Stefan Masure

Gelatinase B functions as regulator and effector in leukocyte biology

Matrix metalloproteinases (MMPs) form a family of enzymes with major actions in the remodeling of extracellular matrix (ECM) components. Gelatinase B (MMP‐9) is the most complex family member in terms of domain structure and regulation of its activity. Gelatinase B activity is under strict control at various levels: transcription of the gene by cytokines and cellular interactions; activation of the pro‐enzyme by a cascade of enzymes comprising serine proteases and other MMPs; and regulation by specific tissue inhibitors of MMPs (TIMPs) or by unspecific inhibitors, such as α2‐macroglobulin. Thus, remodeling ECM is the result of the local protease load, i.e., the net balance between enzymes and inhibitors. Glycosylation has a limited effect on the net activity of gelatinase B, and in contrast to the all‐or‐none effect of enzyme activation or inhibition, it results in a higher‐level, fine‐tuning effect on the ECM catalysis by proteases in mammalian species. Fast degranulation of considerable amounts of intracellularly stored gelatinase B from neutrophils, induced by various types of chemotactic factors, is another level of control of activity. Neutrophils are first‐line defense leukocytes and do not produce gelatinase A or TIMP. Thus, neutrophils contrast sharply with mononuclear leukocytes, which produce gelatinase A constitutively, synthesize gelatinase B de novo after adequate triggering, and overproduce TIMP‐1. Gelatinase B is also endowed with functions other than cleaving the ECM. It has been shown to generate autoimmune neo‐epitopes and to activate pro‐IL‐1β into active IL‐1β. Gelatinase B ablation in the mouse leads to altered bone remodeling and subfertility, results in resistance to several induced inflammatory or autoimmune pathologies, and indicates that the enzyme plays a crucial role in development and angiogenesis. The major human neutrophil chemoattractant, IL‐8, stimulates fast degranulation of gelatinase B from neutrophils. Gelatinase B is also found to function as a regulator of neutrophil biology and to truncate IL‐8 at the aminoterminus into a tenfold more potent chemokine, resulting in an important positive feedback loop for neutrophil activation and chemotaxis. The CXC chemokines GRO‐α, CTAP‐III, and PF‐4 are degraded by gelatinase B, whereas the CC chemokines MCP‐2 and RANTES are not cleaved.

Gelatinase in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological disorders

A substrate conversion assay was used to detect gelatinase activity in the cerebrospinal fluid (CSF) of patients with various neurological disorders. Two main forms of gelatinase with an apparent molecular mass of 65 and 85 kDa, respectively, could be discerned. The high molecular mass gelatinase was detectable only in samples of patients with multiple sclerosis or other inflammatory neurological disorders. A statistically significant correlation was found between the level of the 85-kDa gelatinase and the CSF cytosis. This protease could play a role in the process of demyelination and breakdown of the blood-brain barrier in certain neurological disorders, such as multiple sclerosis.

Resistance of young gelatinase B-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions.

Regulated expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) plays a role in various physiological processes. To determine in vivo how unbalanced expression of these factors can promote or affect the course of pathologies, we knocked out the mouse gelatinase B gene by replacing the catalytic and zinc-binding domains with an antisense-oriented neomycin resistance gene. Adult gelatinase B-deficient mice and wild-type controls could be induced to develop experimental autoimmune encephalomyelitis (EAE) with similar scores for neurologic disease, blood-brain barrier permeability, and central nervous system histopathology. However, whereas diseased control animals showed necrotizing tail lesions with hyperplasia of osteocartilaginous tissue, adult gelatinase B-deficient mice were resistant to this tail pathology. Gelatinase B-deficient mice younger than 4 weeks of age were significantly less susceptible to the development of EAE than were age matched controls and, even as they aged, they remained resistant to tail lesions. These data illustrate that gelatinase B expression plays a role in the development of the immune system and that, in ontogenesis, the propensity to develop autoimmunity is altered by the absence of this MMP.

