Superoxide Anions Induce the Maturation of Human Dendritic Cells
Divisions of Pneumology and Thoracic Surgery, University Hospital, Geneva, Switzerland8wl, http://www.100md.com
Correspondence: Correspondence and requests for reprints should be addressed to Dr. Salomé Kantengwa, Division of Pneumology, University Hospital of Geneva, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland.8wl, http://www.100md.com
ABSTRACT8wl, http://www.100md.com
TOP8wl, http://www.100md.com
ABSTRACT8wl, http://www.100md.com
METHODS8wl, http://www.100md.com
RESULTS8wl, http://www.100md.com
DISCUSSION8wl, http://www.100md.com
REFERENCES8wl, http://www.100md.com
Dendritic cells play a key role in immune responses. There is growing evidence that reactive oxygen species participate in signaling pathways involving nuclear factor (NF)-B, leading to expression of important immune system genes. We found that, unlike H2O2, reactive oxygen species generated by the reaction of oxidase on xanthine induced early phenotypic maturation of dendritic cells by upregulating specific markers CD80, CD83, and CD86 and downregulating mannose receptor–mediated endocytosis. Maturation induced by xanthine oxidase was prevented by allopurinol, an inhibitor of xanthine oxidase activity, and by N-acetylcysteine. The proteasome inhibitor MG-132, which blocks NF-B activation, also inhibited CD86 upregulation, but not endocytosis downregulation by reactive oxygen species. Finally, xanthine-xanthine oxidase enhanced or blocked antigen presentation by dendritic cells depending on whether they had been prepulsed or not with the antigen. Taken together, these results demonstrate that oxidative stress induces phenotypic and functional maturation of dendritic cells, partly through an NF-B–dependent mechanism.
Key Words: reactive oxygen species • dendritic cell • maturationii3+j, 百拇医药
Dendritic cells (DC) are the most potent antigen-presenting cells (APCs). In their immature state, they are located in peripheral nonlymphoid tissues, where they form a surveillance network designed to detect and to capture foreign antigens (1–4). In vivo, after exposure to foreign pathogens, DC acquire the mature phenotype with a strong expression of costimulatory molecules and a high antigen-presenting activity (1, 2). They then migrate to lymph nodes to initiate an immune response in the T cell areas (5). Meanwhile, they lose their endocytic activity and their ability to process antigens. In vitro, immature DC may be differentiated from CD14+ monocytes if cultured in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. Likewise, these cells have a high endocytic activity level but a low T cell stimulatory capacity, and may be matured by bacterial cell wall products such as lipopolysaccharide (LPS) or by cytokines such as tumor necrosis factor- and interleukin-1ß (6).
Oxidative stress results from an oxidant/antioxidant imbalance in favor of oxidants. High levels of oxidants cause tissue injury by peroxidizing membrane lipids, degrading proteins, and oxidizing DNA (7). Low non-toxic concentrations of reactive oxygen species (ROS) are known to participate in signal transduction pathways through their actions on redox-regulated transcription factors such as AP-1 and nuclear factor (NF)-B. Indeed, NF-B activation is regulated by the cell's redox potential and by ROS, and its activation is inhibited by antioxidants such as N-acetylcysteine (NAC) and -lipoic acid (8, 9). NF-B is also involved in the expression of numerous genes that play a major role in immune functions (10), cell cycle progression, and apoptosis (11), as well as in the regulation of DC survival and maturation (12, 13)..f, 百拇医药
DC maturation is a critical process, as only mature DC are able to induce optimal activation of naive T cells. In pathologic situations, DC maturation is triggered by proinflammatory cytokines such as tumor necrosis factor- and interleukin-1ß, and by microbial products such as lipopolysaccharide (LPS) (14). Because both tumor necrosis factor- (15) and LPS (16) stimulate ROS formation, we speculate that ROS might act as mediators in DC maturation. The goal of this study was, therefore, to investigate the effects of superoxide, a frequent product of inflammation, in the phenotypic and functional maturation of human DC. We first found that oxidative stress upregulated CD80, CD86, and CD83 expression, and downregulated mannose receptor–mediated endocytosis in human monocyte–derived DC. We also found that NF-B activation is required for the regulation of the surface expression of CD86, but it is not involved in mannose receptor regulation. Finally, we found that the oxidative stress may either block or enhance antigen presentation, depending on when DC encounter the antigens.
METHODSjg8u, 百拇医药
TOPjg8u, 百拇医药
ABSTRACTjg8u, 百拇医药
METHODSjg8u, 百拇医药
RESULTSjg8u, 百拇医药
DISCUSSIONjg8u, 百拇医药
REFERENCESjg8u, 百拇医药
Cell Isolationjg8u, 百拇医药
Peripheral blood mononuclear cells were isolated by the standard Ficoll-Paque gradient centrifugation from buffy coats obtained from local blood bank. Monocytes were purified by adherence. Nonadherent cells were recovered and T cells were purified on nylon column and frozen until use.jg8u, 百拇医药
DC Differentiation and Maturationjg8u, 百拇医药
Differentiation of DC from monocytes was performed by culture in the presence of granulocyte-macrophage colony-stimulating factor (10 ng/ml) and interleukin-4 (10 ng/ml) as previously described (6). Immature DC were matured with either superoxide (O2- 5 nmol/min/ml) produced by the reaction of xanthine oxidase on xanthine (XaXO) (17) or LPS (100 ng/ml) for 24 or 48 hours. For each experiment, the amount of xanthine oxidase to produce 5 nmol O2-/min/ml was previously determined in vitro by the reduction of cytochrome c in a culture medium containing 2 mM xanthine. H2O2 was used at 100 µM and 1 mM.
Phenotypic Analysis by Flow Cytometry-?|, http://www.100md.com
Cell surface immunophenotype was analyzed by staining the cells with mouse mAbs against CD80 (PharMingen, San Diego, CA), CD83 (Immunotech, Marseille, France), or CD86 (PharMingen), followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody. The samples were analyzed in a fluorescence-activated cell sorter EPICS XL (Beckman Coulter Inc., Fullerton, CA). Cell viability was determined by annexin V-FITC (PharMingen) binding for 10 minutes, followed by propidium iodide (PharMingen) staining immediately before FACS analysis.-?|, http://www.100md.com
Analysis of Endocytic Activity-?|, http://www.100md.com
Mannose receptor–mediated endocytosis was analyzed by uptake of FITC-Dextran at 37°C; control cells were incubated on ice.-?|, http://www.100md.com
Immunofluorescence-?|, http://www.100md.com
Cells were fixed in 4% paraformaldehyde, permeabilized in cold methanol, stained with a rabbit IgG anti–NF-B p65 (Santa Cruz Biotechnology, Santa Cruz, CA), then by a rhodamine-conjugated anti-rabbit IgG (Chemicon, Temecula, CA) and analyzed fuorrescence microscopy.
