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Mast cell tryptase levels in normal canine tissues
Veterinay
Veterinary Immunology and Immunopathology 46(1995)223-235
immunology and immunopathology
Mast cell tryptase levels in normal canine tissues A.D. Myles, R.E.W. Halliwell*, B. Ballauf, H.R.P. Miller Department of Veterinary Clinical Studies, Royal (Dick) School of kterinary Studies. Universityof Edinburgh, Summerhall, Edinburgh EH9 lQH, LX Accepted 7 July I994
Abstract
Levels of canine tryptase from various tissues were quantified using a competition enzyme-linked immunosorbent assay (ELISA). The assay utilises an affinity-purified rabbit anti-tryptase antibody in the solid phase and alkaline-phosphatase conjugated tryptase together with unlabelled tryptase in the fluid phase. The assay will rapidly quantify 40-5000 ng ml-’ of tryptase in tissue extracts. Tissues from the skin, gut, liver and lung were studied, of which canine gut appeared to contain the highest levels of tryptase per milligram wet weight, which may suggest an important role for this enzyme at this site. This assay may prove valuable in assessing the role of mast cells in various disease states in the dog. Abbreviations ELISA=enzyme-linked immunosorbent assay; HSB = high salt buffer: LSB = low salt buffer; SDS-PAGE=sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
1. Introduction Mast cells contain large numbers of metachromatic granules, which store and release the serine proteases chymase and tryptase, simultaneously with other preformed mediators. Two types of chymase have been isolated from rat mast cells, termed rat mast cell protease I (RMCPI ) and RMCPII (Woodbury and Neutrath, 1982 ) , whereas only one chymase has been discovered in humans (Irani et al., 1986 ) and the dog (Schechter et al., 1988) mast cells. Several mouse chymases have also been isolated ( DuBuske et al., 1984). Chymase activity was first reported in human skin mast cells as early at 19 59
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(Benditt and Arase, 1959), but was not noted in dog mast cells until 1962 (Glenner et al., 1962). The presence of tryptase activity localised in both canine and human mast cells was reported by Glenner and Cohen ( 1960). Tryptase has since been purified from human (Schwartz et al., 198 1 ), rat (Kido et al., 1985 ) and dog (Caughey et al., 1987) mast cells, where it can constitute as much as 23% of mast cell protein (Schwartz et al., 198 1). Tryptase is a tetramer of non-covalently associated subunits with an apparent molecular weight (MW) of 132 kDa (Caughey et al., 1987); however, it is thought to be released from mast cells complexed with heparin (Schwartz and Bradford, 1986). Chymase, however, is a monomer of approximately 30 kDa (Caughey et al., 1988). Dog mastocytoma tryptase and chymase are antigenically similar to the corresponding human enzymes as shown by the fact that antibodies raised against human tryptase and kinase cross-react with the corresponding canine enzymes (Schechter et al., 1988). Both enzymes have been amino acid sequenced, demonstrating that dog chymase has approximately 60% sequence homology with that of RMCPI but only 30% homology with dog tryptase (Caughey et al., 1990). Dog chymase is inactivated by inhibitors present in plasma, whereas tryptase remains active in plasma and is resistant to inactivation by circulating inhibitors such as a!,-protease inhibitor (Cromlish et al., 1987). The presence of tryptase in body tissues can thus be used as an indicator of mast cell numbers and activity in the relevant tissues (Schwartz, 1990). Thus it is important to derive values in tissues of normal animals. Furthermore, its presence in body fluids and in blood, where it is more stable than in many other mediators, can be used as an indicator of mast cell degranulation and thus the participation of this cell type in a disease process under investigation. In this study we have developed an assay which will quantify levels of tryptase in tissue extracts and have used it to determine the concentrations of this protease in a variety of canine tissues. This may then provide an insight into the role(s) of this enzyme and the mast cell in disease processes. 2. Materials and methods 2.1. Canine mastocyfomas
Mastocytoma (M 1) was removed from a golden retriever and was provided by Professor N. Gorman (University of Glasgow, School of Veterinary Medicine, Glasgow). Mastocytoma (M2) was removed from a boxer and was provided by S. Milne (Bowler, Lewis and Partners, Evesham, UK). Mastocytomas (M3 ) and (M5) were removed by B. Stanley from a labrador and a boxer respectively (University of Edinburgh, Royal (Dick) School of Veterinary Studies, Edinburgh). Mastocytoma (M4) was also removed from a labrador and was provided by C.R. Chandler (Anicare Veterinary Group, Shoreham by Sea, UK). Mastocytoma (M6 ) was removed from a Weimaraner and was provided by Dr J. Dobson (University of Cambridge, School of Veterinary Medicine, Cambridge).
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2.2. Purification of tryptuse Canine mast cell tryptase was purified from approximately 10 g quantities of frozen mastocytomas employing a method similar to that of Schechter et al. ( 1988) and a FPLC system (Pharmacia, Milton Keynes, UK). Frozen tissue was thinly sliced, resuspended in 0.0 1 M sodium phosphate buffer, pH 7.2 (low salt buffer, LSB) at a ratio of 1 g tissue to 9 ml buffer, and incubated on ice for approximately 30 min. The extract was centrifuged at 2OOOxg for 15 mitt, and the LSB extract discarded. The remaining pellet was resuspended in 0.01 M sodium phosphate buffer, pH 7.2, containing 2 M sodium chloride (high salt buffer, HSB) which dissociates tryptase from heparin. The ratio was 1 g of tissue to 4 ml of HSB. The tissue was chopped more finely, and incubated on ice for a further 30 min with frequent agitation. After further centrifugation at 2OOOxg, the majority of the tryptase activity was found in the supematant. The high salt extract was desalted on a 100 ml Sephadex G25 column (Pharmacia, Milton Keynes, UK) equilibrated in LSB containing 0.4 M NaCl. Major purification of the tryptase was achieved after loading the desalted extract onto a 15 ml heparin-agarose column (Sigma, Poole, UK) and by elution in HSB. Active fractions were desalted into LSB, as described previously, prior to loading on to an anion exchange column ( Econo-Pat Q ) ( Bio-Rad, Hemel Hempstead, UK). One major active fraction was eluted using a 30 ml volume gradient from 0 to 2 M NaCl in LSB, diluted with an equal volume of LSB, and loaded onto the heparin-agarose column for a second time. Tryptase was eluted using a 100 ml volume gradient from 0.4 to 2 M NaCl in LSB. The purified tryptase was concentrated, aliquoted and stored at - 70’ C. Protein concentration was determined by the method of Bradford ( 1976 ) using bovine serum albumin (BSA) (Sigma) as standard. 2.3. Enzyme assays Tryptase activity was determined by cleavage of N-benzoyl-L-Val-Gly-Arg-pnitroanilide (BzVGApNA) (Sigma) during the various stages of purification. Reactions were carried out in 1 ml of 0.06 M Tris-HCl, pH 7.8, at 37°C at a substrate concentration of 80 pg ml- ‘, and monitored spectrophotometrically at 394 nm. A molar extinction coefficient of 11 000 was used to calculate moles of substrate hydrolysed. 2.4. Antibody production Polyclonal antiserum to canine tryptase was produced in New Zealand White rabbits by three subcutaneous injections of approximately 100 pg purified tryptase in 1 ml Freund’s complete adjuvant every 3 weeks, with booster injections as necessary in incomplete adjuvant. Animals were test bled periodically and antisera monitored by immunoblotting.
