Neutrophils present in LNs mainly expressed TNF-α and their depletion resulted in an antigen-specific increase in IL-5 levels. (A-B) Mice immunized with OVA/CFA were injected with PBS or OVA, and 6 hours later LN cells and peripheral blood were obtained and analyzed for intracellular cytokines. Cells were stained with anti–TNF-α, anti–IFN-γ, anti–IL-12, anti–IL-4 (black line), or an isotype-matched control antibody (gray area) (histograms gated on Gr-1^high cells). One typical experiment of 3 performed is shown. (C-D) OVA/CFA-immunized mice were treated with isotype control (undepleted) or RB6-8C5 (NE-depleted) antibody. On day 40 (C) (6 hours after injection of OVA) and on day 42 (D) (48 hours after injection to OVA), LN cells were processed for flow cytometry. Density plots are representative of 3 to 4 mice analyzed. (E) OVA/CFA-immunized mice treated with isotype control (undepleted) or RB6-8C5 (neutrophil-depleted) antibody were injected on day 40 in footpad with OVA, 48 hours later LN cells were removed and then cultured for 72 hours with 100 μg/mL OVA (3.5 × 10⁵ cells/200 μL/well). The supernatants from triplicate cultures were pooled, and cytokine content was measured in triplicate by capture ELISA. *P < .005 compared with undepleted mice. One typical experiment of 3 performed is shown (n = 3-4 mice per group).