628996285

HO-1 INHIBITSDC FUNCTION 1695

BLOOD. 1 SEPTEMBER 2005 • VOLUME 106. NUMBER 5

Mcylan, France). I.ow-density cclls were Lsolatcd by centrifugalion on a Nycodon/ densily gradient (Nycomed, Oslo. Norway). Cells were posi-tively sclccted (>90% purily)¹⁷ using anli-ral 0X62-magnolie-activated celi sorting (MACS) microbeads and MiniMACS selection columns (Mi Ilony i Biolec. Parts, franco).

Rai plasmacytoid DCs (pDCs) were purifietl by spleen digestion in collagenase I). lollowed by centrifugalion over Hcoll-Hypaquo (Amor-sham. I es U lis. France). T-coll and partial B-coll depletion was lhon porformod by Incuhating cells willi the anti-T-cell receptor tv(B (anli-TCRap) and yfc monoclonal anlibodios (mAbs) R7.5 and V65 followed by incubalion willi a mixluroor anti-mouscand auli—rai IgG-eoated magnolie heads(I)ynal Biotoch. Oslo. Norway). Rat CD4⁺CD45RB⁺ pDCs (> 98% purily) were lhon sorted using Iho CD45RB-fluoroscoin isothiocyanale (FUG) and CD4-phycoerythrin (PU) mAbs (BI) Pharmingon. San Diego, CA) willi a lluorcscence-activated celi sorting (FACS) Aria celi sorter (UD Biosciences; donalod by lho Crddit Agricolo).

Rat bono nianow-dorivod DCs (BMI)Cs) were obtaincd as previously described.¹* Briotly. bono manow cells were culturod in medium supplc-montod willi supornatant troili COS cells transfected willi rai II.-4cDNA or rnurine granulocyte-macnophage colony-slinuilaling factor (GM-CSF) cDNA (finał concentration of 4 ng/mL and 1.5 ng/mL of eaoh cylokinc. rcspec-lively). Cultures were red willi GM-CSF and IL-4on days 5 and 6. Al day 8. adherent i m mai u re bonę marnow DCs (iBMDCs; < 1% Tcells. 1% B cells. 1% macrophagos) were used. BMDC maluralion was inducod by a 48-hour trcalment willi 500 U/mL human lumor necrosis factor « (TNf-a: BASF/Knoll, I.udwigshafon Germany), 25 pLg/ml. polyinosinic-polycyii-dylicacid (poly(I:C)),0.5 [ig/mL I.PS (botli from Sigma. Sl I.ouis. MO), 10 p.M cytosino phosphato guanhie (CpG: molil' 2006. Sigma-Genosys), or 0.5 jjig/mLCD40L (Aloxis Biochemicals. San Diego, CA) in the piosence of I g.g/mLonliancer anti-Flag mAb (Sigma). Nonadhorenl maturę DCs wvro collecled lor analysis.

Humań monocyte-derived DCs. Humań iDCs were generated as proviously described.¹⁹ Briolly. monocytes were onrichod by elutrtation (>85% CD14⁺) and cultured 6 days in medium supplomenlod willi IL-4 (40 ng/mL: AbCys. Parts. France), and GM-CSF (500 IU/mL; AbCys). Nexl, iDCs wereharvested and culturod (10⁶ cells/mL) in plates coated willi poly(2-hydroxyolhyl molha erylato) (Sigma) to prevenl cells from adher-ing. Maluralion was inducod by a 48-hour incubalion using 20 ng/mL TNF-a (AbCys). 50 p.g/mL poly(I:C) (Sigma), or 0.5pg/mL CD40L (Alexis) in the piosence of I pig/mL enhancer anti-Flag mAb (Sigma) or l|ig/mLLPS (Sigma).

T cells. Rai lymph nodo T cells were prcparod using nylon wool columns followed by nogal ive soloclion of cells expressing CD45RA (clono 0X53). CD45RU (clone His 24). CDI Ib/c (clone 0X42), and CD 161 (ckre 3.2.5). willi spedfic mAbs (all from BD PharMingen), followed by anti-mouso IgG-coalod magnolie boads (Dynal Uiotocli). Purily was routindy 98% or higher.

Treatment of DCs with metalloprotoporphyrins or IL-10 and LPS

Rat or human iDCs were pulsed for 2 hours willi 50 jxM cobalt protoporphyrin (CoPP) or lin protoporphyrin (SnPP: I\irphyrin Products. Logan. UT), an inducer and an inhibitor of HO-1. respoctiYoly.³³ " The cells were lhon washed lwice and cultured for 16 hours. Protoporphyrins were protecledfrom light ai all limes. This samo protocol has already boen shown to beeflectivc in indueing and inhibiting HO-1 actiWty.²³ Human iDCs were cultured for 18 hours willi 20 ng/mL recombinant human IL-10 (AbCys). DCs treated with metalloprotoporphyrin or IL-10 were lhon cultured for a further 24 houre willi LPS (1 jj-g/mL for human DCs |Escherichia coli 026:B6; Sigma] and 0.5 pig/mL for rai DCs |E coli 011:B4: InvivoGon, San Diego. CA]. Supernalanls and cells were subse-quently harvested for cylokinc and phonolypic analysis, respeetively. DC viabiliiy was analy/od by flow cytomelry by staining willi TO-PRO-3 iodide (Molecular Pnobes, Lugono. OR).

