Exploring the mast cell enigma: a personal reflection of what remains to be done.

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.

Morphological analysis of integrin-mediated adhesion of immature human mast cells to extracellular matrix proteins.

Specific heterodimers of alpha and beta 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, beta(1, )alpha(1)-alpha(6), alpha(v) and alpha(v)beta(5 )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 alpha(4), alpha(5) and beta(1 )chains in fibronectin-adhering cells, and for alpha(v) and alpha(v)beta(5) 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.

Expression of prothrombin, thrombin and its receptors in human scars.

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.

Baseline and stimulated turnover of cell surface c-Kit expression in different types of human 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 in the neurohormonal network: expression of POMC, detection of precursor proteases, and evidence for IgE-dependent secretion of alpha-MSH.

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.

Functional characterization and expression analysis of the proteinase-activated receptor-2 in human cutaneous mast cells.

Proteinase-activated receptor-2 (PAR2) belongs to a new G protein-coupled receptor subfamily activated by serine proteinases. PAR2 has been demonstrated to play a role during inflammation and immune response in different tissues including the skin. We examined whether PAR2 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 PAR2 both at the RNA and protein level. HPMCs and HMC-1 also express PAR1, PAR3, and PAR4. Ca-mobilization studies demonstrate functional PAR2 expressed by human skin mast cells, as shown by natural and synthetic PAR2 agonists. PAR2 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 PAR2 with tryptase in the majority of human skin mast cells. In conclusion, trypsin and tryptase as well as specific agonists for PAR2 were able to induce Ca2+ mobilization in HPMCs, and agonists of PAR2 induce the release of histamine from these cells. Thus, PAR2 may be an important regulator of skin mast cell function during cutaneous inflammation and hypersensitivity.

Hairstyles in the arts of Greek and Roman antiquity.

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.

Evidence for a restricted rather than generalized stimulatory response of skin-derived human mast cells to substance P.

To resolve the controversy regarding substance P (SP) mediated stimulation of mast cells (MC), we demonstrate that SP triggers histamine release from purified human skin MC (sMC), but contrast to stimulation via FcepsilonRI, does not effect the production of TNF-alpha or IL-8. Conversely, both anti-IgE and SP are suppressive in terms of IL-6. By quantitative RT-PCR, the amount of templates at baseline (per 25 ng total RNA) is 2178 (IL-6), 2,665 (IL-8) and 94 (TNF-alpha), and remains unaltered by SP. Contrast to sMC, LAD2 MC respond to SP with stronger histamine release and robust TNF-alpha production in an only partially neurokinin-1R mediated manner, while histamine release of sMC is chiefly mediated by this receptor. We conclude that human sMC are responsive to SP in a selective manner by eliciting degranulation without the induction of cytokines and that SP-triggered cytokine production varies among MC subtypes, likely through differences in signaling mechanisms.

Gene expression and regulation of nerve growth factor in atopic dermatitis mast cells and the human mast cell line-1.

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.

Bivalent effect of UV light on human skin mast cells-low-level mediator release at baseline but potent suppression upon mast cell triggering.

Ultraviolet (UV) irradiation is an established treatment for inflammatory skin diseases, although the precise mode of action is still unclear. Activating and suppressive effects on mast cell (MC) mediator release have been described. The aim of this study was to investigate systematically the effects of UVB, UVA-1, and psoralen plus UVA-1 at therapeutic doses on skin-derived human MC. Baseline and stimulated release of histamine, tryptase, and of interleukin (IL)-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) were examined. In resting MC, UV light induced a slight, yet significant histamine release corresponding to enhanced surface levels of lysosome-associated membrane proteins (LAMP). In contrast, UV pre-treatment caused a marked suppression of the anti-IgE-induced histamine release, accompanied by a diminished, anti-IgE-mediated increase in LAMP expression. The secretion of IL-6, IL-8, and TNF-alpha was inhibited in resting and activated MC, suggesting a different mode of action. Regarding the importance of MC in a variety of allergic and inflammatory processes, our data show a high susceptibility of this cell type towards UV light, which seems to partially depend on the state of cellular activation. Immunosuppressive effects predominate in activated MC, thus corresponding with the beneficial effects in inflammatory diseases, whereas in resting MC, both stimulatory and inhibitory effects are observed.

Protection against melanoma by vaccination with Bacille Calmette-Guerin (BCG) and/or vaccinia: an epidemiology-based hypothesis on the nature of a melanoma risk factor and its immunological control.

