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)