A major role for matrix metalloproteinases in T cell injury in the gut.

Activated lamina propria T cells responding to luminal Ags are thought to be important in celiac disease and Crohn's disease, and T cells responding to foreign MHC products are also important in intestinal graft-vs-host disease and intestinal transplant rejection. However, the mechanism(s) by which T cells mediate damage in the gut is not known. We have previously shown that activation of lamina propria T cells by PWM in explant cultures of second trimester human small intestine produces severe tissue injury, with epithelial cell shedding and loss of villi. In this study, we have investigated the role of matrix metalloproteinases in this system. Organ culture supernatants of explants stimulated with PWM showed a 3-fold increase in the concentration of interstitial collagenase and a 10-fold increase in stromelysin-1 compared with control explant culture supernatants. Tissue inhibitors of metalloproteinase-1 and -2 concentrations were unchanged. Increased metalloproteinase enzymatic activity was detected by gelatin and casein zymography. Western blotting revealed the active forms of interstitial collagenase and stromelysin-1 in PWM-stimulated culture supernatants. Up-regulation of mRNA for interstitial collagenase, stromelysin-1, and gelatinase-B was also seen. Nanomolar amounts of recombinant stromelysin-1 added directly to explants produced rapid severe tissue injury. PWM-induced mucosal injury was inhibited by a synthetic peptidomimetic inhibitor of matrix metalloproteinases. Mesenchymal cells isolated from the mucosa of human fetal small intestine produced increased amounts of interstitial collagenase, gelatinase A, and stromelysin-1 when stimulated with IL-1beta or TNF-alpha. These results suggest that T cell activation in the lamina propria results in increased production of matrix metalloproteinases, which by degrading the lamina propria matrix represent a major pathway by which T cells cause injury in the gut.

Plasma assay of gelatinase B: tissue inhibitor of metalloproteinase complexes in cancer.

BACKGROUND: Matrix metalloproteinases (MMPs), especially gelatinase A and gelatinase B (GLB), are believed to be important components of the metastatic process. Tissue Inhibitors of Metalloproteinases (TIMPs) form complexes with MMPs and inhibit cancer dissemination. After local secretion, MMPs and their complexes with TIMPs leach into the blood stream where their concentration can be measured, thereby serving as surrogate markers of disease. Elevated plasma gelatinase B levels have been detected in gastrointestinal cancer and breast cancer. The goal of this study was to determine whether plasma GLB:TIMP complexes also are increased in cancer and whether these tests have potential use as prognostic tumor markers. METHODS: An enzyme-linked immunosorbent assay (ELISA) was developed to measure the plasma concentration of GLB:TIMP complexes in patients with cancer. Correlation between ELISA results and clinical outcome was sought. RESULTS: Plasma GLB:TIMP complexes were significantly increased in patients with gastrointestinal cancer and gynecologic cancer, but not in patients with breast cancer. When results from plasma GLB:TIMP complexes and plasma GLB assays were combined (GLB/complexes), abnormal levels of one or both assays were found in 36% and 65% of patients with gastrointestinal and gynecologic cancer, respectively. In Stage IV gastrointestinal cancer, patient survival was shorter (P < 0.001) in the group with increased plasma GLB/complexes than for those with normal plasma levels (4 months vs. 20 months, respectively). CONCLUSIONS: The assay of plasma gelatinase B and GLB:TIMP complexes may be clinically useful in predicting survival in subsets of patients with cancer. The possibility of using these assays in early stage cancer to predict metastasis should be studied.

Elevated plasma stromelysin levels in arthritis.

OBJECTIVE: To determine whether plasma concentrations of stromelysin-1 and gelatinase A are increased in patients with various forms of arthritis. METHODS: A sensitive and specific sandwich enzyme linked immunosorbent assay (ELISA), which employs a murine monoclonal antibody and a rabbit polyclonal antibody to human stromelysin-1, was used to measure plasma stromelysin-1 in 53 healthy subjects, 113 patients with various forms of arthritis and connective tissue diseases, and 65 patients with cancer. Gelatinase A was also measured in these patients using specific polyclonal and monoclonal antibodies to gelatinase A in an ELISA: RESULTS: The plasma concentration of stromelysin-1 (X +/- SEM) was significantly increased (p < 0.001) in patients with rheumatoid arthritis (RA) (187 +/- 14 ng/ml) and systemic lupus erythematosus (SLE) (258 +/- 35 ng/ml) as compared to both healthy control subjects (50 +/- 4 ng/ml) or patients with cancer (61 +/- 20 ng/ml). Plasma stromelysin-1 was also significantly increased in smaller groups of men with osteoarthritis (OA) and gout. In contrast, plasma concentrations of gelatinase A were not significantly increased in patients with RA, OA or gout. In healthy subjects, the concentration of stromelysin-1 was significantly higher in men than women. No correlation was noted between plasma stromelysin-1 levels and age. CONCLUSION: The detection of elevated plasma levels of stromelysin-1 in patients with RA is consistent with increased stromelysin-1 concentrations in inflamed synovial tissues in this disease. The origin of increased plasma stromelysin-1 in SLE is speculative. Measurement of plasma stromelysin-1 may be useful in the diagnosis and management of patients with various forms of arthritis.

Immunoassays for the detection of human collagenase, stromelysin, tissue inhibitor of metalloproteinases (TIMP) and enzyme-inhibitor complexes.

Immunoassays have been developed for human collagenase, stromelysin, tissue inhibitor of metalloproteinases (TIMP) and TIMP complexed with both of the active enzymes. Selection of antibodies of defined specificity enabled measurement of both the pro and active forms of the metalloproteinase. Free TIMP was quantified by the selection of a monoclonal antibody which did not recognise TIMP when complexed with metalloproteinases. Detection of enzyme-inhibitor complexes was achieved by capturing the TIMP component of the complex and revealing the metalloenzyme using specific antibodies.

Cell kinetic studies in the epidermis of mouse. III. The percent labelled mitosis (PLM) technique.

Full PLM curves have been obtained for four sites in the mouse. The first peaks have been analysed by computer and the duration of the G2 + M and S phases determined together with their standard deviations. The full curves showed a general similarity for all four sites with no clear second peak. The data are compared with the published data for mouse and human epidermis using the in vivo PLM technique. The timing and shape of the first peak can vary considerably even for one site in mice. Hence, both G2 + M and S can vary in their durations. Cells labelled at one time of day exhibit different kinetic properties to those labelled at another time of day. The duration of G2 + M is shortest in dorsum labelled at 03.00 hours (3 X 2 hr) and longest in tail (up to 7 X 5 hr). The S-phase is shortest in dorsum (6 X 3-7 X 2 hr) and longest in tail or ear (13 X 3-14 X 1 hr). There is also a very large standard deviation in tail and foot. There is little general variability when the psoriatic human data are considered, which is surprising. The general variability amongst the data from experimental mice might also be expected amongst humans which might make comparisons between the cell kinetics of normal and diseased skin difficult.