Somatic activating ARAF mutations in Langerhans cell histiocytosis.

The extracellular signal-regulated kinase (ERK) signaling pathway is activated in Langerhans cell histiocytosis (LCH) histiocytes, but only 60% of cases carry somatic activating mutations of BRAF. To identify other genetic causes of ERK pathway activation, we performed whole exome sequencing on purified LCH cells in 3 cases. One patient with wild-type BRAF alleles in his histiocytes had compound mutations in the kinase domain of ARAF. Unlike wild-type ARAF, this mutant was a highly active mitogen-activated protein kinase kinase in vitro and was capable of transforming mouse embryo fibroblasts. Mutant ARAF activity was inhibited by vemurafenib, a BRAF inhibitor, indicating the importance of fully evaluating ERK pathway abnormalities in selecting LCH patients for targeted inhibitor therapy.

Langerhans Cell Histiocytosis: Emerging Insights and Clinical Implications.

Langerhans cell histiocytosis is a disorder characterized by lesions that include CD207+ dendritic cells along with an inflammatory infiltrate. Langerhans cell histiocytosis has a highly variable clinical presentation, ranging from a single lesion to potentially fatal disseminated disease. The uncertainty as to whether Langerhans cell histiocytosis is a reactive or a neoplastic disease has resulted in a long-standing debate on this question, and the limited understanding of the pathogenesis of the disease has impeded clinical improvement for patients. The current standard of care for multisystem Langerhans cell histiocytosis, empirically derived chemotherapy with vinblastine and prednisone, cures fewer than 50% of patients, and optimal therapies for relapse and neurodegenerative disease remain uncertain. Recent research advances support a model in which Langerhans cell histiocytosis arises due to pathologic activation of the mitogen-activated protein kinase (MAPK) pathway in myeloid precursors. Redefinition of Langerhans cell histiocytosis as a myeloid neoplastic disorder driven by hyperactive ERK supports the potential of chemotherapy with efficacy against immature myeloid cells, as well as mutation-specific targeted therapy.

Rho-family GTPase Cdc42 controls migration of Langerhans cells in vivo.

Epidermal Langerhans cells (LCs) of the skin represent the prototype migratory dendritic cell (DC) subtype. In the skin, they take up Ag, migrate to the draining lymph nodes, and contribute to Ag transport and immunity. Different depletion models for LCs have revealed contrasting roles and contributions of this cell type. To target the migratory properties of DCs, we generated mice lacking the Rho-family GTPase Cdc42 specifically in DCs. In these animals, the initial seeding of the epidermis with LCs is functional, resulting in slightly reduced Langerhans cell numbers. However, Cdc42-deficient LCs fail to leave the skin in steady state as well as upon stimulation, as they do not enter the skin-draining afferent lymph vessels. Similarly, also other Cdc42-deficient migratory DC subsets fail to home properly to the corresponding draining lymph nodes. We used this novel mouse model, in which LCs are locked out, to demonstrate that these cells contribute substantially to priming of Ag-specific CD4 and CD8 T cell responses upon epicutaneous immunization, but could not detect a role in the induction of contact hypersensitivity to various doses of hapten.

Conditional deletion of TGF-ßR1 using Langerin-Cre mice results in Langerhans cell deficiency and reduced contact hypersensitivity.

The critical role of Langerhans cells (LC) in contact hypersensitivity (CHS) was recently questioned in studies using different LC-depletion mouse models. On one hand, inducible ablation of LC led to diminished ear swelling, suggesting functional redundancy between LC and (Langerin(+)) dermal dendritic cells (DC). On the other hand, constitutive or acute depletion of LC resulted in an enhanced reaction, supporting a regulatory role of LC in CHS. To address this controversy by conditional gene targeting, we generated Langerin-Cre knockin mice. Breeding these mice to a Cre-reporter strain demonstrated robust and specific DNA recombination in LC, as well as other Langerin(+) tissue DC. In agreement with the vital requirement of TGF-ß signaling for LC development, crossing Langerin-Cre to mice homozygous for a loxP-flanked TGF-ßR1 allele resulted in permanent LC deficiency, whereas the homeostasis of dermal Langerin(+) DC was unaffected. In the absence of LC, induction of CHS in these Langerin(+) DC-specific TGF-ßR1-deficient mice elicited decreased ear swelling compared with controls. This novel approach provided further evidence against a regulatory function of LC in CHS. Moreover, these Langerin-Cre mice represent a unique and powerful tool to dissect the role and molecular control of Langerin(+) DC populations beyond LC.

