Different molecular weight hyaluronic acid effects on human macrophage interleukin 1β production

Abstract

This study examined the effect of hyaluronan (HA) molecular weight on immune response. HA with molecular weights ranging from the unitary disaccharide unit (400 Da) up to 1.7 × 10(6) Da and with very low endotoxin contamination level (less than 0.03 EU/mg) was used. Primary human monocyte/macrophage cultures were assayed for IL-1β production under a variety of inflammatory conditions with or without HA. Under the highest inflammatory states, production of interleukin 1β (IL-1β) was suppressed in the presence of high molecular weight hyaluronan (HMW-HA) and in the presence of low molecular weight hyaluronan (LMW-HA) at mg/mL concentrations. There was variability in the sensitivity of the response to HA fragments with MW below 5000 Da at micromolar concentrations. There was variability in IL-1β cytokine productions from donor to donor in unstimulated human cell cultures. This study supplements our previous published study that investigated the immunogenic effect of HA molecular weights using murine cell line RAW264.6, rat splenocytes, and rat adherent differentiated primary macrophages. These data support the hypothesis that if the amount of 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

“…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…”

“…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].…”

Bacterial endotoxin detection in hyaluronic acid-based medical devices

“…Possible sources of contamination include water, raw materials that contain lipopeptides, peptidoglycans, endotoxin, or other proteins. 14,22 Bacterial endotoxin is known to bind to other chemicals as well as to many surfaces, 23 revealing lower than tangible levels in the actual device. For example, the LAL assay could be exaggerated (3% HA of OVD) or underestimated (endotoxin-spiked dermal filler), especially in high-viscosity HA solutions or gels.…”

“…12 FDA researchers investigated the causes of TASS outbreaks as adverse events 13 ; and developed new testing methods to determine the inflammatory potential of suspected contamination of subject devices. [14][15][16][17][18][19][20][21] In 2012, Buchen et al evaluated an inflammatory potential of cohesive and dispersive OVDs which were spiked with known endotoxin concentrations, 13 and were measured using the LAL kinetic turbidimetric assay. Rabbits were given known concentrations of bacterial endotoxins mixed with HA and the researchers noted the concentration at which of endotoxin caused inflammation in the eye.…”