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Monoclonal anti-human tryptase antibody G5 (IgGl, kappa) was a gift from Dr L. Schwartz (Medical College of Virginia, Richmond). 2.5. Affinity column chromatography 2.5.1. Neutrophil proteinase afjnity column Both antisera cross-reacted with an antigen in neutrophil granules as shown by immunofluorescence on enriched peripheral blood leucocyte smears and on intestinal tissue followed by counterstaining of adjacent sections. This is most likely a proteinase, and in order to remove this cross-reactivity polyclonal anti-tryptase antiserum R 13 was passed through a neutrophil affinity column. Neutrophils were purified from buffy coat cells collected from 50 ml normal canine blood as described by Barta et al. ( 1984), Briefly, buffy coat cells were centrifuged for 10 min at 6OOxg over a Percoll (Pharmacia) gradient. Fractions containing over 90% neutrophils were frozen and thawed several times in LSB prior to coupling to cyanogen bromide (CNBr) activated sepharose 4B (Sigma) by standard techniques. All cross-reactivity was removed following passage through the column as shown by dot-blots using neutrophil extract. 2.5.2. Tryptase afjnity column Purified antibody was prepared from specific antiserum by absorption and elution from a tryptase affinity column. This was prepared by coupling 500 pg of purified tryptase to 0.5 g CNBr sepharose 4B as described previously. 2.6. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SLSPAGE) SDS-PAGE was performed as described by Russell and Blair ( 1977) using 12.5% mini gels in a mini-protein 11 vertical electrophoresis cell (Bio-Rad). Samples for electrophoresis were either boiled (2 min) after the addition of an equal volume of a solution containing 2% (w/v) SDS, 3.5 M fi-mercaptoethanol, and 0.1% (w/v) bromophenol blue (reducing conditions) or diluted with an equal volume of 2% (w/v) SDS and 0.1% (w/v) bromophenol blue. Apparent molecular masses were determined by comparison with protein standards of known molecular weight (Pharmacia). 2.7. Immunoblotting Electrophoretic transfer of proteins from mini-polyacrylamide gels to nitrocellulose sheets (LKB) was carried out on a semi-dry electroblotter. The blots were blocked with 3% (w/v) gelatin in Tris-buffered saline (TBS) (Bio-Rad) (30 min, 20°C) and washed three times for 10 min each with TBS containing 0.1% (v/v) Tween 20 (TBS-T). Sheets were cut into strips and incubated with either anti-human tryptase monoclonal antibody (G5 ) or purified rabbit anticanine tryptase antibody (R 13 ) diluted in 1% (w/v) gelatin in TBS-T (GTBS-T ) for 2 h at 20°C. After washing three times with TBS-T, either l/3000 dilution of al-
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kaline-phosphatase-goat anti-rabbit antibodies (Bio-Rad) ( AP-GAR) or 1 / 100 dilution of horseradish peroxidase-sheep anti-mouse antibodies (Seralab Ltd., Crawley Down, UK) (HRP-SAM) in GTBS-T was added. After incubation ( 1 h, 20°C) and extensive washing with TBS-T the blots were developed with the appropriate substrate (AP-GAR:-AP developer (Bio-Rad); HRP-SAM: 4% (w/ v ) diaminobenzidine (Sigma) in phosphate buffered saline A (PBS) (Dulbecco and Vogt, 1954) containing 0.06% (v/v) HZ02). 2.8. Alkaline phosphatase conjugation Purified tryptase was conjugated with alkaline-phosphatase VII from bovine intestinal mucosa (Sigma) as described by Engvall and Perlmann ( 1972 ). As the conjugate was unstable when stored at 4°C it was aliquoted and stored in a buffer containing 0.05 M Tris-HCl, pH 7.0, 5% (w/v) BSA, 0.00 1 M MgCl?, 0.02% (w/v) NaN3 and 50% (v/v) glycerol at -70°C.
2.9. Tissue preparation Lung, liver, skin (dorsum, ventrum, lateral) and small intestine (duodenum, ileum) were collected from four normal mongrel dogs postmortem. Four 6-mm punch biopsies were taken from each tissue and tryptase was extracted employing a method similar to that for the tumour, using 1 ml HSB for each biopsy. Quantification of tryptase was carried out after four freeze-thaw cycles which was found to release maximum levels of immunologically reactive tryptase.
2.10. Quantification of mast cells Tissues were fixed in 4% (w/v) paraformaldehyde in PBS for 48 h, prior to being embedded in wax using standard techniques. Sections were stained with Giemsa stain (Sigma) and mast cells were counted in four contiguous 1 mm squares using an eyepiece graticule, starting at the centre of the section and extending down throughout the depth of the biopsy. The concentration of tryptase (ng mg- I wet weight) was plotted against mast cell numbers in an area of 4 mm2 and subjected to regression analysis using a Nimbus microcomputer.
2. Il. Competition enzyme-linked immunosorbent assay (ELBA) Purified antibody was diluted to I 5 ,ug ml- ’ in a 0.05 M carbonate-bicarbonate buffer, pH 9.6 and 100 ~1 was added to each well of a 96-well plate (DynatechImmulon 4, Dynatech Laboratories Ltd., Billingshurst, UK). After overnight incubation and washing with PBS containing 0.1% (v/v) Tween 20 (Sigma), 0.1% (w/v) BSA (PBS-T), all wells were blocked with 200 ~1 1% (w/v) BSA in PBS ( 30 min, 37 oC ). After further washing in PBS-T, standard doubling dilutions of purified tryptase (starting at 1.O lug in 100 ~1) prepared in a suitable dilution of alkaline-phosphatase-conjugated tryptase ( AP-tryptase) (usually l/200 in PBS-
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T) were added to the wells ( 100 ~1 per well). Dilutions of high salt extracts from various canine tissues were also prepared in the same dilution of AP-tryptase and 100 ~1 added to appropriate wells. In order to quantify background levels of alkaline-phosphatase activity present in the tissues which bound non-specifically to the plate, a 1/ 10 dilution in PBST of the tissue extracts ( 100 ~1 per well) was added to the plate. This activity was found to be negligible. The plate was incubated at 37°C for 2 h, after which time it was washed thoroughly in PBS-T and 100 ~1 per well of Sigma 104 phosphatase substrate ( 1 mg ml- ’ ) diluted in 0.92 M diethylamine buffer, pH 9.8 was added. After 2 h incubation in the dark at 2O”C, absorbance measurements at 410 nm were performed on a Dynatech MR5000 plate reader.
3. Results 3. I. Purification of mastocytoma tryptase The use of the FPLC system allowed rapid purification of canine tryptase in an active state, within 4-5 h. On average, 50- 100 ,ug of enzyme was recovered from 5 g of homogenised mastocytoma. The results of a representative purification of mastocytoma (M6) tryptase are shown in Table 1. The crude high salt extract had a specific activity of 0.234, whereas the purified enzyme had a specific activity of 9.7 with a 7% yield. This purification protocol has been repeated many times and with one other tumour, with similar results. 3.2. Electrophoretic and antigenic analysis SDS-PAGE of freshly purified enzyme on a 12.5% mini-gel after boiling in denaturing mix revealed two closely migrating, rather diffuse bands, after staining with Coomassie blue stain, with apparent MW of 30 and 32 kDa (Fig. 1, Lane B). Non-denatured enzyme also revealed the same diffuse banding pattern, Table 1 Purification of mastocytoma
2M Nacl extract Heparin-agarose Econo-Q Heparin-agarose
(I) (2 )
tryptase Protein”
Unitsb
Specific’ activity
% Yield
30 2.4 0.5 0.0499
7.03 5.3 1.61 0.473
0.234 2.21 3.22 9.7
100 75.4 23 7
’ Total milligrams. b Total Units (pmol substrate hydrolysed per min at 37°C). ’ Units mg-’ protein.
