ARTICLE |
Correspondence to: Gordon B. Proctor, Secretory and Soft Tissue Res. Unit, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK.
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Summary |
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We investigated the histochemical characteristics of mast cell tryptase in different mouse tissues. By use of peptide substrates, tryptase activity could be demonstrated in unfixed connective tissue mast cells in different tissues, including the stomach. Tryptase activity was better localized after aldehyde fixation and frozen sectioning, and under such conditions was also demonstrated in mucosal mast cells of the stomach but not in those of the gut mucosa. Double staining by enzyme histochemistry followed by toluidine blue indicated that the tryptase activity was present only in mast cells and that all mast cells in the stomach mucosa contained the enzyme. The peptide substrates z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthlyamide, which are substrates of choice for demonstrating tryptase in other species, were most effective for demonstrating mouse tryptase. The use of protease inhibitors further indicated that activity present in all mast cells was tryptase. Safranin O did not stain stomach mucosal mast cells, suggesting that the tryptase present in these cells was active in the absence of heparin sulfate proteoglycan. (J Histochem Cytochem 47:617622, 1999)
Key Words: enzyme histochemistry, mast cell, mouse, tryptase
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Introduction |
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The importance of mast cells in a number of pathological processes is beyond doubt, but because of their poorly defined physiological functions they have always been a controversial issue. Distinct populations of mast cells are tissue dependent and have been most extensively studied in mouse, rat, and human (
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Materials and Methods |
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Tissues
Gut, lung, prostate, skin, and stomach were removed from adult (12-week-old) male C3H, WHT, and C3A mice under terminal pentobarbital (IP) anesthesia.
Fixation
Pieces of the different tissues were snap-frozen in hexane cooled on solid CO2 and 10-µm frozen sections were cut in a cryostat. Different fixatives were assessed for the fixation of tryptase activity in other pieces of the tissues taken. Aldehyde fixation was performed either by (a) immersion of the material for 618 hr at 04C in formolsucrose containing 4% (w/v) formaldehyde freshly prepared from paraformaldehyde, 7% (w/v) sucrose in 0.05 M cacodylate buffer, pH 7.2, or (b) immersion in a mixture of formaldehydeglutaraldehyde containing 2.5% (w/v) formaldehyde freshly prepared from paraformaldehyde, 0.2% (w/v) distilled glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2. After fixation the tissues were washed overnight at 04C in 0.05 M cacodylate buffer containing 7.5% sucrose, pH 7.2. Nonaldehyde fixation was performed in Carnoy fixative (standard protocol 60% ethanol, 30% chloroform, 10% acetic acid) for 4 hr or overnight or in methanol overnight. Aldehyde-fixed tissues were either paraffin-embedded and sectioned (5 µm) or frozen and cryostat-sectioned (10 µm). Carnoy- and methanol-fixed tissues were paraffin-embedded.
Dye Binding
Sections were stained for 30 min in solutions of either alcian bluesafranin O (0.36% and 0.18%, respectively) in 1 M HClsodium acetate buffer (
Enzyme Histochemistry
For demonstration of tryptase amidolytic activity, tissue sections were incubated in 0.25 mg/ml of the substrate z-Ala-Ala-Lys-4-methoxy-2-naphthylamide (z-AAK-mna; Enzyme System Products; Dublin, CA) in 0.1 M phosphate buffer, pH 6.5, 5.5, or 5.0 for 60120 min at 37C. The incubation medium contained Fast Blue B salt (final concentration 3.2 mM) as a capture reagent and incubation was followed by a 5-min wash in 1% CuSO4. The same protocol was applied for the substrates z-Gly-Pro-Arg-4-methoxy-2-naphthylamide (z-GPR-mna) and D-Val-Leu-Arg-4-methoxy-2-naphthylamide (D-VLR-mna). To demonstrate chymase amidolytic activity with the substrate suc-Ala-Ala-Phe-4-methoxy-2-naphthylamide (s-AAF-mna), the same protocol was followed at pH 7.8 in 0.1 M Tris-HCl, and for chymase esterolytic activity sections were incubated with N-acetyl-L-methionine--naphthyl ester (
-N-O-Met) for 0.5 hr at pH 7.8. After staining, the sections were rinsed in double-distilled water, dehydrated, cleared, and mounted in DPX.