Antigen Presentation+/0, http://www.100md.com
DC were pulsed with tetanus toxoid (TT) for 3 hours, and maintained unstimulated or stimulated with XaXO or LPS for 24 hours. In another set of experiments, DC were prestimulated, then challenged with TT. Increasing numbers of DC were mixed with autologous lymphocytes (1.5 x 105/well) and incubated for 5 days. [Methyl-3H]thymidine (5 µCi/well) was added for the last 18 hours.+/0, http://www.100md.com
Statistical Analysis+/0, http://www.100md.com
Results are expressed as mean ± SEM. Statistical significance was determined by Student's t test. Differences were considered significant when p < 0.05.+/0, http://www.100md.com
RESULTS+/0, http://www.100md.com
TOP+/0, http://www.100md.com
ABSTRACT+/0, http://www.100md.com
METHODS+/0, http://www.100md.com
RESULTS+/0, http://www.100md.com
DISCUSSION+/0, http://www.100md.com
REFERENCES+/0, http://www.100md.com
XaXO Induces Maturation of DC
Immature DC, generated from adherent monocytes cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, were incubated with 5 nmol O2-/ml/min produced by the enzymatic system xanthine (2 mM)-xanthine oxidase (XaXO) for 48 h. For comparison, immature DC were also treated with LPS (100 ng/ml), a bacterial product known to induce DC maturation. The surface expression of molecules involved in the APC function of DC, i.e., CD80 (B7–1), CD86 (B7–2), and the maturation marker CD83, were analyzed by flow cytometry. As shown in , exposure of cells to XaXO resulted in upregulation of the costimulatory molecules CD80 (mean fluorescence intensity [MFI] from 0.77 to 4.5) and CD86 (MFI from 0.15 to 8). The maturation marker CD83 was also strongly induced by XaXO (MFI from 0.12 to 1.7). These effects were strikingly similar to those obtained with LPS. Thus, XaXO was as efficient as LPS in inducing DC maturation phenotype.}k&4[, 百拇医药
fig.ommitted
Figure 1. Expression of cell surface markers during XaXO-induced maturation. (A) Immature DC were either unstimulated or stimulated with O2- (5 nmol/ml/min) or LPS (100 ng/ml). At 48 hours incubation, cells were stained with monoclonal antibodies specific to CD80, CD83, and CD86 (filled histograms), or an isotype-matched antibody (open histograms), then with FITC-conjugated anti-mouse antibody, and analyzed by flow cytometry (fluorescence-activated cell sorter). The values indicated are the MFI recorded on the cells stained with the specific antibodies. (B) Some of the cells were incubated with FITC-Dextran (1 mg/ml) for 1 hour at 37°C and analyzed by fluorescence-activated cell sorter. Unstimulated DC (open histograms), mature DC (filled histograms). One experiment representative of four similar experiments is represented. The data were analyzed by WinMDI software.yw@k, http://www.100md.com
XaXO Downregulates Mannose Receptor–mediated DC Endocytosisyw@k, http://www.100md.com
Immature DC show a high endocytic activity to allow antigen capture. Upon maturation, DC lose their capacity to capture antigen by endocytosis. Endocytic activity was therefore measured in untreated and XaXO-treated DC, by monitoring the uptake of FITC-Dextran. As shown in , untreated DC were able to incorporate FITC-Dextran (open histogram), and XaXO significantly decreased uptake of FITC-Dextran by 80% (filled histogram). A similar downregulation of endocytosis was observed in LPS-treated cells. This finding provides further evidence that XaXO triggers DC maturation phenotype.
XaXO Induces Early Maturation Phenotypea9swzq., http://www.100md.com
The time course of CD86 expression and FITC-Dextran uptake induced by XaXO was then examined . CD86 expression was already detected at 6 hours, by which time approximately 80% of the cells were CD86-positive, and a plateau was reached at 12 hours . Endocytic activity in XaXO-treated DC fell at 40% of the control cells at 6 hours, and decreased progressively thereafter to reach 20% at 48 hours . For both parameters, the effects of XaXO were strikingly similar to those of LPS. Taken together, these data demonstrate that XaXO induced early DC maturation, similarly to LPS.a9swzq., http://www.100md.com
fig.ommitteda9swzq., http://www.100md.com
Figure 2. Time course of CD86 expression (A) and downregulation of endocytosis (B) during maturation of DC. Immature DC were unstimulated (filled circles) or exposed to LPS (open circles) or XaXO (filled diamonds) for different times. At each time point, the cells were analyzed for CD86 expression and endocytic activity (means ± SEM, n = 3).
Viability of DC exposed to XaXO was tested by annexin V–FITC binding and propidium iodide staining to quantify apoptotic and necrotic cells, respectively. Thus, after 24 hours of incubation, 7.1 ± 1.6% of DC were in apoptosis and 12.4 ± 3.3% were necrotic as compared with 4.3 ± 1.6% and 7.1 ± 1.4%, respectively, of the control DC (mean ± SD, n = 3, p = NS).pr:ot), 百拇医药
XaXO-induced Maturation Was Prevented by Allopurinolpr:ot), 百拇医药
To verify that xanthine oxidase activity was required for the maturation events observed, allopurinol, a potent inhibitor of xanthine oxidase, was used. Immature DC were unstimulated or stimulated with XaXO in the absence and presence of 1 mM allopurinol. FITC-Dextran uptake and CD86 expression were analyzed after 48 hours of incubation. As shown in , allopurinol prevented 70% of the decrease in endocytosis and completely prevented CD86 expression induced by XaXO. Therefore, XaXO-triggered DC maturation was related to the enzymatic activity of xanthine oxidase, and thus to the production of superoxide O2-.
fig.ommittedm6#32, 百拇医药
Figure 3. Inhibition of XaXO-induced DC maturation by allopurinol. Stimulation with XaXO was performed in the presence or absence of 1 mM allopurinol. After 48 hours, DC were analyzed for FITC-Dextran uptake or CD86 expression. Results are expressed as means ± SEM (n = 4) . *p < 0.05; **p < 0.001; #p < 0.01.m6#32, 百拇医药
Exogenous H2O2 Failed to Induce DC Maturationm6#32, 百拇医药
Because superoxide O2- dismutates spontaneously to H2O2, we asked whether a bolus addition of H2O2 (100 µM) also induced DC maturation. Immature DC were exposed to 100 µM H2O2 for 48 hours, and cell surface expression of CD86 and endocytic activity were then analyzed. As shown in , XaXO upregulated CD86 expression and downregulated mannose receptor–mediated endocytosis in a manner similar to that of LPS, whereas H2O2 failed to affect these parameters. Even at 1 mM, H2O2 was still unable to induce DC maturation. Because RPMI medium contains 1 mg/L glutathione, H2O2 was also tested in a serum-free medium X-VIVO-15, which was also free of glutathione. Under this condition also, H2O2 did not trigger DC maturation (data not shown).
