628996289

HO-1INHIBITSDC FUNCTION 1699

BLOOD. 1 SEPTEMBER 2005 • VOLUME106. NUMBER 5

populations. Production of the LPS-induced proinflammatory cytokines 1L-12 p40.1L-6. and TNF-a significantly decreased when rat DCs were pulsed with the HO-I inducerCoPP. whereas treatment with SnPP had no effect (Figurę 5A). In contrast, HO-1 induction did not inhibit secretion of the LPS-induced anti-inflammatory cytokine 1L-10 (Figurę 5A).

Similarly to rat DCs. a strong decrease in LPS-induced 1L-12 p70 pmduetion associated with a preservation of IL-10 was also observed following CoPP treatment of human DCs (l^rigurc 5B).

Treatment of immalure rat and human DCs with SnPPor CoPP alone (no LPS) did not induce iDCs to seciete cytokines (Figurę 5A-B).

These results show that HO-1 induction moduiates LPS-induced DC maturation by decreasing pminllammatory cytokines production and preserving IL-10 secretion.

HO-1 induction decreases the capacity of rat and human DCs to stimulate MLRs

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CoPP    •    •    4    •    •    +

LPS    4    4    4    •    •    •

To fuitherassess tlie effect of HO-1 on DC function. we determined whetherHO-I induction modified theT-cell a Most i mula tory capacity of rat and human DCs. Rat BMDCs that had been treated with

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IL-6

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Figurę 6. Induction of H0-1 inhibits ihe allogeneic stimulatory capacity ot rat and human DCs. (A) Rat BMDCs and (B) human DCs were treated or nol with SnPP or Co PP for 2 hours. cu Itured for 16 hours. and then (or a further 24 hours in presence or absence ol LPS. Thereałter. DCs were cultured with allogeneic T cells at dilferenl ratbs for 4 obyś and proliferating T cells were lateled with ^:H-thymidine tors hours bsfore being harvested and counted. Results are shown as means i SD of triplicate values after subtraetbn ol spontaneous ^JH-thymidine. Stalistical significance com-pared with LPS-treated DCs is indicaled as ’P •- .05. Similar results were cbtained in 6 independent expefiments for rat DCs and in 2 tor human DCs. cpm indicatescounts per minutę. (C) In parallel. supematants were collected after 4S hours from rat MLRs and anatyzed by ELISAto determineleve!sof IFN-₇ and IL-10. Data presented are mean i SD oł trplicate anafysis of one exFerimenl and are representative of 5 independent experiments. Statistical significance compared with LPS-treated DCs is indicated as 'P< .01.

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Figura 5. Induction of HO-1 affects cytokiną<?xpression in rat and human DCs. (A) EUSAwasusod to assess Ihe production of IL-12 p40. IL-6.TNF-a. and IL-10 in Iho supematants of rai BMDCs treated or not with SnPP or CoPP for 2 hours. cultured lor 16 hours. and Ihsn lor a further 24 hours in Ihe presence or absence of LPS. Induction ol HO-1 inhibiled expressicn oł IL-12 p40. TNF-a. and IL-6 in DCs but did not alt er IL-10 secretion. Horizcntal bars represent the mean ot indh/idual values. Statislical signifcance compared to LPS-treated cełls is indicated as 'P < .001. (Bi EU SA was used lo assess the production ol IL-12 p70 and IL-10 in the supematants oł human DCs treated a nol with SnPP er CoPP lor 2 hours. cultured for 16 hours. and thenfor 31urther24 hours in the preserce or absence ot LPS. Induction of HO-1 inhibiled expression of IL-12 p70 bul did not aller IL-10 secretion. Dala presenled ara mean i SD of trplicate analysis ol one experimenl and 3re representatń/e ol 3 independent experiments. Stalistical significance corrpared to LPS-trealed cells is indicaled as 'P < .01.

LPS after induction of HO-l by CoPP were poor stimulators in mixed lymphocyte reactions (MLRs) eompaied with eontrol L.PS-

treated DCs. whereas HO-1 inhibition bv SnPP treatment had no

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effect on rat allogeneic T-cell proliferation (Fignie 6A).

Human DCs pretreated with CoPP and then with LPS were also poor stimulators in MLRs compared to LPS-treated DCs. and SnPP-pretreated DCs only inereased proliferation at one T/DC (Figurę 6B). MLR proliferation of rat (data not shown) or human (Figurę 6B) DCs not matured with LPS was unmodified by pretieatment with SnPPor CoPP.

We also investigated cytokine pmduetion in supematants from rat MLRs (Figurę 6C). When DCs were treated with CoPP. IFN-7 production was significantly reduced and IL-10 significantly inereased compared with eontrol LPS-treated DCs. When DCs were treated with SnPP. IFN-7 and IL-10 pmduetion were unmodiłied compared to contml DCs (Figuic 6C). In MLRs using DCs not exposed to LPS but pretreated with CoPP. a reduction in IFN-7 production was obsei-\ed. whereas IL-10 was inereased and SnPP. on the other łiand. had 110 effect (Figure 6C).

These results demonstrate that HO-1 induction not only inhibits the allostimulatory capacity of DCs. but also changes the cytokines produced during DC/T-cell interactions.

HO-1 induction decreases ROS levels in DCs

Inereased levels of reactive oxygen species (ROS) have been shown to be involved in activation of DCs by different stimuli. and antioxidants inhibit DC activation.'^; To assess potential intracellu-lar mechanisms for HO-l inhibition of DC maturation. we analyzed ROS levels in DCs pretreated with CoPP or SnPP. and matured with LPS. a stimulus known to inerease ROS in DCs'^; (Fignie 7). ROS levels were inereased following treatment with LPS. SnPP