Innate immune response mechanisms in the intestinal epithelium: potential roles for mast cells and goblet cells in the expulsion of adult Trichinella spiralis.

SUMMARYGastrointestinal infection with the nematode Trichinella spiralis is accompanied by a rapid and reversible expansion of the mucosal mast cell and goblet cell populations in the intestinal epithelium, which is associated with the release of their mediators into the gut lumen. Both goblet cell and mast cell hyperplasia are highly dependent on mucosal T-cells and augmented by the cytokines IL-4 and IL-13. However, the contribution of both mast and goblet cells, and the mediators they produce, to the expulsion of the adults of T. spiralis is only beginning to be elucidated through studies predominantly employing T. spiralis-mouse models. In the present article, we review the factors proposed to control T. spiralis-induced mucosal mast cell (MMC) and goblet cell differentiation in the small intestine, and focus on some key MMC and goblet cell effector molecules which may contribute to the expulsion of adult worms and/or inhibition of larval development.

Organic dust exposure increases mast cell tryptase in bronchoalveolar lavage fluid and airway epithelium of heaves horses.

BACKGROUND: Mast cell degranulation is believed to act as a key event in initiating and maintaining airway response to allergen challenge in human asthma. It is hypothesized that the mast cell may play a similar role in equine heaves, which shares many similarities with occupational dust-induced asthma. OBJECTIVE: The aim of this study was to quantify the mast cell proteinase tryptase in bronchoalveolar lavage fluid (BALF) from control and heaves-susceptible horses and to investigate tryptase mRNA and protein expression in pulmonary mast cells. METHODS: Equine BALF tryptase concentrations were determined by ELISA from control and heaves-susceptible horses pre and post 24 h hay/straw challenge (HSC). Tryptase mRNA and protein expression were investigated by quantitative PCR and immunohistochemistry in bronchial and bronchiolar tissue samples of control and heaves-susceptible horses. RESULTS: Both control and heaves-susceptible horses had significantly increased BALF tryptase concentrations following HSC (P=0.003 and 0.034, respectively). Increased numbers of tryptase-expressing intra-epithelial mast cells were demonstrated in heaves horses, but not controls, following challenge (P=0.02). Bronchiolar tissue from heaves horses removed from challenge contained significantly lower tryptase transcripts than that from control horses (P=0.02). CONCLUSION: Mast cell degranulation and tryptase release into the airways occur following HSC of control and heaves-susceptible horses. The greater number of mast cells available in the bronchiolar epithelium of heaves horses may be clinically significant in the pulmonary inflammatory response of heaves.

cDNA cloning and substrate specificity of equine tryptase, a possible mediator in equine heaves.

BACKGROUND: Mast cell mediators are believed to play a central role in inflammatory lung disorders such as human allergic and occupational asthma. Equine heaves is characterized by reversible neutrophilic airway inflammation and airway obstruction, primarily due to bronchospasm and mucus hypersecretion, following exposure of susceptible horses to organic stable dusts. As such, heaves shares many similarities with human occupational dust-induced asthma and therefore it is proposed that mast cells may also be implicated in the pathogenesis of heaves. Tryptase, a mast cell-specific proteinase, can be used as an indicator of biological mast cell activity. OBJECTIVE: The aim of this study was to determine the cDNA sequence of equine tryptase and to investigate its substrate specificity in order to rationalize its enzymatic activity. METHODS: RT-PCR cloning was used to sequence equine tryptase. Substrate specificity of equine tryptase was investigated using arginine and lysine containing substrates. RESULTS: The cDNA and deduced amino acid (Aa) sequences for equine tryptase shared strong identity with other tryptases. Unusually for a trypsin-like proteinase however, equine tryptase has alanine at residue 216, rather than glycine, which confers increased arginine substrate specificity in vitro and may restrict fibrinogenolysis in vivo. CONCLUSION: Cloning and sequencing of the mast cell proteinase equine tryptase will allow molecular probing of its expression in the lung of control and heaves-affected horses. Further work is warranted to determine the biological relevance of the unique alanine 216 substitution in the molecular sequence of the equine tryptase substrate-binding pocket.

