Matrix metalloproteinase-2 from bronchial epithelial cells induces the proliferation of subepithelial fibroblasts.

BACKGROUND: In bronchial asthma, subepithelial fibrosis in the conducting airways is associated with increased numbers of subepithelial fibroblasts. OBJECTIVE: This study examined the hypothesis that MMP-2 from airway epithelial cells induces the proliferation of subepithelial fibroblasts. METHODS: Using primary bronchial epithelial cells MMP-2, MT1-MMP and TIMP-2 mRNA expression were assessed by Northern blotting and RT-PCR. Primary bronchial epithelial cells transfected with constructs encoding pro-MMP-2 and MT1-MMP (MMP-14). RESULTS: Transfected cells showed enhanced expression of the appropriate mRNA species by RT-PCR and enhanced MMP-2 or MT1-MMP activity by zymography. Active MMP-2 levels in epithelial supernatants were increased most by cotransfection with pro-MMP-2 and MT1-MMP encoding constructs. By measuring tritiated thymidine incorporation, supernatants from transfected cells were found to enhance DNA synthesis of primary airway fibroblast cultures compared with controls. There was a strong correlation (r = 0.9, P < 0.01) between MMP-2 levels in epithelial cell conditioned media and fibroblast proliferation as indicated by DNA synthesis. The MMP inhibitor 1,10-phenanthroline attenuated the increased proliferation, while the addition of exogenous purified MMP-2 alone also increased fibroblast proliferation. CONCLUSIONS: Our results support a role for MMP-2 in mediating cross-talk between epithelial cells and myofibroblasts.

Extracellular matrix degradation and the role of hepatic stellate cells.

Following liver injury, hepatic stellate cells (HSCs) become activated and express a combination of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs). In the early phases of liver injury (and primary cell culture), HSCs transiently express MMP-3, MMP-13, and uroplasminogen activator (uPA) and exhibit a matrix-degrading phenotype. In the later stages of liver injury and HSC activation, the pattern changes and the cells express a combination of MMPs that have the ability to degrade normal liver matrix, while inhibiting degradation of the fibrillar collagens that accumulate in liver fibrosis. This pattern is characterized by the combination of pro-MMP-2 and membrane type 1 (MT1)-MMP expression, which drive pericellular generation of active MMP-2 and local degradation of normal liver matrix. In addition there is a marked increase in expression of TIMP-1 leading to a more global inhibition of degradation of fibrillar liver collagens by interstitial collagenases (MMP-1/MMP-13). These pathways play a significant role in the progression of liver fibrosis. Following cessation of liver injury, the pattern reverses and TIMP-1 in particular is rapidly downregulated. This phase is characterized by increasing activity of collagenases, degradation of liver matrix, and regression of liver fibrosis.

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo.

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.

Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors.

BACKGROUND: Activated hepatic stellate cells (HSC) are central to the pathogenesis of liver fibrosis, both as a source of fibrillar collagens that characterise fibrosis and matrix degrading metalloproteinases and their tissue inhibitors, the TIMPs. AIMS: To test the hypothesis that HSC apoptosis is critical to recovery from biliary fibrosis and that soluble growth factors may regulate HSC survival and apoptosis. METHODS: Rats (n=15) were subjected to bile duct ligation for 21 days, after which biliodigestive anastomosis was undertaken (n=13). Livers were harvested at fixed time points of recovery for periods of up to 42 days. Numbers of activated HSCs were quantified after alpha smooth muscle actin staining and HSC apoptosis was detected by terminal UDP-nick end labelling (TUNEL) staining and quantified at each time point. HSC apoptosis was quantified in vitro in the presence or absence of insulin-like growth factor (IGF)-1, IGF-2, platelet derived growth factor (PDGF), and transforming growth factor beta1 (TGF-beta1). RESULTS: Following biliodigestive anastomosis after 21 days of bile duct ligation, rat liver demonstrated a progressive resolution of biliary fibrosis over 42 days, associated with a fivefold decrease in activated HSC determined by alpha smooth muscle actin staining. TUNEL staining indicated that loss of activated HSC resulted from an increase in the rate of apoptosis during the first two days post biliodigestive anastomosis. Serum deprivation and culture in the presence of 50 microM cycloheximide was associated with an increase in HSC apoptosis which was significantly inhibited by addition of 10 ng/ml and 100 ng/ml IGF-1, respectively (0.05>p, n=5). In contrast, 1 and 10 ng/ml of TGF-beta1 caused a significant increase in HSC apoptosis compared with serum free controls (p<0.05, n=4). PDGF and IGF-2 were neutral with respect to their effect on HSC apoptosis. CONCLUSION: HSC apoptosis plays a critical role in the spontaneous recovery from biliary fibrosis. Both survival and apoptosis of HSC are regulated by growth factors expressed during fibrotic liver injury.

Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells.

BACKGROUND/AIMS: Connective tissue growth factor is a recently described mitogenic protein implicated in a variety of fibrotic disorders. Connective tissue growth factor may be a downstream mediator of the pro-fibrotic and mitogenic actions of transforming growth factor-beta, promoting extracellular matrix deposition and fibrogenesis. As transforming growth factor-beta is considered important to the pathogenesis of hepatic fibrosis, we examined the possible contribution of connective tissue growth factor to this process. METHODS: Connective tissue growth factor expression was examined in normal and fibrotic human and rat livers using RT-PCR and ribonuclease protection assays, and in primary cultures of rat hepatic stellate cells by Northern and Western blotting. RESULTS: Ribonuclease protection assays demonstrated connective tissue growth factor mRNA was increased 3-5-fold in human fibrotic liver compared with normal. RT-PCR showed this mRNA was increased in carbon-tetrachloride-treated rat liver. Northern analysis showed connective tissue growth factor mRNA was increasingly expressed during progressive activation of cultured rat hepatic stellate cells. Western analysis confirmed that freshly isolated hepatic stellate cells secreted relatively little connective tissue growth factor compared with hepatic stellate cells activated in culture. Hepatic stellate cells stimulated with transforming growth factor-beta showed increased expression of connective tissue growth factor mRNA and protein. CONCLUSIONS: Connective tissue growth factor mRNA is consistently upregulated in human liver cirrhosis of various aetiologies, supporting a role for this growth factor in hepatic fibrogenesis. Our studies suggest that hepatic stellate cells may be an important source of hepatic connective tissue growth factor in vivo, particularly following stimulation with transforming growth factor-beta.

Progelatinase A is produced and activated by rat hepatic stellate cells and promotes their proliferation.

Activated hepatic stellate cells (HSCs) are a potential source of gelatinase A, which accumulates in fibrotic livers. Progelatinase A activation requires its binding to a complex of membrane-type matrix metalloproteinase (MT-MMP) and tissue inhibitor of metalloproteinases (TIMP)-2. These studies examine gelatinase A, MT1-MMP, and TIMP-2 synthesis by HSCs during activation in vitro and the potential role of gelatinase A in promoting HSC proliferation. Gelatinase A, MT1-MMP, and TIMP-2 messenger RNA (mRNA) were all upregulated in HSCs activated on plastic over 5 to 14 days. Gelatinase A expression was maximal at 7 days of culture, coinciding with the peak of HSC proliferation and the onset of procollagen I and alpha-smooth muscle actin (alpha-SMA) mRNA expression. Active forms of gelatinase A of 62 kd and 66 kd were secreted by activated HSCs and reached a maximum of 12.1% of total enzyme in 14-day culture supernatants. Treatment of HSCs with concanavalin A (con A) induced activation of MT1-MMP and enhanced secretion of activated gelatinase A, which reached a maximum of 44.4% of the total enzyme secreted into culture supernatants using 30 microgram/mL con A. [(14)C]-gelatin degradation assays confirmed the presence of gelatinolytic activity in activated HSC supernatants, which reached a maximum level at 7 days of culture. Antisense oligonucleotide inhibition of endogenous progelatinase A production, or the MMP inhibitor 1,10-phenanthroline inhibited (3)H-thymidine incorporation into HSC DNA by greater than 50%. We conclude that HSCs produce progelatinase A during activation in vitro and activate this enzyme coincident with MT1-MMP and TIMP-2 synthesis. Gelatinase A activity is required for maximal proliferation of HSCs in vitro suggesting this metalloproteinase is an autocrine proliferation factor for HSCs.