fig.ommittedhm:m, 百拇医药
TABLE 1. Effects of h2o2 on CD86 expression and dextran uptake by dendritic cellshm:m, 百拇医药
NAC Suppressed the Effects of XaXO on CD86 Expression but Partly Restored Endocytosishm:m, 百拇医药
To confirm the involvement of ROS in XaXO-induced DC maturation, we used the thiol agent NAC, whose antioxidant properties are well established. DC were preincubated with NAC (30 mM) for 1 hour, then stimulated or not with XaXO or LPS for 24 hours. CD86 expression and FITC-Dextran incorporation were analyzed by FACS. NAC inhibited XaXO-induced CD86 upregulation by 80% , but only partly prevented the downregulation of the endocytic activity induced by XaXO . Likewise, NAC markedly inhibited LPS-induced CD86 expression, but was not able to restore the endocytic activity.hm:m, 百拇医药
fig.ommittedhm:m, 百拇医药
Figure 4. Effects of MG-132 and NAC on CD86 expression and endocytosis. Immature DC were preincubated with MG-132 (0.5 µM) or NAC (30 mM) for 1 hour, then maintained in the medium alone or stimulated with LPS or XaXO for 24 hours. Maturation was checked by upregulation of CD86 (A) and downregulation of FITC-Dextran incorporation (B). Results are expressed as means ± SEM (n = 3). (A) LPS versus LPS/MG-132, *p < 0.05; LPS versus LPS/NAC, **p < 0.01; XaXO versus XaXO/MG-132, #p < 0.005; XaXO versus XaXO/NAC, $p < 0.0001. (B) **p < 0.01 between stimulated and inhibited cells.
NO Synthase Inhibition Prevents Part of XaXO Activity)c@5{:, http://www.100md.com
To see to what extent the NO synthase pathway was also involved in the phenomenon, N-nitroarginine was used to inhibit NO formation. In the presence of 1 mM N-nitroarginine, the effects of XaXO on CD86 expression and endocytosis were inhibited by 43.8 ± 9.3% and 64 ± 5.2% in three separate experiments, implying that superoxide production may generate reactive NO-dependent metabolites.)c@5{:, http://www.100md.com
MG-132 Prevented CD86 Expression but Not the Loss of Endocytic Activity Induced by XaXO)c@5{:, http://www.100md.com
The transcription factor NF-B has been shown to be involved in the DC maturation process induced by LPS. Our finding that the effects of XaXO were strikingly similar to those obtained with LPS prompted us to examine whether NF-B was also involved in DC maturation induced by XaXO. To test this hypothesis, we pretreated immature DC for 1 hour before XaXO treatment with the proteasome inhibitor MG-132 (0.5 µM), a compound that inhibits NF-B activation by blocking the proteolytic degradation of IB. As shown in , MG-132 reduced XaXO-induced CD86 expression by 60%, but failed to prevent the endocytosis downregulation induced by XaXO . Likewise, MG-132 partly prevented CD86 expression induced by LPS, but was not able to restore endocytosis. Due to its toxicity, higher concentrations of MG-132 have not been used. These data suggest that NF-B might be involved in the intracellular pathway used by XaXO to upregulate CD86 expression, whereas the endocytic pathway may be regulated by an alternative mechanism.
Activation of NF-B Is Required for XaXO-induced CD86 Upregulation6;3vp, 百拇医药
To further confirm this hypothesis, immunofluorescence was used to examine the translocation of NF-B from the cytoplasm to the nucleus in immature DC and XaXO- or LPS-treated cells in the absence and presence of MG-132 or NAC. Some of the immature DC from the experiments shown in were pretreated with MG-132 or NAC for 1 hour, stimulated with XaXO or LPS for 1 hour, and stained with a polyclonal antibody against NF-B p65 followed by a rhodamine-conjugated anti-rabbit IgG. As shown in , unstimulated cells showed NF-B p65 staining in the cytosol. Incubation of cells with XaXO resulted in a marked nuclear expression of NF-B p65. This translocation was completely prevented in cells pretreated with MG-132 and NAC. The same pattern of response was observed with LPS. As expected, XaXO-induced but not LPS-induced NF-B translocation was prevented when allopurinol was present during the incubation (data not shown). From these results, we conclude that the transcription factor NF-B was involved in XaXO-induced DC maturation at least where CD86 expression is concerned. No significant nuclear translocation of NF-B p65 was observed in H2O2-treated cells (data not shown).
fig.ommitted17h4^, 百拇医药
Figure 5. Inhibition of NF-B translocation by MG-132 and NAC. Some of the cells in were recovered after 1 hour of stimulation and stained with a polyclonal anti–NF-B p65 antibody, then with a rhodamine-conjugated anti-rabbit antibody, and finally analyzed by fluorescence microscopy (magnification: x1,000).17h4^, 百拇医药
XaXO Blocks Antigen Presentation Unless DC Have Been Prepulsed with the Antigen17h4^, 百拇医药
As XaXO upregulates the expression of molecules that are critical for the stimulation of naive T cells by DC, we examined the effects of XaXO on T cell responses. Immature DC were pulsed with TT for 3 hours, then stimulated with XaXO or LPS for 24 hours. As shown in , when DC were previously pulsed with TT, they were able to trigger T cell proliferation in a dose-dependent manner, from 102 DC to 5 x 103 DC per well, whereas TT-pulsed nonstimulated cells were efficient only at 5 x 103 DC. As previously observed with the maturation markers, the effects of XaXO on T cells were strikingly similar to those of LPS. Thus, in addition to the increase of DC maturation–associated costimulatory molecules, XaXO-matured DC showed enhanced capacity to present TT. As expected, antigen presentation by DC exposed to XaXO in the presence of allopurinol was reduced to the level of DC and T cells with antigen alone (0.8 ± 0.05-fold increase compared with untreated DC; mean ± SEM; n = 3). The effect of LPS on T cell proliferation was not significantly altered in the presence of allopurinol.