A.D. Myles et al. / Veterinary Immunology and Immunopathologv 46 (1995) 223-235 A
6
KDa 94.0 67.0
C
D
E
F
229
G
43.0
30.0
20.1
14.4
Fig. 1. Electrophoretic analysis of purified tryptase (approximately 5 pg per track). Lanes A and B: Coomassie stain. (A) molecular mass standards; (B) denatured tryptase. Lanes C, D. E and F: immunoblot. (C) probed with normal rabbit serum ( I / 100 dilution); (D) probed with homologous polyclonal serum (R 13) ( I / 100 dilution); (E) probed with normal mouse serum ( l/ 100 dilution); (F) probed with anti-human tryptase monoclonal antibody (G5) ( I / 100 dilution). Lane G: molecular mass standards stained with amino black after semi-dry blotting.
showing one or more closely associated bands. These appeared to have a slightly lower molecular mass (approximately l-2 kDa) than the denatured subunits. 3.3. Specificity of the polyclonal antiserum After passage through the neutrophil immunoabsorbent column, R 13 detected only tryptase on semipurified mastocytoma extracts (data not shown). Also, mast cell granules alone were detected on immunofluorescence of a wide range of canine tissues. The subunits of canine tryptase were recognised by homologous polyclonal serum R 13 (Fig. 1, Lane D ) and by the antihuman tryptase monoclonal antibody G5 (Fig. 1, Lane F) on western blots. 3.4. Competitive ELISA The assay had a sensitivity range from 500 to 4 ng per 100 ~1 and an example of a standard curve prepared from duplicate samples of purified tryptase ( 1OOO0.78 ng per 100 ,ul) is shown in Fig. 2. Samples were diluted such that the results were calculated using the straight line portion of the curve, i.e. between 125 and 15.63 ng per 100 ~1. Intra-assay variations were determined by the assay of four replicate samples of both skin (dorsum ) and liver tissue extracts. These were found to have a coefficient of variance (%CV= (SD/x) x 100) of 10.1 and 7.1 respectively. Interassay variations were determined by assaying identical aliquots of skin (dorsum) and liver tissue extracts on 3 separate days, on each day absolute values were
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Tryptase Concentration
46 (1995) 223-235
(ng/lOOpl)
Fig. 2. Standard curve, tryptase ( 1000-0.25 ng) diluted in l/200 alkaline-phosphatase tryptase, error bars indicate standard deviations at each point (n = 2 ).
conjugated
calculated from a fresh standard curve. The %CV of skin extracts was 2 1.04 and that of liver was 16.38.
3.5. Quantification of tryptase in canine tissue
Duplicate samples from each of the four biopsies were analysed using the competitive ELBA. Tryptase concentrations in nanograms per milligram wet weight of tissue are shown in Table 2. The concentration of tryptase was highest in the duodenum (93.0 ng mg- ’ ) and ileum (75.0 ng mg- ’ ), followed by the lung (66.0 ng mg- ’ ) and liver (57.0 ng mg- ’ ). The levels in the skin varied from 68.0 ng mg- ’ in the ventrum, to 30 ng mg-’ in the dorsum with the levels in the lateral site falling between the two (46.0 ngmg-‘). The number of mast cells per unit area from each of the seven tissues was com-
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Table 2 Tryptase content of canine tissue Tissue”
Tryptase concentration ngmg-‘+SD
Skin dorsal Skin ventral Skin lateral Lung Liver Duodenum Ileum
30.4 * 3.1 68.42 16.1 45.6? 8.1 66. I + 12.8 56.95? 17.8 92.12 32.3 15.0 + 26.9
a Four biopsies from each tissue.
140 130 i
40
20
1
I
I
'
4
Mast cell counts Fig. 3. The concentration of tryptase (ng mgg’ wet weight) in canine tissue extracts plotted against mast cell numbers in 4 mm2. On regression analysis r= 0.5 I (Pi 0.0 I ).
pared with the appropriate tryptase concentration by regression analysis (Fig. 3 >. A significant correlation between tryptase concentration and mast cell number was noted (P-C 0.0 1).
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Table 3 Tryptase content of six suspected canine mastocytomas Tumour”
Tryptase concentration ng mg-’
Ml M2 M3 M4 M5 M6
211 101 158 206 235 439
a All were confirmed as mast cell tumours except M2, which was a histiocytic tumour.
3.6. Quantification of tryptase in canine mastocytomas Six suspected canine mastocytomas were analysed using the competitive ELBA and tryptase concentrations in nanograms per milligram wet weight of tumour are shown in Table 3. Of the six tumours, all but M2 were confirmed as mast cell tumours. Tumour M2 was later identified as a histiocytic tumour. The concentration of tryptase was highest in mastocytoma M6, which was the tumour used in this study.
4. Discussion The specific activity of the crude high salt extract from the mastocytoma utilised in this study had an activity that was ten fold lower than that of the crude extract used by Caughey et al. ( 1987). This may be due to the fact that these workers used tumours that had been previously passaged through nude mice. The rather low specific activity of the purified enzyme (9.7 vs. 175) and the low yield (7% vs. 28.3%) suggests that, although our purification method is rapid, it may cause substantial loss of activity and yield of enzyme. However, the purified enzyme as reported by Caughey et al. ( 1987), appears to contain contaminating proteins of a higher MW than the tryptase, as shown by these authors on SDSPAGE and Coomassie blue staining. Also, Schechter et al. ( 1988 ) reported that further purification of their canine tryptase resulted in a yield of less than 15%. Thus, the methods used here, which apparently resulted in a purer product, could have been responsible for the reduction in both yield and specific activity. The subunits had an apparent MW of 30 and 32 kDa, but as the enzyme was separated on a mini-gel, molecular mass may not have been accurately determined. However, these results agree with those of Caughey et al. ( 1987), but were slightly lower than the 35 kDa subunits observed by Schechter et al. ( 1988). The diffuse banding pattern observed here, and by Caughey et al. ( 1987) is thought to be due to glycosylation, as glycosidase digestion of tryptase leads to a small drop in MW and band tightening, although some microheterogeneity per-
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sists (Cromlish et al., 1987). As N-terminal sequencing detects only one major sequence this suggests that, if differences in primary structure exist, then the variation in sequence lies internal to the N-terminal 24 amino acids (Caughey et al., 1987). The slight drop in molecular mass of the non-reduced canine tryptase has not been previously reported, but Schwartz and Bradford ( 1986 ) observed a drop in MW of non-reduced human tryptase. This drop in MW may be due to the nonreduced enzyme having a tighter conformational structure than the reduced enzyme. As non-reduced tryptase reveals the same banding pattern on SDS-PAGE as the reduced enzyme, this is supportive of the suggestion that the tryptase subunits are non-covalently bound and that the presence of SDS and/or passage through the gel matrix is enough to separate the native enzyme into its four subunits (Caughey, 1990). The cross-reactivity of anti-human tryptase monoclonal antibody G5 with canine tryptase reflects the 82% sequence homology observed with human and dog tryptase (Reynolds et al., 199 1) . This homology has been previously observed by Schechter et al. ( 1988) using an antihuman tryptase polyclonal serum which also recognised canine tryptase. The competition ELISA developed here is a very rapid and reasonably sensitive assay detecting as little as 4 ng of tryptase per 100 ~1 and is the only assay to date which has been reported to be able to quantitate canine tryptase in various tissue extracts. A more sensitive ELISA has been developed by Wenzel et al. ( 1986), which will detect as little as 0.1 ng of human tryptase per 100 ~1. A similar ELISA was developed by us, also using monoclonal antibody G5, and polyclonal antibody R13 in conjunction with AP-sheep anti-rabbit antibodies, which detected 0.5 ng canine tryptase per 100 ~1. This assay was more time consuming and as normal canine tissue extracts contain over 4 ng per 100 ~1 the detection limits of our competition assay were adequate. Of the tissues studied, the gut contained the highest levels of tryptase per milligram wet weight. The levels of tryptase observed here are similar to those reported for RMCPII in the rat jejunum (Miller et al., 1983). These workers suggested that RMCPII was involved in the expulsion of parasites, as levels or RMCPII increased in response to the presence of parasite antigens. The enzyme may cause an increase in mucosal permeability by promoting the separation and shedding of epithelial cells (Ring and Miller, 1984). Whether the relatively high levels of tryptase in the canine gut indicates a similar role for this enzyme is as yet unknown. Simple regression analysis shows that tryptase concentration is directly related to mast cell density, which suggests that all canine mast cells contain tryptase, as is the case with human mast cells (Irani et al., 1986). Although levels of tryptase per milligram wet weight were lower in the skin than, for example, the gut, relative tryptase concentration to mast cell density was highest in the skin. This suggests that skin mast cells contain a higher concentration of enzyme per individual cell than those in other tissues studied. Of the five confirmed mast cell tumours, Ml, M4, M5 and M6 contained mainly
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tryptase, whereas M3 contained mainly chymase as shown using the substrate Nbenzoyl-L-tyrosine ethyl ester as described by Schechter et al. ( 1988). The use of this assay may prove valuable in assessing the role of mast cells in various disease states in the dog, particularly of allergic origin. In contrast to chymase, there are no known serum inhibitors, which thus permits its measurement, not only in tissues, but also in blood and possibly other body fluids such as lymph following its release. The investigation in this manner of a wide range of diseases of uncertain aetiology is planned to assess the role of mast cells in the inflammatory process.
