(A) TNF- expression by peritoneal neutrophils after thioglycollate challenge. Peritoneal lavage cells were incubated with monesin for 5 hours to block transport of proteins from the golgi apparatus, permeabilized, and stained with FITC-labeled Gr-1 and PE-labeled anti-mouse TNF-. Panel a shows box and whisker plot of geometric mean of fluoroscent intensity as determined by flow cytometry. (▪) Represent wild-type neutrophils, gray boxes represent C/EBPɛ-deficient neutrophils. An asterisk (*) indicates TNF- staining is significantly increased in C/EBPɛ-deficient neutrophils at 4 hours compared with wild-type neutrophils (P < .005, Mann Whitney U). Panel b shows representative dot blots of wild-type and C/EBPɛ-deficient neutrophil samples (+/+ wild-type; −/− C/EBPɛ-deficient). (B) Cytokine expression by ribonuclease protection assay in peritoneal neutrophils after thioglycollate challenge. Arrows show indicated cytokine transcripts as determined by size. L32 and GADPH bands represent controls for sample size. C/EBPɛ-deficient neutrophil RNA (−/−); wild-type (+/+). (C) TNF- expression in circulating, unstimulated neutrophils as determined by intracellular cytokine staining. Results are represented graphically by geometric mean of fluorescent intensity: (□), unlabeled neutrophils; (▨), TNF-–labeled neutrophils. Fluorescent intensity of labeled and unlabeled wild-type neutrophils was not statistically different (P = .999). Asterisk shows indicated statistical significance of TNF- labeling of peripheral blood C/EBPɛ-deficient neutrophils (P < .001, Student’s t-test).