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Biomedical subjects

N M Schechter

Publications and source records attributed to N M Schechter.

At least 55 records · Page 3Linked to original sources
PubMed1989

Structure of the dermal-epidermal junction and potential mechanisms for its degradation: the possible role of inflammatory cells.

Evidence was presented indicating that the DEJ as a basement membrane is highly susceptible to degradation by a variety of neutral proteolytic enzymes with different specificities. The effect of endoglycosidases which degrade heparan sulfate was also discussed. The latter enzymes are capable of removing heparan sulfate from the DEJ, but little gross alteration of structure, such as tissue detachment, appears to result from the loss of this component. Of the proteinases discussed, PMN elastase and probably type IV collagenase are the most destructive. This is likely related to their ability to degrade the type IV collagen network. Even though proteinases with chymotrypsinlike and trypsinlike specificity were not efficient at degrading the lamina densa or removing type IV collagen from intact basement membranes, these proteinases were capable of producing epidermal detachment from the lamina densa. Many inflammatory cells of the immune system contain proteinases and endoglycosidases with the potential to degrade the DEJ and other basement membrane zones, suggesting that these cells may have a significant pathologic role in basement membrane-related diseases. PMNs and mast cells may be of particular interest because they have stored within their secretory granules high concentrations of neutral serine proteinases which have been demonstrated to degrade the DEJ.

Animals↗
PubMed1989

Ultrastructural analysis of maturing human T and TC mast cells in situ.

Mast cells at immature stages of development were identified in human tissues by electron microscopic techniques. General morphologic criteria of immaturity (such as a high apparent nuclear:cytoplasmic ratio and small cell size), the presence of few granules (those present being smaller than those in mature mast cells) and a lack of features of mast cell activation were used together to determine the level of maturity. Mast cells were identified as being of the T or TC type by immunogold staining with polyclonal rabbit IgG anti-chymase and murine monoclonal anti-tryptase primary antibodies and the appropriate gold-labeled secondary antibodies. Only those cells with tryptase-positive granules were recognized as mast cells. Immature T mast cell granules contained the same characteristic discrete scrolls found in their mature counterparts and all stained positive for tryptase. The presence of trace amounts of chymase in a minority of these granules, as in mature T mast cells, could not be ruled out. The majority of granules in immature TC mast cells had one or more amorphous electron-dense cores rather than the grating and lattice substructures characteristic of granules in mature TC mast cells. Secretory granules in immature TC mast cells stained positively for tryptase and chymase. Occasional immature TC mast cells contained a complete granule or a portion of a granule with the substructure characteristic of mature TC mast cells, favoring the concept that these TC mast cell forms are developmentally related. Essentially all mast cells in foreskin of newborns appeared immature, whereas 10, 5, 10, and 15% of the mast cells in adult lung, foreskin, bowel mucosa and bowel submucosa, respectively, appeared immature. The distribution of T and TC types of immature mast cells seemed to parallel that of the mature mast cell types. These compositional and ultrastructural differences between immature T and TC types of mast cells suggest that from the time granule formation begins, and possibly before this time, each type of human mast cell follows a distinct developmental pathway.

Adult↗
PubMed1988

Human skin tryptase: purification, partial characterization and comparison with human lung tryptase.

Human skin tryptase was isolated using stepwise low- and high-salt extraction and further purified 448-fold with 33% yield using octyl-Sepharose CL-4B hydrophobic affinity chromatography, Sephacryl S-200 gel filtration and finally octyl-Sepharose CL-4B or cellulose phosphate ion exchange chromatography. The skin tryptase, which has an apparent Mr of 120,000 by gel filtration in high-salt buffer, consisted of polypeptide chains of Mr 34,000 and 38,000 when resolved on SDS gels. Both polypeptide chains, labelled with [3H]diisopropyl fluorophosphate, indicated that they were representative of subunits and that the native proteinase was an aggregate of subunits. However, in some preparations only one band with Mr 34,000 was seen. In low-salt buffer the enzyme was labile and at least 1.4 M KCl was needed to keep the enzyme stabile when incubated at 37 degrees C for 30 min. Heparin glycosaminoglycan partially stabilized the tryptase but addition of protein (e.g. albumin, 80 micrograms/ml) to the tryptase-heparin mixture was needed to keep the enzyme stabile. Tryptases purified by exactly the same method from human lung tissue and from human skin had identical molecular size in gel filtration and in SDS-polyacrylamide gel electrophoresis. They also revealed identical enzyme kinetic parameters with several synthetic peptide substrates. The inhibition profile was identical for both enzymes, and they also crossreacted completely in immunodiffusion plates. These studies strongly indicate that mast cells found in skin as well as lung contain closely related, possible identical trypsin-like proteinases.

