Cytokine and chemokine expression profiles of maturing dendritic cells using multiprotein platform arrays

Understanding the whole process of dendritic cell (DC) activation might help in the development of more efficient immunotherapeutic strategies for tumor patients. Part of this process is cytokine secretion, which has important effects on innate and adaptive immune response. Here, we cultured circulating monocytes for five days with interleukin-4 and GM-CSF followed by two-day culture with or without CD40 ligand and LPS to create a mature DC (mDC) and an immature DC (iDC) phenotype, respectively, characterized by differential expression of co-stimulatory molecules (CD80, CD83). We then compared the cytokine expression profile of the mDC and iDC using two protein platform arrays. Twelve supernatants from mDC paired with 12 from iDC were compared. The mDC protein expression profile showed significant increases in 16 out of 34 factors tested, including TNFalpha, IL-10, IL-12, IFNgamma, MIP1alpha, MIP1beta, IL-8, MDC, RANTES, and IL-6, which play a crucial role in the regulation of the innate immune response as well as the recruitment and activation of adaptive immune effectors. Interestingly, some of the cytokines expressed during maturation were also found in the gene expression profile identified in tumor metastases following IL-2 therapy using cDNA arrays. This finding suggests a possible role for resident DC maturation as a mediator of systemic IL-2 effects. Most important, the array of cytokines secreted during DC maturation may be considered an important component during adoptive transfer. Further characterization of the kinetics and persistence of their secretion should be undertaken in the future.     

Immunosuppression induced by immature dendritic cells is mediated by TGF-beta/IL-10 double-positive CD4+ regulatory T cells.

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (iDC) is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)-beta and interleukin (IL)-10 double-positive CD4(+) T cells within 1 week of autologous DC/T cell co-cultures. In iDC/T cell cultures, where antigen-specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF-beta and IL-10 secreted by CD4(+)CD25(-)FOXP3(-) T cells. In addition, the suppressive capacity of CD4(+) T cells conditioned by iDC was transferable to already primed antigen-specific CD8(+) T cell cultures. In contrast, addition of CD4(+) T cells conditioned by mDC to primed antigen-specific CD8(+) T cells resulted in enhanced CD8(+) T cell responses, notwithstanding the presence of TGF-beta(+)/IL-10(+) T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine-secreting regulatory T cells. We show that iDC-conditioned CD4(+) T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4(+) T cells. Furthermore, TGF-beta(+)/IL-10(+) T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.     

Regulation of Toll-like receptors in human monocytes and dendritic cells

A number of pathogens induce immature dendritic cells (iDC) to migrate to lymphoid organs where, as mature DC (mDC), they serve as efficient APC. We hypothesized that pathogen recognition by iDC is mediated by Toll-like receptors (TLRs), and asked which TLRs are expressed during the progression of monocytes to mDC. We first measured mRNA levels for TLRs 1-5 and MD2 (a protein required for TLR4 function) by Northern analysis. For most TLRs, message expression decreased severalfold as monocytes differentiated into iDC, but opposing this trend, TLR3 and MD2 showed marked increases during iDC formation. When iDC were induced to mature with LPS or TNF-alpha, expression of most TLRs transiently increased and then nearly disappeared. Stimulation of iDC, but not mDC, with LPS resulted in the activation of IL-1 receptor-associated kinase, an early component in the TLR signaling pathway, strongly suggesting that LPS signals through a TLR. Surface expression of TLRs 1 and 4, as measured by mAb binding, was very low, corresponding to a few thousand molecules per cell in monocytes, and a few hundred or less in iDC. We conclude that TLRs are expressed in iDC and are involved in responses to at least one pathogen-derived substance, LPS. If TLR4 is solely responsible for LPS signaling in humans, as it is in mice, then its extremely low surface expression implies that it is a very efficient signal transducer in iDC.     

