Clinical & Experimental Allergy

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

Authors

  • A. D. Pemberton,

    1. Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK, and
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  • J. K. Brown,

    1. Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK, and
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  • S. H. Wright,

    1. Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK, and
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  • P. A. Knight,

    1. Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK, and
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  • M. L. McPhee,

    1. Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK, and
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  • A. R. McEuen,

    1. Immunopharmacology Group, University of Southampton School of Medicine, Mail Point 837, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK
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  • P. A. Forse,

    1. Immunopharmacology Group, University of Southampton School of Medicine, Mail Point 837, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK
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  • H. R.P. Miller

    1. Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK, and
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Alan D. Pemberton, Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK. E-mail: Alan.Pemberton@ed.ac.uk

Summary

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 μg/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.

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