High PCNA protein levels in neutrophils isolated from G-CSF–treated donors and from patients with systemic inflammation. (A) Neutrophils from a representative G-CSF–treated donor were analyzed either before or during (+G-CSF) cytokine exposure, with PCNA immunodetected in lysates from either freshly isolated (0) or 15-h cultured cells. The percentages of viable neutrophils after culture as in Fig. 3 B are shown in the histogram above the blot. (B) Immunofluorescence analysis by confocal microscopy of neutrophil PCNA detected with the Ab5 pAb. Neutrophils were isolated from a donor before or during (+G-CSF) in vivo G-CSF treatment. Data in A and B are from one G-CSF–treated donor, representative of four different donors. Bars, 10 µm. (C) Total RNA was extracted from neutrophils isolated from healthy donors or G-CSF–treated patients and assayed in triplicates. PCNA and IL-1ra mRNA was normalized to β2m expression and expressed as mean normalized expression (MNE) units. mRNA analysis was performed in three donors before and during G-CSF treatment (*, P < 0.05). (D) Representative Western blots showing neutrophil lysates prepared just after cell isolation from a control subject (CT) or from two patients with sepsis (SP1 and SP2) or Wegener’s granulomatosis (WG1 and WG2). (E) Densitometric analysis of PCNA expression in healthy subjects (CT; n = 7), patients with sepsis (SP; n = 5), and patients with Wegener’s granulomatosis (WG; n = 8). Data are expressed as arbitrary units (AU; means ± SEM; *, P < 0.05; Student’s t test). (F) Analysis of PCNA mRNA expression in neutrophils isolated from sepsis (n = 3) and Wegener’s granulomatosis (n = 5) patients or healthy donors (CT; n = 7). (C and F) Horizontal bars represent the mean value.