fig.ommitted{nv$ft, 百拇医药
Figure 6. XaXO increases antigen presentation. (A) Immature DC pulsed or not with TT for 3 hours were then stimulated with 5 nmol O2-/ml/min or LPS (100 ng/ml) for 24 hours. T cells (1.5 x 105) were cultured with increasing numbers of DC for 5 days, and proliferation was measured by thymidine incorporation. Results are expressed as means ± SEM. Data represent means ± SEM of six separate experiments. Asterisks denote significant differences from control cells (p < 0.05). (B) DC were prestimulated with XaXO or LPS for 24 hours, then challenged with TT (*p < 0.05: unstimulated versus stimulated DC).{nv$ft, 百拇医药
In contrast, when the TT challenge was performed after XaXO or LPS stimulation, DC failed to stimulate T cell proliferation .{nv$ft, 百拇医药
DISCUSSION{nv$ft, 百拇医药
TOP{nv$ft, 百拇医药
ABSTRACT{nv$ft, 百拇医药
METHODS{nv$ft, 百拇医药
RESULTS{nv$ft, 百拇医药
DISCUSSION{nv$ft, 百拇医药
REFERENCES
Maturation of DC is a process of great importance for innate and acquired immune responses, where antigen-capturing immature DC are changed into highly antigen-presenting mature DC able to migrate from peripheral tissues to lymphoid organs and activate naive T cells (2). In this study, we demonstrated that ROS generated by the enzymatic system xanthine-xanthine oxidase (XaXO) induced early maturation of human monocyte–derived DC, via an upregulation of surface markers CD80, CD83, and CD86 and a downregulation of the endocytic activity. These effects were strikingly similar to those observed with LPS..)evn4, 百拇医药
We chose the XaXO system because of its involvement in the ischemia-reoxygenation injury, a common occurrence in solid organ transplantation (18). The XaXO system is a suitable model to produce O2- in vitro. The fact that allopurinol, an XO inhibitor structurally related to purines, significantly inhibited XaXO-induced DC maturation, indicates that XO activity, and thus O2-, were required for XaXO-dependent effects. It is interesting to note that allopurinol immunosuppressive properties have been reported in mice (19), and it is tempting to speculate that this immunosuppressive effect might be related to the inhibition of the XaXO-triggered DC maturation we observed in vitro. The XaXO reaction yields the superoxide radical (O2-) as a primary product, which is highly unstable and spontaneously dismutates into H2O2. In the presence of traces of iron salt, O2- reacts with H2O2 in the Haber-Weiss reaction to produce the highly reactive hydroxyl radical OH·. In our cell system, O2- was probably the first ROS involved in the XaXO-induced DC maturation, because exogenous H2O2 failed to mature DC, at least where CD86 expression and endocytotic activity are concerned. Our finding that H2O2 did not upregulate CD86 corroborates the data of Verhasselt and coworkers (20), but it contradicts the observation of Rutault and colleagues (21), who reported a 2.5-fold increase in CD86 expression in H2O2-treated DC. Both authors reported a slight increase in the surface expression of MHC class II molecules induced by H2O2, but their results were divergent with respect to costimulatory molecules. The discrepancies may be attributed to the different basal maturation states at the time DC were exposed to H2O2. In our hands, H2O2 failed to induce any one of the maturation parameters analyzed. Although H2O2 failed to have such activity, the inhibition of NO synthase was consistently inhibiting part of the maturation observed with XaXO. This could be due to the capacity, for instance, of superoxide anions to generate peroxynitrite from the NO pathway (22). The role of NO itself on DC maturation needs further exploration.
NAC is a thiol agent that exerts its antioxidant properties either by increasing intracellular reduced glutathione (23) or direct scavenging (24). Our observation that NAC significantly suppressed the effects of XaXO on both CD86 expression and endocytosis clearly established that DC maturation induced by XaXO was related to ROS. However, the intracellular signaling pathways underlying these two processes were not the same. Our data obtained with the proteasome inhibitor MG-132 revealed that the ROS-induced expression of CD86 was due to the activation of the transcription factor NF-B, whereas the mannose receptor–mediated endocytosis appears to be dependent on another signal transduction pathway. Likewise, MG-132 prevented the effects of LPS on CD86 expression, but did not affect the inhibitory effect of LPS on endocytosis. Indeed, transduction pathways involving protein kinases in DC maturation have been reported (25). Intriguingly, in our hands, NAC alone inhibited the basal endocytic activity of DC. The reason for this NAC activity is still unknown.
Finally and most importantly, we established the functional relevance of the effects of ROS generated by the XaXO enzyme system on antigen presentation by DC. We found that XaXO-treated DC acquired an enhanced efficiency to activate T cells. As previously observed for the phenotypic maturation markers, the ability of TT-pulsed-XaXO-matured DC to elicit an antigen-depending T cell response was strikingly similar to that of LPS-matured cells.'pp{6, 百拇医药
Inversely, when XaXO-matured DC were challenged with TT, their T cell–stimulating activity was impaired, a result of the maturation-associated low endocytic activity that reduces antigen uptake (26).'pp{6, 百拇医药
Exposure to oxidants is a common event in the airways, as oxidants can be generated by air pollution such as particles or ozone reaching our bronchi (27) and by cigarette smoke (28). The release of oxidants has been well described in the pathogenesis of several lung diseases such as chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis, or cystic fibrosis (29, 30). Our present findings may be of great importance, as the exposure of dendritic cells from our airways to oxidants may amplify T cell activation in the lungs and therefore increase undue inflammatory processes. Drugs like NAC could then be useful to control this process. It is also possible that oxidants, by blocking antigen uptake by dendritic cells, may prevent them from triggering an adequate immune response, e.g., in the case of viral exposure. Oxidants generated after lung transplatation (18) or exposure of the transplanted lungs to oxidants could interfere with either lung tolerance or with infections, which might deserve further investigations.
In conclusion, we have shown that XaXO-generated O2- is as efficient as LPS in inducing phenotypic and functional DC maturation. Our observation may be relevant to a number of situations where organs such as the lungs may be exposed to ROS, either increasing or blocking antigen presentation depending on the time the antigens reach the DC. The same may be true in situations in which hypoxia-reoxygenation occurs, such as transplantation in which increased ROS produced by enhanced XO activity in reoxygenated cells may contribute to either abrogation or, on the contrary, enhancement of cellular immunity.8!{r, 百拇医药
Acknowledgments8!{r, 百拇医药
The authors thank Dr. I. Park for critical review of the manuscript.8!{r, 百拇医药
REFERENCES8!{r, 百拇医药
TOP8!{r, 百拇医药
ABSTRACT8!{r, 百拇医药
METHODS8!{r, 百拇医药
RESULTS8!{r, 百拇医药
DISCUSSION8!{r, 百拇医药
REFERENCES
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Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med 1995;182:389–400.hqr#5]x, 百拇医药
Corda S, Laplace C, Vicaut E, Duranteau J. Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide. Am J Respir Cell Mol Biol 2001;24:762–768.hqr#5]x, 百拇医药
Landmann R, Scherer F, Schumann R, Link S, Sansano S, Zimmerli W. LPS directly induces oxygen radical production in human monocytes via LPS binding protein CD14. J Leukoc Biol 1995;57:440–449.hqr#5]x, 百拇医药
Fridovich I. Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J Biol Chem 1970;245:4053–4057.
Li C, Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 2002;282:C227–C241.*;%, http://www.100md.com
Kato C, Sato K, Wakabayashi A, Eishi Y. The effects of allopurinol on immune function in normal BALB/c SCID mice. Int J Immunopharmacol 2000;22:547–556.*;%, http://www.100md.com
Verhasselt V, Goldman M, Willems F. Oxidative stress up-regulates IL-8 and TNF- synthesis by human dendritic cells. Eur J Immunol 1998;28:3886–3890.*;%, http://www.100md.com
Rutault K, Alderman C, Chain BM, Katz DR. Reactive oxygen species activate human peripheral blood dendritic cells. Free Radic Biol Med 1998;26:232–238.*;%, http://www.100md.com
Crow JP, Beckman JS. The importance of superoxide in nitric oxide-dependent toxicity: evidence for peroxynitrite-mediated injury. Adv Exp Med Biol 1996;387:147–161.*;%, http://www.100md.com
Hashimoto S, Gon Y, Matsumoto K, Takeshita I, Machinoand T, Horie T. Intracellular glutathione regulates tumor necrosis factor-alpha-induced p38 MAP kinase activation and RANTES production by human bronchial epithelial cells. Clin Exp Allergy 2001;31:144–151.
