NKp46 regulates the production of serine proteases and IL-22 in human mast cells in urticaria pigmentosa.

NKp46 (natural cytotoxic receptor 1/CD335) is expressed on natural killer cells and Th2-type innate lymphocytes. However, NKp46 expression in human mast cells has not yet been reported. Here, we explored the expression of, and possible role played by, NKp46 in such cells. NKp46 protein was expressed in human mast cells in urticaria pigmentosa principally of the tryptase-positive/chymase-negative type (MCT), but not in human non-neoplastic skin mast cells of the tryptase-positive/chymase-positive (MCTC) type. NKp46 expression was also evident in the human neoplastic mast cell line HMC1.2. NKp46 knockdown changed the phenotype of this cell line from MCT to MCTC and downregulated GrB production, but did not influence IL-22 production. An agonistic anti-NKp46 antibody upregulated production of GrB and IL-22, but did not change the MCT-like phenotype of HMC1.2 cells. NKp46 was thus involved in the production of serine proteases and IL-22 in human mast cells.

Serum tryptase and SCORMA (SCORing MAstocytosis) Index as disease severity parameters in childhood and adult cutaneous mastocytosis.

BACKGROUND: Skin lesions are the predominant clinical feature of the commonest form of mastocytosis. Mastocytosis is classified according to World Health Organization criteria. Determination of the levels of mast-cell mediators or their metabolites reflects the mast-cell burden. The extent of cutaneous mastocytosis can be assessed clinically using a scoring system (SCORing MAstocytosis; SCORMA Index) that we have developed. OBJECTIVE: Serum tryptase levels were compared with the SCORMA Index in a large group of paediatric and adult patients to investigate whether there was any correlation between the two. METHODS: The SCORMA Index in 64 patients (31 children and 33 adults) was compared with serum tryptase levels. The results of the first visit at which SCORMA and tryptase were evaluated were analysed. RESULTS: There was a positive correlation between the SCORMA Index and serum tryptase levels, indicating the value of the SCORMA Index in the assessment of mastocytosis with skin involvement. CONCLUSION: The results of this study showed that the SCORMA Index is a useful tool for evaluating the severity of cutaneous mastocytosis. The correlation between the SCORMA Index and serum tryptase levels underlines the benefit of the SCORMA Index as a clinical tool. Repeated SCORMA Index measurements can provide a rapid impression of changes in the clinical state of mastocytosis. This is particularly relevant in children, because taking blood samples from this group is much more difficult. The well-established methods for evaluation of disease severity may be expanded by the rapid SCORMA Index method.

Serum tryptase measured with B12 and G5 antibody-based immunoassays in mastocytosis patients and its relation to histamine turnover.

Serum tryptase was measured with the B12 and G5 antibody-based immunoassays in 25 adult patients with mastocytosis and in 18 controls. Twelve patients had uncomplicated cutaneous mastocytosis (urticaria pigmentosa) and 13 had urticaria pigmentosa with systemic symptoms. Tryptase levels were compared with histamine turnover estimated as urinary excretion of the main histamine metabolite tele-methylimidazoleacetic acid. Elevated B12 tryptase levels (> 20 microg/L) were found in most mastocytosis patients, including five of eight patients with only cutaneous manifestations who had a low urinary histamine metabolite excretion. This indicated a higher sensitivity for diagnosing mild mastocytosis on the basis of levels of serum tryptase as opposed to urinary methylimidazoleacetic acid. However, the serum B12 tryptase assay could not differentiate between urticaria pigmentosa patients with and without systemic disease: the measurement of histamine metabolite excretion probably reflects the mast cell burden more accurately. Serum G5 tryptase levels were generally low in both controls and mastocytosis patients.

No relationship between histamine release measured as metabolite excretion in the urine, and serum levels of mast cell specific tryptase in mastocytosis.

To test the hypothesis that histamine release in mastocytosis patients generally occurs without activation of the mast cells, histamine turnover, measured as histamine metabolite excretion in the urine, was compared with the serum level of mast cell specific tryptase, which is released only during active discharge of mast cell granular contents. Twenty mastocytosis patients with a wide range of histamine turnover rates were investigated. Slightly increased levels of tryptase were found in seven patients with no obvious relationship to histamine metabolite excretion. In contrast, there seemed to be a connection between the tryptase level and the severity of symptoms. These results strengthen the view that histamine in mastocytosis is predominantly released from the mast cells without any accompanying active release process. This does not exclude the possibility that, in some mastocytosis patients, a limited number of mast cells, or a subpopulation, may be actively secreting histamine together with tryptase.