The gelatinase inhibitory activity of tetracyclines and chemically modified tetracycline analogues as measured by a novel microtiter assay for inhibitors

A quantitative nonisotopic solution assay for gelatinases and inhibitors was developed using biotinylated gelatin as enzyme substrate. In this assay, residual biotinylated substrate is sandwiched between avidin-coated plates and streptavidin-peroxidase and is quantified by the peroxidase reaction. This assay was useful for measuring gelatinase activities and defining the activities of gelatinase inhibitors. When 23 tetracycline analogues were compared, significant differences in gelatinase B inhibition were found between various compounds. 4-epioxytetracycline base, 4-epichlortetracycline, meclocyclinesulfosalicylate, and unmodified metacycline and minocycline proved to be the most potent gelatinase B (EC 3.4.24.35) inhibitors. The gelatinase B inhibitory activity of tetracyclines was clearly dissociated from their antimicrobial activity. The effect of high-molecular-weight inhibitors, such as monoclonal antibodies, was also demonstrable in the microtiter plate assay. In view of the pathophysiological function of gelatinases, the definition of gelatinase inhibitors with known efficacy, safety, and side effects is crucial for the treatment of diseases such as rheumatoid arthritis and multiple sclerosis. Particular tetracyclines fulfil these criteria and the described assay is useful for defining other gelatinase-inhibiting lead compounds.

HUMAN HEPATOMA CELLS PRODUCE AN 85 KDA GELATINASE REGULATED BY PHORBOL 12-MYRISTATE 13-ACETATE

Several human cell lines were studied for the production of gelatinases. Diploid fibroblasts, the melanoma cell line Bowes, the MG-63 osteosarcoma cell line and the human hepatoma cell line Malavu all constitutively produced a 67 kDa gelatinase. Gelatinolytic enzymes were quantified by a sensitive zymographic substrate conversion assay. Upon induction with phorbol 12-myristate 13-acetate (PMA), the human hepatoma cell line secreted considerable amounts of an 85 kDa gelatinase activity. The induction process was time- and dose-dependent. It represented a true increase in production per individual cell and was associated by a marked change of the cell morphology. The effect of various proteinase inhibitors and the maximal activity of the enzyme near neutral pH demonstrate that it is a neutral metalloproteinase. Characterization studies showed the 85 kDa gelatinase to be transformed to lower molecular weight, active forms by treatment with p-aminophenylmercuric acetate (APMA) or trypsin.

GELATINASE B (MMP-9), BUT NOT ITS INHIBITOR (TIMP-1), DICTATES THE GROWTH RATE OF EXPERIMENTAL THYMIC LYMPHOMA

Dysregulation of metalloproteinase production at tumor sites contributes to the modification of local stromal tissue necessary for tumor development. Gelatinase B (matrix metalloproteinase‐9, MMP‐9) is one of the key enzymes that have been associated with the progression of several tumors. Paradoxically, MMP‐9 expression by tumor cells, most notably by lymphoma cells, is concomitant with the expression of its physiological inhibitor, TIMP‐1. Not only are both genes often co‐expressed in the most aggressive forms of lymphomas but also both are up‐regulated upon contact with stromal cells. Since TIMP‐1 is known to regulate growth in several cell types and some aggressive lymphoma cells express TIMP‐1 constitutively without MMP‐9, it is unclear whether the over‐expression of MMP‐9 is counterbalanced by TIMP‐1 and whether TIMP‐1 expression alone could favor the development of lymphoma. To gain further insight into the respective roles of MMP‐9 and TIMP‐1 in lymphoma, we generated lymphoma cell lines expressing constitutively high levels of MMP‐9 or TIMP‐1 and compared these cells for the ability to form thymic lymphoma in vivo. Moreover, we generated lymphoma cell lines expressing constitutively high levels of both MMP‐9 and TIMP‐1 to reproduce the net physiological balance resulting from the expression of both genes simultaneously and to determine which gene overrides the other. Our results show that mice injected with lymphoma cells expressing MMP‐9 constitutively developed thymic lymphoma more rapidly than those injected with control lymphoma cells. Over‐expression of TIMP‐1 alone did not significantly influence tumor progression of lymphoma nor did it delay the capacity of MMP‐9 to accelerate the development of thymic lymphoma. Int. J. Cancer 82:743–747, 1999.

