Jiang-Hong Gong

Tissue degradation by the matrix metalloproteinase gelatinase A is pivotal to inflammation and metastases. Recognizing the catalytic importance of substrate-binding exosites outside the catalytic domain, we screened for extracellular substrates using the gelatinase A hemopexin domain as bait in the yeast two-hybrid system. Monocyte chemoattractant protein-3 (MCP-3) was identified as a physiological substrate of gelatinase A. Cleaved MCP-3 binds to CC-chemokine receptors-1, -2, and -3, but no longer induces calcium fluxes or promotes chemotaxis, and instead acts as a general chemokine antagonist that dampens inflammation. This suggests that matrix metalloproteinases are both effectors and regulators of the inflammatory response.

Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.

Monocyte chemoattractant protein (MCP)-3 is inactivated upon cleavage by the matrix metalloproteinase (MMP) gelatinase A (MMP-2). We investigated the susceptibility to proteolytic processing of the 4 human MCPs by 8 recombinant MMPs to determine whether MCP-3 is an isolated example or represents a general susceptibility of chemokines to proteolytic inactivation by these important inflammatory proteases. In addition to MMP-2, MCP-3 is efficiently cleaved by membrane type 1 (MT1)-MMP, the cellular activator of MMP-2, and by collagenase-1 and collagenase-3 (MMP-1, MMP-13) and stromelysin-1 (MMP-3). Specificity was shown by absence of cleavage by matrilysin (MMP-7) and the leukocytic MMPs neutrophil collagenase (MMP-8) and gelatinase B (MMP-9). The closely related chemokines MCP-1, MCP-2, and MCP-4 were not cleaved by MMP-2 or MT1-MMP, but were cleaved by MMP-1 and MMP-3 with varying efficiency. MCPs were typically cleaved between residues 4 and 5, but MCP-4 was further processed at Val7-Pro8. Synthetic MCP analogs corresponding to the MMP-cleaved forms bound CC chemokine receptor (CCR)-2 and CCR-3, but lacked chemoattractant activity in pre-B cells transfected with CCR-2 and CCR-3 or in THP-1 monocytic cells, a transformed leukemic cell line. Moreover, the truncated products of MCP-2 and MCP-4, like MCP-3, were potent antagonists of their cognate CC chemokine receptors in transwell cell migration assays in vitro. When they were injected 24 hours after the initiation of carrageenan-induced inflammation in rat paws, their in vivo antagonist activities were revealed by a greater than 66% reduction in inflammatory edema progression after 12 hours. We propose that MMPs have an important role in modulating inflammatory and immune responses by processing chemokines in wound healing and in disease.

An antagonist of human monocyte chemoattractant protein (MCP)-1, which consists of MCP-1(9-76), had previously been characterized and shown to inhibit MCP-1 activity in vitro. To test the hypothesis that, by inhibiting endogenous MCP-1, the antagonist has antiinflammatory activity in vivo, we examined its effect in the MRL-lpr mouse model of arthritis. This strain spontaneously develops a chronic inflammatory arthritis that is similar to human rheumatoid arthritis. Daily injection of the antagonist, MCP-1(9-76), prevented the onset of arthritis as monitored by measuring joint swelling and by histopathological evaluation of the joints. In contrast, controls treated with native MCP-1 had enhanced arthritis symptoms, indicating that the inhibitory effect is specific to the antagonist. In experiments where the antagonist was given only after the disease had already developed, there was a marked reduction in symptoms and histopathology, although individuals varied in the magnitude of the response. The mechanism of inhibition of disease is not known, although the results suggest that it could be more complex than the competitive inhibition of ligand binding that is observed in vitro. The demonstration of the beneficial effects of an MCP-1 antagonist in arthritis suggests that chemokine receptor antagonists could have therapeutic application in inflammatory diseases.

Structural analysis of chemokines has revealed that the alpha/beta structural-fold is highly conserved among both the CXC and CC chemokine classes. Although dimerization and aggregation is often observed, the chemokines function as monomers. The critical receptor binding regions are in the NH2-terminal 20 residues of the protein and are the least ordered in solution. The flexible NH2-terminal region is the most critical receptor binding site and a second site also exists in the loop that follows the two disulfides. The well-ordered regions are not directly involved in receptor binding but, along with the disulfides, they provide a scaffold that determines the conformation of the sites that are critical for receptor binding. These general requirements for function are common to all the chemokines. For the CC chemokines, receptor activation and receptor binding regions are separate within the 10 residue NH2-terminal region. This has allowed identification of high affinity analogs that do not activate the receptor and are potent antagonists.