Pathogen-triggered neutrophil (PMN) recruitment is critical for innate immunity, but aberrant PMN influx is also implicated in the pathogenesis of numerous inflammatory diseases of the gut and lungs. Fucosylated terminal glycans from the Lewis glycan family, such as Lewis-X (Lex) and Sialyl Lewis-X (sLex), have previously been implicated in the regulation of important PMN functions, including Selectin-mediated PMN trafficking. While such glycans based on the type 2 sequence (Galb1-4GlcNAc-R) are abundant on PMNs, the presence of type 1 Galb1-3GlcNAc-R glycans required for the expression of Lewis-A (Lea) have not yet been reported.Methods:
Expression of Lea and related glycosyltransferases by human PMN was determined by PCR and immunoblotting. PNGaseF treatment was used to determine N-glycan linkage of PMN expressed Lea. Specific recognition of Lea glycans by anti-Lea mAbs was confirmed using glycan microarrays from the Consortium for Functional Glycomics (CFG). Expression of Lea in normal and inflamed intestinal mucosa was examined by immunhistochemistry staining. To assess the role of Lea during PMN transepithelial migration (TEM), PMN were incubated with anti-Lea antibodies before migration across T84 and Caco2 intestinal epithelial cells or collagen coated filters was assessed by quantification of MPO levels.Results:
Data revealed PMN expression of both a b1-3 Galactosyltransferase and an α1/4 Fucosyltransferase, glycosyltransferase enzymes required for LeA synthesis, as well as several Lea containing PMN glycoproteins. Immunohistochemistry analyses revealed Lea expression on circulating PMN as well as on PMN within inflamed intestinal mucosa. We further report that antibody mediated ligation of PMN expressed Lea increased PMN chemotaxis across collagen and PMN TEM across model intestinal epithelia. As would be expected treatment of PMN from individuals deficient in α1/4 fucosylation with anti-Lea mAbs did not result in changes in neutrophil trafficking.Conclusions:
These results identify, for the first time, expression of Lea by human PMN and demonstrate its relevance to PMN trafficking within the inflamed intestine. We propose that PMN Lea represents a new target for regulating intestinal innate immunity and modulating inflammation in diseases such as inflammatory bowel disease where dysregulated PMN influx is associated with bystander tissue damage. Furthermore, differential responses of human PMN to physiological ligands of Lea (based on an individuals Lewis phenotype) may provide insights into the mechanisms linking specific Lewis phenotypes with differential susceptibilities to bacterial infections, viral infections and coronary heart disease.