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-Guerin (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.

Altered apoptosis and cell cycling of mast cells in bone marrow lesions of patients with systemic mastocytosis.

Enhanced expression of the apoptosis-preventing protein bcl-xL and the cell cycle-regulating protein p21 was observed in bone marrow infiltrates of systemic mastocytosis. Expression of bcl-2, Ki67, and p53 as well as ISEL apoptosis staining were comparable in patients with mastocytosis and in controls. An altered rate of apoptosis and cell cycling may contribute to accumulation of mast cells in mastocytosis.

Differential expression of mast cell characteristics in human myeloid cell lines.

In order to better understand the mechanisms governing display of mast cell characteristics in human myeloid cells, we have studied the mast cell phenotype in human promyelocytic (HL-60) and myelocytic (U-937, TPH-1) vs. basophilic (KU-812) and mast cell (HMC-1) lines, in part also in skin mast cells and blood monocytes, at mRNA and protein level before and after stimulation with mast cell growth factors. In unstimulated cells, mRNA for the stem cell factor (SCF) receptor c-kit and the gamma chain of the high-affinity IgE receptor (FcepsilonRI) was noted in all cells studied. Like mast and basophilic cells, THP-1 cells expressed the FcepsilonRIalpha and beta chains and weakly histidine decarboxylase (HDC), but they lacked mRNA for mast cell-specific proteases [tryptase, chymase, carboxypeptidase A (CPA)]. In contrast, HL-60 and U-937 cells lacked FcepsilonRIalpha, but expressed tryptase and chymase, HL-60 cells also CPA. KU-812 cells failed to express the basophil-specific marker 2D7. After a 10-day culture with SCF or fibroblast supernatants, baseline mRNA expression of most mast cell characteristics was upregulated, whereas c-kit mRNA expression decreased in all but THP-1 cells. Differential mRNA expression of FcepsilonRI vs. protease (tryptase) was confirmed at protein level by immunocytochemistry and enzymatic activity. KU-812 cells are thus closest to skin mast cells in that they express all molecules studied, except for chymase, followed by THP-1 cells that lack all mast cell proteases. In contrast, HL-60 and U-937 cells fail to express the FcepsilonRIalpha and beta chains but express most mast cell proteases. The selective and differential expression of mast cell characteristics in human myeloid cell lines suggests that induction of the mast cell phenotype is regulated by several independent genes and that mast cells and basophils branch off at early and distinct points of myeloid development.

LAMP-1 and LAMP-2, but not LAMP-3, are reliable markers for activation-induced secretion of human mast cells.

BACKGROUND: Mast cells are resident tissue cells that induce anaphylactic reactions by rapidly releasing mediators after antigen-mediated cross-linking of immunoglobulin E receptors. In the similarly active peripheral blood basophilic leukocyte, lysosome-associated membrane protein 3 (LAMP-3; CD63) has been described as an activation marker, but LAMPs have not been investigated in normal tissue mast cells. METHODS: Intra- and extracellular expressions of LAMP-1 (CD107a), LAMP-2 (CD107b), and LAMP-3 (CD63) were analysed by flow cytometry, immunocytochemistry, and functional assays in unstimulated and stimulated leukemic human mast cell line 1 (HMC-1) and skin mast cells. RESULTS: On flow cytometry, all mast cells expressed LAMP-3 at their cell membranes, whereas LAMP-1 and LAMP-2 were barely detectable (HMC-1 cells) or expressed at low levels (<10% of skin mast cells). After fixation and permeabilisation, high intracellular levels of all three LAMPs were noted in both cell types. After stimulation, a rapid translocation of intracellular LAMPs to the cell membrane, with an associated release of histamine, leukotriene C(4) and prostaglandin D(2), was ascertained in skin mast cells only. CONCLUSION: These results show that LAMP-1 and LAMP-2 are activation markers for normal mast cells. The lack of LAMP translocation after activation of leukemic mast cells may be related to maturation or malignancy-associated defects of these cells.

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, NGFbeta), either alone or in combination, and MC differentiation was monitored by flow-cytometric analysis of c-kit, tryptase, and FcepsilonRIalpha 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 (NGFbeta) 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 FcepsilonRIalpha 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.

Human T lymphocytes and mast cells differentially express and regulate extra- and intracellular CXCR1 and CXCR2.

