Synthetic polysulfated hyaluronic acid is a potent inhibitor for tumor necrosis factor production

Based on the premise that naturally occurring glycosaminoglycans could serve as building blocks for synthesizing nontoxic drugs for suppression of tumor necrosis factor (TNF) production by inflammatory cells, we have chemically modified hyaluronic acid (HA) and tested its effects in blocking TNF-alpha and TNF-beta production in vitro. HA was chosen mainly for its structural simplicity, nonimmunogenicity, and readiness for chemical modifications. When HA was chemically polysulfated to a sulfate/hexosamine molar ratio of 3.9, the sulfated HAs was shown to be a potent inhibitor of TNF-alpha production in lipopolysaccharide (LPS)- or interferon-gamma-activated THP-1 cells. For example, a concentration of HAs as low as 10 ng/ml reduced TNF-alpha production in LPS-activated THP-1 cells more than 50%, whereas achieving a similar extent of reduction required 50 micrograms/ml native HA. By decreasing the extent of polysulfation, the inhibitory effect of HAs on TNF-alpha production was diminished. Other chemical modifications, including deacetylation, thiolation, or reduction of the carboxylic groups, could not increase the efficacy of HA in suppression of TNF-alpha production. Naturally polysulfated glycosaminoglycans, such as chondroitin sulfates, keratan sulfate, heparan sulfate, and heparin, failed to inhibit TNF-alpha production. HAs also restricted TNF-beta (lymphotoxin) secretion in an Epstein-Barr virus-transformed B cell line, Roha-9, which constitutively produces TNF-beta. HAs had no inhibitory effect on the proliferation of THP-1 or Roha-9 cells, which would account for the reduced TNF-alpha or TNF-beta production. Furthermore, time-course metabolic labeling studies revealed that HAs could not restrict overall protein synthesis and secretion in THP-1 cells. However, HAs increased complement C1q secretion in THP-1 in a dose-dependent manner, but it had no effect on biosynthesis of complement C1 inhibitor, factor D, and Fc gamma receptor type II (Fc gamma RII). These results indicate that HA, selectively restricts the production of TNF-alpha, TNF-beta, and probably several other protein species.