Role of 4-hydroxynonenal in the hemozoin-mediated inhibition of differentiation of human monocytes to dendritic cells induced by GM-CSF/IL-4

In falciparum malaria, rupture of parasitized RBC liberates hemozoin (HZ), polymerized heme that contains and generates lipoperoxidation products. In HZ and HZ-loaded monocytes 4-HNE attained approx. 50 and 15 microM, respectively. In malaria, HZ-loaded monocytes are precursors of dendritic cells (DC). Here, the role of 4-HNE as inhibitor of DC differentiation was examined. 4-HNE in HZ was quantified after derivatization by HPLC. DC were differentiated in vitro from human monocytes supplemented with GM-CSF/IL-4 and analyzed for surface antigens and 4-HNE-adducts by FACScan after labelling with specific antibodies. HZ-loading, or treatment with 4-HNE induced large numbers of 4-HNE-protein-adducts on the monocyte membrane. As low as 10 nM 4-HNE inhibited up-regulation of functionally important DC differentiation markers. 1 microM 4-HNE elicited inhibition of up-regulation of DC differentiation markers as follows: MHC-class I and II, -29% and -40%; CD1a, -16%; CD40, -25%; CD54, -27%; and CD83 (the most important DC differentiation marker), -45%, with no signs of apoptosis. The sequence of additions was important, as the inhibitory effect was reduced when 4-HNE was added after GM-CSF/IL-4, indicating that GM-CSF/IL-4 receptors could be modified by 4-HNE. In conclusion, inhibition of DC differentiation by 4-HNE may play a role in malaria immunodepression.     

Standardized generation of fully mature p70 IL-12 secreting monocyte-derived dendritic cells for clinical use

Dendritic cells (DC) have been shown to be efficient antigen-presenting cells (APC) and, as such, could be considered ideal candidates for cancer immunotherapy. Immature DC (iDC) efficiently capture surrounding antigens; however, only mature DC (mDC) prime naive T lymphocytes. Clinical trials using DC-based tumor vaccines have achieved encouraging, but limited, success, possibly due to the use of immature or incompletely mature DC. Thus, it was apparent that a method capable of generating large numbers of fully functional iDC, their pulsing with desired form of tumor antigens and the subsequent complete and reproducible maturation of iDC is needed. Therefore, we compared two different methods of producing large numbers of iDC. Both protocols yielded comparable numbers of cells with an iDC phenotype with phagocytic function. We next determined which of the clinically applicable activators could induce the complete and reproducible maturation of DC, in order to define the most suitable combination for future clinical trials. Only a combination of TNFalpha + Poly (I:C), or a previously described cytokine cocktail of TNFalpha + IL-1beta + IL-6 + prostaglandin E2, induced the complete activation of the whole DC population, as assessed by the cell surface expression of CD83 and costimulatory molecules. The matured DC were functionally superior to iDC in their ability to stimulate the proliferation of allogeneic lymphocytes and autologous keyhole limpet hemocyanin (KLH)-specific T lymphocytes. Furthermore, only the combination of TNFalpha + Poly (L:C) activated DC to produce large amounts of biologically active p70 IL-12. Thus DC maturation by TNFalpha + Poly (I:C) could efficiently bias T cell response towards Th1 response. Implementation of our results into clinical protocols used for DC generation could be beneficial for future immunotherapy trials.     

Monocyte-derived CD1a+ and CD1a- dendritic cell subsets differ in their cytokine production profiles, susceptibilities to transfection, and capacities to direct Th cell differentiation

We describe a phenotypically and functionally novel monocyte-derived dendritic cell (DC) subset, designated mDC2, that lacks IL-12 synthesis, produces high levels of IL-10, and directs differentiation of Th0/Th2 cells. Like conventional monocyte-derived DC, designated mDC1, mDC2 expressed high levels of CD11c, CD40, CD80, CD86, and MHC class II molecules. However, in contrast to mDC1, mDC2 lacked expression of CD1a, suggesting an association between cytokine production profile and CD1a expression in DC. mDC2 could be matured into CD83+ DC cells in the presence of anti-CD40 mAbs and LPS plus IFN-gamma, but they remained CD1a- and lacked IL-12 production even upon maturation. The lack of IL-12 and CD1a expression by mDC2 did not affect their APC capacity, because mDC2 stimulated MLR to a similar degree as mDC1. However, while mDC1 strongly favored Th1 differentiation, mDC2 directed differentiation of Th0/Th2 cells when cocultured with purified human peripheral blood T cells, further indicating functional differences between mDC1 and mDC2. Interestingly, the transfection efficiency of mDC2 with plasmid DNA vectors was significantly higher than that of mDC1, and therefore mDC2 may provide improved means to manipulate Ag-specific T cell responses after transfection ex vivo. Taken together, these data indicate that peripheral blood monocytes have the capacity to differentiate into DC subsets with different cytokine production profiles, which is associated with altered capacity to direct Th cell differentiation.     

