Mast cells are traditionally viewed as effector cells of allergic reactions and parasitic diseases, but their importance in host defense against bacteria, in tissue remodelling, their bone marrow and stem cell origin and a central role of the stem cell factor (SCF) as mast cell growth and chemotactic factor has been worked out only in recent years. Despite this, major aspects about the nature of the cells and their role in disease remain unclear. This holds in particular for the identification of mast cell precursors and the role of growth factors that stimulate specific mast cell commitment from stem cells, such as nerve growth factor, neutrotrophin-3 and certain interleukins, alone and during interaction with SCF. Early data suggesting also an involvement of specific transcription factors need to be expanded in this process. Furthermore, although mast cell proliferative disease (mastocytosis) has been shown to be often associated with SCF receptor c-kit mutations, reasons for the development of this disease remain unclear. This holds also for mast cell release mechanisms in many types of mast cell-dependent urticaria. Exciting new insights are emerging regarding the role of mast cells in bacterial infections, in defense against tumors, in wound healing and in the interplay with the nervous system, with hormones, and in the neurohormonal network. The aim of this reflection is to delineate the many known and unknown aspects of mast cells, with a special focus on their development, and to discuss in detail two mast cell-related diseases, namely mastocytosis and urticaria.

Background Basic mechanisms of pseudoallergic drug reactions as well as a possible role of the psyche are currently unknown.Objective Examination of psychological status and reactions during diagnostic provocation tests in patients with previous pseudoallergic reactions to drugs.Subjects and methods Ten inpatients, admitted for provocation tests, were studied in a double-blind, placebo-controlled setting, with inventories of anxiety and depression being measured at baseline and psychological reactions and symptoms being recorded daily by patients and the attending physician.Results Patients reported more than twice as many symptoms as the physician, independent of the type of exposure. While the basic psychological profile of the patients was normal, anxiety trait and state values were high during testing, with a significant increase depending on whether the patients thought they had received a drug or a placebo. Similarly, frequency of symptoms was dependent on the patients' perception of the type of exposure.Conclusion These findings demonstrate a high level of anxiety during systemic provocation tests in patients with previous pseudoallergic drug reactions, raising serious questions as to the diagnostic validity of the routine application of this testing.

Proteinase-activated receptor-2 (PAR(2)) belongs to a new G protein-coupled receptor subfamily activated by serine proteinases. PAR(2) has been demonstrated to play a role during inflammation and immune response in different tissues including the skin. We examined whether PAR(2) is functionally expressed by cutaneous human primary skin mast cells (HPMC) and the human mast cell line 1 (HMC-1). Reverse transcription-polymerase chain reaction and FACS analysis show expression of PAR(2) both at the RNA and protein level. HPMCs and HMC-1 also express PAR(1), PAR(3), and PAR(4). Ca-mobilization studies demonstrate functional PAR(2) expressed by human skin mast cells, as shown by natural and synthetic PAR(2) agonists. PAR(2) agonists induced histamine release from HPMC indicating a role of PAR2 in regulating inflammatory and immune responses by skin mast cells. Double-immunofluorescence staining reveals colocalization of PAR(2) with tryptase in the majority of human skin mast cells. In conclusion, trypsin and tryptase as well as specific agonists for PAR(2) were able to induce Ca2+ mobilization in HPMCs, and agonists of PAR(2) induce the release of histamine from these cells. Thus, PAR(2) may be an important regulator of skin mast cell function during cutaneous inflammation and hypersensitivity.

