Fibroblasts modulate expression of Thy-1 on the surface of skeletal myoblasts

Thy-1 antigen is a well-characterized cell-surface glycoprotein known to be variably expressed in many different tissues, including lymphocytes, brain, and muscle. Its function remains unknown. In skeletal muscle, both in vivo and in vitro, the antigen has been reported on immature but not on adult tissue, and its disappearance corresponds roughly to the time of myoblast fusion. Using monoclonal H36 antibody to identify myoblasts unambiguously, we demonstrate here that Thy-1 is expressed only on a small (less than 1%) fraction of rat skeletal muscle myoblasts in heterogeneous primary cultures, but the number of myoblasts that express Thy-1 rises to a steady level of about 70% when fibroblasts are removed from secondary cultures. Restitution of fibroblasts or growth of purified myoblasts in medium conditioned by fibroblasts greatly suppresses this increase in myoblast Thy-1 expression. Thus an interaction between fibroblasts and myoblasts, mediated by a soluble nondialyzable molecule, modulates expression of Thy-1 on the myoblast outer membrane.     

Differential expression of Thy-1 on the various components of connective tissue of rat nerve during postnatal development

The glycoprotein Thy-1 is found on the surface of both neurons, and most fibroblasts, in tissue culture of embryonic or neonatal rat nervous tissue. In adult rat nerves, however, we find the antigen restricted in vivo to neurons and their axons (R. Morris, P. Barber, J. Beech, and G. Raisman, 1983, J. Neurocytol., 12, 1017-1039). We show here that this discrepancy is due to a loss of Thy-1 from neural connective tissue during postnatal development. Moreover, the different elements of connective tissue loose Thy-1 at different times. Epineurial fibroblasts, for instance, express Thy-1 for at least 2 weeks after endoneurial fibroblasts lack detectable antigen. As judged by their loss of Thy-1 antigen, therefore, the different components of neural connective tissue mature at different rates. One important practical implication of this is that Thy-1 cannot be used as a cell surface "marker" for all classes of fibroblasts in vitro. Unusual problems encountered in the immunohistochemical staining of Thy-1 on perineurium, but not on the other elements of the nerve, are also described.     

Extracellular-matrix synthesis by skeletal muscle in culture. Major secreted collagenous proteins of clonal myoblasts.

We have previously shown that G8-1, a murine clonal skeletal-muscle cell line, produces a substrate-attached extracellular matrix [Beach, Burton, Hendricks & Festoff (1982) J. Biol. Chem. 257, 11437-11442]. To examine further the expression of extracellular-matrix proteins by muscle cells, we have analysed the collagenous proteins secreted by G8-1 myoblasts. We have found that collagens and/or procollagens, corresponding to genetic types I, III and IV (and possibly V), are produced and secreted by G8-1 myoblasts. The major secreted collagenous polypeptides were identified as alpha 1 type I and its precursors by using pulse-chase studies, pepsin and collagenase digestions and CNBr fragmentation. The presence of lesser amounts of the other collagens was determined by immunoprecipitation. These results demonstrate that clonal skeletal-muscle cells, in the absence of fibroblasts and an exogenous collagen substrate, are able to synthesize and secrete several extracellular-matrix collagenous proteins in proportions similar to those which are commonly found in muscle tissue and mixed cultures of muscle cells and fibroblasts.     

Endocytosis of alpha 2-macroglobulin is developmentally regulated during myogenesis

A monoclonal antibody, H143, reacts with an intracellular antigen present and accumulated in E63 rat myoblasts. H143 is directed against a species-specific determinant on purified equine serum alpha 2-macroglobulin. Immunofluorescence analyses of differentiating myoblasts grown in horse serum demonstrate that the capacity to take up alpha 2-macroglobulin is stage-specific: the rapid uptake of alpha 2-macroglobulin characteristic of myoblasts ceases prior to their fusion to form multinucleate fibers (myotubes). Neither rat fibroblasts nor a developmentally defective mutant of E63 exhibit this change in alpha 2-macroglobulin uptake. The temperature and calcium requirements for the uptake of H143 antigen, and its accumulation as effected by lysosomotropic amines, indicate that alpha 2-macroglobulin is taken up by myoblasts via a developmentally regulated endocytic process. Electron microscopy using equine alpha 2-macroglobulin labeled with colloidal gold supports this finding.     

