activation and maturation markers following exposure to M. leprae. Our data suggest that M. leprae does not induce and probably suppresses in vitro DC maturation/activation, whereas Mtb and BCG are stimulatory.     

Cooperative induction of a tolerogenic dendritic cell phenotype by cytokines secreted by pancreatic carcinoma cells

Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123- myeloid DC (MDC)) or immunosuppressive T cell development (CD11c-,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-gamma. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-beta, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses.     

Glatiramer acetate (copolymer-1, copaxone) promotes Th2 cell development and increased IL-10 production through modulation of dendritic cells

Glatiramer acetate (GA; copolymer-1, Copaxone) suppresses the induction of experimental autoimmune encephalomyelitis and reduces the relapse frequency in relapsing-remitting multiple sclerosis. Although it has become clear that GA induces protective degenerate Th2/IL-10 responses, its precise mode of action remains elusive. Because the cytokine profile of Th cells is often regulated by dendritic cells (DC), we studied the modulatory effects of GA on the T cell regulatory function of human DC. This study shows the novel selective inhibitory effect of GA on the production of DC-derived inflammatory mediators without affecting DC maturation or DC immunostimulatory potential. DC exposed to GA have an impaired capacity to secrete the major Th1 polarizing factor IL-12p70 in response to LPS and CD40 ligand triggering. DC exposed to GA induce effector IL-4-secreting Th2 cells and enhanced levels of the anti-inflammatory cytokine IL-10. The anti-inflammatory effect of GA is mediated via DC as GA does not affect the polarization patterns of naive Th cells activated in an APC-free system. Together, these results reveal that APC are essential for the GA-mediated shift in the Th cell profiles and indicate that DC are a prime target for the immunomodulatory effects of GA.     

A Potential Protein Adjuvant Derived from Mycobacterium tuberculosis Rv0652 Enhances Dendritic Cells-Based Tumor Immunotherapy

A key factor in dendritic cell (DC)-based tumor immunotherapy is the identification of an immunoadjuvant capable of inducing DC maturation to enhance cellular immunity. The efficacy of a 50S ribosomal protein L7/L12 (rplL) from Mycobacterium tuberculosis Rv0652, as an immunoadjuvant for DC-based tumor immunotherapy, and its capacity for inducing DC maturation was investigated. In this study, we showed that Rv0652 is recognized by Toll-like receptor 4 (TLR4) to induce DC maturation, and pro-inflammatory cytokine production (TNF-alpha, IL-1beta, and IL-6) that is partially modulated by both MyD88 and TRIF signaling pathways. Rv0652-activated DCs could activate naïve T cells, effectively polarize CD4⁺ and CD8⁺ T cells to secrete IFN-gamma, and induce T cell-mediated-cytotoxicity. Immunization of mice with Rv0652-stimulated ovalbumin (OVA)-pulsed DCs resulted in induction of a potent OVA-specific CD8⁺ T cell response, slowed tumor growth, and promoted long-term survival in a murine OVA-expressing E.G7 thymoma model. These findings suggest that Rv0652 enhances the polarization of T effector cells toward a Th1 phenotype through DC maturation, and that Rv0652 may be an effective adjuvant for enhancing the therapeutic response to DC-based tumor immunotherapy.     

Distinct responses of splenic dendritic cell subsets to infection with Listeria monocytogenes: maturation phenotype, level of infection, and T cell priming capacity ex vivo

To determine the relative contributions of DC subsets in the development of protective immunity to Listeria monocytogenes we examined the relationship between maturation, bacterial burden, and T cell priming capacity of four well characterized subsets of splenic DC following infection with Lm. CD8α⁺, CD4⁺, and CD8α⁻CD4⁻ DC and the B220⁺ plasmacytoid DC (pDC) were compared for abundance and costimulatory molecule expression at 24, 48, and 72 h post i.v. infection. We further determined the bacterial burden associated with each DC subset and their relative capacities to prime CD8⁺ T cells at 24 hpi. The CD8α⁺ DC displayed the highest level of maturation, association with live bacteria, and T cell activation potential. Second, the CD4⁺ DC were also mature, yet were associated with fewer bacteria, and stimulated T cell proliferation, but not IFN-γ production. The CD8α⁻CD4⁻ DC showed a modest maturation response and were associated with a high number of bacteria, but failed to induce T cell proliferation ex vivo. pDC displayed a strong maturation response, but were not associated with detectable bacteria and also failed to stimulate T cell activation. Finally, we measured the cytokine responses in these subsets and determined that IL-12 was produced predominantly by the CD8⁺ DC, correlating with the ability of this subset DC to induce IFN-γ production in T cells. We conclude that Listeria-specific CD8⁺ T cell activation in the spleen is most effectively achieved by infection-induced maturation of the CD8α⁺ DC subset.     