Inhibition of the growth of Toxoplasma gondii in immature human dendritic cells is dependent on the expression of TNF-alpha receptor 2

An effective immunity to Toxoplasma gondii in humans is dependent on the cellular immune response. Toxoplasma can infect and replicate in almost all nucleated cells, and the most important cytokine regulating the growth in humans is IFN-gamma; however, the role of TNF-alpha has to date been largely described to be synergistic. We show that, compared with mature human dendritic cells (mDC), immature human DC (iDC) demonstrate a reduced parasite proliferation when infected with Toxoplasma. This toxoplasmostasis was only present in iDC after 11 days of culture and was not present in DC that had been matured ex vivo using a cytokine mixture (mDC). Spontaneous toxoplasmostatic activity has previously only been described in fresh human monocytes, and the mechanism involved is as yet unclear. We show that, in comparison with an absence of expression in mDC, TNF-R2 is expressed in both iDC and monocytes infected with Toxoplasma, and furthermore, that blocking the TNF-R2 with Abs abrogates the toxoplasmostasis in the iDC. These findings demonstrate a functional role for TNF-R2 in the newly described spontaneous toxoplasmostasis of iDC.     

Shaping of adaptive immunity by innate interactions

In inflamed tissues, the reciprocal interaction between Natural Killer (NK) cells and Dendritic Cells (DC) results in a potent activating cross talk that leads to DC maturation and NK cell activation with acquisition of NK-mediated cytotoxicity against immature DC (iDC). We focused our studies on NK-mediated killing of monocyte-derived iDC and we provided evidence that NK cells that express CD94/NKG2A but not killer Ig-like receptors (KIR) are able to kill autologous iDC. Indeed HLA-E (i.e. the cellular ligand of CD94/NKG2A) is sharply reduced in iDC, whereas it is partially recovered in mDC. The latter are lysed only by a small fraction of NK clones characterized by low levels of CD94/NKG2A expression. Another NK receptor, whose surface density is crucial for the ability to kill iDC, is represented by NKp30, a member of the NCR (Natural Cytotoxicity Receptor) family. We showed that transforming growth factor beta1 (TGFbeta1) treatment results in specific downregulation of NKp30 expression. This effect profoundly inhibits the NK-mediated killing of DC suggesting a possible mechanism by which TGFbeta1-producing DC may acquire resistance to the NK-mediated attack.     

Dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins

Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic reprogramming. Glycosylation is the most common form of posttranslational modification of proteins and has been implicated in multiple aspects of the immune response. To investigate the involvement of glycosylation in the changes that occur during DC maturation, we have studied the differences in the glycan profile of iDC and mDC as well as their glycosylation machinery. For information relating to glycan biosynthesis, gene expression profiles of human monocyte-derived iDC and mDC were compared using a gene microarray and quantitative real-time PCR. This gene expression profiling showed a profound maturation-induced up-regulation of the glycosyltransferases involved in the expression of LacNAc, core 1 and sialylated structures and a down-regulation of genes involved in the synthesis of core 2 O-glycans. Glycosylation changes during DC maturation were corroborated by mass spectrometric analysis of N- and O-glycans and by flow cytometry using plant lectins and glycan-specific Abs. Interestingly, the binding of the LacNAc-specific lectins galectin-3 and -8 increased during maturation and up-regulation of sialic acid expression by mDC correlated with an increased binding of siglec-1, -2, and -7.     

