(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.