Jayne Lesley

CD44 and Its Interaction with Extracellular Matrix

It is now generally accepted that CD44 is a cell adhesion receptor and that hyaluronan is one of its ligands. Like many cell adhesion receptors, CD44 is broadly distributed, and its ligand, hyaluronan, is a common component of extracellular matrices and extracellular fluids. Yet a great variety of responses has been reported to result from CD44 ligation. These include cell adhesion, cell migration, induction (or at least support) of hematopoietic differentiation, effects on other cell adhesion mechanisms, and interaction with cell activation signals. This diversity of responses indicates that downstream events following ligand binding by CD44 may vary depending on the cell type expressing CD44 and on the environment of that cell. CD44 is expressed on cells in the early stages of hematopoiesis and has been shown to participate in at least some aspects of the hematopoietic process. In mature lymphocytes, CD44 is upregulated in response to antigenic stimuli and may participate in the effector stage of immunological responses. Along with other adhesion receptors that show alterations in expression after activation, CD44 probably contributes to differences in the recirculation patterns of different lymphocyte subpopulations. CD44 ligand-binding function on lymphocytes is strictly regulated, such that most CD44-expressing cells do not constitutively bind ligand. Ligand-binding function may be activated as a result of differentiation, inside-out signaling, and/or extracellular stimuli. This regulation, which in some situations can be rapid and transient, potentially provides exquisite specificity to what would otherwise be a common interaction. CD44 is not a single molecule, but a diverse family of molecules generated by alternate splicing of multiple exons of a single gene and by different posttranslational modifications in different cell types. It is not yet clear how these modifications influence ligand-binding function. The significance of the multiple isoforms of CD44 is not understood, but association of some isoforms with malignancies has been observed. And in at least some experimental systems, a contribution of CD44 isoforms to metastatic behavior has been demonstrated.

Biochemical characterization and cellular distribution of a polymorphic, murine cell-surface glycoprotein expressed on lymphoid tissues

A murine leukocyte surface glycoprotein (Mr = 95 000) has been defined by means of xenogeneic monoclonal antibodies. In normal hematopoietic tissues, the glycoprotein is found in highest amounts in the bone marrow. Flow cytometric analysis shows that essentially all bone-marrow cells express the glycoprotein and that it is a major component of a subpopulation of cells containing predominantly granulocytic precursors. In contrast, only about 5 percent of thymocytes express sufficient glycoprotein to be detected by flow cytometric analysis, although under stringent conditions up to 20 percent of thymocytes are susceptible to complement-mediated cytotoxicity using a monoclonal antibody against the glycoprotein. Functional assays showed that both prothymocytes and colony forming unit-spleen express the glycoprotein which is broadly distributed on murine hematopoietic tumor cell lines. However, although some Thy-I+ (T) cell lymphomas express large amounts of the glycoprotein, others do not express detectable quantities of the molecule. The glycoprotein is not restricted to hematopoietic cells and can be detected on lung, kidney, brain, and liver as well as cultured fibroblasts. Monoclonal antibodies against the glycoprotein cross-react with an antigen present on human cells. As described in the accompanying paper, the glycoprotein exists in two antithetical allelic forms and we show that it is identical to a polymorphic surface molecule independently characterized by Colombatti and co-workers.

Binding of hyaluronic acid to lymphoid cell lines is inhibited by monoclonal antibodies against Pgp-1☆

Recent biochemical and sequence data suggest a possible relationship between Pgp-1 (identical to CD44/Hermes 1/p85) and a hyaluronic acid-binding function. Here, we have studied the hyaluronic acid-binding activity of a series of murine hematopoietic cell lines using several assays: cell aggregation by hyaluronic acid, binding of fluorescein-conjugated hyaluronic acid, and cell adhesion to hyaluronic acid-coated dishes. Certain Pgp-1-positive T and B cell lines show hyaluronic acid binding that is highly specific and is not competed for by other glycosaminoglycans. Monoclonal antibodies against Pgp-1, but not antibodies against other major cell surface glycoproteins, inhibited hyaluronic acid-induced cell aggregation and cell adhesion to hyaluronic acid-coated dishes. Additionally, some anti-Pgp-1 antibodies inhibited binding of fluorescein-hyaluronic acid to hyaluronic acid-binding lines. We found no Pgp-1-negative lines that bound, but many Pgp-1-positive cell lines did not bind hyaluronic acid. Two Pgp-1-positive thymomas that did not bind hyaluronic acid were induced by phorbol ester to bind hyaluronic acid with the same specificity as other hyaluronic acid-binding lines. Normal hematopoietic cells, including those which express high levels of Pgp-1, such as bone marrow myeloid cells and splenic lymphocytes, showed no detectable hyaluronic acid-binding activity. We discuss several models that might account for these observations: (1) the hyaluronic acid receptor is Pgp-1, but it normally exists in an inactive state; (2) hyaluronic acid receptors are a subset of a family of molecules recognized by anti-Pgp-1 antibodies; (3) the hyaluronic acid receptor is not Pgp-1, but is closely associated with Pgp-1 on the surface of cells which express hyaluronic acid-binding activity.

