Gelatinase (type IV collagenase) immunolocalization in cells and tissues: use of an antiserum to rabbit bone gelatinase that identifies high and low Mr forms

Journal of Cell Science, Mar 1989

G. Murphy, R.M. Hembry, A.M. McGarrity, J.J. Reynolds, B. Henderson

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Gelatinase (type IV collagenase) immunolocalization in cells and tissues: use of an antiserum to rabbit bone gelatinase that identifies high and low Mr forms

G. MURPHY 0 1 R. M. HEMBRY 0 1 A. M. McGARRITY 0 1 J. J. REYNOLDS 0 1 0 Pharmacologv Department, Wellcome Research Laboratories , Beckenham, Kent BR3 3BS , UK 1 Cell Physiology Department, Strcwgeways Research Laboratory , Cambridge CBl 4RN , UK - An antiserum was raised to rabbit bone gelatinase (type IV collagenase). It was shown by immunoblotting to detect both the low Mr (72000) enzyme produced by connective tissue cells from rabbit, pig, human and mouse, as well as the high Mr (94000-97000) enzymes secreted by macrophages and polymorphonuclear leucocytes from these species, and by rabbit chondrocytes and endothelial cells. Crossed immunoblotting, antibody inhibition and deglycosylation studies indicated that the high and low Mr forms of gelatinase are immunologically distinct gene products, although their substrate specificity profiles are identical. The anti-gelatinase antiserum was used to immuThe metalloproteinases secreted by stimulated connective tissue cells and some types of peripheral blood leucocytes are thought to be responsible for both normal connective tissue matrix remodelling and the accelerated breakdown associated with tumour development and degradative diseases. Three major types of metalloproteinase have been identified: collagenases, which degrade the fibrillar collagens; gelatinases, which specifically degrade native type IV collagen; and denatured fibrillar collagens and stromelysins, which are more general proteinases degrading many matrix components including proteoglycan, laminin and non-helical regions of collagens (Murphy & Docherty, 1988). Two major forms of gelatinase have been described; a large Mr (>90000) glycosylated species produced by polymorphonuclear leucocytes and monocyte/ macrophages (Murphy et al. 1982; Mainardi et al. 1984; Hibbs et al. 1985), and a form of approximate Mr 72 000 (mainly non-glycosylated) secreted by many connective nolocalize the enzyme. Gelatinase was most efficiently detected in rabbit monocytes and connective tissue cells, but cells derived from the human and pig gave poor immunostaining, although mouse gelatinase stained well. The anti-gelatinase antiserum stained cells of the synovial tissue of rabbits at 14 days after induction of an antigeninduced arthritis, demonstrating its usefulness as a tool to assess the role of this enzyme in degradative events. tissue cells (Seltzer ef al. 1981; Murphy et al. 1981, 1985; Collier et al. 1988). The properties of the two forms are similar, if not identical. They bind avidly to and degrade all denatured forms of collagen, cleave native type IV collagen at a single locus and have some activity against type V and VII collagens. Both proteinases are secreted in an inactive proform and can be activated in vitro in the presence of organomercurials by a process of self-cleavage with an initial fall in Mr of either 9000 (high Mr form) or 6000 (low Mr form) (Collier et al. 1988; Murphy et al. 1985, 1989). Further lower Mr species are subsequently generated with time (Murphy et al. 1989). Unlike the other metalloproteinases, gelatinases are not efficiently activated by either trypsin or plasmin and the question of physiological activation mechanisms remains open. It has recently been shown that the lowMr gelatinase of connective tissue cells is identical to the type IV collagenase produced by some transformed cells and tumour cells, and originally thought to be specific to such cells (Collier et al. 1988). Immunological identity between an Mr 97 000 gelatinase from pig polymorphonuclear (PMN) leucocytes and a glycosylated Mr 68 000 mouse tumour cell type IV collagenase has also been shown (Murphy et al. 1989). However, Hibbs et al. (1985) have shown that an antiserum to high Mr human PMN leucocyte gelatinase does not recognize the low Mr human fibroblast enzyme. It is, therefore, not clear what the precise relationship of the different enzyme forms is, although they are probably related gene products with different degrees of glycosylation (Vartio & Hovi, 1987). To analyse this matter further and to assess the role of these enzymes in physiological and pathological degradation we have developed specific antisera for use in immunohistochemical studies. In this paper we describe the characterization of an antiserum to rabbit gelatinase/type IV collagenase that recognizes high and low Mx forms of the enzyme and demonstrate its use in a rabbit model of arthritis. Materials and methods Most materials were as described in the references cited below. Histopaque and CNBr-activated Sepharose 4B were from Sigma. A'--glycanase was from Boehringer-Mannheim Bio chemicals. Partially purified pig mononuclear cell cytokines were prepared according to Saklatvala et al. (1983). EHS sarcoma type IV collagen was a generous gift from Professor K. Kiihn, prepared by the method of Kleinman et al. (1982). Connective tissue cells were isolated and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) containing 10 (...truncated)


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G. Murphy, R.M. Hembry, A.M. McGarrity, J.J. Reynolds, B. Henderson. Gelatinase (type IV collagenase) immunolocalization in cells and tissues: use of an antiserum to rabbit bone gelatinase that identifies high and low Mr forms, Journal of Cell Science, 1989, pp. 487-495, 92/3,