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SummaryA cell adhesion model was previously used to select a series of monoclonal antibodies (mAbs), which were subsequently found to recognize CD44/Pgp-1 . Interest in these reagents increased with the finding that they totally inhibited production of lymphoid or myeloid cells in long-term bone marrow cultures. Further investigation has now revealed that hyaluronate is a potential ligand for CD44 and that hyaluronate recognition accounts for the adhesion between B lineage hybridoma and stromal cells . The hybridoma cells adhered to hyaluronate-coated plastic wells as well as to monolayers of stromal cells. The adhesion in both cases was inhibited by treatment with hyaluronidases, and did not require divalent cations . Addition of exogenous hyaluronate also diminished binding of lymphoid cells to stromal cells. One of several mAbs to Pgp-1/CD44 was particularly effective at blocking these interactions. Since hyaluronate and Pgp-1/CD44 were present on both cell types, experiments were done to determine the cellular location ofinteracting molecules required for the adhesion process . Treatment oflymphoid cells with an anti-Pgp-1/CD44 antibody was more inhibitory than antibody treatment ofthe stromal cells. Conversely, hyaluronidase treatment of stromal cells reduced subsequent binding more than treatment of the lymphoid cells . Adhesive interactions that involve hyaluronate and CD44 could contribute to a number of cell recognition processes, including ones required for normal lympho-hemopoiesis .

Abstract. The hyaluronan receptor belongs to the polymorphic family of CD44 glycoproteins, which have been implicated in a variety of cellular functions including adhesion to hyaluronan and collagen, the binding of lymphocytes to high endothelial cells during extravasation, and conferring metastatic potential to carcinoma cells . Here, we demonstrate that the receptor also participates in the uptake and degradation of hyaluronan by both transformed fibroblasts (SV3T3 cells) and alveolar macrophages . These cells were incubated with isotopically labeled hyaluronan for various periods of time, and the extent of degradation was determined by either molecular-sieve chromatography or centrifugation through Centricon 30 microconcentrators. The macrophages degraded the hyaluronan at a faster rate than the SV3T3 cells, which may reflect the fact that they contained a greater number of receptors . More importantly, in T HE hyaluronan receptor is a cell-surface glycoprotein of 85-100 kD, which recognizes a six sugar sequence of hyaluronan and mediates the divalent-cation independent aggregation of cells (32,33,34,35,39) . Indeed, mAbs to this receptor specifically block the hyaluronaninduced aggregation of both cultured fibroblasts and macrophages (12) . Biochemical studies of this receptor have revealed that it is an elliptical glycoprotein which is phosphorylated and has a high negative charge (4, 33, 39) . In addition, its cytoplasmic domain appears to be associated with actin filaments (16), perhaps through an ankyrin-like molecule (15). This receptor is present on a wide variety of cells including most epithelial cells, macrophages and lymphocytes (l, 11, 33) . Interestingly, in the case of epithelia, it is preferentially expressed on those cells undergoing active cell division (1).Recently, a number of studies have shown that the hyaluronan receptor is identical to CD44, which has been implicated in the homing oflymphocytes to mucosal lymphoid tissues (2,4,19,22) . CD44 on the surfaces of lymphocytes binds to a protein termed mucosal addressin, which is present on the high-endothelial cells of gut-associated lymphoid tissues (14,23,28,30) . However, the ability of CD44 to bind addressin is distinct from its ability to bind hyaluronan . In-© The Rockefeller University Press, 0021-9525/92/02/1055/8 $2 .00 The Journal of Cell Biology, Volume 116, Number 4, February 1992 1055-1062 both cell types, the degradation of hyaluronan was specifically blocked by antibodies directed against the receptor. However, the receptor by itself did not have the ability to degrade hyaluronan, since preparations of SV3T3 membranes containing the receptor did not break down hyaluronan. Subsequent experiments revealed that macrophages can internalize fluoresceintagged hyaluronan, and this process was blocked by antibodies against the receptor. Furthermore, the subsequent degradation of hyaluronan was inhibited by agents that block the acidification of lysosomes (chloroquine and NH4C1) . Thus, the most likely explanation for these results is...

Abstract. The present study was undertaken to determine the relationship between the hyaluronate receptor and CD44 (H-CAM), cell-surface glycoproteins of similar molecular weights that have been implicated in cell adhesion. In initial experiments, a panel of monoclonal antibodies directed against CD44 were tested for their ability to cross react with the hyaluronate receptor. These antibodies immunoprecipitated [3H]hyaluronate binding activity from detergent extracts of both mouse and human cells, indicating that the hyaluronate receptor is identical to CD44. In addition, one of these antibodies (KM-201 to mouse CD44) directly blocked the binding of labeled hyaluronate to the receptor and inhibited hyaluronate dependent aggregation of SV-3T3 cells.

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In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85-200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan.

Chronic sun exposure leads to structural and functional alterations in exposed skin. Photoageing is a process distinct from the changes taking place due to chronological ageing. Unique alterations in the dermal extracellular matrix occur as a result of photoageing and are responsible for many of these physiological changes taking place in sun-damaged skin. Accompanying the deposition of abnormal elastic tissue, or solar elastosis, are significant alterations in dermal glycosaminoglycans (GAGs). Accumulation of GAGs as a result of photoageing, as demonstrated in both humans and animal models of photoageing, seems almost paradoxical in view of the large amounts of GAGs present in the skin of newborns, making their skin well hydrated and supple, in sharp contrast to the weathered appearance of photoaged skin. We investigate the relative GAG content of photoaged skin using immunoperoxidase stains specific for hyaluronic acid and chondroitin sulphate, and determine the location of these GAGs using confocal laser scanning microscopy. Our results demonstrate significant increases in GAG staining in sun-damaged vs. sun-protected skin from the same individuals, as measured by computer-based image analysis. Furthermore, confocal laser scanning microscopy reveals that the increased dermal GAGs in sun-damaged skin are deposited on the elastotic material of the superficial dermis of photodamaged skin, and not between collagen and elastic fibres as in normal skin. The abnormal location of GAGs on these fibres may explain the apparent paradoxical weathered appearance of photodamaged skin despite increased GAGs.