Straface E, Matarrese P, Gambardella L, Forte S, Carlone S, Libianchi E, Schmid G, Malorni W. N-Acetylcysteine counteracts erythrocyte alterations occurring in chronic obstructive pulmonary disease. Biochem Biophys Res Commun 2000;279:552–556.9g, 百拇医药
Bouchon A, Hernandez-Munain C, Cella M, Colonna M. A DAP12-mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendritic cells. J Exp Med 2001;194:1111–1122.9g, 百拇医药
Cella M, Engering A, Pinet V, Pieters J, Lanzavecchia V. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature 1997;388:782–787.9g, 百拇医药
Stringer B, Kobzik L. Environmental particulate-mediated cytokine production in lung epithelial cells (A549): role of pre-existing inflammation and oxidant stress. J Toxicol Environ Health 1998;55:31–44.9g, 百拇医药
Pryor WA, Prier DG, Church DF. Electron-spin resonance study of mainstream and sidestream cigarette smoke: nature of the free radicals in gasphase smoke and in cigarette tar. Environ Health Perspect 1983;47:345–355.9g, 百拇医药
Rahman I, Morrison D, Donaldson K, MacNee W. Systemic oxidative stress in asthma, COPD, and smokers. Am J Respir Crit Care Med 1996;154:1055–1060.9g, 百拇医药
Roum JH, Borok Z, McElvaney NG, Grimes GJ, Bokser AD, Buhl R, Crystal RG. Glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis. J Appl Physiol 1999;87:438–443.(Salomé Kantengwa, Lan Jornot, Christiane Devenoges and Laurent P. Nicod)
Correspondence: Correspondence and requests for reprints should be addressed to Dr. Salomé Kantengwa, Division of Pneumology, University Hospital of Geneva, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland.8wl, http://www.100md.com
ABSTRACT8wl, http://www.100md.com
TOP8wl, http://www.100md.com
ABSTRACT8wl, http://www.100md.com
METHODS8wl, http://www.100md.com
RESULTS8wl, http://www.100md.com
DISCUSSION8wl, http://www.100md.com
REFERENCES8wl, http://www.100md.com
Dendritic cells play a key role in immune responses. There is growing evidence that reactive oxygen species participate in signaling pathways involving nuclear factor (NF)-B, leading to expression of important immune system genes. We found that, unlike H2O2, reactive oxygen species generated by the reaction of oxidase on xanthine induced early phenotypic maturation of dendritic cells by upregulating specific markers CD80, CD83, and CD86 and downregulating mannose receptor–mediated endocytosis. Maturation induced by xanthine oxidase was prevented by allopurinol, an inhibitor of xanthine oxidase activity, and by N-acetylcysteine. The proteasome inhibitor MG-132, which blocks NF-B activation, also inhibited CD86 upregulation, but not endocytosis downregulation by reactive oxygen species. Finally, xanthine-xanthine oxidase enhanced or blocked antigen presentation by dendritic cells depending on whether they had been prepulsed or not with the antigen. Taken together, these results demonstrate that oxidative stress induces phenotypic and functional maturation of dendritic cells, partly through an NF-B–dependent mechanism.
Key Words: reactive oxygen species • dendritic cell • maturationii3+j, 百拇医药
Dendritic cells (DC) are the most potent antigen-presenting cells (APCs). In their immature state, they are located in peripheral nonlymphoid tissues, where they form a surveillance network designed to detect and to capture foreign antigens (1–4). In vivo, after exposure to foreign pathogens, DC acquire the mature phenotype with a strong expression of costimulatory molecules and a high antigen-presenting activity (1, 2). They then migrate to lymph nodes to initiate an immune response in the T cell areas (5). Meanwhile, they lose their endocytic activity and their ability to process antigens. In vitro, immature DC may be differentiated from CD14+ monocytes if cultured in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. Likewise, these cells have a high endocytic activity level but a low T cell stimulatory capacity, and may be matured by bacterial cell wall products such as lipopolysaccharide (LPS) or by cytokines such as tumor necrosis factor- and interleukin-1ß (6).
Oxidative stress results from an oxidant/antioxidant imbalance in favor of oxidants. High levels of oxidants cause tissue injury by peroxidizing membrane lipids, degrading proteins, and oxidizing DNA (7). Low non-toxic concentrations of reactive oxygen species (ROS) are known to participate in signal transduction pathways through their actions on redox-regulated transcription factors such as AP-1 and nuclear factor (NF)-B. Indeed, NF-B activation is regulated by the cell's redox potential and by ROS, and its activation is inhibited by antioxidants such as N-acetylcysteine (NAC) and -lipoic acid (8, 9). NF-B is also involved in the expression of numerous genes that play a major role in immune functions (10), cell cycle progression, and apoptosis (11), as well as in the regulation of DC survival and maturation (12, 13)..f, 百拇医药
DC maturation is a critical process, as only mature DC are able to induce optimal activation of naive T cells. In pathologic situations, DC maturation is triggered by proinflammatory cytokines such as tumor necrosis factor- and interleukin-1ß, and by microbial products such as lipopolysaccharide (LPS) (14). Because both tumor necrosis factor- (15) and LPS (16) stimulate ROS formation, we speculate that ROS might act as mediators in DC maturation. The goal of this study was, therefore, to investigate the effects of superoxide, a frequent product of inflammation, in the phenotypic and functional maturation of human DC. We first found that oxidative stress upregulated CD80, CD86, and CD83 expression, and downregulated mannose receptor–mediated endocytosis in human monocyte–derived DC. We also found that NF-B activation is required for the regulation of the surface expression of CD86, but it is not involved in mannose receptor regulation. Finally, we found that the oxidative stress may either block or enhance antigen presentation, depending on when DC encounter the antigens.
METHODSjg8u, 百拇医药
TOPjg8u, 百拇医药
ABSTRACTjg8u, 百拇医药
METHODSjg8u, 百拇医药
RESULTSjg8u, 百拇医药
DISCUSSIONjg8u, 百拇医药
REFERENCESjg8u, 百拇医药
Cell Isolationjg8u, 百拇医药
Peripheral blood mononuclear cells were isolated by the standard Ficoll-Paque gradient centrifugation from buffy coats obtained from local blood bank. Monocytes were purified by adherence. Nonadherent cells were recovered and T cells were purified on nylon column and frozen until use.jg8u, 百拇医药
DC Differentiation and Maturationjg8u, 百拇医药
Differentiation of DC from monocytes was performed by culture in the presence of granulocyte-macrophage colony-stimulating factor (10 ng/ml) and interleukin-4 (10 ng/ml) as previously described (6). Immature DC were matured with either superoxide (O2- 5 nmol/min/ml) produced by the reaction of xanthine oxidase on xanthine (XaXO) (17) or LPS (100 ng/ml) for 24 or 48 hours. For each experiment, the amount of xanthine oxidase to produce 5 nmol O2-/min/ml was previously determined in vitro by the reduction of cytochrome c in a culture medium containing 2 mM xanthine. H2O2 was used at 100 µM and 1 mM.
