In situ detection of the mast cell proteases chymase and tryptase in human lung tissue using light and electron microscopy.

Scroll-rich, "mucosal" mast cells are the predominant human lung mast cell type. It has been proposed that these mast cells store tryptase but are mostly chymase deficient. We present a detailed immunolocalisation study of chymase and tryptase in lung specimens of eight patients. Using monoclonal antibody B7 in a conventional tissue processing method for light microscopy, chymase-positive mast cells were much fewer than tryptase-positive ones. However, they approached the number of tryptase-positive cells when optimised processing was used. Two different monoclonal antibodies, B7 and CC1, were used to visualise chymase in purified lung mast cells of two patients using ultrastructural immunogold labelling. Immunoabsorption controls demonstrated a reactivity of B7 with both tryptase and chymase, but indicated specificity of CC1 for chymase. On the ultrastructural level, all of more than 1,400 lung mast cells evaluated labelled for chymase. Reactivity was seen in cytoplasmic granules, cytoplasm and vesicles, but not elsewhere. Tryptase labelling using monoclonal antibody G3 was also present in all mast cells detected, and was retained in altered granules (=activated mast cells), where B7 labelling was sparse. The average labelling density was approximately sixfold higher than for chymase. In summary, chymase may be more abundant in human lung mast cells than hitherto thought.

Mast cell granule composition and tissue location--a close correlation.

This review provides a survey on mast cell heterogeneity, with aspects differing in humans and rodents or which are subject of conflicting evidence being discussed in greater detail. Mast cell subsets have been first defined in rats by their fixation and dye-binding properties, and detailed studies in humans and pigs reveal very similar observations. The dye-binding properties of rat mast cell subsets are causally related to the absence or presence of heparin in their granules. In humans, this relation has not been shown. Rodent mast cell subsets store different chymase-isoforms. In contrast, just a single chymase has been defined in humans, and mast cells are classified by the presence or relative absence of this chymase. Different investigators find quite different proportions of chymase-positive to chymase-negative mast cells. Tryptase(s) are found in most or every human mast cell, but in rodents, they have hitherto been essentially localised to mast cells in connective tissues. Human mast cell subsets may also be defined by their expression of receptors such as C5aR and possibly the beta-chemokine receptor CCR3; the CCR3 expression seems to be related to the human mast cell chymase expression. Ultrastructural studies are helpful to distinguish human mast cell subsets, and allow to distinguish between chronic and acute activation. The phenotypical characteristics may change in association with inflammation or other disease processes. Studies in humans and pigs show changed dye-binding and fixation properties of the granules. Experimental rodent infection models reveal similar changes of chymase isoform expression. Human lung mast cells have been reported to strongly upregulate their chymase content in pulmonary vascular disease. This line of evidence can explain some inconsistent information on mast cell heterogeneity and may help to understand the physiological role of mast cells.

Phenotypic and functional characterization of mast cells derived from renal tumor tissues.

Mast cells (MCs) originate from multipotent hematopoietic progenitor cells. However, MCs in various organs are heterogenous in terms of mediator or receptor expression and response to diverse stimuli. We characterized the phenotype and functional properties of human renal mast cells (HRMCs). Tissue was obtained from 17 patients suffering from renal tumors (transitional cell carcinoma, n = 4; renal cell carcinoma, n = 13). HRMCs were isolated by collagenase digestion. Double staining with toluidine blue and immunofluorescence using monoclonal antibodies (mAbs) revealed expression of stem cell factor (SCF)-receptor (c-kit/CD117), CD9, CD29, CD33, CD43, CD44, CD54, and CD63 on HRMCs. In contrast, HRMCs were not recognized by mAbs to CD2, CD3, CD4, CD11b, CD14, CD15, CD16, CDw17, CD19, or CD23. HRMCs were also negative for CD116 (granulocyte-macrophage colony-stimulating factor [GM-CSF] receptor alpha), CD123 (interleukin [IL]-3Ralpha), CD121a (IL-1R type I), CD122 (IL-2Rbeta), and CD127 (IL-7R) and were also found to lack C5aR (CD88). Ligand-induced activation of HRMCs through immunoglobulin (Ig)E-R or SCF-R (c-kit) resulted in histamine secretion (control: <10%; alphaIgE, 1 microg/mL: 50.12 +/-5.18%; rhSCF, 100 ng/mL: 29.24 +/- 22.39), whereas recombinant C5a, erythropoietin (EPO), IL-1 through 10, and GM-CSF exerted no effects. As determined by in situ staining, HRMCs contained tryptase, but only low or undetectable amounts of chymase. Electron microscopy confirmed the presence of MCs in renal tissues and revealed a scroll-rich granule population in HRMCs. Together, HRMCs are tryptase+, C5aR- mast cells exhibiting phenotypic and functional properties similar to those of lung MCs.

