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Published ahead of print on April 30, 2003, doi:10.1164/rccm.200211-1289OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 215-221, (2003)
© 2003 American Thoracic Society

Original Article

Effect of Diesel on Chemokines and Chemokine Receptors Involved in Helper T Cell Type 1/Type 2 Recruitment in Patients with Asthma

Stéphanie Sénéchal, Patricia de Nadai, Natacha Ralainirina, Arnaud Scherpereel, Han Vorng, Philippe Lassalle, André-Bernard Tonnel, Anne Tsicopoulos and Benoît Wallaert

INSERM U-416, Institut Pasteur de Lille; and Clinique des Maladies Respiratoires et Centre Hospitalier Régional et Universitaire de Lille, Lille, France

Correspondence and requests for reprints should be addressed to Anne Tsicopoulos, M.D., U-416, Institut Pasteur de Lille, BP 245, 59 019 Lille Cedex, France. E-mail: anne.tsicopoulos{at}pasteur-lille.fr


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to evaluate if diesel exhausts could favor helper T cell type (Th) 2-associated allergic reactions either through an increased production of Th2-associated chemokines and of their associated receptors or through a decrease of Th1-attracting chemokines and chemokine receptors. Diesel but not allergen exposure of peripheral blood mononuclear cells from subjects with allergy induced a release of I-309, whereas both diesel and Der p 1 induced an early but transient release of monokine induced by IFN-{gamma} and a late release of pulmonary and activation–regulated chemokine. Although both Th1- and Th2-attracting chemokines were induced, the resulting effect was an increased chemotactic activity on Th2 but not Th1 cells. Surprisingly, diesel induced a late increase in the expression of the Th1-associated CXC receptor 3 and CC receptor 5. T cell CXC receptor 3 upregulation was not associated with an increased migration to its ligands. These two antagonistic effects have been previously reported as a scavenger mechanism to clear chemokines. Altogether, these results suggest that diesel, even without allergen, may amplify a type 2 immune response but that it can also increase late Th1-associated chemokine receptor expression, perhaps as a scavenger mechanism to clear pro-Th1 chemokines and promote the Th2 pathway.

Key Words: chemokines • diesel • helper T cell type 1/type 2 • allergy • asthma

Air pollutants including diesel exhaust particles and in particular their associated polyaromatic hydrocarbons (DEP-PAH), have been shown to enhance allergic responses (1–7), which are characterized by a predominant Th2-type cytokine profile (8).

Together with adhesion molecules, chemokines and their receptors are believed to be essential for leukocyte trafficking from the circulation into inflammatory tissues (9). Recently, certain chemokines and their receptors have been shown to be associated with Th1-type and Th2-type–mediated immune response (10). Th2 cells preferentially express the chemokine receptors CC receptor 3 (CCR3), CC receptor 4 (CCR4), and CC receptor 8 (CCR8) and are recruited by their ligands, eotaxin for CCR3, macrophage-derived chemokine (MDC) and thymus and activation–regulated chemokine for CCR4, and I-309 for CCR8. Th1 cells preferentially express CXC receptor 3 (CXCR3) and CC receptor 5 (CCR5) (11–14) and are mainly recruited by IFN-{gamma}–induced protein-10 (IP-10) and monokine induced by IFN-{gamma} (MIG) through CXCR3 binding.

In a previous study performed on peripheral blood mononuclear cells (PBMC) obtained from individuals with atopy and asthma (15), we showed that diesel exposure alone decreased the IP-10 production, whereas it further augmented allergen-induced MDC production, resulting in a significantly increased capacity to chemoattract human Th2 but not Th1 clones. However, Th2 cell recruitment was not completely abolished by using a neutralizing anti-MDC antibody, suggesting the involvement of other pro-Th2 mediators. We therefore tested the hypothesis that diesel exposure might favor Th2 recruitment either through increased production of Th2-attracting chemokines and increased expression of their receptors or through a decrease of Th1-attracting chemokines and of their receptors.

