Skip to main content
. Author manuscript; available in PMC: 2019 Sep 18.
Published in final edited form as: Immunity. 2018 Aug 28;49(3):449–463.e6. doi: 10.1016/j.immuni.2018.07.012

Figure 4: CCL8 is produced in the dLN by CD169+SIGN-R1+ LN macrophages that interact with CD301b+ DCs.

Figure 4:

(A) Confocal immunofluorescence of whole popliteal LN (top) and area of interest (bottom) 24 hrs after Th2-skewing immunization in WT or Ccl8−/−Ccl12−/− mice as indicated. Panels show single stains of CCL8 (white), CD169 (blue), CD301b (red) and overlay. Scale bars in top panels represent 200μm, scale bars in bottom panels represent 10μm. Images are representative of 3 mice in each of 3 independent experiments. (B) Confocal immunofluorescence of whole popliteal LN with insets showing CD169+SIGN-R1+ macrophage populations in medullary regions (med) and IFR (IFR), as well as CD169+SIGN-R1− SCS macrophages in the SCS (SCS). CD169 (red), SIGN-R1 (white), gp38 (green), and B220 (blue). Scale bar in whole LN represents 200 μm, those in insets represent 10 μm. (C) QPCR analysis of total cells (Pre) or MACS positively selected SIGN-R1+ or SIGN-R1− cells from popliteal LNs. (D) Gating scheme to identify macrophage populations from total viable lymphocytes obtained 24 hours after Th2-immunization from dLN, and QPCR analysis of the indicated populations showing Ccl8 expression. Colored histograms correspond to colored gates. (E) QPCR from total LNs of control liposome- or clodronate-treated mice 24 hrs after Th2-mediated immunization. Data points are from individual mice (4-6) in one representative experiment out of three independent trials. (F) Decreased percentage and number of CD301b+ DCs in Ccl8−/−Ccl12−/− mice after Th2-skewing immunization, based on flow cytometry of viable CD11c+CD301b+ cells. Data points are from dLNs of individual mice (3-4) from one of three independent trials. Cells in (C) and (D) were obtained using enhanced enzymatic digestion, while those in (F) were obtained using routine enzymatic digestion. QPCR data presented as copies of indicated transcript over Gapdh. Bar graphs represent mean value of indicated data points, error bars represent SEM. * p<0.05, ** p<0.01, *** p<0.001, Student’s T test. See also Figure S4 and S5.