Quantitation of mast cell heparin by flow cytofluorometry.

Mast cells can be automatically identified in a mixed cell population by flow cytofluorometry after Berberine sulphate staining. Volume specific counts of the total number of cells and number of mast cells, as well as frequency distributions of fluorescence intensities of mast cells, based on a large number of cells, can be rapidly obtained. Results obtained by microscope fluorometry of cells identified by phase contrast microscopy showviously published results it may be inferred that the fluorescence intensity of individual mast cells is proportional to mast cell heparin content. The automated cell counts correlated very well with manual hemocytometer counts. Both cell counts and the determination of mean mast cell fluorescence showed excellent reproducibility.     

Degradation of human anaphylatoxin C3a by rat peritoneal mast cells: a role for the secretory granule enzyme chymase and heparin proteoglycan

Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0 degrees C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared with 37 degrees C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with bis (sulfosuccinimidyl) suberate (BS3) covalently cross-linked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC by using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan.     

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.     

Altered processing of fibronectin in mice lacking heparin. a role for heparin-dependent mast cell chymase in fibronectin degradation

We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2(-/-) mice show reduced levels of various mast cell mediators such as histamine and various heparin-binding mast cell proteases, including chymases, tryptases, and carboxypeptidase A. In this work we have addressed the possible functional consequences of the lack of sulfated heparin. Peritoneal cells were harvested from normal and NDST-2(-/-) mice. After culturing the cells, conditioned media were collected and were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Several differences in the protein patterns were observed, including the presence of large amounts of a approximately 250-kDa protein in medium from NDST-2(-/-) mice that was absent in normal controls. Peptide microsequencing revealed identity of this protein with fibronectin. Western blot analysis showed the presence of fibronectin degradation products in cell cultures from normal mice, which were absent in cultures from NDST-2(-/-) animals. Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2(+/+) mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner. This report thus indicates a biological function for chymase/heparin proteoglycan complexes in fibronectin turnover.     

Mast cell chymase regulates dermal mast cell number in mice

Chymase inhibitor reduced the increase in the number of dermal mast cells in 2,4-dinitrofluorobenzene-induced dermatitis in a dose-dependent manner. Intradermal injection of human chymase to mouse ear significantly increased histamine content, the marker for mast cell number in the skin. These results suggest that chymase released by mast cells may participate in local mast cell accumulation in a positive feedback fashion. Immunohistochemical analysis revealed that the intradermal injection of chymase reduces expression of stem cell factor (SCF) on surface of the skin keratinocytes. In addition, incubation of human keratinocytes with chymase in vitro resulted in release of SCF into the culture medium. Since soluble SCF is thought to regulate mast cell number, the chymase-induced mast cell accumulation may occur via the ability of chymase to process membrane-bound SCF on the epidermal keratinocytes.     

Inactivation of bradykinin and kallidin by cathepsin G and mast cell chymase

Human neutrophil cathepsin G and human skin chymase can inactivate bradykinin by cleavage at the carboxy terminal phenylalanyl-arginyl peptide bond of this polypeptide. The mast cell enzyme is far more effective than cathepsin G, the rates of hydrolysis being comparable to that found for angiotensin I to angiotensin II conversion (C.F. Reilly, D. Tewksbury, N. Schechter, and J. Travis, J. Biological Chemistry 257:8619-8622). This ability to both inactivate bradykinin and accelerate the production of angiotensin II may be of significance in the development of biochemical events associated with inflammation.     

Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase. Implications for HDL function

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL(3) by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL(3) revealed that proteolysis for up to 24 h did not alter the integrity of the alpha-migrating HDL, whereas a minor peak containing particles of smaller size with prebeta mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the alphaHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL(3) with chymase inhibited binding of Mab A-I-9 (specific for prebeta(1)HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL(3) to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL(3) by chymase failed to reduce the ability of HDL(3) to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation. This differential sensitivity of the two key functions of HDL(3) to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for prebeta(1)HDL to promote the high-affinity efflux of cellular cholesterol, but not for the alpha-migrating HDL particles to activate LCAT.     

