Figure 6: CCL8 is exclusively produced by CD169⁺ macrophages in response to sterile and non-sterile inflammatory stimuli.

(a) CCL8 concentration in the culture medium of colon explant was quantified by ELISA and divided by the dry weight of the explant. Average and s.d. of three mice. Representative data from two independent experiments. *P<0.05, Student’s t-test. (b) LP myeloid cells from the colon of indicated mice were cultured overnight in vitro. CCL8 concentrations in the culture medium were quantified. Average and s.d. of three mice. *P<0.05, two-way analysis of variance (ANOVA). (c) CD11b⁺, CD169⁺ and CD11b⁺, CD169⁻ cells from the colon of WT naive or colitis mice were fractionated by a cell sorter. Different numbers of fractionated macrophages were cultured in vitro. CCL8 concentrations in the culture supernatant were quantified. Representative data from two independent experiments. Error bar marks s.d. *P<0.05, two-way ANOVA. (d–f) Bone marrow (BM) cells were cultured for 5 days in the presence of M-CSF (black bar) or GM-CSF (white bar) to induce or not to induce CD169 expression. Those cells were treated in vitro with various stimulants for 24 h. CCL8 (d), TNFα (e) and IL-6 (f) concentrations in the culture medium. Average values and s.d. of a triplicate experiment. Statistical differences relative to unstimulated BM cells (−) were determined. *P<0.05, Student’s t-test. Representative data of two independent experiments. (g) Immunohistochemistry of colon sections from DSS-fed WT mice. Arrowheads indicate contact of CD169⁺ cells with TUNEL⁺ cells. Original magnification, × 20. Scale bars, 100 μm. (h) CCL8 attracts monocytic WEHI-3 cells in vitro. WEHI-3 cells were seeded in the upper chamber, whereas the lower chamber was supplied with serum-free medium with or without 100 nM CCL8. The number of cells in the lower chamber at 4 h was enumerated. Average values and s.d. of triplicate experiment. *P<0.05, Student’s t-test. Data are representative of three independent experiments. (i) Cells that migrated towards CCL8 in vivo showed mononuclear morphology. Cells retrieved from the Matrigel plugs supplemented with CCL8 were cytospun onto a glass slide, and stained with modified Giemsa. Scale bar, 10 μm. (j) The number of cells per milligram Matrigel plug was quantified by flow cytometry. Average values and s.d. of three mice. *P<0.05, Student’s t-test. Data are representative of three independent experiments. DAPI, 4,6-diamidino-2-phenylindole.