Human and rat hepatic stellate cells produce stem cell factor: a possible mechanism for mast cell recruitment in liver fibrosis.

BACKGROUND/AIMS: Mast cell numbers are markedly increased in advanced liver fibrosis. Stem cell factor may recruit mast cells to the liver following injury as it induces mast cell proliferation, survival and differentiation from resident tissue precursors. This study examines stem cell factor production in human fibrotic liver and by hepatic stellate cells during culture in vitro. METHODS: Stem cell factor production was examined in human fibrotic livers by ELISA and in human and rat hepatic stellate cell cultures using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, Western blotting and immunocytochemistry. Co-culture studies examined adhesion between hepatic stellate cells and purified mast cells. RESULTS: RT-PCR showed stem cell factor mRNA was more consistently expressed in fibrotic human livers relative to normal, and ELISA confirmed this by showing stem cell factor protein was significantly increased 2-3-fold in homogenates of human cirrhotic liver (primary biliary cirrhosis, primary sclerosing cholangitis) relative to normal. RT-PCR detected stem cell factor mRNA in human and rat hepatic stellate cells activated by culture on plastic. This was confirmed by Western blotting, which showed that freshly isolated hepatic stellate cells expressed relatively little 30 kD stem cell factor compared to late primary culture activated hepatic stellate cells (14 day) and passaged hepatic stellate cells. As assessed by fluorescence immunocytochemistry, stem cell factor protein was homogeneously expressed by populations of culture-activated rat hepatic stellate cells. During co-culture, purified human skin mast cells adhered to hepatic stellate cell monolayers on plastic, and this adherence was inhibited >50% by addition of antibodies against stem cell factor. CONCLUSIONS: Hepatic stellate cells activated in vitro produce stem cell factor. These cells may play an important role in recruiting mast cells to liver during injury and fibrosis.

Control of the tissue inhibitor of metalloproteinases-1 promoter in culture-activated rat hepatic stellate cells: regulation by activator protein-1 DNA binding proteins.

In the injured liver hepatic stellate cells (HSCs) undergo a dramatic phenotypic transformation known as "activation" in which they become myofibroblast-like and express high levels of the tissue inhibitor of metalloproteinase 1 (TIMP-1). HSC activation is accompanied by transactivation of the TIMP-1 promoter. Truncation mutagenesis studies delineated a minimal active promoter consisting of nucleotides -102 to +60 relative to the major start site for transcription. Removal of an AP-1 site located at nucleotides -93 to -87 caused almost a complete loss of promoter activity. Analysis of AP-1 DNA binding activities during culture activation of HSCs initially indicated transient expression of proteins capable of forming a low mobility AP-1 DNA binding complex (LMAP-1). LMAP-1 was maximally induced at 24 hours of culture and then fell to undetectable levels at 120 hours. Western blot studies showed that both c-Fos and c-Jun underwent similar transient inductions. These temporal changes in c-Fos and c-Jun activities were unexpected because TIMP-1 mRNA expression is not detected in HSCs until culture day 3 to 5 and is thereafter sustained at a high level. Previous work in other cell lineages has established a key role for Pea3 binding proteins (Ets-1) in AP-1 mediated transactivation of the TIMP-1 promoter. We show that HSCs express relatively low levels Ets-1 and Ets-2 and show that mutagenesis of the Pea3 DNA binding site in the TIMP-1 promoter has less than a twofold effect on its activity in activated HSCs. Further analysis of AP-1 DNA binding activities in 7- to 14-day culture activated HSCs led to the discovery of high mobility AP-1 complexes (HMAP-1). HMAP-1 DNA binding activities were sequence specific with respect to AP-1 and absent from freshly isolated HSCs. Supershift EMSA and Western blot studies identified JunD, Fra2, and FosB as potential components of the HMAP-1. Mutations of the AP-1 site of the TIMP-1 promoter that prevented formation of HMAP-1 caused a 70% loss of activity in transfected activated HSCs. Taken together the data indicate that sustained upregulation of TIMP-1 gene expression may be at least partially controlled by a novel AP-1 dependent regulation of TIMP-1 promoter activity.

Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.

Liver fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSC), which proliferate during fibrotic liver injury. We have studied a model of spontaneous recovery from liver fibrosis to determine the biological mechanisms mediating resolution. Livers were harvested from rats at 0, 3, 7, and 28 d of spontaneous recovery from liver fibrosis induced by 4 wk of twice weekly intraperitoneal injections with CCl4. Hydroxyproline analysis and histology of liver sections indicated that the advanced septal fibrosis observed at time 0 (peak fibrosis) was remodeled over 28 d of recovery to levels close to control (untreated liver). alpha-Smooth muscle actin staining of liver sections demonstrated a 12-fold reduction in the number of activated HSC over the same time period with evidence of HSC apoptosis. Ribonuclease protection analysis of liver RNA extracted at each recovery time point demonstrated a rapid decrease in expression of the collagenase inhibitors TIMP-1 and TIMP-2, whereas collagenase mRNA expression remained at levels comparable to peak fibrosis. Collagenase activity in liver homogenates increased through recovery. We suggest that apoptosis of activated HSC may vitally contribute to resolution of fibrosis by acting as a mechanism for removing the cell population responsible for both producing fibrotic neomatrix and protecting this matrix from degradation via their production of TIMPs.

The plasminogen-activating system in hepatic stellate cells.

Urokinase plasminogen activator (uPA) generates plasmin, a process inhibited by plasminogen-activator inhibitor (PAI)-1 and localized to the cell surface by binding of uPA to a specific receptor. Plasmin degrades extracellular matrix (ECM) both directly and by activation of matrix metalloproteinases (MMPs). Because stellate cells play a central role in the pathogenesis of liver fibrosis both via production of ECM proteins and through secretion of MMPs, their contribution to plasmin generation was assessed. Stellate cells were prepared from rat liver and cultured on plastic. Northern analysis showed cellular expression of messenger RNA (mRNA) for PAI-1, uPA, and uPA receptor. Zymography/reverse zymography identified cell-surface-associated uPA activity and uPA and PAI-1 in culture media. Net uPA activity in culture media was maximal after 7 days in culture and then declined, whereas PAI-1 antigen levels remained consistently elevated between 7 and 21 days in culture. Stellate cell-mediated plasmin generation was also seen in in vitro cultures supplemented with plasminogen. Because hepatic stellate cells (HSCs) contain retinoids and release them on activation, the effect of retinoic acid on the plasminogen-activating system was also assessed. Treatment of cultured HSCs with retinoic acid (1 micromol/L) increased uPA secretion 2.6-fold but did not alter PAI-1. We conclude that stellate cells synthesize key components of the plasminogen-activating system and generate plasmin and therefore have the ability to regulate MMP activation. Upregulation of uPA synthesis by retinoic acid may have implications in matrix remodeling in sites of stellate cell activation in which high concentrations of retinoids may be achieved.

Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis.

Liver fibrosis results from a relative imbalance between synthesis and degradation of matrix proteins. We have previously described release of the protein collagenase inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), by culture-activated human hepatic stellate cells (HSCs). In this study, we have investigated the relative expression of TIMP-1 and interstitial collagenase in culture-activated rat HSCs and rat models of liver injury and fibrosis. The complementary DNA (cDNA) for rat TIMP-1 was obtained by homology polymerase chain reaction (PCR) and sequenced. By Northern analysis using this probe, TIMP-1 messenger RNA (mRNA) expression was up-regulated with HSC activation by culture on plastic as defined by cellular expression of procollagen-1. Interstitial collagenase mRNA was expressed in early 1. Interstitial collagenase mRNA was expressed in early culture (<4 days) but became undetectable in more activated cells (7-21 days). By activity assay of serum-free cell-conditioned media, TIMP-1 was found to be released in increasingly concentrations with duration of culture on plastic. Expression of TIMP-1 interstitial collagenase, and procollagen-1 mRNAs were studied in rat models of biliary and parenchymal injury (bile duct ligation and CC14 administration) by ribonuclease protein assay. TIMP-1 mRNA expression was increased at 6, 24 hours, and 3 days after bile duct ligation and was also shown to rise in acute CC14 liver injury and remain elevated as the liver became fibrotic. TIMP-1 expression preceded procollagen-1 expression in both models. In contrasts, interstitial collagenase mRNA levels remained similar to control values throughout both models of liver injury. Total cellular RNA from hepatocytes, HSCs, and kupffer cells freshly isolated from livers after acute CC14 injury was subjected to Northern analysis. TIMP-1 transcripts were observed in nonparenchymal cells only. We suggest that increased expression of TIMP-1 relative to interstitial collagenase by HSCs may promote progression of liver fibrosis in these rat models by preventing degradation of secreted collagens.

