CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia.

Abstract

Abstract. Transepithelial migration of neutrophils (PMN) is a defining characteristic of active inflammatory states of mucosal surfaces. The process of PMN transepithelial migration, while dependent on the neutrophi1132 integrin CDllb/CD18, remains poorly understood. In these studies, we define a monoclonal antibody, C5/D5, raised against epithelial membrane preparations, which markedly inhibits PMN migration across polarized monolayers of the human intestinal epithelial cell line T84 in a bidirectional fashion. In T84 cells, the antigen defined by C5/D5 is upregulated by epithelial exposure to IFN-% and represents a membrane glycoprotein of ~60 kD that is expressed on the basolateral membrane. While transepithelial migration of PMN was markedly inhibited by either C5/D5 IgG or C5/D5 Fab fragments, the antibody failed to inhibit both adhesion of PMN to T84 monolayers and adhesion of isolated T84 cells to the purified PMN integrin, CDllb/CD18. Thus, epithelial-PMN interactions blocked by C5/D5 appear to be downstream from initial CDllb/CD18-mediated adhesion of PMN to epithelial cells. Purification, microsequence analysis, and cross-blotting experiments indicate that the C5/D5 antigen represents CD47, a previously cloned integral membrane glycoprotein with homology to the immunoglobulin supeffamily. Expression of the CD47 epitope was confirmed on PMN and was also localized to the basolateral membrane of normal human colonic epithelial cells. While C5/D5 IgG inhibited PMN migration even in the absence of epithelia, preincubation of T84 monolayers with C5/D5 IgG followed by antibody washout also resulted in inhibition of transmigration. These results suggest the presence of both neutrophil and epithelial components to CD47-mediated transepithelial migration. Thus, CD47 represents a potential new therapeutic target for downregulating active inflammatory disease of mucosal surfaces. TIVE inflammation of surfaces lined by columnar epithelia is histologically defined by transmigration of neutrophils (PMN) 1 across such epithelial monolayers and subsequent collection of PMN in the lumen (26,53). Recently, neutrophils have been recognized not only to influence epithelial function during transmigration, but also to interact with biochemically distinct apical domains after translocation to the lumenal compartment, thus further modifying key epithelial processes (33, 51). For example, in intestinal epithelia it appears that PMN transepithelial migration may reversibly influence epithelial barrier function (19,37,44), while arrival in the lumenal space may result in interactions promoting electrogenic C1-secretion (32, 33), the known basis for secretory diarrhea

Neutrophil-Epithelial Interactions

“…Specifically, the rate of polymorphonuclear neutrophil (PMN) transepithelial migration has been shown to be regulated, in part, by interactions between PMN-expressed signal-regulatory protein a and CD47 expressed on the basolateral membrane of intestinal epithelial cells. 43,63 Attractive candidate adhesion molecules within the apical junctional complex include the adherens junction proteins E cadherin and Nectins. Within the TJ, potential neutrophil-interacting proteins include junctional adhesion molecules (JAMs), as well as transmembrane TJassociated molecules, including the claudins and occludin.…”

Neutrophil Migration across Tight Junctions Is Mediated by Adhesive Interactions between Epithelial Coxsackie and Adenovirus Receptor and a Junctional Adhesion Molecule-like Protein on Neutrophils

“…For transmigration experiments, cells were grown on collagen-coated permeable filters (5-m pore size; Costar, Cambridge, MA) as previously described in detail (Parkos et al, 1996b). PMN were isolated from whole blood of normal human volunteers using Ficoll/dextran sedimentation and resuspended in cold Hanks' balanced salt solution (HBSS) devoid of calcium or magnesium (HBSS Ϫ ) as previously described (Parkos et al, 1996a).…”

“…PMN transepithelial migration experiments were performed using confluent, high-resistance T84 cell monolayers cultured in an inverted manner on collagen-coated transwells (inverts) as previously described in detail (Parkos et al, 1996b;Liu et al, 2001). With this setup, fMLP-directed PMN migration was assessed in the physiologically relevant basolateral to apical (b-to-a) direction.…”

“…Freshly isolated PMN were added to fusion protein-coated wells (ϳ5 ϫ 10 5 cells/well in a total volume of 300 l) followed by stationary incubation (30 min, 37°C) in the presence or absence of inhibitors. After three washes, PMN adhesion was quantified by direct visualization using digitalized microscopy and by myeloperoxidase (MPO) assay (Parkos et al, 1996b;Liu et al, 2001). …”

Neutrophil transepithelial migration: role of toll-like receptors in mucosal inflammation

“…Efforts aimed at characterizing proteins important in the regulation of PMN transepithelial migration identified a membrane protein termed CD47 (Parkos et al 1996). CD47 is a multiple membrane spanning immunoglobulin superfamily member (Lindberg et al 1993) with several cytoplasmic tail splice variants that we have shown to play a significant role in facilitating PMN migration after initial β2 integrin dependent adhesion (Parkos et al 1996).…”