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Pro- and Anti-Inflammatory Factors Cooperate to Control Hyaluronan Synthesis in Lung Fibroblasts

Department of Vascular Biology, Hope Heart Institute, Seattle; and Department of Medicine, Division of Allergy and Infectious Disease, and Department of Pathology, University of Washington, Seattle, Washington

Address correspondence to: T. N. Wight, Ph.D., Department of Vascular Biology, The Hope Heart Institute, 1124 Columbia Street, Seattle, WA 98104-2046. E-mail: twight{at}hopeheart.org

Hyaluronan (HA) is an important constituent of the extracellular matrix and accumulates during inflammatory lung diseases like asthma. Little is known about the factors that regulate HA synthesis by lung cells. Accordingly, we investigated the effect of T-helper 1 (T_H1) and 2 (T_H2) cytokines and the anti-inflammatory agents fluticasone and salmeterol on HA synthesis in human lung fibroblasts. Interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF)- were the most potent stimulators of HA synthesis and when combined, caused synergistic increases in HA accumulation. Time-course analysis of HA accumulation and [³H]-glucosamine incorporation into HA demonstrated continued synthesis over the 24 h of stimulation. Peak synthesis at 6–12 h coincided with an increased proportion of high molecular weight HA. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that IL-1ß and TNF- induced HA synthase-2 messenger RNA (mRNA) 3 h following stimulation and remained elevated throughout the 24-h stimulation period. Fluticasone inhibited IL-1ß and TNF- induced HA synthesis (44.5%) whereas salmeterol had no effect. When combined, fluticasone and salmeterol inhibited HA synthesis to a greater extent (85.2%). Further, fluticasone attenuated IL-1ß and TNF- stimulated hyaluronan synthase-2 messenger RNA (mRNA), and the addition of salmeterol cooperatively enhanced this inhibition. These results indicate that enhanced synthesis of HA by the proinflammatory cytokines IL-1ß and TNF- can be abrogated by specific corticosteroid and ß₂ blocker combinations shown to be effective in the treatment of asthma.

Abbreviations: biotinylated proteoglycan, bPG • bovine serum albumin, BSA • complimentary DNA, cDNA • enzyme-linked immunosorbent assay, ELISA • extracellular matrix, ECM • ethylenediaminetetraacetic acid, EDTA • fetal bovine serum, FBS • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • high molecular weight, HMW • hyaluronan, HA • hyaluronan synthase, HAS • Interferon, IFN • interleukin, IL • messenger RNA, mRNA • nuclear factor B, NF-B • reverse transcriptase polymerase chain reaction, RT-PCR • T-helper type 1, T_H1 • T-helper type 2, T_H2 • tumor necrosis factor , TNF-

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