Mast cells,neuropeptides, histamine,and prostaglandins in UV-induced systemic immunosuppression

There is a direct correlation between dermal mast cell prevalence in dorsal skin of different mouse strains and susceptibility to UVB-induced systemic immunosuppression; highly UV-susceptible C57BL/6 mice have a high dermal mast cell prevalence while BALB/c mice, which require considerable UV radiation for 50% immunosuppression, have a low mast cell prevalence. There is also a functional link between the prevalence of dermal mast cells and susceptibility to UVB- and cis-urocanic acid (UCA)-induced systemic immunosuppression. Mast cell-depleted mice are unresponsive to UVB or cis-UCA for systemic immunosuppression unless they are previously reconstituted at the irradiated or cis-UCA-administered site with bone marrow-derived mast cell precursors. cis-UCA does not stimulate mast cell degranulation directly. Instead, in support of studies showing that neither UVB nor cis-UCA was immunosuppressive in capsaicin-treated, neuropeptide-depleted mice, cis-UCA-stimulated neuropeptide release from sensory c-fibers which, in turn, could efficiently degranulate mast cells. Studies in mice suggested that histamine, and not tumor necrosis factor alpha (TNF-alpha), was the product from mast cells that stimulated downstream immunosuppression. Histamine receptor antagonists reduced by approximately 60% UVB and cis-UCA-induced systemic immunosuppression. Indomethacin administration to mice had a similar effect which was not cumulative with the histamine receptor antagonists. Histamine can stimulate keratinocyte prostanoid production. We propose that both histamine and prostaglandin E(2) are important in downstream immunosuppression; both are regulatory molecules supporting the development of T helper 2 cells and reduced expression of type 1 immune responses such as a contact hypersensitivity reaction.     

Cutaneous mast cell depletion and recovery in murine graft-vs-host disease

Mast cells as studied by light microscopy with metachromatic staining, have been noted to "disappear" from the skin of mice with chronic graft-vs-host disease (GVHD) produced across minor histocompatibility barriers. This mast cell disappearance is accompanied by ultrastructural evidence of loss of granule contents. In this study, we followed cutaneous mast cells in chronic GVHD over 9 mo by three methods: Light microscopy of toluidine blue-stained sections showed that mast cells not seen at day 42 reappeared between days 94 and 125, were supramaximal at days 146 and 164, and returned to normal levels at days 195 and 280. Double immunofluorescent staining of mast cells for the presence of surface IgE receptors and cytoplasmic granules (avidin) revealed IgE receptor-bearing cells that lacked avidin-binding granules at the time when mast cells were not apparent on light microscopy. By electron microscopy, reappearing mast cells have the morphology of immature dermal mast cells. Ultrastructural abnormalities of mast cells persist some 150 days after GVHD induction. The possible relationship of these mast cell changes to the development of dermal fibrosis in chronic GVHD is discussed.     

Mast cell chymase regulates dermal mast cell number in mice

Chymase inhibitor reduced the increase in the number of dermal mast cells in 2,4-dinitrofluorobenzene-induced dermatitis in a dose-dependent manner. Intradermal injection of human chymase to mouse ear significantly increased histamine content, the marker for mast cell number in the skin. These results suggest that chymase released by mast cells may participate in local mast cell accumulation in a positive feedback fashion. Immunohistochemical analysis revealed that the intradermal injection of chymase reduces expression of stem cell factor (SCF) on surface of the skin keratinocytes. In addition, incubation of human keratinocytes with chymase in vitro resulted in release of SCF into the culture medium. Since soluble SCF is thought to regulate mast cell number, the chymase-induced mast cell accumulation may occur via the ability of chymase to process membrane-bound SCF on the epidermal keratinocytes.     