The coagulation system is thought to play a pivotal role during the initial phase of wound healing, but mechanisms and cells involved are only partly understood. We have therefore examined human scars for the expression of thrombin, its precursor prothrombin and the thrombin receptors, thrombomodulin (TM) and protease-activated receptor-1 (PAR-1), compared with normal skin. Biopsies of scars were obtained from primary excision sites of melanoma patients (n = 20) and were compared with normal skin distant from the scar (n = 10), using immunohistochemistry. In addition, polymerase chain reaction analyses were performed on scar versus normal tissue and on cultured keratinocytes, fibroblasts and endothelial cells before and after stimulation with selected cytokines known to be active in wound healing. Normal epidermis was stained for prothrombin, thrombin, TM and PAR-1, and dermal tissue was stained only for TM and PAR-1. In scar tissue, thrombin and TM were upregulated in the epidermis and all four molecules in the dermis, independent of the age of the scars. In tissue extracts, mRNA expression of PAR-1 and prothrombin expression were, however, unchanged and TM even slightly decreased in scars, compared with normal skin. On analysis of cultured cells, keratinocytes expressed mRNA for PAR-1, TM and prothrombin, endothelial cells for PAR-1 and TM, and fibroblasts for PAR-1. An upregulation of PAR-1 mRNA was induced in fibroblasts on exposure to tumor necrosis factor-alpha (TNF-alpha), while it remained unchanged in endothelial cells in response to TNF-alpha. A downregulation of TM was induced in endothelial cells on exposure to TNF-alpha. These findings, showing a marked modulation of thrombin, PAR-1 and TM even in older human scar tissue, suggest that the coagulation system is not only involved during clotting, but also during the inflammatory and tissue remodelling phases of wound healing.

Specific heterodimers of α and β integrins are implicated in mediating adhesion and functional activation of mast cells to extracellular matrix (ECM) proteins, determining thus homing, secretion and tissue distribution of these cells. In the present study, we have examined integrin expression and associated morphological features of mast cells adhering to ECM, also depending on cell activation and under the influence of protein kinase C (PKC) inhibitors. Unstimulated and PMA-activated human leukaemic mast cells (HMC-1 line) were allowed to adhere to fibronectin or vitronectin-coated surfaces. Cells were specifically stained for actin, β1, α1–α6, αv and αvβ5 integrins and were evaluated by fluorescence microscopy and confocal laser scan microscopy. Spontaneously adhering cells rapidly assumed an oblong shape, with pronounced formation of filopodia, whereas PMA-stimulated cells were round in shape. Clustering of integrins on filopodia and on comma-like shapes at the cell circumference in rounded cells was noted only for α4, α5 and β1 chains in fibronectin-adhering cells, and for αv and αvβ5 chains in vitronectin-adhering cells. On double staining, clustered integrins co-localized with each other and with actin at the cell membrane and along intracellular tension lines of actin filaments. PKC inhibitors affected the shape of cells, but adhesion was maintained. These data provide a morphological correlate to previously reported functional studies, demonstrating clustering of selected integrins during ECM adhesion at the cell membrane. This was associated with alignment of integrins along actin filaments within the cytoplasm, PKC signalling and changes in shape and activation of mast cells.

The receptor tyrosine kinase c-Kit is fundamental to mast cell (MC) development and maintenance. Its regulation can occur at various levels, but nothing is known about how this is accomplished in normal human tissue MC. Likewise, the baseline turnover of c-Kit has not been addressed yet. We used mature MC from human skin, along with the MC lines LAD-2 and HMC-1 and treated them with stem cell factor (SCF), cycloheximide, actinomycin D (AD) and combinations thereof, and determined expression levels of c-Kit and other surface receptors by flow cytometry. Ligand-induced internalization of c-Kit was found to be a universal mechanism and detectable in all MC subtypes. By Western blot analysis of LAD-2 cells, c-Kit was found to nearly disappear 3 h after the addition of SCF to slowly recover thereafter. Investigations into the baseline turnover of c-Kit expression revealed that c-Kit is strongly affected by the inhibition of de novo translation in all MC subsets, while a suppression of transcription had a weaker effect and displayed greater cell-to-cell variation. Only a minor impact on other cell surface receptors (CD29, CD50 and CD54) was noted. On combined treatment, cycloheximide, AD and SCF displayed additive effects, resulting in a complete disappearance of c-Kit from the cell surface. In conclusion, c-Kit represents a rapidly cycling cell surface receptor. It is not only immediately internalized upon binding of its ligand, but it is also heavily affected by the inhibition of translation or transcription when viewed against an average background. Interestingly, c-Kit regulation seems largely independent of the MC subtype.

