Neutrophils and MDMs are the relevant IL-1β–producing cells in the acute phase of EAE. (A) Representative immunoblots for bioactive IL-1β and Casp-1 performed on cell lysate extracts obtained from the lumbar spinal cord of EAE mice at different days after immunization. (B and C) Densitometric quantification of immunoblot revealed that the bioactive forms of IL-1β (p17; B) and Casp-1 (p26; C) were converted from their respective pro-forms beginning at the onset of EAE. Results were normalized with β-actin. (D and E) Flow cytometric analysis of whole spinal cords of pIl1b-DsRed mice collected at 14 dpi (n = 4). Individual leukocyte populations were immunophenotyped based on their expression (or lack thereof) of CD11b, CD3, Ly6C, and Ly6G, whereas expression of the fluorescent reporter DsRed was used to identify pro–IL-1β–producing cells. The percentage of all DsRed-expressing cells identified as microglia (CD45^dimCD11b^dimLy6G^–), T lymphocytes (CD45^hiCD11b⁻CD3⁺), MDMs (CD45⁺CD11b⁺CD3⁻Ly6C^int-hiLy6G⁻), and neutrophils (CD45⁺CD11b⁺CD3⁻Ly6C^intLy6G^hi) is shown in D. (F) To further correlate the presence of DsRed with IL-1β, DsRed⁺ neutrophils and MDMs, as well as DsRed⁻ MDMs and T cells, were sorted, and Il-1β and Casp-1 mRNA levels were analyzed by real-time quantitative RT-PCR. (G and H) Flow cytometric analysis of bone marrow (G) and blood (H) samples from pIl1b-DsRed mice at various days after immunization (n = 5 mice/time). The percentage of total neutrophils and DsRed⁺ cells are expressed as a ratio of total CD45⁺ leukocytes. (I) Percentage of DsRed⁺ cells in the blood that are neutrophils, monocytes, lymphocytes, or other cells (n = 5 mice/time). (J) Percentage of neutrophils producing pro–IL-1β (DsRed) in the bone marrow, blood, and spinal cord at the peak of clinical score (14 dpi). All data are expressed as mean ± SEM. **, P < 0.01; ***, P < 0.001, compared with naive mice; one-way ANOVA followed by Dunnett’s post-hoc test (B, C, G, and H).