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Biomedical subjects

J Grabbe

Publications and source records attributed to J Grabbe.

At least 37 records · Page 2Linked to original sources
PubMed2000

Erythema elevatum diutinum--evidence for disease-dependent leucocyte alterations and response to dapsone.

Erythema elevatum diutinum (EED) is a type of leucocytoclastic vasculitis of unknown aetiology. We report a patient with unusually widespread and disabling EED that had been unresponsive to corticosteroids and antibiotics, but resolved on dapsone. Biopsies of fresh lesions showed typical features of leucocytoclastic vasculitis, with prominent neutrophil infiltration, marked expression of the beta(2)-integrins CR3 and LFA-1, and increased mast cell numbers. Older lesions exhibited granulation tissue and fibrosis, macrophages were more dominant, beta(2)-integrins were expressed less markedly, and mast cell numbers were lower. In vitro chemotaxis of the patient's peripheral blood neutrophils prior to treatment showed increased random migration and directed migration towards interleukin-8 (by 424%), but a profoundly decreased responsiveness towards the bacterial peptide analogue N-formyl-methionyl-leucyl-phenylalanine (fMLP) (by 98%). These values returned to normal after dapsone treatment and clinical improvement 5 months later. These findings support the concept that in EED, activation via cytokines such as interleukin-8 allows a selective recruitment of leucocytes to tissue sites, while immune complexes and bacterial peptides sustain the persistent local inflammatory infiltrate and the leucocytoclastic vasculitis.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

Nerve growth factor-beta induces mast-cell marker expression during in vitro culture of human umbilical cord blood cells.

Nerve growth factor-beta (NGF) is known as a growth factor for human basophils and murine mast cells and has recently been shown to also up-regulate mast cell characteristics in human leukaemic mast cells. We have examined here the effect of NGF on the differentiation of normal human mast cells from cord blood progenitors during culture with stem cell factor (SCF), NGF alone or in combination, or fibroblast supernatants. All these supplements induced mast cell immunoreactivity against tryptase, c-Kit and FcepsilonRIalpha, but none of the cells reacted against the basophil specific antibody 2D7 before or during culture. Intracellular tryptase activity increased as well, with maximal levels on combined culture with SCF and NGF. On reverse transcription-polymerase chain reaction (RT-PCR), cells lacked tryptase and chymase and expressed low levels of FcepsilonRI and c-Kit mRNA prior to culture, with marked up-regulation of FcepsilonRI and c-Kit, and with de novo expression of mast-cell specific alpha- and beta-tryptase by week 3, and of chymase by week 5. Only the TrkA and not the p75 NGF receptor was detected at m-RNA and protein level, and only the TrkA NGF receptor was up-regulated during NGF-driven culture. These findings show therefore that, like SCF, NGF is another growth factor that can induce and regulate human mast-cell development and differentiation.

Biomarkers↗
PubMed2000

Mast cell and myeloid marker expression during early in vitro mast cell differentiation from human peripheral blood mononuclear cells.

UNLABELLED: In order to characterize the phenotype of human mast cell precursors in the peripheral blood mononuclear fraction and its alterations during in vivo mast cell differentiation, cells were studied before and during culture with stem cell factor or stem cell factor-containing cell supernatants. Prior to culture, 86% of cells were immunoreactive for the monocytic marker CD14, slightly fewer for CD11b and CD64, < 10% expressed FcepsilonRIalpha, rare cells were CD34 + ( < 0,1%), and none stained for CD1, CD33, c-Kit, and tryptase. After 2 wk of culture, there was de novo expression of c-Kit (14% - 43% positive cells), tryptase (26% - 79%), CD33 (57%), and CD64 (64%), an upregulation of FcepsilonRIalpha (23% - 52%), CD11b (93%), and CD68 (95%), but no expression of CD34. Levels of mRNA for FcepsilonRIalpha and c-Kit were detectable prior to culture and increased during culture, together with de novo expression of tryptase. Double staining after 2 wk of culture showed that FcepsilonRIalpha-positive cells were mostly CD14 + (90%), CD64 + (82%), and CD68 + (52%) on flow cytometry. Intracellular tryptase activity was first detectable after 1 wk of culture, increased FcepsilonRIalpha expression was only detectable by week 2. Cultured cells acquired the ability to release histamine during IgE-dependent stimulation, and culture with the c-Kit antibody YB5.B8 resulted in a downregulation of tryptase and FcepsilonRIalpha, but not of c-Kit. These data show that human mast cells develop from c-Kit- and tryptase-negative precursors in the myelomonocytic fraction of peripheral blood and that they upregulate, maintain, and share many phenotypic characteristics of cells from the monocyte/macrophage lineage during early phases of in vitro differentiation. KEYWORDS: c-kit/FcepsilonRI/SCF/tryptase.