We focused our attention on other chemokines involved in the Th1 or Th2 pattern. The CC chemokine I-309 binds to CCR8, preferentially expressed on Th2 cells (16, 17) and is produced by activated monocytes and by T cells (18). Its secretion is independent of interleukin (IL)-4 and IFN-{gamma} but is inhibited by IL-12 (18). Pulmonary and activation–regulated chemokine (PARC) is also associated with the Th2 pattern. Indeed, this chemokine is induced by Th2-type cytokines such as IL-4, IL-13, and IL-10 (19) and inhibited by IFN-{gamma}. Among the Th1-associated chemokines, MIG is a related member of the CXC chemokine subfamily, which as IP-10 binds to CXCR3, and which is produced by monocytes (20), macrophages (21), neutrophils (22) and human umbilical vein endothelial cells (23) in response to IFN-{gamma} (22, 24). MIG is negatively regulated by Th2-type cytokines (22) and is chemotactic for Th1 lymphocytes, natural killer cells, and eosinophils (25). For all these reasons, we evaluated the effect of diesel exposure on the production of these chemokines by PBMC from patients with allergy as well as on the expression of chemokine receptors associated with the Th1 or Th2 phenotype.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Diesel Exhaust and Extraction of DEP-PAH
DEPs were obtained and DEP-PAH were extracted in dichloromethane solvent, as described previously (26) (Table E1, online supplement).

Subjects
Venous blood was collected from patients with allergy and asthma who were sensitive to house dust mite.

All patients had a clinical history of asthma and exhibited positive skin-prick tests toward Dermatophagoides pteronyssinus allergen, positive RAST (RAST class >= 3), and elevated serum IgE levels (mean level = 677 ± 287 KU/L). None had received oral or inhaled corticosteroids within the 2 months before the sample collection. Patients were under ß2 agonists as required. All patients signed an informed consent form.

Cell Culture
PBMC from 18 patients were prepared from blood (26), cultured with complete Roswell Park Memorial Institute ± dichloromethane, DEP-PAH, or Der p 1 (100 ng/ml) (Indoor Biotechnologies, Manchester, UK). Time course and dose–response showed that the effect of DEP-PAH on I-309, PARC, and MIG productions was optimal at a dose of 50 ng/ml after a culture period of 24 to 48 hours. Kinetics experiments were also performed for messenger RNA (mRNA) and flow cytometry studies.

Generation of polarized human Th1 and Th2 cells.
T cells were polarized in vitro toward a Th1 or a Th2 cytokine profile (27, 28). Cells were considered as correctly polarized when more than 50% of the cells expressed IL-4 or IFN-{gamma}.

Quantification of Chemokine Levels in Cell Culture Supernatants
Concentrations of I-309, MIG (R&D Systems, Abington, UK), and PARC (Biosource International, Camarillo, CA) were measured by ELISA.

Semiquantitative Reverse Transcription–Polymerase Chain Reaction
After extraction, total cellular RNA was treated with ribonuclease-free deoxribonuclease (Stratagene, La Jolla, CA) (15), and RT-PCR performed as described previously (29). The sequences of the primers (purchased from Eurogentec, Seraing, Belgium), the number of cycles, and the cycle temperatures are indicated in Table E2 in the online supplement.

T Cell Chemotaxis Assays
To evaluate the resulting biologic chemotactic activity of the supernatants, chemotaxis assays were performed on polarized Th1 and Th2 cells (27), as described previously (15). For neutralization assays, samples were incubated for 30 minutes at room temperature with anti–I-309 (R&D Systems), and/or anti-MDC neutralizing antibodies (R&D Systems).

In additional experiments, the functionality of T cell chemokine receptors associated with the Th1 phenotype was evaluated on PBMC exposed to DEP-PAH ± Der p 1 during 48 hours. Monocytes were separated from lymphocytes by plastic adherence, and lymphocyte migration toward IP-10 and MIG was evaluated in the chemotaxis assay as described previously.

Flow Cytometry Analysis
Monocyte and T cell surface expression of chemokine receptors was analyzed using three-color immunolabeling. Cells were collected after 1, 3, 6, 12, 24, or 48 hours of stimulation and incubated with different monoclonal antibodies: Cy-chrome–conjugated anti-CD3 and fluorescein isothiocyanate–conjugated (Becton Dickinson, Mountain View, CA) or phycoerythrine-conjugated anti-CD14 (Immunotech, Marseille, France) were used to gate lymphocytes and monocytes. Additional labeling was performed using phycoerythrine-conjugated anti-CCR4 antibody, fluorescein isothiocyanate–conjugated anti-CCR5 (Becton Dickinson), anti-CXCR3 (R&D), and anti-CCR3 antibodies (RDI, Flanders, ND). fluorescein isothiocyanate/phycoerythrine/Cy-chrome–conjugated mouse IgG1, fluorescein isothiocyanate/phycoerythrine–conjugated mouse IgG2a, fluorescein isothiocyanate–conjugated rat IgG2a (Becton Dickinson) were used as negative controls.