Increase in CD30 ligand/CD153 and TNF-α expressing mast cells in basal cell carcinoma

Mast cells are a significant source of tumor necrosis factor (TNF) superfamily members, such as TNF-α, CD30 ligand/CD153 (CD30L) and CD40L/CD154. Furthermore, the expression of some of these proteins in mast cells has been associated with tumorigenesis, and mast cells have been found to be increased in number in the basal cell carcinoma (BCC) lesion. In this study, we have examined the expression of TNF-α, CD30L and CD40L immunoreactivity in mast cells in the healthy-looking skin and lesional skin of ten patients with superficial spreading BCC. Also, the counterparts of these molecules, TNF receptor (TNFR) I and II as well as CD30 and CD40, were analysed immunohistochemically. We found that numbers of mast cells and Kit-positive cells were significantly increased in the dermal BCC lesion. The percentage of CD30L-positive mast cells and the number of CD30-positive cells were significantly increased in the upper dermis of the BCC lesion as well. In addition, the numbers of TNF-α-positive mast cells and cells with TNFRI and TNFRII were markedly increased in the upper lesional dermis. In contrast, no mast cells positive for CD40L could be detected, even though the lesional dermis contained increased numbers of CD40 positive cells. The BCC epithelium was positive for TNFRI, TNFRII and CD40, but not for CD30, though the larger basal buds appeared to be less intensely stained for TNFRI and CD40. In conclusion, mast cells positive for CD30L and TNF-α, but not CD40L, are increased in number in the lesional dermis in BCC. These data suggest plausible pathways whereby mast cells can be activated and to interact with other cells and thereby contribute to the tumorigenesis in BCC.     

Effect of hemp fiber on UVB-induced epidermal cell proliferation and PCNA expression

Ultraviolet radiation commonly causes serious skin diseases, and skin cell death. The UVB-blocking effect of hemp fabric which known to be a powerful agent against UVB was evaluated using mouse auricle skin. Based on UVB irradiation and the use of different fabrics, four mouse groups were evaluated in this study: two experimental groups, Group 1 (UVB exposed hemp fabric-shield site, EHFS), Group 2 (UVB exposed polyester fabric-shield site, EPFS), and two control groups, Group 3 (UVB exposed non-fabric-shield site, ENFS), and Group 4 (UVB unexposed non-fabric-shield site, UNFS). Except for UNFS all samples were exposed to UVB for 28 h and showed clear histologic changes in epidermis and dermis. After 45 h chronic irradiation, epidermal thickness was doubled in EHFS, roughly tripled in EPFS, and more than quadrupled in ENFS over that of UNFS. Based on the thickness of the altered epidermis, the blocking effect of hemp fabric was 50% higher than that of polyester fabric. Additionally, immunohistochemical analysis revealed expression of proliferating cell nuclear antigen (PCNA) in ENFS and EPFS throughout the hyperplasia of keratinocytes and sebocytes. After 45 h irradiation, the sebocytes of sebaceous glands, observed in sectioned images, increased on average to 14 cells in ENFS, 9 cells in EPFS, and 7 cells in EHFS, as compared to 8 cells in UNFS. In contrast, the cell area of adipose tissue was decreased by half in EHFS, one-fourth in EPFS, and one-tenth in ENFS, and mostly replaced with fibroblasts and other supporting cells. These results suggest that UVB irradiation directly affects epidermal and dermal tissues, and induces abnormal proliferation of keratinocytes and hyperplasia of sebocytes consuming fats in adipose tissue. For skin health, hemp fabric is a better material for protecting the skin against UVB than polyester fabric.     

Mast cell heterogeneity in normal man

Thermographic analysis of the skin of the forearm of normal men submitted to nasal instillation of 1 ml of a solution of 48/80, 1 X 10(-2), demonstrates that the skin vessels undergo local vasodilatation. Erythema and wheals sometimes appear, due to the stimulation of the dermal mast cells. Mast cells of the nasal mucosa are never stimulated by such instillation. The differences between dermal and nasal mast cell reactions are in accordance with the concept of mast cell heterogeneity.     