Phenotypic Analysis by Flow Cytometry-?|, http://www.100md.com
Cell surface immunophenotype was analyzed by staining the cells with mouse mAbs against CD80 (PharMingen, San Diego, CA), CD83 (Immunotech, Marseille, France), or CD86 (PharMingen), followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody. The samples were analyzed in a fluorescence-activated cell sorter EPICS XL (Beckman Coulter Inc., Fullerton, CA). Cell viability was determined by annexin V-FITC (PharMingen) binding for 10 minutes, followed by propidium iodide (PharMingen) staining immediately before FACS analysis.-?|, http://www.100md.com
Analysis of Endocytic Activity-?|, http://www.100md.com
Mannose receptor–mediated endocytosis was analyzed by uptake of FITC-Dextran at 37°C; control cells were incubated on ice.-?|, http://www.100md.com
Immunofluorescence-?|, http://www.100md.com
Cells were fixed in 4% paraformaldehyde, permeabilized in cold methanol, stained with a rabbit IgG anti–NF-B p65 (Santa Cruz Biotechnology, Santa Cruz, CA), then by a rhodamine-conjugated anti-rabbit IgG (Chemicon, Temecula, CA) and analyzed fuorrescence microscopy.
Antigen Presentation+/0, http://www.100md.com
DC were pulsed with tetanus toxoid (TT) for 3 hours, and maintained unstimulated or stimulated with XaXO or LPS for 24 hours. In another set of experiments, DC were prestimulated, then challenged with TT. Increasing numbers of DC were mixed with autologous lymphocytes (1.5 x 105/well) and incubated for 5 days. [Methyl-3H]thymidine (5 µCi/well) was added for the last 18 hours.+/0, http://www.100md.com
Statistical Analysis+/0, http://www.100md.com
Results are expressed as mean ± SEM. Statistical significance was determined by Student's t test. Differences were considered significant when p < 0.05.+/0, http://www.100md.com
RESULTS+/0, http://www.100md.com
TOP+/0, http://www.100md.com
ABSTRACT+/0, http://www.100md.com
METHODS+/0, http://www.100md.com
RESULTS+/0, http://www.100md.com
DISCUSSION+/0, http://www.100md.com
REFERENCES+/0, http://www.100md.com
XaXO Induces Maturation of DC
Immature DC, generated from adherent monocytes cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, were incubated with 5 nmol O2-/ml/min produced by the enzymatic system xanthine (2 mM)-xanthine oxidase (XaXO) for 48 h. For comparison, immature DC were also treated with LPS (100 ng/ml), a bacterial product known to induce DC maturation. The surface expression of molecules involved in the APC function of DC, i.e., CD80 (B7–1), CD86 (B7–2), and the maturation marker CD83, were analyzed by flow cytometry. As shown in , exposure of cells to XaXO resulted in upregulation of the costimulatory molecules CD80 (mean fluorescence intensity [MFI] from 0.77 to 4.5) and CD86 (MFI from 0.15 to 8). The maturation marker CD83 was also strongly induced by XaXO (MFI from 0.12 to 1.7). These effects were strikingly similar to those obtained with LPS. Thus, XaXO was as efficient as LPS in inducing DC maturation phenotype.}k&4[, 百拇医药
fig.ommitted
Figure 1. Expression of cell surface markers during XaXO-induced maturation. (A) Immature DC were either unstimulated or stimulated with O2- (5 nmol/ml/min) or LPS (100 ng/ml). At 48 hours incubation, cells were stained with monoclonal antibodies specific to CD80, CD83, and CD86 (filled histograms), or an isotype-matched antibody (open histograms), then with FITC-conjugated anti-mouse antibody, and analyzed by flow cytometry (fluorescence-activated cell sorter). The values indicated are the MFI recorded on the cells stained with the specific antibodies. (B) Some of the cells were incubated with FITC-Dextran (1 mg/ml) for 1 hour at 37°C and analyzed by fluorescence-activated cell sorter. Unstimulated DC (open histograms), mature DC (filled histograms). One experiment representative of four similar experiments is represented. The data were analyzed by WinMDI software.yw@k, http://www.100md.com
XaXO Downregulates Mannose Receptor–mediated DC Endocytosisyw@k, http://www.100md.com
Immature DC show a high endocytic activity to allow antigen capture. Upon maturation, DC lose their capacity to capture antigen by endocytosis. Endocytic activity was therefore measured in untreated and XaXO-treated DC, by monitoring the uptake of FITC-Dextran. As shown in , untreated DC were able to incorporate FITC-Dextran (open histogram), and XaXO significantly decreased uptake of FITC-Dextran by 80% (filled histogram). A similar downregulation of endocytosis was observed in LPS-treated cells. This finding provides further evidence that XaXO triggers DC maturation phenotype.
XaXO Induces Early Maturation Phenotypea9swzq., http://www.100md.com
The time course of CD86 expression and FITC-Dextran uptake induced by XaXO was then examined . CD86 expression was already detected at 6 hours, by which time approximately 80% of the cells were CD86-positive, and a plateau was reached at 12 hours . Endocytic activity in XaXO-treated DC fell at 40% of the control cells at 6 hours, and decreased progressively thereafter to reach 20% at 48 hours . For both parameters, the effects of XaXO were strikingly similar to those of LPS. Taken together, these data demonstrate that XaXO induced early DC maturation, similarly to LPS.a9swzq., http://www.100md.com
fig.ommitteda9swzq., http://www.100md.com
Figure 2. Time course of CD86 expression (A) and downregulation of endocytosis (B) during maturation of DC. Immature DC were unstimulated (filled circles) or exposed to LPS (open circles) or XaXO (filled diamonds) for different times. At each time point, the cells were analyzed for CD86 expression and endocytic activity (means ± SEM, n = 3).