Human mast cells have been shown to release histamine in response to the neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH), but it is unknown whether these cells express proopiomelanocortin (POMC) or POMC-derived peptides. We therefore examined highly purified human skin mast cells and a leukemic mast cell line-1 (HMC-1) for their ability to express POMC and members of the prohormone convertase (PC) family known to process POMC. Furthermore, we investigated whether these cells store and secrete alpha-MSH. Reverse transcriptase-PCR (RT-PCR) analysis revealed that both skin mast cells and HMC-1 cells express POMC mRNA and protein. Expression of the POMC gene at the RNA level in HMC-1 cells could be confirmed by Northern blotting. Transcripts for both PC1 and furin convertase were detectable in skin-derived mast cells and HMC-1 cells, as shown by RT-PCR. In contrast, PC2 transcripts were detected only in skin mast cells, whereas transcripts for paired basic amino acid converting enzyme 4 (PACE4) were present only in HMC-1 cells. Radioimmunoassays performed on cell lysates and cell culture supernatants from human skin-derived mast cells disclosed immunoreactive amounts of alpha-MSH in both fractions. Stimulation with an anti-IgE antibody significantly reduced intracellular alpha-MSH and increased extracellular levels, indicating IgE-mediated secretion of this neuropeptide. Our findings show that human mast cells are active players in the cutaneous POMC system. Mast cell-derived alpha-MSH may contribute to cutaneous hyperpigmentation as seen in patients with urticaria pigmentosa. Moreover, IgE-dependent release of alpha-MSH suggests an immunomodulatory role of this neurohormone during inflammatory and allergic reactions of the skin.

BACKGROUND:All-trans retinoic acid (ATRA) inhibits IgE synthesis from anti-CD40 plus IL-4 stimulated human B lymphocytes.OBJECTIVE:To study the underlying mechanisms, we examined here molecules which are known to have an impact on IgE production, namely CD23, CD54 and IL-6.METHODS:Human anti-CD40 plus IL-4 stimulated B cells were cultured in the absence and presence of ATRA (10(-6)-10(-10) M). ELISAs were performed to determine soluble (s) CD23 and sCD54, IL-6 and IgE-levels. CD23 and CD54 surface expression were determined by flow cytometric analysis. Semiquantitative-RT-PCR was employed to analyse IL-6, CD23 and CD54 mRNA expression.RESULTS:ATRA induced a dose-dependent increase of percent CD23 (3.4 fold) or CD54 (1.6 fold) positive B cells. At the mRNA level, this was reflected by a modest increase of CD54 mRNA (46.5 +/- 15.8%) only. By contrast, levels of sCD54 were decreased dose-dependently in the presence of ATRA (56.6 +/- 7.6%). Cytokine analysis showed that IL-6 secretion was significantly inhibited by ATRA (53.6 +/- 0.6%) and also IL-6 mRNA synthesis was reduced (66.3 +/- 11.6%). The observed inhibition of IgE production mediated by ATRA was significantly reversed to 90.5 +/- 12% by the addition of 100 pg/mL recombinant IL-6.CONCLUSIONS:ATRA interferes through several pathways with the anti-CD40 plus IL-4 mediated B cell activation, namely IL-6, CD23 and CD54.

