Collagen measured in primary cultures of normal rat hepatocytes derives from lipocytes within the monolayer.

The cellular origin of hepatic collagen is under active investigation. Several recent studies using cells in primary culture suggest that hepatocytes are the source of much of the collagen in normal rat liver. In view of other data indicating that lipocytes produce substantial amounts of this protein, we have reexamined collagen biosynthesis in hepatocyte cultures that have been carefully characterized with respect to the presence of lipocytes. We find that routinely prepared hepatocyte isolates contain, by number, approximately 10% lipocytes. Lipocytes in early culture are difficult to visualize by phase-contrast microscopy but after 4 d proliferate and eventually replace the parenchymal cells. The size of the lipocyte subpopulation in these cultures correlates positively with collagen production. Similarly, removal of lipocytes by further processing of the initial hepatocyte isolate significantly reduces collagen production. Moreover, the only cells within hepatocyte cultures that display type I collagen by immunohistochemistry are lipocytes. We conclude that lipocytes are the principal source of collagen in primary hepatocyte cultures. The findings indicate also that these cells are the previously described "fibroblast" that appear in relatively long-term hepatocyte cultures.

Role for cyclic AMP in the postreceptor control of cytokine-stimulated stromal cell growth factor production.

We examined the role of augmented formation of intracellular cyclic AMP (cAMP) in the mediation of stromal cell growth factor production that occurs constitutively or upon cytokine stimulation. Clonal murine marrow adherent cell lines were stimulated under serum-free conditions by interleukin-1 (IL-1) or lipopolysaccharide (LPS) and one (+/+ -1.LDA11) was found to produce low quantities of granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF identity was confirmed by the ability of supernatants from stromal cells to promote proliferation of the factor-dependent cell line FDC-P1, neutralization of this activity by antiserum to GM-CSF, and by Northern blot analysis. However, optimal concentrations of IL-1 and tumor necrosis factor-alpha (TNF-alpha), in combination, led to synergistic (greater than 5-fold higher quantity) GM-CSF production compared with either stimulus alone in the +/+ -1. LDA11 cell line, capable of GM-CSF production after only single stimulation with IL-1 or LPS. In addition, synergistic stimulation by IL-1 and TNF-alpha led to equivalent high amounts of GM-CSF in another cell line incapable of GM-CSF production after induction with only IL-1 or LPS. Any of several means to raise intracellular cAMP levels, including addition of 8-bromo-cyclic AMP (8Br cAMP) (0.25-1mM), pertussis toxin (20-100 ng/ml), or addition of prostaglandin E1 (PGE1) (1 microM), failed to stimulate GM-CSF production alone and strongly inhibited GM-CSF production in stromal cells stimulated by IL-1, LPS, or the synergistic combination of IL-1 and TNF-alpha. In addition, PGE1 and pertussis intoxication were agonists of adenylate cyclase in membranes of marrow adherent cells, whereas IL-1 and LPS were not. The role for regulators of intracellular cAMP was specific because any of the cAMP agonists alone, or in the presence of cytokine stimulators of stromal cells, strongly enhanced IL-6 production, an event known to be cAMP-responsive. Thus, acute formation of intracellular cAMP is a negative regulator of stromal cell GM-CSF production mediated by cytokines, but positively regulates IL-6 production and may be an important determinant of cytokine-directed marrow microenvironmental function. These findings on the requirement for augmentation versus inhibition of cytokine-mediated production of hemopoietic growth factors might be applied to an analysis of marrow stromal cell heterogeneity.

Interactions between fibronectin, glycosaminoglycans and native collagen fibrils: an EM study in artificial three-dimensional extracellular matrices.

The interactions between plasma fibronectin (FN), glycosaminoglycans (GAGs) and native collagen (COL) fibrils were studied in artificial three-dimensional extracellular matrices. FN-COL binding was investigated using rotary shadowed platinum-carbon replicas, EM immunocytochemistry and FN-gold complexes. GAGs were visualized using cationic dyes and anti-GAG antibodies. The specificity of the reactions was evaluated with dot blots. FN showed a low affinity for the surface of native COL fibrils. In contrast, it attached extensively to incompletely/irregularly polymerized COL. Where associated with native fibrils, FN showed preferential affinity for the dark cross-striations, particularly with the a and b bands. Like FN, GAGs bound mostly to the zones of incomplete/irregular COL polymerization. However, they also extensively decorated native fibrils, particularly over the dark cross-striations The d band in the gap zone was not a preferential binding site for either FN or GAGs. The pattern of FN-COL fibril binding was not influenced by GAGs. Dot blots confirmed that, under physiological conditions, FN shows no significant affinity for GAGs other than heparin. However, it does bind to all GAGs in low salt conditions. Results suggest that some of the roles ascribed to FN-COL interactions, particularly structural ones, must be reevaluated.