Mixed leukocyte reaction

Incieasing numhers of treated DCs were cultured in triplicate in round-bottom 96-wvll plates with 10⁵ allogeneic rai T lymphocytes or human poriphoral blood lymphocytes (PBLs; < 1% CI)14⁺ cells puriiied by clutriation). Proliforalion was determined 4 days lider by uptake of ³H-thymidine (Amorsham. Oisay. France: 0.5 pCi/woll [0.0185 MBq|) during the last 8 hours of culturc.

Immunohistology and confocal image analysis

Human chronically infected tonsils and healthy. nor mai skin (kindly providod by Dr K. Renaudoau and Dr B. Dreno. CHU do Nantes) were ombeddod in oni ling compound (Tissue-Tek. Flkhart, IN), frozen in isopentane and cryosectioned (5 pm). Tissue sections and DC cytospins were fixod willi 4%, paraformaldchyde (PI-A) in phosphale-bulfered salino (PBS). pH 7.4. Slidos were incubaled willi anti-MHCcIass II. anti-SIRP-1 (0X41). anti-CD5 (OX 19) (all from the liuropoan Collection of Coli Cullure. Salisbury. United Kingdom), anli-CD4. anti-Q)45RB (bolh from BD PharMingen), anli-DC-SIGN (R&D Systems. Minneapolis. MN), or PI>coupled anti-DC-LAMP (Immunotoch. Marseille. France) Abs. Slidos were next incubaled willi a biotinylaled anli-mouso Ab and a strcptavidin-Aloxa 56S conjugate (excepl for anti-DC-LAMP). I issuo sections incu-bated willi luiti-DC-SIGN and biotinylaled anti-mouse Abs were firsl incubaled willi 10% mouse serum. Nogalive Controlsincludedanti-human CD16 mAb (IgG 1 )and a mouse IgG 1 isotypo conlrol (BI) PharMingen) for rai cells and human cells/lissuos. respectiwly. Cells or tissues were postfixed willi 4% PFA. pormoabilized willi 0.5% saponin, incubaled with a rabbil anli-ral HO-1 or mouse anti-human HO-1 Ab (Stressgen. Vicioria. BC. Canada). followed by incubalion with secondaiy FITC-labeled Abs. Coli nucie i were countcrctained with TO-PRO-5 iodide (Molecular Probos) and slidos were mounted in ProLong Ant i Fa de reagonl (Molecular Probos). Slidos were analy/od with a Lei ca confocal microscope (Heidelburg. Germany) and lho Leica TCS NT software. Positivo cells (a minimum of 400 cells) were counted manually and coexpression of lho different celi suiface markera wasassossedin oach of lhose cells.

The antibilirubin mAb²⁴ (kindly provided by M. Suemalsu. Koio Univeraily. Tokyo. Japan) was used to analyze HO-1 enzymatic activity in 0.1% Triton-pormeabili/ed IX’s. Staining was performed as doscribed.

Celi extracts and Western biot analysis

Western biot analysis was performed as previous!y described.⁵ Briolly. coli protein exiracts were boilod. electrophoresed on a sodium dodocyl sulfate-polyacrylamide gol and blotlod. Mombranos were blockod and incubaled willi eithera rabbil anli-ral. a mouse anti-human HO-1 (Stressgen) Ab or a mouse anti-(3 lubulin mAb (Calbiochom, San Diego. CA). Mombranos were lhon incubaled with horseradish peroxidase-labolod secondary Abs (Jackson ImmunoReseareh. West Grove. PA) and detection performed by onhancod chemoluminosconce (Amorsham).

Quantitative real-time RT-PCR

Tolal RN A was isolaled using TRI/ol (Imilrogon. Cergy Ponloise, France). RN A was treated with DNAso and rcvorse Iranscribed. Reverse iranscrip-tion (RT) quantitative polymerase Chain reaction (PCR) was performed willi a GonAmp 7700 scquencc detection system (Applied Biosystems. Fostor City. CA) using SYBR Greon PCR core reagonts (Applied Biosystems). Rai and human primer sequonces were used to largel HO-1 and hypoxanthlne-guanine phosphoribosyltransfera.se (HPRT).^&25 The PCR method and the 2^_AAn quanlificalion melhod. after nonnali/alion to HPRf values. liavo boen previouslv doscribed.⁶ Iho transcript arcumulalion index (TAI) is dofinod as the fold change in mRNAlevels in a givon sample (Q) relalive to lovols in a calibrator (CB)—in this case iDCs. The calibrator is the IX exprossion of oach gono, The TAI is calculated as follows: TAI = 2^_AAQ. whero AAC, = (C,^, - Qhprt)q - (C,-^, - C,hprt>cb- Spe-cific amplifications were chockod by amplicon melting cuiyos.