Figure 9.

The polymorphonuclear neutrophil granulocytes (PMN)-derived immunostimulatory activity is present in the <3 kDa fraction and quite heat-stable. (A) PMNs were incubated for 72 h in the presence or absence of the arginase inhibitor INCB001158 (100 μM). The PMN supernatant (PMN-SN) was harvested (total) and also further subjected to step-wise ultrafiltration with molecular weight cut off filters of 10 kDa and 3 kDa. T cells were stimulated with anti-CD3/anti-CD28-tagged beads and cultured in the different PMN-SNs (+10% dialyzed FCS) for 48 h. T cell proliferation was then determined by [³H]thymidine incorporation over 16 h (n=3) and values of stimulated cells in the presence of arginine were set as 100%. Statistical analysis refers to the control conditions of activated T cells in the presence of L-arginine and INCB001158. Statistical calculations were performed with one-way ANOVA and Tukey´s post hoc test (*p < 0.05). (B) PMNs were incubated for 72 h in the presence or absence of the arginase inhibitor nor-NOHA (1 mM). The PMN supernatant (PMN-SN) was harvested and further incubated for 30 min at different temperatures, as indicated. As controls, normal RPMI cell culture media were treated accordingly. T cells were then stimulated with anti-CD3/anti-CD28-tagged beads and cultured in the different control media or PMN-SNs for 48 h. T cell proliferation was then determined by [³H]thymidine incorporation over 16 h (n=3) and the values of stimulated cells in control medium at 37°C were set as 100%. Statistical analysis refers to the hyperactivated T cells in PMN-SN in the presence of nor-NOHA at 37°C. Statistical calculations were performed with one-way ANOVA and Tukey´s post hoc test (** p < 0.01, *p < 0.05).