Figure 2.

CXCL1 drives aging-associated neutrophil reverse TEM

(A–D) Young and aged mice were treated i.s. with PBS or IL-1β. (A) Inflammatory mediator analysis in homogenized cremaster muscles as assayed by protein array (n = 3 mice/condition).

(B) CXCL1 levels in cremaster muscles (n = 4-7 mice/group) or (C) plasma (n = 4–8 mice/group) as quantified by ELISA.

(D) Frequency of neutrophil rTEM in Y→Y or Y→A chimeras (generated as detailed in Figure 1H) treated i.v. with isotype control, anti-CXCL1 or anti-CXCL2 blocking mAbs (n = 3–5 mice/group).

(E) Representative confocal images of mast cells (MCs; Avidin) associated with post-capillary venules (CD31) in young and aged unstimulated WT cremaster muscles (scale bar: 20 μm) and quantification in (F) cremaster muscles, and (G) ear skin (n = 5-7 mice/group).

(H–I) Analysis of CXCL1 expression in MCs of young and aged IL-1β-stimulated cremasteric tissues by confocal microscopy with (H) showing representative images and (I) quantification by MFI (scale bar: 5 μm; n = 3–7 mice/group).

(J) Representative confocal images of MCs (CD117) in young and aged unstimulated WT ear skin (scale bar: 10 μm) and quantification of MC volume (n = 4 mice/group). (K) Peritoneal MCs acquired from unstimulated young and aged mice assayed for SA-β-galactosidase activity by flow cytometry (n = 6-13 mice/group). (L) Frequency of neutrophil rTEM in control and MC depleted IL-1β-stimulated cremaster muscles of aged chimeras (see Figure 1H; n = 4-5 mice/group). (M) Frequency of neutrophil rTEM in IL-1β-stimulated ear skin of aged MC deficient (Mcpt5-Cre-R-DTA) mice and littermate controls (n = 5 mice/group). Means ± SEM, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 relative to controls, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 as indicated.

See also Figure S2.