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

Hyaluronan (or hyaluronic acid; HA), a component of the extracellular matrix (ECM), is composed of repeating polymeric disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine linked by a glucuronide bond. Hyaluronan is abundant in a variety of human tissues, including skin, umbilical cord, synovial fluid, cartilage, and skeletal tissues, and is turned over through the catabolic activity of hyaluronidases [1]. In addition to its structural role in the ECM, both intact high molecular weight (HMW) HA (>1,000 kDa) and its catabolites (low molecular weight (LMW) HA; 25-1,000 kDa or smaller oligoand disaccharides) exert various biologic effects [2]. A number of biologic activities have been attributed to HA, including stimulation of inflammation and tumor growth [1,3]. Various reports in the literature suggest different roles for HMW and LMW HA in processes such as inflammation and wound healing. For example, antiinflammatory effects have been suggested for HMW-HA based on its inhibition of leukocyte migration in the murine air pouch model [4], reduction in gene expression levels of cytokines and matrix metalloproteases in arthritis models [5,6], and reduced production of interleukins in a model of ultraviolet (UV) light-induced corneal damage [7]. In contrast, small oligosaccharides derived by in vitro digestion of HA using hyaluronidases have been reported to stimulate inflammation in cell culture [8], and it has been suggested that LMW HA fragments which accumulate at sites of tissue injury and repair may stimulate inflammatory gene expression in macrophages [9]. In addition, a recent report suggests that hyaluronidase may increase inflammation and angiogenesis to promote wound healing in an animal model [10]. Taken together, these reports have raised concerns about potential inflammatory adverse effects when hyaluronidases, such as recombinant human PH20 (rHuPH20), are used therapeutically. However, the true effects of LMW HA and hyaluronidases on inflammation are unclear and, in many cases, the reported effects may be attributable (at least in part) to proinflammatory contaminants, particularly endotoxins and peptidoglycans, in many commercially available hyaluronidase reagents which are often used to generate HA fragments in experimental models. Indeed, the available evidence shows that the pro-inflammatory activity of commonly used test reagents is directly correlated with their level of endotoxin and peptidoglycan contamination (Table 1) [2].