Gene for an extracellular matrix receptor protein from Pneumocystis carinii.

An initial and crucial step in the establishment of many microbial infections is the attachment of the pathogen to the host cells. Thus, adherence of Pneumocystis carinii (Pc) to type I pneumocytes is believed to be important in the induction of Pc pneumonia. Little is known about the nature of the attachment of Pc to type I cells, although extracellular matrix (ECM) proteins, such as fibronectin and laminin, have been implicated in the process. We report here the isolation of a Pc gene encoding a receptor protein that binds both fibronectin and laminin in vitro. A cDNA clone encoding the Pc ECM receptor was isolated from a Pc cDNA library and identified on the basis of sequence homology to the human colon carcinoma laminin receptor. Southern blot analysis of Pc genomic DNA confirmed that the cDNA was of Pc origin. Northern blot analysis of Pc total RNA showed a predominant mRNA of approximately 1400 nucleotides that hybridized to the ECM receptor gene. The ECM receptor predicted from the cDNA sequence is 295 amino acid residues long, with a molecular mass of 32.8 kDa. The C-terminal third of the polypeptide is highly negatively charged, whereas the N-terminal two-thirds contains hydrophobic segments that may play a role in membrane association. Sequence analysis and alignment of the N terminus with the laminin receptor cDNA sequence of human colon carcinoma support the conclusion that the Pc ECM receptor cDNA clone is a full-length clone. A Western blot of the overexpressed ECM receptor protein bound both laminin and fibronectin in vitro. Antibodies raised to the overexpressed receptor protein interacted with a 33-kDa protein in total Pc cell lysates. These findings raise the possibility that the Pc ECM receptor protein may mediate the organism's attachment to type I pneumocytes and, thus, may play a crucial role in Pc pathogenesis.     

The extracellular release of granulocyte collagenase and elastase during phagocytosis and inflammatory processes.

Human granulocytes release 25-30% of the granular neutral proteases, collagenase and elastase, to the exterior of the cell during phagocytosis of yeast cells or immune complexes. Similar amounts of myeloperoxidase and lactoferrin are released. Crossed immunoelectrophoresis demonstrated that collagenase and elastase released extracellularly formed complexes with serum alpha1-antitrypsin. The presence of alpha1-antitrypsin complexes with granulocyte collagenase and elastase were also demonstrated in inflammatory processes, e.g. in the peritoneal exudate of acute peritonitis. The reactivity of neutrophil proteases with natural plasma protease inhibitors must be considered in assessing the role of these proteases as the etiologic agent of tissue damage and degradation during the inflammatory process.     

Identification of CD9 extracellular domains important in regulation of CHO cell adhesion to fibronectin and fibronectin pericellular matrix assembly

CD9, a 24-kDa member of the tetraspanin family, influences cellular growth and development, activation, adhesion, and motility. Our investigation focuses on the hypothesis that the CD9 second extracellular loop (EC2) is important in modulating cell adhesive events. Using a Chinese hamster ovary (CHO) cell expression system, we previously reported that CD9 expression inhibited cell adhesion to fibronectin and fibronectin matrix assembly. For the first time, a functional epitope on CD9 EC2 that regulates these processes is described. Binding of mAb7, an EC2-specific anti-CD9 monoclonal antibody, reversed the CD9 inhibitory activity on CHO cell adhesion and fibronectin matrix assembly. This reversal of cell phenotype also was observed in CHO cells expressing CD9 EC2 truncations. Furthermore, our data showed that the EC2 sequence (173)LETFTVKSCPDAIKEVFDNK(192) was largely responsible for the CD9-mediated CHO cell phenotype. Two peptides, (135)K-V(172) (peptide 5b) and (168)P-I(185) (peptide 6a), selectively blocked mAb7 binding to soluble CD9 and to CD9 on intact cells. These active peptides reversed the influence of CD9 expression on CHO cell adhesion to fibronectin. In addition, confocal microscopy revealed that CD9 colocalized with the integrin alpha(5)beta(1) and cytoskeletal F-actin in punctate clusters on the cell surface, particularly at the cell margins. Immunoprecipitation studies confirmed CD9 association with beta(1) integrin. The cellular distribution and colocalization of focal adhesion kinase and alpha-actinin with cytoskeletal actin was also influenced by CD9 expression. Thus, CD9 may exhibit its effect by modulating the composition of adhesive complexes important in facilitating cell adhesion and matrix assembly.     