Expression profiles of mRNAs and proteins for various transcription factors were determined for human skin mast cells (sMCs), leukemic HMC-1 MCs, monocytes and granulocytes. By quantitative RT-PCR, sMCs expressed lower levels of c-fos, PU.1, C/EBPα, and C/EBPɛ than monocytes and granulocytes, but higher levels of MITF, SCL, GATA-1 and GATA-2. At the protein level, MITF, SCL, GATA-2, Elf-1 and c-fos were clearly detectable in sMCs. With the exception of c-fos, these proteins were absent or expressed only slightly in monocytes and granulocytes. The expression of NF-E2p45, GATA-1, PU.1, Ets-1, C/EBPα and C/EBPɛ was below the detection limit in sMCs, but detectable in other myelocytes. The high expression of SCL and GATA-2 in sMCs is reminiscent of stem cells. The absence of C/EBPɛ in sMCs, but strong expression in HMC-1, suggests it may impair MC maturation. In summary, mature human MCs can be characterized as C/EBPαlow, C/EBPɛ−, PU.1low, GATA-1low, GATA-2+, SCL+, MITFhigh.

The gene expression and regulation of nerve growth factor (NGF) in atopic dermatitis (AD) and the human mast cell line (HMC)-1 was investigated at the molecular level. NGF-stimulation of HMC-1 cells resulted in increases in tryptase activity and histamine contents, paralleled by an increase of tryptase and histamine at the transcriptional level. Also, an increased expression of NGF was found in AD lesions, in association with increased systemic NGF plasma levels. Further cutaneous sources for increased NGF levels were keratinocytes and fibroblasts. These findings demonstrate an increased expression of NGF in AD and effects on tryptase and histamine. Mast cells may be major mediators of neurotrophin effects in AD.

BACKGROUND:Angiotensin AT1 and AT2 receptors are expressed in human skin. Furthermore, AT2 receptors have been reported to be upregulated during tissue repair and remodelling in various noncutaneous human tissues.OBJECTIVES:Detection of alterations in angiotensin II receptor expression during wound healing in human skin.METHODS:Three models were employed. (i) Primary human keratinocytes were razor scraped in culture flasks and alterations in the expression of angiotensin receptor mRNA determined by semiquantitative reverse transcription-polymerase chain reaction for 1-12 h thereafter. (ii) Early wound healing (48 h after cutting) was studied in punch biopsies from human skin ex vivo by means of immunohistochemical staining using polyclonal antibodies against the AT1 or AT2 receptor. (iii) In vivo wound healing was studied in sections of human cutaneous scars by immunohistochemistry to determine receptor expression early (2 days) and late (2 weeks-3 months) after surgery.RESULTS:In all experimental settings, an upregulation of both receptor subtypes was noticed after wounding. Immunohistochemically stained skin sections showed a stronger expression of AT2 than of AT1 receptors within the area of scarring. Enhanced receptor expression was detectable as early as 24 h after injury and lasted for up to 3 months.CONCLUSIONS:From these data, we conclude that angiotensin AT1 and AT2 receptors are upregulated in human cutaneous wounds, giving further support to the concept that angiotensin II plays a role even at an early stage during cutaneous wound healing.

BACKGROUND:The transcription factor activator protein (AP)-2 regulates cell-type specific gene expression during development and differentiation, but its role in mast cell development has so far not been explored.METHODS:Gene expression and regulation of AP2 was assessed in normal skin, diseases with increased mast cell numbers, and in vitro models of mast cell differentiation.RESULTS:AP-2alpha-protein was not detectable in normal skin but in mastocytoma lesional mast cells. AP-2alpha-mRNA and -protein were also detected in leukemic mast cells (HMC-1), in the adherent fraction of peripheral blood (PBMC) and umbilical cord blood mononuclear cells (CBMC), and AP-2alpha-mRNA at low levels in isolated-purified mast cells. During culture with fibroblast supernatants or SCF, AP-2alpha-mRNA was de novo expressed in KU812-cells, maintained at about the same level in PBMC and CBMC, and upregulated in HMC-1-cells. On extended culture, a down-regulation was noted at mRNA and/or protein levels. In contrast, tryptase expression increased in all cells throughout culture, as did c-Kit in normal cells, whereas in both leukemic cell lines, c-Kit was maintained unchanged at about the same level.CONCLUSIONS:These findings suggest a continuous activation of AP-2alpha in mastocytomas and mast cell leukemia and its transient upregulation during c-Kit dependent early steps of normal mast cell differentiation.