Rapid lineage-specific diversification of the mast cell chymase locus during mammalian evolution

Serine proteases constitute the major protein granule content of cells of several hematopoietic cell lineages. A subgroup of these proteases, including the mast cell chymases, neutrophil cathepsin G, and T cell granzymes B to F and N, are in all investigated mammals encoded in one locus, the chymase locus. It is interesting to note that this locus has diversified greatly during the last 95 Myr of mammalian evolution. This divergence is exemplified by the presence of Mcpt8-related genes and multiple β-chymases in the mouse and rat, which lack direct counterparts in primates and in seven functional granzyme genes in the mouse where the human locus has only two. To study the expansion of the locus during rodent evolution and to better understand the evolutionary origin of β-chymases and the Mcpt8-family, we have performed a detailed analysis of the chymase locus of four mammalian species, i.e., human, dog, mouse, and rat. As a result, we report here a second chymase-like gene in dog, Cma2, which clusters with β-chymases in phylogenetic analyses. This finding supports a duplication of the common ancestor for α- and β-chymases before the major radiation of placental mammals, and a loss of the ancestral β-chymase gene sometime during primate evolution. Moreover, we show that in the rat, the Mcpt8-family diversified relatively recently together with sequences related to the β-chymase Mcpt2. Eight novel genes were identified in the duplication region, four of which are predicted to be functional. Duplications of rat granzyme B- and C-like sequences occurred seemingly independently within a similar time frame, but did not give rise to functional genes. Due to the duplications in rat and deletions in the carnivore/primate lineage, the rat chymase locus is approximately 15 and 9 times larger than its counterparts in dog and human, respectively. These findings illustrate the importance of gene duplications in conferring rapid changes in mammalian genomes.     

Mast cell proteoglycans modulate the secretagogue, proteoglycanase, and amidolytic activities of dog mast cell chymase.

Chymase, a potent secretagogue for airway gland serous cells, is stored in secretory granules and released from mast cells together with proteoglycans. To investigate the hypothesis tha tproteoglycans modulate chymase-induced effects, we studied the influence of proteoglycans purified from dog mastocytoma cells on chymase-induced secretion from cultured bovine airway gland serous cells. Heparin proteoglycans reduced the chymase-induced secretory response, whereas glycosaminoglycans and chondroitin sulfate proteoglycans had less of an effect. Chymase released together with proteoglycans from activated mast cells caused secretion comparable to that caused by purified chymase reconstituted with purified proteoglycans. Despite partial inhibition by exocytosed proteoglycans, the secretagogue activity of chymase remains substantial compared to that of histamine. However, proteoglycans virtually abolished chymase-induced degradation of the products of serous cell secretion. Although the secretagogue and proteoglycanase activities of chymase are inhibited by most classes of mast cell granule-associated glycans, the amidolytic activity of chymase toward tripeptide 4-nitroanilide substrates is augmented. These findings suggest that mast cell proteoglycans modulate the secretagogue, proteoglycanase, and peptidase activity of chymase, and the results predict that the extent of this modulation in vivo depends on the nature of the proteoglycans with which chymase is released from mast cells.     

Oxidative degradation of rat mast-cell heparin proteoglycan.

The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90%) and Glu-plasminogen (10%), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and and alpha 2-antiplasmin. In contrast, when alpha 2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30%) and released to the soluble phase. In the presence of alpha 2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10(-3), 2.0 x 10(-3) and 1.8 x 10(-3) (delta A405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-alpha 2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and alpha 2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Glu-plasminogen, are generated as well.     

Serglycin proteoglycan promotes apoptotic versus necrotic cell death in mast cells

The mechanisms that govern whether a cell dies by apoptosis or necrosis are not fully understood. Here we show that serglycin, a secretory granule proteoglycan of hematopoietic cells, can have a major impact on this decision. Wild type and serglycin(-/-) mast cells were equally sensitive to a range of cell death-inducing regimens. However, whereas wild type mast cells underwent apoptotic cell death, serglycin(-/-) cells died predominantly by necrosis. Investigations of the underlying mechanism revealed that cell death was accompanied by leakage of secretory granule compounds into the cytosol and that the necrotic phenotype of serglycin(-/-) mast cells was linked to defective degradation of poly(ADP-ribose) polymerase-1. Cells lacking mouse mast cell protease 6, a major serglycin-associated protease, exhibited similar defects in apoptosis as observed in serglycin(-/-) cells, indicating that the pro-apoptotic function of serglycin is due to downstream effects of proteases that are complex-bound to serglycin. Together, these findings implicate serglycin in promoting apoptotic versus necrotic cell death.     