Fungal proteases induce Th2 polarization through limited dendritic cell maturation and reduced production of IL-12

Allergens are capable of polarizing the T cell immune response toward a Th2 cytokine profile in a process that is mediated by dendritic cells (DCs). Proteases derived from Aspergillus species (Aspergillus proteases; AP) have been shown to induce a Th2-like immune response when administered directly to the airway and without adjuvant or prior priming immunizations at sites remote from the lung in models of allergic airway disease. To explore mechanisms that underlie the Th2 immune response, we have investigated the effect of AP on DC function. We found that human DCs derived from CD14(+) monocytes from healthy donors underwent partial maturation when incubated with AP. Naive allogeneic T cells primed with AP-activated DCs proliferated and displayed enhanced production of IL-4 and reduced expression of IFN-gamma as compared with naive T cells primed with LPS-activated DCs. Global gene expression analysis of DCs revealed relatively low expression of IL-12p40 in AP-activated DCs as compared with those activated by LPS, and this was confirmed at the protein level by ELISA. Exogenous IL-12p70 added to cocultures of DCs and T cells resulted in reduced IL-4 and increased IFN-gamma expression when DCs were activated with AP. When the proteolytic activity of AP was neutralized by chemical inactivation it failed to up-regulate costimulatory molecules on DCs, and these DCs did not prime a Th2 response in naive T cells. These findings provide a mechanism for explaining how proteolytically active allergens could preferentially induce Th2 responses through limited maturation of DCs with reduced production of IL-12.     

Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses

The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite.     

IL-32gamma induces the maturation of dendritic cells with Th1- and Th17-polarizing ability through enhanced IL-12 and IL-6 production

IL-32, a newly described multifunctional cytokine, has been associated with a variety of inflammatory diseases, including rheumatoid arthritis, vasculitis, and Crohn's disease. In this study, we investigated the immunomodulatory effects of IL-32γ on bone marrow-derived dendritic cell (DC)-driven Th responses and analyzed the underlying signaling events. IL-32γ-treated DCs exhibited upregulated expression of cell-surface molecules and proinflammatory cytokines associated with DC maturation and activation. In particular, IL-32γ treatment significantly increased production of IL-12 and IL-6 in DCs, which are known as Th1- and Th17-polarizing cytokines, respectively. This increased production was inhibited by the addition of specific inhibitors of the activities of phospholipase C (PLC), JNK, and NF-κB. IL-32γ treatment increased the phosphorylation of JNK and the degradation of both IκBα and IκBβ in DCs, as well as NF-κB binding activity to the κB site. The PLC inhibitor suppressed NF-κB DNA binding activity and JNK phosphorylation increased by IL-32γ treatment, thereby indicating that IL-32γ induced IL-12 and IL-6 production in DCs via a PLC/JNK/NF-κB signaling pathway. Importantly, IL-32γ-stimulated DCs significantly induced both Th1 and Th17 responses when cocultured with CD4(+) T cells. The addition of a neutralizing anti-IL-12 mAb abolished the secretion of IFN-γ in a dose-dependent manner; additionally, the blockage of IL-1β and IL-6, but not of IL-21 or IL-23p19, profoundly inhibited IL-32γ-induced IL-17 production. These results demonstrated that IL-32γ could effectively induce the maturation and activation of immature DCs, leading to enhanced Th1 and Th17 responses as the result of increased IL-12 and IL-6 production in DCs.     

