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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI042818-02
Application #
6137256
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Kraemer, Kristy A
Project Start
1999-01-01
Project End
2002-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
2
Fiscal Year
2000
Total Cost
$245,894
Indirect Cost
Name
University of Maryland Baltimore
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Rifat, Salahaldin; Kang, Tae Jin; Mann, Dean et al. (2008) Expression of sialyltransferase activity on intact human neutrophils. J Leukoc Biol 84:1075-81
Stamatos, Nicholas M; Liang, Feng; Nan, Xinli et al. (2005) Differential expression of endogenous sialidases of human monocytes during cellular differentiation into macrophages. FEBS J 272:2545-56
Sakarya, Serhan; Rifat, Salahaldin; Zhou, Jie et al. (2004) Mobilization of neutrophil sialidase activity desialylates the pulmonary vascular endothelial surface and increases resting neutrophil adhesion to and migration across the endothelium. Glycobiology 14:481-94
Stamatos, Nicholas M; Curreli, Sabrina; Zella, Davide et al. (2004) Desialylation of glycoconjugates on the surface of monocytes activates the extracellular signal-related kinases ERK 1/2 and results in enhanced production of specific cytokines. J Leukoc Biol 75:307-13
Cross, Alan S; Sakarya, Serhan; Rifat, Salahaldin et al. (2003) Recruitment of murine neutrophils in vivo through endogenous sialidase activity. J Biol Chem 278:4112-20