Injury to a peripheral nerve is followed by a remodeling process consisting of axonal degeneration and regeneration. It is not known how Schwann cell–derived basement membrane is preserved after injury or what role matrix metalloproteinases (MMPs) and their inhibitors play in axonal degeneration and regeneration. We showed that the MMPs gelatinase B (MMP-9), stromelysin-1 (MMP-3), and the tissue inhibitor of MMPs (TIMP)-1 were induced in crush and distal segments of mouse sciatic nerve after injury. TIMP-1 inhibitor activity was present in excess of proteinase activity in extracts of injured nerve. TIMP-1 protected basement membrane type IV collagen from degradation by exogenous gelatinase B in cryostat sections of nerve in vitro. In vivo, during the early phase (1 d after crush) and later phase (4 d after crush) after injury, induction of TNF-α and TGF-β1 mRNAs, known modulators of TIMP-1 expression, were paralleled by an upregulation of TIMP-1 and gelatinase B mRNAs. At 4 days after injury, TIMP-1, gelatinase B, and TNF-α mRNAs were localized to infiltrating macrophages and Schwann cells in the regions of nerve infiltrated by elicited macrophages. TIMP-1 and cytokine mRNA expression was upregulated in undamaged nerve explants incubated with medium conditioned by macrophages or containing the cytokines TGF-β1, TNF-α, and IL-1α. These results show that TIMP-1 may protect basement membrane from uncontrolled degradation after injury and that cytokines produced by macrophages may participate in the regulation of TIMP-1 levels during nerve repair.
Using 35S-methionine metabolic labeling, we studied de novo synthesis and secretion of proteins by activated polymorphonuclear neutrophils (PMN) from two different sources. PMN isolated from inflammatory synovial fluid of patients with inflammatory joint disease were first analyzed. The protein synthetic activity of these cells was compared with that of nonactivated PMN isolated from the peripheral blood of the same patient. Similar studies were conducted on glycogen-activated PMN from the peritoneal cavity of rabbits and results were compared with nonactivated peripheral blood PMN isolated from the same rabbit. Cells were labeled for a period of 16 to 20 h and supernatants were analyzed by one and two dimensional gel electrophoresis. In both models, the activated PMN showed a marked increase in the synthesis and secretion of thrombospondin as identified by immunoisolation with antibodies to this protein. The production of thrombospondin by activated cells paralleled a similar increase in production of another extracellular matrix and cell adhesion protein, fibronectin. The proportion of thrombospondin synthesis and secretion relative to total protein was approximately 1% in both human- and rabbit-activated PMN. For fibronectin, this proportion was in the 0.02% range. Although fibronectin mRNA accumulation in activated PMN could be demonstrated by Northern blots, we were not able to obtain similar results for thrombospondin mRNA. This could be caused by the rapid turnover of this transcript because it is known to contain an adenine uridine-rich 3' untranslated sequence. We conclude that activated PMN are capable of producing thrombospondin. Furthermore, glycogen-activated rabbit peritoneal fluid PMN represent a valuable and relevant source of activated PMN for studying the protein synthetic events of these cells in the context of inflammation.
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