Only 10-15% of unseparated thymocytes adhered to culture plates precoated with fibronectin (FN), but 60-70% of the CD4-8-(double-negative) thymocyte population did. This population bound to FN but not to collagen, laminin, or vitronectin. Its binding to FN was inhibited by anti-FN antibody or a mixture of synthetic peptides corresponding to two different sites of EN, termed the V10 sequence and the RGDS (Arg-Gly-Asp-Ser) sequence, which interact, respectively, with the VLA-4 and VLA-5 FN receptors expressed on T-lineage cells. CD4-8-thymocytes also adhered to a monolayer of a thymic stromal cell clone, MRL104.8a, that induces growth-maintenance and differentiation of such thymocytes. . Most important, blocking the adhesion of CD4-8-thymocytes to the thymic stromal cell monolayer resulted in potent inhibition of the differentiation of these thymocytes, which was otherwise induced toward the expression of CD4 and/or CD8 molecules. These results indicate that immature CD4-8-thymocytes adhere to thymic stromal cells preferentially through FN-FN receptor interaction and that such adhesion has a critical role in inducing and/or supporting the differentiation of these thymocytes.Lymphocytes express a number of surface molecules that mediate the adhesion of cells to one another or to extracellular matrix (ECM) components (1-4). For example, cell-cell interaction molecules such as CD2 and LFA-3 are used to augment specific adhesion of T cells to antigen-presenting cells (5), and other cell-cell interaction molecules function as homing receptors (6, 7). Thus, these cell surface molecules have been recognized as regulating the migration of lymphocytes as well as the interactions of activated lymphocytes during immune responses.Another type of cell adhesion mechanism involves the interaction of cells with ECM (2, 4). While the importance of such cell-cell interactions in developmental biology has been documented (8-12), few studies have investigated the role of cell-ECM adhesion mechanisms in lymphocyte development in lymphopoietic microenvironments such as the bone marrow and thymus. This may be ascribed to the lack of in vitro systems for such analyses and to the complexity arising from the expression of various forms of ECM molecules regulated in a tissue-and cell-specific fashion, as exemplified by fibronectin (FN) molecules (13-15). In this context, several lines of marrow and thymic stromal cells have been isolated to provide tools for analyzing the interaction between developing lymphocytes and stromal cells (16). We have also established a thymic stromal cell clone that is capable of providing an in vitro model for intrathymic T-cell development (17-19).The present study investigates the role of FN molecules expressed on thymic stromal cells in thymocyte-stromal cell adhesion and thymocyte differentiation. The results demonstrate that CD4-8-("double-negative") thymocytes adhere to thymic stromal cells through FN molecules on the stromal cells. Molecular analyses revealed that two adhesion sites, the classic...

We studied the regulation of the L L-galactoside K K2,6-sialyltransferase (hST6Gal I) gene during HL-60 cell differentiation induced with dimethyl sulfoxide (DMSO), all transretinoic acid (ATRA), and phorbol myristate acetate (PMA). During HL-60 cell line differentiation, cell surface levels of K K2,6-sialic acids expression decreased, as measured by flow cytometric analysis using Sambucus nigra agglutinin (SNA). Activities of hST6Gal I and levels of hST6Gal I mRNA dramatically decreased after 1 day of stimulation. Using reverse transcription polymerase chain reaction (PT-PCR), we found the major hST6Gal I mRNA isoform in HL-60 cells contains 5P-untranslated exons Y and Z. These results suggest that the expression of cell surface K K2,6-sialic acids is controlled at the mRNA level, which is regulated by a promoter located 5P-upstream of exon Y.z 1998 Federation of European Biochemical Societies.

