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Affinity cytochemistry analysis of mast cells in skin lesions: a possible tool to assess the timing of lesions after death.

The histamine content in vital wounds is known to increase, with a zenith after 3 h, and then decrease until 24 h after wounding. We addressed whether this biochemical alteration has a morphological counterpart. Since the main source of skin histamine are mast cells, the distribution and number of these cells was assessed upon labeling with fluorescent avidin and with antibodies to the mast cell specific enzymes, chymase and tryptase. Analyses were performed on skin from 15 healthy controls (from surgical biopsies), from 15 post-mortem lesions and 75 vital lesions, obtained at autopsy from subjects who had survived from a few seconds to 24 h. The number of mast cells per unit area of section surface increased progressively with survival time, up to a maximum in subjects who survived 1-3 h ( p<0.01), and decreased thereafter becoming less than in the controls if lesions had occurred earlier than 6 h before death ( p<0.01). Samples from post-mortem lesions had significantly fewer mast cells than those of any other groups of samples ( p<0.01). We suggest that in association to other histological and circumstantial evidence the analysis of mast cells by affinity cytochemistry can help to discriminate vital from post-mortem lesions and to estimate survival time after lesions.

Adult↗

Mast cells and macrophages in normal C57/BL/6 mice.

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.

Animals↗

Chronic urticaria is associated with mast cell infiltration in the gastroduodenal mucosa.

Chronic urticaria (CU) is characterized by recurrent itching skin eruptions caused by mast cell degranulation. Relapses can be provoked by food intake. The aim of this study was to investigate if the mast cell number in the gastroduodenal mucosa is increased in CU patients, and whether mast cell counting by pathologists is clinically useful. We defined two study groups: 50 disease controls (16 Belgians and 34 Italians) and 43 Belgian CU patients. Mast cells were detected using immunohistochemistry for tryptase and CD117. The mast cell number in the disease controls was 20.2/high-power filed (HPF; 133.3/mm2) in the stomach and 32.5/HPF (209.2/mm2) in the duodenum. There was no difference between Belgian and Italian controls, indicating that dietary habits have no influence on the normal gastroduodenal mast cell number. In CU patients, mast cell numbers were significantly higher: 32.4/HPF (186.0/mm2) in the stomach (P<0.0001) and 44.8/HPF (246.0/mm2) in the duodenum (P=0.0002). CU is thus associated with mast cell infiltration in the gastroduodenal mucosa, even if patients do not have gastrointestinal symptoms. Mast cell counting in gastroduodenal biopsies of CU patients can be useful in selecting patients who may respond to a therapy with intestinal mast-cell-stabilizing agents.

Adolescent↗

Mast cells in chronic rejection of human renal allografts.

An increased number of mast cells (MCs) is found in renal specimens of patients with diseases associated with persistent chronic inflammation. MCs proliferation is partly dependent on the presence of T lymphocytes. Both chronic inflammation and T-lymphocytes are essential in the development of chronic rejection (CR), and probably for the infiltration of MCs. MC-derived products such as heparin, histamine, and serine proteases may be responsible for endothelial proliferation and excess collagen production by fibroblasts. In this study, a quantitative evaluation of the MCs infiltration in kidney allografts with CR is performed. The extent of renal fibrosis was analysed in samples stained with Masson's trichrome. To evaluate the potential relationship between MCs and fibrosis in CR we analysed 30 kidneys with CR (25 from nephrectomies and 5 from autopsies). Ten transplanted kidneys obtained from patients died by causes not related with rejection were used as controls. CR was graded according to the Banff schema, which assesses the degree of vasculopathy, tubular atrophy, interstitial fibrosis and transplantation glomerulopathy. Giemsa-stained sections and immunohistochemistry using anti-MC tryptase and c-kit monoclonal antibodies were used to detect MCs. The mean number of MCs per 20 high-power fields (HPF) in the transplanted kidney with CR was 101.8+/-15.3 in the renal cortex and 46.60+/-6.52 in the medulla. MCs were significantly more numerous in CR with respect to normal kidneys, both in the cortex (P<0.01; Mann-Whitney U test) and in the medulla (P<0.01; Mann-Whitney U test). There was a positive correlation between the number of MCs and extent of fibrosis (P<0.01; Kruskal-Wallis one-way anova test) and tubular atrophy (P<0.01). These results suggest that MCs may play a role in the process of development of interstitial fibrosis in CR.