Acknowledgements
This work was supported by a grant from the Wellcome Trust. We would also like to thank C.S. Hussell for her technical assistance and also the staff of the Wellcome animal unit.
References Barta, O., Shaffer, L.M. and Huang, L-J., 1984. Separation of lymphocytes, monocytes and neutrophils. In: 0. Barta (Editor), Laboratory Techniques of Veterinary Clinical Immunology. C. Thomas, Springfield, IL, Chap. B 1. Benditt, E.P. and Arase, M., 1959. An enzyme in mast cells with properties like chymotrypsin. J. Exp. Med., 110:451-460. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem., 72:248-254. Caughey, G.H., 1990. Ttyptase and chymase in dog mast cells. In: L.B. Schwartz (Editor), Neutral Proteases of Mast Cells. Monograph Allergy. Karger, Base], Vol. 27, pp. 67-89. Caughey, G.H., Viro, N.F., Ramachandran, J., Lazarus, SC., Borson, D.B. and Nadel, J.A., 1987. Dog mastocytoma tryptase: affinity purification, characterisation and aminoterminal sequence. Arch. B&hem. Biophys., 258555-563. Caughey, G.H., Viro, N.F., Lazarus, S.C. and Nadel, J.A., 1988. Purification and characterisation of dog mastocytoma chymase: identification of an octapeptide conserved in chymotryptic leukocyte proteinases. Biochim. Biophys. Acta, 952: 142-l 49. Caughey, G.H., Raymond, W.W. and Vanderslice, P., 1990. Dog mast cell chymase: molecular cloning and characterisation. Biochemistry, 29:5 166-5 17 1. Cromlish, J.A., Seidah, N.G., Marcinkiewicz, M., Hamelin, J., Johnson, D.A. and Chretien, M., 1987. Human pituitary tryptase: molecular forms NH,-terminal sequence, immunocytochemical localization, and specificity with prohormone and lluorogenic substrates. J. Biol. Chem., 262: 13631373. DuBuske, L., Austen, K.F., Czop, J. and Stevens, R.L., 1984. Granule-associated serine neutral proteases of the mouse bone marrow-derived mast cell that degrade libronectin: Their increase after sodium butyrate treatment of the cells. J. Immunol., 133: 1535- I54 1. Dulbecco, R. and Vogt, P., 1954. Formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med., 99:167-169. Engvall, E. and Perlmann, P., 1972. Enzyme-linked immunosorbent assay (ELISA) III. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulins in antigen coated tubes. J. Immunol., 109: 129-135.
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235
Glermer, G.G. and Cohen, L.A., 1960. Histochemical demonstration of a species-specific trypsin-like enzyme in mast cells. Nature, 185:846-847. Glermer, G.G., Hopsu, V.K. and Cohen, L.A., 1962. Histochemical demonstration of a trypsin-like esterase activity in mast cells. J. Histochem. Cytochem., 10: 109-l 10. Irani, A.A., Schecter, N.M., Craig, S.S., DeBlons, G. and Schwartz, L.B., 1986. Two types of human mast cells that have distinct neutral protease compositions. Proc. Natl. Acad. Sci., 83:4464-4468. Kido, H., Fukusen, N. and Katunma, N., 1985. Chymotrypsin-type and trypsin-type serine proteases in rat mast cells: properties and functions. Arch. Biochem. Biophys., 239: 436-443. King, S.J. and Miller, H.R.P., 1984. Anaphylactic release of mucosal mast cell protease and its relationship to gut permeability in Nippostrongylus-primed rats. Immunology, 5 1: 653-660. Miller, H.R.P., Woodbury, R.G., Huntley, J.F. and Newlands, G., 1983. Systemic release of mucosal mast-cell protease in primed rats challenged with Nippostrongy/mhrasiliensis.Immunology, 49147I 479. Reynolds, D.S., Gurley, D.S., Austen, K.F. and Serafins, W.E., 199 1. Cloning of the cDNA and gene of mouse mast cell protease-6. J. Biol. Chem., 266:3847-3853. Russell, W.C. and Blair, G.E., 1977. Polypeptide phosphorylation in adenovirus-infected cells. J. Gen. Virol., 34:19-35. Schechter, N.M., Slavin, D., Fetter, R.D., Lazarus, G.S. and Fraki, I.E., 1988. Purification and identification of two serine class proteinases from dog mast cells biochemically and immunologically similar to human proteinases tryptase and chymase. Arch. Biochem. Biophys., 262:232-244. Schwartz, L.B., 1990. Tryptase from human mast cells: biochemistry, biology and clinical utility. Monogr. Allergy, 27:90-l 13. Schwartz, L.B. and Bradford, T.R., 1986. Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer. J. Biol. Chem., 261:7372-7379. Schwartz, L.B., Lewis, R.A. and Austen, K.F., 198 1. Tryptase from human pulmonary mast cells. J. Biol. Chem., 256:11939-l 1943. Wenzel, S., Irani, A-MA., Sanders, J.M., Bradford, T.R. and Schwartz, L.B., 1986. Immunoassay of tryptase from human mast cells. J. Immunol. Methods, 86: 139- 142. Woodbury, R.G. and Neutrath, H., 1982. Structure, specificity and localisation of the serine proteases of connective tissue. FEBS Lett., I 14: 189-l 96.
