LPS-activated Dendritic Cells Research Articles

IL-33–activated dendritic cells induce an atypical T H2-type response

IL-33, a recently discovered IL-1 family cytokine, is implicated in the development of T(H)2-type responses in vivo. However, the cellular targets for IL-33 are poorly understood. We tested the hypotheses that dendritic cells (DCs) respond to IL-33 and that IL-33-activated DCs prime naive CD4(+) T cells to produce T(H)2-type cytokines. Dendritic cells were derived from mouse bone marrow, and their expression of the IL-33 receptor, ST2, was examined by fluorescence-activated cell sorting and real-time RT-PCR. The DCs' responses to IL-33 were examined by fluorescence-activated cell sorting (MHC-II and CD86 expression) and by ELISA (IL-6 and IL-12 production). The ability of IL-33-activated DCs to prime naive T cells was assessed by coculture with isolated CD4(+) T cells and by measuring cytokines in the supernatants. ST2 mRNA was detectable in highly purified DCs. ST2 protein was abundant within DCs, but was barely detectable on their cell surfaces. Incubation of DCs with IL-33 increased their expression of MHC-II and CD86 and production of IL-6, but IL-12 was not produced. Anti-ST2 antibody inhibited IL-6 production from IL-33-activated DCs by approximately 60%; anti-ST2 did not affect IL-6 production from LPS-activated DCs. When incubated with naive CD4(+) T cells alone, IL-33 failed to stimulate cytokine production. In contrast, naive CD4(+) T cells incubated with IL-33-activated DCs showed robust production of IL-5 and IL-13, but IL-4 and IFN-gamma were undetectable. Dendritic cells respond directly to IL-33 through ST2. The IL-33 and DC interaction may represent a new pathway to initiate T(H)2-type immune responses.

ICOS/B7h Bidirectional Signal to Induce Secretion of IL-1β and IL-17 (46.3)

Abstract B7h is constitutively expressed by antigen presenting cells (APC) and binds ICOS expressed by activated T cells. ICOS triggering costimulates T helper (TH) cell activation and modulates their cytokine secretion, whereas the effect of B7h triggering in APC is unknown. This work investigated B7h reverse signalling in dendritic cells (DC). We induced DC maturation with LPS in the presence or absence of a soluble ICOS-Ig fusion protein; then, both cytokine secretion and allostimolatory activity were evaluated. B7h stimulation substantially modulated LPS-induced DC maturation by influencing cytokine secretion. The most striking effect was 80-fold increase of IL-1b secretion, but other changes were 3-fold increase of IL-10 and 2-fold decrease of TNF-a. In IL-1b secretion, B7h triggering acted as a "second signal" by activating caspase-1, required to cleave pro-IL-1b whose synthesis was induced by LPS. B7h triggering on LPS-activated DC also modulated their capacity to activate allogeneic T cells in mixed lymphocyte cultures (MLC) by decreasing the proliferative response and changing cytokines secretion, including 20-fold decrease of IFN-g and 5-fold increase of IL-17. This effect was partly ascribable to the cytokine milieu secreted by DC and particularly to the high IL-1b levels, known to be involved in differetiation of human Th17 cells. However, ICOS triggering too played a role in IL-17 secretion since blocking of the B7h/ICOS interaction inhibited IL-17 secretion in MLC with DC. Moreover, in naive Th cells activated with anti-CD3 antibodies, ICOS stimulation induced IL-17 secretion in the presence of exogenous IL-1b. In this work we showed that B7h-mediated reverse signaling induced IL-1b by DCs which in turn favors IL-17 secretion by Th cells. On the other side, we demonstrated that ICOS costimulation directly favors Th17 differentiation.

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.

MHC class II stabilization at the surface of human dendritic cells is the result of maturation-dependent MARCH I down-regulation

In response to Toll-like receptor ligands, dendritic cells (DCs) dramatically enhance their antigen presentation capacity by stabilizing at the cell-surface MHC II molecules. We demonstrate here that, in human monocyte-derived DCs, the RING-CH ubiquitin E3 ligase, membrane-associated RING-CH I (MARCH I), promotes the ubiquitination of the HLA-DR beta-chain. Thus, in nonactivated DCs, MARCH I induces the surface internalization of mature HLA-DR complexes, therefore reducing their stability and levels. We further demonstrate that the maturation-dependent down-regulation of MARCH I is a key event in MHC class II up-regulation at the surface of LPS-activated DCs. MARCH I is, therefore, a major regulator of HLA-DR traffic, and its loss contributes to the acquisition of the potent immunostimulatory properties of mature human DCs.