Chromatography, Gel↗
PubMed1988

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase.

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.

Animals↗
PubMed1988

Ultrastructural analysis of human T and TC mast cells identified by immunoelectron microscopy.

Tryptase and chymase were localized in human mast cells by immunoelectron microscopy, enabling the T (tryptase positive, chymase negative) and TC (tryptase positive, chymase positive) types of mast cells to be identified and ultrastructurally characterized. A double immunogold staining procedure was performed on samples of human skin, small intestine, and lung with rabbit polyclonal IgG anti-chymase and mouse monoclonal IgG anti-tryptase primary antibodies and gold-conjugated secondary antibodies. Approximately 225 mast cells were examined in this fashion; comparable sections from 170 of these mast cells along with approximately 200 additional mast cells also were examined using techniques optimized for ultrastructural detail. Each secretory granule of TC mast cells contained both tryptase and chymase; secretory granules of T mast cells stained strongly positive for tryptase alone. Extremely small amounts of chymase appeared to be present in an occasional T mast cell granule. Staining for the neutral proteases was more intense over electron-dense regions of the granules, particularly noticeable over the characteristic discrete scrolls of T mast cells. T and TC mast cells each had large numbers of cytoplasmic granules, nuclei with peripherally condensed chromatin and low nuclear/cytoplasmic ratios, indicating maturity of both cell types. TC mast cell granules generally were more uniformly electron dense, larger and more numerous than T mast cell granules, which were more variable in shape. Compact solid-core scrolls, peripheral parallel lamellae and amorphous electron-dense material were found in granules of both cell types. Only TC mast cells had granules with grating and lattice substructures; only T mast cells had granules containing discrete scrolls. Less commonly, T mast cells were detected containing granules with a characteristic beaded or particulate ultrastructure. The ultrastructural features noted above were observed in T and TC mast cells regardless of the tissue in which they were examined and thereby permit T and TC mast cells to be distinguished by ultrastructure alone.

Cell Nucleus↗
PubMed1987

Deficiency of the tryptase-positive, chymase-negative mast cell type in gastrointestinal mucosa of patients with defective T lymphocyte function.

The distribution and concentration of human T (tryptase-positive, chymase-negative) and TC (tryptase-positive, chymase-positive) mast cells were examined in Carnoy's-fixed specimens of the gastrointestinal tract of normal individuals, patients with inflammatory bowel diseases, and patients with immunodeficiency disorders. In normal specimens, T mast cells predominated in the mucosa (89%), with a mean concentration of 17,850 +/- 4,998 per mm3 (+/- SD, n = 16), whereas TC mast cells predominated in the submucosa (90%) with a mean concentration of 7,516 +/- 1,227 per mm3 (+/- SD, n = 16). The concentrations of T and TC mast cells in specimens of ileum from five patients with active Crohn's disease and of colon from three patients with active ulcerative colitis were not significantly different (p greater than 0.4) from normal values. Three patients with combined immunodeficiency disorders demonstrated a marked decrease in the concentration of the T mast cells in the intestinal mucosa, to 540 +/- 630, and a corresponding decrease in the percentage of T mast cells to 9%. Concentrations of TC mast cells were unchanged, both in the mucosa and in the submucosa. In three patients with acquired immunodeficiency syndrome, a similar deficiency of the T mast cell type was observed in the ileal mucosa, with a mean concentration of 788 +/- 534 T mast cells per mm3, but not in the appendiceal and colonic mucosa of one of the three patients. These findings indicate a role for functional T lymphocytes in the development of the T mast cell type in humans, and suggest divergent pathways for development of T and TC mast cells.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Human skin chymotrypsin-like proteinase chymase. Subcellular localization to mast cell granules and interaction with heparin and other glycosaminoglycans.