Single-locus control of the mast cell population in mouse skin

The number of mast cells in connective tissue from dorsal skin varied markedly among mouse strains. Inbred strains of mice were typed into three groups, high (NC and NZB mice), low (B6, B10, and BALB/c mice), and intermediate (C3H/He and DBA/2 mice), by their mast cell content in the skin. However, the strain differences in the number of mast cells was marginal at the age of weaning but became distinct with age. This could be explained mainly by the frequently observed clustering of mast cells in adult NC and NZB mice and the rarely observed clustering in younger mice as well as in adult B10 and BALB/c mice. The breeding experiment revealed that the difference in the number of mast cells between NC and B10 mice was controlled by a single autosomal dominant locus, for which we propose the designation Mcr (mast cell regulator). The role of the Mcr locus with regard to the frequency of the mast cell population in connective tissue is discussed.     

Ultraviolet B radiation induces a transient appearance of IL-4+ neutrophils, which support the development of Th2 responses

UVB irradiation can cause considerable changes in the composition of cells in the skin and in cutaneous cytokine levels. We found that a single exposure of normal human skin to UVB induced an infiltration of numerous IL-4(+) cells. This recruitment was detectable in the papillary dermis already 5 h after irradiation, reaching a peak at 24 h and declining gradually thereafter. The IL-4(+) cells appeared in the epidermis at 24 h postradiation and reached a plateau at days 2 and 3. The number of IL-4(+) cells was markedly decreased in both dermis and epidermis at day 4, and at later time points, the IL-4 expression was absent. The IL-4(+) cells did not coexpress CD3 (T cells), tryptase (mast cells), CD56 (NK cells), and CD36 (macrophages). They did coexpress CD15 and CD11b, showed a clear association with elastase, and had a multilobed nucleus, indicating that UVB-induced infiltrating IL-4(+) cells are neutrophils. Blister fluid from irradiated skin, but not from control skin, contained IL-4 protein as well as increased levels of IL-6, IL-8, and TNF-alpha. In contrast to control cultures derived from nonirradiated skin, a predominant type 2 T cell response was detected in T cells present in primary dermal cell cultures derived from UVB-exposed skin. This type 2 shift was abolished when CD15(+) cells (i.e., neutrophils) were depleted from the dermal cell suspension before culturing, suggesting that neutrophils favor type 2 T cell responses in UVB-exposed skin.     

Effects of ultraviolet light on melanocyte differentiation: studies with mouse neural crest cells and neural crest-derived cell lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L-3,4-dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT- or DOPA-positive cells between the UV-irradiated cultures and the non-irradiated cultures. We then examined the effects of UV light on KIT-positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase-positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with alpha-melanocyte-stimulating hormone (alpha-MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase-negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal-regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as alpha-MSH and/or endothelin-1.     

Lectin histochemistry of the mast cell: Heterogeneity of rodent and human mast cell populations

Summary Mast cell granules contain a variety of N-linked saccharides. Heterogeneity of the expression of these saccharides in mast cells was studied in rodent and human tissues which were so selected as to contain all the mast cell subsets previously identified using other criteria. Dermal and intestinal mucosal mast cells were stained with lectins using an avidin-biotin system. It was found that dermal and subepidermal mast cells in the rat and mouse, and mucosal and dermal human mast cells showed very similar lectin binding properties to each other, with a fine variation in the inlensity of staining with certain lectins. Rat mucosal mast cells, however, showed a distinctive pattern of lectin binding which was not seen in mast cells from any other tissue studied. The chemical basis of the differences seen were deduced and the possible significance of these structural variations is discussed.     