Exovesicles from Human Activated Dendritic Cells Fuse with Resting Dendritic Cells, Allowing Them to Present Alloantigens

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

IL‐2 production by dendritic cells is not critical for the activation of cognate and innate effectors in draining lymph nodes

Dendritic cells (DC) are unique antigen-presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo. Microbial stimulation induces early IL-2 production by mouse DC. Previous reports demonstrated that IL-2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC-derived IL-2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone marrow-derived DC results in early IL-2 production unless IL-4 is introduced in DC cultures. Here we show that IL-2 produced by LPS-activated DC is dispensable for cognate T cell responses since IL-2 loss of function DC elicit OVA-specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL-2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL-2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.

Autoreactive human peripheral blood CD8+ T cells with a regulatory phenotype and function

Despite substantial advances in our understanding of CD4+ CD25+ regulatory T cells, a possible equivalent regulatory subset within the CD8+ T cell population has received less attention. We now describe novel human CD8+/TCR alphabeta+ T cells that have a regulatory phenotype and function. We expanded and cloned these cells using autologous LPS-activated dendritic cells. The clones were not cytolytic, but responded in an autoreactive HLA class I-restricted fashion, by proliferation and production of IL-4, IL-5, IL-13 and TGFbeta1, but not IFN-gamma. They constitutively expressed CD69 and CD25 as well as molecules associated with CD4+ CD25+ regulatory T cells, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and Foxp3. They suppressed IFN-gamma production and proliferation by CD4+ T cells in vitro in a cell contact-dependent manner, which could be blocked using a CTLA-4-specific mAb. They were more readily isolated from patients with ankylosing spondylitis and may therefore be up-regulated in response to inflammation. We suggest that they are the CD8+ counterparts of CD4+ CD25+ regulatory T cells. They resemble recently described CD8+ regulatory cells in the rat that were able to abrogate graft-versus-host disease. Likewise, human HLA-restricted CD8+ regulatory T cells that can be cloned and expanded in vitro may have therapeutic applications.

Polyunsaturated Fatty Acids Block Dendritic Cell Activation and Function Independently of NF-κB Activation

Polyunsaturated fatty acids (PUFAs) modulate immune responses leading to clinically significant beneficial effects in a variety of inflammatory disorders. PUFA effects on T cells have been extensively studied, but their influence on human dendritic cells (DCs), which are the most potent antigen-presenting cells and play a key role in initiating immune responses, has not been elucidated so far. Here we show that PUFAs of the n-3 and n-6 series (arachidonic and eicosapentaenoic acid) affect human monocyte-derived DC differentiation and inhibit their activation by LPS, resulting in altered DC surface molecule expression and diminished cytokine secretion. Furthermore, the potency to stimulate T cells was markedly inhibited in PUFA-treated DCs. The PUFA-mediated block in LPS-induced DC activation is reflected by diminished TNF-alpha, IL-12p40, CD40, and COX-2 mRNA levels. Strikingly, typical LPS-induced signaling events such as degradation of IkappaB and activation of NF-kappaB were not affected by PUFAs, even though DC membrane lipid composition was markedly altered. Arachidonic and eicosapentaenoic acid both altered DC prostaglandin production, but inhibitors of cyclooxygenases and lipoxygenases did not abolish PUFA effects, indicating that the observed PUFA actions on DCs were independent of autoregulation via eicosanoids. These data demonstrate a unique interference with DC activation and function that could significantly contribute to the well known anti-inflammatory effects of PUFAs.

Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections

CD1d-restricted natural killer T (NKT) cells are innate-like lymphocytes that express a conserved T-cell receptor and contribute to host defence against various microbial pathogens. However, their target lipid antigens have remained elusive. Here we report evidence for microbial, antigen-specific activation of NKT cells against Gram-negative, lipopolysaccharide (LPS)-negative alpha-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata. We have identified glycosylceramides from the cell wall of Sphingomonas that serve as direct targets for mouse and human NKT cells, controlling both septic shock reaction and bacterial clearance in infected mice. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. These findings identify two novel antigenic targets of NKT cells in antimicrobial defence, and show that glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall.

Distinct mechanisms are involved in tumoristatic and tumoricidal activities of monocyte-derived dendritic cells

Monocyte-derived dendritic cells (DC) were found to inhibit proliferation of different tumor cell lines. LPS-induced maturation of DC strongly increased their capacity to inhibit tumor cell growth. We observed that tumoristatic activity of LPS-activated DC was independent of their cytotoxic potential. Indeed, LPS-activated DC were able to inhibit growth of caspase-8-deficient or Bcl-2-overexpressing Jurkat cells whereas they were not cytotoxic towards the same targets. On the other hand, we found that supernatant derived from LPS-activated DC exerted a significant anti-proliferative activity against Jurkat cells while it did not induce any cytotoxic effect. Tumor necrosis factor (TNF) was shown to critically contribute to tumor growth inhibition in this system.

Effects of histamine on functional maturation of dendritic cells

There is increasing evidence that histamine affects dendritic cell (DC) activation, maturation, and preference for Th1/Th2 differentiation. In this paper we report that histamine affects interleukin (IL)-12 and IL-6 production in an immature DC (iDC) line derived from murine spleen. Histamine treatment of iDC significantly increased the IL-12 p40 mRNA and protein levels compared to histamine untreated iDC. In the presence of tumor necrosis factor (TNF)-alpha histamine also increased IL-12 p40 and IL-6 production. However, histamine significantly decreased IL-12 p40 production by lipopolysaccharide (LPS)-stimulated DC in a concentration dependent manner. When expressions of histamine H1 (H1R) and H2 (H2R) receptors in DC were analyzed by RT-PCR, both receptors were down-regulated after LPS or TNF-alpha stimulation compared to unstimulated iDC. Histamine treatment significantly increased the expression of H2R mRNA in iDC and H1R mRNA in LPS-activated DC. However, histamine treatment decreased the expression of both histamine receptors in TNF-alpha-stimulated DC. Similar results were obtained by flow cytometry with FITC-conjugated histamine. These results demonstrate that histamine can regulate the expression of its own receptors and activate iDC, which may influence subsequent functional states of mature DC in a maturation signal-dependent manner. Consequently, histamine may contribute to an immune response outcome.

Ganglioside G D1a impedes lipopolysaccharide-induced maturation of human dendritic cells

Immunosuppressive membrane gangliosides are released by tumor cells and inhibit normal antigen presenting cell (APC) function. To better understand this process, we have studied the effect of gangliosides on lipopolysaccharide (LPS)-induced maturation of human dendritic cells (DCs). Immature DCs were generated in vitro from human peripheral blood monocytes and were exposed for 72 h to a highly purified ganglioside, G D1a. During the last 24 h, LPS was added to effect maturation. As assessed by fluorescence activated cell sorting (FACS) analysis, incubation in 50 μM G D1a significantly blunted the LPS-induced maturation of the dendritic cells. The expected up-regulation of expression of the co-stimulatory molecules CD80 and CD86 was ablated and that of CD40 was reduced, as were surface CD83 expression and intracellular CD208 production. In addition, ganglioside pretreatment of DC markedly inhibited the allostimulatory capacity and partially prevented the down-regulation of FITC–dextran uptake characteristic of LPS-activated DC. Furthermore, ganglioside-exposed DC also evidenced a broad down-regulation of the cytokine release that is normally initiated by LPS exposure, i.e., there was no increase in IL-1β, IL-6, IL-10, IL-12, or tumor necrosis factor (TNF)-α release. That a common mechanism may underlie these defects was suggested by the finding that G D1a exposure of DC also inhibited the nuclear binding of NF-κB that is normally induced by LPS. These results suggest that tumor gangliosides may blunt the anti-tumor immune response in vivo by binding and interfering with dendritic cell maturation.