Endothelial cell compatibility of fluoroquinolone solutions for intravenous use.

One local side-effect closely related to the use of parenteral fluoroquinolones is phlebitis. The occurrence of this phenomenon is largely thought due to the damage of endothelial cells with subsequent inflammation. In order to evaluate the effect of ciprofloxacin, fleroxacin, and ofloxacin on the viability of human umbilical venous endothelial cells (HUVEC), intracellular ATP levels were measured by a luciferin-luciferase assay. Prostacyclin (PGI2) and thromboxane A2 (TXA2) were determined by means of direct radioimmunoassay. Commercially available preparations of ciprofloxacin (2 mg/ml) and fleroxacin (4 mg/ml) reduced the intracellular ATP content by 75.9 +/- 1.9% and 82.1 +/- 0.6%, respectively, within 20 minutes, indicating severe damage of endothelial cells. Incubation with ofloxacin (2 mg/ml) did not have any detrimental effect. All fluoroquinolones were tolerated well by endothelial cells at low concentrations up to 20 micrograms/ml. Concentrations between 100-200 micrograms/ml gradually led to functional alterations such as increased PGI2 release. The tolerance of intravenously applied antibiotics has been tested in animal models. Use of human venous endothelial cells for testing antibiotic solutions for intravenous application provides a valuable alternate model for tolerability.

Number, fixation properties, dye-binding and protease expression of duodenal mast cells: comparisons between healthy subjects and patients with gastritis or Crohn's disease.

There is an accumulation of evidence to suggest that mast cells may play a key role in gastrointestinal inflammation. We have investigated the numbers and heterogeneity in staining properties of mast cells in biopsies of the duodenum of normal subjects (n = 10), and of normal duodenum from patients with Crohn's disease of the ileum and/or colon (n = 7) or with Helicobacter-associated gastritis of the antrum/corpus (n = 6). In normal donors, two subsets of mast cells, one located in the duodenal mucosa and the other in the submucosa, were clearly distinguished by their morphology and dye-binding properties. Whereas submucosal mast cells stained metachromatically with Toluidine Blue after neutral formalin fixation and emitted a yellow fluorescence after staining with Berberine sulphate, those in the mucosa were invisible using these stains. In patients with gastritis or Crohn's disease, there were marked changes in the numbers of mucosal mast cells compared with control subjects even though the duodenal biopsies were from apparently uninvolved tissue. Gastritis was associated with increased mucosal mast cell numbers (controls: 187 +/- 23 cells mm-2; gastritis: 413 +/- 139 cells mm-2; p = 0.0004), but mean mucosal mast cell counts in the uninvolved duodenum of Crohn's patients were actually decreased (34 +/- 30 cells mm-2, p = 0.0147). The clear differentiation between mucosal and submucosal mast cells on the basis of metachromasia with Toluidine Blue was not seen in biopsies from the patients with gastritis or Crohn's disease. Previous studies which have suggested that there are no distinct mucosal and submucosal mast cell subsets in the human intestine may, therefore, have been affected by the use of tissue from diseased subjects. Heterogeneity in the expression of mast cell tryptase and chymase was seen by immunohistochemistry using specific antibodies, but the relative numbers of mast cell subsets were critically dependent on the methods used. Using a sensitive staining procedure, the majority of mucosal mast cells stained positively for chymase as well as for tryptase, an observation confirmed by immunoelectron microscopy and immunoabsorption studies. Our findings suggest that early stages in intestinal inflammation may be reflected in changes in mast cell numbers and in their staining properties, and call for a reappraisal of mast cell heterogeneity in the human intestinal tract.

