Activation of the D prostanoid receptor 1 regulates immune and skin allergic responses

The mobilization of Langerhans cells (LCs) from epithelia to the draining lymph nodes is an essential process to initiate primary immune responses. We have recently shown that in mice, PGD2 is a potent inhibitor of epidermal LC emigration. In this study, we demonstrate that activation of the D prostanoid receptor 1 (DP1) impedes the TNF-alpha-induced migration of human LCs from skin explants and strongly inhibits the chemotactic responses of human LC precursors and of maturing LCs to CC chemokine ligands 20 and 19, respectively. Using a murine model of atopic dermatitis, a chronic Th2-type allergic inflammatory disease, we demonstrate that the potent DP1 agonist BW245C dramatically decreases the Ag-specific T cell activation in the skin draining lymph nodes and markedly prevents the skin lesions following repeated epicutaneous sensitization with OVA. Interestingly, analysis of the local response indicates that BW245C treatment strongly reduces the recruitment of inflammatory cells into the dermis and disrupts the Th1/Th2 balance, probably through the increased production of the immunoregulatory cytokine IL-10, in the skin of sensitized mice. Taken together, our results suggest a new function for DP1 in the regulation of the immune and inflammatory responses. We propose that DP1 activation by specific agonists may represent a strategy to control cutaneous inflammatory Th2-associated diseases.     

CD4 T cell-induced, bid-dependent apoptosis of cutaneous dendritic cells regulates T cell expansion and immune responses

The fate of dendritic cells (DCs) after Ag presentation may be DC subset-specific and controlled by many factors. The role of activation-induced apoptosis in regulating DC function is not clear. We investigated the fate of cutaneous DCs (cDCs), specifically Langerhans cells (LCs), and observed that they undergo apoptosis after successful Ag presentation to CD4 T cells. Caspase-specific inhibitors revealed that LC lines use a type II apoptosis pathway in response to CD4 T cells. In support of this, BH3-interacting domain (Bid) protein was present at high levels and specifically cleaved in the presence of Ag-specific T cells. Significant resistance to apoptosis by OT-2 CD4 cells was also observed for Bid knockout (KO) LCs in vitro. To test whether Bid was required to regulate LC function in vivo, we measured contact sensitization and topical immunization responses in Bid KO mice and observed markedly enhanced ear swelling and proliferation responses compared with wild-type mice. Furthermore, when Ag-pulsed Bid KO migratory cDCs were inoculated into wild-type recipients, an increase in both the rate and percentage of expanded OT-2 T cells expressing IFN-gamma was observed. Thus, enhanced Ag presentation function was intrinsic to Bid KO cDCs. Therefore, Bid is an important regulator of LC viability and Ag presentation function.     

Skin langerin+ dendritic cells transport intradermally injected anti-DEC-205 antibodies but are not essential for subsequent cytotoxic CD8+ T cell responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8α(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8α(+) DCs are sufficient to subsequent CD8(+) T cell responses.     

Modulation of dendritic cell trafficking to and from the airways

We investigated the fate of latex (LX) particles that were introduced into mice intranasally. Macrophages acquired the vast majority of particles and outnumbered LX particle-bearing airway dendritic cells (DCs) by at least two orders of magnitude. Yet alveolar macrophages were refractory to migration to the draining lymph node (DLN), and all transport to the DLN could be ascribed to the few LX(+) airway DCs. Upon macrophage depletion, markedly greater numbers of DCs were recruited into the alveolar space. Consequently, the number of DCs that carried particles to the DLN was boosted by 20-fold. Thus, a so far overlooked aspect of macrophage-mediated suppression of airway DC function stems from the modulation of DC recruitment into the airway. This increase in DC recruitment permitted the development of a robust assay to quantify the subsequent migration of DCs to the DLN. Therefore, we determined whether lung DCs use the same molecules that skin DCs use during migration to DLNs. Like skin DCs, lung DCs used CCR7 ligands and CCR8 for emigration to DLN, but the leukotriene C(4) transporter multidrug resistance-related protein 1 did not mediate lung DC migration as it does in skin, indicating that pathways governing DC migration from different tissues partially differ in molecular regulation.     