Biomarkers↗
PubMed2000

Altered expression of mast cell chymase and tryptase and of c-Kit in human cutaneous scar tissue.

In order to explore a possible involvement of mast cells during human wound healing, we studied sections from scars (4-369-d-old) (N = 20) and normal skin (N = 10) for mast-cell-specific tryptase and chymase by enzyme histochemistry, for the stem cell factor receptor c-Kit and the melanosomal marker TA99 by immunohistochemistry, and for simultaneous c-Kit expression and avidin fluorescence by double staining. Enzyme activities and mRNA expression were also studied in tissue extracts. Chymase-reactive mast cell numbers as well as chymase activity and mRNA expression were reduced in all scars, whereas overall numbers of tryptase-reactive cells did not differ from normal skin, although tryptase activity and mRNA expression were increased in scar extracts. In contrast, numbers of c-Kit positive cells were significantly increased in old scars, and in the mid and lower dermis of all scars. A marked reduction of c-Kit reactivity was noted, however, in avidin-positive dermal mast cells and in epidermal basal cells, despite unchanged numbers of melanosome-positive cells, with an associated overall decrease of c-Kit mRNA in scar extracts. These data thus show that numbers of resident mast cells are very low in human cutaneous scars, suggesting massive mediator release from these cells into fresh wounds. Downregulation of stem cell factor receptors may also prevent these cells from increasing in number even in old scars. Instead, scar tissue is populated by a mast cell subpopulation that is chymase-, avidin-, tryptase +, c-Kit +, reflecting most probably an increased immigration and/or proliferation of immature mast cells and their precursors.

Chymases↗
PubMed2000

Adult rhabdomyoma of the extremity: a case report and review of the literature.

The adult rhabdomyoma is a rare, benign skeletal muscle neoplasm that usually occurs in the head and neck. A case report of an adult rhabdomyoma arising in the thigh is presented with a review of the literature. This is the first case of an extremity adult rhabdomyoma to be reported. It is also the largest at 13 centimeters. Distinction from a highly differentiated rhabdomyosarcoma is important. Recent chromosomal studies suggest that the adult rhabdomyoma is a true neoplasm. Total resection is curative but the lesion may recur if incompletely excised.

Aged↗
PubMed2000

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.

Alternative Splicing↗
PubMed1999

Inhibitors of PI 3-kinase and MEK kinase differentially affect mediator secretion from immunologically activated human basophils.

Effects of inhibitors of PI 3-kinase and MEK kinases were investigated on histamine, leukotriene C4(LTC4), and cytokine release from human basophils stimulated with anti-IgE. The PI 3-kinase antagonists wortmannin (> 10 nM) and LY 294002 (>1 microM) strongly inhibited anti-IgE-induced release of all mediators by 40-100%. This was contrasted by the effects of the MEK kinase inhibitor PD 098059, which weakly inhibited histamine, interleukin (IL)-4, and IL-13 release but was substantially more efficacious at blocking LTC4 production (>70% at 10 microM). Previous studies have shown that arachidonic acid synthesis is controlled by MEK kinases. We observed that wortmannin, LY 294002, and PD 098059 reduce basophil ERK-1,2 activation, thus implying that, with regard to arachidonic acid metabolism, MEK kinases are a downstream target for PI-3-kinase. Our results demonstrate a universal regulatory role played by PI 3-kinases in basophil mediator production and release, whereas MEK kinase signaling is largely limited to controlling arachidonic acid metabolism.

Antibodies, Anti-Idiotypic↗
PubMed1999

Human mast cells produce and differentially express both soluble and membrane-bound stem cell factor.

