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Expression and distribution of Endoglycan on B cells Lam, Mindy Ching Wan

Abstract

Endoglycan is the third and newest member of the CD34-family (CD34, Podocalyxin and Endoglycan) of sialomucins. Although all three molecules are markers of early hematopoietic progenitors and vascular associated tissues, they also have additional unique distribution patterns. In most tissues, CD34 and Podocalyxin have been shown to act as anti-adhesives, most likely due to the strong negative charges conferred by terminal sialic acid residues on their highly glycosylated mucin domains (Doyonnas et al., 2001 and unpublished data). We have demonstrated that ectopic expression of CD34 on murine mast cells, or Podocalyxin in breast cancer cells, induces a profound reduction in homotypic aggregation/adhesion. Surprisingly, ectopic expression of Endoglycan in similar assays does not decrease aggregation, suggesting that it may have a different function. In addition, most mucins (including CD34 and Podocalyxin) do not show a high degree of interspecific sequence conservation. However, Endoglycan shows a strikingly high degree of conservation across species. Therefore, the conservation in the Endoglycan sequence may reflect the maintenance of a specific binding domain for extracellular ligand(s). Hence, we propose that Endoglycan has a pro-adhesive function and act as an antagonist of Podocalyxin and CD34. Using an anti-Endoglycan monoclonal antibody (F4B10) generated in our lab, along with RT-PCR data, we determined the general distribution of Endoglycan. In addition to early hematopoietic progenitors and vascular associated tissues, Endoglycan is expressed on macrophages, thymocytes and lipopolysaccharide (LPS)-stimulated B cells. A survey of splenic B cells and immature B cells showed that Endoglycan is expressed on lgM[sup med/hi]IgD[sup Io]CD11b[sub med]CD21[sup med/hi]CD22[sup hi]CD23[sup Io] subset of Splenocytes. These cells are characteristic of marginal zone B cells, which have the phenotype: lgM[sup hi], lgD'°, CD21[sup hi], CD22[sup hi], CD23[sup Io] and CD1[sup hi] . Furthermore, I did not detect Endoglycan expression on immature or pre-B cells. My results show that Endoglycan is up-regulated on LPSstimulated B220-positive splenocytes. In these cultures, Endoglycan is expressed on a subset of plasma cells (as indicated by the marker syndecan-1 (SDC1)), and all Endoglycan-positive cells express pi-integrin. We have evaluated the time course of Endoglycan induction by LPS using FACS and RT-PCR analysis, cell surface expression peaks at 48hr and Endoglycan transcript levels peaks at 24hr. The induction of Endoglycan is specific to TLR-stimulations and is not induced by other B cell activators, such as anti-IgM, anti-CD40 or BAFF. Since T cell cytokines play a large role in the regulation of B cell activation and maturation, I examined the effect of T cell secreted cytokines on Endoglycan expression. I found that IL-2, IL-4, IL-5 and TGFp reduce the up-regulation of Endolgycan by LPS on splenic B cells. On the other hand, IL-ip increases Endoglycan expression. The regulated expression of Endoglycan suggests that this molecule may serve an important role on activated B cells. The data in this thesis provide the first description of Endoglycan expression and regulation on B cells. Endoglycan is a highly specific marker for TLR-activated B cells. Expression of pi-integrin on these Endoglycan-positive plasma cells suggests that these cells may home to specific niches for the establishment of long-term antibody repertoire. Therefore, Endoglycan may have a role in homing and migration of these cells.

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