Exogenous dendritic cell homing to draining lymph nodes can be boosted by mast cell degranulation

Dendritic cells (DCs), as potent antigen presenting cells, are increasingly used for immunotherapeutic approaches, predominantly in oncology. Low efficiency of injected Ag-pulsed DC homing to draining lymph nodes (DLNs) is one of the factors that affect the efficacy of therapy. As Langerhans cell emigration was enhanced after skin mast cell degranulation, we investigated the effect of local mast cell activation on exogenous bone marrow-derived DCs (BM-DCs) homing to DLNs. Product of activated MC/9 mast cells enhanced chemotaxis of BM-DCs to CCL21 in vitro. Intradermal injection of compound 48/80 (c48/80) induced local skin mast cell obvious degranulation and boosted exogenous BM-DC homing to DLNs. Both Ag-specific lymphocyte proliferation and TH1/TH2 cytokine production increased after HBsAg-pulsed BM-DC was injected into c48/80 pretreated mice. These results suggest that transferred DC homing to DLNs promoted by local mast cell degranulation may have potential application to improve DC-based immunotherapy.     

Dermal dendritic cells, and not Langerhans cells, play an essential role in inducing an immune response

Langerhans cells (LCs) serve as epidermal sentinels of the adaptive immune system. Conventional wisdom suggests that LCs encounter Ag in the skin and then migrate to the draining lymph nodes, where the Ag is presented to T cells, thus initiating an immune response. Platelet-activating factor (PAF) is a phospholipid mediator with potent biological effects. During inflammation, PAF mediates recruitment of leukocytes to inflammatory sites. We herein tested a hypothesis that PAF induces LC migration. Applying 2,4-dinitro-1-fluorobenzene (DNFB) to wild-type mice activated LC migration. In contrast, applying DNFB to PAF receptor-deficient mice or mice injected with PAF receptor antagonists failed to induce LC migration. Moreover, after FITC application the appearance of hapten-laden LCs (FITC+, CD11c+, Langerin+) in the lymph nodes of PAF receptor-deficient mice was significantly depressed compared with that found in wild-type mice. LC chimerism indicates that the PAF receptor on keratinocytes but not LCs is responsible for LC migration. Contrary to the diminution of LC migration in PAF receptor-deficient mice, we did not observe any difference in the migration of hapten-laden dermal dendritic cells (FITC+, CD11c+, Langerin-) into the lymph nodes of PAF receptor-deficient mice. Additionally, the contact hypersensitivity response generated in wild-type or PAF receptor-deficient mice was identical. Finally, dermal dendritic cells, but not LCs isolated from the draining lymph nodes after hapten application, activated T cell proliferation. These findings suggest that LC migration may not be responsible for the generation of contact hypersensitivity and that dermal dendritic cells may play a more important role.     

Visualizing dendritic cell migration within the skin

Dendritic cells (DCs) within the skin are a heterogeneous population of cells, including Langerhans cells of the epidermis and at least three subsets of dermal DCs. Collectively, these DCs play important roles in the initiation of adaptive immune responses following antigen challenge of the skin as well as being mediators of tolerance to self-antigen. A key functional aspect of cutaneous DCs is their migration both within the skin and into lymphatic vessels, resulting in their emigration to draining lymph nodes. Here, we discuss our current understanding of the requirements for successful DC migration in and from the skin, and introduce some of the microscopic techniques developed in our laboratory to facilitate a better understanding of this process. In particular, we detail our current use of multi-photon excitation (MPE) microscopy of murine skin to dissect the migratory behavior of DCs in vivo. B. Roediger and L. G. Ng contributed equally to this work.     