Reconstruction of thyroid follicles from isolated porcine follicle cells in three-dimensional collagen gel culture.

Thyroid follicles, an essential functional unit of the thyroid, are ball-like structures and exist in the extracellular matrix in vivo. Thus far, the follicles have not been reconstructed in any culture system. The presumed reason for that was that the in vitro environment for the follicle cells in monolayer culture markedly differed from their environment in vivo. We, therefore, considered that isolated follicle cells had to be localized in a three-dimensional environment of extracellular matrix, specifically collagen, to reconstruct thyroid follicles in vitro. At first, follicle cells were completely isolated. These cells were cultured in the three-dimensional collagen gel. An intracytoplasmic cavity first developed in individual cells. A single cell with the cavity then underwent cell division, and the follicle consisting of two cells was reconstructed. This gradually grew to be a large ball-like structure through proliferation of the component cells, and they exhibited morphological polarity specific for thyroid follicle cells. In addition, these cells clearly produced thyroid hormones. To the best of our knowledge, this report is the first instance of reconstruction of thyroid follicles in an in vitro culture system. This culture system is more useful than the monolayer culture system in that this system provides a more physiological environment for investigations of differentiation of follicle cells. Further experiments using this method will probably provide a new clue to the mechanism of thyroid folliculogenesis.

Differentiation of extracellular matrix in the cellular cartilage ("Zellknorpel") of the mouse pinna.

Differentiation of cellular cartilage was studied in the mouse pinna with particular reference to matrix material. Fixation of glycosaminoglycans was performed by the use of acridine orange and elastin was identified by staining thin sections with tannic acid and uranyl acetate. Condensation of mesenchymal cells ("prechondroblasts") initiates the formation of a blastema of cartilaginous tissue at postnatal day 4. The synthesis of acidic glycosaminoglycans begins at postnatal day 8 when prechondroblasts transform to chondroblasts. Glycosaminoglycans can be detected within secretory vesicles of chondroblasts at postnatal day 8, in the extracellular space at postnatal day 13. Delicate collagen fibrils and elastic fiber microfibrils are seen between prechondroblasts and chondroblasts. Deposition of elastin begins at postnatal day 11. A network of elastic fibers and lamellae is formed, which replaces both collagen fibrils and elastic fiber microfibrils. In the interstice of mature cellular cartilage only elastin and proteoglycans are present (postnatal day 21). These findings indicate that cellular cartilage represents an independent kind of supporting tissue, which may serve as a progenitor of hyaline or elastic cartilage ("transitional cellular cartilage") but does not differentiate from hyalin cartilage.

Interleukin-1, stromal cells, granulopoiesis, and the inflammatory response.

A number of in vitro studies carried out in our laboratory over the past ten years have led to some clarification of the role of mononuclear phagocytes in hematopoietic regulation. The results of these studies have demonstrated that mononuclear phagocytes produce proteins, notably interleukin-1 (IL-1), that induce the expression of multilineage hematopoietic growth factors by human vascular endothelial cells, fibroblasts, T-lymphocytes, and thymic epithelial cells. More recently we and others have identified these induced factors as G-CSF, GM-CSF, IL-6, and IL-1. Although IL-1 seems to stimulate expression of these genes by inducing the accumulation of gene transcripts, interestingly the accumulation results from prolongation of mRNA half-life. We propose that the inductive capacity of IL-1 results from its activation of ribonuclease inhibitory activity in the cytoplasm of IL-1 induced cells and hypothesize that this may be a general mechanism by which IL-1 induces gene expression.

Application of microwave irradiation to immunohistochemistry: preservation of antigens of the extracellular matrix.

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodate-lysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.

Cell traction models for generating pattern and form in morphogenesis.