Expression of tissue inhibitor of metalloproteinases 1 and 2 is increased in fibrotic human liver.

BACKGROUND & AIMS: Tissue inhibitors of metalloproteinases may contribute to liver fibrosis by preventing remodeling of fibrillar collagens by interstitial collagenase. This hypothesis was investigated by comparing the relative expression of messenger RNA for interstitial collagenase, gelatinase A, and tissue inhibitor of metalloproteinases 1 and 2 in fibrotic and normal human liver. METHODS: Hepatic expression of metalloproteinases and their inhibitors was examined using ribonuclease protection assay, immunocytochemistry, and immunoassay. Lipocyte expression of tissue inhibitors of metalloproteinases was examined using Northern blotting and reverse zymography. RESULTS: Messenger RNA levels for tissue inhibitors of metalloproteinase 1 and 2 were elevated to 260%-526% of levels in normal liver in biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis. In fibrotic livers, tissue inhibitor of metalloproteinase 1 protein was 367%-724% of normal. Gelatinase A messenger RNA level increased to 324%-430% of normal values in fibrotic liver, but interstitial collagenase messenger RNA level was not significantly altered. Normal human liver lipocytes activated by culture on plastic expressed messenger RNA and protein for tissue inhibitor of metalloproteinase 1 and 2. CONCLUSIONS: Increased expression of tissue inhibitors of metalloproteinases relative to interstitial collagenase may promote deposition of interstitial collagens in liver fibrosis. Our studies further suggest that lipocytes are an important source of tissue inhibitors of metalloproteinases in progressive liver fibrosis.

Tissue inhibitor of metalloproteinase-I and interstitial collagenase expression in autoimmune chronic active hepatitis and activated human hepatic lipocytes.

1. Activated hepatic lipocytes are central to the pathogenesis of liver fibrosis as the principal source of both interstitial collagens and matrix-degrading metalloproteinases. In progressive fibrosis there is a failure to degrade interstitial collagens with a reported decrease in collagenase activity. In these studies we investigate expression of the potent collagenase inhibitor, tissue inhibitor of metalloproteinase-1, and interstitial collagenase in end-stage autoimmune chronic active hepatitis and activated human hepatic lipocytes in culture. 2. Messenger RNA transcripts for interstitial collagenase and tissue inhibitor of metalloproteinase-1 in explanted human liver were quantified by ribonuclease protection assay and densitometric analysis. This indicated that tissue inhibitor of metalloproteinase-1 and interstitial collagenase expression in autoimmune chronic active hepatitis were also coordinately up-regulated. 3. Using Northern analysis of RNA from human lipocytes in primary culture on plastic, mRNA for interstitial collagenase could not be detected in unstimulated cells but was present after stimulation with tumour necrosis factor alpha. Tissue inhibitor of metalloproteinase-1 mRNA was present in unstimulated lipocytes and up-regulated fivefold in response to tumour necrosis factor alpha. Using activity assay of serum-free conditioned media, interstitial collagenase could not be detected in unstimulated primary cultures, primary cultures stimulated with tumour necrosis factor alpha or transforming growth factor beta-1 (n = 3 and n = 4 respectively) or in passaged lipocytes (n = 6). In contrast, free tissue inhibitor of metalloproteinase-1 activity was present in unstimulated and passaged cultures and this was increased in response to tumour necrosis factor alpha and transforming growth factor beta-1.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effects of H1-antihistamines on histamine and PGD2 release from mast cells of human lung, tonsil, and skin.