CD13 regulates dendritic cell cross-presentation and T cell responses by inhibiting receptor-mediated antigen uptake

Dendritic cell (DC) Ag cross-presentation is generally associated with immune responses to tumors and viral Ags, and enhancement of this process is a focus of tumor vaccine design. In this study, we found that the myeloid cell surface peptidase CD13 is highly and specifically expressed on the subset of DCs responsible for cross-presentation, the CD8(+) murine splenic DCs. In vivo studies indicated that lack of CD13 significantly enhanced T cell responses to soluble OVA Ag, although development, maturation, and Ag processing and presentation of DCs are normal in CD13KO mice. In vitro studies showed that CD13 regulates receptor-mediated, dynamin-dependent endocytosis of Ags such as OVA and transferrin but not fluid-phase or phagocytic Ag uptake. CD13 and Ag are cointernalized in DCs, but CD13 did not coimmunoprecipitate with Ag receptors, suggesting that CD13 does not control internalization of specific receptors but regulates endocytosis at a more universal level. Mechanistically, we found that phosphorylation of the endocytic regulators p38MAPK and Akt was dysregulated in CD13KO DCs, and blocking of these kinases perturbed CD13-dependent endocytic uptake. Therefore, CD13 is a novel endocytic regulator that may be exploited to enhance Ag uptake and T cell activation to improve the efficacy of tumor-targeted vaccines.     

Dendritic cells in distinct oral mucosal tissues engage different mechanisms to prime CD8+ T cells

Although oral dendritic cells (DCs) were shown to induce cell-mediated immunity, the identity and function of the various oral DC subsets involved in this process is unclear. In this study, we examined the mechanisms used by DCs of the buccal mucosa and of the lining mucosa to elicit immunity. After plasmid DNA immunization, buccally immunized mice generated robust local and systemic CD8(+) T cell responses, whereas lower responses were seen by lining immunization. A delayed Ag presentation was monitored in vivo in both groups; yet, a more efficient presentation was mediated by buccal-derived DCs. Restricting transgene expression to CD11c(+) cells resulted in diminished CD8(+) T cell responses in both oral tissues, suggesting that immune induction is mediated mainly by cross-presentation. We then identified, in addition to the previously characterized Langerhans cells (LCs) and interstitial dendritic cells (iDCs), a third DC subset expressing the CD103(+) molecule, which represents an uncharacterized subset of oral iDCs expressing the langerin receptor (Ln(+)iDCs). Using Langerin-DTR mice, we demonstrated that whereas LCs and Ln(+)iDCs were dispensable for T cell induction in lining-immunized mice, LCs were essential for optimal CD8(+) T cell priming in the buccal mucosa. Buccal LCs, however, failed to directly present Ag to CD8(+) T cells, an activity that was mediated by buccal iDCs and Ln(+)iDCs. Taken together, our findings suggest that the mechanisms engaged by oral DCs to prime T cells vary between oral mucosal tissues, thus emphasizing the complexity of the oral immune network. Furthermore, we found a novel regulatory role for buccal LCs in potentiating CD8(+) T cell responses.     

TLR activation of Langerhans cell-like dendritic cells triggers an antiviral immune response

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.     

Large-scale culture and selective maturation of human Langerhans cells from granulocyte colony-stimulating factor-mobilized CD34+ progenitors

Dendritic cells (DCs) play a critical role as APCs in the induction of the primary immune response. Their capacity for Ag processing and presentation is tightly regulated, controlled by a terminal developmental sequence accompanied by striking changes in morphology, organization, and function. The maturation process, which converts DCs from cells adapted for Ag accumulation to cells adapted for T cell stimulation, remains poorly understood due in part to difficulties in the culture and manipulation of DCs of defined lineages. To address these issues, we have devised conditions for the culture of a single DC type, Langerhans cells (LCs), using CD34+ cells from G-CSF-mobilized patients. Homogenous populations of LCs, replete with abundant immunocytochemically demonstrable Birbeck granules, could be stably maintained as immature DCs for long periods in culture. Unlike other human DC preparations, the LCs remained fully differentiated after cytokine removal. Following exposure to TNF-alpha, LPS, or CD40 ligand, the LCs could be synchronously induced to mature. Depending on the agent used, distinct types of LCs emerged differing in their capacity for T cell stimulation, IL-12 production, intracellular localization of MHC products, and overall morphology. Most interestingly, the expression of different sets of Toll family receptors is induced or down-regulated according to the maturation stimulus provided. These results strongly suggest that different proinflammatory stimuli might drive distinct developmental events.     