After washing, the cells were fixed in phosphate-buffered saline–1% paraformaldehyde and analyzed using a FACSCalibur (Becton Dickinson).

Statistical Analysis
Differences in chemokine production, in chemotaxis assays, and in the kinetics of chemokine receptor expression were first evaluated within the subgroups using the Kruskal–Wallis test and subsequently analyzed by using the Wilcoxon test for diluent/stimuli pairs.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DEP-PAH but Not Der p 1 Induce I-309 Chemokine Release from PBMC
Exposure of PBMC from patients with asthma to 50 ng/ml DEP-PAH during 24 hours induced a significant production of the Th2-attracting chemokine I-309, whereas Der p 1 alone had no effect (Figure 1A) . The addition of Der p 1 to DEP-PAH induced a similar increase in I-309 production (+127 vs. +109% for DEP-PAH alone). At 48 hours of culture, I-309 production was further increased after DEP-PAH exposure (+162%) as well as after the combined DEP-PAH and Der p 1 exposure (+252%) (Figure 1A). I-309 mRNA expression was modified in a similar way to what was observed at the protein level at 24 and 48 hours (Figure 2) . These results show that diesel alone is able to induce a sustained production of the Th2-attracting chemokine I-309 by PBMC from patients with allergies.



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  		Figure 1. Chemokine release by peripheral blood mononuclear cells (PBMC) from patients with allergy, stimulated with diesel exhaust particles–polyaromatic hydrocarbons (DEP-PAH) and/or Der p 1. Production of I-309 (A), monokine induced by IFN-{gamma} (MIG) and pulmonary and activation-regulated chemokine (PARC) (B) by PBMC from patients with allergy to house dust mite, stimulated with 50 ng/ml DEP-PAH and/or 100 ng/ml Der p 1 for 24 or 48 hours. Control samples were stimulated with dichloromethane (CH2Cl2) used to dissolve DEP-PAH. Results are expressed as mean ± SEM for n = 10 patients. *p Value less than 0.01 versus control samples. White bars, CH2Cl2; gray bars, DEP PAH; hatched bars, Der p 1; black bars, DEP-PAH + Der p 1.

 


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  		Figure 2. Analysis of messenger (mRNA) expression in peripheral blood mononuclear cells (PBMC) from patients with allergy stimulated with diesel exhaust particles–polyaromatic hydrocarbons (DEP-PAH) and/or Der p 1. I-309, pulmonary and activation-regulated chemokine (PARC), and glyceraldehyde phosphate dehydrogenase (GAPDH) housekeeping gene mRNA expression was determined by semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR), after stimulation of PBMC with control dichloromethane (CH2Cl2) (C), 50 ng/ml DEP-PAH (1), 100 ng/ml Der p 1 (2), or DEP-PAH and Der p 1 (3) at 24 and 48 hours. One representative experiment out of three is shown.

 
Both DEP-PAH and Der p 1 Induce MIG and PARC Release from PBMC
Exposure of PBMC to DEP-PAH or Der p 1 induced an early (24 hour) increase in the Th1-attracting chemokine MIG (+57% for DEP-PAH; +59% for Der p 1) (Figure 1B). However, this effect was completely abolished at 48 hours with no variation in MIG production between the different conditions of cell stimulation (data not shown). At the gene level, MIG mRNA was not detected whatever the time point, although IFN-{gamma}–stimulated PBMC showed a positive signal.

In contrast, the level of the Th2-induced chemokine PARC remained unchanged at 24 hours in all culture conditions (data not shown), whereas it increased 48 hours after exposure of PBMC to either DEP-PAH or Der p 1 (+79 and +95%, respectively) (Figure 1B). No variation of PARC mRNA was observed at 24 hours, whereas it was upregulated at 48 hours in the presence of DEP-PAH and/or Der p 1 (Figure 2). The combined DEP-PAH and Der p 1 exposure also increased MIG and PARC productions, but no additional effect of the two stimuli was observed. All together, these results show that diesel exposure can kinetically regulate the production of chemokines involved in the Th2 as well as the Th1 pattern.