Veterinary Immunology and Immunopathology 46(1995)223-235
immunology and immunopathology
Mast cell tryptase levels in normal canine tissues A.D. Myles, R.E.W. Halliwell*, B. Ballauf, H.R.P. Miller Department of Veterinary Clinical Studies, Royal (Dick) School of kterinary Studies. Universityof Edinburgh, Summerhall, Edinburgh EH9 lQH, LX Accepted 7 July I994
Abstract
Levels of canine tryptase from various tissues were quantified using a competition enzyme-linked immunosorbent assay (ELISA). The assay utilises an affinity-purified rabbit anti-tryptase antibody in the solid phase and alkaline-phosphatase conjugated tryptase together with unlabelled tryptase in the fluid phase. The assay will rapidly quantify 40-5000 ng ml-’ of tryptase in tissue extracts. Tissues from the skin, gut, liver and lung were studied, of which canine gut appeared to contain the highest levels of tryptase per milligram wet weight, which may suggest an important role for this enzyme at this site. This assay may prove valuable in assessing the role of mast cells in various disease states in the dog. Abbreviations ELISA=enzyme-linked immunosorbent assay; HSB = high salt buffer: LSB = low salt buffer; SDS-PAGE=sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
1. Introduction Mast cells contain large numbers of metachromatic granules, which store and release the serine proteases chymase and tryptase, simultaneously with other preformed mediators. Two types of chymase have been isolated from rat mast cells, termed rat mast cell protease I (RMCPI ) and RMCPII (Woodbury and Neutrath, 1982 ) , whereas only one chymase has been discovered in humans (Irani et al., 1986 ) and the dog (Schechter et al., 1988) mast cells. Several mouse chymases have also been isolated ( DuBuske et al., 1984). Chymase activity was first reported in human skin mast cells as early at 19 59
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(Benditt and Arase, 1959), but was not noted in dog mast cells until 1962 (Glenner et al., 1962). The presence of tryptase activity localised in both canine and human mast cells was reported by Glenner and Cohen ( 1960). Tryptase has since been purified from human (Schwartz et al., 198 1 ), rat (Kido et al., 1985 ) and dog (Caughey et al., 1987) mast cells, where it can constitute as much as 23% of mast cell protein (Schwartz et al., 198 1). Tryptase is a tetramer of non-covalently associated subunits with an apparent molecular weight (MW) of 132 kDa (Caughey et al., 1987); however, it is thought to be released from mast cells complexed with heparin (Schwartz and Bradford, 1986). Chymase, however, is a monomer of approximately 30 kDa (Caughey et al., 1988). Dog mastocytoma tryptase and chymase are antigenically similar to the corresponding human enzymes as shown by the fact that antibodies raised against human tryptase and kinase cross-react with the corresponding canine enzymes (Schechter et al., 1988). Both enzymes have been amino acid sequenced, demonstrating that dog chymase has approximately 60% sequence homology with that of RMCPI but only 30% homology with dog tryptase (Caughey et al., 1990). Dog chymase is inactivated by inhibitors present in plasma, whereas tryptase remains active in plasma and is resistant to inactivation by circulating inhibitors such as a!,-protease inhibitor (Cromlish et al., 1987). The presence of tryptase in body tissues can thus be used as an indicator of mast cell numbers and activity in the relevant tissues (Schwartz, 1990). Thus it is important to derive values in tissues of normal animals. Furthermore, its presence in body fluids and in blood, where it is more stable than in many other mediators, can be used as an indicator of mast cell degranulation and thus the participation of this cell type in a disease process under investigation. In this study we have developed an assay which will quantify levels of tryptase in tissue extracts and have used it to determine the concentrations of this protease in a variety of canine tissues. This may then provide an insight into the role(s) of this enzyme and the mast cell in disease processes. 2. Materials and methods 2.1. Canine mastocyfomas
Mastocytoma (M 1) was removed from a golden retriever and was provided by Professor N. Gorman (University of Glasgow, School of Veterinary Medicine, Glasgow). Mastocytoma (M2) was removed from a boxer and was provided by S. Milne (Bowler, Lewis and Partners, Evesham, UK). Mastocytomas (M3 ) and (M5) were removed by B. Stanley from a labrador and a boxer respectively (University of Edinburgh, Royal (Dick) School of Veterinary Studies, Edinburgh). Mastocytoma (M4) was also removed from a labrador and was provided by C.R. Chandler (Anicare Veterinary Group, Shoreham by Sea, UK). Mastocytoma (M6 ) was removed from a Weimaraner and was provided by Dr J. Dobson (University of Cambridge, School of Veterinary Medicine, Cambridge).
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2.2. Purification of tryptuse Canine mast cell tryptase was purified from approximately 10 g quantities of frozen mastocytomas employing a method similar to that of Schechter et al. ( 1988) and a FPLC system (Pharmacia, Milton Keynes, UK). Frozen tissue was thinly sliced, resuspended in 0.0 1 M sodium phosphate buffer, pH 7.2 (low salt buffer, LSB) at a ratio of 1 g tissue to 9 ml buffer, and incubated on ice for approximately 30 min. The extract was centrifuged at 2OOOxg for 15 mitt, and the LSB extract discarded. The remaining pellet was resuspended in 0.01 M sodium phosphate buffer, pH 7.2, containing 2 M sodium chloride (high salt buffer, HSB) which dissociates tryptase from heparin. The ratio was 1 g of tissue to 4 ml of HSB. The tissue was chopped more finely, and incubated on ice for a further 30 min with frequent agitation. After further centrifugation at 2OOOxg, the majority of the tryptase activity was found in the supematant. The high salt extract was desalted on a 100 ml Sephadex G25 column (Pharmacia, Milton Keynes, UK) equilibrated in LSB containing 0.4 M NaCl. Major purification of the tryptase was achieved after loading the desalted extract onto a 15 ml heparin-agarose column (Sigma, Poole, UK) and by elution in HSB. Active fractions were desalted into LSB, as described previously, prior to loading on to an anion exchange column ( Econo-Pat Q ) ( Bio-Rad, Hemel Hempstead, UK). One major active fraction was eluted using a 30 ml volume gradient from 0 to 2 M NaCl in LSB, diluted with an equal volume of LSB, and loaded onto the heparin-agarose column for a second time. Tryptase was eluted using a 100 ml volume gradient from 0.4 to 2 M NaCl in LSB. The purified tryptase was concentrated, aliquoted and stored at - 70’ C. Protein concentration was determined by the method of Bradford ( 1976 ) using bovine serum albumin (BSA) (Sigma) as standard. 2.3. Enzyme assays Tryptase activity was determined by cleavage of N-benzoyl-L-Val-Gly-Arg-pnitroanilide (BzVGApNA) (Sigma) during the various stages of purification. Reactions were carried out in 1 ml of 0.06 M Tris-HCl, pH 7.8, at 37°C at a substrate concentration of 80 pg ml- ‘, and monitored spectrophotometrically at 394 nm. A molar extinction coefficient of 11 000 was used to calculate moles of substrate hydrolysed. 2.4. Antibody production Polyclonal antiserum to canine tryptase was produced in New Zealand White rabbits by three subcutaneous injections of approximately 100 pg purified tryptase in 1 ml Freund’s complete adjuvant every 3 weeks, with booster injections as necessary in incomplete adjuvant. Animals were test bled periodically and antisera monitored by immunoblotting.