Cloning and molecular modeling of duodenase with respect to evolution of substrate specificity within mammalian serine proteases that have lost a conserved active-site disulfide bond.

Mammalian serine proteases such as the chromosome 14 (Homo sapiens, Mus musculus) located granzymes, chymases, cathepsin G, and related enzymes including duodenase collectively represent a special group within the chymotrypsin family which we refer to here as "granases". Enzymes of this group have lost the ancient active-site disulfide bond Cys191-Cys220 (bovine chymotrypsinogen A numbering) which is strongly conserved in classic serine proteases such as pancreatic, blood coagulation, and fibrinolysis proteases and others (granzymes A, M, K and leukocyte elastases). We sequenced the cDNA encoding bovine (Bos taurus) duodenase, a granase with unusual dual trypsin-like and chymotrypsin-like specificity. The sequence revealed a 17-residue signal peptide and two-residue (GlyLys) activation peptide typical for granases. Production of the mature enzyme is apparently accompanied by further proteolytic processing of the C-terminal pentapeptide extension of duodenase. Similar C-terminal processing is known for another dual-specific granase, human cathepsin G. Using phylogenetic analysis based on 39 granases we retraced the evolution of residues 189 and 226 crucial for serine protease primary specificity. The analysis revealed that while there is no obvious link between mutability of residue 189 and the appearance of novel catalytic properties in granases, the mutability of residue 226 evidently gives rise to different specificity subgroups within this enzyme group. The architecture of the extended substrate-binding site of granases and structural basis of duodenase dual specificity based on molecular dynamic method are discussed. We conclude that the marked selectivity of granases that is crucial to their role as regulatory proteases has evolved through the fine-tuning of specificity at three levels--primary, secondary, and conformational.

Purification and characterization of mouse mast cell proteinase-2 and the differential expression and release of mouse mast cell proteinase-1 and -2 in vivo.

BACKGROUND: Gastrointestinal nematode infection is associated with mucosal mast cell (MMC) hyperplasia. In the mouse, this is accompanied by the release of substantial quantities of the chymase mouse mast cell proteinase-1 (mMCP-1) into the gut lumen and peripheral bloodstream. Expression of mMCP-1 is largely restricted to intraepithelial MMC and is thought to play a role in the regulation of epithelial permeability. MMCs also express mouse mast cell proteinase-2 (mMCP-2), but less is known about the expression or biological function of this proteinase. OBJECTIVES: (1) To purify and characterize mMCP-2. (2) To compare the expression and release of mMCP-2 and mMCP-1 in vivo using specific antibodies. METHODS: Bone marrow-derived mast cells (mBMMCs) were generated from mMCP-1(-/-) BALB/c mice. mMCP-2 was purified, characterized and used to generate rat and sheep polyclonal antibodies. The expression and systemic release of mMCP-1 and -2 were compared in vivo by immunohistochemistry and ELISA. RESULTS: mMCP-2 was successfully purified from mMCP-1(-/-) mBMMC and its identity confirmed by N-terminal amino acid sequencing. mMCP-2 bound [3H]-labelled DFP, indicating the presence of an active serine proteinase catalytic site, but showed little evidence of chymotryptic activity. MMC expressed comparable levels of mMCP-1 and -2 in the jejunum but not in the gastric mucosa, where mMCP-2 was more abundant. Expression of both proteinases increased substantially during primary Nippostrongylus brasiliensis infection and this was accompanied by a substantial increase in peripheral blood levels of mMCP-1 (70 microg/mL on day 12). By contrast, mMCP-2 was not detected in the serum of uninfected mice and only increased to approximately 25 ng/mL on day 12. CONCLUSION: As in the case of mMCP-1, mMCP-2 expression is restricted to MMC. However, mMCP-2 lacks chymase activity, is expressed at higher levels in gastric MMC and appears to be differentially released into the peripheral bloodstream.

Peripheral blood mononuclear cell responses to major and minor Dermatophagoides allergens in canine atopic dermatitis.

