Arthur Z. Eisen

Neutral metalloproteinases produced by human mononuclear phagocytes. Enzyme profile, regulation, and expression during cellular development.

Mononuclear phagocytes are developmentally and functionally complex cells that play critical roles in extracellular matrix remodeling. We hypothesized that differentiated mononuclear phagocytes, typified by alveolar macrophages, use a spectrum of metalloproteinases to degrade various matrix macromolecules. To test this hypothesis, we have evaluated synthesis and secretion of four metalloproteinases (interstitial collagenase, stromelysin, 72-kD type IV collagenase, and 92-kD type IV collagenase) by human mononuclear phagocytes with regard to (a) the effect of cellular differentiation, (b) regulation of secretion, and (c) comparisons/contrasts with a prototype metalloproteinase-secretory cell, the human fibroblast. We found that regulated secretion of greater quantities and a wider spectrum of metalloenzymes correlated with a more differentiated cellular phenotype. As extreme examples, the 92-kD type IV collagenase was released by peripheral blood monocytes and uninduced U937 monocyte-like cells, whereas stromelysin was secreted only by lipopolysaccharide-stimulated alveolar macrophages. Macrophage production of interstitial collagenase, stromelysin, and 72-kD type IV collagenase was approximately 20%, 10%, and 1-2%, respectively, of that from equal numbers of fibroblasts; secretion of the 92-kD type IV collagenase was not shared by fibroblasts. This work confirms the potential of macrophages to directly degrade extracellular matrix via secreted metalloproteinases in a manner that differs both qualitatively and quantitatively from that of fibroblasts. Moreover, varying regulation of metalloenzyme synthesis, evidenced by distinct patterns of basal and stimulated secretion during differentiation, can be studied at a molecular level in this model system.

Integrin α2β1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells

The capacity of cells to organize and contract collagen fibrils is fundamental to processes as diverse as embryogenesis and wound healing. We analyzed different beta 1 integrins on diploid fibroblasts for their role in modifying the tertiary structure of collagen matrices. Using monoclonal antibodies that block the interaction of integrins with their ligands, evidence was obtained that alpha 2 beta 1 integrin is required for the contraction of a type I collagen matrix. Further supporting the role of alpha 2 beta 1, cell lines expressing minimal levels of this integrin uniformly failed to contract collagen matrices. In addition, transfection of a full-length alpha 2 cDNA into one such cell line led to enhanced cell surface expression of alpha 2 beta 1 and conferred the de novo capacity to contract collagen matrices.

Scleroderma: INCREASED BIOSYNTHESIS OF TRIPLE-HELICAL TYPE I AND TYPE III PROCOLLAGENS ASSOCIATED WITH UNALTERED EXPRESSION OF COLLAGENASE BY SKIN FIBROBLASTS IN CULTURE

To assess potential abnormalities in collagen metabolism in systemic scleroderma, skin fibroblast lines from patients with this disease were established and compared to control cell lines derived from healthy subjects. For studies on the biosynthesis of procollagen, the cells were incubated with [(14)C]proline in a medium supplemented with ascorbic acid and beta-aminopropionitrile, and the synthesis of nondialyzable [(14)C]hydroxyproline, in relation to DNA or cell protein, was taken as an index of procollagen formation. Five of eight scleroderma fibroblast cell lines demonstrated procollagen biosynthesis rates significantly higher than the controls, and the mean rate of procollagen synthesis by scleroderma fibroblasts was about twice that of the control cells. Control experiments demonstrated that the specific activity of the intracellular free proline was not different in scleroderma and control fibroblasts, and the mean population doubling times of the scleroderma and the control fibroblast cell lines were the same. The relative synthesis of the genetically distinct procollagens was examined by isolating type I and type III procollagens from the cell culture medium using DEAE-cellulose chromatography. The ratios of type I/III procollagens in scleroderma cell lines did not differ from the controls. The helical stability of the collagenous portion of type I and type III procollagens, estimated by the resistance of (14)C-collagen to limited proteolytic digestion with pepsin under nondenaturing conditions, was the same in both scleroderma and control cultures. The capacity of the cells to synthesize enzymatically active and immunologically reacting collagenase was also studied; no marked differences in these parameters could be observed. The results suggest that cultured skin fibroblasts from patients with scleroderma demonstrate a metabolic abnormality expressed as increased synthesis of type I and type III procollagens in a normal ratio. This abnormality may play a role in the excessive accumulation of collagen in the skin and other organs affected in scleroderma.