CD44 in inflammation and metastasis.

CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronan (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 binding of HA is regulated by the cells in which it is expressed. Thus, CD44 expression alone does not correlate with HA binding activity. CD44 is subject to a wide array of post-translational carbohydrate modifications, including N-linked, O-linked and glycosaminoglycan side chain additions. These modifications, which differ in different cell types and cell activation states, can have profound effects on HA binding function and are the main mechanism of regulating CD44 function that has been described to date. Some glycosaminoglycan modifications also affect ligand binding specificity, allowing CD44 to interact with proteins of the extracellular matrix, such as fibronectin and collagen, and to sequester heparin binding growth factors. It is not yet established whether the HA binding function of CD44 is responsible for its proposed involvement in inflammation. It has been shown, however, that CD44/HA interactions can mediate leukocyte rolling on endothelial and tissue substrates and that CD44-mediated recognition of HA can contribute to leukocyte activation. Changes in CD44 expression (mainly up-regulation, occasionally down-regulation, and frequently alteration in the pattern of isoforms expressed) are associated with a wide variety of cancers and the degree to which they spread; however, in other cancers, the CD44 pattern remains unchanged. Increased expression of CD44 is associated with increased binding to HA and increased metastatic potential in some experimental tumor systems; however, in other systems increased HA binding and metastatic potential are not correlated. CD44 may contribute to malignancy through changes in the regulation of HA recognition, the recognition of new ligands and/or other new biological functions of CD44 that remain to be discovered. Abbreviations:aa, amino acid(s); CS, chondroitin sulfate; CSPG, chondroitin sulfate containing proteoglycan; CD44H, ‘hematopoietic’, also called ‘standard’, isoform of CD44 which contains none of the alternatively spliced variant exons; CD44-Rg, CD44 receptor globulin, a secreted chimaeric protein composed of the external domain of the adhesion receptor CD44 and the hinge, CH2 and CH3 regions of human immunoglobulin-G heavy chain; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; KS, keratan sulfate; PB, peripheral blood; PBL, peripheral blood lymphocytes

Expression of Transferrin Receptor on Murine Hematopoietic Progenitors

We have used a monoclonal antibody against the murine transferrin receptor to study the expression of the transferrin receptor on the hematopoietic progenitor cells (BFU-E, CFU-E, and CFU-C) present in mouse bone marrow. Elutriation and cell-sorting data are consistent with the hypothesis that most CFU-E are transferrin receptor positive while most BFU-E express much less transferrin receptor. CFU-C comprise both transferrin-receptor-positive and -negative cells.

Effects of cytotoxic monoclonal antibody specific for T200 glycoprotein on functional lymphoid cell populations

Abstract A monoclonal antibody against T200 glycoprotein is selectively cytotoxic for thymocytes and mature thymus-dependent (T) lymphocytes. All T-cell functions assayed, cell-mediated cytotoxicity, helper cell activity, and proliferation in response to T-cell mitogens or allogeneic cells were abolished by prior treatment of spleen cells with anti-T200 antibodies and complement. In contrast, thymus-independent (B) cell responses to lipopolysaccharide (LPS) and other B-cell mitogens were unaffected. Although treatment of spleen cells with anti-T200 antibodies and complement markedly reduced their capacity to mount an in vitro antibody response to sheep red blood cells (SRBC), responsiveness could be restored by the addition of SRBC-primed T-helper cells. Treatment of bone marrow cells with anti-T200 antibodies and complement did not eliminate either in vivo colony-forming units-spleen (CFU-S) or prothymocytes. It is concluded that T lymphocytes become sensitive to complement-mediated lysis by anti-T200 antibodies as a consequence of cell-surface modifications occurring shortly before or just after their entry into the thymus. In contrast to Thy-1 antigen, the selective killing by antibody against T200 glycoprotein cannot be readily accounted for by quantitative differences in the expression of T200 glycoprotein on the cell surface. Fluorescence-activated cell analysis showed that T200 glycoprotein was expressed in similar amount on the majority of all thymocytes, spleen, and bone marrow cells.