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro characteristics of rat mesangial cells in comparison with aortic smooth muscle cells and dermal fibroblasts

Rat glomerular mesangial cells were cultured and their antigens were compared with those of aortic vascular smooth muscle cells and dermal fibroblasts. Glomeruli, aortic, and dermal explants were cultured for 3 weeks and subcultured in the same conditions. These cultured cells were evaluated by indirect immunofluorescence studies using antibodies against Thy-1 antigen, desmin, and chicken gizzard actin. Most of mesangial cells were positive for Thy-1, desmin, and actin. On the other hand, fibroblasts were negative for desmin, and smooth muscle cells stained Thy-1 scarcely, and were negative for desmin. In the latter two cells, actin-positive fibrils were thinner and fainter than mesangial cells. These results indicated that mesangial cells could be distinguished in vitro from vascular smooth cells and fibroblasts by immunofluorescence microscopy.     

Differential expression of the human Thy-1 gene in rodent-human somatic cell hybrids [corrected

Thy-1 is a cell surface differentiation marker which shows distinct patterns of tissue-specific expression in different species. In man, the Thy-1 antigen is encoded by chromosome 11. We have examined the regulatory signals determining human Thy-1 expression through serologic analysis of rodent-human somatic cell hybrids retaining human chromosome 11 in which the fusion partners belong to distinct differentiation lineages. Cell surface expression of human Thy-1 was determined by mixed hemadsorption assays with two monoclonal antibodies (mAb), K117 and L127, shown to detect authentic human Thy-1 through analysis of COS-7 monkey kidney cells transfected with a cloned human Thy-1 gene. Three different patterns of human Thy-1 expression were observed when hybrid cells, constructed with different human and rodent cell types, were tested with mAb K117 and L127. Hybrids formed between Thy-1+ human neuroblastoma cells and Thy-1- mouse neuroblastoma cells, or hybrids between Thy-1+ human fibroblasts and the Thy-1- mouse kidney carcinoma, RAG, retain human Thy-1 expression. In contrast, hybrids formed between either Thy-1+ human neuroblastoma cells or Thy-1+ human fibroblasts and Thy-1- mouse L cells lose expression of human Thy-1 even though chromosome 11 is retained. Finally, hybrids formed between Thy-1- human peripheral lymphocytes or a Thy-1- lymphoblastoid B cell line and Thy-1- Chinese hamster fibroblasts begin to express human Thy-1. These studies suggest that both positive and negative trans-acting signals may play a role in the tissue-specific regulation of the human Thy-1 gene.     

Adhesive multiplicity in the interaction of embryonic fibroblasts and myoblasts with extracellular matrices

Neff et al. (1982, J. Cell Biol., 95:654-666) have described a monoclonal antibody, CSAT, directed against a cell surface antigen that participates in the adhesion of skeletal muscle to extracellular matrices. We used the same antibody to compare and parse the determinants of adhesion and morphology on myogenic and fibrogenic cells. We report here that the antigen is present on skeletal and cardiac muscle and on tendon, skeletal, dermal, and cardiac fibroblasts; however, its contribution to their morphology and adhesion is different. The antibody produces large alterations in the morphology and adhesion of skeletal myoblasts and tendon fibroblasts; in contrast, its effects on the cardiac fibroblasts are not readily detected. The effects of CSAT on the other cell types, i.e., dermal and skeletal fibroblasts, cardiac muscle, 5-bromodeoxyuridine-treated skeletal muscle, lie between these extremes. The effects of CSAT on the skeletal myoblasts depends on the calcium concentration in the growth medium and on the culture age. We interpret these differential responses to CSAT as revealing differences in the adhesion of the various cells to extracellular matrices. This interpretation is supported by parallel studies using quantitative assays of cell-matrix adhesion. The likely origin of these adhesive differences is the progressive display of different kinds of adhesion-related molecules and their organizational complexes on increasingly adhesive cells. The antigen to which CSAT is directed is present on all of the above cells and thus appears to be a lowest common denominator of their adhesion to extracellular matrices.     

Phenotypic variation in clonal Abelson virus lymphoma cells

Two clonal A-MuLV lymphoma cell lines have the capacity to generate phenotypic variants when grown in vivo as ascites tumors. Variant lines differed from parental lymphoma cells in their expression of enzymatic or cell surface differentiation markers. Parental lines expressed the B220 and Lyb-2 glycoproteins characteristic of pre-B cells and bound B220-specific monoclonal antibodies such as 14.8. The parental cells expressed low levels of TdT activity but did not synthesize detectable mu-heavy chain, a cellular phenotype that may correspond to lymphoid progenitor cells. Three classes of phenotypic variants were recovered from the Thy-1- parental lines: 1) 14.8+, Lyt-1+, Thy-1- cells; 2) 14.8 +/-, Lyt-1+, Thy-1+ cells, and 3) 14.8-, Lyt-1+, Thy-1+ cells. Cell cloning experiments indicated that Thy-1+ variant cells can be recovered within 14 days of in vivo inoculation as a minor proportion (1/10(6] of the tumor cell population and subsequently become the predominant tumor cell population. These clonal tumor lines provide a model for the study of cellular and molecular alterations that occur during neoplastic differentiation and progression in the lymphoid system.     