Regulation of antigen presentation machinery in human dendritic cells by recombinant adenovirus

Recombinant adenoviral vectors (AdV) are potent vehicles for antigen engineering of dendritic cells (DC). DC engineered with AdV to express full length tumor antigens are capable stimulators of antigen-specific polyclonal CD8+ and CD4+ T cells. To determine the impact of AdV on the HLA class I antigen presentation pathway, we investigated the effects of AdV transduction on antigen processing machinery (APM) components in human DC. Interactions among AdV transduction, maturation, APM regulation and T cell activation were investigated. The phenotype and cytokine profile of DC transduced with AdV was intermediate, between immature (iDC) and matured DC (mDC). Statistically significant increases in expression were observed for peptide transporters TAP-1 and TAP-2, and HLA class I peptide-loading chaperone ERp57, as well as co-stimulatory surface molecule CD86 due to AdV transduction. AdV transduction enhanced the expression of APM components and surface markers on mDC, and these changes were further modulated by the timing of DC maturation. Engineering of matured DC to express a tumor-associated antigen stimulated a broader repertoire of CD8+ T cells, capable of recognizing immunodominant and subdominant epitopes. These data identify molecular changes in AdV-transduced DC (AdV/DC) that could influence T cell priming and should be considered in design of cancer vaccines.     

Mature but not immature Fas ligand (CD95L)-transduced human monocyte-derived dendritic cells are protected from Fas-mediated apoptosis and can be used as killer APC

Several in vitro and animal studies have been performed to modulate the interaction of APCs and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells in an Ag-specific manner. However, due to the difficulties in vector generation and low transduction frequencies, similar studies with primary human APC are still lacking. To evaluate whether Fas ligand (FasL/CD95L) expressing killer APC could be generated from primary human APC, monocyte-derived dendritic cells (DC) were transduced using the inducible Cre/Loxp adenovirus vector system. Combined transduction of DC by AdLoxpFasL and AxCANCre, but not single transduction with these vectors, resulted in dose- and time-dependent expression of FasL in >70% of mature DC (mDC), whereas <20% of immature DC (iDC) expressed FasL. In addition, transduction by AdLoxpFasL and AxCANCre induced apoptosis in >80% of iDC, whereas FasL-expressing mDC were protected from FasL/Fas (CD95/Apo-1)-mediated apoptosis despite coexpression of Fas. FasL-expressing mDC eliminated Fas(+) Jurkat T cells as well as activated primary T cells by apoptosis, whereas nonactivated primary T cells were not deleted. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC and cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC, cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. The present results demonstrate that FasL-expressing killer APC can be generated from human monocyte-derived mDC using adenoviral gene transfer. Our results support the strategy to use killer APCs as immunomodulatory cells for the treatment of autoimmune disease and allograft rejection.     

Malaria‐derived hemozoin exerts early modulatory effects on the phenotype and maturation of human dendritic cells

Plasmodium falciparum (P. falciparum)‐induced effects on the phenotype of human dendritic cells (DC) could contribute to poor induction of long‐lasting protective immunity against malaria. DC ability to present antigens to naïve T cells, thus initiating adaptive immune responses depends on complex switches in chemokine receptors, production of soluble mediators and expression of molecules enabling antigen‐presentation and maturation. To examine the cellular basis of these processes in the context of malaria, we performed detailed analysis of early events following exposure of human monocyte‐derived DC to natural hemozoin (nHZ) and the synthetic analog of its heme core, β‐hematin. DC exposed to either molecule produced high levels of the inflammatory chemokine MCP‐1, showed continuous high expression of the inflammatory chemokine receptor CCR5, no upregulation of the lymphoid homing receptor CCR7 and no cytoskeletal actin redistribution with loss of podosomes. DC partially matured as indicated by increased expression of major histocompatibility complex (MHC) class II and CD86 following nHZ and β‐hematin exposure, however there was a lack in expression of the maturation marker CD83 following nHZ but not β‐hematin exposure. Overall our data demonstrate that exposure to nHZ partially impairs the capacity of DC to mature, an effect in part differential to β‐hematin.     

Modulation of the development of human monocyte‐derived dendritic cells by lithium chloride