Anterior pituitary hormone release in vitro inversely related to extracellular osmolarity.

Release of hormones from bovine anterior pituitary tissue in vitro was inveresly related to the osmolarity of the incubation medium. Addition of each of several ionic or non-ionic solutes to Krebs-Ringer-bicarbonate (KRB) or to beef serum inhibited hormone release. If the value of 286 mOsm/liter of KRB is considered as 100%, the osmolarity of the medium was altered from -15% to +10% of control. Over this range, an increase in osmolarity of 10% resulted in the following percentage inhibition: ACTH, 47; PRL, 43; TSH, 36; LH, 23; GH, 18; maximal percentage inhibition over this range was as follows: ACTH, 80; PRL, 60; TSH, 60; LH, 45; GH, 40. The inverse relationship between extracellular oxmolarity and secretion of ACTH and PRL would appear to be appropriate in view of the salt and water retaining actions of these hormones. The sensitivity of the response to osmotic changes suggests a possible role of body osmolarity in the regulation of adenohypophysial secretion.     

T-lymphocyte differentiation and the extracellular matrix: identification of a thymocyte subset that attaches specifically to fibronectin.

A population of murine thymocytes adheres specifically to fibronectin but not to vitronectin, laminin, or collagen type I. The interaction of these thymocytes with fibronectin could be inhibited by the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which comprises the previously identified cell-attachment determinant of the molecule, suggesting that the cell attachment site on fibronectin is recognized by these cells. A similar peptide, in which the aspartate residue had been replaced with glutamate, had no effect on this adhesion. The fibronectin-adherent thymocytes were found to be cortisone-sensitive; to bind peanut agglutinin; to have a Thy-1.2+, Ia- surface phenotype; and to express H-2 antigen only weakly on their surface. In addition, approximately 80% of the fibronectin-adherent cells expressed L3T4 and 80% expressed Ly-1 on their surface, whereas greater than 95% were positive for Ly-2. The data suggest that these cells, which constitute 10% of all thymic lymphocytes, are cortical thymocytes. We propose that their adhesion to fibronectin may be important for their differentiation. The binding to fibronectin provides a means to selectively isolate these cells for study.     

Hypoxic injury to human alveolar macrophages accelerates release of previously bound neutrophil elastase. Implications for lung connective tissue injury including pulmonary emphysema

Human neutrophil elastase and other neutrophil granule constituents are internalized by human alveolar macrophages in vitro via receptor-mediated endocytosis, and immunoreactive neutrophil elastase is detectable within alveolar macrophages freshly harvested from human smokers. To gain insight into the potential role of neutrophil elastase bound by alveolar macrophages in the pathogenesis of connective tissue proteolysis, we have chosen hypoxia as a model of macrophage injury and have studied its effect upon the fate of bound neutrophil elastase. We found (1) that in a 3-h incubation after brief exposure to neutrophil elastase, control alveolar macrophages partially degraded bound enzyme, but they also released intact, enzymatically active, elastase in small amounts; (2) that release of TCA-insoluble radiolabeled elastase and elastase activity was enhanced fivefold and twofold over control, respectively, by alveolar macrophage injury during a 3-h incubation in humidified nitrogen; (3) that enzymatic activity of bound neutrophil elastase was largely masked by human neutrophil elastase-inhibitory activity of macrophage cell extracts. The data suggest (1) that the fate of neutrophil elastase bound to alveolar macrophages may be modulated by the local tissue environment; (2) that noxious agents may cause proteolytic tissue injury in the vicinity of alveolar macrophages by enhancing release of bound neutrophil elastase; (3) that alveolar macrophages may participate in the pathogenesis of centrilobular pulmonary emphysema by serving as a vector for neutrophil elastase, even if elastase activity is not detectable in alveolar macrophage lysates.     

Permissive effect of the extracellular matrix on cell proliferation in vitro.

