Figure 5.

Human CRP triggers iROS production by human peripheral blood neutrophils and grants them the capacity to suppress the proliferation of autologous T cells. Peripheral blood neutrophils were isolated from five healthy human donors (see the Materials and Methods) to assess CRP's impact on their production of iROS and suppression of T cell proliferation. (A) ROS generated by human neutrophils stimulated with PMA (100 nM) vs. human CRP (100 μg/ml) was measured by luminol assay. Similar to mouse MDSCs (see Figure 2A), PMA stimulated human neutrophils showed a strong respiratory burst and those stimulated with CRP did not. Untreated controls are shown in the inset. (B) Intracellular ROS generated by human neutrophils stimulated with PMA (100 nM) vs. human CRP (1–100 μg/ml) was measured with the cell-permeant redox-sensing fluorescent dye H₂DCFDA. Similar to mouse MDSCs (see Figure 2B), human neutrophils exhibited a significant increase in iROS-dependent fluorescence intensity when stimulated with 100 nM PMA or high concentrations of human CRP. The data shown are the mean ± SEM for two donors each done in triplicate and the asterisks indicate significantly higher RFI than untreated controls (shown in the inset) by Sidak's multiple comparisons tests; ^*p < 0.05 at 60 min, ^**p < 0.05 at 30 min, and ^***p < 0.05 at 14 min for CRP, and ^#p < 0.05 at 12 min for PMA. (C) Representative CFSE dilution histograms (normalized to mode) for autologous human CD3⁺ T cells stimulated with anti-CD3 and anti-CD28 mAbs. From bottom to top the T cells received no further treatment, neutrophils (Neu at 1E:1T), or Neu plus 10–100 μg/ml human CRP. The addition of CRP bestowed suppressive capacity to the autologous neutrophils in a dose-dependent fashion, as indicated by the rightward shift in the CFSE histograms. (D) CD4⁺ T cell proliferation in the presence of Neu performed as in (C). The data shown are mean + SEM pooled across donors and are normalized to CD3/CD28 stimulated T cells in the presence of Neu with no CRP added (cross-hatched bar). The asterisk indicates significantly more suppression of T cell proliferation (p < 0.05 for Sidak's test) in the presence of CRP than in its absence.