DEP-PAH–stimulated PBMC Supernatants Attract Th2 Cells
To examine the net selective chemotactic activity of PBMC supernatants on human Th cell subpopulations, polarized human Th1 and Th2 cells were generated and used in a chemotaxis assay. Supernatants from PBMC incubated for 24 hours (Figure 3A) with DEP-PAH, Der p 1, or both compounds exhibited no enhanced capacity to attract Th1 cells as compared with control supernatants, despite the presence of the Th1-attracting chemokine MIG.



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  		Figure 3. Capacity of peripheral blood mononuclear cells (PBMC) from patients with allergy stimulated with diesel exhaust particles–polyaromatic hydrocarbons (DEP-PAH) and/or Der p 1 to induce chemotaxis in Th1 and Th2 polarized cells. Supernatants of PBMC from patients with allergy, stimulated with dichloromethane (CH2Cl2) in the absence or presence of 50 ng/ml DEP-PAH and/or 100 ng/ml Der p 1 were harvested after 24 hours (A) and 48 hours (B) and used in an in vitro chemotaxis assay with either Th1 or Th2 polarized cells. Recombinant human macrophage-derived chemokine (MDC) or IFN-{gamma}–induced protein-10 (IP-10) at a final concentration of 10-7 M were used as control samples. Results are expressed as mean ± SEM of five experiments. *p Value less than 0.05. (C) Inhibitory effect of chemokine neutralizing antibodies on Th2 cell recruitment induced by DEP-PAH and Der p 1–stimulated PBMC supernatants from patients with allergy at 24 and 48 hours, using neutralizing antibodies against I-309 and MDC. Results are expressed as the percentage of inhibition of Th2 cell recruitment (mean ± SEM of four experiments).

 
In contrast, supernatants harvested 24 hours after exposure to DEP-PAH alone or associated with Der p 1, displayed a significant increased capacity to attract Th2 cells as compared with control supernatants. The same pattern of chemoattraction was observed with 48-hour supernatants (Figure 3B).

To evaluate the part of each chemokine in the Th2 chemotactic activity, experiments were performed with neutralizing antibodies directed against chemokines known to attract Th2 cells and present in the supernatants such as I-309 and MDC, previously shown to be upregulated by DEP-PAH and Der p 1 (15). The addition of a neutralizing anti–I-309 antibody to supernatants from PBMC exposed to DEP-PAH and Der p 1 before the assay reduced the Th2 cell-attracting activity of 24-hour supernatants by 57%, and for 48-hour supernatants by 75%. The neutralizing anti-MDC antibody reduced the Th2 chemotactic activity of supernatants from cells exposed to both DEP-PAH and Der p 1 for 24 hours by 72% and for 48 hours by 80%. Finally, the addition of the two different antibodies totally abolished the Th2 chemoattractant activity of 48-hour supernatants, whereas a 83% inhibition was observed for 24-hour supernatants (Figure 3C).

DEP-PAH Exposure Modulates Th1 but Not Th2-associated Chemokine Receptor Expression
T cell subset-associated chemokine receptor expression was analyzed kinetically in PBMC by RT-PCR and flow cytometry. Surprisingly, mRNA transcripts for the Th1-associated chemokine receptors CCR5 and CXCR3 were enhanced after exposure of total PBMC to either DEP-PAH or DEP-PAH and Der p 1 but only at 48 hours of culture, whereas the Th2-associated chemokine receptors CCR3, CCR4, and CCR8 transcripts were not modified irrespective of the time point or of the conditions of stimulation (Figure 4) . Chemokine receptor cell surface expression was also analyzed by flow cytometry. To distinguish between T cell and monocyte surface expression, cells were gated on CD14+ monocytes and on CD3+ lymphocytes. Lymphocyte chemokine receptor baseline expression was low at 1, 3, and 6 hours (data not shown) and started to increase at 12 hours of culture. Similar to the mRNA data, diesel exposure induced an increase of CCR5 and CXCR3 expression that was statistically significant at 48 hours (Figures 5A and 5B) as compared with the solvent control. There was no variation of the Th2-associated chemokine receptors CCR3 and CCR4 after diesel exposure. Stimulation of PBMC with Der p 1 alone did not modify lymphocyte chemokine receptor expression, whereas the combination of DEP-PAH and Der p 1 or DEP-PAH alone upregulated CCR5 and CXCR3 T cell surface expression at 48 hours (Figure 5C).