A fine-structural analysis of normal and modulated cells in myogenic cultures.

It has formerly been reported that muscle and nonmuscle progenitor cells in clonal culture could be distinguished from one another on the basis of their distinctive morphologies. In light of recent studies critical of this interpretation, this investigation was concerned with identifying whether one could truly discriminate between myoblasts and fibroblast-like cells. The results reveal that not only is cell morphology a reliable criterion, but myoblasts and fibroblasts can also be distinguished on the basis of their fine-structural characteristics. Cytologically, spindle-shaped myoblasts exhibit an unusually dense concentration of free ribosomes and polysomes, a Golgi of variable dimensions, and a paucity of rough endoplasmic reticulum (RER). Flattened, stellate fibroblasts show an extensive elaboration of RER, multiple Golgi complexes, actomyosin stress fibers, and dense accumulations of intermediate (10 nm) filaments. A third cell type, the mesenchyme-like cell, exhibits the morphology of the fibroblast and has a myoblast-like cytology. The morphological and ultrastructural characteristics of myoblasts are dependent upon culture conditions. In less than optimum culture environments, myoblast morphology, cytology, and differentiation can be reversibly altered or “modulated.” Myoblast modulations induced by both bromodeoxyuridine and by culture conditions which simulate an ischemic environment are analyzed. Myoblast modulation and its implication to muscular dystrophy are discussed.     

Thrombospondin-1-induced focal adhesion disassembly in fibroblasts requires Thy-1 surface expression, lipid raft integrity, and Src activation

The hep I peptide of thrombospondin-1 is known to induce the disassembly of focal adhesions, a critical step in regulating cellular adhesive changes needed for cell motility. Fibroblasts that are heterogeneous with respect to the surface expression of Thy-1 differ markedly in morphology, cytoskeletal organization, and migration, suggesting differential regulation of focal adhesion dynamics. Here we demonstrate that disassembly of focal adhesions mediated by both full-length thrombospondin-1 and the hep I peptide in fibroblasts requires the expression of Thy-1, although it does not appear to function as a stable member of the hep I receptor complex. Consistent with a known function of Thy-1 in regulating lipid raft-associated signaling, intact lipid rafts are necessary for hep I-mediated focal adhesion disassembly. Furthermore, we establish Src family kinase (SFK) activation as a novel component required for hep I-induced signaling leading to focal adhesion disassembly. hep I induces transient phosphorylation of SFKs in Thy-1-expressing fibroblasts only. Therefore, we conclude that Thy-1 surface expression is required for thrombospondin-1-induced focal adhesion disassembly in fibroblasts through an SFK-dependent mechanism. This represents a novel role for Thy-1 in the regulation of fibroblast-matrix interactions critical to tissue homeostasis and remodeling.     

Molecular evidence that SJL reticulum cell sarcomas are derived from pre-B cell. Clonal rearrangement of heavy chain but not of light chain immunoglobulin genes

In order to investigate the clonal origin of SJL reticulum cell sarcoma (RCS), two-color cell membrane-staining and molecular biologic analyses were performed. Flow cytometric analysis revealed that the SJL RCS consists of about 50 to 60% Thy-1-positive and 20 to 30% B220-positive cells and that the majority of the Thy-1-positive cells are L3T4-positive, whereas a few are Lyt-2-positive. In spite of this pleomorphic nature of SJL RCS shown by cell membrane analysis, when the immunoglobulin heavy chain J segment (JH) was used to probe the DNA obtained from the tumor, we observed clonal rearrangements of the gene. Results of the cell-sorting experiment combined with Southern hybridization using the JH gene probe confirmed that those clonally expanding cells are Ia and B220 antigen-positive. Furthermore, all tumors derived from a given mouse showed the same rearrangement pattern. However, no clonal rearrangement was observed when the DNA was probed with the T cell receptor beta-chain gene or with immunoglobulin kappa- or lambda-light chain genes. From these data, we conclude that SJL RCS is a tumor of B cells and that the neoplastic event takes place at an immature B cell stage.     