During early development migratory mesenchymal cells navigate to distant sites where they aggregate to form a variety of embryonic organ rudiments. We present here a new model for mesenchymal cell morphogenesis based on the mechanical interaction between motile cells and their extracellular environment. The model is based on two properties of motile cells: (a) they are capable of generating large traction forces which can deform the extracellular matrix through which they move, and (b) the deformations they produce in their environment affect the direction of their movements. We derive field equations which describe the motion of cells in an elastic extracellular matrix and show that these equations can generate a variety of spatial patterns, such as the formations of skin organ primordia, especially feather germs, cartilage condensation patterns which presage bone formation in limb development, and melanocyte density patterns which form animal coat patterns.

[Molecular mechanisms of cellular adhesion]. / Mécanismes moléculaires de l'adhésion cellulaire.

Regeneration of connective tissue structures lost as a result of periodontal disease remains the major goal of periodontal therapy. The ability of cells of the periodontium to adhere to the tooth surface is central to a number of phenomenons. Among these are cellular migration, morphogenesis and wound healing. Several classes of molecules appear to mediate the ability of cells to adhere. These cells utilize a group of receptors called Integrins to anchor themselves to the extra-cellular matrix. The receptors are transmembrane heterodimers which serve as bridges which communicate informations between the extra-cellular matrix and the cytoskeleton.

Lineage- and stage-specific adhesion of human hematopoietic progenitor cells to extracellular matrices from marrow fibroblasts.

Local regulation of hematopoietic differentiation in the marrow requires close interactions with components of the microenvironment. In this study, we explored the capacity of human marrow hematopoietic progenitor cells to adhere in vitro to the extracellular matrix (ECM) secreted by human marrow fibroblasts. When marrow mononuclear cells were incubated on ECM-coated dishes, all types of progenitors adhered to this substrate through an active process requiring divalent cations and serum factors. The proportion of erythropoietic progenitors attached to ECM in two hours was at least twofold higher than that of granulopoietic progenitors. Moreover, in the erythroid lineage, the capacity to adhere to ECM increased with the degree of differentiation of the progenitor: 28% of CFU-E adhered to ECM as compared with 13% of immature BFU-E. Thus, ECM-mediated adherence varied both with the cell lineage and the maturation stage of the progenitor. Purified fibronectin could substitute for ECM in the adhesion assay, and ECM-mediated adhesion of CFU-E and BFU-E was partially inhibited by a polyclonal antifibronectin antibody, which implies that fibronectin may be one ECM component involved in progenitor cell adhesion. Incomplete inhibition of progenitor adhesion to ECM by the antifibronectin antibody, however, as well as the lower proportion of precursors attaching to purified fibronectin as compared with ECM suggest that other matrix molecules may also mediate erythroid progenitors attachment to ECM. These observations support the idea that hematopoietic progenitor cells may regulate their differentiation in part through the modulation of adhesive interactions with a number of constituents of the microenvironment.

Extracellular matrix organized in embryonic cavities during induction of the embryonic axis in chick embryo.

Extracellular matrix (ECM) is detected as short, disorganized fibrils in the forming embryonic extracellular spaces shortly prior to the first morphogenetic cellular movements and interactions in the early chick embryo. As development progresses, the ECM is organized into an intricate network spanning the embryonic cavities. This dynamic entity undergoes relatively rapid changes in its organization pattern during the developmental period from morula to the induction of the neural plate. The ECM seems to preserve the exquisite architecture of the embryo and could guide migrating cells into defined pathways in the early embryo.

Epithelial cytodifferentiation and extracellular matrix formation in enamel-free areas of the occlusal cusp during development of mouse molars: light and electron microscopic studies.

Mandibular first molars in mice ranging in age from 18 days prenatal to 5 days postnatal were used for light and electron microscopic examinations of the enamel-free area (EFA) during development of the occlusal cusp (mesiobuccal cusp). Notable morphological changes in the inner enamel epithelium and the cells of the stratum intermedium were observed. At prenatal age of 18 days, the inner enamel epithelium of the EFA (EFA epithelium) was composed of a layer of columnar cells and covered by the cells of the stratum intermedium. Two days after birth, the EFA epithelium was made up largely of preameloblasts, with mitochondria located in the proximal side of the cells toward the stratum intermedium. The cells of the stratum intermedium were irregularly shaped, with wide intercellular spaces between them. At a postnatal age of 3 days, most of the EFA epithelial cells resembled maturation-stage ameloblasts, being short and columnar in shape and having nuclei located in their proximal side. Distal cell membranes were folded, and mitochondria were scattered throughout the cytoplasm. In 4-day-old mice, the EFA epithelium was found to be formed of short columnar or cuboidal cells with distinct intercellular spaces. The cells of the stratum intermedium could no longer be detected, and cells of the EFA epithelium could not be distinguished from those of the stellate reticulum. Odontoblasts of the EFA were arranged and polarized parallel to the basal lamina, and odontoblastic processes extended toward the cusp tip. The orientation of thin and thick collagen fibers within predentin and dentin was also parallel to the basal lamina. Even after dentin mineralization, disrupted basal lamina and long, aperiodic, fine fibrils were found between the epithelium and the dentin. Following the disappearance of the basal lamina and fine fibrils, stippled material and crystals appeared on the dentin surface. The mineralized matrix, which x-ray microanalytical energy peaks identified as containing calcium and phosphorus, was continuous with enamel in the distal slope of the cusp at the cusp tip. Thus, the inner enamel epithelium of the EFA differentiated into secretory cells capable of enamel-like matrix formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Monocyte adhesion to subendothelial components.

