Mast cell lines HMC‐1 and LAD2 in comparison with mature human skin mast cells – drastically reduced levels of tryptase and chymase in mast cell lines

Please cite this paper as: Mast cell lines HMC‐1 and LAD2 in comparison with mature human skin mast cells – drastically reduced levels of tryptase and chymase in mast cell lines. Experimental Dermatology 2010; 19 : 845–847. Abstract: To circumvent the costly isolation procedure associated with tissue mast cells (MC), two human MC lines, i.e. HMC‐1 and LAD2, are frequently employed, but their relation to mature MC is unknown. Here, we quantitatively assessed their expression of MC markers in direct comparison to skin MC (sMC). sMC expressed all lineage markers at highest and HMC‐1 cells at lowest levels. LAD2 cells expressed comparable high‐affinity IgE receptor α (FcεRIα) and FcεRIγ but less FcεRIβ than sMC and displayed slightly reduced, but robust FcεRI‐mediated histamine release. Only minor differences were found for total histamine content and c‐Kit expression. Huge, and to this level unexpected, differences were found for MC tryptase and chymase, with sMC >>> LAD2 > HMC‐1. Taken together, HMC‐1 cells represent very immature malignantly transformed MC, whereas LAD2 cells can be considered intermediately differentiated. Because of the minute levels of MC proteases, MC lines can serve as surrogates of tissue MC to a limited degree only.     

A possible mechanism of mast cell proliferation in mastocytosis

The abnormality in mastocytosis is the excessive accumulation of mast cells in the affected tissue. The growth and differentiation of human mast cells are quite dependent on stem cell factor (SCF), the ligand for the protein products of c-kit. Recent studies have demonstrated that all adult patients examined so far carry c-kit point mutations, leading to SCF-independent autophosphorylation of the receptor and autonomous cell growth. On the other hand, typical pediatric patients have been found to bear no activating Asp816Val mutation in c-kit. Although most mastocytosis patients are children, the mechanism by which mast cells proliferate in these pediatric patients remains unclear. Recently, were reported that human mast cells obtained from adult skin could dramatically proliferate when cultured with SCF. From these experimental results, it is speculated that local excessive production of SCF results in the mast cell proliferation in pediatric patients.     

Non-neuronal kappa-opioid receptor activation enhances epidermal keratinocyte proliferation,and modulates mast cell functions in human skin ex vivo

Kappa-opioid receptor (KOR) activation reportedly elicits anti-inflammatory responses and can downregulate neuropeptide release from sensory nerve fibers. While this renders KOR agonists (KORAs) potentially interesting therapeutics in skin diseases associated with neurogenic inflammation, it remains poorly understood how KOR agonists impact on human skin and dermal mast cells (MCs) ex vivo, in the absence of functional innervation. The KORA 5a was administrated to the culture medium (200 nmol/L and 1 µmol/L) in human skin organ culture, thus mimicking a “systemic” mode of application. We show that KORA significantly increased epidermal thickness and upregulated the number and proliferation of epidermal keratinocytes. Unexpectedly, it also stimulated epidermal keratinocyte apoptosis in situ, compared with vehicle. Moreover, KORA significantly decreased the number of c-Kit-positive MCs, but did not significantly alter the number or degranulation of mature (tryptase- or toluidine blue-positive) MCs. These pilot observations render the tested KORA (5a) an interesting candidate for the management of inflammatory dermatoses in which MC-dependent neurogenic skin inflammation plays an important role (e.g. atopic dermatitis, psoriasis).     

The immunohistopathologic study of scabies by the PAP method--identification of IgE positive mast cells

Skin biopsy specimens taken from 18 cases of scabies were studied immunohistopathologically using the PAP method. In 12 of 18 cases, membrane-bound, IgE positive mast cells were observed in the dermis and subcutaneous tissues of the scabies lesions. In a Norwegian scabies, IgE deposits were found not only on the surface of scabies mites but also on feces-like granules. We conclude that the membrane-bound IgE mast cells may play an important role in immune skin reactions of scabies.     

