Essential role of EP3 subtype in prostaglandin E2-induced adhesion of mouse cultured and peritoneal mast cells to the Arg-Gly-Asp-enriched matrix

Accumulating evidence has indicated that mast cells can modulate a wide variety of immune responses. Migration and adhesion play a critical role in regulation of tissue mast cell function, in particular, under inflammatory conditions. We previously demonstrated that prostaglandin (PG) E(2) stimulates adhesion of a mouse mastocytoma cell line, P-815, to the Arg-Gly-Asp (RGD)-enriched matrix through cooperation between two PGE(2) receptor subtypes: EP3 and EP4 (Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. J Biol Chem 278: 17977-17981, 2003). We here investigated PGE(2)-induced adhesion of IL-3-dependent bone marrow-derived cultured mast cells (BMMCs). In contrast to the elevated cAMP-dependent adhesion of P-815 cells, EP3-mediated Ca(2+) mobilization plays a pivotal role in PGE(2)-induced adhesion of BMMCs. Adhesion and Ca(2+) mobilization induced by PGE(2) were abolished in the Ptger3(-/-) BMMCs and were significantly suppressed by treatment with pertussis toxin, a phospholipase C inhibitor, U-73122, and a store-operated Ca(2+) channel inhibitor, SKF 36965, indicating the involvement of G(i)-mediated Ca(2+) influx. We then investigated PGE(2)-induced adhesion of peritoneal mast cells to the RGD-enriched matrix. EP3 subtype was found to be the dominant PGE receptor that expresses in mouse peritoneal mast cells. PGE(2) induced adhesion of the peritoneal mast cells of the Ptger3(+/+) mice, but not that of the Ptger3(-/-) mice. In rat peritoneal mast cells, PGE(2) or an EP3 agonist stimulated both Ca(2+) mobilization and adhesion to the RGD-enriched matrix. These results suggested that the EP3 subtype plays a pivotal role in PGE(2)-induced adhesion of murine mast cells to the RGD-enriched matrix through Ca(2+) mobilization.     

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.     

IgE-mediated mast cell activation induces Langerhans cell migration in vivo

Langerhans cells and mast cells are both resident in large numbers in the skin and act as sentinel cells in host defense. The ability of mast cells to induce Langerhans cell migration from the skin to the draining lymph node in vivo was examined. Genetically mast cell-deficient (W/Wv) mice and control mice were sensitized with IgE Ab in the ear pinna. Seven to 14 days later, mice were challenged with Ag i.v. After a further 18-24 h, epidermal sheets and draining auricular lymph nodes were examined using Langerin/CD207 immunostaining. In mast cell-containing mice, a significant decrease in the number of Langerhans cells was observed at epidermal sites of mast cell activation. A significant increase in total cellularity and accumulation of Langerin-positive dendritic cells was observed in the auricular lymph nodes, draining the sites of IgE-mediated mast cell activation. These changes were not observed in W/Wv mice, but were restored by local mast cell reconstitution. Treatment of mast cell-containing mice with the H2 receptor antagonist cimetidine significantly inhibited the observed IgE/Ag-induced changes in Langerhans cell location. In contrast, Langerhans cell migration in response to LPS challenge was not mast cell dependent. These data directly demonstrate the ability of mast cells to induce dendritic cell migration to lymph nodes following IgE-mediated activation in vivo by a histamine-dependent mechanism.     

Prostaglandin D2 plays an essential role in chronic allergic inflammation of the skin via CRTH2 receptor

PGD(2) plays roles in allergic inflammation via specific receptors, the PGD receptor designated DP and CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells). We generated mutant mice carrying a targeted disruption of the CRTH2 gene to investigate the functional roles of CRTH2 in cutaneous inflammatory responses. CRTH2-deficent mice were fertile and grew normally. Ear-swelling responses induced by hapten-specific IgE were less pronounced in mutant mice, giving 35-55% of the responses of normal mice. Similar results were seen in mice treated with a hemopoietic PGD synthase inhibitor, HQL-79, or a CRTH2 antagonist, ramatroban. The reduction in cutaneous responses was associated with decreased infiltration of lymphocytes, eosinophils, and basophils and decreased production of macrophage-derived chemokine and RANTES at inflammatory sites. In models of chronic contact hypersensitivity induced by repeated hapten application, CRTH2 deficiency resulted in a reduction by approximately half of skin responses and low levels (63% of control) of serum IgE production, although in vivo migration of Langerhans cells and dendritic cells to regional lymph nodes was not impaired in CRTH2-deficient mice. In contrast, delayed-type hypersensitivity to SRBC and irritation dermatitis in mutant mice were the same as in wild-type mice. These findings indicate that the PGD(2)-CRTH2 system plays a significant role in chronic allergic skin inflammation. CRTH2 may represent a novel therapeutic target for treatment of human allergic disorders, including atopic dermatitis.     