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Monoclonal anti-human tryptase antibody G5 (IgGl, kappa) was a gift from Dr L. Schwartz (Medical College of Virginia, Richmond). 2.5. Affinity column chromatography 2.5.1. Neutrophil proteinase afjnity column Both antisera cross-reacted with an antigen in neutrophil granules as shown by immunofluorescence on enriched peripheral blood leucocyte smears and on intestinal tissue followed by counterstaining of adjacent sections. This is most likely a proteinase, and in order to remove this cross-reactivity polyclonal anti-tryptase antiserum R 13 was passed through a neutrophil affinity column. Neutrophils were purified from buffy coat cells collected from 50 ml normal canine blood as described by Barta et al. ( 1984), Briefly, buffy coat cells were centrifuged for 10 min at 6OOxg over a Percoll (Pharmacia) gradient. Fractions containing over 90% neutrophils were frozen and thawed several times in LSB prior to coupling to cyanogen bromide (CNBr) activated sepharose 4B (Sigma) by standard techniques. All cross-reactivity was removed following passage through the column as shown by dot-blots using neutrophil extract. 2.5.2. Tryptase afjnity column Purified antibody was prepared from specific antiserum by absorption and elution from a tryptase affinity column. This was prepared by coupling 500 pg of purified tryptase to 0.5 g CNBr sepharose 4B as described previously. 2.6. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SLSPAGE) SDS-PAGE was performed as described by Russell and Blair ( 1977) using 12.5% mini gels in a mini-protein 11 vertical electrophoresis cell (Bio-Rad). Samples for electrophoresis were either boiled (2 min) after the addition of an equal volume of a solution containing 2% (w/v) SDS, 3.5 M fi-mercaptoethanol, and 0.1% (w/v) bromophenol blue (reducing conditions) or diluted with an equal volume of 2% (w/v) SDS and 0.1% (w/v) bromophenol blue. Apparent molecular masses were determined by comparison with protein standards of known molecular weight (Pharmacia). 2.7. Immunoblotting Electrophoretic transfer of proteins from mini-polyacrylamide gels to nitrocellulose sheets (LKB) was carried out on a semi-dry electroblotter. The blots were blocked with 3% (w/v) gelatin in Tris-buffered saline (TBS) (Bio-Rad) (30 min, 20°C) and washed three times for 10 min each with TBS containing 0.1% (v/v) Tween 20 (TBS-T). Sheets were cut into strips and incubated with either anti-human tryptase monoclonal antibody (G5 ) or purified rabbit anticanine tryptase antibody (R 13 ) diluted in 1% (w/v) gelatin in TBS-T (GTBS-T ) for 2 h at 20°C. After washing three times with TBS-T, either l/3000 dilution of al-
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kaline-phosphatase-goat anti-rabbit antibodies (Bio-Rad) ( AP-GAR) or 1 / 100 dilution of horseradish peroxidase-sheep anti-mouse antibodies (Seralab Ltd., Crawley Down, UK) (HRP-SAM) in GTBS-T was added. After incubation ( 1 h, 20°C) and extensive washing with TBS-T the blots were developed with the appropriate substrate (AP-GAR:-AP developer (Bio-Rad); HRP-SAM: 4% (w/ v ) diaminobenzidine (Sigma) in phosphate buffered saline A (PBS) (Dulbecco and Vogt, 1954) containing 0.06% (v/v) HZ02). 2.8. Alkaline phosphatase conjugation Purified tryptase was conjugated with alkaline-phosphatase VII from bovine intestinal mucosa (Sigma) as described by Engvall and Perlmann ( 1972 ). As the conjugate was unstable when stored at 4°C it was aliquoted and stored in a buffer containing 0.05 M Tris-HCl, pH 7.0, 5% (w/v) BSA, 0.00 1 M MgCl?, 0.02% (w/v) NaN3 and 50% (v/v) glycerol at -70°C.
2.9. Tissue preparation Lung, liver, skin (dorsum, ventrum, lateral) and small intestine (duodenum, ileum) were collected from four normal mongrel dogs postmortem. Four 6-mm punch biopsies were taken from each tissue and tryptase was extracted employing a method similar to that for the tumour, using 1 ml HSB for each biopsy. Quantification of tryptase was carried out after four freeze-thaw cycles which was found to release maximum levels of immunologically reactive tryptase.
2.10. Quantification of mast cells Tissues were fixed in 4% (w/v) paraformaldehyde in PBS for 48 h, prior to being embedded in wax using standard techniques. Sections were stained with Giemsa stain (Sigma) and mast cells were counted in four contiguous 1 mm squares using an eyepiece graticule, starting at the centre of the section and extending down throughout the depth of the biopsy. The concentration of tryptase (ng mg- I wet weight) was plotted against mast cell numbers in an area of 4 mm2 and subjected to regression analysis using a Nimbus microcomputer.
2. Il. Competition enzyme-linked immunosorbent assay (ELBA) Purified antibody was diluted to I 5 ,ug ml- ’ in a 0.05 M carbonate-bicarbonate buffer, pH 9.6 and 100 ~1 was added to each well of a 96-well plate (DynatechImmulon 4, Dynatech Laboratories Ltd., Billingshurst, UK). After overnight incubation and washing with PBS containing 0.1% (v/v) Tween 20 (Sigma), 0.1% (w/v) BSA (PBS-T), all wells were blocked with 200 ~1 1% (w/v) BSA in PBS ( 30 min, 37 oC ). After further washing in PBS-T, standard doubling dilutions of purified tryptase (starting at 1.O lug in 100 ~1) prepared in a suitable dilution of alkaline-phosphatase-conjugated tryptase ( AP-tryptase) (usually l/200 in PBS-
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T) were added to the wells ( 100 ~1 per well). Dilutions of high salt extracts from various canine tissues were also prepared in the same dilution of AP-tryptase and 100 ~1 added to appropriate wells. In order to quantify background levels of alkaline-phosphatase activity present in the tissues which bound non-specifically to the plate, a 1/ 10 dilution in PBST of the tissue extracts ( 100 ~1 per well) was added to the plate. This activity was found to be negligible. The plate was incubated at 37°C for 2 h, after which time it was washed thoroughly in PBS-T and 100 ~1 per well of Sigma 104 phosphatase substrate ( 1 mg ml- ’ ) diluted in 0.92 M diethylamine buffer, pH 9.8 was added. After 2 h incubation in the dark at 2O”C, absorbance measurements at 410 nm were performed on a Dynatech MR5000 plate reader.
3. Results 3. I. Purification of mastocytoma tryptase The use of the FPLC system allowed rapid purification of canine tryptase in an active state, within 4-5 h. On average, 50- 100 ,ug of enzyme was recovered from 5 g of homogenised mastocytoma. The results of a representative purification of mastocytoma (M6) tryptase are shown in Table 1. The crude high salt extract had a specific activity of 0.234, whereas the purified enzyme had a specific activity of 9.7 with a 7% yield. This purification protocol has been repeated many times and with one other tumour, with similar results. 3.2. Electrophoretic and antigenic analysis SDS-PAGE of freshly purified enzyme on a 12.5% mini-gel after boiling in denaturing mix revealed two closely migrating, rather diffuse bands, after staining with Coomassie blue stain, with apparent MW of 30 and 32 kDa (Fig. 1, Lane B). Non-denatured enzyme also revealed the same diffuse banding pattern, Table 1 Purification of mastocytoma
2M Nacl extract Heparin-agarose Econo-Q Heparin-agarose
(I) (2 )
tryptase Protein”
Unitsb
Specific’ activity
% Yield
30 2.4 0.5 0.0499
7.03 5.3 1.61 0.473
0.234 2.21 3.22 9.7
100 75.4 23 7
’ Total milligrams. b Total Units (pmol substrate hydrolysed per min at 37°C). ’ Units mg-’ protein.