Collagenase production by human skin fibroblasts.

Abstract Normal human skin fibroblasts, when cultured in serum free medium, produce collagenase in an inactive form. The enzyme in the crude medium can be activated by a brief preincubation with trypsin or by autoactivation. Once activated, the fibroblast collagenase is identical in its mechanism of action to human skin collagenase obtained from organ cultures. In addition, an inhibitor of collagenase is also present in the medium of fibroblast cultures. The inhibitor appears to be produced by the cells and its molecular weight is slightly higher than that of the enzyme. The presence of this inhibitor may account for previous inability to detect collagenase in human skin fibroblast cultures. It is also possible that some of the inactive enzyme exists in the medium in the form of a proenzyme.

Connective tissue nevi of the skin: Clinical, genetic, and histopathologic classification of hamartomas of the collagen, elastin, and proteoglycan type

Connective tissue nevi of the skin are hamartomatous lesions consisting predominantly of one of the components of the extracellular matrix, namely, collagen, elastin, or glycosaminoglycans. On the basis of clinical, histopathologic, and genetic considerations, the connective tissue nevi can be classified into defined categories. Association with extracutaneous features allows further delineation of these disease entities and aids in establishing an accurate diagnosis.

Human Skin Fibroblast Collagenase: Interaction with Substrate and Inhibitor

Human skin fibroblasts secrete collagen, procollagenase and a collagenase inhibitor. This study addresses the nature of the interaction between these important fibroblast products. The binding of procollagenase and of active collagenase to native collagen in solution was examined by employing Sephadex G-150 gel-filtration chromatography to separate bound versus unbound enzyme. Active enzyme bound readily to collagen; the equilibrium constant of binding, Kd, was calculated to be 5.1 to 10(-7)M. Thus, collagenase binds with nearly equal affinity to both monomeric collagen and aggregated fibrils (Kd = 9 X 10(-7)M; [Welgus et al., 1980]). Furthermore, since Kd congruent to Km congruent to 10(-6)M, the ratio k2/k1 must be extremely small, directly implicating the catalytic step represented by the rate constant k2, and not the binding of enzyme to substrate, as the rate-limiting step of collagenase action. In contrast, procollagenase demonstrated no capacity to bind to collagen. The interaction of procollagenase and of active collagenase with inhibitor was examined utilizing both conventional and high-precision liquid gel-filtration chromatography. A higher molecular weight complex could be demonstrated consisting of active collagenase and inhibitor; no such interaction occurred between procollagenase and the inhibitory protein. Analysis of Lineweaver-Burk plots showed that inhibition was accompanied by a corresponding change in Vmax; Km remained unchanged. Such results are indicative of a noncompetitive mechanism of inhibition and are consistent with the formation of an enzyme-inhibitor complex. The Ki of enzyme-inhibitor binding was determined to be less than 10(-9)M. The data indicate that procollagenase can neither interact with its specific inhibitor nor bind to collagen. Extracellular activation of the collagenase zymogen is thus a critical event, which can be followed either by binding to substrate or interaction with inhibitor.

Structure and organization of mammalian stratum corneum.