Testosterone exerts selective anti‐inflammatory effects on human skin mast cells in a cell subset dependent manner

Androgens are known to exert anti‐inflammatory effects but their impact on mast cells (MCs) remains to be determined. Here, we show that MCs isolated from human foreskin samples (male) and those from breast skin (female) express the androgen receptor, albeit with a 10‐fold difference between the subsets. While fundamental MC properties (FcεRI, c‐Kit, tryptase; histamine release upon FcεRI cross‐linking) were unaffected or slightly reduced (chymase) by testosterone, the hormone had a more profound impact on the production of cytokines, with IL‐6 being a target (reduction by 53%). Interestingly, this effect was limited to breast skin MCs (15 of 16 donors displayed this phenomenon), but was not reproduced by foreskin MCs. Collectively, effector functions of human skin MCs are modulated by androgens in a gene‐selective and MC subset‐specific fashion. Possibly, MCs from women are more susceptible to testosterone. We also demonstrate that MC IL‐6 production is highly variable among individuals.     

Indolent systemic mast cell disease: immunophenotypic characterization of bone marrow mast cells by flow cytometry

BACKGROUND: Mast cell disease has a low prevalence and is difficult to diagnose in the absence of the characteristic skin lesions that usually accompany the condition. Extracutaneous involvement is not easy to assess. There are reports in the recent literature on the use of tryptase as a reliable immunohistochemical marker as well as on the study of the immunophenotype of bone marrow mast cells. The latter is of great help for the diagnosis of systemic involvement as, after the skin, the bone marrow is the organ most commonly affected by the disease. OBJECTIVE: We describe two cases of indolent systemic mast cell disease (SMCD) where flow cytometry was used to identify immunophenotypical characteristics of bone marrow mast cells. Recent advances in the application of this technique prove it can be a good diagnostic tool for assessing systemic involvement of the disease. PATIENTS AND METHODS: Two adult subjects with indolent SMCD had multiple clinical symptoms. Cutaneous lesions were the clue to the diagnosis but, subsequently, in the presence of disturbing symptoms, involvement of other organ systems was confirmed. In both cases, the authors used flow cytometry techniques, as described by Escribano et al. (1998) to define the immunophenotype of bone marrow mast cells. RESULTS: Both patients were diagnosed with indolent SMCD with cutaneous and bone marrow involvement. Also, they presented visible clues to the presumptive bone, cardiovascular and nervous system involvement. Gastrointestinal manifestations were documented in one case. CONCLUSIONS: The use of flow cytometry on bone marrow samples from patients with mastocytosis reveals immunophenotypic differences that can serve to allow classification of these subjects in the category of indolent SMCD even though involvement of another organ system may not be thoroughly confirmed.     

The significance of mast cells associated with basal cell carcinoma

In the stroma of basal cell carcinoma (BCC), the numbers of mast cells are increased, but their significance remains unknown. In a retrospective histological study of 74 cases, we investigated the significance of mast cells related to BCC and the possible relationship between increased numbers of mast cells and clinical and histological parameters including the patients' age, sex, subtype of BCC, and severity of peritumoral inflammation. We found a significant increase in peritumoral mast cell numbers over those in uninvolved skin (p<0.0001). Morpheaform BCCs had significantly more mast cells than did the other variants (p=0.047). No significant relationship was found between mast cell number and the degree of peritumoral inflammation, patient age, or gender. Our results support previous reports indicating that mast cell numbers are increased in RCC, particularly in its morphoeic variant, implying a contributory role for mast cells in the aggressiveness of BCC.     

Regulation of mast cell characteristics by cytokines: divergent effects of interleukin-4 on immature mast cell lines versus mature human skin mast cells

Mast cells (MC) are of hematopoietic origin but complete their differentiation exclusively within tissues. The mediators that positively or negatively affect the maturation process are incompletely defined. Here, the human MC line HMC-1 (subclone 5C6) was used along with several treatments (IL-4, IL-6, NGF), either alone or in combination, and MC differentiation was monitored by flow-cytometric analysis of c-kit, tryptase, and FcRI expression. Of the different treatments, IL-4 displayed the clearest effects by suppressing the expression of the three markers and inhibiting cellular growth, while the other cytokines had no (NGF) or negligible (IL-6) effects only. The downregulating effects of IL-4 could not be overcome by any other treatment. There is some controversy in the literature as to the impact of IL-4 on the MC lineage. To determine whether the effects from IL-4 were differentiation stage dependent, two further human MC subsets (skin MC and LAD 2 cells) were investigated. No effects on c-kit and FcRI expression were noted when terminally differentiated skin MC were used as target cells, while a modest downregulation of c-kit was observed with intermediately matured LAD 2 cells. In sharp contrast to HMC-1 5C6 cells, the survival of skin MC was significantly enhanced by IL-4 treatment. Our data therefore imply that at a lower maturation stage, IL-4 acts as a negative regulator of the MC lineage, but that this property disappears or is even reversed upon terminal differentiation of the cell. Our study provides direct proof that the effects of IL-4 vary substantially in the course of MC maturation.     