T cell receptor gamma delta expression of Thy-1+ dendritic epidermal cells--an update

Thy-1+ dendritic epidermal cells (Thy-1+DEC) are present in the murine epidermis. They are morphologically dendritic and express Thy-1, CD3 and asialoGM1, but not CD4 or CD8. T cell receptor (TCR) of Thy-1+DEC is TCR gamma delta. Allison et al and Tonegawa et al recently found that TCR of Thy-1+DEC is V gamma 5 J gamma C gamma -V delta 1D2J2C delta and has no junctional diversity. This TCR gamma delta of Thy-1+DEC is identical to TCR expressed on the earliest fetal thymocytes. It is distinct from that of other epithelial associated lymphocytes or other thymocytes. The ligand of Thy-1+DEC is not known, although TCR gamma delta of adult type could recognize allogenic major histocompatibility complex(MHC) class I or class II and mycobacterium antigen, especially heat shock protein. The TCR of Thy-1+DEC may not be the homing receptor to epidermis. The further studies are needed to elucidate the ligands or functions of Thy-1+DEC.     

Surface molecules and cell interactions

Many of the cell surface molecules of lymphocytes or their precursors are expressed in an unpredictable way on a limited set of other cell types. This often seems to involve expression on lymphoid and brain cells. The Thy-1 antigen is in this category, being a major glycoprotein of murine neuronal cells, fibroblasts and thymocytes. Structural studies show that this molecule is homologous with immunoglobulin domains which are the structural sub-units of all immunoglobulin polypeptides. Thy-1 is the size of one immunoglobulin domain and its sequence is most homologous with variable regions of immunoglobulins.It is suggested that Thy-1 is one of a set of surface molecules concerned with triggering interactions between cells and that this is the primitive function of the immunoglobulin domain. Cell interactions could be mediated by domain-like structures and receptors for them in a way which parallels the triggering of immunological effector reactions by the interaction of receptors with immunoglobulin constant regions. If this is so then the structure seen in the immunoglobulin domain would have evolved along with the evolution of cell organisation. The genes specifying the cell interaction molecules could then have provided the genetic material for the evolution of antibody and histocompatibility antigen at the time of vertebrate emergence.     

The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells.

Stimulation of murine T cells by engagement of the multi-component T cell antigen receptor or by cross-linking the Thy-1 molecule leads to a similar response characterized by lymphocyte activation and lymphokine production. The early biochemical events induced by engaging these molecules also are similar and begin with activation of a tyrosine kinase pathway and tyrosine phosphorylation of a comparable set of substrates. Previous work demonstrates that the protein tyrosine kinase p60fyn is associated with the antigen receptor and therefore it may participate in the tyrosine phosphorylations that are observed with antigen receptor signaling. In this study we demonstrate that the Thy-1 molecule is also associated with p60fyn in a murine T cell hybridoma and in murine thymocytes. The interaction is independent of antigen receptor expression. Thy-1 is a member of the class of molecules anchored to the plasma membrane by a glycophosphatidylinositol (GPI) group. The association of Thy-1 with p60fyn is dependent on the GPI linkage, since cleavage of the GPI anchor disrupts the interaction. The association of Thy-1 and p60fyn suggests a means by which Thy-1 cross-linking leads to tyrosine phosphorylation and T cell activation.     

The activities of MMP‐9 and total gelatinase respond differently to substrate coating and cyclic mechanical stretching in fibroblasts and myoblasts

The current study was designed to investigate whether the activities of TGC (total gelatinase and collagenase) as well as MMP‐9 (matrix metalloproteinase‐9, gelatinase B) secreted by the cultured fibroblasts and myoblasts were influenced by the specific extracellular substrates and by cyclic mechanical strain. Fibroblasts (Rat 2) and myoblasts (C2C12) were cultured with either fibronectin, laminin or collagen type I for 24 h and applied with or without a biaxial deformation at 1 Hz using the Flexcell FX‐4000 system. MMP‐9 activity was increased in fibroblasts when the cells were in contact with fibronectin and laminin, while in myoblasts, enhanced activity of the secreted enzyme was only observed when collagen was present. TGC activity expressed from myoblasts was increased in cells growing on all three types of extracellular proteins in response to the mechanical stimulation, but in fibroblasts, such an increase was only observed in cells grown on the laminin coating. In summary, our data demonstrate that the activities of MMP‐9 synthesized by fibroblasts tend to be regulated by the specific extracellular protein the cells are in contact with, whereas the gelatinolytic actions of proteases produced by myoblasts are more responsive to the mechanical deformation.