Sialic acids are amino sugars that are present on mammalian cells as glycoconjugates and impart to these cells a negative surface charge. The loss of sialic acid occurs during both myeloid maturation and activation and is accompanied by enhanced motility, deformability and adhesiveness. Sialidases are enzymes that rapidly remove sialic acid residues from glycoconjugates and on neutrophil activation are rapidly mobilized to plasma membrane. In contrast, by adding sialic acid residues back onto glycoconjugates, sialyltransferases (ST) may restore glycoconjugates to the preactivated state. Thus, the amount and distribution of sialic acid residues on both neutrophils and perhaps endothelial cells (EC) may represent a rapidly adaptable mechanism by which neutrophils respond to local inflammatory stimuli and leave the circulation.
In Specific Aim I the PI will examine the effect of sialic acid modulation at each stage of neutrophil diapedeis: rolling adhesion, firm adhesion and transendothelial migration (TEM). Resting or activated human neutrophils will be added to EC monolayers that are either resting or activated with short-acting (histamine) or long acting (TNF) stimuli or treated with neutroaminidase. Neutrophil adherence and TEM will be measured in response to either IL-8 or to buffer (spontaneous). The role of PMN sialidase will be assessed with the use of pharmacologic inhibitors of sialidase and a polyclonal antisialidase antibody previously shown to inhibit neutrophil sialidase. The expression of PMN ST relative to sialidase will be determined.
In Specific Aim II the applicant will determine the role of sialic acid modulation of EC monolayers in neutrophil adherence and TEM. Preliminary evidence for an endogenous EC sialidase will be extended by both enzymatic activity and immune detection.
in Specific AIM III he will determine the degree of homology of neutrophil sialidase with a recently described human sialidase cloned from the MHC region of lymphoblastoid cells and which may play a role in T cell activation. Neutrophil sialidase will be purified and sequenced from lysates eluded from antisialidase antibody bound to a solid matrix. These sequences will be used to construct primers from cloning of the sialidase from a human HL-60 cell library. Endothelial cell sialidase will be isolated, cloned and compared to neutrophil sialidase in Aim III. These studies will define a new mechanism for a rapidly adaptable, temporally- restricted local response to inflammation. This proposal will provide new targets and additional approaches to the treatment of inflammation.