Atopic dermatitis is a chronic inflammatory and pruritic skin disease commonly seen in dogs and humans. Most cases involve hypersensitivity to the house dust mites (HDM) Dermatophagoides farinae and Dermatophagoides pteronyssinus. Human atopic dermatitis is associated with the HDM derived allergens Der f 1 and 2, and Der p 1 and 2. Serological data, however, suggest that a 98/104kD protein is the most important allergen in dogs with atopic dermatitis. The aim of this study was to characterise the specificity of circulating T-cells in canine atopic dermatitis for HDM derived allergens. Peripheral blood mononuclear cells (PBMCs) from dogs with atopic dermatitis that were skin test positive for D. farinae and D. pteronyssinus were cultured with crude extracts of D. farinae, D. pteronyssinus and D. microceras, a 98/104kD allergen purified from D. farinae, Der f 1 and Der f 2. There was significantly greater responsiveness of PBMCs to the D. farinae and D. pteronyssinus extracts compared to the D. microceras extract, and similarly to the purified 98/104kD allergen compared to Der f 1 and Der f 2. The close association between serological findings and PBMC proliferation implies that the 98/104kD HDM protein is a major target of immune recognition and that T-cells also participate in the pathogenesis of canine atopic dermatitis by supporting IgE production.

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.

Local lung responses following local lung challenge with recombinant lungworm antigen in systemically sensitized sheep.

BACKGROUND: Chronic mast cell-mediated inflammation may contribute significantly towards the extensive tissue remodelling that is a feature of lungworm infection in ruminants. Understanding the factors that control tissue remodelling is a necessary step toward effective management and treatment of conditions that feature such pathology. OBJECTIVE: We sought to define in a novel ovine model system, the cellular, immune and mast cell phenotypic events that occur following local lung challenge with a recombinant protein antigen, DvA-1, derived from the ruminant lungworm nematode, Dictyocaulus viviparus. METHODS: Two spatially disparate lung segments in systemically sensitized sheep were challenged on three occasions with DvA-1 (3xDVA) and two further segments were challenged with saline (3xSAL). Two months after the third challenge, one of the two segments previously repeatedly challenged with DvA-1 was challenged again with DvA-1 (3xDVA:DVA) whilst the other was challenged with saline (3xDVA:SAL). A similar protocol was followed with the saline challenged segments (3xSAL:SAL and 3xSAL:DVA). Bronchoalveolar lavage fluid (BALF) (n = 16) and tissue (n = 3) were collected after the last challenge. RESULTS: Cellular changes 24 h after the fourth challenge were characterized by an increase in the absolute numbers of neutrophils and eosinophils in BALF from 3xDVA:DVA and 3xSAL:DVA segments. Local antibody production was implied through increased levels of antibody in both 3xDVA:DVA and 3xDVA:SAL segments, with the latter being unaffected by inflammation. Levels of active transforming growth factor beta-1 (TGF-beta(1)) were significantly increased in 3xDVA:SAL segments and a trend towards an increase was apparent in 3xDVA:DVA segments. Total TGF-beta1 levels were significantly correlated with eosinophil counts in all except the 3xDVA:SAL segments. Such changes in the bronchoalveolar space were complemented by increased ratios of sheep mast cell proteinase-1 expressing cells and tryptase expressing cells, to toluidine blue positive cells in airways from 3xDVA:DVA segments. CONCLUSION: Mast cell phenotypic events occurring as a consequence of antigen challenge were limited to segments in which changes in BALF were characterized by neutrophil influx and increased local antibody production.

cDNA sequence of two sheep mast cell tryptases and the differential expression of tryptase and sheep mast cell proteinase-1 in lung, dermis and gastrointestinal tract.