Mast cell-lineage versus basophil lineage involvement in myeloproliferative and myelodysplastic syndromes: diagnostic role of cell-immunophenotyping.

Mast cells and blood basophils are distinct hemopoietic cells. They can be distinguished from each other and from all other lymphohemopoietic cells using antibodies against surface receptors or stored cytoplasmic molecules. In patients with myelodysplastic syndromes (MDS) or myeloproliferative syndromes (MPS), an elevation of metachromatically granulated cells (MCS) is frequently seen. These cells can be classified as basophils or mast cells using monoclonal antibodies (mAbs) against leukocyte antigens, including mast cell tryptase, c-kit (= mast cell growth factor [MGF] receptor), interleukin-3 receptor alpha chain (IL-3R alpha = CD123), and CD11b (C3biR). In a stable phase of MDS or MPS, the circulating MCS usually are basophils (histamine+, tryptase-, c-kit-, IL-3R alpha +, CD11b+). In an accelerated or terminal phase of disease, however, mast cell lineage involvement and circulating mast cell precursors (histamine+, tryptase+, c-kit+, IL-3R alpha-, CD11b-) are found in a subset of patients. The use of mAbs against mast cell antigens and granulocyte antigens is diagnostic in these patients.

Tumor necrosis factor alpha immunoreactivity of rat peritoneal mast cell granules decreases during early secretion induced by compound 48/80: an ultrastructural immunogold morphometric analysis.

We used fast (seconds) and ultrafast (milliseconds) microwave energy-assisted chemical fixation protocols, postembedding immunogold staining, and a morphometric analysis to investigate the early morphological changes and the TNF-alpha immunoreactivity in the cytoplasmic granules of rat peritoneal mast cells that had been stimulated to secrete by exposure to compound 48/80. Exposure to compound 48/80 induced the development of increased numbers of cytoplasmic granules that exhibited decreased electron density; these granules often also appeared swollen. These granule alterations were accompanied by a significantly decreased proportion of granules that were positive for TNF-alpha immunoreactivity. We also calculated the density of TNF-alpha labeling/mu 2 in both dense (unaltered) and altered granules in specimens. TNF-alpha immunoreactivity was present in dense granules (regardless of whether or not the specimens had been stimulated with compound 48/80) and in cells that were fixed with either fast or ultrafast microwave energy. However, altered granules exhibited a decreased density of TNF-alpha label. These findings show that changes in the immunolocalization and/or density of TNF-alpha immunoreactivity occur very rapidly upon stimulation of rat peritoneal mast cells with compound 48/80.

A case of malignant mastocytosis with circulating mast cell precursors: biologic and phenotypic characterization of the malignant clone.

