Hyaluronic acid induces tumour necrosis factor-alpha production by human macrophages in vitro

Foetal wounds heal with minimal or no scar formation. High levels of hyaluronic acid (HA) have been implicated as a contributory factor. Macrophages are essential for normal wound healing, a role facilitated by secretion of an array of cytokines. Of these, tumour necrosis factor alpha (TNF-alpha) has been shown to reduce wound collagen levels and thus scarring. This study examines the ability of HA to stimulate TNF-alpha production by human macrophages. The human U937 myelomonocytic cell line was differentiated into DU937 adherent macrophages. DU937 monolayers were exposed to HA at concentrations of 0.1, 1, 10 and 100 micrograms/ml. Conditioned media from HA-exposed monolayers were assayed for TNF-alpha activity using a standard L929 fibroblast bioassay. TNF-alpha activities of HA-exposed DU937 culture supernatants were compared to those of controls and expressed as % cytotoxicity. Exposure of macrophages to HA at concentrations of 10 micrograms/ml and 100 micrograms/ml significantly stimulated TNF-alpha production, as demonstrated by % cytotoxicities expressed as median (interquartile range) of 33.5 (29-34.5)% (P = 0.03) and 77.5 (67-85)% (P = 0.029) respectively (Mann-Whitney U test). This effect was specifically associated with TNF-alpha generated during HA exposure, as these cytotoxic effects could be abolished by addition of anti-TNF-alpha antibody, reducing cytotoxicity to 9 (6.5-13.5)% and 8.5 (6-12)% respectively. These observations indicate that HA stimulates TNF-alpha production by human macrophages. TNF-alpha is known to downregulate fibroblastic collagen synthesis within experimental wounds. We suggest that the high levels of HA within foetal wounds may play a part in limiting fibroplasia, and thereby limit scarring, via an upregulation of TNF-alpha production from wound macrophages.