BACKGROUND: Mast cell tryptases are a family of serine proteinases which are implicated in the proliferation of smooth muscle cells and fibroblasts, upregulation of interleukin-8 synthesis by endothelial cells, and recruitment of neutrophils and eosinophils. Trials in sheep showed that administration of a specific tryptase inhibitor reduced the late-phase response to inhaled allergen. OBJECTIVES: The aim of this study was to characterize the sequence and distribution of sheep tryptase(s), to validate the sheep model of allergic lung disease. METHODS: Reverse transcriptase PCR cloning was used to obtain cDNA sequences for two sheep tryptases. Lung and gut extracts were used as a source of tryptase for partial purification and characterization of the protein. The distribution of tryptase in skin, lung and gut was determined by immunohistochemistry, and compared with the distribution of sheep mast cell proteinase-1 (sMCP-1). RESULTS: Two highly similar cDNA sequences encoding sheep tryptase were found, indicating the presence of a 28 amino acid leader sequence, and a mature peptide of 245 amino acids. Partial purification of a putative sheep tryptase from lung and gut extracts was achieved using heparin-Sepharose affinity chromatography. Rabbit antihuman skin tryptase antiserum recognized the putative sheep tryptase on Western blot (approximate Mr 32-34 000) and paraformaldehyde-fixed tissue sections. Tryptase was detected in all lung, skin and gut mast cells by this antibody, and transcripts for tryptase were detected in all three tissues by RT PCR. Sheep mast cell proteinase-1, detected by a specific monoclonal antibody, was present in all intestinal and gastric mucosal mast cells, but was not found in mast cells of the muscularis, thus defining at least two mast cell phenotypes in the gut. Whereas all dermal and pulmonary mast cells were tryptase positive, only a low proportion in the lung, and almost none in the dermis, were positive for sMCP-1. CONCLUSION: In view of the structural and functional similarities of sheep and human tryptases, and their similarity in tissue distribution in normal sheep, the sheep lung appears to be a good model for in vivo studies relating to human tryptase.

Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation.

Man and horses both suffer from neutrophil mediated pulmonary diseases however there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to human respiratory disease it is not a major feature of the common equine neutrophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain less elastase than equivalent human cells and therefore there is a reduced risk of excess and/or uninhibited elastase activity, which is considered the major cause of pulmonary emphysema in man, in the horse lung. In previous studies equine neutrophil elastase (ENE) has been assayed by measuring elastinolytic activity whereas human neutrophil elastase content has been determined using immunological techniques. Neutrophils contain several intracellular protease inhibitors therefore measurement of elastase activity may underestimate the total NE content. The aim of the current study was to develop immunological techniques to allow investigation of the cellular content, distribution and release of ENE from purified equine neutrophils. Equine neutrophil elastase 2A (ENE 2A), the most abundant elastase in equine neutrophils, and equine alpha-1-proteinase inhibitor (API), the main inhibitor of elastase were found to be present at 0.813 pg +/- 0.179 and 0.021 pg +/- 0.003 (mean +/- SEM, n = 11 individual horses) per neutrophil, respectively. This represents twice as much elastase as previously found in the equine neutrophil and a comparable amount to that reported in human neutrophils. Immunolocalisation demonstrated that ENE 2A has a granular distribution within the cytosol of neutrophils, whereas API exhibits a uniform non-granular cytoplasmic appearance. In addition the kinetics of simultaneous generation and release of superoxide anions (SOA) and release of ENE 2A from equine neutrophils, stimulated in vitro by zymosan-activated serum (ZAS) in the presence and absence of the cation chelator ethylene glycol-N,N,N',N'-tetraacetic acid (EGTA), showed a close relationship between total SOA generation and total ENE 2A release during the initial 90 min post-ZAS stimulation and the dependence of both events on extracellular cations. In conclusion these studies have shown that horse and human neutrophil elastase content and mediator release functions are more closely matched than was previously thought. This suggests that the species differences in pathology resulting from neutrophil-mediated respiratory disease are determined by other factors such as differences in the abundance and function of intra- and extra-cellular protease inhibitors.

Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity.