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  		Figure 4. Messenger RNA expression of chemokine receptors in peripheral blood mononuclear cells stimulated under various conditions (control dichloromethane [CH2Cl2] [C], 50 ng/ml DEP-PAH [1], 100 ng/ml Der p 1 [2], or diesel exhaust particles–polyaromatic hydrocarbons (DEP-PAH) and Der p 1 [3]) at 48 hours. Chemokine receptor expression was determined by semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR). One representative experiment out of three is shown.

 


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  		Figure 5. Expression of T cell chemokine receptors on lymphocytes from patients with allergy stimulated with diesel exhaust particles–polyaromatic hydrocarbons (DEP-PAH) and/or Der p 1. (A) Expression of CC receptor 5 (CCR5) and CXC receptor 3 (CXCR3) on T cells stimulated with diesel extracts during 12, 24, or 48 hours. Cells were stained with the stated antibody (thick line) or isotype-matched monoclonal antibody (thin line) and their cell surface expression analyzed by flow cytometry. Data are shown as histograms. (B) Kinetics of surface expression of Th1- (CCR5 and CXCR3) and Th2- (CCR3 and CCR4) associated chemokine receptors on DEP-PAH–stimulated T cells. Results are expressed as percentage of variation of the mean fluorescence intensity (MFI) as compared with the control diluent (mean ± SEM of six patients). *p Value less than 0.05. (C) Expression of Th1 (CCR5 and CXCR3) associated chemokine receptors on DEP-PAH and/or Der p 1–stimulated T cells at 48 hours. Results are expressed as mean percentage of variation of the MFI compared with the control dichloromethane (CH2Cl2) (n = 6 experiments). *p Value less than 0.05.

 
Monocytes did not express CCR3 but showed variable expression of CCR4, CCR5, and CXCR3. However, there was no difference of expression for these three receptors on monocytes between the different conditions of cell stimulation or at the different time points (data not shown).

DEP-PAH–induced Increase in Th1-associated Chemokine Receptor Expression Is Not Associated with Increased Migration
To evaluate the functionality of the upregulation of the Th1-associated chemokine receptor CXCR3 under diesel stimulation, 48-hour–stimulated T cells were used in a chemotaxis assay toward CXCR3 ligands, IP-10, and MIG (Figure 6) . Unexpectedly, lymphocytes obtained from PBMC incubated for 48 hours with DEP-PAH alone or in combination with Der p 1, which exhibited an increased expression of CXCR3, did not show an enhanced migration but rather a trend toward a decreased migration to IP-10 and MIG, as compared with control or Der p 1–stimulated lymphocytes, whereas they responded similarly to the positive control stromal cell-derived factor-1, suggesting an uncoupling of the CXCR3 receptor from the migratory transduction pathway.



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  		Figure 6. T cell upregulation of the Th1-associated chemokine receptor CXC receptor 3 (CXCR3), on diesel stimulation does not lead to increased migration to its ligands. Lymphocytes were purified from PBMC from patients with allergy cultured for 48 hours in the absence or presence of 50 ng/ml DEP-PAH and/or 100 ng/ml Der p 1. T cell migration was assessed in an in vitro chemotaxis assay toward CXCR3 ligands, IP-10, and MIG. (A) Recombinant human (rh) stromal cell-derived factor-1 (SDF-1) was used as a positive control, (B) rh IP-10 and (C) rh MIG were used at a final concentration of 10-7 M. Results are expressed as mean ± SEM (n = 5). White bars, CH2Cl2; gray bars, DEP PAH; hatched bars, Der p 1; black bars, DEP-PAH + Der p 1.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
DEPs and their associated DEP-PAH are known to enhance allergic responses. Diesel has been reported to increase the specific allergic IgE response (30) to modulate T cell response (31), in particular to favor the production of Th2-type cytokines (4, 32) and to induce the recruitment of inflammatory cells such as neutrophils (33) and eosinophils (34, 35) by modulating the chemokine pathway (26, 36–39). In a previous study, we have shown that diesel exhausts could also dysregulate some particular chemokines resulting in the attraction of type 2 T cells (15). In particular, diesel exposure was shown to increase allergen-induced MDC but not eotaxin and thymus and activation–regulated chemokine production by PBMC from patients with allergy and to decrease IP-10 constitutive expression, resulting in a significantly increased capacity to chemoattract human Th2 but not Th1 clones. However, Th2 cell recruitment was not completely abolished by using a neutralizing anti-MDC antibody, suggesting the involvement of other pro-Th2 mediators. To further elucidate the mechanisms involved in this effect, we conducted another study analyzing the effect of diesel exposure on other chemokines and chemokine receptors associated with the Th1 and Th2 pattern in patients with allergy. The reason for this choice was that in our previous study, diesel alone without the allergen had no effect on Th2-associated chemokines.