Host-derived Langerhans cells persist after MHC-matched allografting independent of donor T cells and critically influence the alloresponses mediated by donor lymphocyte infusions

Mouse models of minor histocompatibility Ag-mismatched bone marrow transplantation were used to study donor dendritic cell (DC) reconstitution after conditioning, variables influencing the persistence of residual host DCs in different compartments, their phenotype, and their role in governing donor lymphocyte infusion (DLI)-mediated alloresponses. Reconstitution of all splenic DC subsets occurred rapidly after bone marrow transplantation and before T cell reconstitution. However, in contrast to MHC-mismatched chimeras, residual host-derived DCs persisted in the cutaneous lymph nodes (CLNs) of MHC-matched chimeras despite the presence or addition of donor T cells to the graft. The phenotype of these residual host-derived DCs in CLNs was consistent with Langerhans' cells (LCs). We confirmed their skin origin and found near-complete preservation of host-derived LCs in the skin. Host-derived LCs retained their ability to continuously traffic to the CLNs, expressed homogeneously increased levels of costimulatory molecules, and could capture and carry epicutaneously applied Ags. To determine the role of residual host LCs in governing DLI-mediated alloresponses, we administered DLI alone or after topical application of the TLR7 ligand imiquimod, which is known to enhance the LC emigration from the skin. DLI administration resulted in a decrease in host-derived DCs in the CLNs and increased recruitment of donor-derived DCs to the skin, whereas imiquimod augmented their alloreactivity. These results suggest uniqueness of the MHC-matched setting in relation to the persistence of host-derived DCs in the skin and points to a previously unrecognized role of host-derived LCs in the induction of DLI-mediated graft-vs-host alloresponses.     

Targeting Viral Antigens to CD11c on Dendritic Cells Induces Retrovirus-Specific T Cell Responses

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.     

A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation

Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.     

Exacerbation of granuloma formation in IL-1 receptor antagonist-deficient mice with impaired dendritic cell maturation associated with Th2 cytokine production

Dendritic cell (DC) maturation at the site of inflammation and migration into draining lymph nodes is fundamental to initiate Ag-specific immune responses. Although several proinflammatory cytokines, including IL-1, are known to promote DC maturation in vitro, their contributions to DC activation and migration within peripheral inflamed tissue compartments are not yet fully understood. We show here that endogenous IL-1 receptor antagonist (IL-1ra) controls the activation state of liver-recruited DCs and their migration in a Propionibacterium acnes-induced murine granulomatous liver disease model. After P. acnes treatment, formation of portal tract-associated lymphoid tissue was conversely impaired in IL-1ra-deficient mice. IL-1ra-deficient mice developed hepatic granulomas within 3 days after P. acnes administration and showed a more pronounced granuloma formation than wild-type mice. Although sinusoidal granulomas contained numerous CD11c+ DCs at day 7, expressions of CCR7, IL-12p40 by these DCs were dramatically decreased in IL-1ra-deficient mice, suggesting aberrant DC maturation and sinusoid portal migration in the absence of endogenous IL-1ra. This was accompanied with enhanced intrahepatic Th2 cytokine production and severe hepatocellular damage. Thus, hepatocyte-derived IL-1ra may control optimal activation and migration of inflammatory DCs within the liver and thereby determine the local immune responses in granulomatous liver disease.     

