Dynamic Imaging of the Effector Immune Response to Listeria Infection In Vivo

Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30–1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ [CD8 superscript +] T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ [CD8 superscript +] T cells in effector sites.National Institutes of Health (U.S.) (Grant P01AI-071195

Development of a Solid-Phase Assay for Analysis of Matrix Metalloproteinase Activity

Proteases play fundamentally important roles in normal physiology and disease pathology. Methods for detection of active proteolysis may greatly aid in the diagnosis of disease progression, and suggest modes of therapeutic intervention. Most assays for proteolytic potential are limited by a lack of specificity and/or quantification. We have developed a solid-phase activity assay for members of the matrix metalloproteinase (MMP) family that is specific and can be used to quantify active enzyme concentration. The assay has two principal components: a capture antibody that immobilizes the MMP without perturbing the enzyme active site, and a fluorescence resonance energy transfer substrate for monitoring proteolysis at low enzyme concentrations. The assay was standardized for MMP-1, MMP-3, MMP-13, and MMP-14. The efficiency of the assay was found to be critically dependent upon the quality of the antibodies, the use of substrates exhibiting high specific activities for the enzymes, and enzyme samples that are fresh. The assay was applied to studies of constitutive and induced MMP activity in human melanoma cells. Analysis of several melanoma cell lines, and comparison with prior studies, correlated higher constitutive MMP-13 activity with higher levels of the cell surface receptor CD44. Ligands to two different melanoma cell surface receptors (the α2β1 integrin or CD44) were found to induce different proteolytic profiles, suggesting that the extracellular matrix can modulate melanoma invasion. Overall, the solid-phase MMP activity assay was found to be valuable for analysis of protease activity in cellular environments. The solid-phase assay is suitably flexible to allow studies of virtually any proteolytic enzyme for which appropriate substrates and antibodies are available

Modulation of Tumor Progression by Glycosylated Triple-helical Ligands

Specific tumor cell interactions with basement membrane (type IV) or fibrillar (types I–III) collagen have been shown previously to represent a regulatory step in metastasis [Nat. Rev. Cancer3, 422 (2003)]. Interestingly, numerous tumor cell receptors, such as integrins, CD44 proteoglycan, and DDR Tyr kinases, bind sites within distinct triple-helical regions of collagen that are glycosylated. The present study has refined methodology for constructing galactosylated hydroxylysine [Hyl(O-ß-D-galactopyranosyl)]-containing triple-helical ligands, and utilized these ligands to evaluate binding and activation of (a) 2ß 1 and 3ß 1 integrins by 1(IV)382–393 and 1(V)531–543, respectively, (b) CD44/CSPG by 1(IV)1263–1277, and (c) DDR2 by 1(I)79–93. The possible effect of glycosylation on ligand conformation, and hence the correlation of structure with activity, has been studied by CD spectroscopy. Initial results showed that glycosylation significantly decreased CD44-mediated adhesion and spreading of melanoma cells [J. Biol. Chem. 278, 14321 (2003)]. This was the first demonstration of the prophylactic effects of glycosylation on tumor cell interaction with the basement membrane, and suggested a possible cryptic sites mechanism associated with tumor cell invasion

Collagenase unwinds triple-helical collagen prior to peptide bond hydrolysis

Breakdown of triple-helical interstitial collagens is essential in embryonic development, organ morphogenesis and tissue remodelling and repair. Aberrant collagenolysis may result in diseases such as arthritis, cancer, atherosclerosis, aneurysm and fibrosis. In vertebrates, it is initiated by collagenases belonging to the matrix metalloproteinase (MMP) family. The three-dimensional structure of a prototypic collagenase, MMP-1, indicates that the substrate-binding site of the enzyme is too narrow to accommodate triple-helical collagen. Here we report that collagenases bind and locally unwind the triple-helical structure before hydrolyzing the peptide bonds. Mutation of the catalytically essential residue Glu200 of MMP-1 to Ala resulted in a catalytically inactive enzyme, but in its presence noncollagenolytic proteinases digested collagen into typical 3/4 and 1/4 fragments, indicating that the MMP-1(E200A) mutant unwinds the triple-helical collagen. The study also shows that MMP-1 preferentially interacts with the α2(I) chain of type I collagen and cleaves the three α chains in succession. Our results throw light on the basic mechanisms that control a wide range of biological and pathological processes associated with tissue remodelling