Influx of OVA-FITC⁺ neutrophils in LNs increases with the number of immunizations and requires an antigen-specific response. (A-C) Mice were immunized with OVA/CFA on days 0, 15, and 30. (A) After the first (day 15), second (day 30), or third (day 40) immunization, mice were injected with OVA-FITC (▴) or PBS (▪), and 6 hours later LNs were obtained and the percentages of OVA-FITC⁺ NEs were measured by flow cytometry. Each point represents the mean ± SD. (B) The percentage of NEs in smears of peripheral blood stained with May-Grünwald-Giemsa was evaluated on days 15, 30, and 40. Each point represents the mean ± SD. The value of NEs in peripheral blood from unimmunized mice was 20% ± 5%. (C) Plasma from individual immune mice was assayed for anti–OVA IgG by ELISA on days 15, 30, and 40. Each point represents the mean of specific antibody titers (log₁₀) ± SD. IgG antibody titers were calculated as the reciprocal of the last plasma dilution that yielded an A₄₉₀ above that of the double-mean value of preimmune plasma. (D) OVA-antibody complexes or soluble OVA-FITC were injected in unimmunized or PBS/CFA-immunized animals. Six hours after injection, LN cells were counted, and fluorescent cells were analyzed by flow cytometry. Results are expressed as the mean number of OVA-FITC⁺ NEs/LNs ± SD. (E) OVA/CFA-immunized or KLH/CFA-immunized mice (day 40) were injected in the footpad with OVA or KLH. As the control, a group of mice were immunized with PBS/CFA and injected (day 40) with OVA. After 6 hours, LNs were obtained, and the cells were stained with anti–Gr-1 and analyzed by flow cytometry. The percentage of NEs (Gr-1^high) is indicated (black line); the gray area corresponds to cells stained with isotype-matched control antibody. One typical experiment of 4 performed is shown (n = 3-4 mice per group).