Prevention of acute autoimmune encephalomyelitis and abrogation of relapses in murine models of multiple sclerosis by the protease inhibitor D-penicillamine

Thein vitro activity of gelatinase B, an enzyme whose appearance in the cerebrospinal fluid is associated with inflammatory diseases of the central nervous system, was dose-dependently inhibited by the antirheumatic D-penicillamine. Inhibition of gelatinase B in electrophoretically pure preparations and in cell culture supernatants and human body fluids was obtained at dosages reached in the circulation of patients treated with a peroral dosis of 750mg D-penicillamine per day. In mice, developing acute demyelination, D-penicillamine significantly reduced the mortality and morbidity rates of experimental allergic encephalomyelitis (EAE). In chronic relapsing EAE in Biozzi AB/H mice, an animal model for relapses in multiple sclerosis (MS), it attenuated the exacerbations, even when the treatment was started after the primary full-blown disease had developed. We infer protease inhibition as the mechanism of action of D-penicillamine and suggest that its use may be effective as peroral treatment for MS.

The cytokine-protease connection: Identification of a 96-kD THP-1 gelatinase and regulation by interleukin-1 and cytokine inducers

The induction of proteolytic enzymes is an important mechanism in the migration of monocytes into tissues and body fluids. The monocytic cell line THP-1 was used as a model system to study the production of a particular gelatinase. Upon stimulation with phorbol myristate acetate (PMA) the cells differentiated to the adherent phenotype and produced significant amounts of a 96-kD gelatinase in a dose-dependent way. The secretion rate was maximal between 12 and 24 h after induction. Study of gelatinase mRNA steady state levels showed that the synthesis of THP-1 gelatinase is regulated by PMA at transcriptional or posttranscriptional levels. Stimulation of signal transduction pathways with other substances, including calcium ionophore A 23187, dibutyryl cyclic AMP, and dexamethasone, were ineffective in inducing gelatinase mRNA or enzyme activity. However, THP-1 cells were responsive to the cytokine interleukin (IL)-1 beta, to bacterial lipopolysaccharide (LPS), and the lectin concanavalin A (Con A), the kinetics of gelatinase induction being similar to those of induction by PMA. The THP-1 cells did not synthesize and/or secrete detectable levels of IL-6 after stimulation with PMA, Con A, LPS, or IL-1 beta. The 96-kD monocytic THP-1 gelatinase was shown to be a neutral metalloproteinase that cross-reacted with hepatoma-derived and neutrophil gelatinases in immunoprecipitation experiments. The active enzyme produced by THP-1 cells consistently showed, however, a molecular mass different from that of normal granulocyte-, monocyte-, and tumor cell-derived gelatinases.(ABSTRACT TRUNCATED AT 250 WORDS)

Natural human monocyte gelatinase and its inhibitor

Gelatinases produced by stimulated peripheral blood monocytes were detected by substrate zymography and were compared with those derived from tumor cells. Stimulated monocytes were found to produce an 85 kDa gelatinase which co‐migrated upon electrophoretic separation and cross‐reacted in immunoprecipitation experiments with a phorbol ester inducible metalloprotease from human tumor cells. The intact natural gelatinase (85 kDa), a high molecular weight and complexed gelatinase as well as a proteolytic fragment (25 kDa) were purified by substrate‐ and antibody‐affinity chromatography techniques. Aminoterminal sequence analysis showed that natural monocyte gelatinase occurs as a truncated form of tumor cell gelatinase/type IV collagenase. Furthermore, peripheral blood monocytes were found to also produce a tissue inhibitor of metalloproteases (TIMP). TIMP was co‐purified with gelatinase on gelatin sepharose and identified by microsequencing. The balanced and regulated production of gelatinase and TIMP might be important in monocyte migration and tissue remodeling.

Cytokine-mediated proteolysis in tissue remodelling

Proteolytic enzymes play a key role in a variety of physiological processes in which the degradation of macromolecules is essential: angiogenesis, embryogenesis, bone and tissue remodelling, blood hemostasis and cell migration. The action of these enzymes is also crucial in the development of many pathological conditions such as wound healing, neoplasia, inflammation and arthritic disorders. the activity of proteases is negatively affected by specific protease-inhibitors. Various growth factors and other cytokines modulate the synthesis and secretion of both proteases and protease-inhibitors. The study of this regulation results in a better insight into (patho)physiology at the molecular level and promises to result in alternative treatment strategies.