Induction of anti-chlamydial mucosal immunity by transcutaneous immunization is enhanced by topical application of GM-CSF

Transcutaneous immunization (TCI) involves the direct application of antigen plus adjuvant to skin, taking advantage of the large numbers of Langerhans cells and other resident skin dendritic cells, that process antigen then migrate to draining lymph nodes where immune responses are initiated. We have used this form of immunization to protect mice against genital tract and respiratory tract chlamydial infection. Protection was associated with local antibody responses in the vagina, uterus and lung as well as strong Th1 responses in the lymph nodes draining the reproductive tract and lungs respectively. In this study we show that topical application of GM-CSF to skin enhances the numbers and activation status of epidermal dendritic cells. Topical application of GM-CSF also increased the immune responses elicited by TCI. GM-CSF supplementation greatly increased cytokine (IFNgamma and IL-4) gene expression in lymph node and splenic cells compared to cells from animals immunized without GM-CSF. IgG responses in serum, uterine lavage and bronchoalveolar lavage and IgA responses in vaginal lavage were also increased by topical application of GM-CSF. The studies show that TCI induces protection against genital and respiratory tract chlamydial infections and that topical application of cytokines such as GM-CSF can enhance TCI-induced antibody and cell-mediated immunity.     

Mast cell migration from the skin to the draining lymph nodes upon ultraviolet irradiation represents a key step in the induction of immune suppression

The UV radiation in sunlight is the primary cause of skin cancer. UV is also immunosuppressive and numerous studies have shown that UV-induced immune suppression is a major risk factor for skin cancer induction. Previous studies demonstrated that dermal mast cells play a critical role in the induction of immune suppression. Mast cell-deficient mice are resistant to the immunosuppressive effects of UV radiation, and UV-induced immune suppression can be restored by injecting bone marrow-derived mast cells into the skin of mast cell- deficient mice. The exact process however, by which mast cells contribute to immune suppression, is not known. In this study, we show that one of the first steps in the induction of immune suppression is mast cell migration from the skin to the draining lymph nodes. UV exposure, in a dose-dependent manner, causes a significant increase in lymph node mast cell numbers. When GFP(+) skin was grafted onto mast cell-deficient mice, we found that GFP(+) mast cells preferentially migrated into the lymph nodes draining the skin. The mast cells migrated primarily to the B cell areas of the draining nodes. Mast cells express CXCR4(+) and UV exposure up-regulated the expression of its ligand CXCL12 by lymph node B cells. Treating UV-irradiated mice with a CXCR4 antagonist blocked mast cell migration and abrogated UV-induced immune suppression. Our findings indicate that UV-induced mast cell migration to draining lymph nodes, mediated by CXCR4 interacting with CXCL12, represents a key early step in UV-induced immune suppression.     

Involvement of aquaporin-7 in the cutaneous primary immune response through modulation of antigen uptake and migration in dendritic cells

Dendritic cells (DCs) have the ability to present antigen and play a critical role in the induction of the acquired immune response. Skin DCs uptake antigen and subsequently migrate to regional draining lymph nodes (LNs), where they activate naive T cells. Here we show that the water/glycerol channel protein aquaporin 7 (AQP7) is expressed on epidermal and dermal DCs and involved in the initiation of primary immune responses. AQP7-deficient DCs showed a decreased cellular uptake of low-molecular-mass compounds (fluorescein isothiocyanate and Lucifer yellow) and high-molecular-mass substances (ovalbumin and dextran), suggesting that AQP7 is involved in antigen uptake. AQP7-deficient DCs also exhibited reduced chemokine-dependent cell migration in comparison to wild-type DCs. Consistent with these in vitro results, AQP7-deficient mice demonstrated a reduced accumulation of antigen-retaining DCs in the LNs after antigen application to the skin, which could be attributed to decreased antigen uptake and migration. Coincidentally, AQP7-deficient mice had impaired antigen-induced sensitization in a contact hypersensitivity model. These observations suggested that AQP7 in skin DCs is primarily involved in antigen uptake and in the subsequent migration of DCs and is responsible for antigen presentation and the promotion of downstream immune responses.     