The chemokine I-309 selectively attracts Th2 cells through CCR8 triggering (13) and is secreted by T lymphocytes (40), in particular by T cell receptor–activated Th1 and Th2 cells (18), and by activated human peripheral blood monocytes (41). In the present study, we show that the exposure of PBMC to diesel for 24 and 48 hours resulted in an increase of synthesis and secretion of I-309, in a significant chemoattraction of polarized Th2 cells by cell supernatants and in an inhibition of this chemotactic activity by specific anti–I-309 neutralizing antibodies. Hitherto, diesel effect has been observed only as an amplifying mechanism of allergen-induced chemokine production. The fact that diesel alone but not Der p 1 increased I-309 synthesis and secretion strongly suggests that diesel exposure may not only amplify an ongoing allergic reaction but also trigger it. Although I-309 appears as a possible candidate involved in the allergic reaction (42), expression of I-309 was not observed in the airway mucosa or in the bronchial epithelial cells of individuals with atopy and asthma, whereas thymus and activation-regulated chemokine, MDC, and CCR8 immunoreactivities were found (43). Therefore, the precise role of I-309 in the allergic reaction has still to be determined. Nevertheless, in our study the combination of anti-MDC and anti–I-309 neutralizing antibodies totally abolished the Th2-attracting effect of diesel and allergen-stimulated PBMC supernatants at 48 hours, showing that MDC and I-309 are the main chemokines involved in this effect.

Among chemokines involved in the Th1/Th2 pattern, the chemokine PARC is of interest. PARC is expressed constitutively at high levels in human lung (44) and is known to be induced by Th2-type cytokines (19). PARC is mainly produced by antigen-presenting cells such as activated macrophages, monocytes, and dendritic cells. Its receptor is still unknown (44). PARC has been found in atheromatosis (45) and in contact hypersensitivity reactions (46). Although no previous study has evaluated the involvement of PARC in allergic reactions, our data suggest that in patients with asthma, PARC is induced at the protein and mRNA levels after allergen and diesel stimulation and may play a role in such reactions. PARC was produced at late time points, which is in agreement with the literature (47, 48), suggesting a potential role in the perpetuation of the allergic reaction after allergen and/or diesel exposure. To date, the only known stimuli for PARC production are Th2-type cytokines. However, in our study no modification was observed in the levels of IL-4 and IL-13 by PBMC from patients with allergy stimulated with allergen or diesel, which remained very low in all conditions of stimulation. Furthermore, the neutralizing antibody against IL-4 and IL-13 did not affect PARC production (data not shown), suggesting the involvement of other pathways. The functional relevance of PARC production is unclear. PARC has been shown to chemoattract naive CD45RA+ T cells (48, 49). The attraction of such cells at late time points of the allergic reaction might serve as an amplifying mechanism.

We previously showed that diesel exposure induced a decrease of the Th1-attracting chemokine IP-10 by PBMC from patients with allergy. However, in the present study another Th1-attracting chemokine, MIG, was found to be upregulated at the protein level by diesel and/or allergen stimulation. This chemokine is produced by different cell types in response to IFN-{gamma} (50) and has been implicated in Th1 reactions (51), in bacterial infections in humans (52), and in contact dermatitis reactions (53). As MIG is a surrogate marker for IFN-{gamma} producing cells (54), we measured IFN-{gamma} levels in PBMC supernatants. No modification was observed in the levels of IFN-{gamma} by PBMC from patients with allergy (data not shown), which remained very low in all conditions of stimulation. Furthermore, a neutralizing antibody against IFN-{gamma} did not inhibit MIG production. On the other hand, there was no MIG mRNA expression whatever the kinetics (3, 6, 12, 24, or 48 hours of stimulation; data not shown) and the stimulation (DEP-PAH or Der p 1). These data show a discrepancy between MIG mRNA expression and protein levels. However, despite the presence of MIG protein, there was no Th1 chemoattraction, suggesting that MIG was not functional.

