Cheryl L. Scudamore

Constitutive expression of mouse mast cell protease-1 in normal BALB/c mice and its up-regulation during intestinal nematode infection.

Rodent intestinal mucosal mast cells (IMMC) store and secrete soluble granule serine proteases, the β‐chymases, which may promote epithelial permeability during intestinal hypersensitivity reactions. The β‐chymase mouse mast cell protease‐1 (mMCP‐1) is generally considered to be expressed late in the in vitro differentiation of mast cells. The purpose of this study was to determine the kinetics of mMCP‐1 transcription and expression in vivo during nematode‐induced IMMC hyperplasia. Concentrations of mMCP‐1 in blood and jejunum of BALB/c mice were quantified by enzyme‐linked immunosorbent assay before and at various stages after infection with the intestinal nematode Nippostrongylus brasiliensis. Mature mMCP‐1 enzyme was detected in jejunal homogenate (194 ng/mg soluble protein) and in blood (8·3 ng/ml serum) from normal uninfected BALB/c mice. Maximal IMMC hyperplasia occurred 7–14 days post infection and was significantly correlated with increased levels of mMCP‐1 in jejunum (r=0·58, P<0·001) and with raised concentrations of mMCP‐1 in serum (r=0·66, P<0·001). Transcription of the mMCP‐1 gene was detected by RNA blotting in normal, uninfected jejunum, but transcription was up‐regulated after infection with maximal transcription occurring on days 7 and 14. In conclusion, mMCP‐1 transcription, storage and secretion occur constitutively in normal BALB/c jejunum but this basal secretion is up‐regulated during nematode infection, suggesting both a physiological and pathological function for this protease.

Chemokine and cytokine expression in murine intestinal epithelium following Nippostrongylus brasiliensis infection

Infection of mice with the nematode parasite Nippostrongylus brasiliensis results in a well characterized intestinal mastocytosis with intraepithelial migration of mucosal mast cells (MMC). The molecules mediating this response are unknown. We examined expression of several putative mast cell chemoattractants in intestinal epithelium following N. brasiliensis infection. Expression of the chemokines monocyte chemoattractant protein‐1 (MCP‐1), macrophage inflammatory protein‐1α(MIP‐1α), RANTES (regulated on activation normal T‐cell expressed and secreted), fractalkine, and thymocyte expressed chemokine (TECK); and the cytokines stem cell factor (SCF) and transforming growth factor β1 (TGFβ1), was constitutive and no alteration was detected following infection. MCP‐1 expression was also constitutive but at much lower levels and increased expression was detected on days 7 and 14 postinfection. Expression of MCP‐1 in whole jejunum was at much higher levels than in epithelium. Constitutive expression of MCP‐1, MIP‐1α and TGFβ1 was also detected in cultured bone marrow‐derived homologues of MMC. In an intestinal epithelial cell line (CMT‐93), there was constitutive expression of SCF, TGFα1, fractalkine and MCP‐1. The results show that, in vivo, epithelium is a potentially important source of mast cell chemoattractants.

TGF-β1 Regulates Adhesion of Mucosal Mast Cell Homologues to Laminin-1 Through Expression of Integrin α7

Mucosal mast cells (MMC) or their precursors migrate through the intestinal lamina propria to reside intraepithelially, where expression of mouse mast cell protease-1 indicates the mature phenotype. Alterations in expression of integrins that govern cell adhesion to the extracellular matrix may regulate this process. As the key cytokine mediating differentiation of mouse mast cell protease-1-expressing MMC homologues in vitro, TGF-β1 was considered a likely candidate for regulation of the integrins that facilitate intraepithelial migration of MMC. Therefore, we examined adhesion of bone marrow-derived mast cells cultured with and without TGF-β1 to laminin-1, fibronectin, and vitronectin along with expression of integrins likely to regulate this adhesion. Adhesion of PMA-stimulated cultured mast cells to laminin-1 increased from 5.3 ± 3.6% (mean ± SEM) in the absence of TGF-β1 to 58.7 ± 4.0% (p < 0.05) when cultured mast cells had differentiated into MMC homologues in the presence of TGF-β1. Increased adhesion of MMC homologues to laminin-1 was also stimulated by FcεRI cross-linking and the calcium ionophore A23187. Expression of the laminin-binding integrin α7 by MMC homologues grown in the presence of TGF-β1 was demonstrated by RT-PCR and flow cytometry, and preincubation of MMC homologues with the α7-neutralizing Ab 6A11 inhibited adhesion to laminin-1 by 98% (p < 0.05), demonstrating a novel role for this molecule in adhesion of a hemopoietic cell to laminin-1.