Changes in dendritic cell migration and activation during SIV infection suggest a role in initial viral spread and eventual immunosuppression

Dendritic cells (DC) serve an essential function in linking the innate and acquired immune responses to antigen. Peripheral DC acquire antigen and migrate to draining lymph nodes, where they localize to the T cell-rich paracortex and function as potent antigen presenting cells. We examined the effects of human immunodeficiency virus (HIV) infection on DC function in vivo using the rhesus macaque/simian immunodeficiency virus (SIV) model. Our data show that during acute SIV infection, Langerhans cell density is reduced in skin and activated DC are increased in proportion in lymph nodes, whereas during AIDS, DC migration from skin and activation within lymph nodes are suppressed. These findings suggest that changes in DC function at different times during the course of infection may serve to promote virus dissemination and persistence: early during infection, DC mobilization may facilitate virus spread to susceptible lymph node T cell populations, whereas depressed DC function during advanced infection could promote generalized immunosuppression.     

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.     

Lymph node resident rather than skin-derived dendritic cells initiate specific T cell responses after Leishmania major infection

Langerhans cells have been thought to play a major role as APCs for induction of specific immune responses to Leishmania major. Although their requirement for control of infection has been challenged recently, it remains unclear whether they can transport Ag to lymph nodes and promote initiation of T cell responses. Moreover, the role of dermal dendritic cells (DCs), another population of skin DCs, has so far not been addressed. We have investigated the origin and characterized the cell population responsible for initial activation of L. major-specific T cells in susceptible and resistant mice. We found that Ag presentation in draining lymph nodes peaks as early as 24 h after infection and is mainly mediated by a population of CD11c(high)CD11b(high)Gr-1-CD8-langerin- DCs residing in lymph nodes and acquiring soluble Ags possibly drained through the conduit network. In contrast, skin-derived DCs, including Langerhans cells and dermal DCs, migrated poorly to lymph nodes and played a minor role in early T cell activation. Furthermore, prevention of migration through early removal of the infection site did not affect Ag presentation by CD11c(high) CD11b(high) DCs and activation of Leishmania major-specific naive CD4+ T cells in vivo.     

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz-(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.     

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.     

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.     

A critical role for the proapoptotic protein bid in ultraviolet-induced immune suppression and cutaneous apoptosis

Apoptosis plays an important role in eliminating UV-damaged keratinocytes, but its role in UV-induced immune suppression is not clear. Langerhans cells (LCs) may function as inducers of immune suppression. We have shown that LCs derived from mice deficient in the proapoptotic Bid (BH3-interacting death domain protein) gene (Bid KO) resist apoptosis and induce amplified immune responses. In this report, we examined responses in Bid KO mice to UVB exposure. Acute UV exposure led Bid KO mice to develop fewer apoptotic cells and retain a greater fraction of LCs in the epidermal layer of skin in comparison to wild-type mice. Bid KO mice were also markedly resistant to local and systemic UV tolerance induction to hapten sensitization and contact hypersensitivity responses. Elicitation responses and inflammation at skin sensitization sites in UV-treated Bid KO mice were equal to or greater than nonsuppressed control responses. In Bid KO mice, LCs accumulated in lymph nodes to greater numbers, demonstrated longer lifespans, and contained fewer DNA-damaged cells. These studies provide evidence that Bid activation is a critical upstream mediator in UV-induced keratinocyte and LC apoptosis and that its absence abrogates UV-induced immune tolerance.     

Langerhans cells: target cells in immune complex reactions.

Skin of normal, cobra venom extract-treated, and C₄-deficient guinea pigs was injected with ferritin-antiferritin or with peroxidase-antiperoxidase immune complexes. Skin and draining lymph nodes were studied to compare the phagocytosis of these immune complexes by Langerhans cells (LC) and by macrophages. When complement was present, immune complexes were damaging to LC, and uptake of the immune complexes, although present, was limited. When components of complement were absent or diminished, increased numbers of LC in lymph nodes were seen, but damage to LC was absent or decreased. However no detectable change in the amount of phagocytosis by LC was noted. Since some LC can carry antigen from skin to lymph nodes and may be involved in the presentation of antigen to lymphoid cells in some cell-mediated immune responses, impairment or abolition of LC function by immune complexes could represent a mechanism through which the local presence of antibodies might interfere with the induction and elicitation of cellular immunity by antigen. Moreover, damage to LC and subsequent release of intracellular (lysosomal?) substances may constitute a general mechanism of response in the skin to injury and may be an integral part of inflammatory and allergic skin reactions.     