Dendritic cells express hematopoietic prostaglandin D synthase and function as a source of prostaglandin D2 in the skin.

Prostaglandin D2 (PGD2), an arachidonic acid metabolite, has been implicated in allergic responses. A major source of PGD2 in the skin is mast cells that express hematopoietic PGD synthase (H-PGDS). In this study, we show the expression of H-PGDS in human dendritic cells (DCs) and the regulatory mechanisms by which DCs produce PGD2. We detected H-PGDS in epidermal Langerhans cells, dermal DCs, plasmacytoid DCs, and myeloid DCs. Monocyte-derived DCs rapidly secreted PGD2 when stimulated with the calcium ionophore A23187. More importantly, pretreatment of monocyte-derived DCs with PMA (phorbol 12-myrisate 13-acetate) synergistically enhanced the rapid PGD2 secretion induced by A23187, whereas PMA alone did not induce PGD2 secretion. Lipopolysaccharide (LPS) reduced H-PGDS expression, but interferon-gamma followed by LPS induced significant PGD2 production in a delayed time course at 6 hours. This effect was associated with inhibition of LPS-induced H-PGDS reduction. Interestingly, an irritant compound, SDS, also induced a rapid PGD2 release. PGD2 synergistically enhanced CCL22/macrophage-derived chemokine synthesis in interferon-gamma-treated human keratinocytes. In addition, bone marrow-derived DCs from wild-type mice stimulated lymph node cells to produce higher amounts of interleukin-17 than did DCs from mice lacking the H-PGDS gene. Thus, DCs could be an important source of skin PGD2 and may mediate or regulate skin inflammation by releasing PGD2 in response to various stimuli, contributing to the innate and/or acquired immune responses.

CD39 is the dominant Langerhans cell-associated ecto-NTPDase: modulatory roles in inflammation and immune responsiveness.

CD39, the endothelial ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), regulates vascular inflammation and thrombosis by hydrolyzing ATP and ADP. Although ecto-NTPDase activities have been used as a marker of epidermal dendritic cells (DCs) known as Langerhans cells, the identity and function of these activities remain unknown. Here we report that Langerhans cells in CD39-/- mice express no detectable ecto-NTPDase activity. Irritant chemicals triggered rapid ATP and ADP release from keratinocytes and caused exacerbated skin inflammation in CD39-/- mice. Paradoxically, T cell-mediated allergic contact hypersensitivity was severely attenuated in CD39-/- mice. As to mechanisms, T cells increased pericellular ATP concentrations upon activation, and CD39-/- DCs showed ATP unresponsiveness (secondary to P2-receptor desensitization) and impaired antigen-presenting capacity. Our results show opposing outcomes of CD39 deficiency in irritant versus allergic contact dermatitis, reflecting its diverse roles in regulating extracellular nucleotide-mediated signaling in inflammatory responses to environmental insults and DC-T cell communication in antigen presentation.

Identification of the molecular target for the suppression of contact hypersensitivity by ultraviolet radiation.

This study was conducted to explore the involvement of DNA damage in the suppression of contact hypersensitivity (CHS) by UV irradiation. The opossum, Monodelphis domestica, was used because cells of these marsupials have an enzyme that is activated by visible light (photoreactivating enzyme) and repairs ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA. A single dose of 1,500 J/m2 of UVB (280-320 nm) radiation, representing 2 minimal erythema doses, was administered to the dorsal skin of opossums. This treatment prevented the opossums from developing a CHS response to dinitrofluorobenze (DNFB) applied either at the site of irradiation or an unirradiated site. In addition, this dose of UVR decreased the number of ATPase+ epidermal Langerhans cells in the dorsal epidermis to approximately 3% of that in unirradiated skin at the time of DNFB application. Treatment of the animals with wavelengths that activate the repair enzyme (320-500 nm, photoreactivating light, PRL) for 120 min immediately after UV irradiation inhibited the UVR-induced suppression of CHS almost completely. Exposure to PRL before UVR did not prevent UVR-induced suppression of CHS. PRL treatment after UV irradiation also prevented the decrease in the number of ATPase+ Langerhans cells. Measurements of lesions in DNA indicated that PRL treatment removed around 85% of the UVR-induced pyrimidine dimers. These data provide direct evidence that DNA, and most likely, the pyrimidine dimer, is the primary molecular target for the UVB-induced suppression of contact hypersensitivity to haptens applied to irradiated or unexposed skin.