Potentiation of the extracellular release of equine neutrophil elastase and alpha-1-proteinase inhibitor by a combination of two bacterial cell wall components: fMLP and LPS.

REASONS FOR PERFORMING STUDY Lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-like peptides are Gram-negative bacterial cell wall components which, when released into the peripheral circulation in endotoxaemia, have the potential to activate leucocytes. In vitro, equine neutrophils require priming with LPS in order to generate reactive oxygen intermediates (ROI) in response to fMLP. OBJECTIVES The aim of this study was to examine whether the release of other neutrophil products is similarly dependent on prior priming with LPS. In particular, neutrophil elastase (NE), a potent proteolytic enzyme, and its major inhibitor, alpha-1 proteinase inhibitor, were investigated. METHODS Neutrophils were isolated from equine peripheral blood (n = 5) by discontinuous Percoll gradient preparative centrifugation and primed with LPS prior to stimulation with fMLP. ROI were measured by lucigenin dependent chemiluminescence (LDCL). Concentrations of NE and API were determined by ELISA on cell free supernatants taken at 0, 2, 10, 30, 60 and 90 mins post stimulus. Data was analysed by Kruskal-Wallis and Mann-Whitney Tests. RESULTS Sequential exposure of Percoll purified equine blood neutrophils in vitro to LPS followed by fMLP resulted in the greatest release of NE from equine neutrophils and was required for ROI generation. However, LPS or fMLP stimulation alone resulted in an increase in NE release compared to unstimulated control cells. In contrast, significant API release was only induced by LPS stimulation or fMLP stimulation only after LPS priming, not fMLP on its own. CONCLUSIONS These results suggest that different stimuli (fMLP or LPS) are capable of invoking similar responses from equine neutrophils with respect to NE release yet different ones with respect to API release. POTENTIAL RELEVANCE In addition, demonstration of elastase release induced by LPS and/or fMLP suggests that monitoring serum elastase levels is a potential diagnostic tool for detecting the early onset of endotoxaemia in the horse.

Characterisation of tryptase and a granzyme H-like chymase isolated from equine mastocytoma tissue.

Mast cell proteinases are important inflammatory mediators in man and other species, but until now there has been no investigation of the nature of equine mast cell proteinases. These studies describe the purification and characterisation of two proteolytic components from equine mastocytoma tissue, detected using chromogenic substrates for trypsin and chymotrypsin. Following chromatographic purification, the trypsin-like component was found to be equine mast cell tryptase by N-terminal amino acid sequencing, showing a close similarity with human tryptase-beta (85% identity over 20 residues). It also had similar subunit molecular size (34-36kDa by SDS-PAGE) and substantially similar cleavage specificity to human tryptase-beta with the substrates tested. A 32kDa chymotrypsin-like component was also purified from mastocytoma extract, and termed equine mast cell proteinase-1 (eqMCP-1). The N-terminal amino acid sequence of eqMCP-1 was very similar to human granzyme H (95% over 19 residues). Rabbit antisera directed against tryptase and eqMCP-1 both detected equine mast cells by immunohistochemistry, and will be of use in future clinical studies of the relevance of mast cell proteinases in equine allergic disease.

Measurement by ELISA of equine alpha-1-proteinase inhibitor in uterine flushings from mares

An enzyme linked immunosorbent assay (ELISA) was developed and used to estimate the concentrations of the serine proteinase inhibitor, alpha-1 proteinase inhibitor (API), in uterine flushings recovered from mares at different stages of the oestrous cycle and before and after the induction of experimental endometritis. There was a significant increase in the concentrations of API and albumin relative to total protein in flushings recovered during oestrus compared with dioestrus but no difference was observed in the concentrations of these proteins relative to total protein before and after the induction of endometritis. A regression analysis revealed a significant correlation between the concentrations of albumin and API in the flushings examined, suggesting that the API was derived entirely from serum and was not produced locally in the uterus.

Neutrophil chemotaxis in the horse is not mediated by a complex of equine neutrophil elastase and equine alpha-1-proteinase inhibitor

Studies have demonstrated that as a result of proteolytic inactivation or complex formation (with neutrophil elastase), human alpha-1-proteinase inhibitor (API) becomes a potent chemoattractant for human neutrophils. The present study aimed to investigate the in vitro chemotactic response of equine neutrophils to an equivalent complex of equine API and neutrophil elastase. No evidence of neutrophil migration was observed towards purified complex derived from equine neutrophil elastase and the Spi 1 isoform of equine API, or to crude mixtures of porcine pancreatic elastase and unseparated equine API isoforms, although the same neutrophil preparations actively migrated towards zymosan activated plasma. It was concluded that, in the horse, complexes of API are not involved in the migration of neutrophils to sites of inflammation.