Adolescent↗

Localization of vascular endothelial growth factor in synovial membrane mast cells: examination with "multi-labelling subtraction immunostaining".

Mast cells are believed to play a novel part in the development of destructive synovial pannus in rheumatoid arthritis (RA). This study was undertaken to investigate the localization of vascular endothelial growth factor (VEGF) in the synovial membrane using a unique immunostaining technique. Synovial specimens of RA patients were examined immunohistochemically and were compared with specimens from non-RA controls. Multi-labelling subtraction immunostaining, a modification of double- and triple-labelling immunostaining, revealed that the VEGF-positive cells were identical to tryptase-positive cells (mast cells). No other cell types were found to be positive for VEGF. The synovium of RA patients showed a larger number of VEGF-positive mast cells than that of non-RA controls (P<0.001). The study suggests that mast cell-derived VEGF may contribute to the development of synovial pannus in RA.

Arthritis, Rheumatoid↗

Mast cells in human bile duct obstruction.

Surgical biopsy specimens obtained from 50 patients with secondary cholangitis caused by obstruction of the common bile duct were studied immunohistochemically. Data on the number and ultrastructural appearances of mast cells positive for tryptase, chymase, vasointestinal polypeptide (VIP), and substance P (SP) were obtained. The bile ducts from patients presenting combined chronic exacerbated cholangitis and chronic sclerotic cholangitis showed significantly higher numbers of mast cell types compared to the controls (P < 0.0001). Cases with sclerotic cholangitis alone had significantly lower number of cells than patients with chronic exacerbated cholangitis alone (P < or = 0.0001). Morphometric measurements of electron micrographs showed that mast cell granules containing VIP, SP and chymase were commensurable in size. Electron-lucent granules without reaction product (altered granules) and granules with focal distribution of the reaction product were observed in all types of mast cells. Furthermore, some nerve fibers positive for SP and VIP and serotonin-positive endocrine cells were observed in close proximity to the mast cells. In conclusion, the results of our study demonstrate the existence of different populations of mast cells, nerve structures and endocrine cells in the lower part of the human large bile duct, and suggest their participation in the development of pathological processes.

Adult↗

Preformed enzymes in mast cell granules and their potential role in allergic rhinitis.

Mast cells not only function as effector cells but also influence nearly every other cell involved in causing allergic rhinitis. Mast cell-derived mediators such as histamine, bradykinin, tryptase, and the arachidonic acid derivatives produce the symptoms of the early-phase reaction of allergic rhinitis and also attract and activate other leukocytes involved in the late-phase reaction. In addition, activated mast cells are known to secrete a number of cytokines, both preformed and newly synthesized, that can modulate T- and B-cell function, propagate the early- and late-phase reactions, and contribute to tissue remodeling. Most currently available therapies work by antagonizing the mediators secreted by mast cells and other leukocytes. With the possible exception of immunotherapy, these therapies do not provide long-term protection against allergic disorders. Exciting new developments in gene-based therapies seem promising in both reducing and reversing the development of allergic rhinitis.

Animals↗

New targets for modifying mast cell activation in asthma.

Mast cells play a central role in innate immunity and in orchestrating the asthmatic response. Current medication relies on beta-agonists to relieve bronchoconstriction and steroids to reduce inflammation. However, recently drugs such as leukotriene-receptor antagonists and anti-immunoglobulin E have come on to the market. In this paper, a number of potential targets for modifying mast cell activation in asthma are reviewed. Some are already under study, including clinical trials (eg, tryptase inhibitors); others are more speculative (eg, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity). In each case, where data are available, the action of the agents on human lung mast cells is described.

