Francis J Descamps

Analysis of gelatinases in complex biological fluids and tissue extracts.

G elatinase B (matrix metalloproteinase-9 [MMP-9]) and gelatinase A (MMP-2) are two closely related members of the MMP family that efficiently degrade denatured collagens or gelatins (Van den Steen et al, 2002). Specific MMPs play a major role in physiological processes, including angiogenesis, wound healing, bone remodeling, and cell migration. Moreover, MMP-9 and MMP-2 are key effector molecules in inflammation, autoimmunity (Opdenakker and Van Damme, 1994), and cancer (Sehgal et al, 1998). Analysis of both enzymes in complex biological samples, especially those with low gelatinase content, therefore is essential. We combined a miniaturized gelatin affinity chromatography with gelatin zymography and Western blot analysis. This strategy allows extremely sensitive and unambiguous detection of gelatinases. Gelatinases are often detected with specific antibodies or by substrate conversion assays. Gelatinase activity assays measure overall gelatinase activity often with the use of labeled gelatins (Paemen et al, 1996). Because these do not discriminate between gelatinase A and B and even other gelatin-degrading enzymes, specificity is low, especially in the analysis of complex biological samples. Introducing affinity prepurification with the use of monoclonal antibodies enhances the specificity (Hanemaaijer et al, 1998). Moreover, only activated enzymes are recognized. ELISA detects specific forms of MMP-9 or MMP-2. Unfortunately, this method does not necessarily differentiate between pro-enzyme and activated forms. Discrimination between different gelatin-degrading enzymes and their respective activation status may be achieved by zymography or Western blot analysis. Gelatin zymography detects picogram levels of MMP-9 (Masure et al, 1991). The sensitivity of Western blot analysis is usually lower and depends on the antibody affinity for MMP-9. In our hands, a combination of two monoclonal antibodies against mouse MMP-9 resulted in a detection limit of 100 pg of MMP-9. Nevertheless, such low detection limits are frequently not attained because the ratio of MMP-9 versus total protein is generally extremely low in crude samples, whereas the total protein load per lane is limited to 25 g so as not to distort the electrophoresis. After protein overloading, gelatinolysis may be eclipsed in zymography. Interference by gelatinolytic activity from other (abundant) enzymes constitutes an additional problem for zymography. For instance, in stomach extracts, pepsin is abundantly present and because this and other proteases also cleave gelatin, it will mask the gelatinases. To improve the ratio of MMP-9 versus total protein and to exclude interfering gelatinolytic activity, we developed a simple strategy for optimal preparation of complex samples, including tissue extracts. We used a miniaturized affinity chromatography purification step, taking advantage of gelatinases’ strong affinity for gelatin (Masure et al, 1991). For rapid and reproducible purification of the samples, we made use of mini-spin columns (Bio-Rad Laboratories, Hercules, California) and gelatin-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). Equilibration buffer was composed of 50 mM of Tris (pH 7.5), 0.5 M of NaCl, 10 mM of CaCl2, 0.01% Tween 20, and 5 mM of o-phenanthroline; washing buffer 1 had a similar composition except that the concentration of Tween 20 was increased to 0.05%. Washing buffer 2 was with omission of NaCl because high salt concentrations hinder the electrophoresis. The o-phenanthroline was added to the samples as a gelatinase inhibitor to prevent the gelatinolytic activity from breaking down the gelatin from the Sepharose beads, which are used for the affinity purification of the enzymes. The binding of gelatinases to gelatin-Sepharose is not disturbed by the presence of o-phenanthroline because this inhibitor acts by the chelation of the catalytic Zn , whereas binding to gelatin is mediated by the three fibronectin type II–like repeats. Elution buffer was at once the electrophoresis loading buffer and was composed of 100 mM of Tris/HCl (pH 6.8), 4% sodium dodecyl sulfate, 20% glycerol, and 200 g/ml of bromophenol blue as tracking dye. DOI: 10.1097/01.LAB.0000038556.54069.73

Gelatinase B is diabetogenic in acute and chronic pancreatitis by cleaving insulin

Genetic, endocrine, and environmental factors contribute to the development of diabetes. Much information has been gathered on the homeostasis mechanisms of glucose regulation by insulin‐producing pancreatic β cells. Here we demonstrate high expression levels of gelatinase B (matrix metalloproteinase‐9, MMP‐9) by neutrophils in acute pancreatitis and by ductular epithelial cells in chronic pancreatitis. Because gelatinase B processes cytokines and chemokines, we investigated whether and how gelatinase B cleaves insulin. Pure human neutrophil gelatinase B was found to destroy insulin by cleavage at 10 sites. Pancreatic islet and ductular cells are relatively spared in comparison with the complete destruction of acinar cells of the exocrine pancreas in chronic pancreatitis. High expression levels of gelatinase B are maintained in the immediate proximity of insulin‐secreting β cells. Consequently, diabetes may be worsened by enzymatic degradation of insulin by gelatinase B and by the consequent enhancement of the autoimmune process. Gelatinase B is diabetogenic in acute and chronic pancreatitis by cleaving insulin.