Mast cells from different anatomic sites differ in cytochemistry and response to various secretory stimuli. We have investigated whether responsiveness to the second-generation H1-receptor antagonists, which are important first-line drugs for the relief of symptoms in patients with chronic urticaria and allergic rhinoconjunctivitis, also differs according to the site of origin of mast cells. The effects of terfenadine, ketotifen, and cetirizine were therefore examined in relation to the IgE-dependent release of histamine and prostaglandin D2 (PGD2) from dispersed human lung, tonsil, and skin mast cells. Terfenadine had a biphasic effect on lung and skin mast cells: at low concentrations, a concentration-dependent inhibition of histamine release from lung and skin mast cells was observed, whereas at higher concentrations the drug stimulated mediator release. Even at a high concentration, terfenadine inhibited mediator release from tonsil mast cells. Ketotifen had low potency as an inhibitor of mediator release from lung and tonsil mast cells. In skin mast cells, no inhibition of mediator release was observed below 1.0 microM, and above that concentration it induced mediator release. Cetirizine, a much less lipophilic drug than the others tested, did not induce mediator release from mast cells even at concentrations up to 100 microM. This drug showed concentration-dependent inhibition of IgE-dependent mediator release from lung and tonsil mast cells only. Our results show that human mast cells are heterogeneous with respect to modulation of mediator release by these H1-antihistamines. In particular, differences were observed between skin mast cells and those dispersed from lung and tonsils.

Analysis of human skin mast cell proteins by two-dimensional gel electrophoresis. Identification of tryptase as a sialylated glycoprotein.

Proteins of mast cells purified from human foreskin were separated by 2-D polyacrylamide gel electrophoresis using either nonequilibrium pH gradient electrophoresis or isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. Silver staining showed that a major feature of skin mast cell 2-D protein maps was a variety of relatively abundant proteins in the m.w. range of 29 to 37 kDa and covering a broad pH range from 5.0 to 8.5. Tryptase was identified on Western blots of 2-D-separated proteins by its binding of mAb and of 3H-diisopropylfluorophosphate. The precise distribution of tryptase varied among individuals but this protein generally occupied a continuum of molecular weights between 28 and 37 kDa and ranged in isoelectric point between 5.0 and 6.5. Tryptase was one of a number of mast cell proteins that bound the lectin concanavalin A as well as lectins specific for sialic acid, demonstrating that this enzyme is a sialylated glycoprotein. The diffuse m.w. distribution of skin mast cell tryptase (31 to 36 kDa) observed after SDS-PAGE was reduced to a single band of 30 kDa after treatment with protein-N-glycosidase F to remove asparagine-linked oligosaccharides. This finding suggests that intrinsic m.w. heterogeneity of tryptase in skin mast cells is largely a result of the addition of variable amounts of oligosaccharide to the tryptase polypeptide.

Inhibition profiles of sodium cromoglycate and nedocromil sodium on mediator release from mast cells of human skin, lung, tonsil, adenoid and intestine.

We have studied an aspect of the functional heterogeneity of human mast cells, namely responsiveness to the inhibitory effects of sodium cromoglycate and nedocromil sodium. The effects of these drugs were examined on the release of histamine and PGD2 from mast cells of human skin, lung, tonsils, adenoids and intestine. A high concentration, 1000 microM, of sodium cromoglycate was required to significantly inhibit histamine release from lung and tonsillar mast cells. Nedocromil sodium, 1000 microM, was more effective than sodium cromoglycate against histamine release from lung, tonsillar and adenoidal cells. Both compounds showed tachyphylaxis in lung and tonsillar mast cells but not in adenoidal and intestinal mast cells. In contrast, in intestinal mast cells, the effect of nedocromil sodium was weaker and more variable than sodium cromoglycate. Skin mast cells differed from mast cells of the other anatomical sites in being unresponsive to sodium cromoglycate and nedocromil sodium. Our results confirm that high concentrations of sodium cromoglycate and nedocromil sodium are required to achieve even modest inhibition of mediator release from human mast cells under in vitro conditions. Notwithstanding this, the results also indicate that differences exist among skin, lung, tonsillar, adenoidal and intestinal mast cells with respect to their sensitivity to sodium cromoglycate and nedocromil sodium, thus extending our knowledge of functional heterogeneity within the human mast cell populations.