The subcellular localization of human skin chymase to mast cell granules was established by immunoelectron microscopy, and binding of chymase to the area of the dermo-epidermal junction, a basement membrane, was demonstrated immunocytochemically in cryosections incubated with purified proteinase prior to immunolabeling. Because heparin and heparan sulfate proteoglycans are major constituents of mast cell granules and basement membranes, respectively, the ability of chymase to bind to glycosaminoglycans (GAG) was investigated. Among a variety of GAGs, only binding of chymase to heparin and heparan sulfate appears physiologically significant. Binding was ionic strength-dependent, involved amino groups on the proteinase, and correlated with increasing GAG sulfate content, indicating a predominantly electrostatic association. Interaction with heparin was observed in solutions containing up to 0.5 M NaCl, and interaction with heparan sulfate was observed in solutions containing up to 0.3 M NaCl. Binding of heparin did not detectably affect catalysis of peptide substrates, but may reduce accessibility of proteinase to protein substrates. Measurements among a series of serine class proteinases indicated that heparin binding was a more common property of mast cell proteinases than proteinases stored in other secretory granules. Binding of chymase to heparin is likely to have a storage as well as a structural role within the mast cell granule, whereas binding of chymase to heparan sulfate may have physiological significance after degranulation.

Chymases↗
PubMed1987

Quantitation of histamine, tryptase, and chymase in dispersed human T and TC mast cells.

Levels of histamine, chymase, and tryptase were assessed in preparations of dispersed human TC (tryptase+, chymase+) mast cells obtained from foreskin and of dispersed human T (tryptase+, chymase-) mast cells obtained from lung. Consistent with previous immunohistochemical results, extracts of T mast cells, the predominant mast cell type in lung (93% T and 7% TC mast cells), were deficient in human chymase (less than 0.3 microgram and 0.04 U/10(6) mast cells) but not tryptase (10.8 micrograms and 0.3 U/10(6) mast cells) by corresponding immunologic and enzymatic (suc-L-ala-ala-pro-phe-p-nitroanilide in the presence of aprotinin and tosyl-L-gly-pro-lys-p-nitroanilide in the presence of soybean trypsin inhibitor, respectively) assays. The minor presence of chymase activity in lung could be accounted for by the minor presence of lung TC mast cells. Extracts of TC mast cells, the predominant mast cell type (1% T and 99% TC mast cells) in foreskin, contained both proteases. However, TC mast cells from adult foreskin contained eightfold to 10-fold higher levels of chymase (4.5 micrograms and 1.01 U/10(6) mast cells) and twofold to threefold higher levels of tryptase (11.5 micrograms and 0.27 U/10(6) mast cells) than did TC mast cells from newborn foreskin (less than 0.6 microgram and 0.09 U of chymase and 35 micrograms and 0.62 U of tryptase/10(6) mast cells). In contrast, histamine levels were not significantly different in adult foreskin TC (1.9 microgram/10(6) mast cells), newborn foreskin TC (1.6 microgram/10(6) mast cells), and adult lung T (1.5 microgram/10(6) mast cells) mast cells. The relative ratio of each mediator in newborn foreskin mast cells to that in adult foreskin mast cells is highest for histamine, followed by tryptase and then chymase. Tryptase from TC and T mast cells had identical subunit compositions by Western blot analysis and similar apparent specific activities. This study extends the previously reported immunohistochemical distinction between human T and TC mast cells in tissue sections by direct quantitation of chymase and tryptase in dispersed preparations of T and TC mast cells.

Adult↗
PubMed1987

Aggressive topical corticosteroid therapy: a novel approach to mast-cell-dependent cutaneous disorders.

Topical corticosteroids are utilized in the treatment of a wide variety of skin diseases, primarily those involving an inflammatory component. Recent investigations have revealed that one of the effects of long-term usage of steroids is the depletion of skin mast cells. This led to the treatment of patients with urticaria pigmentosa with topical high potency corticosteroids for 6 weeks. At the end of treatment there was a marked reduction in tissue histamine and an absence of mast cells as well as a disappearance of pruritus and Darier's sign. Treated areas remained clinically improved for at least 9-12 months. Observations that corticosteroids profoundly affect mast cells in vivo provides a rationale to devise new treatment regimens for mast-cell-related diseases.

Administration, Topical↗
PubMed1986

Identification of a chymotrypsin-like proteinase in human mast cells.