IL-4 modulates the histamine content of mast cells in a mast cell/fibroblast co-culture through a Stat6 signaling pathway in fibroblasts

IL-4 plays a crucial role in the pathogenesis of allergic diseases, such as the induction of IgE synthesis and the development of mast cells. To further understand the effect of IL-4 on mast cells in skin, we utilized a mast cell/fibroblast co-culture system as an in vitro model of dermal mast cells. IL-4 induced mast cell growth in the culture with fibroblasts. Immunoblot analysis revealed that IL-4 activated Stat6 in both mast cells and fibroblasts. The over-expression of dominant-negative Stat6 in fibroblasts in the presence of IL-4 decreased the histamine content per mast cell, but not the number of mast cells. In contrast, the over-expression of constitutively-active Stat6 in fibroblasts increased the histamine content per mast cell, indicating that the activation of Stat6 in fibroblasts supports the maturation of mast cells co-cultured with fibroblasts.     

Histamine release from mouse and rat mast cells cultured with supernatants from chronic murine graft-vs-host splenocytes.

There is growing interest in studying pathways of mast cell activation. In a mouse model of chronic graft-vs-host disease (cGVHD) extensive mast cell activation and degranulation occurs in vivo coincident with the development of dermal fibrosis. An interesting feature of this model is that the mast cell reaction is slow to develop, occurring over a period of weeks and waning by 300 days. The aim of our work was to investigate the effects of supernatants from splenocytes of such cGVHD mice (cGVHD sups) on mouse and rat peritoneal mast cells cocultured with 3T3 skin fibroblasts. We found that cGVHD sups are able to release histamine from both mouse and rat cultured mast cells in a slow fashion. Histamine release became evident only after 5-8 days of coculture of the mast cells with the cGVHD supernatants and thereafter decreased to basal levels. Mast cell activation due to cGVHD supernatants was a noncytotoxic event as demonstrated by mast cell counts in the cocultures and by the ability of mast cells to exclude trypan blue. Mast cells that had been activated by incubation with the cGVHD sups were as responsive to stimulation with either anti-IgE antibodies or compound 48/80 as were mast cells incubated with control sups. Supernatants from mice early in GVHD (Days 11-28) were most active in promoting histamine release. Supernatants from spleens of mice which had GVHD for 290 days and where the mast cells had returned to full granulation in vivo were inactive. This is the first in vitro study demonstrating slow mast cell histamine release instituted by other cells, namely the splenocytes of cGVHD mice.     

Chronic graft-versus-host disease as a model for scleroderma. II. Mast cell depletion with deposition of immunoglobulins in the skin and fibrosis

We explored the pathologic changes in the skin of mice undergoing a chronic graft-versus-host (GVH) reaction. In rodents and in man, chronic GVH includes the deposition of excess collagen in the skin-a reaction which resembles idiopathic scleroderma. GVH disease across minor histocompatibility barriers was produced by injecting B10.D2 cells into irradiated BALB/c mice. These strains are identical at the H-2 and Mls loci but differ in minor histocompatibility antigens. Control BALB/c mice received irradiation and BALB/c cells. Serial skin biopsies were taken and studied for histological changes characteristic of chronic GVHD, for mast cell density, and for the deposition of immunoreactants. GVHD was produced in B10.D2----BALB/c mice as measured by body weight loss and the production of skin changes including dermal fibrosis, loss of fat and appendages, and a mononuclear cell infiltrate. Dermal mast cells, assessed by toluidine blue staining, were normal at Day 11, but had disappeared by Days 21-63 and returned to normal by Day 104. Immunoglobulins IgG, IgA, and IgM appeared at the dermo-epidermal junction and along the basement membrane zone of hair follicles. This deposition was maximal at Day 42 and waned thereafter. Thus the appearance of immunoglobulins in the skin was maximal when mast cell staining was minimal. The changes in this GVHD model leading to a scleroderma-like picture in the skin are compatible with an immune etiology for the fibrosis. Vasodilation following liberation of mast cell mediators would facilitate the deposition of immunoglobulins. The disappearance of mast cell staining may be caused by extensive degranulation. We postulate an interaction between GVHD-activated T cells, mast cell stimulation, fibroblast activation, and fibrosis.     