Altogether, this study and the previous one (15) suggest that diesel exposure of PBMC from patients with asthma may trigger and intensify the allergic reaction by inducing I-309 and MDC synthesis thereby favoring the recruitment of Th2 cells.

Another point of this study was to evaluate if diesel and allergen exposure may regulate the expression of chemokine receptors associated with the Th1 or Th2 pattern. Our hypothesis was that such exposure might upregulate Th2 and/or downregulate Th1-associated chemokine receptors resulting in increased recruitment of Th2 cells. CCR4, CCR3, and CCR8 have been shown to be preferentially expressed on Th2 cells (12, 55, 56). These receptors can be modulated in vitro, in particular through T cell receptor stimulation (57, 58) or through the cytokine environment (11, 59). This is why we did not look at the effect of diesel and/or allergen on already differentiated and committed Th1 or Th2 cells but evaluated their effects on total PBMC throughout stimulation. No variation of either CCR3 and CCR4 expression at the cell surface or of CCR8 at the mRNA level was observed on T cells stimulated with diesel or allergen, irrespective of the time point. The lack of upregulation of Th2-associated chemokine receptors in response to allergen, which mimics T cell receptor stimulation, might be related to the small percentage of circulating Th2 cells in patients with allergy not allowing significant variations among total T cells.

CCR5 and CXCR3 have been shown to be preferentially expressed on Th1 cells (12, 55, 60). Unexpectedly, we here observed a late increase in CXCR3 and CCR5 expression on T cells stimulated with diesel extracts but not with allergen, suggesting a mechanism specific to diesel activation. Th1-associated chemokine receptors can be regulated by different stimuli. In particular, engagement of CD3 and CD28 can upregulate CXCR3 in peripheral blood T cells (61) as well as CCR5 expression (62). This mechanism can be ruled out in the present study because the allergen did not induce such upregulation. It has also to be pointed out that the preferential expression of some chemokine receptors on Th1/Th2 cells has been mainly determined in vitro but is not exclusive. Recent reports have shown that CXCR3 and CCR5 can be expressed on Th2 cells but thus are always coexpressing CCR4 in vivo (60, 63). Three-color immunolabeling performed on 48-hour diesel–stimulated PBMC showed that the majority of CXCR3+ CD3+ cells did not coexpress CCR4 (data not shown), suggesting that they were indeed of the Th1 phenotype.

In our study, we assessed the functionality of the increased CXCR3 expression. Lymphocytes from patients with allergy stimulated during 48 hours with diesel extracts did not show an increased migration toward IP-10 or MIG, suggesting that CXCR3 was uncoupled from the migration pathway. Such an increase in chemokine receptor expression associated with a lack of migration has been already described as a scavenger mechanism to clear the corresponding ligands (64). In our study, the increase in Th1-associated receptors, especially CXCR3, might result in a local increase of the clearance of its ligands, IP-10 and MIG, and thus might explain the decrease of IP-10 and MIG chemokines at late time points of diesel exposure in favor of a net Th2-selective chemotactic activity.

In conclusion, this study shows that diesel exposure even without the simultaneous presence of the allergen is not only able to induce pro-Th2 chemokines leading to the recruitment of Th2 cells but also able to increase Th1-associated chemokine receptor expression maybe as a scavenging mechanism to clear pro-Th1 chemokines.


   Acknowledgments
 
The authors thank the team of the Pneumology Department of the Calmette Hospital of Lille for recruitment of patients with allergy and the staff of Renault Lardy for providing the diesel extracts and for the HPLC analysis. They are grateful to Catherine Duez for critical review of this work.