Human monocytes have been shown to penetrate the endothelial layer of large blood vessels and to adhere to the subendothelial basement membrane. To determine the active components of this process, we have studied the ability of monocytes to adhere to isolated components of the subendothelial matrix. Using a quantitative dot-blot adhesion assay, we find that monocytes adhere preferentially to immobilized laminin and elastin. The monocytes adhere less well to fibronectin and bind poorly or not at all to collagen types I and IV, or to heparan sulfate. Monocyte binding to elastin requires an intact, crosslinked molecule as no binding was observed to soluble, acid-alcohol elastin extracts, to pepsin or elastase digests of elastin, to tropoelastin monomer, or to desmosine/isodesmosine crosslinks. Similar binding profiles to elastin, laminin, and fibronectin were seen with the established human leukocyte cell line U937. The promyelocytic cell line HL60 adhered equally well to laminin but showed slightly reduced adhesion to elastin when compared with the fresh monocytes or U937 cells. Freshly isolated human erythrocytes did not demonstrate significant adhesion to fibronectin, laminin, or elastin.

Cell adhesion to extracellular matrix in normal Rana pipiens gastrulae and in arrested hybrid gastrulae Rana pipiens female X Rana esculenta male.

Rana pipiens eggs fertilized by Rana esculenta sperm (ESC) hybrid embryos develop until gastrulation in control Rana pipiens embryos (PIP) and then show morphogenetic arrest. After arrest, ESC do not gastrulate but live for 5 days as blastula-like embryos. We studied the distribution of fibronectin (FN)-containing fibrils and integrin (INT) in PIP and ESC. There are many FN-fibrils in PIP organized in anastomosing networks radiating away from the center of individual cells and across intercellular boundaries. ESC have fewer fibrils compared to PIP. These fibrils are first located between cells in disorganized arrays. After arrest in ESC, when PIP are Stage 14 neurulae, many more FN-fibrils appear. INT-staining occurs in both embryos in similar patterns. In xenoplastic transplantations, we found that the extracellular matrix on the inner surface of the ESC blastocoel roof serves as a substratum for PIP cell migration. In an in vitro assay, we found more cell adhesion to FN-substrata in PIP than in ESC. Cell locomotion rates on FN-substrata were 1.70 +/- 0.85 microns/min for PIP but only 0.46 +/- 0.56 microns/min for ESC. We also found that the inner surface of the blastocoel roof from ESC can not promote cell adhesion and locomotion when Stage 11 fragments are used for conditioning but that Stage 14 fragments can deposit a FN-fibril-rich extracellular matrix which supports PIP mesodermal cell migration at a rate of 1.26 +/- 0.38 microns/min.

The inhibitory effect of neonatal rat smooth muscle cells and elastic extracellular matrix on development of the newly seeded cell population in culture.

Neonatal smooth muscle cells were seeded in standard plastic Falcon flasks, on top of another 2-month-old culture of the same cell population or on top of an acellular matrix prepared by removal of these cells. The effect of both complete and acellular layers on the production of elastin, collagen and total extracellular matrix (EM) proteins as well as on cell division was measured. Compared with the standard population grown on plastic, the complete cell layer almost completely prevented the newly seeded cells from dividing. The acellular matrix did not affect cell doubling but caused a distinct decrease in the production of EM components.

Extracellular matrix (ECM) proteoglycans produced by cultured bovine corneal endothelial cells.