Background: Atopic dermatitis skin lesions are characterized by inflammatory changes and epithelial hyperplasia requiring angiogenesis. As mast cells may participate in this process via bidirectional secretion of tissue‐damaging enzymes and pro‐angiogenic factors, the present study aimed to assess the occurrence and possible function of mast cells in the papillary dermis and in epidermal layers of atopic dermatitis lesions.Methods: Semi‐thin and serial sections in combination with immunohistochemistry, histochemistry and proliferating cell nuclear antigen (PCNA)‐activity assays were used and related to epidermal thickness and targeted gene expression studies.Results: Mast cells were located in the papillary dermis and migrated through the basal lamina into the epidermis of atopic dermatitis lesions. An increased PCNA‐activity in cells of superficial epidermal layers indicated an activation of keratinocytes and stimulation of endothelial growth. Only ∼30% of the papillary mast cells stained with the tryptase were toluidin‐blue‐positive, and ∼80% were chymase positive. A high number of mast cells expressed c‐kit. Most papillary and epidermal mast cells were localized close to endothelial cells. Vascular expression of endoglin (CD105) demonstrated neoangiogenic processes. Mast cells stimulation led to the expression of proangiogenic factors. Also, gene expression of tissue‐damaging factors such as matrix metalloproteinases was increased.Conclusions: These data suggest that in atopic dermatitis, mast cells are abundantly localized close to and within the epidermis where they may stimulate neoangiogenesis. Via the new vessels, inflammatory cells, together with complement components and antibodies, can be transported to the epidermis to aid in the defense against environmental antigens and to maintain chronic inflammation.

Styling one's hair seems to be an innate desire of humans to emphasize their beauty and power. As reviewed here, hairstyles were influenced by preceding cultures, by religion, by those depicted for gods and emperors on sculptures and coins. In addition, they were determined by aspects of lifestyle such as sports, wealth, and the desire to display inner feelings. The historical changes in fashions can be exemplarily followed by a visitor to an art collection of Graeco-Roman antiquity. The study of hairstyles permits an insight into very basic aspects of the self-conception of individuals and of the respective societies.

A multicentre case-control study conducted by the FEBrile Infections and Melanoma (FEBIM) group has demonstrated a reduced risk of melanoma associated with Bacille Calmette-Guérin (BCG) and/or vaccinia vaccination in early childhood and/or with infectious diseases later in life. This has led to the recognition of a new risk indicator of melanoma; namely ‘not being vaccinated with either with BCG or vaccinia’. On the basis of these findings, we propose a hypothesis of immune surveillance for melanoma induced or enhanced by prior contacts with pathogens unexpectedly cross-reactive to a cellular ‘marker of melanoma risk’. The reduced risk of melanoma due to BCG and vaccinia, as well as certain common causes of infectious disease, is shown to be associated with antigenic determinants exhibiting sequence homologies with the HERV-K-MEL-antigen. The latter is a product of a pseudo-gene that is closely associated with the env-gene of the endogenous human retrovirus K (HERV-K). A suppressive immune reaction appears to inhibit the expression of endogenous retroviral genes, such as the HERV-K env-gene, that could otherwise result in malignant transformation years or even decades later. The HERV-K env-protein has homologous amino acid sequences with the human nuclear factor Oxygen Responsive Element Binding Protein (OREBP) that controls the expression of glutathione peroxidase. The formation of this and other redox-enzymes, needed to maintain appropriate levels of the normal intracellular redox potential, seems to be suppressed by the OREBP-homologous protein. The present hypothesis is in accordance with the concept that immune dysregulation due to adverse environmental impacts is a risk factor not only for some autoimmune disorders, as previously described, but also for certain malignancies such as melanoma.