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Blood, Vol. 112, Issue 10, 4117-4127, November 15, 2008
Resolution-phase macrophages possess a unique inflammatory phenotype that is controlled by cAMP
Blood Bystrom et al.
112: 4117
Supplemental materials for: Bystrom et al
Files in this Data Supplement:
- Figure S1. Dynamics of macrophage trafficking in resolving inflammation (PDF, 177 KB) -
In the first set of experiments, peritoneal inflammatory cells from animals stimulated with (A) 0.1 or (B) 10mg zymosan were double labelled with Ly6C and F4/80 and analysed by FACS which revealed a transition from monocytes early in the response to fully differentiated macrophages from 24h onwards, panels (I–V). Given that monocytes differentiated into macrophages at 24h, animals bearing inflammation elicited by either (C) 0.1 or (D) 10 mg zymosan were then injected intraperitoneally at this time point with the macrophage marker PKH26-PCLred and samples analysed by FACS at 72h. Panel C/D (II), shows macrophages labelling positively with F4/80 (R1) compared to the isotype control, panel C/D (I). Similarly, panel C/D(IV) shows populations of cells labelling positively for PKH26-PCLred (R2) compared to cells taken from animals injected with PKH26-PCLred dye diluent. Panel C/D (V) shows cells in the top right quadrant labelling positively for PKH26-PCL also labelled for the macrophage marker, F4/80. 12h after PKH26-PCLred injection animals were given a second injection of PKH26-PCLgreen i.p. the idea being that subsequent macrophage trafficking would label positively for PKH26-PCLgreen only. This resulted in few macrophages labelling positively for green only in resolving inflammation (E, top left quadrant) while about 15% in the 10mg model (F, top left quadrant) labelled for green only. These data indicate that in resolving inflammation rM are derived from a common Ly6C-positive monocyte precursor that migrated into the peritoneum by 24h while in non-resolving inflammation there is a more prolonged influx of monocytes over a broader time frame than when inflammation is self-limiting. n=6–8 mice per group with data expressed as mean ± SEM.
- Figure S2. Proteomic analysis of resolving inflammatory exudates (PDF, 444 KB) -
(A) Exudates from either resolving inflammation (0.1mg zymosan) or from mice injected with 10mg zymosan i.p. that progressed to systemic inflammation were depleted of serum albumin and IgG. 100µg of protein from these exudates were run on 10% (LHS lanes) or 7.5% (RHS lanes) PAGE gels. Gels were silver stained and bands of interest excised from the gel with resultant proteins identified by Mass spectrometry. (B) 50 µg protein from exudates of mice injected with 0.1 mg (upper gel) or 10 mg (lower gel) zymosan were run using 2 Dimensional Electrophoresis. Gels were silver stained and spots of interest were picked and analysed by Mass spectrometry. In addition, (C) western blotting, which was controlled for by (D) pre-absorbing the primary antibody with blocking peptide (5-fold higher concentration compared to primary antibody), revealed the differential expression of the pro-inflammatory protein HMGB1 being absent from resolving inflammation but present at high levels in ongoing inflammation. These experiments highlight a differential profile of proteins specific to resolving inflammation that are considerably lower or absent from ongoing inflammation. Moreover, given the abundance of these factors we found that although the phenotype of macrophages from 10mg zymosan-induced peritonitis can be altered by (E) cAMP elevating agents (rolipram, 30mg/kg or theophylline 100mg/kg), this change in phenotype has no effect on (F) resolution as defined by peritoneal total cell numbers. n=6–8 mice per group with data expressed as mean ± SEM.
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