Lack of effect of carbohydrate depletion on some properties of human mast cell chymase

Human chymase from vascular tissues was purified to homogeneity by heparin affinity and gel filtration chromatography. Treatment of human chymase with endoglycosidase F resulted in cleavage of the carbohydrate moiety yielding a deglycosylation product that did not lose its catalytic activity. This enzymatic deglycosylation product was enough to explore possibilities that N-glycan might modify some properties of human chymase. Substrate specificity, optimum pH and the elution profile from the heparin affinity gel were not affected by the deglycosylation. Only a slight but significant difference was observed in the Km value for conversion of angiotensin I to angiotensin II. Other kinetic constants such as kcat were not influenced. The kinetics of conversion of big endothelin-1 to endothelin-1(1-31) were not significantly affected. The deglycosylated human chymase was more susceptible to deactivation under alkaline pH and thermal stress. Even at physiological temperature and pH, the activity of glycosylated human chymase was more stable. From these results, it appears that the N-glycan of human chymase contributes to the stability of this enzyme but not to its functional properties.     

Cooperation between mast cell carboxypeptidase A and the chymase mouse mast cell protease 4 in the formation and degradation of angiotensin II

The octapeptide angiotensin II (Ang II) exerts a wide range of effects on the cardiovascular system but has also been implicated in the regulation of cell proliferation, fibrosis, and apoptosis. Ang II is formed by cleavage of Ang I by angiotensin-converting enzyme, but there is also evidence for non-angiotensin-converting enzyme-dependent conversion of Ang I to Ang II. Here we address the role of mast cell proteases in Ang II production by using two different mouse strains lacking mast cell heparin or mouse mast cell protease 4 (mMCP-4), the chymase that may be the functional homologue to human chymase. Ang I was added to ex vivo cultures of peritoneal cells, and the generation of Ang II and other metabolites was analyzed. Activation of mast cells resulted in marked increases in both the formation and subsequent degradation of Ang II, and both of these processes were strongly reduced in heparin-deficient peritoneal cells. In the mMCP-4(-/-) cell cultures no reduction in the rate of Ang II generation was seen, but the formation of Ang-(5-10) was completely abrogated. Addition of a carboxypeptidase A (CPA) inhibitor to wild type cells caused complete inhibition of the formation of Ang-(1-9) and Ang-(1-7) but did not inhibit Ang II formation. However, when the CPA inhibitor was added to the mMCP-4(-/-) cultures, essentially complete inhibition of Ang II formation was obtained. Taken together, the results of this study indicate that mast cell chymase and CPA have key roles in both the generation and degradation of Ang II.     

A role for serglycin proteoglycan in mast cell apoptosis induced by a secretory granule-mediated pathway

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin(-/-) cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis.     

Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.     

Cytofluorometric quantitation of 5-hydroxytryptamine and heparin in individual mast cell granules.

Cytofluorometric quantitation of 5-hydroxytryptamine (5-HT) and heparin in individual mast cell granules is described. The technique is based on micromanipulation of intact mast cells reacted with formaldehyde or stained with Berberine sulfate and the use of a cytofluorometer equipped with a sensitive peak detecting device. The quantities of 5-HT and heparin contained in mast cell granules which are of the order of 10(-16) and 10(-13) g, respectively were expressed as relative fluorescence guanta. The results of measurements on representative samples of mast cell granules indicate that all granules contain heparin as well as 5-HT, and that there are large variations in both 5-HT and heparin content within the granule populations of individual cells. A dose dependent increase in 5-HT content in both cells and individual mast cell granules occurred 24 hr after the injection of 10--50 mg L-5-hydroxytryptophan/kg intraperitoneally. There was no evidence for an increase in the heparin content of granules or cells, indicating that a new synthesis of granular macromolecules is not required for the 5-HT uptake. The results further suggest that 5-HT may be stored initially in a cytoplasmic extragranular pool and then taken up in the mast cell granules.     

Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6

Tryptase, a serine protease with trypsin-like substrate cleavage properties, is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6xHis-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6xHis-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6xHis-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant approximately 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present paper thus indicates a crucial role for heparin in the formation of active mast cell tryptase.