Pia Welker

Human skin mast cells rapidly release preformed and newly generated TNF‐α and IL‐8 following stimulation with anti‐IgE and other secretagogues

Abstract: Several groups have previously reported that rodent or human leukemic mast cells produce inflammatory cytokines such as TNF‐α and IL‐8 as well as the pro‐allergic cytokines IL‐4, IL‐5 and IL‐13. Comparatively little is known, however, regarding the ability of normal human skin mast cells to secrete these factors following either IgE‐dependent or IgE‐independent modes of activation. We therefore investigated whether normal human skin mast cells produce these cytokines following stimulation by a variety of secretagogues. Enriched isolated skin mast cells released both TNF‐α and IL‐8 following activation with either anti‐IgE, SCF, substance P, compound 48/80 or A23187. This release was dose‐ and time‐dependent, with maximal levels being reached within 4 h of stimulation involving, in part, the secretion of preformed stores of both cytokines. In accordance with this, using lysates of highly purified (>90%) skin mast cells, we could demonstrate that both TNF‐α and IL‐8 mRNA and protein were present in both unstimulated as well as stimulated mast cells. In stark contrast to these results, no significant levels of either IL‐4, IL‐5 or IL‐13 were detected, regardless of the secretagogue used or the period of stimulation. These results show that human skin mast cells are capable of rapidly secreting pro‐inflammatory cytokines like TNF‐α and IL‐8 following IgE‐dependent activation and stimulation by the neuropeptide substance P, SCF and the basic polypeptide analogue compound 48/80. In contrast to other types of human mast cells however, human skin mast cells were incapable of secreting IL‐4, IL‐5 or IL‐13 in these settings.

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.

Mast cells and vasculature in atopic dermatitis - potential stimulus of neoangiogenesis

Background:u2002 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.

GM-CSF downmodulates c-kit, FcεRIα and GM-CSF receptor expression as well as histamine and tryptase levels in cultured human mast cells

Abstract GM-CSF is known primarily as a hematopoietic growth factor, but it has also been shown to inhibit mast cell differentiation in vitro. In order elucidate the mechanisms involved, we investigated the effects of GM-CSF in vitro on the differentiation of human leukemic mast cells (HMC-1 cells) and normal cord blood-derived mast cells (CBMC) under the influence of SCF, NGF, and fibroblast supernatant (FS). Under all culture conditions, GM-CSF induced a dose- and time-dependent reduction in intracellular histamine levels, tryptase activity, and numbers of cells immunoreactive for c-Kit and FcÂRI·. This effect leveled off between 10–100 ng/ml and after 4 days of culture. There was an associated decrease in mRNA expression for c-kit, FcÂRI· and tryptase. In contrast, no significant changes in the expression of the NGF receptor TrkA were noted under the same conditions. The GM-CSF receptor was found in HMC-1 cells and CBMC at both the mRNA and protein levels, but its expression decreased during culture with FS, and even more markedly during culture with GM-CSF. GM-CSF thus selectively inhibits in vitro induction and/or upregulation of all major mast cell characteristics in HMC-1 cells and CBMC irrespective of the growth factors present, and a concomitant downregulation of GM-CSF receptors can counteract these effects. GM-CSF may therefore function as a regulatory factor in mast cell growth and differentiation under normal and pathological conditions.

Expression of SCF splice variants in human melanocytes and melanoma cell lines : potential prognostic implications

Stem cell factor (SCF), the ligand for c-Kit, is known to regulate developmental and functional processes of haematopoietic stem cells, mast cells and melanocytes. Two different splice variants form predominantly soluble (sSCF or SCF-1) and in addition some membrane-bound SCF (mSCF or SCF-2). In order to explore the prognostic significance of these molecules in melanoma, total SCF, SCF splice variants and c-Kit expression were studied in normal skin melanocytes and in 11 different melanoma cell lines, using reverse transcription polymerase chain reaction, immunocytochemistry and enzyme-linked immunosorbent assay. Nine of the 11 melanoma cell lines expressed SCF-1 mRNA, only two of them SCF-2, and these two also SCF-1. Coexpression of both SCF-1 and c-Kit was noted in five cell lines, and only one cell line as well as normal melanocytes expressed both SCF-1 and SCF-2 as well as c-Kit. Corresponding results were obtained on immunocytochemical staining. Of three exemplary melanoma cell lines studied, two expressing SCF mRNA also released SCF spontaneously and on stimulation, whereas the line lacking SCF and c-kit mRNA (SK-Mel-23) failed to do so. These data demonstrate thus that melanoma cell lines, particularly those known to metastasize in vivo, lose the ability to express SCF-2 mRNA, suggesting that this molecule may serve, next to c-Kit, as a prognostic marker for malignant melanoma.

Induction of MHC class II antigen expression on human HMC-1 mast cells

Mast cells are not only the primary effector cells of immediate type immune reactions, but they have recently also been considered to contribute to the induction of an immune response. Data on the ability of the cells to express major histocompatibility complex (MHC) antigens and the mechanisms involved are however controversial or unclear. We have therefore studied the expression of MHCI and the induction of MHCII molecules on leukemic HMC-1 mast cells by immunohistochemistry. Cells were incubated for up to 72 h in the presence of IFN gamma, TNF alpha and IL-4, and immunohistochemical staining was done with monoclonal antibodies with specificity for HLA class I heavy chain, HLA-DQ (Tü22), HLA-DR (Tü36) and HLA-DQ, -R and -P (Tü35). All unstimulated mast cells expressed MHC class I, but almost no class II antigens. Incubation with IFN gamma caused a rapid, dose-dependent induction of MHC class II molecules, with Tü35 staining maximally one third of the cells within 24 h at the highest dose tested (100 IU/ml), with decline on extended culture. TNF alpha (2 ng/ml) was less effective but caused more persistent induction with time. IL-4 (200 ng/ml) had hardly any effects at all. Staining with Tü22 and Tü36 was always lower than with Tü35, and additive or even synergistic results were obtained when cells were stimulated with a combination of IFN gamma and TNF alpha. These findings support the concept that mast cells can facultatively participate in immune recognition processes depending on the type of pathological conditions in their microenvironment which allow expression of MHC class II molecules.

Effect of granulocyte macrophage colony-stimulating factor in a patient with benign systemic mastocytosis.

We report the in vitro and in vivo effects of granulocyte macrophage colony stimulating factor (GM‐CSF), a known inhibitor of in vitro mast cell differentiation, in a patient with benign, adult‐onset systemic mastocytosis. In vitro effects of GM‐CSF on bone marrow cultures before the start of treatment showed a marked inhibition of mast cell marker expression [tryptase, Kit, and high‐affinity IgE receptor (FcεRIα)] at both protein and mRNA levels. Therefore, the patient was treated with daily injections of GM‐CSF for 10 weeks. After an initial improvement, increasing worsening of clinical symptoms was noted, and the patient refused further treatment. Lesional skin biopsies showed an increase of toluidine blue‐positive mast cells, compared with uninvolved skin, with further significant increase after treatment. Similar results were obtained on staining for mast cell‐specific tryptase and Kit, as well as for CD1a and FcεRIα. These findings show that GM‐CSF inhibits human bone marrow mast cell differentiation in vitro, and also in mastocytosis. However, GM‐CSF apparently enhances recruitment of mast cell as well as dendritic cell precursors into the tissue during systemic treatment. These findings and the observed adverse clinical effects in the present patient make it unlikely that GM‐CSF monotherapy will be beneficial for the treatment of mastocytosis.