CXCL8 plays a major role in cell recruitment to sites of inflammation. Apart from neutrophils, little is known, however, about the cellular distribution and regulation of CXCL8 receptors in cells involved in acquired and adaptive immune responses. In previous studies, we have demonstrated the extracellular expression and function of CXCR1/2 on mast cells and also detected an intracellular pool of CXCR1/2. Here, we have addressed the question of receptor regulation during stimulation of human mast cells (HMC-1 cell line) and have studied T cells in comparison. Cell permeabilization was performed to detect both surface and possible intracellular receptor pools. HMC-1 cells stained positive for both receptors on the cell surface (CXCR1, 50%; CXCR2, 51%) and also after cell permeabilization (CXCR1, 86%; CXCR2, 74%). Similarly, T cells exhibited both cell-surface receptor expression (CXCR1, 30%; CXCR2, 23%) and higher total receptor expression (CXCR1, 50%; CXCR2, 36%), although overall values were lower than that in HMC-1 cells. On immunoblot, molecular weights of extra- and intracellular receptors on mast cells were the same, excluding altered receptor glycosylation. On stimulation with phorbol 12-myristate 13-acetate plus calcium ionophore, a time-dependent decrease of surface-membrane receptors was observed in both cell types, while total receptor remained the same, suggesting that receptor shedding is not involved. The kinetics of membrane receptor internalization and replenishment differed for the two cell types. Furthermore, receptor internalization was associated with decreased F-actin polymerization, a basic prerequisite for cell migration. These findings demonstrate for the first time the expression of extra- and intracellular CXCR1/2 receptors on T cells and delineate the dynamics of CXCR1/2 receptors on mast cells and T cells. Furthermore, they suggest a cell-type-specific and finely tuned regulation of chemokine responses at the receptor level in the context of inflammation.

Human skin mast cells express H2 and H4, but not H3 receptors.

Mast cells generate and release histamine during anaphylactic reactions, and there is pharmacological evidence that histamine regulates this process via specific receptors. Therefore, we examined human leukemic (HMC-1) and normal skin mast cells for the expression of all four currently known histamine receptors. Both cell types expressed H2 and H4 receptors at mRNA and protein levels, whereas H3 receptor specific mRNA and receptor protein was undetectable. Similarly, immunohistochemistry of cutaneous tissue showed an absence of H3 receptor in these cells. Despite transcription of mRNA, H1 receptor protein was only moderately expressed in HMC-1 cells and was virtually absent in skin mast cells. Furthermore, only H1, H2, and H4 receptors were detectable by Western blot analysis of HMC-1 cells. Radiolabeled histamine binding was strongly inhibited only by H2 (ranitidine)- and H3/H4 (FUB 108)-specific antagonists. Histamine-induced increase of cAMP was inhibited by the H2 receptor antagonist famotidine, whereas induction of IP3 was not observed, making signaling via the H1 receptor unlikely. These data show that human mast cells constitutively express primarily H2 and H4 receptors and that H2 receptors are functionally linked to cellular processes. They provide new insights into the mechanisms that govern auto- and paracrine histamine-induced mast cell functions.

Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures.

The vanilloid receptor subtype 1 (VR1)/(TRPV1), binding capsaicin, is a non-selective cation channel that recently has been shown in human keratinocytes in vitro and in vivo. However, a description of VR1 localization in other cutaneous compartments in particular cutaneous nerve fibers is still lacking. We therefore investigated VR1 immunoreactivity as well as mRNA and protein expression in a series (n = 26) of normal (n = 7), diseased (n = 13) [prurigo nodularis (PN) (n = 10), generalized pruritus (n = 1), and mastocytosis (n = 2)], and capsaicin-treated human skin (n = 6). VR1 immunoreactivity could be observed in cutaneous sensory nerve fibers, mast cells, epidermal keratinocytes, dermal blood vessels, the inner root sheet and the infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts, and the secretory portion of eccrine sweat glands. Upon reverse transcriptase-polymerase chain reaction and Western blot analysis, VR1 was detected in mast cells and keratinocytes from human skin. In pruritic skin of PN, VR1 expression was highly increased in epidermal keratinocytes and nerve fibers, which was normalized after capsaicin application. During capsaicin therapy, a reduction of neuropeptides (substance P, calcitonin gene-related peptide) was observed. After cessation of capsaicin therapy, neuropeptides re-accumulated in skin nerves. In conclusion, VR1 is widely distributed in the skin, suggesting a major role for this receptor, e.g. in nociception and neurogenic inflammation.