Secretion of interleukin-10 or interleukin-12 by LPS-activated dendritic cells is critically dependent on time of stimulus relative to initiation of purified DC culture

Dendritic cells (DC) are key regulators of adaptive immunity with the potential to induce T cell activation/immunity or T cell suppression/tolerance. DC are themselves induced by "maturation" signals such as bacterial lipopolysaccharide (LPS). We demonstrate here that LPS can stimulate DC to display similar maturation phenotypes but to differentiate toward an interleukin (IL)-10(high)- or IL-12(high)-secretor profile depending on the timing of maturation signal induction. Immediate/early administration of LPS induced purified bone marrow-derived DC (BMDC) to differentiate as IL-10(high)IL-12(low)-secreting cells, termed early DC (eDC). Conversely, delayed administration of LPS altered the DC cytokine profile to IL-10(low)IL-12(high), termed later DC (lDC). The presence of IL-4 enhanced the yield and maturation of BMDC but inhibited LPS-induced IL-10 production by eDC. In contrast, interferon-gamma reduced the yield of DC but promoted the level of LPS-induced IL-10 production by lDC. Our data provide new evidence that ex vivo manipulation and the cytokine environment regulate DC maturation status and cytokine-secretor phenotype with implications for the control of T cell differentiation and function via DC-based immunotherapeutic strategies.

Trypanosoma cruzi infects human dendritic cells and prevents their maturation: inhibition of cytokines, HLA-DR, and costimulatory molecules.

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas' disease. Despite the many immune system disorders recognized in this infection and the crucial role played by dendritic cells (DC) in acquired immune responses, it was not known whether these cells could be infected by T. cruzi trypomastigotes and the consequences of such an infection on their immune functions. We now provide evidence that human monocyte-derived DC can be infected by T. cruzi and can support its intracellular multiplication. Interestingly, this infection has functional consequences on immature DC and on their maturation induced by lipopolysaccharide (LPS). First, after T. cruzi infection, the basal synthesis of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) was impaired. Furthermore, the process of maturation of DC induced by LPS was drastically affected by T. cruzi infection. Indeed, secretion of cytokines such as IL-12, TNF-alpha, and IL-6, which are released normally at high levels by LPS-activated DC, as well as the up-regulation of HLA-DR and CD40 molecules, was significantly reduced after this infection. The same effects could be induced by T. cruzi-conditioned medium, indicating that at least these inhibitory effects were mediated by soluble factors released by T. cruzi. Taken together, these results provide new insights into a novel efficient mechanism, directly involving the alteration of DC function, which might be used by T. cruzi to escape the host immune responses in Chagas' disease and thus might favor persistent infection.

Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CD14-dependent pathway.

To investigate the responses of dendritic cells (DC) during Gram-negative infections, we analyzed the effects of graded doses of LPS on the cytokine profile, phenotype, and allostimulatory potential of human DC generated by culturing plastic-adherent PBMC in presence of IL-4 and granulocyte-macrophage-CSF. First, we found that LPS stimulates the production of high levels of TNF-alpha, IL-6, IL-8, IL-12 by DC and up-regulates their expression of HLA-DR, B7-1, B7-2, and CD40. The effects of LPS were dose dependent, with a significant stimulatory effect already observed at a concentration of 0.1 ng/ml and a plateau being reached at 10 ng/ml. These phenotypic changes correlated with increased allostimulatory properties of LPS-activated DC because DC treated with LPS were significantly more efficient than untreated DC in eliciting IL-2 and IFN-gamma synthesis by alloreactive T cells and stimulating their proliferation. Experiments using neutralizing anti-IL-12 mAb indicated that LPS-induced IL-12 is responsible for the increased production of IFN-gamma but not for the increased proliferation during MLR. Finally, we observed that the DC responses to low levels of LPS (1 ng/ml) were dramatically inhibited by a blocking anti-CD14 mAb, although DC do not express CD14 molecules on their membrane. Experiments using serum depleted of soluble CD14 (sCD14) and sCD14 either purified from human serum or in recombinant form further established that DC respond to LPS via a soluble CD14-dependent pathway.