寻常型银屑病皮损肥大细胞密度及IL-8表达的研究

目的：探讨肥大细胞和IL-8在寻常型银屑病中的作用。方法：采用免疫组化技术(SABC法)观察寻常型银屑病皮损处MC分布和IL-8在表皮中的表达情况。结果：寻常型银屑病皮损处MC密度明显高于皮肤血管炎组和健康对照组；寻常型银屑病皮损处KC中IL-8的表达明显高于两对照组。结论：结果提示MC和IL-8参与寻常型银屑病的致病过程，并且两者之间存在相关性。     

Alterations in ganglioside expression during the differentiation of human mast cells

Gangliosides are physiological components of the outer cell membrane. In the present study, the role of ganglioside expression during differentiation of human mast cells was evaluated. After 11 days of culture in medium known to induce mast cell differentiation, 70% of peripheral blood mononuclear cells (PBMC) showed positive staining for the high affinity IgE receptor and tryptase on immunocytochemistry and an associated 20-fold increase of ganglioside GM3 expression. Furthermore, exogenous addition of GM3 during cultivation of PBMC in medium containing low levels of growth factors induced an increase of mast cell specific tryptase. The association of ganglioside expression with mast cell differentiation was confirmed by experiments with the human mast cell line HMC-1. FcepsilonRI-positive cultured cells enriched with immunobeads exhibited a 3-fold higher expression of GM3, compared to FcepsilonRI negative HMC-1 cells. Furthermore, measurable amounts of the gangliosides GM2, GM1 and GD1a were found only in the FcepsilonRI positive cells. A corresponding transient increase of mRNA for GalNAcT, the key enzyme in the production of these latter gangliosides, could be detected preceding the expression of these gangliosides and the FcepsilonRI by RT-PCR. Taken together, these data point to a functional role of gangliosides in the differentiation of human mast cells.     

Pattern analysis of human cutaneous mast cell populations by total body surface mapping

BACKGROUND: Mast cells (MCs), critical effector cells in allergic inflammation and innate immunity to bacteria, are located in large numbers in tissues that interface the external environment, including the skin. However, little is known about the distribution and numbers of human skin MCs. OBJECTIVES: To assess the influence of age, sex and skin region on size and spatial distribution of MC populations in normal human skin. METHODS: Biopsies of healthy skin were obtained from 150 male and female individuals (age range 10-86 years). MCs were quantified and mapped planimetrically by histomorphometry in 15 anatomical sites (abdomen, thorax, lower and upper back, lower and upper arm, lower and upper leg, foot, hand, chin, nose, cheek, forehead, back of head). RESULTS: No differences in skin MC numbers or distribution were found when comparing skin obtained from male or female and from young or old individuals. At all skin sites, regardless of age or sex, MC numbers were highest in the most superficial skin layers where up to 10-fold more MCs were found as compared with the subcutis, which consistently contained the lowest numbers of MCs. Interestingly, MC numbers were highest at peripheral skin sites (maximum: chin and nose) and lowest at central skin sites (minimum: abdomen). Thus, healthy human skin exhibits a proximal/distal and a central/peripheral MC gradient and 'skin MC numbers' may vary by a factor of more than 20, depending on the skin layer and skin site analysed. CONCLUSIONS: These findings support the recently identified role of MCs in the elicitation of protective immune responses against infectious microorganisms. One possible explanation for these unexpected findings is that skin site/layer-specific factors (e.g. the density of nerves or vessels, exposure to ultraviolet irradiation, frequency of minimal trauma) are involved in the regulation of skin MC numbers and distribution.     

Acute exposure of human skin to ultraviolet or infrared radiation or heat stimuli increases mast cell numbers and tryptase expression in human skin in vivo