Prostaglandin E2-EP3 Axis in Fine-Tuning Excessive Skin Inflammation by Restricting Dendritic Cell Functions

Prostaglandin E₂ (PGE₂) is produced in the skin and is suggested to play a role in the regulation of cutaneous immune homeostasis and responses. However, the multifaceted functions of PGE₂ continue to elude our understanding, especially because of the multiplicity of PGE₂ receptors—EP1, EP2, EP3, and EP4. While cAMP-elevating EP4 is known to activate the functions of cutaneous dendritic cells (DCs), including Langerhans cells (LCs) and dermal DCs, the role of cAMP-suppressing EP3 in this process remains unknown. Here we demonstrated that an EP3 receptor selective agonist, ONO-AE-248, inhibited chemotaxis and co-stimulatory molecule expressions of DCs in vitro. A suboptimal dose of antigen was sufficient to induce contact hypersensitivity in EP3-deficient mice. Intriguingly, EP3 deficiency did not impair skin inflammation at all when the antigen dose was sufficiently high. EP3 limited the functions of cutaneous DCs only when the antigen dose was low. In contrast to EP4, the observed unappreciated function of EP3 may stabilize the cutaneous DCs to halt the impetuous response to a suboptimal dose of antigen. Taken together, PGE₂-EP3 signaling is essential for fine-tuning excessive skin inflammation by restricting DC functions.     

Cyclooxygenase-2/prostaglandin E2 accelerates the healing of gastric ulcers via EP4 receptors

We examined the involvement of cyclooxygenase (COX)-1 as well as COX-2 in the healing of gastric ulcers and investigated which prostaglandin (PG) EP receptor subtype is responsible for the healing-promoting action of PGE2. Male SD rats and C57BL/6 mice, including wild-type, COX-1(-/-), and COX-2(-/-), were used. Gastric ulcers were produced by thermocauterization under ether anesthesia. Gastric ulcer healing was significantly delayed in both rats and mice by indomethacin and rofecoxib but not SC-560 given for 14 days after ulceration. The impaired healing was also observed in COX-2(-/-) but not COX-1(-/-) mice. Mucosal PGE2 content increased after ulceration, and this response was significantly suppressed by indomethacin and rofecoxib but not SC-560. The delayed healing in mice caused by indomethacin was significantly reversed by the coadministration of 11-deoxy-PGE1 (EP3/EP4 agonist) but not other prostanoids, including the EP1, EP2, and EP3 agonists. By contrast, CJ-42794 (selective EP(4) antagonist) significantly delayed the ulcer healing in rats and mice. VEGF expression and angiogenesis were both upregulated in the ulcerated mucosa, and these responses were suppressed by indomethacin, rofocoxib, and CJ-42794. The expression of VEGF in primary rat gastric fibroblasts was increased by PGE2 or AE1-329 (EP4 agonist), and these responses were both attenuated by coadministration of CJ-42794. These results confirmed the importance of COX-2/PGE2 in the healing mechanism of gastric ulcers and further suggested that the healing-promoting action of PGE2 is mediated by the activation of EP4 receptors and is associated with VEGF expression.     

Prostaglandin E2 protects lower airways against bronchoconstriction

Prostaglandin E2 (PGE2), similar to beta-adrenergic receptor agonists, can protect airways from bronchoconstriction and resulting increase in airway resistance induced by a number of agents, including cholinergic receptor agonists and antigen. We examined the impact of sustained alterations in PGE2 pathways on changes in airway resistance. Genetic methods were utilized to alter PGE2 metabolism and signal transduction in the murine lung. PGE2 levels were elevated by generating mice lacking 15-hydroxyprostaglandin (Hpgd-/-), the major catabolic enzyme of PGE2, and by generating a transgenic line in which mouse PGE2 synthase (Ptges) expression is driven by a human lung-specific promoter, hSP-C. Conversely, to determine the impact of loss of PGE2 on airway reactivity, we examined mice lacking this synthase (Ptges-/-) and receptors that mediate the actions of PGE2, particularly the PGE2 EP2 receptor (Ptger2). Diminished capacity to produce and respond to PGE2 did not alter the response of mice to cholinergic stimuli. In contrast, the responsiveness to cholinergic stimulation was dramatically altered in animals with elevated PGE2 levels. The Hpgd-/- and hSP-C-Ptges transgenic lines both showed attenuated airway responsiveness to methacholine as measured by lung resistance. Thus, whereas compromise of the Ptges/PGE2/Ptger2 pathway does not alter airway responsiveness, genetic modulation that elevates PGE2 levels in the lung attenuates airway responsiveness.