Low molecular weight hyaluronic acid effects on murine macrophage nitric oxide production

Abstract

Hyaluronic acid (HA) is increasingly used for a number of medical device applications. Since the chemical structure of HA is identical no matter its bacterial or animal origin, it should be the ideal biomaterial. However, short term transient inflammatory reactions are common, while rare long-term adverse events may correlate with subclinical chronic inflammation. Concern has been raised that low molecular weight components or degradation fragments from implanted HA may directly stimulate inflammatory reactions. This study examined a panel of HA molecular weights from the unitary disaccharide up to 1.7 x 10(6) Dalton lengths, in which endotoxin was assayed at a very low level (less than 0.03 EU/mg). The murine cell line RAW 264.7, rat splenocytes, and rat adherent differentiated primary macrophages were assayed for nitric oxide production under a variety of inflammatory conditions plus or minus HA. Under the highest inflammatory states, nitric oxide production was mildly suppressed by HMW-HA while slightly augmented by LMW-HA at mg/mL concentrations. However, at micromolar concentrations fragments below 5000 Daltons, thought to have drug-like qualities, were without effect. These data support the hypothesis that if endotoxin is reduced to an extremely low level, LMW-HA may not directly provoke normal tissue macrophage-mediated inflammatory reactions.

Recombinant Human Hyaluronidase PH20 Does Not Stimulate an Acute Inflammatory Response and Inhibits Lipopolysaccharide-Induced Neutrophil Recruitment in the Air Pouch Model of Inflammation

“…In this study, we demonstrate that LMW HA fragments do not bind or stimulate TLR2 or TLR4 on the cell surface, consistent with a recently published observation that LMW HA fragments are not TLR ligands (31). Applying LMW HA fragments to a murine macrophage cell RAW 264.7 failed to stimulate production of MIP-2, a classic proinflammatory cytokine, in agreement with previous reports that LMW HA did not stimulate production of IL-1b or NO (28,29).…”

“…1D). Our data are consistent with recent publications reporting that LMW HA does not stimulate chemokine production in the RAW 264.7 cell line, rat splenocytes, rat adherent differentiated primary macrophages, or primary human macrophages (28,29). rHuPH20 or LMW HA do not stimulate production of cytokines/chemokines in the air pouch model…”

“…LMW HA has been reported to stimulate inflammation (24,45,50), but endotoxin-free HA fragments at the same molecular sizes failed to stimulate an inflammatory response in vitro (28,29). The present study provides in vitro and in vivo evidence to demonstrate that 1) LMW HA or highly purified rHuPH20 do not stimulate inflammation and 2) in BTH preparations extracted from animal tissue, endotoxin, but not PH20, is a major contaminant that stimulates inflammation.…”

“…Recent evidence that supports skepticism in this area is the observation that recombinant human hyaluronidase PH20 (rHuPH20), which degrades HMW to LMW HA fragments, did not stimulate IL-8 production in HUVECs (27). Other publications also showed that low endotoxin-containing HA fragments ranging from Mr 1,000 to 1,500,000 did not stimulate IL-1a production in primary human macrophages (28), or NO production in the murine cell line RAW 264.7, rat splenocytes, or rat adherent differentiated primary macrophages (29). HA at both high and low m.w.…”

The Activity of Hyaluronan and Hyaluronidase PH20 in Inflammation-A Role by Reagent Contaminants?

“…However, there is no direct evidence that LMW HA binds to TLR2 or TLR4, and some reports have suggested that if endotoxin (a putative stimulator of inflammation) is reduced to extremely low levels in purified preparations, LMW HA does not trigger normal tissue macrophage-mediated inflammatory reactions [15,16]. Our research clearly demonstrates that endotoxin-free LMW HA fragments (molecular weight range: 5-500 kDa) do not bind either TLR2 or TLR4; nor do these fragments stimulate TLR2-or TLR4-mediated nuclear factor-kappa-B (NF-kB) pathway signaling [3].…”