Microglia Are Mediators of Borrelia burgdorferi–Induced Apoptosis in SH-SY5Y Neuronal Cells

Inflammation has long been implicated as a contributor to pathogenesis in many CNS illnesses, including Lyme neuroborreliosis. Borrelia burgdorferi is the spirochete that causes Lyme disease and it is known to potently induce the production of inflammatory mediators in a variety of cells. In experiments where B. burgdorferi was co-cultured in vitro with primary microglia, we observed robust expression and release of IL-6 and IL-8, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES), but we detected no induction of microglial apoptosis. In contrast, SH-SY5Y (SY) neuroblastoma cells co-cultured with B. burgdorferi expressed negligible amounts of inflammatory mediators and also remained resistant to apoptosis. When SY cells were co-cultured with microglia and B. burgdorferi, significant neuronal apoptosis consistently occurred. Confocal microscopy imaging of these cell cultures stained for apoptosis and with cell type-specific markers confirmed that it was predominantly the SY cells that were dying. Microarray analysis demonstrated an intense microglia-mediated inflammatory response to B. burgdorferi including up-regulation in gene transcripts for TLR-2 and NFκβ. Surprisingly, a pathway that exhibited profound changes in regard to inflammatory signaling was triggering receptor expressed on myeloid cells-1 (TREM1). Significant transcript alterations in essential p53 pathway genes also occurred in SY cells cultured in the presence of microglia and B. burgdorferi, which indicated a shift from cell survival to preparation for apoptosis when compared to SY cells cultured in the presence of B. burgdorferi alone. Taken together, these findings indicate that B. burgdorferi is not directly toxic to SY cells; rather, these cells become distressed and die in the inflammatory surroundings generated by microglia through a bystander effect. If, as we hypothesized, neuronal apoptosis is the key pathogenic event in Lyme neuroborreliosis, then targeting microglial responses may be a significant therapeutic approach for the treatment of this form of Lyme disease

Identification and characterization of L-selectin ligands in porcine lymphoid tissues

A human L-selectin–ZZ fusion protein was used to screen porcine inguinal lymph nodes for the presence of monoclonal antibody (mAb) MECA 79-negative ligands. Fractionation of lymph node-conditioned medium by anion-exchange chromatography revealed two distinct L-selectin-binding fractions, one containing a MECA 79 non-reactive species and the second containing two MECA 79 reactive species of ≈84 000 and 210 000 molecular weight. The MECA 79 non-reactive species exhibited Ca(2+)- and lectin-dependent binding to L-selectin–ZZ in a solid-phase capture enzyme-linked immunosorbent assay (ELISA), and this was specifically disrupted by the addition of EDTA, mannose-6-phosphate and the blocking anti-L-selectin mAb, DREG-56. Enzymatic characterization of the ligand by trypsin, O-sialoglycoprotease endopeptidase, heparinases I and III, or chondroitinase ABC lyase digestion indicated that L-selectin binding was predominantly dependent upon a chondroitin sulphate-modified glycoprotein determinant. Although Coomassie Blue staining of sodium dodecyl sulphate (SDS) polyacrylamide gels did not reveal a detectable protein species, carbohydrate-specific staining using GlycoTrack™ revealed a single, heavily glycosylated protein of high molecular weight (> 220 000). These studies have revealed the existence of a MECA 79 non-reactive, chondroitin sulphate glycosaminoglycan-modified ligand, termed csgp>220, which is secreted by peripheral lymph nodes and may play a role in leucocyte trafficking to the lymph node

E-selectin ligands recognised by heca452 induce drug resistance in myeloma, which is overcome by the e-selectin antagonist, gmi-1271

Multiple myeloma (MM) is characterized by the clonal expansion and metastatic spread of malignant plasma cells to multiple sites in the bone marrow (BM). Recently, we implicated the sialyltransferase ST3Gal-6, an enzyme critical to the generation of E-selectin ligands, in MM BM homing and resistance to therapy. Since E-selectin is constitutively expressed in the BM microvasculature, we wished to establish the contribution of E-selectin ligands to MM biology. We report that functional E-selectin ligands are restricted to a minor subpopulation of MM cell lines which, upon expansion, demonstrate specific and robust interaction with recombinant E-selectin in vitro. Moreover, an increase in the mRNA levels of genes involved in the generation of E-selectin ligands was associated with inferior progression-free survival in the CoMMpass study. In vivo, E-selectin ligand-enriched cells induced a more aggressive disease and were completely insensitive to Bortezomib. Importantly, this resistance could be reverted by co-administration of GMI-1271, a specific glycomimetic antagonist of E-selectin. Finally, we report that E-selectin ligand-bearing cells are present in primary MM samples from BM and peripheral blood with a higher proportion seen in relapsed patients. This study provides a rationale for targeting E-selectin receptor/ligand interactions to overcome MM metastasis and chemoresistance