The present study investigates the expression of VLA-4 on thymocytes at various stages of maturation and their capacity to adhere to thymic stromal cells. Whole thymocytes were stained with anti-CD4 and anti-CD8, as well as anti-VLA-4 antibodies. Flow microfluorometric analyses revealed that a) most of CD4-8- (double negative DN) and CD4-8intermediate thymocyte populations expressed large amounts of VLA-4, b) the levels of VLA-4 were considerably and markedly reduced on CD4+8+ (double positive DP) and single positive (SP) (CD4+8- or CD4-8+) populations, respectively. This contrasted with an increase in the levels of LFA-1 along with thymocyte maturation. DN, DP, and SP subsets were isolated and examined for their capacity to express VLA-4 and to adhere to fibronectin (FN) molecules as well as thymic stromal cells expressing FN. DN, DP, and SP subsets were confirmed to express the respective high, low, and very low levels of VLA-4, respectively. Approximately 70% of DN thymocytes became bound to FN-precoated culture plates, whereas 30 to 40% of DP and only 10 to 20% of SP cells adhered to FN. Similar patterns of adhesion were observed between these thymocyte subsets and thymic stromal monolayers. The binding of the DN subset to FN-plates or thymic stromal monolayers was inhibited only marginally by the RGDS peptide, but was efficiently inhibited by V10 peptide (cell-binding sequence that is located in the V region on FN and reacts with the VLA-4 integrin) or anti-VLA-4 antibody. Anti-VLA-4 antibody plus RGDS peptide strongly inhibited DN cell binding to FN-coated plates and thymic stromal monolayers. These results indicate that i) VLA-4 expressed on DN thymocytes functions as an important integrin for interacting with thymic stromal cells; ii) the expression level of this integrin decreases with the progress of thymocyte maturation, and iii) most of the mature thymocytes (SP) are rendered less adhesive to thymic stromal cells by reducing the level of VLA-4 expression.

IL-6 is a cytokine secreted in normal individuals by monocytes, fibroblasts, and endothelial cells. We have found increased levels of IL-6 in the sera from MH134 hepatoma- and CSA1M fibrosarcoma-bearing mice. Concerning the capacity of these tumor cells themselves to produce IL-6 in vitro, they exhibited the distinct contrast, i.e., the MH134 tumor cells produced high levels of IL-6 whereas the CSA1M generated a marginal level of IL-6. It was, however, demonstrated that appreciably enhanced IL-6 production was observed in spleen cell culture supernatants from both types of tumor-bearing mice when compared to those obtained from normal mice. More importantly, in contrast to the production of IL-6 by non-T cell compartment of normal spleen cells, enhanced IL-6 production of spleen cells from tumor-bearing mice was ascribed to T cell compartment. Analysis of T cell phenotype has revealed that enhanced IL-6 production was mediated predominantly by Lyt-2+ but not by L3T4+ T cell subset. Thus, these results indicate that increased circulating IL-6 is elicited in the tumor-bearing state and that irrespective of the potential of tumor cells themselves to produce IL-6, T cells, especially Lyt-2+ T cells from tumor-bearing mice are responsible for such a high level of IL-6 production.

The present study investigates the nature of humoral component(s) generated in tumor-bearing hosts to induce immune dysfunction of T cells. Cell-free ascitic fluid and culture supernatant (SN) were obtained from the ascites and cultures allowing MH134 hepatoma cells to grow. These ascites and SN samples were tested for their abilities to influence the generation of CTL responses to TNP and alloantigens. The generation of the anti-TNP CTL responses that require self H-2-restricted CD4+ Th cells was markedly suppressed by addition of the ascites or SN under conditions in which these samples did not inhibit anti-allo CTL responses capable of using alternate pathways of allo-restricted CD4+ and CD8+ Th. The activation of CD8+ CTL precursors and CTL activity were also resistant to the ascites or SN. The ascites- or SN-induced suppressive effect to which CD4+ Th were most susceptible was found to be mediated by transforming growth factor-beta (TGF-beta) activity, because: 1) the TGF-beta activity was detected in the MH134 ascites and culture SN; 2) the suppression of CD4+ Th function required for anti-TNP CTL responses was almost completely prevented by addition of anti-TGF-beta antibody to cultures and; 3) rTGF-beta also induced similar patterns of immunosuppression to those observed by ascites or SN. These results indicate that TGF-beta produced by tumor cells induces deleterious effects on T cell, especially on the CD4+ Th subset, and provide an explanation for the molecular mechanism underlying the previously observed CD4+ Th-selective suppression in the tumor-bearing state.