Viability of DC exposed to XaXO was tested by annexin V–FITC binding and propidium iodide staining to quantify apoptotic and necrotic cells, respectively. Thus, after 24 hours of incubation, 7.1 ± 1.6% of DC were in apoptosis and 12.4 ± 3.3% were necrotic as compared with 4.3 ± 1.6% and 7.1 ± 1.4%, respectively, of the control DC (mean ± SD, n = 3, p = NS).pr:ot), 百拇医药
XaXO-induced Maturation Was Prevented by Allopurinolpr:ot), 百拇医药
To verify that xanthine oxidase activity was required for the maturation events observed, allopurinol, a potent inhibitor of xanthine oxidase, was used. Immature DC were unstimulated or stimulated with XaXO in the absence and presence of 1 mM allopurinol. FITC-Dextran uptake and CD86 expression were analyzed after 48 hours of incubation. As shown in , allopurinol prevented 70% of the decrease in endocytosis and completely prevented CD86 expression induced by XaXO. Therefore, XaXO-triggered DC maturation was related to the enzymatic activity of xanthine oxidase, and thus to the production of superoxide O2-.
fig.ommittedm6#32, 百拇医药
Figure 3. Inhibition of XaXO-induced DC maturation by allopurinol. Stimulation with XaXO was performed in the presence or absence of 1 mM allopurinol. After 48 hours, DC were analyzed for FITC-Dextran uptake or CD86 expression. Results are expressed as means ± SEM (n = 4) . *p < 0.05; **p < 0.001; #p < 0.01.m6#32, 百拇医药
Exogenous H2O2 Failed to Induce DC Maturationm6#32, 百拇医药
Because superoxide O2- dismutates spontaneously to H2O2, we asked whether a bolus addition of H2O2 (100 µM) also induced DC maturation. Immature DC were exposed to 100 µM H2O2 for 48 hours, and cell surface expression of CD86 and endocytic activity were then analyzed. As shown in , XaXO upregulated CD86 expression and downregulated mannose receptor–mediated endocytosis in a manner similar to that of LPS, whereas H2O2 failed to affect these parameters. Even at 1 mM, H2O2 was still unable to induce DC maturation. Because RPMI medium contains 1 mg/L glutathione, H2O2 was also tested in a serum-free medium X-VIVO-15, which was also free of glutathione. Under this condition also, H2O2 did not trigger DC maturation (data not shown).
fig.ommittedhm:m, 百拇医药
TABLE 1. Effects of h2o2 on CD86 expression and dextran uptake by dendritic cellshm:m, 百拇医药
NAC Suppressed the Effects of XaXO on CD86 Expression but Partly Restored Endocytosishm:m, 百拇医药
To confirm the involvement of ROS in XaXO-induced DC maturation, we used the thiol agent NAC, whose antioxidant properties are well established. DC were preincubated with NAC (30 mM) for 1 hour, then stimulated or not with XaXO or LPS for 24 hours. CD86 expression and FITC-Dextran incorporation were analyzed by FACS. NAC inhibited XaXO-induced CD86 upregulation by 80% , but only partly prevented the downregulation of the endocytic activity induced by XaXO . Likewise, NAC markedly inhibited LPS-induced CD86 expression, but was not able to restore the endocytic activity.hm:m, 百拇医药
fig.ommittedhm:m, 百拇医药
Figure 4. Effects of MG-132 and NAC on CD86 expression and endocytosis. Immature DC were preincubated with MG-132 (0.5 µM) or NAC (30 mM) for 1 hour, then maintained in the medium alone or stimulated with LPS or XaXO for 24 hours. Maturation was checked by upregulation of CD86 (A) and downregulation of FITC-Dextran incorporation (B). Results are expressed as means ± SEM (n = 3). (A) LPS versus LPS/MG-132, *p < 0.05; LPS versus LPS/NAC, **p < 0.01; XaXO versus XaXO/MG-132, #p < 0.005; XaXO versus XaXO/NAC, $p < 0.0001. (B) **p < 0.01 between stimulated and inhibited cells.
NO Synthase Inhibition Prevents Part of XaXO Activity)c@5{:, http://www.100md.com
To see to what extent the NO synthase pathway was also involved in the phenomenon, N-nitroarginine was used to inhibit NO formation. In the presence of 1 mM N-nitroarginine, the effects of XaXO on CD86 expression and endocytosis were inhibited by 43.8 ± 9.3% and 64 ± 5.2% in three separate experiments, implying that superoxide production may generate reactive NO-dependent metabolites.)c@5{:, http://www.100md.com
MG-132 Prevented CD86 Expression but Not the Loss of Endocytic Activity Induced by XaXO)c@5{:, http://www.100md.com
The transcription factor NF-B has been shown to be involved in the DC maturation process induced by LPS. Our finding that the effects of XaXO were strikingly similar to those obtained with LPS prompted us to examine whether NF-B was also involved in DC maturation induced by XaXO. To test this hypothesis, we pretreated immature DC for 1 hour before XaXO treatment with the proteasome inhibitor MG-132 (0.5 µM), a compound that inhibits NF-B activation by blocking the proteolytic degradation of IB. As shown in , MG-132 reduced XaXO-induced CD86 expression by 60%, but failed to prevent the endocytosis downregulation induced by XaXO . Likewise, MG-132 partly prevented CD86 expression induced by LPS, but was not able to restore endocytosis. Due to its toxicity, higher concentrations of MG-132 have not been used. These data suggest that NF-B might be involved in the intracellular pathway used by XaXO to upregulate CD86 expression, whereas the endocytic pathway may be regulated by an alternative mechanism.
Activation of NF-B Is Required for XaXO-induced CD86 Upregulation6;3vp, 百拇医药
To further confirm this hypothesis, immunofluorescence was used to examine the translocation of NF-B from the cytoplasm to the nucleus in immature DC and XaXO- or LPS-treated cells in the absence and presence of MG-132 or NAC. Some of the immature DC from the experiments shown in were pretreated with MG-132 or NAC for 1 hour, stimulated with XaXO or LPS for 1 hour, and stained with a polyclonal antibody against NF-B p65 followed by a rhodamine-conjugated anti-rabbit IgG. As shown in , unstimulated cells showed NF-B p65 staining in the cytosol. Incubation of cells with XaXO resulted in a marked nuclear expression of NF-B p65. This translocation was completely prevented in cells pretreated with MG-132 and NAC. The same pattern of response was observed with LPS. As expected, XaXO-induced but not LPS-induced NF-B translocation was prevented when allopurinol was present during the incubation (data not shown). From these results, we conclude that the transcription factor NF-B was involved in XaXO-induced DC maturation at least where CD86 expression is concerned. No significant nuclear translocation of NF-B p65 was observed in H2O2-treated cells (data not shown).
fig.ommitted17h4^, 百拇医药
Figure 5. Inhibition of NF-B translocation by MG-132 and NAC. Some of the cells in were recovered after 1 hour of stimulation and stained with a polyclonal anti–NF-B p65 antibody, then with a rhodamine-conjugated anti-rabbit antibody, and finally analyzed by fluorescence microscopy (magnification: x1,000).17h4^, 百拇医药
XaXO Blocks Antigen Presentation Unless DC Have Been Prepulsed with the Antigen17h4^, 百拇医药
As XaXO upregulates the expression of molecules that are critical for the stimulation of naive T cells by DC, we examined the effects of XaXO on T cell responses. Immature DC were pulsed with TT for 3 hours, then stimulated with XaXO or LPS for 24 hours. As shown in , when DC were previously pulsed with TT, they were able to trigger T cell proliferation in a dose-dependent manner, from 102 DC to 5 x 103 DC per well, whereas TT-pulsed nonstimulated cells were efficient only at 5 x 103 DC. As previously observed with the maturation markers, the effects of XaXO on T cells were strikingly similar to those of LPS. Thus, in addition to the increase of DC maturation–associated costimulatory molecules, XaXO-matured DC showed enhanced capacity to present TT. As expected, antigen presentation by DC exposed to XaXO in the presence of allopurinol was reduced to the level of DC and T cells with antigen alone (0.8 ± 0.05-fold increase compared with untreated DC; mean ± SEM; n = 3). The effect of LPS on T cell proliferation was not significantly altered in the presence of allopurinol.
