Abstract. It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril. major question in cell biology is how individual macromolecules combine to form the often large and complex supramolecular structures of the extracellular matrix. Approaches to this problem include the direct microscopic visualization of tissue sections aided by chemical stains and immunological tools, reconstitution and binding studies with purified components in solution followed by analysis of the resulting products, and, where possible, direct x-ray analysis of highly ordered arrays in situ.This strategy is particularly well exemplified by the many detailed studies of collagen fibrils and fibrillogenesis. This work has resulted in an understanding of the interactions that govern lateral association of fibrillar collagens (for recent reviews see references 3, 7, and 19). The quarter stagger model that arose from these studies has been considerably refined since its introduction, but still remains as the cornerstone of current models as it explains how fibrils formed in vitro from purified collagen molecules give rise to the characteristic staining patterns observed in the electron microscope. Although the fibril staining patterns produced in vitro match those seen in vivo, the diameter regulation of collagen fibrils in vivo and their complexity, including association with other components, especially proteoglycans, are features not reproduced by mixing solutions of collagens in vitro. In summary, two critical questions remain: how is the construction of fibrils regulated in vivo and what role might other molecules play in these processes?This problem is particularly well illustrated in cartilage. The morphology of fibrils reconstituted from purified type II collagen in solution is vastly different from that of cartilage fibrils in situ. Large tactoidal aggregates with d-periodic staining patterns are formed under appropriate reconstitution conditions (21). In contrast, fibrils from chicken embry...
Polymyositis (PM) and dermatomyositis (DM) are the prototypical inflammatory diseases of skeletal muscle. In PM, CD8 ϩ T cells invade and destroy muscle fibers, whereas humoral effector mechanisms prevail in DM. We studied the expression of the cytotoxic mediator perforin in inflammatory cells in PM and DM muscle by semiquantitative PCR, immunohistochemistry and confocal laser microscopy. Similar levels of perforin mRNA were expressed in PM and DM, and abundant perforin-expressing CD3 ϩ CD8 ϩ and CD3 ϩ CD4 ϩ T cells were observed in both diseases. However, there was a striking difference in the intracellular localization of perforin. In DM, perforin was distributed randomly in the cytoplasm of the inflammatory T cells. In contrast, 43% of the CD8 ϩ T cells that contacted a muscle fiber in PM showed perforin located vectorially towards the target muscle fiber. The results suggest ( a ) that the random distribution of perforin in the cytoplasm of muscle-infiltrating T cells observed in DM reflects nonspecific activation, and ( b ) that the vectorial orientation observed only in PM reflects the specific recognition via the T cell receptor of an antigen on the muscle fiber surface, pointing to a perforin-and secretion-dependent mechanism of muscle fiber injury. ( J. Clin. Invest. 1996. 97:2905-2910.)
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