The nature of the proteoglycan(s) (PG) found in the extracellular matrix (ECM) layer produced by cultured bovine corneal endothelial (BCE) cells is analyzed. The PG(s) account for approximately 5 to 6% of the dry weight of the ECM, regardless of the amount of extracellular soluble PG available in the medium. A 4 M guanidinium chloride (GuCl) extract of ECM was separated on a dissociative cesium chloride (CsCl) gradient (1.25 g/cm3 starting density). Results showed one main peak of PG substance(s) comprising 91% of the total labelled substance and uronic acid, banding at a specific buoyant density of 1.29 g/cm3. The molecular weight of this major PG(s) as estimated by gel filtration on Sepharose CL-4B ranged from 0.5 to 0.7 X 10(6). Further chemical analysis of the main PG(s) band revealed a protein moiety accounting for 45% of the weight and carbohydrates-glycosaminoglycans (GAG) accounting for the remaining 55%. Analysis of the GAG chains (over the entire gradient) showed a composition, based on the susceptibility of the PG substance(s) to degrading enzymes, of 50% heparan sulfate, 43.5% dermatan sulfate, and 6.5% chondroitin 4- and 6-sulfate chains. BCE cell cultures grown in the presence of beta-D-xyloside produced an ECM lacking more than 90% of the GAG content found in the control ECM. The medium-soluble GAG chains, produced in vast excess in cultures grown in the presence of beta-D-xyloside, are composed mainly of chondroitin 4- and 6-sulfates.

Development of avascularity during cartilage differentiation in the embryonic limb. An exclusion model.

The differentiation of cartilage and muscle in limb-bud mesenchyme has been interpreted by some investigators in terms of a vascular pre-pattern model. It has been argued that a pre-pattern of the early limb vasculature compartmentalises the mesenchyme into specific microenvironmental areas in which, depending on the oxygen tension and nutrient supply, cartilage or muscle will differentiate. However, recent analyses of the development and differentiation of blood vessels in limbs have shown that regional variations in vascularization develop co-incidentally with the earliest indication of cartilage formation or mesenchymal condensation. The simple model described in the present study suggests that the mechanical compression/tension forces generated by the condensing mesenchyme are sufficient to constrict and eventually close off the thin-walled undifferentiated vessels caught in the condensation foci, thus leading to the avascularity of cartilage rudiments. This view suggests that the vasculature has no major function in governing the pattern of cartilage differentiation.

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.

Cellular stages in cartilage formation as revealed by morphometry, radioautography and type II collagen immunostaining of the mandibular condyle from weanling rats.

The role played by cell addition, cell enlargement, and matrix deposition in the endochondral growth of the condyle was assessed in weanling rats by four approaches making use of the light microscope: morphometry, 3H-thymidine radioautography, 3H-proline radioautography, and immunostaining for the cartilage-specific type II collagen. From the articular surface down, the condyle may be divided into five layers made up of cells embedded in a matrix: 1) the articular layer composed of static cells in a matrix rich in fibers presumed to be of type I collagen, 2) the polymorphic cell layer including the progenitor cells from which arise the cells undergoing endochondral changes, 3) the flattened cell layer in which cells produce a precartilagenous matrix devoid of type II collagen while undergoing differentiation in two stages: a "chondroblast" stage and a short "flattened chondrocyte" stage when intracellular type II collagen elaboration begins, 4) the upper hypertrophic cell layer, in which cells are "typical chondrocytes" that enlarge at a rapid rate, actively produce type II collagen, and deposit it into a cartilagenous matrix, and 5) the lower hypertrophic cell layer, composed of chondrocytes at a stage of terminal enlargement while the cartilagenous matrix is adapting for mineralization. 3H-thymidine radioautographic results indicate that the turnover time of progenitor cells in the polymorphic cell layer is about 2.9 days. The time spent by cells at each stage of development is estimated to be 1.4 days as chondroblasts, 0.5 days as flattened chondrocytes, 2.3 days as the chondrocytes of the upper hypertrophic cell layer, and 1.1 days as those of the lower hypertrophic cell layer. Calculations referring to a 1 x 1-mm square-sided column extending from the articular surface to the zone of vascular invasion provide the daily rate of cell addition (0.0077 mm3), extracellular matrix deposition (0.0127 mm3), and cell enlargement (0.0302 mm3). Hence the respective contribution of the three factors to condyle growth is in a ratio of about 1:1.6:4. This result emphasizes the role played by cell enlargement in the overall growth of the condyle.