Murine CXCL14 is dispensable for dendritic cell function and localization within peripheral tissues

Dendritic cells (DCs) have long been recognized as key regulators of immune responses. However, the process of their recruitment to peripheral tissues and turnover during homeostasis remains largely unknown. The chemokine CXCL14 (BRAK) is constitutively expressed in skin and other epithelial tissues. Recently, the human chemokine was proposed to play a role in the homeostatic recruitment of macrophage and/or DC precursors toward the periphery, such as skin. Although so far no physiological function could be demonstrated for the murine CXCL14, it shows a remarkable homology to the human chemokine. In order to elucidate the in vivo role of CXCL14, we generated a mouse defective for this chemokine. We studied various components of the immune system with emphasis on monocytes/macrophages and DC/Langerhans cell (LC) populations in different tissues during steady state but did not find a significant difference between knockout (CXCL14(-)(/)(-)) and control mice. Functionally, LCs were able to become activated, to migrate out of skin, and to elicit a delayed type of hypersensitivity reaction. Overall, our data indicate that murine CXCL14 is dispensable for the homeostatic recruitment of antigen-presenting cells toward the periphery and for LC functionality.     

The HGF Receptor/Met Tyrosine Kinase Is a Key Regulator of Dendritic Cell Migration in Skin Immunity

The Met tyrosine kinase has a pivotal role in embryonic development and tissue regeneration, and deregulated Met signaling contributes to tumorigenesis. After binding of its cognate ligand hepatocyte growth factor, Met signaling confers mitogenic, morphogenic, and motogenic activity to various cells. Met expression in the hematopoietic compartment is limited to progenitor cells and their Ag-presenting progeny, including dendritic cells (DCs). In this study, we demonstrate that Met signaling in skin-resident DCs is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. By using a conditional Met-deficient mouse model (Met(flox/flox)), we show that Met acts on the initial step of DC release from skin tissue. Met-deficient DCs fail to reach skin-draining lymph nodes upon activation while exhibiting an activated phenotype. Contact hypersensitivity reactions in response to various contact allergens is strongly impaired in Met-deficient mice. Inhibition of Met signaling by single-dose epicutaneous administration of the Met kinase-specific inhibitor SU11274 also suppressed contact hypersensitivity in wild-type mice. Additionally, we found that Met signaling regulates matrix metalloproteinase MMP2 and MMP9 activity, which is important for DC migration through extracellular matrix. These data unveil Met signaling in DCs as a critical determinant for the maintenance of normal immune function and suggest Met as a potential target for treatment of autoimmune skin diseases.     

A novel DC therapy with manipulation of MKK6 gene on nickel allergy in mice

BACKGROUND: Although the activation of dermal dendritic cells (DCs) or Langerhans cells (LCs) via p38 mitogen-activated protein kinase (MAPK) plays a crucial role in the pathogenesis of metal allergy, the in vivo molecular mechanisms have not been identified and a possible therapeutic strategy using the control of dermal DCs or LCs has not been established. In this study, we focused on dermal DCs to define the in vivo mechanisms of metal allergy pathogenesis in a mouse nickel (Ni) allergy model. The effects of DC therapy on Ni allergic responses were also investigated. METHODS AND FINDING: The activation of dermal DCs via p38 MAPK triggered a T cell-mediated allergic immune response in this model. In the MAPK signaling cascade in DCs, Ni potently phosphorylated MAP kinase kinase 6 (MKK6) following increased DC activation. Ni-stimulated DCs could prime T cell activation to induce Ni allergy. Interestingly, when MKK6 gene-transfected DCs were transferred into the model mice, a more pronounced allergic reaction was observed. In addition, injection of short interfering (si) RNA targeting the MKK6 gene protected against a hypersensitivity reaction after Ni immunization. The cooperative action between T cell activation and MKK6-mediated DC activation by Ni played an important role in the development of Ni allergy. CONCLUSIONS: DC activation by Ni played an important role in the development of Ni allergy. Manipulating the MKK6 gene in DCs may be a good therapeutic strategy for dermal Ni allergy.