An antiserum was produced against a chymotryptic proteinase purified from human skin. The antiserum did not cross-react with human leukocyte cathepsin G and elastase, rat mast cell proteinase I, and human skin tryptase. Indirect immunofluorescent staining of frozen skin sections to localize the proteinase showed cytoplasmic staining of cells scattered about the papillary dermis and around blood vessels and appendages. Restaining these sections with toluidine blue revealed that the fluorescently stained cells contained metachromatically staining granules, the major distinguishing feature of mast cells. A similar correlation was found in lung tissue. Ultrastructural studies employing the ferritin bridge technique to immunologically identify the proteinase additionally localized the proteinase to mast cell granules. Biochemical and immunochemical characterization of chymotryptic activity solubilized from isolated human lung mast cells identified a chymotryptic proteinase that may be identical to the skin chymotryptic proteinase. These studies establish that human skin mast cells contain a chymotrypsin-like proteinase that is a granule constituent and provide evidence that indicates a comparable proteinase is also present in lung mast cells.

Chymases↗
PubMed1986

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.

Biopsy↗
PubMed1986

Two types of human mast cells that have distinct neutral protease compositions.

Two human mast cell types were identified by immunohistochemical techniques in skin, lung, and small intestine. One type contains the neutral proteases, tryptase and chymotryptic proteinase, and is termed the TC mast cell. The second type contains only tryptase and is termed the T mast cell. Both types are fixed better by Carnoy's fluid than by formalin. The percentage of mast cells accounted for by the T type was 12 in skin; 98 in mucosa and 13 in submucosa of small intestine; and 77 in bronchial/bronchiolar subepithelium, about 97 in bronchial/bronchiolar epithelium, and 93 in alveoli of lung. Dispersed lung cells contained 90% T mast cells. The mean area of TC mast cells (76 micron2) was slightly larger than that of T mast cells (66 micron2); however, there was such extensive overlap that individual mast cells belonging to different types could not be distinguished on the basis of size. The recognition of human mast cell types with distinct protease compositions suggests a higher level of complexity of human mast cell-mediated reactions than heretofore appreciated.

Endopeptidases↗
PubMed1986

A human lung mast cell chymotrypsin-like enzyme. Identification and partial characterization.

We have used a high performance liquid chromatography assay, which detects chymotryptic cleavage of the phe8-his9 bond of angiotensin I to yield angiotensin II, in order to examine human lung mast cells for the presence of chymotryptic activity. Mast cells, purified from human lung by enzymatic dispersion, countercurrent elutriation, and Percoll gradient centrifugation, were lysed or challenged with goat anti-human IgE. In multiple experiments angiotensin II-converting activity was detected in lysates of 10-99% pure mast cell preparations. Regression analysis of net percent release values of histamine and the angiotensin I-converting activity from dose-response experiments demonstrated a correlation between the two parameters, indicating that the chymotrypsin-like enzyme is a constituent of the mast cell secretory granule. The chymotryptic activity was completely inhibited by 10(-3) M phenylmethylsulfonylfluoride but not by 10(-3) M Captopril, and the pH optimum of activity was 7.5-9.5. Gel filtration of released material separated the activity from tryptase and demonstrated an approximate molecular weight of 30-35,000. The mast cell enzyme, like a human skin chymotrypsin-like proteinase, can be distinguished from leukocyte cathepsin G by lack of susceptibility to inhibition by bovine pancreatic trypsin inhibitor. Thus, an enzyme with limited chymotryptic specificity is present in human lung mast cells. The Michaelis constant of the enzyme for angiotensin I of 6.0 X 10(-5) M is similar to that of endothelial cell angiotensin-converting enzyme and is consistent with a reaction of physiologic importance.

Angiotensin I↗
PubMed1985

Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors.

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.

Amino Acid Chloromethyl Ketones↗
PubMed1985

Cutaneous mast cell depletion results from topical corticosteroid usage.