Sheep mast-cell proteinase-1 (sMCP-1) is a serine proteinase expressed predominantly by mucosal mast cells, with specificity for cleavage C-terminal to basic and hydrophobic amino acid residues. A cDNA encoding sMCP-1 has been cloned using reverse transcriptase (RT)-PCR. It appears to be translated as a pre-proenzyme with a 17-amino-acid signal peptide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic domain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constructed for both enzymes using coordinates for the refined X-ray structures of human cathepsin G, chymase and rat mast-cell proteinase-2. The model for sMCP-1 suggests that the acidic Asp-226 side chain extends into the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moiety in this position would favour interaction with basic substrate residues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance between chymotryptic and tryptic activities of sMCP-1 was found to be sensitive to salt concentration, with increasing univalent cation concentration favouring chymotryptic activity relative to the tryptic. Using a peptide substrate representing residues 36-59 of the human thrombin receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.

Differential inhibition of mast cell chymases by secretory leukocyte protease inhibitor.

The major physiological role of human secretory leukocyte protease inhibitor (SLPI), a low molecular weight inhibitor present in mucus, is the rapid formation of a tight-binding inhibitory complex with neutrophil elastase. It is also the most effective known inhibitor of human mast cell chymase. The inhibitory efficacy of recombinant SLPI towards three other mast cell chymases was therefore investigated. Rat mast cell proteinases-1 and -2 (rMCP-1 and -2, respectively) and sheep mast cell proteinase-1 (sMCP-1), a chymase with additional tryptase-like properties, were treated with the inhibitor. SLPI inhibited rMCP-1 very efficiently in the absence of heparin, with a low dissociation constant, Ki = 3 x 10(-10) M and high second order association constant, kass = 8.0 x 10(6) M(-1) s(-1), and inhibition was enhanced when heparin was present. rMCP-2 was not inhibited by SLPI in the presence or absence of heparin, and did not degrade SLPI on prolonged incubation. SLPI inhibited sMCP-1 very poorly in the absence of heparin (Ki = 9 X 10(-6) M). However, in the presence of heparin, the Ki for inhibition of sMCP-1 by SLPI was reduced to the nanomolar range. sMCP-1 was observed to cleave SLPI with chymase-like specificity at Leu72-Met73 on prolonged incubation in the absence of heparin, but increasing concentrations of heparin reduced the extent of cleavage.

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.

Sheep mast cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery fibroblasts.

Sheep mast cell proteinase-1 (sMCP-1), a serine proteinase with dual chymase/tryptase activity, is expressed in gastrointestinal mast cells, and released systemically and on to the mucosal surface during gastrointestinal nematode infection. The potential for native plasma proteinase inhibitors to control sMCP-1 activity was investigated. Sheep alpha1-proteinase inhibitor (alpha1PI) inhibited sMCP-1 slowly, with second-order association rate constant (kass) 1. 1x10(3) M-1.s-1, whereas sheep contrapsin inhibited trypsin (kass 2.2x10(6) M-1.s-1) but not sMCP-1. Western-blot analysis and gel filtration showed that when added to serum or plasma, sMCP-1 was partitioned between alpha1PI and alpha2-macroglobulin. The possibility that significant cleavage of plasma proteins could occur before sMCP-1 was inhibited was investigated using gel filtration and SDS/PAGE after adding sMCP-1 to plasma. Cleavage of ovine fibrinogen occurred in the presence of excess alpha1PI and alpha2-macroglobulin, the alpha-chain being cleaved C-terminally and the beta-chain at the putative Lys-27. In addition, sMCP-1 was found to be mitogenic for bovine pulmonary artery fibroblasts, but was not mitogenic in the presence of soya-bean trypsin inhibitor. In terms of fibrinogen cleavage and fibroblast stimulation, sMCP-1 shows functional similarities to mast cell tryptase.

Sheep mast cell proteinase-1: characterization as a member of a new class of dual-specific ruminant chymases.

