628996287

BLOOD. 1 SEPTEMBER 2005 • YOLUME 106. NUMBER 5

HO-1 INHIBITSDC FUNCTION 1697

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Figurą 2. HO-1 expression decreases during rat and human DC maturation. (A) Western biot analysis of HO-1 expression in rat immalure (IM) BMDCs and foliowi ng maturation with TNF-a. poly(l:C). CpG. CD40L. and LPS was carried out using an anti-HO-1 anlibody. Anti-p tutulin was used as a loading conlrol. Bar graph shcws densitorretry analysis i SDof HO-1 signalsafter normalization with p-tubulin trem 3 differenl experiments. (B) Western biot analysis of HO-1 expression in human immature (IM) monocyte-derrved DCsand human DCsfollowing maturation induclion with TNF-u. poly(l:C). CD40L. and LPS. performed using an anti-HO-1 antibody. Anti-p tubulin was used as a loading control. Bar graph shows densitometry analysis i SD oł HO-1 signals. ałter normalization with p-tubulin. trom 2 differenl experiments. (C) Time-course Western biot analysis ot HO-1 expression in human immature (IM) monccyte-derrved DCsand human DCsłollowing maturation induclion with LPS. performed with an anti-HO-1 antibody. Anti-p tubulin was used as a loading control. Bar graph shows densitometry analysis of HO-1 signals after normalization with p-tututin. (D) Human immature (IM) ironocyle-derived DCs were cullured with or withoul 20 ng''mL ol IL-10 for 1S hours and then with or withou! 1 p-g^mL oł LPS lor 24 hours. Western biot was perlormed with an anti-HO-1. Anti-p tubulin was used as a loading control. Histograms show densitometry analysis r SD of HO-1 signals after normalization with p titoulin. from 2 different experirrents.

HO-1 expression decreases during rat and human DC maturation

HO-1 expression was analyzed after maturation of rat and human iDCs induced by poly(I:C). LPS. or CpG. as well as by stimulation with inflammatory and lymphocyte stimuli, such as TNF-a or CD40L. respectively. DC maturation was confinned in eoch experiment by an enhanced expression of MHC II. CD80. and CD86, and by secretion of tlie cytokines IL-12, IL-6. TNF-a. and IL-10. HO-1 expression by rat BMDCs was significantly decreased compaied to control untreated DCs after maturation induced by all of the stimuli tested (mean decreose % ± SD): TNF-a. 83% ± 1%: poly(I:C), 82% ± 7.4%: CpG. 69% ± 2%: CD40L. 81% ± 2.5%: and LPS. 73 ± 15% (n = 3 for each, /^} < .05; Figurę 2A). Inhibition of HO-1 expression on DC maturation was also observed in rat spienić OX62⁺ DCs and pDCs (data not shown).

As obsened in rat BMDCs, HO-1 expression in human monocyte-derived DCs was strongly decreased (mean decrease % ± SD) during DC maturation in response to TNF-a (90% ± 8.5%), poly(LC) (81% ±7%), TNF-a plus poly(LC) (98.5% ± 4%). LPS (98.5% ± 2%), and CD40L (97 ± 3%; n = 2 for each: Figurę 2B).

Kinetic analysis of HO-1 levels after LPS stimulation of human DCs showed Ievels compai able to tliose of untreated DCs at I hour and drastically redueed levels at 6. 12. 24. and 48 hours of stimulation (20%, 11%, 6%, and 2% versus untreated cells; Figurę 2C).

Moreover. expression of HO-1 in rat and human iDCs and its strong deciease following maturation (> 85%. P < .05. n = 3)

was conłirmed by measuring HO-1 niRNA by quantitative real-time RT-PCR (Figurę S2).

These data demonstrate that HO-1 expression in rat and human DCs is inhibited at the transcriptional or mRNA (orboth) levels by Toll-like receptor (TLR), inflammatory. and lymphocyte stimuli.

HO-1 expression is inereased by IL-10

Because IL-10 is potent at maintaining DCs in an immature State.^lfx:T we analyzed whetlier HO-1 expression could be inereased by IL-10. As compared to LPS-treated DCs or untieated iDCs (100%: n = 2). pretreatment with IL-10 inereased HO-1 expres-sion levels (mean % ± SD) in iDC’s (378% ± 23%) and in IL-10 plus LPS-treated DCs (240% ± 113%: Figurę 2D). The inhibitory effect of IL-10 on LPS-induced DC phenotypic maturation was confinned by FACS (Figurę S3).

These data demonstrate that inhibition of DC maturation by IL-10 is associated with an inerease in HO-1 expression.

HO-1 is expressed by immature DC-SIGN⁺ but not by maturę DC-LAMP+ DCs in human tissues

We next investigated whetlier this dichotomy in HO-1 expression between iDCs and matmę DCs also exists in vivo using chionically infected tonsils and healthy skin. Two specilic DC markers were used to identify DCs in human tissue sections: DC-SIGN. a C-type lectin expiessed in interstitial iDCs but not Langerhan cells.^2s"'' and DC-LAMP. a marker of maturę DCs.³⁰ As previously de-scribed,²⁸‘^V) human skin displayed many DC’-SIGN ‘ cells in the dermis but not in the epidennis (Figurę 3) and DC-LAMP⁴ cells were absent (data not shown). Many HO-1⁺ cells were observed in tlie dennis and an almost complete overlap was obsei ved between HO-1⁺ and DC’-S1GN⁺ cells (Figurę 3).

In contrast. and as previously desciibed for human cluonically infected tonsils.^{28 3}" DC-LAMP was expressed by numerous cells (Figurę 3), whereas DC-SIGN was nearly absent (data not shown). Only a few weakly HO-l⁺ cells were found in this tissue (Figurę

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Figurą 3. Dątection of H0-1^f and HO-1- DCs in human tissues. (A) Conlocal micrograpbs of cryoslat sections from human skin show HO-1 cells (grsen fluorescence). DC-SIGN celis (red llucrescerce). and rrerged images with dual labeling. Derma I iDCs show complete overlap ot HO-1 and DC-SIGN expression. The epidermis (‘) shoivs v«y \an or absent expression of toth HO-1 and DC-SIGN. (objective. x 4Qh .0). (B) Conłccal micrographs of cryostal sectbns from a chrccti-calfy infected human lonsil show feiv weakly HO-1 cells (green fluorescence. arrow). DC-LAMP cells (red fluorescence). and rrerged images with diet labeling. Tonsil malure DCs expressing DC-LAMP do nol express HO-1 (ob)ective. x 63/1.4). Results are representatrv'e oł 3 differenl skin and tonsils anafyzed in the same way.