Stem cell factor (SCF), characterized as mast cell growth factor, is known to be produced by fibroblasts, keratinocytes and endothelial cells. Two different splice variants encode for either a soluble (SCF-1) or a membrane-bound (SCF-2) form. In order to explore whether mast cells themselves can produce SCF, we examined cultured cord blood (CBMC) and peripheral-blood-derived mast cells (PBMC), mast/basophil cell lines (HMC-1 and KU-812), and skin mast cells for SCF expression. On immunocytochemistry, cytoplasmatic SCF-reactivity was observed in HMC-1 cells, with additional cell membrane staining in KU-812, skin and cultured mast cells. Low amounts of SCF could be detected by ELISA in lysates of isolated and unstimulated mast cells and in supernatants of skin cells stimulated with anti-IgE or Ca-ionophore A23187. SCF mRNA was detected in all cells, although marked quantitative differences were observed among the various cell types. SCF-2 mRNA expression was low in HMC-1 cells while it was marked in skin mast cells, KU-812 cells, CBMC and PBMC. A time-dependent, increasing induction of both SCF forms was seen in CBMC and PBMC during culture. After stimulation with A23187 and phorbol myristate acetate, an up-regulation of SCF mRNA was noted in HMC-1 and KU-812 cells, without changes in the relationship of the two splice variants. The differential expression of SCF-specific mRNA splice variants in immature and mature human mast cells and the secretion of this molecule by these cells may play a role in autocrine stimulation, maintenance of survival and the differentiation of tissue mast cells.

Cell Line↗
PubMed1999

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.

Basophils↗
PubMed1998

Regulation and possible function of axl expression in immature human mast cells.

The receptor tyrosine kinase Axl which expresses extracellular domains reminiscent of cell adhesion molecules, is involved in homotypic binding as well as in intracellular signaling of myeloid progenitor cells. In order to investigate factors which might influence differentiation pathways through changes of the adhesive properties of cells, we analyzed the expression of axl in immature basophil and mast cell lines and in cultured basophil and mast cell precursors. Axl expression was induced by interferon-alpha in the human leukemic mast cell line HMC-1 and in cultured mast cells derived from CD34+ peripheral blood cells. Axl induction was dose dependent, appeared within 1 h, and was independent of de novo protein synthesis. IFNalpha-treated HMC-1 cells expressing axl formed large cell aggregates within 40 h while untreated cells did not. HMC-1 cells also expressed gas6, the putative ligand of axl, which has been shown to induce axl-mediated homotypic binding. Gas6 expression was independent of interferon treatment in HMC-1 cells. The present results suggest that axl-mediated changes of cellular adhesive properties in mast cells may be important in mast cell differentiation as well as in mast cell-associated inflammation.

Cell Line↗
PubMed1998

Induction of human leukaemic mast cell differentiation by fibroblast supernatants, but not by stem cell factor.

In order to explore the potential existence of human mast cell growth factors other than stem cell factor (SCF), we have compared SCF to L-cell fibroblast supernatants (LCS) during in vitro mast cell differentiation, using human leukaemic mast cells (HMC-1 cells) which contain a gain-of-function mutated SCF receptor (c-Kit) as model. At baseline, cells exhibited an immature phenotype, with <25% being metachromatic or chloroacetate esterase, tryptase and FcepsilonRIalpha positive. Intracellular levels of histamine, tryptase, TNF-alpha and chymase were low, whereas 83% of cells were c-Kit positive. During a 10 day culture with 30% LCS, a significant, time-dependent increase of all mast cell markers, except for chymase and c-Kit, was observed at the protein and for tryptase and FcepsilonRIalpha also at the mRNA level. Cytoplasmatic granulation and stimulated histamine and leukotriene C4 release were increased as well. In contrast to LCS, rhSCF induced none of these changes in HMC-1 cells. On Sephadex G100 fractionation of LCS, HMC-1 cells increased tryptase activity with fractions between 40 and 60, and below 10 kDa, away from the SCF peak. These data show that HMC-1 cells fail to differentiate in response to SCF and that in addition to SCF, LCS contains other human mast cell growth factors.

Animals↗
PubMed1998

Identification of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a possible explanation for divergent clinical behavior.