Sheep mast cell proteinase 1 (SMCP-1), which is abundantly expressed in gastrointestinal but not skin mast cells, was isolated and its substrate specificity was investigated. Peptide substrates, including angiotensin I, substance P, bradykinin and oxidized insulin B chain were hydrolysed at P1 Phe, Leu or Tyr residues, conforming to the known chymotrypsin-like properties of the enzyme. However, SMCP-1 was found to hydrolyse some chromogenic substrates with P1 Lys and Arg residues. The enzyme also demonstrated trypsin-like activity against protein substrates, cleaving BSA at Lys114-Leu115, Lys238-Val239, Lys260-Tyr261 and Lys376-His377. Bovine fibrinogen beta-chain was cleaved at Lys28-Lys29. To ensure homogeneity of the enzyme, the ratio of chymotrypsin-like to trypsin-like activity was observed; it was found to be constant during purification and between different preparations of SMCP-1. Treatment of SMCP-1 with a range of inhibitors decreased chymotrypsin-like and trypsin-like activities by similar extents, supporting the assertion that both activities are the property of a single enzyme. In terms of activity, and by N-terminal amino acid sequencing, SMCP-1 strongly resembles the similarly dual-specific bovine duodenal proteinase, duodenase. It is proposed that SMCP-1 and duodenase represent a new class of ruminant chymases with unusual dual specificities.

Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases.

We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species.

Decrease in the alpha 1-proteinase inhibitor Spi3 in equine bronchoalveolar lavage fluid.

The alpha 1-proteinase inhibitors of trypsin, Spi1, Spi3A, and Spi3B, in bronchoalveolar lavage fluid (BALF) and serum of horses were separated by electrophoresis, and their proportions were quantified in 12 control horses and 12 with chronic obstructive pulmonary disease (COPD). A significantly lower proportion of Spi3B (P < 0.05) and higher proportion of Spi1 (P < 0.02 to P < 0.01) were detected in BALF, compared with serum, in control and COPD-affected horses and appeared to be attributable to reduced Spi3 activity in BALF. There was no significant difference between the control and COPD groups in this respect, indicating that the decrease in Spi3 may be a physiologic phenomenon. The differences observed may be associated with proteolytic damage to or preferential complex formation by Spi3.

Quantitation of alpha-1 proteinase inhibitor in the pulmonary epithelial lining fluid of horses with chronic obstructive pulmonary disease.

The concentration of alpha-1 proteinase inhibitor (API) was measured in the pulmonary epithelial lining fluid (PELF) of horses with chronic obstructive pulmonary disease (COPD) while they had clinical signs and while they had none. The concentrations of total protein, albumin and API were significantly higher in the PELF of animals with clinical signs of COPD. The correlation between albumin and API in the PELF suggested that most of the API was derived from the serum.

Molecular weight alterations of alpha-1 proteinase inhibitor in equine bronchoalveolar lavage fluid.

The equine alpha-1 proteinase inhibitor (alpha 1PI) system differs from that of man in that the equine system consists of four closely-linked genes (Spi1-Spi4) whereas in man, a single gene encodes for alpha 1PI. We have previously found differences in the proportion of the Spi proteins in equine serum and bronchoalveolar lavage fluid (BALF). We therefore wished to determine whether, as reported in man, there was any molecular weight difference between the Spi proteins in serum and BALF. alpha 1PI and albumin from equine BALF migrated further towards the anode compared with serum alpha 1PI on native polyacrylamide gel electrophoresis (PAGE) although the difference was only significant for alpha 1PI. Sodium dodecyl sulphate-PAGE (SDS-PAGE) showed that a mean decrease in molecular weight of 1.5 kDa for alpha 1PI and 1.3 kDa for albumin had occurred in BALF. These findings were observed in control animals and in those with symptomatic or asymptomatic chronic obstructive pulmonary disease. The mechanism of this decrease in molecular weight of alpha 1PI is likely to differ from reports of alpha 1PI cleavage by bacterial proteinases in man since the molecular weight change was relatively small and loss of trypsin inhibitory activity did not occur. Nor, in our system, was there evidence of bacterial infection. Damage by endogenous proteinases or glycosidases at a site other than the reactive site may be involved but the resultant effect on the efficiency of the antiproteinase screen of the lower respiratory tract is uncertain.

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.