Determination of the primary structures of human skin chymase and cathepsin G from cutaneous mast cells of urticaria pigmentosa lesions.

This study establishes the primary structure of human skin chymase and provides further evidence for the presence of a cathepsin G-like proteinase within human mast cells. The amino acid sequence of human skin chymase was established by protein methods and by analysis of PCR amplification products obtained with cDNA-derived from urticaria pigmentosa (UP) lesions. UP is a disease characterized by skin lesions containing high numbers of mast cells. Proteolytic digests of human chymase purified from normal skin yielded 10 resolvable peptides that were sequenced by automated Edman degradation. The amino acid sequences for these peptides combined with the sequence obtained for the protein's NH2-terminal region (35 residues) accounted for 137 residues of the human skin chymase sequence. This partial amino acid sequence corresponded to the sequence of human heart chymase, a proteinase isolated from heart tissue with immunologic and hydrolytic properties similar to skin chymase. PCR amplification of UP-derived cDNA with primers based on the cDNA structure of heart chymase demonstrated a single amplification product of expected size which was subcloned and sequenced. The amino acid sequence (135 residues) deduced from this product was identical to that of heart chymase in the region between the primers. This sequence, along with that established for the purified protein, constituted 99% of the heart chymase primary structure, strongly indicating that human skin and heart chymases have identical primary structures. Amplification of the same UP-cDNA with primers coding for the NH2- and COOH-terminal sequences of human neutrophil cathepsin G also produced a specific amplification product which was sequenced. The deduced amino acid sequence between the primers was identical to that reported for neutrophil cathepsin G, indicating that the protein of cutaneous mast cells previously shown to be immunologically cross-reactive with neutrophil cathepsin G has a comparable amino acid sequence. UP-cDNA demonstrating amplification products for cathepsin G did not demonstrate amplification products for human neutrophil elastase, suggesting that the cathepsin G PCR amplification product was not derived from neutrophils or monocytes possibly contaminating the lesion. These studies provide further evidence that human skin mast cells contain two different chymotrypsin-like proteinases.

Increased tryptase levels in suction-blister fluid from patients with urticaria.

The levels of tryptase in the suction-blister fluid from patients with chronic urticaria, urticaria pigmentosa, cholinergic urticaria, urticarial dermographism, prurigo of unknown origin, eczema, psoriasis, atopic dermatitis, and from healthy controls were studied. The blister fluid from controls contained up to 15 micrograms/l of tryptase, whereas that from patients with active urticaria contained greater than 50 micrograms/l. This study demonstrates that patients with urticaria have mast cells that readily release tryptase in both the lesional and non-lesional areas of skin.

Plasma esterase activity in patients with aspirin-sensitive asthma or urticaria.

Plasma aspirin esterase activity and cholinesterase activity were reduced in patients with aspirin sensitive asthma and aspirin sensitive urticaria compared to asthmatic and dermatological controls. Phenylacetate (non specific) esterase activities, were however unaltered in these patients. The reason for the lower activity is uncertain but it does not appear to be due to genetically determined lower cholinesterase or due to the avoidance of aspirin by sensitive patients. A low aspirin esterase activity may be a contributory factor in precipitating these aspirin sensitive reactions.

Comparative cytochemical study of dipeptidyl aminopeptidase (DAP) II and IV in normal and malignant haemic cells.