Conjugated avidin binds to mast cell granules

The glycoprotein, avidin, conjugated either to the enzyme horseradish peroxidase, or to the fluorochrome dyes, fluorescein or rhodamine, identifies the granules of mast cells in both tissues and cell suspensions. In the absence of prior fixation, mast cells were not identified with conjugated avidin; however, granules released from these cells were stained with this labeled glycoprotein. The specificity of avidin for mast cells was confirmed by the absence of conjugated avidin-positive cells in the skin of mice (S1/S1d) deficient in mature dermal mast cells. Electron microscopic studies confirmed that avidin binds specifically to individual mast cell granules rather than to other cellular structures. Rodent and human mast cells were readily stained with avidin conjugated to horseradish peroxidase or to either of the fluorochrome dyes. The conjugated avidin staining technique is a reliable and simple method for identifying rodent and human mast cells, one that is useful as both an investigative and a clinical tool.     

Absence of BRAF Gene Mutation in Non-Melanoma Skin Tumors

Basal cell carcinoma (BCC) is the most common skin cancer, and its incidence is increasing. It was proposed that the RAS oncogene significantly contributes to skin cancer development. Numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines. For the first time, in the present study, tumor biopsy specimens from 78 patients with BCC were screened for BRAF mutation within exons 11 and 15. Our results indicate that the BRAF gene does not appear to be frequently mutated in non-melanoma skin tumors such as BCC. These data suggest that other gene alterations may cause tumor development.     

YAP and TAZ are essential for basal and squamous cell carcinoma initiation

YAP and TAZ are key downstream regulators of the Hippo pathway, regulating cell proliferation and differentiation. YAP and TAZ activation has been reported in different cancer types. However, it remains unclear whether they are required for the initiation of major skin malignancies like basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Here, we analyze the expression of YAP and TAZ in these skin cancers and evaluate cancer initiation in knockout mouse models. We show that YAP and TAZ are nuclear and highly expressed in different BCC types in both human and mice. Further, we find that cells with nuclear YAP and TAZ localize to the invasive front in well‐differentiated SCC, whereas nuclear YAP is homogeneously expressed in spindle cell carcinoma undergoing EMT. We also show that mouse BCC and SCC are enriched for YAP gene signatures. Finally, we find that the conditional deletion of YAP and TAZ in mouse models of BCC and SCC prevents tumor formation. Thus, YAP and TAZ are key determinants of skin cancer initiation, suggesting that targeting the YAP and TAZ signaling pathway might be beneficial for the treatment of skin cancers.     

Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W/Wv mice: evidence that a common precursor can give rise to both "connective tissue-type" and "mucosal" mast cells

We investigated the issue of mast cell heterogeneity by cloning mast cell colonies from peritoneal cells in methylcellulose, injecting the cloned cells into the skin and stomach of mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice, and staining the mast cells that developed in these sites with Berberine sulfate, a fluorescent dye that identifies heparin-containing mast cells. When peritoneal cells of nontreated WBB6F1-+/+ mice were plated in methylcellulose containing pokeweed mitogen-stimulated spleen cell conditioned medium, pure mast cell colonies developed. In contrast, the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice lacked the progenitor cells that made mast-cell colonies. The clonal nature of the mast cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when mixture of peritoneal cells of C57BL/6-bgJ/bgJ mice and C57BL/6-+/+ mice were plated, all of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. Individual mast c 11 colonies of WBB6F1-+/+ mouse origin were divided into two parts; one part was directly injected into the wall of the glandular stomach of a WBB6F1-W/Wv mouse, and another part was injected into the skin of the same W/Wv mouse. Injections of 14 of 46 such colonies resulted in development of mast cells in both the "connective tissues" (skin or stomach muscle or both) and the stomach mucosa. Mast cells in the connective tissues were stained with Berberine-sulfate, indicating that they contained heparin, whereas mast cells in the stomach mucosa were not. These results suggest that a single precursor cell can give rise to both "connective tissue-type" and "mucosal" mast cells.     

