Notes: Drop-like, transient blisters of miliaria crystallina may develop with focal intensity of heat within the skin, such as occurs in tropical climates or during febrile episodes. Miliaria crystallina develops due to a transient poral closure of the sweat duct opening, resulting in obstruction of free flow of eccrine sweat and retention in a vesicle below the skin surface. Dual cholinergic and adrenergic sweat gland innervation is influenced by a variety of medications used in intensive care patients. We present two febrile intensive care patients in whom enhanced α-adrenergic stimulation of sweat gland myoepithelia may have led to miliaria crystallina.

Notes: Abstract: In order to shed further light on the potential role of mast cells during tissue turnover, we have investigated the number of mast cells containing only tryptase and those storing both tryptase and chymase by enzyme histochemistry in normal versus healing skin. Furthermore, we have studied the in vitro effect of these enzymes on the mitogenesis of subconfluent quiescent fibroblast and HaCaT keratinocyte cultures, using flowcytometric DNA analysis. Chymase-containing mast cell numbers were markedly decreased in scars (P〈0.001), whereas the overall number of tryptase-containing mast cells was not decreased, although these cells were smaller and stained more faintly in scars. Chymase (5 to 300 mU/ml) induced a marked, dose-dependent in vitro mitogenic response in 3T3 fibroblasts, whereas the effects of tryptase, at up to 60 nM, were only moderate, compared to the known fibroblast mitogens EGF, TGF-α, α-thrombin and trypsin at optimal concentrations. Coincubation of either protease with EGF or α-thrombin had additive effects. In contrast to fibroblasts, keratinocytes showed only minor mitogenic responses to tryptase and chymase, also in comparison to other known mitogenic stimuli, and responses to EGF and α-thrombin were inhibited on costimulation of cells with the proteases. These findings document for the first time a potential role of mast cell chymase in connective tissue repair, with tryptase being less active on fibroblasts, and with inhibitory effects of both mast cell proteases on keratinocytes.

Notes: Abstract: H1-type antihistamines have recently been reported to inhibit cytokine secretion from human and murine mast cells and basophils. In order to confirm and expand these studies, we have compared several H1-blockers and the H2-blocker ranitidine for their effect on TNF-α, IL-3, 6, 8 and GM-CSF release from human leukemic mast (HMC-1) and basophilic (KU812) cells, compared to dexamethasone. Cells were stimulated for 24 h with phorbol myristate acetate (25 ng/ml) and calcium ionophore A 23187 (2.5×10−7 M) alone or with the drugs added at 10−4 to 10−15 M, and production of cytokines was measured by ELISA. All antihistamines caused a dose-dependent inhibition of TNF-α release from HMC-1 cells, with maximal effects at 10−12 M for azelastine, 10−9 M for loratadine and cetirizine, and 10−8 M for ranitidine. The inhibitory potency of H1-blockers on cytokines from HMC-1 cells was TNF-α 〉IL-8≥IL-6≥IL-3, with no significant effects on GM-CSF. In KU812 cells which failed to secrete TNF-α and GM-CSF, the sequence was IL-6 〉IL-8 after preincubation. Dexamethasone inhibited all cytokines, but ranitidine only TNF-α and IL-3. Antihistamines had no effect on calcium flux in resting or stimulated cells. At the mRNA level, inhibition was only seen with KU812 cells and IL-8 in the presence of azelastine at 10−10 M. These data show thus distinct inhibitory patterns for different antihistamines during cytokine production from human mast cells and basophils which may contribute to the anti-inflammatory effects of these drugs during treatment of allergic diseases.

Notes: Mast cells are bone marrow-derived, ubiquitous connective tissue resident cells. However, their mechanisms of migration, the distribution of immature and mature cells and their interaction with other inflammatory cells are largely unclarified. Possibly, β2-integrins play an important role in these processes. In the present investigation, the authors studied the expression and regulation of the β2-integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18) and of the LFA-1/Mac-1 counter-receptor intercellular adhesion molecule-1 (ICAM-1; CD54) on leukaemic (HMC-1 cell subclone 5C6) and on normal mature human skin mast cells. The HMC-1 cells clearly expressed CD11a, CD18 and CD54, while expression of CD11b and CD11c was low. The apparent molecular weights were 180 kDa (CD11a), 95 kDa (CD18) and 90 kDa (CD54) as determined by Western blot analysis. Phorbol myristate acetate (PMA) induced a time- and dose-dependent up-regulation of CD11a, CD11b, CD11c, CD18 and CD54 that was inhibited by cycloheximide, suggesting a dependence on de novo protein synthesis. Enhanced expression of CD11a, CD11b, CD11c and CD18 could also be confirmed at the gene level as demonstrated by semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR). Increased expression of LFA-1/ICAM-1 in response to PMA was accompanied by strong enhancement of homotypic cell aggregation, suggesting that newly synthesized LFA-1/ICAM-1 is functionally active. In order to determine a physiologically relevant way of mast cell β2-integrin modulation, several cytokines and chemotactic mediators (interleukin-4, IL-4; nerve growth factor β, NGFβ; C5a; and leukotriene B4, LTB4) were tested for their influence on adhesion molecule cell surface density. Only LTB4 was shown specifically to up-regulate CD11a and CD18, but not CD11b or CD11c. The presence of CD11a, CD11c and CD18 could be confirmed on a low percentage of normal skin mast cells by immunofluorescence, using a double staining technique. In comparison to normal skin, a significantly higher percentage of CD18+ mast cells was found in inflammatory dermatoses such as psoriasis vulgaris, atopic dermatitis and lichen planus. Therefore, mast cell β2-integrins possibly play an important role during homing of immature mast cells as well as during the interaction of activated mast cells with other inflammatory cells.