Modified cytochemical methods were used to show dipeptidyl aminopeptidases (DAP) II and IV in peripheral blood buffy coat preparations and bone marrow smears. In 23 normal buffy coats both enzymes were confined to lymphocytes. DAP II was found in T and B lymphocytes (about 80%) while DAP IV was restricted to T lymphocytes only (around 46%). In 11 normal bone marrows DAP II was found in 53% of the lymphocytes, as well as in plasma cells, macrophages, and occasional myeloblasts. DAP IV was found only in lymphocytes (around 32%). DAP II activity, but not DAP IV activity, was present in all of the mast cells in a case of systemic mastocytosis. Whereas DAP II was found, to a variable extent, in leukaemic myeloblasts, monoblasts, proerythroblasts, and in megakaryoblasts in 52 cases of acute myeloid leukaemia, DAP IV was not shown. Variable positivity to DAP II and DAP IV was found in the lymphoblasts in seven cases of acute lymphoblastic leukaemia, in 14 cases of B chronic lymphocytic leukaemia, and in three cases of non-Hodgkin's lymphoma. DAP II activity was variable compared with DAP IV activity, which was constantly reduced. Virtually all of the myeloma cells (96%) all of the myeloma cells (96%) in five cases of multiple myeloma and two cases of plasma cell leukaemia were DAP II positive and DAP IV negative. In 10 cases of hairy cell leukaemia most hairy cells were positive to DAP II (74%) with no demonstrable DAP IV activity. In a single case of Sézary's syndrome around 90% of the helper T cells were positive to DAP II with no DAP IV activity.

Serine proteinases in human cutaneous mastocytosis.

The main chymotryptic and tryptic proteinases of human skin were found in high-salt extracts of human dermis. The levels of these enzymes were markedly increased in salt extracts of human cutaneous mastocytosis as compared to the levels found in extracts of involved skin from the same patients, human cutaneous hemangiomas, and normal human skin. These data suggest that the chymotryptic and tryptic proteinases of human skin are primarily of mast-cell origin.

Peroxidase activity in human cutaneous mast cells: an ultrastructural demonstration.

An intense and reproducible peroxidase staining in the cutaneous mast cells of two patients with systemic mast cell disease and urticaria pigmentosa is demonstrated at the ultrastructural level. This enzyme activity was demonstrated by use of a cytochemical technique employing 3,3'- diaminobenzicine (DAB) as an oxidizable substrate, after fixation by a tannic acid-aldehyde mixture. Enzyme activity was localized in the perinuclear cisterna and strands of endoplasmic reticulum. Granules appeared unreactive. This peroxidase activity appears sensitive to fixation by aldehydes; it is inhibited by 3-amino-1,2,4-triazole (AMT) and by lack of H2O2 or DAB in the incubation medium. These characteristics are fundamentally different from the peroxidase activity of basophils, and the demonstration of this enzyme is therefore not a further argument for a common ontogenetic origin of both cells. On the other hand, the cytochemical characteristics of this enzyme are very similar to those of platelet peroxidase (P-PO), which has been connected to the synthesis by platelets of prostaglandins. Since the mast cell is known to generate prostaglandins, the relationship between the enzyme described and prostaglandin synthesis by mast cells is discussed.

[Occurrence of naphthol-AS-D-chloracetate-esterase-activity in mast cells with special references to cutaneous mastocytoses (author's transl)]. / Nachweis der Naphthol-AS-D-Chloracefat-Esterase-Aktivität in Mastzellen unter besonderer Berücksichtigung cutaner Mastocytosen.

The enzyme naphthol-AS-D-chloracetate esterase (NASDCE) was demonstrated histochemically in mast cells of tissues of laboratory animals and men under normal and pathological conditions using the method after Leder (1964). Investigating the possible action of different fixations as Bouin's and Carnoy's fluid as formaldehyde also cryostate sections were studied for comparison. After tissue fixation the activity of the NASDCE is localized strongly in the granules of mast cells. The type of fixation is without influence on the histochemical reaction and localization. Differences concerning the investigated species could not be demonstrated. In benign neoplasias of mast cells such as diffuse (urticaria pigmentosa) and localized cutaneous mastocytosis as well as benign systemic mastocytosis with skin involvement (Sagher) the granule bound reaction is very intensive. The NASDCE can be recommended as a marker for the microscopical localization of mast cells, especially in the histological diagnosis of their benign and malignant neoplasias.

[Peroxidase deficiency in neutrophils in systemic mastocytosis (author's transl)]. / Déficit en peroxydase des granulocytes neutrophiles au cours d'une mastocytose diffuse.

A case associating a systemic mastocytosis and an acquired myeloperoxidase deficiency is reported. The myeloperoxydase deficiency is studied by cytochemical techniques in optical and electron microscopy and confirmed by biochemical measures. An important defect in bactericidal and candidacidal activity is demonstrated in vitro in P.N.M. The authors discuss the links between the two anomalies which might bring one more argument for the common origin of both granulocytes and mastocytes.