Lithium has been used or explored to treat psychiatric and neurodegenerative diseases that are frequently associated with an abnormal immune status. It is likely that lithium may work through modulation of immune responses in these patients. Because dendritic cells (DC) play a central role in regulating immune responses, this study investigated the influence of lithium chloride (LiCl) on the development and function of DC. Exposure to LiCl during the differentiation of human monocyte‐derived immature DCs (iDC) enhances CD86 and CD83 expression and increases the production of IL‐1β, IL‐6, IL‐8, IL‐10, and TNF‐α. However, the presence of LiCl during LPS‐induced maturation of iDC has the opposite effect. During iDC differentiation, LiCl suppresses the activity of glycogen synthase kinase (GSK)‐3β, and activates PI3K and MEK. In addition, LiCl activates peroxisome proliferator‐activated receptor γ (PPARγ) during iDC differentiation, a pathway not described before. Each of these signaling pathways appears to have distinct impact on the differentiating iDC. The enhanced CD86 expression by LiCl involves the PI3K/AKT and GSK‐3β pathway. LiCl modulates the expression of CD83 in iDC mainly through MEK/ERK, PI3K/AKT, and PPARγ pathways, while the increased production of IL‐1β and TNF‐α mainly involves the MEK/ERK pathway. The effect of LiCl on IL‐6/IL‐8/IL‐10 secretion in iDC is mediated through inhibition of GSK‐3β. We have also demonstrated that PPARγ is downstream of GSK‐3β and is responsible for the LiCl‐mediated modulation of CD86/83 and CD1 expression, but not IL‐6/8/10 secretion. The combined influence of these molecular signaling pathways may account for certain clinical effect of lithium. J. Cell. Physiol. 226: 424–433, 2011. © 2010 Wiley‐Liss, Inc.     

Inverse correlation of maturity and antibacterial activity in human dendritic cells

Dendritic cells (DCs) are a key part of host defense against microbial pathogens, being part of the innate immune system, but also instructing the adaptive T cell response. This study was designed to evaluate whether human DCs directly contribute to innate immunity by killing intracellular bacteria, using tuberculosis as a model. DCs were detected in bronchoalveolar lavage samples indicating that DCs are available for immediate interaction with Mycobacterium tuberculosis (M. Tb) after inhalation of the pathogen. The phenotype of DC in bronchoalveolar lavage closely resembles monocyte-derived immature DC (iDC) according to the expression of CD1a, CD83, and CCR7. The antimicrobial activity of iDC against intracellular M. Tb inversely correlated with TNF-alpha-release and was enhanced by treatment with anti-TNF-alpha Abs. Differentiation of iDC into mature DC by addition of TNF-alpha or activation via Toll-like receptors further reduced killing of M. Tb. The antibacterial activity against intracellular M. Tb of all DCs was significantly lower than alveolar macrophages. Therefore, the maintenance of a pool of DCs at the site of disease activity in tuberculosis, and the maturation of these DC by TNF-alpha provides a mechanism by which M. Tb escapes the innate immune system.     

Matrix protein mutant of vesicular stomatitis virus stimulates maturation of myeloid dendritic cells

Matrix (M) protein mutants of vesicular stomatitis virus have recently been used as oncolytic viruses for tumor therapies and are being developed as vaccine vectors for heterologous antigens. Because dendritic cell (DC) maturation is an important correlate of tumor immunosurveillance and vaccine efficacy, we sought to determine the ability of a recombinant M protein mutant virus (rM51R-M virus) to mature DC in vitro. We have previously shown that rM51R-M virus is defective at inhibiting host gene expression in several cell lines compared to its recombinant wild-type counterpart, rwt virus. Therefore, rM51R-M virus allows the expression of genes involved in antiviral responses, such as the type I interferon (IFN) gene. Our results demonstrate that, in contrast to the rwt virus, rM51R-M virus induced the maturation of myeloid DC (mDC) populations, as indicated by an increase in the surface expression of CD40, CD80, and CD86 as well as the secretion of interleukin-12 (IL-12), IL-6, and type I IFN. In addition, mDC infected with rM51R-M virus effectively activated na?ve T cells in vitro, whereas rwt virus-infected mDC were defective in antigen presentation. The inability of rwt virus to induce mDC maturation was correlated with the inhibition of host gene expression in rwt virus-infected cells. Our studies also indicated that the production of costimulatory molecules on mDC by rM51R-M virus was dependent on the type I IFN receptor, while maturation induced by this virus was largely independent of MyD88. These data indicate that rM51R-M virus effectively stimulates the maturation of mDC and has the potential to promote effective T-cell responses to vector-expressed antigens, activate DC at tumor sites during therapy, and aid in tumor immunosurveillance and destruction.     