Background  Mast cells are key effector cells in diverse immunological and pathological processes. It is still unclear why there are more mast cells at peripheral and sun-exposed skin sites than at sun-protected sites.
Objectives  To investigate changes in mast cell numbers associated with natural ageing and photoageing, and to observe the effects of ultraviolet (UV) and infrared (IR) radiation and heat on the prevalence of mast cells and tryptase expression in human skin in vivo .
Methods  Sun-exposed and sun-protected skin samples were taken from individuals in four different age groups. UV, IR or heat-treated buttock skin of young volunteers was also obtained. Mast cells were quantified by immunohistochemical staining of mast cell-specific tryptase and chymase. The expression of tryptase was determined by Western blotting.
Results  Both sun-exposed and sun-protected skin showed a gradual decrease in total mast cells (MC_Total) number with ageing. The number of mast cells in sun-exposed skin was significantly higher than that in sun-protected skin. After UV irradiation (2 minimal erythema doses), MC_Total and mast cells expressing tryptase and chymase were significantly increased at 24 and 48 h postirradiation. After IR irradiation (3 minimal heating doses) and heat treatment (43 °C for 90 min), MC_Total reached peak induction at 8 and 48 h after stimulation, respectively. Tryptase expression was also clearly upregulated by UV, IR and heat.
Conclusions  Our data demonstrate that mast cell numbers decreased with ageing in human skin. Also, mast cells may be activated and recruited by UV, IR and heat. These findings should further our understanding of the reason for the high prevalence of mast cells at peripheral sun-exposed skin sites.     

银屑病皮损区肥大细胞分布和血管内皮细胞CD34表达的研究

目的探讨肥大细胞(MC)和血管内皮细胞在银屑病发病中的作用.方法采用组织化学和免疫组化的方法,观察寻常性银屑病皮损处MC和CD34标记血管内皮细胞的分布情况.结果经甲苯胺蓝特殊染色发现,银屑病患者真皮区的MC密度在进行期(33.07±14.63)个/mm2,高于静止期(21.80±4.86)个/mm2,静止期又高于正常对照组(15.85±6.93)个/mm2,它们之间的差异均有显著性;免疫组化观察发现,银屑病真皮区CD34标记的微血管密度在进行期(2931±4.04)个/mm2,明显高于静止期(2231±2.07)个/mm2,而静止期又明显高于正常对照组(18.81±2.59)个/mm2.结论寻常性银屑病皮损处真皮内肥大细胞和血管内皮细胞密度明显增加,且与病情变化有关.     

Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase

Proteinase-activated receptor-2 (PAR-2) is a G-protein coupled receptor. Tryptic proteases cleave PAR-2 exposing a tethered ligand (SLIGKV), which binds and activates the receptor. Although PAR-2 is highly expressed by cultured keratinocytes and is an inflammatory mediator, its precise localization in the normal and inflamed human skin is unknown, and the proteases that activate PAR-2 in the skin have not been identified. We localized PAR-2 in human skin by immunohistochemistry, examined PAR-2 expression by RT-PCR and RNA blotting, and investigated PAR-2 activation by mast cell tryptase. PAR-2 was localized to keratinocytes, especially in the granular layer, to endothelial cells, hair follicles, myoepithelial cells of sweat glands, and dermal dendritic-like cells. PAR-2 was also highly expressed in keratinocytes and endothelial cells of inflamed skin. PAR-2 mRNA was detected in normal human skin by RT-PCR, and in cultured human keratinocytes and dermal microvascular endothelial cells by Northern hybridization. Trypsin, tryptase and a peptide corresponding to the tethered ligand (SLIGKVNH2) increased [Ca2+]i in keratinocytes, measured using Fura-2/AM. Although tryptase-containing mast cells were sparsely scattered in the normal dermis, they were numerous in the dermis in atopic dermatitis, and in the dermis, dermal-epidermal border, and occasionally within the lower epidermis in psoriasis. Tryptase may activate PAR-2 on keratinocytes and endothelial cells during inflammation.     

Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE‐independent mast cell degranulation elicited by neuromuscular blocking agents

Please cite this paper as: Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE‐independent mast cell degranulation elicited by neuromuscular blocking agents. Experimental Dermatology 2010; 19: 302–304. Abstract: Women predominate in the anaphylactic reactions to neuromuscular blocking agents (NMBA). The expression of oestrogen receptors has been demonstrated in mast cells and oestrogen treatment can enhance mast cell degranulation, but the influence of androgens remains largely unclear. Our immunocytochemical study showed the expression of androgen receptor (AR) in mast cells isolated from human foreskin as well as in two human mast cell lines, HMC‐1 and LAD2. The amount of AR was most abundant in human skin mast cells as determined by real‐time polymerase chain reaction analysis. Treatment of the HMC‐1 mast cells with testosterone or 17β‐oestradiol, alone or in combination with different NMBA, did not affect mast cell degranulation as measured by the release of β‐hexosaminidase. Our study shows for the first time the expression of AR in human skin mast cells. Further studies using primary human mast cell cultures are needed to understand whether and how sex hormones can influence mast cell activation.