Animals↗

Plasma protein leakage and local secretion of proteins assessed in sputum in asthma and COPD. The effect of inhaled corticosteroids.

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation with cell infiltration, increased plasma exudation and abnormal local secretion of proteins. We have analysed whether sputum differs in this respect between asthma (n = 9) and COPD (n = 9), and whether inflammatory markers in sputum are affected by treatment. In non-smoking asthma patients there was more plasma protein leakage, based on the relative coefficient of excretion Q alpha 2macroglobulin/QIgG (P = 0.03). There was less local secretion of sIgA and lactoferrin than in COPD (P < 0.05). Tryptase was slightly higher in sputum from asthma than from COPD (P < 0.05), whereas eosinophil cationic protein and myeloperoxidase were similar. After treatment with glucocorticosteroids, there was a reduction in the Q alpha 2macroglobulin/Qalbumin (P < 0.015), but no effect was seen on the levels of products from local cells. We conclude that sputum analysis is useful to study the local inflammatory process in asthma and COPD.

Administration, Inhalation↗

The modulation by nedocromil sodium of proteases released from rat peritoneal mast cells capable of degrading vasoactive intestinal peptide and calcitonin gene-related peptide.

Tryptase and chymase released from activated mast cells degrade the neuropeptides calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) to peptide fragments. We have examined whether nedocromil sodium can modulate the ability of rat activated peritoneal mast cells to degrade 125I-CGRP and 125I-VIP. Mast cell-dependent degradation of both 125I-CGRP and 125I-VIP was observed with compound 48/80 (0.03-1 microgram/ml) and in the case of 125I-VIP with anti-IgE (1-20 micrograms/ml). Nedocromil sodium (10(-6)-10(-4) M) caused significant inhibition of neuropeptide degradation, with the most effective inhibition observed against anti-IgE-induced degradation of 125I-VIP. Nedocromil sodium had no inhibitory effect on the ability of lysed mast cells, bovine trypsin or chymotrypsin to breakdown 125I-VIP. These results suggest that nedocromil sodium inhibits mast cell-dependent degradation of neuropeptides, such as VIP, as a secondary consequence of inhibiting the release of mast cell proteases.

Animals↗

Immunological characterization and functional importance of human heart mast cells.

Mast cells are present in normal and even more abundant in diseased human heart tissue and their localization is of particular relevance to their function. Within heart tissue mast cells lie between myocytes and in close contact with blood vessels. They are also found in the coronary adventitia and in the shoulder regions of a coronary atheroma. The density of cardiac mast cells is markedly higher in some patients with myocarditis and dilated cardiomyopathy than in accident victims without cardiovascular diseases. More importantly, in some of these conditions there is in situ evidence of mast cell activation. We have described an original technique to isolate and purify HHMC for in vitro study. This procedure gives viable cells and after stimulation with immunological or non-immunological stimuli they release performed (histamine and tryptase) and newly generated mediators (PGD2 and LTC4). We have demonstrated that HHMC differ from those in other anatomical districts in that they are activated by specific immunological and non-immunological stimuli, and in their relation to the arachidonic acid metabolism, suggesting that the local microenvironment can influence their phenotypic and biochemical characteristics. Our own and other findings suggest that HHMC have complex and significant roles in different pathophysiological conditions involving the cardiovascular system. Direct activation of HHMC by therapeutic and diagnostic substances injected intravenously explains some of the anaphylactoid reactions caused by these agents. HHMC possess Fc epsilon RI and IgE bound to the surface and C5a receptors, which could explain how cardiac mast cells are involved in systemic and cardiac anaphylaxis. Cardiac mast cells and those in human coronary arteries also play a role in the early and late stages of atherogenesis and during ischemic myocardial injury. In conclusion, although studies of HHMC are in their infancy, their in vitro isolation may be useful in identifying additional mediators synthesized and released, stimuli relevant to human pathophysiology, and pharmacological agents selectively modulating the activation of these cells and their mediators. Drugs specifically acting on HHMC or on their mediators may eventually be useful in treating different cardiovascular diseases.