Mastocytosis represents a mast cell proliferative disease that generally runs a benign clinical course, with spontaneous remissions mostly by puberty in childhood-onset disease, although rare forms, particularly in adult-onset disease, can be associated with (pre)malignant hematologic disorders and very rarely present as mast cell leukemia or malignant mastocytosis. Reasons for this divergent clinical behavior of childhood- versus adult-onset disease are unknown. Recently, two activating mutations in the intracellular domain of the proto-oncogene c-kit, which encodes a tyrosine kinase receptor for the mast cell growth factor stem cell factor, have been detected in the human leukemic mast cell line HMC-1. We have therefore studied lesional skin biopsies from patients with adult- and childhood-onset indolent mastocytosis for the presence of these codon 560 and 816 mutations. C-kit coding DNA sequences were amplified and analyzed by mutation-specific restriction analyses, and mutated polymerase chain reaction products were additionally cloned and sequenced. The codon 816 mutation was found in all six samples from adult patients, but not in any of the 11 specimens from children. In addition, the codon 560 mutation could be demonstrated for the first time in indolent mastocytosis, namely in two of four specimens from adult patients, but not in those from two children. These data thus provide a possible explanation for the divergent clinical behavior of adult- versus childhood-onset indolent mastocytosis, with the first being associated with an activating mutation, possibly as part of a neoplastic process, and the latter representing most likely a reactive process of an as yet unknown pathogenesis.

Adult↗
PubMed1998

Genotype and phenotype of N-acetyltransferase 2 (NAT2) polymorphism in patients with contact allergy.

We investigated whether patients with contact allergy differed from non-contact-allergic, non-atopic controls with regard to genotype and phenotype of the polymorphic enzyme N-acetyltransferase 2 (NAT2). 55 contact-allergic patients recruited from the Information Network of Departments of Dermatology (IVDK) were compared to 85 controls from among local health care personnel. NAT2 activity was calculated from HPLC analysis of the ratio of the caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (1MX) in the urine. NAT2 genotype was determined by polymerase chain reaction (PCR). A statistically significantly increased proportion of rapid acetylators was found in contact-allergic patients. This may have 2 possible implications: acetylation may enhance contact sensitization; or NAT2 status may be a genetic marker for contact sensitizability.

Acetylation↗
PubMed1998

Effects of nerve growth factor (NGF) and other fibroblast-derived growth factors on immature human mast cells (HMC-1).

We have previously shown that fibroblast and keratinocyte supernatants up-regulate expression of mast cell characteristics in the human immature mast cell line HMC-1. This effect could not be induced in HMC-1 cells by the well-known mast cell growth factor stem cell factor (SCF), probably due to mutations of the SCF receptor c-Kit in these cells. Here we report the effects of several known fibroblast- and keratinocyte-derived growth factors, namely nerve growth factor (NGF), basic fibroblast growth factor, platelet-derived growth factor and transforming growth factor-beta, on mast cell differentiation, using HMC-1 cells as a model. NGF, at 0.1-50 ng/ml concentrations, caused a marked, dose-dependent up-regulation of tryptase, Fc epsilon RI and histamine within 10 days of culture, associated with an enhanced expression of mRNA for Fc epsilon RI and mast cell tryptase. On restriction analysis, only mast cell beta-tryptase, but not alpha-tryptase, could be demonstrated. Furthermore, the high-affinity NGF receptor (TrkA) was found at both the transcriptional and protein levels, while expression of the low-affinity NGF receptor was detectable at the mRNA level only. None of the other growth factors caused a significant alteration of the mast cell markers studied when added to HMC-1 cells at concentrations known to be biologically active in other culture systems. Immature human mast cells are thus induced to assume a more mature phenotype in vitro in response to NGF, most probably via stimulation of the high-affinity NGF receptor expressed on these cells. Besides SCF, NGF should therefore be considered as an additional mast cell growth factor that contributes to human mast cell maturation at tissue sites.

Biomarkers↗
PubMed1997

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.

Antibodies, Monoclonal↗
PubMed1997

Demonstration of the high-affinity IgE receptor (Fc epsilon RI) on Langerhans' cells of diseased nasal mucosa.

Langerhans' cells in the skin have recently been shown to bind IgE molecules via the high-affinity IgE receptor (Fc epsilon RI). Using two highly specific antibodies against the antibody-binding alpha-chain of this receptor, 29C6 and 6F7, we demonstrate by immunohistochemistry and immunoelectron microscopy that Langerhans' cells of diseased nasal mucosa can express the Fc epsilon RI. Tissue sections from hyperplastic nasal conchae and nasal polyps of atopic and nonatopic patients have shown no basic differences in epithelial Fc epsilon RI-bearing cells. Only a few cells expressed the low-affinity IgE receptor (Fc epsilon RII) (Tü1 antibody) in some sections. These findings suggest that Langerhans' cells play an important role in the induction of transepithelial IgE-mediated allergy and in the mediation of inflammation of the nasal mucosa via their Fc epsilon RI.

Biopsy↗