Inhibitors
Selective inhibitors were used to characterize the enzyme activity detected (
Double Staining
To estimate the number and distribution of tryptase-containing mast cells, sections were stained with z-Ala-Ala-Lys-mna, dehydrated, mounted, photographed, and afterwards were hydrated again and stained with 1% toluidine blue in 0.5 M HCl, pH 0.5, for 30 min, followed by dehydration, mounting, and photomicrography.
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Results |
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Enzyme Histochemistry
With the use of amidolytic substrates, tryptase activity could be demonstrated in connective tissue mast cells in unfixed frozen sections of all tissues. Of the substrates tested, z-GPR-mna (Figure 1A) gave intense staining, and similar results were obtained with z-AAK-mna, whereas inferior faint staining was obtained with D-VLR-mna. Occasional diffuse staining, suggestive of mast cells, was seen in the unfixed stomach mucosa using z-AAK-mna and z-GPR-mna (Figure 1B). When tissues were fixed with formaldehydeglutaraldehyde and frozen-sectioned, tryptase activity in connective tissue mast cells, and particularly in stomach mucosal mast cells, was better localized within distinct granules and the mucosal mast cells were seen to be present in intraepithelial sites (Figure 2A and Figure 2B). Tryptase activity could not be demonstrated in aldehyde-, Carnoy-, or methanol-fixed tissues after paraffin embedding. Tryptase-containing mucosal mast cells were not observed in either unfixed or fixed gut. The tryptase-containing mucosal mast cells in the stomach were examined in more detail. Like tryptase in connective tissue mast cells, the enzyme in stomach mucosal mast cells was best demonstrated with the substrates z-AAK-mna and z-GPR-mna, and when the pH of the enzyme histochemical incubation was lowered from 6.3 to 5.5 or 5.0 the intensity of the reaction product was decreased in mucosal mast cells of the stomach as in connective tissue mast cells (Figure 3). Incubation of the tissue sections with Tos-Lys-chloromethylketone, an inhibitor of trypsin-like enzymes, blocked the reaction with substrates z-Ala-Ala-Lys-mna, z-Gly-Pro-Arg-mna, and D-Val-Leu-Arg-mna because mast cells remained unstained (not shown). The amidolytic substrate s-AAF-mna and the esterolytic substrate -N-O-Met demonstrated chymase activity in mast cells and the intensity of the reaction product was unaffected by incubation with the trypsin-like enzyme inhibitor Tos-Lys-chloromethylketone (Figure 4). However, such chymase activity was greatly reduced by the specific chymotryptic enzyme inhibitor Gly-Lys-Pro-chloromethylketone (not shown).
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Dye Binding
To examine whether the tryptase-containing mast cells also contain heparin sulfate PG in their granules, aldehyde- and Carnoy-fixed tissues were stained with alcian bluesafranin O or Berberine sulfate. All connective tissue mast cells showed safranin or Berberine sulfate affinity, whereas the mucosal mast cells, including the tryptase-positive stomach mucosal mast cells, were negative (not shown). Both connective tissue and mucosal mast cells were demonstrated by toluidine blue staining of aldehyde-fixed, frozen-sectioned tissues. When used after enzyme histochemistry in a double staining protocol, toluidine blue staining showed that all tryptase activity was present only in mast cells and that all toluidine blue-positive stomach mucosal mast cells contained active tryptase (Figure 5A and Figure 5B).
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Discussion |
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The amidolytic substrates z-AAK-mna and z-GPR-mna have previously been shown to be effective in specific demonstration of tryptase in mast cells of cat, human, ox, and rat, whereas the substrate D-VLR-mna has been shown to be less effective (
Human tryptase activity is dependent on heparin sulfate, with which it forms a complex (
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Acknowledgments |
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Supported by Grants from the International Journal of Experimental Pathology and the European Federation of Experimental Morphology.
We thank Katherine Paterson and Robert Hartley for their assistance.
Received for publication October 16, 1998; accepted December 31, 1998.
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