Mast cell-dependent down-regulation of antigen-specific immune responses by mosquito bites

While probing host skin to search for blood vessels, the female Anopheles mosquito delivers Plasmodium parasites in the presence of saliva. Saliva from various blood-feeding vectors which contains several pharmacologically active components is believed to facilitate blood feeding as well as parasite transmission to the host. Recently, we found that mosquito saliva has the capacity to activate dermal mast cells and to induce local inflammatory cell influx. Our main objective in the present work is to investigate whether saliva, through mosquito bites, controls the magnitude of Ag-specific immune responses and whether this control is dependent on the mast cell-mediated inflammatory response. Using a mast cell knockin mouse model, we found that mosquito bites consistently induced MIP-2 in the skin and IL-10 in draining lymph nodes, and down-regulate Ag-specific T cell responses by a mechanism dependent on mast cells and mediated by IL-10. Our results provide evidence for new mechanisms which may operate during Plasmodium parasite transmission by mosquito bites.     

Differential Effects of Doses and Forms of Dietary Selenium on Immune Cell Numbers in the Skin of Ultraviolet-irradiated and Unirradiated Mice

The effect of three different doses of dietary l-selenomethionine (SM) and sodium selenite (SS) on skin selenium (Se) content, glutathione peroxidase (GPx) activity, Langerhans cell (LC) and mast cell numbers in ultraviolet radiation-B (UVB)-irradiated and unirradiated C3H/HeN mice was determined. After weaning, groups of mice were given Se-deficient, Se-adequate, or Se-high diets. Six weeks later, some animals in each group were exposed to a single UVB dose (acute), while others were exposed three times weekly for the following 40 weeks (chronic). The skin Se content and GPx activity increased in all the Se-supplemented groups, and the latter was not altered by UVB exposure. Generally, the Se-containing diets caused an increase in LC numbers at 6 weeks and a further rise at 40 weeks, but did not prevent the loss induced by acute or chronic UVB radiation. Skin mast cell numbers were highest in animals fed the Se-deficient diet after 6 and 40 weeks. Acute and chronic UVB radiation decreased the mast cell number and dietary Se did not prevent the reduction. While the present study shows that Se plays an important role in governing the number of LCs and mast cells in the skin, no protective effect against the immunomodulating properties of UVB radiation on these cell types was observed. However, this conclusion may only apply to the experimental conditions chosen, and additional studies at different Se dosages and reduced intensities of chronic UVB exposure are required to confirm the results.     

Mast cell "disappearance" in chronic murine graft-vs-host disease (GVHD)-ultrastructural demonstration of "phantom mast cells"

Mast cells were studied during the induction of chronic graft-vs-host disease (GVHD) induced in mice across minor histocompatibility barriers. B10.D2 spleen cells (or control BALB/c cells) were injected into irradiated (600 rad) BALB/c recipients. Serial skin biopsies were taken over 26 days, during which time changes occurred resembling scleroderma, namely, dermal fibrosis, a mononuclear cell infiltrate, and loss of fat and appendages. Mast cells, when stained with toluidine blue, "disappeared" from GVHD, but not from control skin. Ultrastructural analysis showed that mast cells in GVHD skin were indeed present but underwent degranulation. Some mast cells showed only pale expanded sacs, indicating granule depletion. Because these cells could not be seen by toluidine blue staining but were plainly present, we have called them "phantom mast cells." Cellular activation occurred in many GVHD mast cells as shown by increased cytoplasmic activity, with numerous Golgi complexes, ribosomes, granular endoplasmic reticulum, and small vesicles. No identifiable mast cells were seen after day 19. No significant changes were seen in the mast cells of syngeneic control mice. We believe that immunologic processes in chronic GVHD cause a slow release of mast cell granule contents, which is different from anaphylactic degranulation. The depleted mast cells (invisible by toluidine blue staining) are also activated, perhaps in an attempt to replete their stores of granule contents. We discuss the relation of mast cell changes to fibrosis.