The phenotypic and biologic properties of malignant cells in a case of aggressive mastocytosis with multi-organ involvement, circulating mast cell precursors and absence of skin infiltrates were analyzed. Circulating mast cell precursors were detected by immunostaining using antibodies against mast cell tryptase as well as by electron microscopy. These progenitors were tryptase+/chymase- (MCT) and accounted for 10 to 20% of nucleated mononuclear blood cells (MNC). A subset of them contained metachromatic granules. As assessed by combined toluidine blue/immunofluorescence staining, the granulated mast cell precursors were found to express CD9 (P24), CD33 (gp67) and CD44 (Pgp-1), but not basophil-related markers (CD11b (C3biR), CDw17 (lactosylceramide), CD123 (il-3R alpha))or monocyte-related antigens (CD14, CD15). Expression of the mast cell growth factor (MGF) receptor, c-kit(CD117), was also demonstrable, whereas the skin mast cell marker C5aR (CD88) could not be detected on mast cell precursors. The ligand of c-kit, recombinant human (rh) stem cell factor (SCF = MGF), induced histamine release from circulating mast cell progenitors, whereas rhC5a, a potent skin mast cell-/basophil-agonist, was ineffective over the dose-range (10(-9) to 10(-7(M)) tested. Analysis of mast cell antigens in malignant mastocytosis or mast cell leukemias may be helpful to establish a diagnosis and to determine the phenotype of the clone.

Ultrastructural immunogold localization of subcellular sites of TNF-alpha in colonic Crohn's disease.

Tumor necrosis factor-alpha, a proinflammatory cytokine, might have an important role(s) in initiating, modifying, and/or sustaining chronic inflammatory processes such as those that characterize Crohn's disease, an inflammatory bowel disease of unknown etiology. We used an immunogold ultrastructural morphometric approach to localize tumor necrosis factor-alpha in colonic Crohn's disease biopsies. Tumor necrosis factor-alpha was present in seven cell types (fibroblasts, eosinophils, mast cells, macrophages, colonic epithelial absorptive cells, Paneth cells, neutrophils). Tumor necrosis factor-alpha-containing subcellular organelles included lipid bodies (fibroblasts, eosinophils, macrophages, mast cells, colonic epithelial cells, neutrophils), secretory granules (eosinophils, Paneth cells), phagolysosomes (macrophages, colonic epithelial cells), and Golgi structures and vesicle membranes (neutrophils). A gradient of extracellular tumor necrosis factor-alpha immunoreactivity surrounded eosinophils, mast cells, and macrophages. P values of gold counts/microns2 were significant for all cells, organelles, and extracellular spaces measured, and all positive structures significantly exceeded the background labeling density/microns2. Specificity controls (normal rabbit serum, tumor necrosis factor-alpha-absorbed primary antibody) either failed to label these sites or gave markedly reduced specific tumor necrosis factor-alpha labeling, respectively. These findings represent the first ultrastructural localization of the subcellular sites of TNF-alpha in vivo in seven cell lineages in human colonic tissues.

Ultrastructural immunogold localization of tumor necrosis factor-alpha to the cytoplasmic granules of rat peritoneal mast cells with rapid microwave fixation.

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional, proinflammatory cytokine, which can be produced by mast cells and several other cell types. We used a newly developed microwave energy-assisted aldehyde fixation method to prepare purified rat peritoneal mast cells for the postembedding immunogold ultrastructural localization of TNF-alpha. These fixation methods were superior to chemical fixation alone in preserving both the ultrastructural morphology and immunoreactive TNF-alpha in rat mast cells. The percent of TNF-alpha-positive mast cells in samples prepared with microwave-assisted fixation in low (84%) and standard (81%) glutaraldehyde concentrations exceeded that for low (56%) and standard (15%) glutaraldehyde concentrations without the assistance of microwave energy. TNF-alpha was identified in the large storage granules of rat mast cells. The percent of positive granules in microwave-assisted standard (44%) and low (40%) glutaraldehyde samples was considerably higher than the percent of positive granules in standard (5%) and low (10%) glutaraldehyde-fixed samples without microwave assistance. This location of TNF-alpha in rat peritoneal mast cells suggests that this cytokine can use the regulated secretory route(s) for release from appropriately stimulated rat mast cells into the microenvironment.

Ultrastructural immunogold localization of tumor necrosis factor-alpha to the matrix compartment of eosinophil secondary granules in patients with idiopathic hypereosinophilic syndrome.

