628996286

1696 CHAUVEAU et al

BLOOD. 1 SEPTEMBER 20C6 • VOLUME 106. NU MB ER 5

Flow cytometry

Rat !)Cs were stained willi the following PE- or FITC-conjugated mAbs: anti-MHC class II. anti-CDSO. anti-CD86 (all from BI) PharMingen) and anti-CD54 (Serotec. Oxford. United Kingdom). Humań DCs were stained willi the following PE- or FITC-conjugated mAhs: anti-CD80. anti-CD83. anti-CD86. and anti-HLA-ABC (all from Im mu no te di). Staining was assessed using a FACSCallbur flow cy tonie ter and CellQucst software (Becton Dickinson. Pont de Claix. I rance). PE- or HTC-labeled niou.se anti-IgG 1 Abs (Immunotech) wene used as negative Controls.

Cytokine measurement

Rat and human IX's were culturcd for 2 days willi various stimuli. Supernatants were serially dilutcd. and cytokine concentralion assessed in duplicate by enzyme-linked immunosorbent assay (MI.ISA). Rat KI.ISA kits lor II.-6. II.-IO, TNF-a (OptKIA set: BI) PharMingen) and II.-12 p40 (Biosource. Camarillo. CA) as well as human II.-12p70 and II.-10 KI.ISA kits (BI) PharMingen) were used.

Measurement of reactive oxygen species

Rat iBMDCs or human iDCs were treated with CoPP or SnPP. culturcd for 48 hours, and incubated or not willi KPS for 15 minutes. DCs weie incubated willi 10 mM iV-acetyI-t.-cysteine (NAC) for 45 minutes prior to incubation willi KPS. DCs were Hien incubated willi 5 |xM of the oxidalive sensitiee dye CM-H2DCIDA (Molecular Prohes) for 15 minutes. Samples were then washed and analyzcd by cytofluorimetiy.

Statistical analysis

Stalistical significance was assessed using the nonparametric one-way AN()VA test with a post test. Differences were considered signiflcant when P < .05.

Results

Expression of HO-1 in rat and human iDCs

To ineestigate the capacity of DCs to express HO-1. we analyzed several previously desciibed rat spienić DC subpopulntions¹'²⁶ and BMDCs.^,8ns well as human monocyte-derived DCs. Western biot analysis showed that freshly isolated and purified rat spienić OX62⁴ DCs and pDCs (OX62“). and rat iBMDCs expressed HO-1 (Figurę 1 A). Spienić B cells were used as negative Controls (Figurę 1A). Purified rat OX62⁴ DCs include 2 distinct subsets.¹⁷ A subpopulation of CD4“CD1 lb+SIRP-l “CD5“ DCs. which are effectiee at presenting antigens to CD4⁴ but not to CD8⁴ T cells. produce Iarge amounts of IL-12 but not interferon o (IFN-«). and induce Th 1 responses.¹⁷ The second subpopulation of OX62⁴ DCs me CD4⁺CD1 Ib⁴ S1RP-1⁺CD5⁺ cells that are effective at presenting antigens to CD4⁴ and CD8⁴ T cells, produce Iow amounts of IL-12 and IFN-a. and induce Th2/Th0 responses.¹⁷ Intracellular staining with an anti-HO-1 Ab revealed that the CD4" DC population did not express HO-1 and that HO-1 expression was restricted to the CD4⁴ DC population (32% ± 12% expressed HO-1, n = 3; Figurę IB). HO-l-expressing cells also expressed MHC class II antigens and were only found aniong SIRP-1⁴ and CD5⁴ cells (Figurę SI: see tlie Supplemental Materials link at the top of the online aiticle. at the Blood website). HO-1 expression %vas detected in 74% ± 12% (n = 2) of freshly isolated CD45RB⁴ pDCs (CD4⁴CDI Ib^-26; Figurę IB). HO-1 expression was also detected in 80% ± 20% (n = 5) of iBMDCs (Figuro IB). All rat iBMDCs that expressed HO-1 also expressed bilirubin, which can only derive from heme degradation (Figurę IC). Bilirubin was

52 kDi —    HO-1

»I.D« -    — — ^ Tulwlłi

Rat spienić DCs

	HO-1	CD4	mcigcri
0X62+
			o
	HO-1	CD45R	merged
pDCs	• * .		V.
	HO-1	WHC class II	merged
BMDCs
	/		/
c Rat BMDCs
	HO-1	bilirubin	merged
	9 «		ę V
			a i \ 1 ’f‘
D Human DCs
	HO-1	bilirubin	mergerl 1

Figurę i. Rat and human DCsexpress funclional HO-1. (A) Western ttot analysis of HO-1 expression in rat freshly isolated spieni: 0X62 DCs. freshly isolated płasmacytdd dendriti: cells (pDCs). and BMDCs cultured fors days was performed using an anti-HO-1 antibody. Purified spienić B cells were used as a necptń/e contrcK An anli-p lubulin antibcdy was used as a loading control. (B) Confocal micrographs show rat HO-1 cells (green fluorescence): CD4 -. CD45RB . or MHC class II oells (red fluorescence): and merged images with dual labeling. Celi nuclei were counter-staired withTO-PRO-3 (blue: cbjeclive. x 63/1.4). (C) Rat immalure bonę rrerrow-derived DCs express funclional HO-1 as shown by the preserce of HO-1 cells (green fluorescence) also reactwe with an antibilirubin mAb (red fluorescence). Merged images display dual labeling. Celi nuclei were counterslained with TO-PRO-3 (blue: objecth/e. x 63/1.4). (D) Human immature monccyte-derived DCs express furclional HO-1 as shown by the presence of HO-1 cells (green fluorescence) also reactive with an antibilirubin mAb (red fluorescence). Merged images display dual labeling. Celi nuclei were counterslained with TO-PRO-3 (blue: ob)ective. x 63/1.4). Simila r results were obta ined for each DC celi type i n 2 to 5 independent experiments.

undetectable in other cells Incking HO-1, such as 293 cells (data not shown).

HO-1 expression was also detected in most immature human monocyte-derived DCs as well as intracellular staining of bilirubin, demonstrnting HO activity (Figuro ID).

In conclusion. tliese data establish that certain but not all rat iDC subsets and human nionocyte-derived iDCs express functional HO-1.