A.D. Myles et al. / Veterinary Immunology and Immunopathologv 46 (1995) 223-235 A
6
KDa 94.0 67.0
C
D
E
F
229
G
43.0
30.0
20.1
14.4
Fig. 1. Electrophoretic analysis of purified tryptase (approximately 5 pg per track). Lanes A and B: Coomassie stain. (A) molecular mass standards; (B) denatured tryptase. Lanes C, D. E and F: immunoblot. (C) probed with normal rabbit serum ( I / 100 dilution); (D) probed with homologous polyclonal serum (R 13) ( I / 100 dilution); (E) probed with normal mouse serum ( l/ 100 dilution); (F) probed with anti-human tryptase monoclonal antibody (G5) ( I / 100 dilution). Lane G: molecular mass standards stained with amino black after semi-dry blotting.
showing one or more closely associated bands. These appeared to have a slightly lower molecular mass (approximately l-2 kDa) than the denatured subunits. 3.3. Specificity of the polyclonal antiserum After passage through the neutrophil immunoabsorbent column, R 13 detected only tryptase on semipurified mastocytoma extracts (data not shown). Also, mast cell granules alone were detected on immunofluorescence of a wide range of canine tissues. The subunits of canine tryptase were recognised by homologous polyclonal serum R 13 (Fig. 1, Lane D ) and by the antihuman tryptase monoclonal antibody G5 (Fig. 1, Lane F) on western blots. 3.4. Competitive ELISA The assay had a sensitivity range from 500 to 4 ng per 100 ~1 and an example of a standard curve prepared from duplicate samples of purified tryptase ( 1OOO0.78 ng per 100 ,ul) is shown in Fig. 2. Samples were diluted such that the results were calculated using the straight line portion of the curve, i.e. between 125 and 15.63 ng per 100 ~1. Intra-assay variations were determined by the assay of four replicate samples of both skin (dorsum ) and liver tissue extracts. These were found to have a coefficient of variance (%CV= (SD/x) x 100) of 10.1 and 7.1 respectively. Interassay variations were determined by assaying identical aliquots of skin (dorsum) and liver tissue extracts on 3 separate days, on each day absolute values were
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Tryptase Concentration
46 (1995) 223-235
(ng/lOOpl)
Fig. 2. Standard curve, tryptase ( 1000-0.25 ng) diluted in l/200 alkaline-phosphatase tryptase, error bars indicate standard deviations at each point (n = 2 ).
conjugated
calculated from a fresh standard curve. The %CV of skin extracts was 2 1.04 and that of liver was 16.38.
3.5. Quantification of tryptase in canine tissue
Duplicate samples from each of the four biopsies were analysed using the competitive ELBA. Tryptase concentrations in nanograms per milligram wet weight of tissue are shown in Table 2. The concentration of tryptase was highest in the duodenum (93.0 ng mg- ’ ) and ileum (75.0 ng mg- ’ ), followed by the lung (66.0 ng mg- ’ ) and liver (57.0 ng mg- ’ ). The levels in the skin varied from 68.0 ng mg- ’ in the ventrum, to 30 ng mg-’ in the dorsum with the levels in the lateral site falling between the two (46.0 ngmg-‘). The number of mast cells per unit area from each of the seven tissues was com-
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Table 2 Tryptase content of canine tissue Tissue”
Tryptase concentration ngmg-‘+SD
Skin dorsal Skin ventral Skin lateral Lung Liver Duodenum Ileum
30.4 * 3.1 68.42 16.1 45.6? 8.1 66. I + 12.8 56.95? 17.8 92.12 32.3 15.0 + 26.9
a Four biopsies from each tissue.
140 130 i
40
20
1
I
I
'
4
Mast cell counts Fig. 3. The concentration of tryptase (ng mgg’ wet weight) in canine tissue extracts plotted against mast cell numbers in 4 mm2. On regression analysis r= 0.5 I (Pi 0.0 I ).
pared with the appropriate tryptase concentration by regression analysis (Fig. 3 >. A significant correlation between tryptase concentration and mast cell number was noted (P-C 0.0 1).
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Table 3 Tryptase content of six suspected canine mastocytomas Tumour”
Tryptase concentration ng mg-’
Ml M2 M3 M4 M5 M6
211 101 158 206 235 439
a All were confirmed as mast cell tumours except M2, which was a histiocytic tumour.
3.6. Quantification of tryptase in canine mastocytomas Six suspected canine mastocytomas were analysed using the competitive ELBA and tryptase concentrations in nanograms per milligram wet weight of tumour are shown in Table 3. Of the six tumours, all but M2 were confirmed as mast cell tumours. Tumour M2 was later identified as a histiocytic tumour. The concentration of tryptase was highest in mastocytoma M6, which was the tumour used in this study.
4. Discussion The specific activity of the crude high salt extract from the mastocytoma utilised in this study had an activity that was ten fold lower than that of the crude extract used by Caughey et al. ( 1987). This may be due to the fact that these workers used tumours that had been previously passaged through nude mice. The rather low specific activity of the purified enzyme (9.7 vs. 175) and the low yield (7% vs. 28.3%) suggests that, although our purification method is rapid, it may cause substantial loss of activity and yield of enzyme. However, the purified enzyme as reported by Caughey et al. ( 1987), appears to contain contaminating proteins of a higher MW than the tryptase, as shown by these authors on SDSPAGE and Coomassie blue staining. Also, Schechter et al. ( 1988 ) reported that further purification of their canine tryptase resulted in a yield of less than 15%. Thus, the methods used here, which apparently resulted in a purer product, could have been responsible for the reduction in both yield and specific activity. The subunits had an apparent MW of 30 and 32 kDa, but as the enzyme was separated on a mini-gel, molecular mass may not have been accurately determined. However, these results agree with those of Caughey et al. ( 1987), but were slightly lower than the 35 kDa subunits observed by Schechter et al. ( 1988). The diffuse banding pattern observed here, and by Caughey et al. ( 1987) is thought to be due to glycosylation, as glycosidase digestion of tryptase leads to a small drop in MW and band tightening, although some microheterogeneity per-
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sists (Cromlish et al., 1987). As N-terminal sequencing detects only one major sequence this suggests that, if differences in primary structure exist, then the variation in sequence lies internal to the N-terminal 24 amino acids (Caughey et al., 1987). The slight drop in molecular mass of the non-reduced canine tryptase has not been previously reported, but Schwartz and Bradford ( 1986 ) observed a drop in MW of non-reduced human tryptase. This drop in MW may be due to the nonreduced enzyme having a tighter conformational structure than the reduced enzyme. As non-reduced tryptase reveals the same banding pattern on SDS-PAGE as the reduced enzyme, this is supportive of the suggestion that the tryptase subunits are non-covalently bound and that the presence of SDS and/or passage through the gel matrix is enough to separate the native enzyme into its four subunits (Caughey, 1990). The cross-reactivity of anti-human tryptase monoclonal antibody G5 with canine tryptase reflects the 82% sequence homology observed with human and dog tryptase (Reynolds et al., 199 1) . This homology has been previously observed by Schechter et al. ( 1988) using an antihuman tryptase polyclonal serum which also recognised canine tryptase. The competition ELISA developed here is a very rapid and reasonably sensitive assay detecting as little as 4 ng of tryptase per 100 ~1 and is the only assay to date which has been reported to be able to quantitate canine tryptase in various tissue extracts. A more sensitive ELISA has been developed by Wenzel et al. ( 1986), which will detect as little as 0.1 ng of human tryptase per 100 ~1. A similar ELISA was developed by us, also using monoclonal antibody G5, and polyclonal antibody R13 in conjunction with AP-sheep anti-rabbit antibodies, which detected 0.5 ng canine tryptase per 100 ~1. This assay was more time consuming and as normal canine tissue extracts contain over 4 ng per 100 ~1 the detection limits of our competition assay were adequate. Of the tissues studied, the gut contained the highest levels of tryptase per milligram wet weight. The levels of tryptase observed here are similar to those reported for RMCPII in the rat jejunum (Miller et al., 1983). These workers suggested that RMCPII was involved in the expulsion of parasites, as levels or RMCPII increased in response to the presence of parasite antigens. The enzyme may cause an increase in mucosal permeability by promoting the separation and shedding of epithelial cells (Ring and Miller, 1984). Whether the relatively high levels of tryptase in the canine gut indicates a similar role for this enzyme is as yet unknown. Simple regression analysis shows that tryptase concentration is directly related to mast cell density, which suggests that all canine mast cells contain tryptase, as is the case with human mast cells (Irani et al., 1986). Although levels of tryptase per milligram wet weight were lower in the skin than, for example, the gut, relative tryptase concentration to mast cell density was highest in the skin. This suggests that skin mast cells contain a higher concentration of enzyme per individual cell than those in other tissues studied. Of the five confirmed mast cell tumours, Ml, M4, M5 and M6 contained mainly
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tryptase, whereas M3 contained mainly chymase as shown using the substrate Nbenzoyl-L-tyrosine ethyl ester as described by Schechter et al. ( 1988). The use of this assay may prove valuable in assessing the role of mast cells in various disease states in the dog, particularly of allergic origin. In contrast to chymase, there are no known serum inhibitors, which thus permits its measurement, not only in tissues, but also in blood and possibly other body fluids such as lymph following its release. The investigation in this manner of a wide range of diseases of uncertain aetiology is planned to assess the role of mast cells in the inflammatory process.