Anaphylaxis↗

Correlation of gingival crevicular fluid proteases with clinical and radiological measurements of periodontal attachment loss.

Probing attachment loss and radiographical measurements of bone loss were made on 20 untreated chronic periodontitis patients. At a second visit, gingival crevicular fluid was collected on filter paper strips from the deepest accessible interdental probing site of each tooth. Gingival crevicular fluid volumes were determined and the samples eluted into buffer. Protease activities in the resulting eluates were assayed with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin (AFC). Cathepsin B/L-like activity was determined with Bz-Val-Lys-Lys-Arg-AFC, elastase-like activity with MeOSuc-Ala-Ala-Pro-Val-AFC, tryptase-like activity with Z-Ala-Ala-Lys-AFC, trypsin-like activity with Z-Gly-Gly-Arg-AFC and dipeptidyl peptidase IV-like activity with Ala-Pro-AFC. Total enzyme activities and enzyme concentrations correlated positively with probing attachment loss and bone loss in linear regression analysis. This was true at both a patient level, using mean patient values, and a site level, using either individual patient or pooled patient data. All of these correlations were highly statistically significant for site comparisons. In inter- and intra-patient comparisons the proportion of significant correlations was greater for total enzyme activity than concentration. Clinical and radiological measurements of attachment loss showed generally similar levels of correlation. Total enzyme activities had good specificity and sensitivity as indicators of attachment loss in this cross-sectional study. The results support further investigation of the diagnostic potential of gingival crevicular fluid proteases in evaluation of the periodontal condition.

Adult↗

Investigations on plasma levels of mast cell mediators in acute atopic dermatitis.

Skin mast cells have been proposed to be involved in the pathogenesis of acute and chronic phases of atopic dermatitis (AD). The aim of the present study was to investigate the significance of different mast cell mediators during acute exacerbation of this frequent skin disease. Plasma levels from 19 patients with AD were screened for elevation of the mast cell-specific protease tryptase, the biogene amine histamine, and the arachidonic acid metabolite prostaglandin D2. None of the patients showed elevated plasma levels of these three mediators, whereas the mean serum IgE level was strongly elevated. We conclude that the investigated mediators are either only active on the cutaneous level or that other mediators are responsible for the development of the acute eczematous and pruritic skin reactions. Alternatively, the assays could have been insufficiently sensitive since some studies have demonstrated increased plasma histamine levels, e.g. after food challenge of such patients.

Acute Disease↗

Requirement of c-jun transcription factor on the mouse mast cell protease-6 expression in the mast cells.

Mast cell tryptases may be a key mediator in mast cell-mediated inflammatory reactions, and these expressions can be regulated by microenvironmental factors of tissues, particularly stem cell factor. In the present study, we investigated whether the transcription of mouse mast cell protease-6 (mMCP-6) gene was caused by SCF-mediated c-jun. We observed that mMCP-6 mRNA was expressed by overexpression of c-jun in the immature mast cell line in which both mMCP-6 and c-kit receptor are negative. The c-jun increased synergistically the luciferase activity of mMCP-6 promoter through the direct interaction with mi transcription factor (MITF). The synergic effect of c-jun with MITF was abolished by deletion of sequence between nt -171 and -151 in the mMCP-6 promoter. Furthermore, the level of mMCP-6 mRNA in mast cells was attenuated by the introduction of dominant negative c-jun (TAM-67) and the treatment of Jun N-terminal kinase inhibitor, SP600125. These results show that c-jun might play a role in regulating the transcription of mMCP-6 gene in mast cells stimulated by SCF.

Animals↗

IL-6 levels predict disease variant and extent of organ involvement in patients with mastocytosis.

