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Cell-Bound IL-8 Increases in Bronchial Epithelial Cells after Arylsulfatase B Silencing due to Sequestration with Chondroitin-4-Sulfate

¹ Department of Medicine, University of Illinois at Chicago, Chicago, Illinois; ² Jesse Brown VA Medical Center, Chicago, Illinois; ³ Rensselaer Polytechnic Institute, Troy, New York; and ⁴ Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, Illinois

Correspondence and requests for reprints should be addressed to Joanne K. Tobacman, M.D., University of Illinois at Chicago, Department of Medicine, 840 S. Wood Street, CSN440, M/C 718 Chicago, IL 60612. E-mail: jkt{at}uic.edu

The chemokine IL-8 is critically important in inflammatory processes in human tissues, and IL-8 interactions with sulfated glycosaminoglycans have been implicated in modification of inflammatory responses in bronchial epithelium. To determine the role of chondroitin-4-sulfate (C4S) in mediating effects of IL-8, we silenced the enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B [ASB]) that removes the 4-sulfate group from C4S, in the IB3-1 and C38 bronchial epithelial cell lines and in normal primary bronchial epithelial cells. When ASB was silenced and IL-8 production stimulated by exposure to TNF-, ASB activity declined by roughly 75%, cellular C4S content increased by over 7.5 µg/mg protein, cell-bound IL-8 increased by over 530 pg/mg protein, and secreted IL-8 declined by over 520 pg/mg protein in all cell lines (P < 0.001). When cell lysates were immunoprecipitated with C4S antibody after ASB silencing and TNF-, the IL-8 content of the immunoprecipitate was approximately 500 pg/mg protein, indicating that most of the cell-bound IL-8 was associated with C4S. Cell fractionation demonstrated that the IL-8 content associated with the cell membranes was about twice that of the cytosolic fraction. Also, ASB appeared to localize in the cell membrane, as well as in lysosomes. Neutrophil attraction to the cell lysates increased after ASB silencing, consistent with increased attraction to the cell-bound IL-8. These findings provide evidence for the influential role of ASB and C4S in the regulation of IL-8 secretion, and suggest that changes in ASB activity and C4S content may have a significant impact on IL-8–mediated inflammatory responses.

Key Words: IL-8 • chondroitin-4-sulfate • arylsulfatase B • glycosaminoglycan • cystic fibrosis

CLINICAL RELEVANCE This research may contribute to better understanding of mechanisms of inflammation and chronic infection in cystic fibrosis (CF). The enzyme arylsulfatase B, which appears to have extra-lysosomal localization, modifies chondroitin sulfation, leading to reduced secretion of IL-8 and retention of IL-8 by bronchial epithelial cells. Better understanding of these mechanisms may help to explain the pathophysiology of CF and improve therapeutic interventions.