The structure of horny cells and their spatial relationship in the stratum corneum of several mammals, including man, have been studied by the combined use of Nomarski differential interference microscopy, transmission electron microscopy, and scanning electron microscopy. In the body skin of these mammals the horny cells and cells in the stratum granulosum are organized in neatly stacked vertical columns. The cells in each stack interdigitate at their edges with the cells in adjacent stacks by means of a steplike shallow depression which encircles the superficial aspect of each cell, thus maintaining contiguity and uniform thicknesss for the stratum corneum as a whole. The columned stack arrangement is quite likely responsible for the hexagonal shape of the individual cell. In the palmar and plantar skin of the mammals studied, the horny cells of the stratum corneum apparently lack a specific organization as well as the hexagonal shape. The horny cells from all areas are characterized by abundant villous-like processes which are consistent in size, shape, and distribution with attachment sites of desmosomes.

Morphea and lichen sclerosus et atrophicus: Clinical and histopathologic studies in patients with combined features

Ten patients with skin lesions clinically consistent with morphea and lichen sclerosus et atrophicus were studied. In all cases, histopathologic changes of both morphea and lichen sclerosus et atrophicus were also present. The coexistence of morphea and lichen sclerosus et atrophicus in the same patient suggests that these lesions represent a spectrum which may reflect similar etiologic events or closely related pathologic processes in these two diseases.

Matrix metalloproteinase (MMP)-9 type IV collagenase/gelatinase implicated in the pathogenesis of Sjogren's syndrome

Type IV collagenases/gelatinases (matrix metalloproteinases MMP-2 and MMP-9) in labial salivary glands (LSG) and saliva in Sjögrens syndrome (SS) and healthy controls were studied. Zymograms and Western blots disclosed that SS saliva contained 92/82 kD MMP-9/type IV collagenase duplex. Specific activity measurement disclosed 53.1+/-9.8 U/mg protein MMP-9 in SS compared to 16.5+/-2.6 U/mg in healthy controls (p=0.01). MMP-2 did not differ between SS and controls. In SS salivary glands, MMP-2 and MMP-9 were also expressed, in addition to stromal fibroblasts and occasional infiltrating neutrophils, respectively, in acinar end piece cells. In addition, an effective proMMP-9 activator, human trypsin-2 (also known as tumor-associated trypsin-2 or TAT-2), was found in acinar end piece cells and in saliva. Interestingly, proteolytically processed MMP-9 was found in saliva (vide supra), and in vivo activated MMP-9 was significantly higher in SS than in controls (p=0.002). LSGs, particularly in SS, were characterized ultrastructurally by areas containing small cytoplasmic vesicles in the basal parts of the epithelial cells associated with areas of disordered and thickened basal lamina. Based on our results, we conclude here that SS saliva contains increased concentrations of MMP-9, which is of glandular origin in part. Pro MMP-9 is to a large extent proteolytically activated. This is probably mediated by the most potent pro MMP-9 activator found in vivo thus far, namely trypsin-2. Therefore, the MMP 9/trypsin-2 cascade may be responsible for the increased remodelling and/or structural destruction of the basement membrane scaffolding in salivary glands in SS. Due to the role of basal lamina as an important molecular sieve and extracellular matrix-cell signal, these pathological changes may contribute to the pathogenesis of the syndrome.

Regulation of human skin collagenase activity by hydrocortisone and dexamethasone in organ culture.

Abstract Hydrocortisone and dexamethasone (9α-fluoro, 16α-methyl prednisolone) prevent the appearance of collagenase in cultures of normal human skin, human rheumatoid synovium and rat uterus. Hydrocortisone is maximally inhibiting at 10−7M and dexamethasone at 10−8M in culture medium. Neither steroid is an inhibitor of enzyme activity. The loss of collagenase activity in cultured tissue is not accompanied by detectable inhibition of protein synthesis. Reduction of enzyme activity in culture medium is concomitant with a parallel cessation of tissue collagen degradation, indicating that the tissue fails to produce active collagenase in the presence of physiologic levels of glucocorticoids.