Peripheral blood eosinophils from two normal donors and two patients with the hypereosinophilic syndrome (HES) were analyzed with a post-embedding immunogold method to detect the substructural location of tumor necrosis factor-alpha (TNF-alpha). In eosinophils of HES patients, TNF-alpha was localized to the matrix compartment of 64% of the specific secondary granules. Other structures in the HES eosinophils were unlabeled. No TNF-alpha was detected in eosinophils of normal donors. These studies document the first ultrastructural subcellular localization of any cytokine within the major population of secretory granules in human eosinophils and support other lines of evidence indicating that the expression of TNF-alpha may be greater in the eosinophils of HES patients than in those of normal donors.

Human eosinophils can express the cytokines tumor necrosis factor-alpha and macrophage inflammatory protein-1 alpha.

By in situ hybridization, 44-100% of the blood eosinophils from five patients with hypereosinophilia and four normal subjects exhibited intense hybridization signals for TNF-alpha mRNA. TNF-alpha protein was detectable by immunohistochemistry in blood eosinophils of hypereosinophilic subjects, and purified blood eosinophils from three atopic donors exhibited cycloheximide-inhibitable spontaneous release of TNF-alpha in vitro. Many blood eosinophils (39-91%) from hypereosinophilic donors exhibited intense labeling for macrophage inflammatory protein-1 alpha (MIP-1 alpha) mRNA, whereas eosinophils of normal donors demonstrated only weak or undetectable hybridization signals for MIP-1 alpha mRNA. Most tissue eosinophils infiltrating nasal polyps were strongly positive for both TNF-alpha and MIP-1 alpha mRNA. By Northern blot analysis, highly enriched blood eosinophils from a patient with the idiopathic hypereosinophilic syndrome exhibited differential expression of TNF-alpha and MIP-1 alpha mRNA. These findings indicate that human eosinophils represent a potential source of TNF-alpha and MIP-1 alpha, that levels of expression of mRNA for both cytokines are high in the blood eosinophils of hypereosinophilic donors and in eosinophils infiltrating nasal polyps, that the eosinophils of normal subjects express higher levels of TNF-alpha than MIP-1 alpha mRNA, and that eosinophils purified from the blood of atopic donors can release TNF-alpha in vitro.

Differences in the onset of the inflammatory response to cutaneous leishmaniasis in resistant and susceptible mice.

Sites of cutaneous infection with Leishmania major in genetically susceptible (BALB/c) and resistant (C57B1/6) mice were investigated for the early inflammatory response (6 h to 12 days) by electron microscopy combined with enzyme-histochemical methods. Susceptible BALB/c mice spontaneously recruited only polymorphonuclear leukocytes (PMNs) at the site of infection. Infiltrating mononuclear phagocytes (and eosinophils) were first observed at day 1 in a ratio equal to the influx of PMNs (about 40%). This pattern persisted during the following 11 days of infection. In the resistant C57/B16 mice, the first cellular infiltrate at the infected site contained mononuclear phagocytes (25%) and eosinophils (15%) besides PMNs (60%). Within 3 days after infection, mononuclear phagocytes became the dominant population of cells in cutaneous lesions (up to 80%). It was found in situ that L. major accumulated and replicated in immature macrophages, that is, intermediate stages between monocytes and resident macrophages, which were found in lesions of both strains. The burden of parasites was, however, degraded more rapidly by the infiltrating cells of the resistant mice than by those of the susceptible ones. Within the first 4 days of infection, the parasites were found in PMNs, mononuclear phagocytes, and extracellular spaces in both strains. In susceptible mice this distribution pattern persisted up to 12 days after infection; in resistant C57B1/6 mice parasites accumulated inside mononuclear phagocytes within this period. It is concluded that the features of acute inflammation during leishmaniasis in BALB/c mice are sustained over a prolonged period that is ineffective in the elimination of L. major.