Size-dependent cytotoxic effects of amorphous silica nanoparticles on Langerhans cells.

Amorphous silica nanoparticles (nSPs), are widely used in medicines, cosmetics and food. However, due to their reduced particle size they are suspected to pose new risks induced by changes in biological reactivity and kinetics, which differ from those of bulk materials. In a previous study, we showed that silica particles with a diameter of 70 nm penetrated the stratum corneum (SC) of mouse skin and were taken up by living cells such as keratinocytes and Langerhans cells. To clarify the relationship between particle size, distribution and cellular response, we have evaluated size-dependent intracellular localization and cytotoxicity of silica particles, using the mouse epidermal Langerhans cell line XS52. On treatment with silica particles of diameters 70, 300, and 1000 nm, cellular uptake and cytotoxicity increased with reduction in particle size. These results suggest that smaller sized silica particles induced greater cytotoxicity against Langerhans cells, which was correlated with the quantity of particle uptake into the cells.

Low-frequency low-intensity ultrasounds do not influence the survival and immune functions of cultured keratinocytes and dendritic cells.

Low-frequency ultrasounds (US) are used to enhance drug transdermal transport. Although this phenomenon has been extensively analyzed, information on US effects on the single skin cell components is limited. Here, we investigated the possible effects of low-frequency US on viability and immune functions of cultured human keratinocytes and dendritic cells (DC), skin cells involved in the regulation of many immune-mediated dermatoses. We demonstrated that US, employed at low-frequency (42 KHz) and low-intensity (0.15 W/cm(2)) values known to enhance drug and water transdermal transport, did not affect extracellular-signal-regulated-kinase (ERK)1/2 activation, cell viability, or expression of adhesion molecules in cultured keratinocytes. Moreover, US at these work frequency and intensity did not influence the keratinocyte expression and release of immunomodulatory molecules. Similarly, cultured DC treated with low-frequency low-intensity US were viable, and did not show an altered membrane phenotype, cytokine profile, nor antigen presentation ability. However, intensity enhancement of low-frequency US to 5 W/cm(2) determined an increase of the apoptotic rate of both keratinocytes and DC as well as keratinocyte CXCL8 release and ERK1/2 activation, and DC CD40 expression. Our study sustains the employment of low-frequency and low-intensity US for treatment of those immune skin disorders, where keratinocytes and DC have a pathogenetic role.

The diverse functions of Src family kinases in macrophages.

Macrophages are key components of the innate immune response. These cells possess a diverse repertoire of receptors that allow them to respond to a host of external stimuli including cytokines, chemokines, and pathogen-associated molecules. Signals resulting from these stimuli activate a number of macrophage functional responses such as adhesion, migration, phagocytosis, proliferation, survival, cytokine release and production of reactive oxygen and nitrogen species. The cytoplasmic tyrosine kinase Src and its family members (SFKs) have been implicated in many intracellular signaling pathways in macrophages, initiated by a diverse set of receptors ranging from integrins to Toll-like receptors. However, it has been difficult to implicate any given member of the family in any specific pathway. SFKs appear to have overlapping and complementary functions in many pathways. Perhaps the function of these enzymes is to modulate the overall intracellular signaling network in macrophages, rather than operating as exclusive signaling switches for defined pathways. In general, SFKs may function more like rheostats, influencing the amplitude of many pathways.

Characterization of langerhans cells in epidermal sheets along the body of Armadillo (Dasypus novemcinctus).

Armadillos are apparently important reservoirs of Mycobacterium leprae and an animal model for human leprosy, whose immune system has been poorly studied. We aimed at characterizing the armadillo's langerhans cells (LC) using epidermal sheets instead of tissue sections, since the latter restrict analysis only to cut-traversed cells. Epidermal sheets by providing an en face view, are particularly convenient to evaluate dendritic morphology (cells are complete), spatial distribution (regular vs. clustered), and frequency (cell number/tissue area). Lack of anti-armadillo antibodies was overcome using LC-restricted ATPase staining, allowing assessment of cell frequency, cell size, and dendrites extension. Average LC frequency in four animals was 528 LC/mm(2), showing a rather uniform non-clustered distribution, which increased towards the animal's head, while cell size increased towards the tail; without overt differences between sexes. The screening of antibodies to human DC (MHC-II, CD 1a, langerin, CD86) in armadillo epidermal sheets, revealed positive cells with prominent dendritic morphology only with MHC-II and CD86. This allowed us to test DC mobilization from epidermis into dermis under topical oxazolone stimulation, a finding that was corroborated using whole skin conventional sections. We hope that the characterization of armadillo's LC will incite studies of leprosy and immunity in this animal model.