Notes: Abstract: Although chronic urticaria is generally thought to be mostly idiopathic, we have recently provided convincing evidence that in the majority of patients, food ingredients provoke the symptoms and sustain the disease. On a diet largely avoiding preservatives, dyes and natural pseudoallergens, 73% of patients experienced remission of more than 6 months duration, starting within the first 3 weeks after initiation of the diet. This response rate is clearly higher than the reported 24% spontaneous remission rate over the same time period. The specificity of the dietary effect was proven 1) by double-blind provocation with pureed pseudoallergen-low versus -rich food and 2) by induction of a clinical response to a 3-week diet low in pseudoallergens, but not to a standard diabetes diet in 3 patients studied in a double-blind crossover design. On double-blind, placebo controlled oral provocation, only 18% of diet-responsive patients reacted to known food preservatives and dyes, but 71% to pureed tomatoes and 44% to their steem extracts. These findings identify naturally occurring pseudoallergens in food as major elicitors of chronic urticaria. In contrast, autoantibodies against Fc-RIα have been identified in only about 30% of chronic urticaria patients, and evidence for their truly causative role is still lacking since therapeutic measures work in patients irrespective of the presence or absence of the autoantibodies. For both food intolerance and Fc-RIα-autoantibodies in chronic urticaria, the associated pathomechanisms are however still in need of clarification. Meanwhile, the diet-responsiveness in the majority of patients opens new perspectives for the management of chronic urticaria.

Notes: Abstract Since the presence of major histocompatibility complex (MHC) antigens has recently been reported on murine and human mast cells under various conditions, we have investigated their expression on mast cells in different types of cutaneous inflammation. Cryostat sections from lesional biopsies of patients with psoriasis, atopic eczema, chronic urticaria, lichen planus, bullous pemphigoid and urticaria pigmentosa were immunohistochemically stained with monoclonal antibodies against MHC class I and class II antigens using a double staining APAAP/toluidine blue methodology. While strongly positive staining with the antibody directed against MHC class I antigens was found on nearly all mast cells in normal skin and in inflammatory dermatoses, reactivity for HLA-DR and HLA-DQ antigens on mast cells could not be detected, except for less than 2% of cells with doubtful staining. Human mast cells therefore probably play no significant rôle as antigen-presenting cells in the conditions studied.

Notes: 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.

Notes: Abstract: Until recently, mast cells have been viewed primarily as harmful because of their key role as effector cells of allergic and potentially lethal anaphylactic reactions. Their contribution to human health appeared instead to be limited to the elimination of parasites. There is, however, growing evidence for additional beneficial functions of mast cells, particularly regarding the initiation of acquired immune reactions. Thus, mast cells can phagocytize diverse particles, take up antigens, and express a number of receptors, particularly MHC class I and II antigens, ICAM-1 and -3, CD43, CD80, CD86 and CD40L which allow them to interact with T and B lymphocytes. They can also secrete numerous cytokines that induce and enhance recruitment and functions of lymphocytes. Finally, there is good evidence that mast cells present e.g. pollen and bacterial antigens, respond to bacterial superantigens, but fail to react to endogenously produced antigens or superantigens. Mast cells can also activate B cells directly to produce IgE, but this activity and the ability to produce IL-4 or IL-13 is restricted primarily to basophil leukocytes and mucosal mast cells. Finally, recent evidence attributes a pivotal role to the cells in natural immunity to bacteria. There is also emerging evidence that mast cells can downmodulate the immune response. While these data require further clarification, the basic ability of mast cells to initiate innate and acquired immune reactions can no longer be questioned.

Notes: Abstract: Different subpopulations of mast cells are characterized by their abundant contents of either tryptase or in addition chymase. These two neutral proteases are found in mast cells and may thus hold a key to the understanding of mast cell dependent reactions. Such studies are however hampered by the lack of readily available supplies of chymase. We have therefore studied the simultaneous purification of both proteases from hairless moro hr/hr mouse skin, using a sequence of salt extractions and chromatographic separations. After three steps of extraction, a 13-fold purification with an 82% yield was obtained for tryptase and a 15-fold purification with a 90% yield for chymase. Further one step purification on conventional sephadex, sephacryl and octyl sepharose columns was unsatisfactory because of further protein contamination of the fractions. Heparin affinity chromatography caused a high loss of tryptase and residual protein contamination. Gradient elution on a benzamidine sepharose 6B column resulted however in a single, low yield (17.9%) tryptase peak and a broader, high yield (〉90%) chymase peak, and a 34% yield high purity fraction. The proteases thus purified exhibited their typical inhibitor profile. On Western blot analysis and on autoradiography in the presence of the serine protease inhibitor diisopropylfuorophosphate (DFP), only one 28 kD molecule with chymase activity was identified, whereas a broad 32-38 kD band of tryptase monomers was noted. Taken together, these data show that, after salt extraction and a single benzamidine affinity chromatography step, both mast cell chymase and tryptase can be separated and in case of chymase also highly purified, allowing thus for the study of biological activities of this molecule.

Notes: Abstract: 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. FcɛRI-positive cultured cells enriched with immunobeads exhibited a 3-fold higher expression of GM3, compared to FcɛRI negative HMC-1 cells. Furthermore, measurable amounts of the gangliosides GM2, GM1 and GD1a were found only in the FcɛRI 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 FcɛRI by RT-PCR. Taken together, these data point to a functional role of gangliosides in the differentiation of human mast cells.