Acknowledgements
This work was supported by a grant from the Wellcome Trust. We would also like to thank C.S. Hussell for her technical assistance and also the staff of the Wellcome animal unit.
References Barta, O., Shaffer, L.M. and Huang, L-J., 1984. Separation of lymphocytes, monocytes and neutrophils. In: 0. Barta (Editor), Laboratory Techniques of Veterinary Clinical Immunology. C. Thomas, Springfield, IL, Chap. B 1. Benditt, E.P. and Arase, M., 1959. An enzyme in mast cells with properties like chymotrypsin. J. Exp. Med., 110:451-460. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem., 72:248-254. Caughey, G.H., 1990. Ttyptase and chymase in dog mast cells. In: L.B. Schwartz (Editor), Neutral Proteases of Mast Cells. Monograph Allergy. Karger, Base], Vol. 27, pp. 67-89. Caughey, G.H., Viro, N.F., Ramachandran, J., Lazarus, SC., Borson, D.B. and Nadel, J.A., 1987. Dog mastocytoma tryptase: affinity purification, characterisation and aminoterminal sequence. Arch. B&hem. Biophys., 258555-563. Caughey, G.H., Viro, N.F., Lazarus, S.C. and Nadel, J.A., 1988. Purification and characterisation of dog mastocytoma chymase: identification of an octapeptide conserved in chymotryptic leukocyte proteinases. Biochim. Biophys. Acta, 952: 142-l 49. Caughey, G.H., Raymond, W.W. and Vanderslice, P., 1990. Dog mast cell chymase: molecular cloning and characterisation. Biochemistry, 29:5 166-5 17 1. Cromlish, J.A., Seidah, N.G., Marcinkiewicz, M., Hamelin, J., Johnson, D.A. and Chretien, M., 1987. Human pituitary tryptase: molecular forms NH,-terminal sequence, immunocytochemical localization, and specificity with prohormone and lluorogenic substrates. J. Biol. Chem., 262: 13631373. DuBuske, L., Austen, K.F., Czop, J. and Stevens, R.L., 1984. Granule-associated serine neutral proteases of the mouse bone marrow-derived mast cell that degrade libronectin: Their increase after sodium butyrate treatment of the cells. J. Immunol., 133: 1535- I54 1. Dulbecco, R. and Vogt, P., 1954. Formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med., 99:167-169. Engvall, E. and Perlmann, P., 1972. Enzyme-linked immunosorbent assay (ELISA) III. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulins in antigen coated tubes. J. Immunol., 109: 129-135.
A.D. Myles et al. / Veterinary Immunology and Imrnunopathology 46 (1995) 223-235
235
Glermer, G.G. and Cohen, L.A., 1960. Histochemical demonstration of a species-specific trypsin-like enzyme in mast cells. Nature, 185:846-847. Glermer, G.G., Hopsu, V.K. and Cohen, L.A., 1962. Histochemical demonstration of a trypsin-like esterase activity in mast cells. J. Histochem. Cytochem., 10: 109-l 10. Irani, A.A., Schecter, N.M., Craig, S.S., DeBlons, G. and Schwartz, L.B., 1986. Two types of human mast cells that have distinct neutral protease compositions. Proc. Natl. Acad. Sci., 83:4464-4468. Kido, H., Fukusen, N. and Katunma, N., 1985. Chymotrypsin-type and trypsin-type serine proteases in rat mast cells: properties and functions. Arch. Biochem. Biophys., 239: 436-443. King, S.J. and Miller, H.R.P., 1984. Anaphylactic release of mucosal mast cell protease and its relationship to gut permeability in Nippostrongylus-primed rats. Immunology, 5 1: 653-660. Miller, H.R.P., Woodbury, R.G., Huntley, J.F. and Newlands, G., 1983. Systemic release of mucosal mast-cell protease in primed rats challenged with Nippostrongy/mhrasiliensis.Immunology, 49147I 479. Reynolds, D.S., Gurley, D.S., Austen, K.F. and Serafins, W.E., 199 1. Cloning of the cDNA and gene of mouse mast cell protease-6. J. Biol. Chem., 266:3847-3853. Russell, W.C. and Blair, G.E., 1977. Polypeptide phosphorylation in adenovirus-infected cells. J. Gen. Virol., 34:19-35. Schechter, N.M., Slavin, D., Fetter, R.D., Lazarus, G.S. and Fraki, I.E., 1988. Purification and identification of two serine class proteinases from dog mast cells biochemically and immunologically similar to human proteinases tryptase and chymase. Arch. Biochem. Biophys., 262:232-244. Schwartz, L.B., 1990. Tryptase from human mast cells: biochemistry, biology and clinical utility. Monogr. Allergy, 27:90-l 13. Schwartz, L.B. and Bradford, T.R., 1986. Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer. J. Biol. Chem., 261:7372-7379. Schwartz, L.B., Lewis, R.A. and Austen, K.F., 198 1. Tryptase from human pulmonary mast cells. J. Biol. Chem., 256:11939-l 1943. Wenzel, S., Irani, A-MA., Sanders, J.M., Bradford, T.R. and Schwartz, L.B., 1986. Immunoassay of tryptase from human mast cells. J. Immunol. Methods, 86: 139- 142. Woodbury, R.G. and Neutrath, H., 1982. Structure, specificity and localisation of the serine proteases of connective tissue. FEBS Lett., I 14: 189-l 96.