Corneal endothelial cells maintained in tissue culture retain the ability to synthesize and secrete an extracellular matrix (ECM) along their basal cell surface. Treatment of confluent cultures with 0.5% Triton X-100 results in the removal of the cell monolayer, thereby exposing the ECM, which adheres strongly to the tissue culture dish. Dishes coated with ECM were used to study the permissive effect of such a substrate on cell proliferation. The proliferation of bovine granulosa and adrenal cortex cells maintained on plastic tissue culture dishes was compared to that on dishes coated with ECM. Neither cell type, even when exposed to optimal serum concentration, replicated when seeded at low cell density on plastic. In contrast, when seeded on ECM they proliferated actively. None of the cultures maintained on ECM required fibroblast growth factor in order to reach confluence, although when maintained on plastic they were totally dependent on fibroblast growth factor for proliferation. Because cells maintained on plastic do not respond to factors present in serum or plasma, although they do so respond when maintained on ECM, it is likely that the close contact of the cells with the ECM restores their sensitivity to agents present in serum and plasma.     

Proteoglycans contribution to association of Lp(a) and LDL with smooth muscle cell extracellular matrix.

Lp(a) interference with fibrinolysis could contribute to atherothrombosis. Additionally, accumulation of Lp(a) and LDLs, could lead to cholesterol deposition and foam cell formation in atherogenesis. The interactions between Lp(a) and LDL could cause their entrapment in the extracellular matrix of lesions. We found that association of Lp(a) with matrix secreted by cultured human arterial smooth muscle cells increased 2 to 3 times the subsequent specific binding of radioactive LDL. Chondroitin sulfate proteoglycans seem responsible for formation of the specific matrix-Lp(a) and matrix-LDL aggregates. The proteoglycans appeared also to participate in a cooperative increase of radioactive LDL binding to matrix pretreated with Lp(a). In the matrix preincubated with LDL, approximately 50% of the additional lipoprotein was bound by ionic interactions. In the matrix preincubated with Lp(a), 20% of the additional LDL was held by ionic bonds, and the rest was held by strong nonionic associations. Binding analysis in physiological solutions confirmed that chondroitin sulfate-rich proteoglycans from the smooth muscle cell matrix have a high affinity for Lp(a) and LDL. The results provide an explanation to the observed localization of Lp(a) and LDL in the extracellular matrix of arterial lesions and suggest a mechanism for their cooperative accumulation there.     

Degradation of extracellular matrix proteins (fibronectin, vitronectin and laminin) by serine-proteinases isolated from Lonomia achelous caterpillar hemolymph.

Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are > or = 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.     

Participance of fibronectin and various collagen types in the formation of fibrous extracellular matrix in cardiosclerosis

Investigation of the extracellular matrix composition of the left heart ventricle was carried out on autopsy material of subjects, aged from 60 to 70 years, in a number of cases, including: (1) tissue without cardiosclerosis; (2) granulation tissue formed 2 weeks after infarction; (3) post-infarctial fibrous scars; (4) diffuse cardiosclerosis in consequence of stenotic coronary atherosclerosis. Cryostat sections treated with highly specific antibodies to fibronectin and types I, III, IV and V collagens were examined by the indirect immunofluorescence technique. Fibronectin and the mentioned collagenous proteins were detected in the extracellular matrix of granulation tissue. In contrast, fibronectin and collagen type IV were not revealed in post-infarctial fibrous scars. Collagen types III and V were diffusely distributed in fibrous tissue, whereas collagen type I was demonstrated to accumulate preferentially in the deeper regions of post-infarctial scars. Fibronectin and collagen types I, III, V, but never type IV, were also found in the connective tissue in diffuse cardiosclerosis. The significance of type V collagen in the extracellular matrix is discussed.     

An in vitro model for the study of acute release of von Willebrand factor from human endothelial cells

An in vitro model is described which utilizes human umbilical vein endothelial cells cultured on plastic microcarrier spheres and perfused with serum-free medium. This model was used to study the acute release of von Willebrand factor following stimulation of the cells with putative agonists. Thrombin, plasmin and interleukin-1 were found to release von Willebrand factor. Adrenaline and bradykinin also stimulated release but only at high dosage. 1-desamino-8-D-arginine vasopressin (DDAVP) was inactive.     

Calcium pyrophosphate crystal deposition. An in vitro study using a gelatin matrix model

Deposition of crystalline triclinic (t) and monoclinic (m) calcium pyrophosphate dihydrate (CPPD) in fibrocartilage and articular cartilage is the hallmark of chondrocalcinosis. Using biologic grade gelatin to model this crystal growth process, t-CPPD, m-CPPD, amorphous calcium pyrophosphate, orthorhombic calcium pyrophosphate tetrahydrate (o-CPPT), and 3 mixed calcium/sodium pyrophosphate salts were grown at physiologic pH. Amorphous and o-CPPT appeared to be kinetic precursor crystals in the formation of t-CPPD and m-CPPD. Optimal concentration ranges for the different crystals were determined.     