The effect of long-term topical application of corticosteroids on human cutaneous mast cells was examined. Two potent corticosteroids, clobetasol-17-propionate and fluocinonide, produced a greater than 85% decrease in histamine content over a 6-wk treatment period, whereas betamethasone valerate, a less potent corticosteroid, produced a 66% decrease. Electron microscopic examination of the biopsies taken from sites after 6 wk of treatment indicate that the reduced levels of histamine were caused by the depletion of mast cells, as evidenced by: the inability to identify any cells representative of mast cells by detailed electron microscopy of the biopsies; and the marked acellularity around the vasculature where mast cells are certain to be detected. Histamine levels did not begin to decline until after 3 wk of corticosteroid treatment, indicating that corticosteroids are not immediately harmful to mast cells. Electron microscopic examination of biopsies taken at the beginning of treatment and 1 wk later showed normal-appearing mast cells, whereas at 3 wk, a small population of mast cells was detected with features usually associated with degenerating or dying cells. These observations suggest that protracted application of corticosteroids to skin is toxic to mast cells. After discontinuation of treatment, the drug-related atrophy associated with chronic application of potent corticosteroids to skin is rapidly reversed, and skin structure returns to near normal by 14 days. Over this time period, however, histamine levels did not increase and mature mast cells could not be observed by electron microscopy. At 14 days post-steroid treatment, the first signs of cells containing sparse amounts of granules having the characteristics of mast cell granules were seen. We interpret this to represent new mast cells beginning to mature in the skin. By 3 mo, histamine levels returned to normal, demonstrating the reversibility of the steroid-induced mast cell depletion. The studies presented here establish the deleterious effects of long-term topical corticosteroid treatment on cutaneous mast cells, and begin to establish a system in which the development of mast cells in tissue can be investigated.

Administration, Topical↗
PubMed1985

Treatment of urticaria pigmentosa with corticosteroids.

Based on a previous observation that the long-term application of potent topical corticosteroids under occlusion to normal skin resulted in the loss of mast cells, we investigated the effects of intralesional and topical steroids in urticaria pigmentosa (UP). Three patients with UP had lesions that were injected with triamcinolone acetonide. Four weeks after injection, all patients showed a loss of Darier's sign, and, by eight weeks after injection, there was a dramatic clearing of the plaques and a decrease in brown hyperpigmentation. By 12 weeks, mast cells were undetectable by light microscopy and transmission electron microscopy (TEM). The injection sites remained dramatically improved for as long as one year after treatment, and histamine content was reduced 95% in one patient 48 weeks after injection. In six patients, the topical application of 0.05% betamethasone dipropionate under occlusion to limited areas induced almost complete clearing of UP lesions. Lesions treated with an emollient under occlusion, as a control, demonstrated no change. After treatment, no mast cells were seen by light microscopy or TEM, and this persisted for at least 24 weeks. There was also a significant decrease in tissue histamine levels in the treated areas. The treated areas remained clinically improved for at least nine to 12 months. These data indicate that steroid therapy dramatically decreases the excess number of normal-appearing mast cells in UP as well as induces a prolonged resolution of UP lesions. Local corticosteroids thus are a useful therapeutic modality for UP.

Administration, Topical↗
PubMed1984

Degradation of the epidermal-dermal junction by proteolytic enzymes from human skin and human polymorphonuclear leukocytes.

The degradation of normal human skin by the human polymorphonuclear leukocyte proteinases cathepsin G and elastase, and by a human skin chymotrypsin-like proteinase that appears to be a mast cell constituent, was examined. Enzymes were incubated with fresh, split-thickness skin for up to 8 h; the tissue was examined ultrastructurally and immunohistochemically using antibodies to known basement membrane constituents. In all cases, the primary damage observed was at the epidermal-dermal junction. Elastase degraded the lamina densa leaving scattered and disorganized anchoring fibrils, dermal microfibril bundles, and normal-appearing collagen fibers. Immunohistochemically, type IV collagen, laminin, KF1 antigen, and EBA antigen were absent. The bullous pemphigoid antigen was present and localized on the basal cells. Epidermal-dermal separation produced by the chymotrypsin-like proteinases, cathepsin G, and the human skin proteinase, was confined to the lamina lucida. The lamina densa and sub-lamina densa fibrillar network remained intact. The human skin chymotrypsin-like proteinase produced extensive epidermal-dermal separation, while cathepsin G, at comparable concentrations, produced only focal separations. Immunohistochemically, all antigens were present after incubation with enzyme. The bullous pemphigoid antigen, however, was found on the epidermal side of the split, while laminin was found on the dermal side. These results show that the epidermal-dermal junction is highly susceptible to neutral serine proteinases located in mast cells and polymorphonuclear leukocytes. Although all the proteinases produce epidermal-dermal separation, the patterns and extent of degradation are different. The distinctive patterns of degradation may provide a clue to the involvement of these proteinases in skin diseases.

Animals↗