Mastocytosis is often associated with organ involvement and hematological disorders. Patients may also exhibit elevated levels of plasma IL-6. To gain insight into the relevance of this observation, we correlated plasma levels of IL-6 and soluble IL-6 receptor (sIL-6R) with multiple disease parameters in 29 patients with mastocytosis. Mean plasma IL-6 levels were elevated in patients compared to healthy controls (P < 0.0001). Disease category significantly correlated with plasma IL-6 levels, as did severity of bone marrow pathology, organomegaly, and extent of skin involvement. In plasma, there was a positive correlation of IL-6 to total tryptase, alkaline phosphatase, IgM, white blood cell count, prothrombin time, partial thromboplastin time, and neutrophil numbers. There was an inverse correlation to hemoglobin. sIL-6R levels were not elevated. These observations demonstrate that IL-6 is a useful surrogate marker of severity of hematologic disease and suggest that IL-6 contributes to pathology.

Adult↗

Antioxidant defence during cardiopulmonary bypass surgery.

OBJECTIVE: Cardiac surgery may lead to severe oxidative stress due to formation of oxidation products generated during ischemia and reperfusion. We investigated to which extent oxidative stress influences a number of endogenous antioxidants and markers of cellular activation. METHODS: At six time points blood was withdrawn from patients undergoing coronary artery bypass grafting, using the on-pump procedure. RESULTS: Both glutathione peroxidase and superoxide dismutase show a gradual and strong increase in activity during surgery (40 and 30%, respectively), returning to baseline values 24 h after surgery. The total antioxidant capacity has a maximum increase of 60%. Markers of cellular activation, such as eosinophil cationic protein and tryptase also increase during the procedure. CONCLUSION: Cardiac surgery results in systemic inflammation accompanied or caused by severe oxidative stress. The human body has a strong innate oxidative defence screen, which is probably not sufficient to fully compensate for the total amount of oxidative damage.

Aged↗

Hymenoptera sting-induced Kounis syndrome: effects of aspirin and beta-blocker administration.

Kounis syndrome is the concurrence of acute coronary syndromes with conditions associated with activation of interacting inflammatory cells including allergic or hypersensitivity and anaphylactic or anaphylactoid insults. It is caused via inflammatory mediators released during inflammatory cell activation. A variety of conditions, drugs, and environmental exposures can induce Kounis syndrome. A patient suffering from coronary artery disease and taking metoprolol and aspirin was stung by wasps and developed cutaneous allergic signs including rash, urticaria and orbital oedema. This was followed by retrosternal pain, chest discomfort and electrocardiographic changes compatible with acute myocardial ischemia. Cardiac enzymes, troponins and blood pressure remained normal but serum tryptase was raised. The clinical implications and pathophysiology of this rare association are discussed.

Adrenergic beta-Antagonists↗

Diagnosis and classification of mast cell proliferative disorders: delineation from immunologic diseases and non-mast cell hematopoietic neoplasms.

In mast cell (MC) disorders (mastocytosis), clinical symptoms are caused by the release of chemical mediators from MCs, the pathologic infiltration of neoplastic MCs in tissues, or both. Cutaneous mastocytosis is a benign disease in which MC infiltration is confined to the skin. In pediatric cases cutaneous mastocytosis might regress spontaneously. Systemic mastocytosis (SM) is more frequently diagnosed in adults and is a persistent (clonal) disease of bone marrow-derived myelomastocytic progenitors. The somatic c-kit mutation D816V is found in the majority of such patients. The natural clinical course in SM is variable. Whereas most patients remain at the indolent stage for many years, some have aggressive SM (ASM) at diagnosis. Other patients have an associated clonal hematologic non-MC lineage disease (AHNMD). MC leukemia (MCL) is a rare disease variant characterized by circulating MCs and fatal disease progression. The diagnoses of ASM, SM-AHNMD, and MCL might be confused with a variety of endocrinologic, vascular, or immunologic disorders. It is therefore of particular importance to be aware of the possibility of an underlying (malignant) MC disease in patients with unexplained vascular instability, unexplained (anaphylactoid) shock, idiopathic flushing, diarrhea, headache, and other symptoms that might be mediator related. An important diagnostic clue in such cases is an increased serum tryptase level. The current review provides an overview of mastocytosis and its subvariants and a practical guide that might help to delineate mastocytosis from unrelated systemic disorders.

Adult↗