INT-HA induced early activation of monocytes and subsequent formation of immunosuppressive Mφ. (A) HMW-HA (lane 1) was digested by exposure to hyaluronidase for 30 (lane 2) or 60 (lane 3) minutes, and the resulting INT-HA fragments were separated by agarose gel electrophoresis and visualized with Stain-All. HA monomer was loaded in lane 4. (B) Monocytes were cultured for 18 hours in the presence of 10 (open bars), 25 (shaded bars), or 50 (solid bars) μg/mL of the different HA preparations. Levels of TNF-α and IL-10 in the medium were determined by enzyme-linked immunosorbent assay. (C-D) Cells were cultured for 6 days in medium alone (solid lines) or with 50 μg/mL INT-HA from 60-minute digestion (dashed lines). Thereafter, Mφ were left untreated (Med) or were stimulated with LPS or IFN-γ for 18 hours. Expression of cell surface markers and release of cytokines were determined as described in Figure 1. Data given are means (± SE; n = 4); **P < .01, compared with Mφ cultured in medium alone.