Gelatinase B/matrix metalloproteinase-9 provokes cataract by cleaving lens βB1 crystallin

Cataract is a common cause of blindness and results from destruction of the microarchitecture of the lens. It is observed in many genetic syndromes, infections, inflammatory diseases and during aging. Fluctuations in lens density and light scattering by altered refraction index form the physical basis for this process, but the pathogenesis is poorly understood. Increased levels of gelatinase B/matrix metalloprotein‐ ase‐9 have been reported for cataract‐associated disor¬ders such as eye inflammation and diabetes. We dem¬onstrate that incubation of lenses with gelatinase B leads immediately to cataract. In complete eye extracts, βB1 crystallin was identified as the major gelatinase B substrate by combination of proteomics, mass spec¬trometry, and Edman degradation analysis. The cleav¬age of βB1 crystallin was also observed in vivo after endogenous gelatinase B‐induction by the chemokine granulocyte chemotactic protein‐2 in wild‐type mice but not in gelatinase B−/− mice.—Descamps, F. J., Mar¬tens, E., Proost, P., Starckx, S., Van den Steen, P. E., Van Damme, J., Opdenakker, G. Gelatinase B/matrix metalloproteinase‐9 provokes cataract by cleaving lens βB1 crystallin. FASEB J. 19, 29‐35 (2004)

In vivo activation of gelatinase B/MMP‐9 by trypsin in acute pancreatitis is a permissive factor in streptozotocin‐induced diabetes

Matrix metalloproteinases, in particular gelatinase B/MMP‐9, are key mediators in autoimmune diseases like multiple sclerosis and rheumatoid arthritis, but their pathogenic roles in diabetes are not well established. Gelatinase B has previously been shown to be upregulated in pancreas tissue from patients with acute and chronic pancreatitis and was suggested to exacerbate diabetes by cleaving insulin. In this study, the role of gelatinase B in diabetes was investigated using two streptozotocin‐induced animal models of type I diabetes. In both a hyperacute and a subacute model, gelatinase B upregulation was found to be associated with disease activity. However, gelatinase B deficiency did not significantly protect against diabetes development, and wild‐type and gelatinase B‐deficient animals behaved similarly in terms of β‐cell apoptosis or necrosis. The fact that gelatinase B was found almost exclusively as the inactive pro‐enzyme in most of the streptozotocin‐induced diabetic animals may explain the lack of a gelatinase B effect. On the contrary, gelatinase B was completely activated in a minority (15%) of wild‐type animals. This coincided with exocrine pancreatic inflammation, as revealed by the presence of active trypsin. The discovery of in vivo activation of progelatinase B by trypsin in acute pancreatitis is extended in a model of caerulein‐induced pancreatitis. In the latter model, trypsinogen activation is systematically achieved and gelatinase B is found in its active form. In conclusion, gelatinase B itself is not a causative factor but, when activated by endogenous trypsin, is a permissive factor for insulin degradation and diabetes. Copyright

Interphotoreceptor retinoid-binding protein as biomarker in systemic autoimmunity with eye inflictions

Autoimmune diseases of the eye, exemplified by Beh cet disease and Vogt‐Koyanagi‐Harada disease, are a major cause of blindness. We studied interphotoreceptor retinoid‐binding protein (IRBP), a dominant autoimmune antigen in the eye. Aqueous humour samples from 28 patients with active uveitis were analysed for immunoglobulin G (IgG) content as a marker for blood‐ocular barrier breakdown and by gelatinase B zymography for the detection of inflammation. The data were correlated with the presence of intact IRBP (≈140 kD) as determined by Western blot analysis and with the clinical disease activity. Aqueous humour samples from control eyes and eyes with low disease activity showed positive immunoreactivity for intact IRBP. The IRBP signal weakened or disappeared with higher disease activity. Significant positive correlations were observed between disease activity and levels of gelatinase B/matrix metalloproteinase‐9 (MMP‐9) (rs= 0.713; P < 0.001) and IgG (rs= 0.580; P= 0.001). Significant negative correlations were found between levels of IRBP and disease activity (rs=−0.520; P= 0.005) and levels of MMP‐9 (rs=−0.727; P < 0.001) and of IgG (rs=−0.834; P < 0.001). Whereas neutrophil elastase converted intact IRBP into an immunoreactive 55 kD peptide in vitro, the conversion by neutrophil degranulates resembled more the in vivo context with a complete degradation of IRBP. Reversal of inflammation with immunosuppressive therapy was accompanied with reappearance of intact IRBP and disappearance of IgG and MMP‐9. The analysis of IRBP proteolysis is useful as a biomarker for uveitis and suggests that inhibition of proteinases might become a therapeutic strategy in an inflammatory context of a damaged blood‐ocular barrier.