Improved hepatic and pancreatic localisation of the equine alpha-1-proteinase inhibitor family of serpins using an antigen enhancement technique and a monoclonal antibody.

Equine alpha-1-proteinase inhibitor (API) consists of three, occasionally four, serum glycoproteins. This study investigated the immunohistochemical localisation of equine API in paraformaldehyde fixed, paraffin embedded equine tissue samples of liver, lung, stomach, pancreas, jejunum and colon in five horses using affinity purified sheep polyclonal and protein A purified mouse monoclonal antibodies, whose specificities were verified by Western blotting. Exposing tissue sections to boiling citrate buffer greatly enhanced antigen recovery and improved immunostaining with both antibodies, resulting in discovery of novel tissue distribution patterns for the horse. In the horses studied, all hepatocytes showed some degree of cytoplasmic staining, many having perinuclear intense granular inclusions. This finding is contrary to findings in human studies where hepatocytes of Pi MM phenotype have proven difficult to stain for human API, despite evidence at the molecular level suggesting hepatocytes as the major source of serum API. This discrepancy may be due to the use of different tissue fixation and antigen recovery techniques. In all other tissues examined, the distribution of equine API was similar to human studies.

Comparative studies of the Spi1 proteins of three equine alpha-1-proteinase inhibitor haplotypes following isolation by affinity chromatography

1. Antiproteinase deficiency can result in excessive proteinase-induced tissue damage. The major anti-elastase (Spi1) protein of equine alpha 1-proteinase inhibitor (alpha 1-PI) was isolated from the plasma/serum of three common haplotypes (I, L and U). 2. The N-terminal amino acid sequences of the three inhibitors were identical, but were only approx 65-77% homologous with two other published equine Spi1 sequences. 3. All three inhibitors complexed quickly and irreversibly with equine leucocyte proteinase 2A (kass = 2 x 10(7) M-1 sec-1). They were also efficient inhibitors of chymase (rat mast cell proteinase-II; kass = 2 x 10(5) M-1 sec-1; Ki = 2 x 10(-10) M). There was therefore no evidence of deficient inhibition in the Spi1 variants of the I,L and U haplotypes.

Serum alpha‐1‐proteinase inhibitor concentration in 2 Quarter Horse foals with idiopathic pyogranulomatous panniculitis

Sterile nodular panniculitis has been recognised as a relatively uncommon dermatological condition in a wide variety of species including man, dogs, cats and horses (Jubb et al. 1993). It is considered in many cases to be an idiopathic condition although it can also be a manifestation of a variety of underlying disease processes including immune-mediated disorders, nutritional deficiencies and neoplasia. In man, some cases of sterile panniculitis are known to be associated with a specific deficiency of the serum glycoprotein, α1-antitrypsin (Pittlekow et al. 1988) more correctly known as α1proteinase inhibitor (API) because, in addition to inhibiting trypsin, it plays a major role in the inhibition of neutrophilderived neutral proteases, particularly elastase. It also has a broad spectrum of activity against a variety of other proteases involved in the regulation of a diverse range of biological processes including inflammation, fibrinolysis, coagulation, phagocytosis, activation of zymogens and release of peptide hormones (Breit et al. 1985; Smith et al. 1989). The importance of API in controlling such a wide range of biological processes is demonstrated by the well-documented deficiency state in man which is associated with a variety of diseases including premature emphysema, cirrhosis and vasculitis in addition to panniculitis (Crystal 1996). The pathogenesis of API deficiency panniculitis is obscure but may involve unregulated neutrophil protease-mediated damage to connective tissue, as occurs in the development of emphysema in API deficient individuals, or alterations in lymphocyte and phagocyte activation (Black and Cunliffe 1998). Only one study has so far investigated the API status of animals with panniculitis and this failed to show any evidence of deficiency in 9 dogs with the condition (Hughes et al. 1996). Unlike human API, which is transcribed from a single gene producing one plasma glycoprotein (Crystal 1989), equine API consists of 4 or 5 plasma glycoproteins controlled by 4 closely linked loci named serine proteinase inhibitor (Spi) 1, 2, 3A, 3B and 4 (the latter of which is only expressed in 5 of the 22 known haplotypes). Limited sequence data suggests that these 4 loci are all derived from the identical ancestral gene that gave rise to human API and biochemical studies suggest that the Spi1 isoform is the closest functional analogue of human API (Patterson and Bell 1989; Patterson e t a l . 1991; Potempa et al. 1991). In the current study, 2 foals from the same Quarter Horse mare developed apparent idiopathic pyogranulomatous panniculitis within the first few months postpartum. The aim was to determine whether a relative, absolute or functional deficiency of API could be implicated in the pathogenesis of panniculitis in these foals.