The pro- and anti-inflammatory properties of human antigen-presenting cells expressing the high affinity receptor for IgE (Fc epsilon RI).

Almost 20 years after the first description of IgE on the surface of epidermal Langerhans cells (LC) and the subsequent characterization of the trimeric Fc epsilon RI on human antigen-presenting cells (APC), we have gained profound insights into the receptor responsible for this binding. Fc epsilon RI may act as a pro-inflammatory structure on some APC such as inflammatory dendritic epidermal cells (IDEC) in the skin of patients with atopic dermatitis while it can also be an important instrument in mechanisms leading to tolerance on other APC such as LC of the oral mucosa. By virtue of Fc epsilon RI, APC can initiate inflammation by secretion of a wide spectrum of pro-inflammatory cytokines and chemokines. Fc epsilon RI+DC can induce either Th2 or Th1 profile in T-cells. In contrast, the production of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) as well as IL-10 and TGFss may contribute to the tolerogenic properties of DC.

Alterations in Langerhans cells and Thy-1+ dendritic epidermal cells in murine epidermis during the evolution of ultraviolet radiation-induced skin cancers.

To understand the role of cutaneous immune cells in host resistance to the induction and growth of skin cancer, we investigated the number and morphology of murine dendritic epidermal cells (dEC) during the evolution of ultraviolet (UVA) UV-induced skin cancers. Female C3H/HeN mice were treated topically with 8-methoxypsoralen followed by ultraviolet A (UVA) radiation 3 times/week or irradiated with UVB radiation 3 times/week. In both psoralen plus UVA- and UVB-treated mice, ATPase+ and Ia+ Langerhans cells almost completely disappeared from the treated skin during the early latency period of tumor development (4 weeks) but reappeared in the epidermis late in the latency period (between 15 and 22 weeks). The ATPase+ cells that reappeared in the epidermis had a rounder, less dendritic morphology than normal Langerhans cells. Thy-1+ dEC were totally depleted from the epidermis in both treatment groups at the end of first week of treatment and were nearly absent from the skin during the entire latency period. After tumors appeared (29 weeks), Thy-1+ dEC were still absent or detected only in small numbers in skin surrounding the tumors. ATPase+ and Ia+ cells present in skin around the tumors constituted 60 to 80% of the number in nonirradiated skin. Mice that received UVA radiation alone developed no tumors. ATPase+ and Ia+ Langerhans cells and Thy-1+ dEC were detected in UVA-treated epidermis after 22 weeks and 43 weeks, although the numbers were lower than those in unirradiated mice. Most psoralen plus UVA-induced tumors (81%) were squamous cell carcinomas, whereas only 24% of UVB-induced tumors were of this histological type. Our results demonstrate that UV-induced skin cancers developed in the presence of ATPase+ and Ia+ cells in the epidermis and in the absence of Thy-1+ dEC.

The Langerhans cell.

In all mammalian species so far examined, Langerhans cells or their precursors are the only epidermal cells expressing Ia antigens or their equivalents. In man, xeno-antisera raised in rabbits against purified B-lymphocyte cell membrane antigens were utilized to stain the Langerhans cells by either fluorescent or immunoferritin methods. As high proportion of the indeterminate cells in the epidermis also expressed HLA-DR antigens, and a relationship to Langerhans cells is suggested. Confirmation of these results was obtained in mouse. Alloantisera raised against I-A and I-EC subregion products again stained only Langerhans cells. Fluorescent, immunoperoxidase, and immunoferritin methods were used and confirmation of the specificity of the reaction was achieved at the electron microscope level. Langerhans cells were shown by ATPase staining to be absent from the epithelium of the central cornea, but present in the limbus. Population of the entire corneal epithelium surface was induced by application of irritants or contact sensitizing agents such as DNCB. Grafting of corneas either deficient or populated with Langerhans cells, to skin beds, may answer the question of the influence of such cells on allograft rejection.