Dendritic cell maturation requires STAT1 and is under feedback regulation by suppressors of cytokine signaling

In this study we show that activation of STAT pathways is developmentally regulated and plays a role in dendritic cell (DC) differentiation and maturation. The STAT6 signaling pathway is constitutively activated in immature DC (iDC) and declines as iDCs differentiate into mature DCs (mDCs). However, down-regulation of this pathway during DC differentiation is accompanied by dramatic induction of suppressors of cytokine signaling 1 (SOCS1), SOCS2, SOCS3, and cytokine-induced Src homology 2-containing protein expression, suggesting that inhibition of STAT6 signaling may be required for DC maturation. In contrast, STAT1 signaling is most robust in mDCs and is not inhibited by the up-regulated SOCS proteins, indicating that STAT1 and STAT6 pathways are distinctly regulated in maturing DC. Furthermore, optimal activation of STAT1 during DC maturation requires both IL-4 and GM-CSF, suggesting that synergistic effects of both cytokines may in part provide the requisite STAT1 signaling intensity for DC maturation. Analyses of STAT1(-/-) DCs reveal a role for STAT1 in repressing CD86 expression in precursor DCs and up-regulating CD40, CD11c, and SOCS1 expression in mDCs. We further show that SOCS proteins are differentially induced by IL-4 and GM-CSF in DCs. SOCS1 is primarily induced by IL-4 through a STAT1-dependent mechanism, whereas SOCS3 is induced mainly by GM-CSF. Taken together, these results suggest that cytokine-induced maturation of DCs is under feedback regulation by SOCS proteins and that the switch from constitutive activation of the STAT6 pathway in iDCs to predominant use of STAT1 signals in mDC is mediated in part by STAT1-induced SOCS expression.     

Endocytosis of micro- and nanosized particles in vitro by human dendritic cells

The ability of human dendritic cells (DC) to uptake synthetic micro- and nanosized particles was assessed by flow cytometry and fluorescent microscopy. DCs were differentiated in vitro from blood monocytes in the presence of recombinant cytokines. Further maturation of DC in culture after the addition of maturation factors resulted in the increased expression of HLA-DR and co-stimulatory molecules CD80, CD83, CD86, in comparison with immature DC. Active internalization of Fluoresbrite-YG fluorescent microbeads (0.2 μm) was noted for immature but not mature DCs. The decrease of endocytic activity after DC maturation correlated with the reduced expression of CD209, the surface membrane receptor participating in phagocytosis. Unlike microparticles, the uptake of nanoscale Quantum dots-655 did not depend on the stage of DC maturation and probably was mediated by a different endocytosis mechanism.     

A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-alpha, and contact sensitizers

We investigated the involvement of mitogen-activated protein kinases (MAPKs) in the maturation of CD83(-) dendritic cells (DC) derived from human blood monocytes. Maturating agents such as LPS and TNF-alpha induced the phosphorylation of members of the three families of MAPK (extracellular signal-regulated kinase l/2, p46/54 c-Jun N-terminal kinase, and p38 MAPK). SB203580, an inhibitor of the p38 MAPK, but not the extracellular signal-regulated kinase l/2 pathway blocker PD98059, inhibited the up-regulation of CD1a, CD40, CD80, CD86, HLA-DR, and the DC maturation marker CD83 induced by LPS and TNF-alpha. In addition, SB203580 inhibited the enhancement of the allostimulatory capacity and partially prevented the down-regulation of FITC-dextran uptake induced by LPS and TNF-alpha. Likewise, SB203580 partially prevented the up-regulation of IL-1alpha, IL-1beta, IL-lRa, and TNF-alpha mRNA upon stimulation with LPS and TNF-alpha, as well as the release of bioactive TNF-alpha induced by LPS. DC maturation induced by the contact sensitizers 2,4-dinitrofluorobenzene and NiSO(4), as seen by the up-regulation of CD80, CD86, and CD83, was also coupled to the phosphorylation of p38 MAPK, and was inhibited by SB203580. The irritants SDS and benzalkonium chloride that do not induce DC maturation did not trigger p38 MAPK phosphorylation. Together, these data indicate that phosphorylation of p38 MAPK is critical for the maturation of immature DC. These results also suggest that p38 MAPK phosphorylation in DC may become useful for the identification of potential skin contact sensitizers.