Identification and characterization of the human Pgp-1 glycoprotein

Two monoclonal antibodies have been raised against human Pgp-1 by the immunization of mice with human fibroblasts. The human molecule, like the previously identified mouse counterpart, is an abundant membrane protein (Mr approximately 95 000) with a broad tissue distribution. Pgp-1 is phosphorylated, and phosphoamino acid analysis demonstrates that this occurs exclusively on serine residues. A major difference between the mouse and the human is that 50–60% of human thymocytes are Pgp-1+ compared to 5–10% of mouse thymocytes at an equivalent stage in development. Immunofluorescence studies of cryostat sections showed that the majority of human medullary thymocytes are strongly stained with Pgp-1-specific antibody, whereas the expression of Pgp-1 on cortical thymocytes is much more heterogenous.

Evidence that the Pgp-1 glycoprotein is expressed on thymus-homing progenitor cells of the thymus

The Pgp-1 glycoprotein is found on the bone marrow prothymocyte; however, only a few percent of cells within the normal thymus express significant quantities of Pgp-1 glycoprotein. One hypothesis is that some or all of these Pgp-1+ thymocytes represent thymocyte progenitors or the immediate descendents of the bone marrow-derived prothymocyte. A cell present in the thymus which is able to home back to the thymus and to transiently repopulate it represents one class of thymocyte progenitor. Thymocyte populations enriched in this thymus-homing progenitor are enriched in Pgp-1+ cells. Treatment of these enriched populations with anti-Pgp-1 antibody inhibits activity of the thymus-homing progenitor. These results are consistent with the hypothesis that the thymus-homing progenitor bears Pgp-1 on its surface.

Genetic characterization of a polymorphic murine cell-surface glycoprotein

As described in the preceding paper, monoclonal antibodies have been raised by immunization of rats with mouse hematopoietic cells which detect a major cell-surface glycoprotein (Mr=95 000) of mouse bone-marrow cells of the granulocytic series. While most of the monoclonal antibodies detect this molecule on bone-marrow and spleen cells of all mouse strains, two antibodies recognize alternative allelic forms of the molecule. One alloantigen is expressed in all the remaining inbred strains examined. The alloantigens are codominantly expressed on the cells of F1 mice. Backcrosses of DBA/2 and C57BL/6 with F1 mice (B6D2F1) confirmed that a single genetic locus is involved in the expression of the two antigenic forms and demonstrated linkage to Ly-m11 which has previously been mapped to mouse chromosome 2. These genetic mapping experiments and the biochemical properties of the glycoprotein suggested that it might be identical to a glycoprotein first identified on murine fibroblasts by Hughes and August and designated Pgp-1. This has been firmly established by exchange of monoclonal antibody reagents and sequential immunoprecipitations.

Modulation of transferrin receptor expression and function by anti-transferrin receptor antibodies and antibody fragments☆

It has been suggested that effects of anti-transferrin receptor antibodies on cell growth and receptor expression are the result of varying degrees of receptor crosslinking by bi- and multivalet binding agents. In order to study this question directly, we have cultured murine lymphoma cells in mono- and divalent fragments from IgG and IgM monoclonal anti-transferrin receptor antibodies and in intact antibodies. The studies presented here demonstrate that effects of antibody binding on transferrin receptor distribution, metabolism, and function depend, at least in part, on antibody valence, and therefore on the degree of crosslinking of receptors by antibody. We found that monovalent antibody fragments did not significantly alter cell growth, receptor surface expression, intracellular localization, or degradation. Diavalent antibody caused a uniform down-regulation of cell-surface receptor expression, which was accompanied by increased degradation only when antibody Fc was present. Normal receptor cycling apparently continued, despite the reduction in surface expression. Culture in multivalent IgM antibody, however, resulted in accumulation of antibody-complexed receptor on the cell surface without internalization and caused profound inhibition of cell growth. Thus, we show two mechanisms by which different degrees of antibody crosslinking can influence transferrin receptor function: by receptor down-regulation and blocking internalization.