In vitro transformation of mesenchymal cells derived from embryonic muscle into cartilage in response to extracellular matrix components of bone.

Subcutaneous implantation of demineralized diaphyseal bone matrix into rats induces cartilage and bone formation in vivo. When minced skeletal muscle is cultured on hemicylinders of demineralized bone in vitro, mesenchymal cells are transformed into chondrocytes. In the present investigation, the potential of extracellular matrix components of bone to trigger cartilage differentiation in vitro was examined. Extraction of bone hemicylinders with 6 M guanidine X HCl resulted in the absence of chondrogenesis in vitro and endochondral bone formation in vivo. Biologically inactive hemicylinders of bone were then reconstituted with the guanidine extract and also with partially purified components extracted from bone matrix and bioassayed. Reconstitution completely restored the ability to elicit chondrogenesis in vitro and endochondral bone differentiation in vivo. Reconstitution of the whole guanidine extract on Millipore filters coated with gels of tendon collagen (type I) and subsequent culture with minced skeletal muscle also resulted in cartilage induction in vitro. These observations show that the extracellular matrix of bone is a repository of factors that govern local cartilage and bone differentiation.     

Retinoids and phorbol esters alter release of fibronectin from enucleated cells.

Addition of 12-tetradecanoylphorbol 13-acetate (TPA) to cultures of intact Swiss mouse 3T3 fibroblasts induced a dose-dependent increase in ornithine decarboxylase (OrnDCase) activity. Over the same concentration range, 10(-9) to 10(-6) M, TPA induced the release of radioactively labeled fibronectin (FN) from the cells into the culture medium. Retinoic acid, a derivative of vitamin A, inhibited in a dose-dependent manner both the increase in OrnDCase activity and the release of FN induced by TPA. To examine the effects of TPA and retinoic acid in enucleated cells, the cells were treated with 7.5 micrograms of cytochalasin B per ml of medium during centrifugation at 10,000 X g for 35 min at 37 degrees C. In a series of five experiments, the treated cells were 94.7 +/- 4.8% (+/- SEM) enucleated as measured by [3H]thymidine incorporation and verified by Giesma staining for nuclei. In the enucleated cells, TPA did not induce increased OrnDCase activity but did induce FN release in a dose-dependent fashion over the same concentration range effective for FN release from intact cells. Moreover, addition of retinoic acid to the enucleated cells inhibited the phorbol ester-induced release of FN in a dose-dependent manner. A series of phorbol ester derivatives showed the same order of activity in causing FN release from the enucleated cells as reported for inducing OrnDcase activity in intact cells or in promoting mouse skin tumors in vivo. Similarly, several retinoids were tested for their ability to inhibit the phorbol ester-induced release of FN from enucleated cells. The efficacy of the retinoids in preventing FN release paralleled their activity in inhibiting phorbol ester-induced OrnDCase activity and skin tumor promotion, as reported in the literature. We conclude that at least one aspect of tumor promotion induced by phorbol esters--the loss of FN--does not require the cell nucleus, and further, that retinoids can inhibit this aspect of tumor promotion without nuclear involvement.     

Isolation of human small intestinal brush border membranes using polyethylene glycol and effect of exposure to various oxidants in vitro

This study presents a method of brush border membrane (BBM) preparation from the human small intestine using polyethylene glycol (PEG) precipitation and also looks at the effect of in vitro oxidant exposure on structural and functional alterations in the membrane. Isolated BBM were relatively pure as judged by 10- to 14-fold enrichment of marker enzymes with less than 1% contamination by other subcellular organelles. These membranes showed uphill transport of glucose and lipid analysis showed a cholesterol–phospholipid (C/P) ratio of 1.19. Isolated BBM were found to be susceptible to superoxide generated by xanthine oxidase (XO), resulting in lipid and protein oxidation along with altered glucose uptake. Superoxide exposure also resulted in phospholipid alterations, especially generation of lyso phospholipids. These changes were prevented by inhibiting XO by allopurinol or scavenging superoxide by superoxide dismutase (SOD). Other oxidants studied did not have significant affect on these membranes. These studies suggest that PEG can be used for preparation of BBM from the human small intestine and these membranes undergo structural and functional alterations on exposure to superoxide.