fig.ommitted{nv$ft, 百拇医药
Figure 6. XaXO increases antigen presentation. (A) Immature DC pulsed or not with TT for 3 hours were then stimulated with 5 nmol O2-/ml/min or LPS (100 ng/ml) for 24 hours. T cells (1.5 x 105) were cultured with increasing numbers of DC for 5 days, and proliferation was measured by thymidine incorporation. Results are expressed as means ± SEM. Data represent means ± SEM of six separate experiments. Asterisks denote significant differences from control cells (p < 0.05). (B) DC were prestimulated with XaXO or LPS for 24 hours, then challenged with TT (*p < 0.05: unstimulated versus stimulated DC).{nv$ft, 百拇医药
In contrast, when the TT challenge was performed after XaXO or LPS stimulation, DC failed to stimulate T cell proliferation .{nv$ft, 百拇医药
DISCUSSION{nv$ft, 百拇医药
TOP{nv$ft, 百拇医药
ABSTRACT{nv$ft, 百拇医药
METHODS{nv$ft, 百拇医药
RESULTS{nv$ft, 百拇医药
DISCUSSION{nv$ft, 百拇医药
REFERENCES
Maturation of DC is a process of great importance for innate and acquired immune responses, where antigen-capturing immature DC are changed into highly antigen-presenting mature DC able to migrate from peripheral tissues to lymphoid organs and activate naive T cells (2). In this study, we demonstrated that ROS generated by the enzymatic system xanthine-xanthine oxidase (XaXO) induced early maturation of human monocyte–derived DC, via an upregulation of surface markers CD80, CD83, and CD86 and a downregulation of the endocytic activity. These effects were strikingly similar to those observed with LPS..)evn4, 百拇医药
We chose the XaXO system because of its involvement in the ischemia-reoxygenation injury, a common occurrence in solid organ transplantation (18). The XaXO system is a suitable model to produce O2- in vitro. The fact that allopurinol, an XO inhibitor structurally related to purines, significantly inhibited XaXO-induced DC maturation, indicates that XO activity, and thus O2-, were required for XaXO-dependent effects. It is interesting to note that allopurinol immunosuppressive properties have been reported in mice (19), and it is tempting to speculate that this immunosuppressive effect might be related to the inhibition of the XaXO-triggered DC maturation we observed in vitro. The XaXO reaction yields the superoxide radical (O2-) as a primary product, which is highly unstable and spontaneously dismutates into H2O2. In the presence of traces of iron salt, O2- reacts with H2O2 in the Haber-Weiss reaction to produce the highly reactive hydroxyl radical OH·. In our cell system, O2- was probably the first ROS involved in the XaXO-induced DC maturation, because exogenous H2O2 failed to mature DC, at least where CD86 expression and endocytotic activity are concerned. Our finding that H2O2 did not upregulate CD86 corroborates the data of Verhasselt and coworkers (20), but it contradicts the observation of Rutault and colleagues (21), who reported a 2.5-fold increase in CD86 expression in H2O2-treated DC. Both authors reported a slight increase in the surface expression of MHC class II molecules induced by H2O2, but their results were divergent with respect to costimulatory molecules. The discrepancies may be attributed to the different basal maturation states at the time DC were exposed to H2O2. In our hands, H2O2 failed to induce any one of the maturation parameters analyzed. Although H2O2 failed to have such activity, the inhibition of NO synthase was consistently inhibiting part of the maturation observed with XaXO. This could be due to the capacity, for instance, of superoxide anions to generate peroxynitrite from the NO pathway (22). The role of NO itself on DC maturation needs further exploration.
NAC is a thiol agent that exerts its antioxidant properties either by increasing intracellular reduced glutathione (23) or direct scavenging (24). Our observation that NAC significantly suppressed the effects of XaXO on both CD86 expression and endocytosis clearly established that DC maturation induced by XaXO was related to ROS. However, the intracellular signaling pathways underlying these two processes were not the same. Our data obtained with the proteasome inhibitor MG-132 revealed that the ROS-induced expression of CD86 was due to the activation of the transcription factor NF-B, whereas the mannose receptor–mediated endocytosis appears to be dependent on another signal transduction pathway. Likewise, MG-132 prevented the effects of LPS on CD86 expression, but did not affect the inhibitory effect of LPS on endocytosis. Indeed, transduction pathways involving protein kinases in DC maturation have been reported (25). Intriguingly, in our hands, NAC alone inhibited the basal endocytic activity of DC. The reason for this NAC activity is still unknown.
Finally and most importantly, we established the functional relevance of the effects of ROS generated by the XaXO enzyme system on antigen presentation by DC. We found that XaXO-treated DC acquired an enhanced efficiency to activate T cells. As previously observed for the phenotypic maturation markers, the ability of TT-pulsed-XaXO-matured DC to elicit an antigen-depending T cell response was strikingly similar to that of LPS-matured cells.'pp{6, 百拇医药
Inversely, when XaXO-matured DC were challenged with TT, their T cell–stimulating activity was impaired, a result of the maturation-associated low endocytic activity that reduces antigen uptake (26).'pp{6, 百拇医药
Exposure to oxidants is a common event in the airways, as oxidants can be generated by air pollution such as particles or ozone reaching our bronchi (27) and by cigarette smoke (28). The release of oxidants has been well described in the pathogenesis of several lung diseases such as chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis, or cystic fibrosis (29, 30). Our present findings may be of great importance, as the exposure of dendritic cells from our airways to oxidants may amplify T cell activation in the lungs and therefore increase undue inflammatory processes. Drugs like NAC could then be useful to control this process. It is also possible that oxidants, by blocking antigen uptake by dendritic cells, may prevent them from triggering an adequate immune response, e.g., in the case of viral exposure. Oxidants generated after lung transplatation (18) or exposure of the transplanted lungs to oxidants could interfere with either lung tolerance or with infections, which might deserve further investigations.
In conclusion, we have shown that XaXO-generated O2- is as efficient as LPS in inducing phenotypic and functional DC maturation. Our observation may be relevant to a number of situations where organs such as the lungs may be exposed to ROS, either increasing or blocking antigen presentation depending on the time the antigens reach the DC. The same may be true in situations in which hypoxia-reoxygenation occurs, such as transplantation in which increased ROS produced by enhanced XO activity in reoxygenated cells may contribute to either abrogation or, on the contrary, enhancement of cellular immunity.8!{r, 百拇医药
Acknowledgments8!{r, 百拇医药
The authors thank Dr. I. Park for critical review of the manuscript.8!{r, 百拇医药
REFERENCES8!{r, 百拇医药
TOP8!{r, 百拇医药
ABSTRACT8!{r, 百拇医药
METHODS8!{r, 百拇医药
RESULTS8!{r, 百拇医药
DISCUSSION8!{r, 百拇医药
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