FIGURE 7.

The effect of IαI on TSG-6 enhancement of HA binding to CD44. A, representative histograms showing the distribution of the fluorescence of 10,000 cells for fl-HA binding to CD44 in the absence of TSG-6 (sample (i)), in the presence of TSG-6 (sample (ii)), and in the presence of TSG-6 together with IαI (samples (iii) and (v)). TSG-6 and IαI in samples (sample (iii) and (v)) were incubated together at different sample preparation stages. In sample (iii), the TSG-6 was incubated with IαI prior to the addition of fl-HA and cells. In samples (iv) and (v), TSG-6 was incubated with HA. IαI was subsequently added after the addition of cells in sample (iv), i.e., after the formation of a TSG-6 enhanced film, and before the addition of cells in sample (v). Final concentrations of 1 μg/ml fl-HA, 0.3 μm TSG-6, 1 μm IαI, and ∼5 × 10⁵ AKR1/CD44+ positive cells were used throughout; fl-HA, TSG-6, and IαI were incubated for 30 min, and cells were incubated for 60 min, all at 37 °C. B, mean fluorescence intensity for the negative control sample (i.e., PBS alone) and for each sample (samples (i)–(v); defined above). IαI reverses the enhancement of the CD44/HA interaction induced by TSG-6 in all assays, yet the degree of reversal depends on the incubation sequence. The data are plotted as means of three independent experiments performed in triplicate (± S.E.).