Regulation of T cell cytokine production by dendritic cells generated in vitro from hematopoietic progenitor cells

When dendritic cells (DC) present antigens to T cells, reciprocal cellular interactions occur that lead to cytokine production. This cytokine response is regulated by specific properties of DC, notably their maturation/activation status and perhaps their origin. The latter possibility prompted us to determine if DC produced along distinct developmental pathways induced distinct T cell responses. Hematopoietic progenitor cells with the potential to differentiate into multiple lineages of cells were induced to differentiate into DC along two pathways. One leads to the formation of lymphoid-related DC but not of monocyte-derived DC and is induced by culture of CD34(+) cells with flt-3 ligand (F), c-kit ligand (K), GM-CSF (Gm), IL-1beta ("1"), and IL-7 ("7") (FKGm17). Another pathway with distinct molecular requirements supports in part monocyte-derived DC and is induced by the cytokines F, K, Gm, TNF-alpha (T), and IL-4 ("4") (FKGmT4). DC produced along these two pathways were isolated by flow cytometry and compared. They differed only slightly in phenotype and morphology and both induced Th1-type cytokine production in MLR (mixed lymphocyte reactions). However, on a cell-per-cell basis, FKGm17-DC produced more IL-18 or IL-12 and induced more IFN-gamma by T cells in MLR. Such superior properties were not intrinsically determined by the origin of the DC but were induced by FKGm17 cytokines. We conclude that lymphoid-related DC have the potential to induce Th1 T cell responses but that environmental signals strongly influence T-cell-stimulating properties of DC.     

Comparative roles of IL-4, IL-13, and IL-4Ralpha in dendritic cell maturation and CD4+ Th2 cell function

IL-4 and IL-13 play key roles in Th2 immunity and asthma pathogenesis. Although the function of these cytokines is partially linked through their shared use of IL-4Ralpha for signaling, the interplay between these cytokines in the development of memory Th2 responses is not well delineated. In this investigation, we show that both IL-4 and IL-13 influence the maturation of dendritic cells (DC) in the lung and their ability to regulate secretion of IFN-gamma and Th2 cytokines by memory CD4(+) T cells. Cocultures of wild-type T cells with pulmonary DC from allergic, cytokine-deficient mice demonstrated that IL-4 enhanced the capacity of DC to stimulate T cell secretion of Th2 cytokines, whereas IL-13 enhanced the capacity of DC to suppress T cell secretion of IFN-gamma. Because IL-4Ralpha is critical for IL-4 and IL-13 signaling, we also determined how variants of IL-4Ralpha influenced immune cell function. T cells derived from allergic mice expressing a high-affinity IL-4Ralpha variant produced higher levels of IL-5 and IL-13 compared with T cells derived from allergic mice expressing a low-affinity IL-4Ralpha variant. Although DC expressing different IL-4Ralpha variants did not differ in their capacity to influence Th2 cytokine production, they varied in their capacity to inhibit IFN-gamma production by T cells. Thus, IL-4 and IL-13 differentially regulate DC function and the way these cells regulate T cells. The affinity of IL-4Ralpha also appears to be a determinant in the balance between Th2 and IFN-gamma responses and thus the severity of allergic disease.     

Induction of CTL and nonpolarized Th cell responses by CD8alpha(+) and CD8alpha(-) dendritic cells

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second, we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However, IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8(+) T cells, while Th2 cytokines were produced by CD4(+) T cells. Thus, this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets.     

Pertussis toxin enhances Th1 responses by stimulation of dendritic cells

Pertussis toxin (PTX) has been widely used as an adjuvant to induce Th1-mediated organ-specific autoimmune diseases in animal models. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that dendritic cells (DC) stimulated with PTX (PTX-DC) were able to substitute for PTX to promote experimental autoimmune uveitis (EAU). EAU induced by PTX-DC revealed a typical Th1 response, characterized by high uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP)-specific IFN-gamma and IL-12 production in the draining lymph nodes, as well as increased levels of anti-IRBP IgG2a and decreased levels of anti-IRBP IgG1 in the serum of IRBP-immunized mice. Furthermore, PTX-DC preferentially induced T cells to produce the Th1 cytokine, IFN-gamma. After being stimulated with PTX, DC exhibited up-regulation of MHC class II, CD80, CD86, CD40, and DEC205. PTX-DC had also increased allostimulatory capacity and IL-12 and TNF-alpha production. Serum IL-12 was increased in naive mice that received PTX-DC i.p. In addition, PTX activated extracellular signal-regulated kinase in DC. Following the inhibition of extracellular signal-regulated kinase signaling, the maturation of PTX-DC was reduced. Subsequently, the ability of PTX-DC to promote IFN-gamma production by T cells in vitro and to induce EAU in vivo was blocked. The results suggest that PTX might exert an adjuvant effect on DC to promote their maturation and the production of proinflammatory cytokines, thereby eliciting a Th1 response.