Implication of the MAPK pathways in the maturation of human dendritic cells induced by nickel and TNF-alpha.

After application of haptens to the skin, immature dendritic cells (DC), also named Langerhans cells (LC), come into a maturation process, which include disappearance of specific molecules such as E-cadherin and Langerin and the expression of new molecules such as CD83, CD86 and CCR7. The involvement of p38 MAPK in DC maturation induced by haptens and TNF-alpha has already been shown, however, the role of the other MAPK, ERK and JNK, is less described. In this study, we demonstrated on human CD34(+)-derived DC that the three MAPK are participating to the expression of CD83, CD86 and CCR7 induced by nickel (NiSO(4)) but not to the down-regulation of E-cadherin and Langerin. In contrast, following TNF-alpha stimulation, only p38 MAPK is involved in CD83 and CCR7 expressions and ERK inhibits DC maturation while JNK inhibition had no effect. Our results also suggest that activation of p38 MAPK by TNF-alpha could partially suppress ERK activation and abrogates the inhibitory effect of ERK on DC maturation. In summary, the three MAPK pathways regulate DC maturation induced by haptens while only p38 MAPK seems to play a key role after TNF-alpha addition.

Photoreversal of the ultraviolet radiation-induced disappearance of ATPase-positive Langerhans cells in the epidermis of Monodelphis domestica.

The present study was undertaken to explore the possible causes of ultraviolet radiation (UVR)-induced disappearance of ATPase-positive, epidermal Langerhans cells (LC). Monodelphis domestica was used because it has the capacity for photoreactivation of UVR-induced pyrimidine dimers in epidermal DNA. Single, 330 J/m2 (ears) or 500 J/m2 (back) UVR exposures (FS-40 sunlamps) reduced the numbers of ATPase-positive epidermal LC in M. domestica ears to approximately 15% of those in unirradiated ears and approximately 37% of those in unirradiated dorsal skin. Immediate 90-minute exposures to photoreactivating light (PRL, 320-400 nm) post-UVR reversed the effects of UVR, resulting in ATPase-positive LC numbers not being significantly different from controls. Exposure to PRL immediately preceeding UVR did not prevent ATPase-positive LC disappearance. The photoreactivation of UVR-induced ATPase-positive LC disappearance indicates that DNA damage (pyrimidine dimers) is involved in the loss of ATPase-positive LC.

Segregation of two glutaminase isoforms in islets of Langerhans.

Despite the importance of glutamatergic signalling in the co-ordination of hormone secretion, the identity of the enzyme for the production of glutamate in beta-cells is still unresolved. We have found that the endocrine pancreas co-expresses two isoforms of GA (glutaminase), denoted as kidney-type (KGA) and liver-type (LGA), with a complementary cellular pattern of expression. Whereas KGA was mainly present in alpha-cells, LGA was very abundant in beta-cells. This spatial segregation may have important functional implications, facilitating a differential regulation of glutamate production in insulin- and glucagon-secreting cells.

Initial human myeloid/dendritic cell progenitors identified by absence of myeloperoxidase protein expression.

Myeloperoxidase (MPO) represents an early-appearing and highly reliable intracellular myeloid lineage marker molecule. MPO protein can be detected in a subset of human hematopoietic bone marrow progenitor cells and in granulomonopoietic (GM) cells. However, other myeloid-related cell types such as epidermal Langerhans-type dendritic cells (LC) lack MPO. Therefore, human myeloid progenitors might be subdivided based on MPO protein expression into functional subsets. Here we identified two consecutive myelopoietic cell stages, i.e., early myeloid progenitors that lack MPO, as well as their immediate MPO+ progeny. MPO- myeloid progenitors possess previously described granulomonocyte (GM) progenitor-associated cell-surface characteristics (CD34+CD45RA+CD13+lin-). They are specifically recruited and can be expanded in cultures of CD34+ cord blood cells in response to early-acting hematopoietic cytokines. Furthermore, cell fractions enriched in MPO- myeloid progenitors efficiently developed along Langerhans-type dendritic cell (LC) and granulomonocytic (GM) lineages, whereas progeny enriched in MPO+ cells showed diminished LC potential. In line with this, peripheral blood progenitors, known to possess LC differentiation potential, lacked MPO expression. We conclude that differential expression of MPO therefore further characterizes cells with myeloid or LC potential.