   FOOTNOTES
 
Supported by a grant from Agence de l'Environnement et de la Maîtrise de l'Energie (ADEME), Conseil régional Nord-Pas-De-Calais, PRIMEQUAL 2 (02-0000-70, from Ministère de l'Environnement), and Comité National contre les Maladies Respiratoires et de la Tuberculose.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form November 6, 2002; accepted in final form April 24, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fujimaki H, Ui N, Ushio H, Nohara K, Endo T. Roles of CD4+ and CD8+ T cells in adjuvant activity of diesel exhaust particles in mice. Int Arch Allergy Immunol 2001;124:485–496.[CrossRef][Medline]
  2. van Zijverden M, van der Pijl A, Bol M, van Pinxteren FA, de Haar C, Penninks AH, van Loveren H, Pieters R. Diesel exhaust, carbon black, and silica particles display distinct Th1/Th2 modulating activity. Toxicol Appl Pharmacol 2000;168:131–139.[CrossRef][Medline]
  3. Wang M, Saxon A, Diaz-Sanchez D. Early IL-4 production driving Th2 differentiation in a human in vivo allergic model is mast cell derived. Clin Immunol 1999;90:47–54.[CrossRef][Medline]
  4. Diaz-Sanchez D, Tsien A, Fleming J, Saxon A. Combined diesel exhaust particulate and ragweed allergen challenge markedly enhances human in vivo nasal ragweed-specific IgE and skews cytokine production to a T helper cell 2-type pattern. J Immunol 1997;158:2406–2413.[Abstract]
  5. Diaz-Sanchez D, Penichet-Garcia M, Saxon A. Diesel exhaust particles directly induce activated mast cells to degranulate and increase histamine levels and symptom severity. J Allergy Clin Immunol 2000;106:1140–1146.[CrossRef][Medline]
  6. Miyabara Y, Ichinose T, Takano H, Lim HB, Sagai M. Effects of diesel exhaust on allergic airway inflammation in mice. J Allergy Clin Immunol 1998;102:805–812.[CrossRef][Medline]
  7. Miyabara Y, Ichinose T, Takano H, Sagai M. Diesel exhaust inhalation enhances airway hyperresponsiveness in mice. Int Arch Allergy Immunol 1998;116:124–131.[CrossRef][Medline]
  8. Maggi E, Biswas P, Del Prete G, Parronchi P, Macchia D, Simonelli C, Emmi L, De Carli M, Tiri A, Ricci M, et al. Accumulation of Th-2-like helper T cells in the conjunctiva of patients with vernal conjunctivitis. J Immunol 1991;146:1169–1174.[Abstract]
  9. Teran LM, Davies DE. The chemokines: their potential role in allergic inflammation. Clin Exp Allergy 1996;26:1005–1019.[CrossRef][Medline]
  10. Homey B, Zlotnik A. Chemokines in allergy. Curr Opin Immunol 1999;11:626–634.[CrossRef][Medline]
  11. Sallusto F, Mackay CR, Lanzavecchia A. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 1997;277:2005–2007.[Abstract/Free Full Text]
  12. Bonecchi R, Bianchi G, Bordignon PP, D'Ambrosio D, Lang R, Borsatti A, Sozzani S, Allavena P, Gray PA, Mantovani A, et al. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J Exp Med 1998;187:129–134.[Abstract/Free Full Text]
  13. Zingoni A, Soto H, Hedrick JA, Stoppacciaro A, Storlazzi CT, Sinigaglia F, D'Ambrosio D, O'Garra A, Robinson D, Rocchi M, et al. The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells. J Immunol 1998;161:547–551.[Abstract/Free Full Text]
  14. Siveke JT, Hamann A. T helper 1 and T helper 2 cells respond differentially to chemokines. J Immunol 1998;160:550–554.[Abstract/Free Full Text]
  15. Fahy O, Senechal S, Pene J, Scherpereel A, Lassalle P, Tonnel AB, Yssel H, Wallaert B, Tsicopoulos A. Diesel exposure favors Th2 cell recruitment by mononuclear cells and alveolar macrophages from allergic patients by differentially regulating macrophage-derived chemokine and IFN-gamma-induced protein-10 production. J Immunol 2002;168:5912–5919.[Abstract/Free Full Text]
  16. Haque NS, Fallon JT, Taubman MB, Harpel PC. The chemokine receptor CCR8 mediates human endothelial cell chemotaxis induced by I-309 and Kaposi sarcoma herpesvirus-encoded vMIP-I and by lipoprotein(a)-stimulated endothelial cell conditioned medium. Blood 2001;97:39–45.[Abstract/Free Full Text]
  17. Inngjerdingen M, Damaj B, Maghazachi AA. Human NK cells express CC chemokine receptors 4 and 8 and respond to thymus and activation-regulated chemokine, macrophage-derived chemokine, and I-309. J Immunol 2000;164:4048–4054.[Abstract/Free Full Text]
  18. Iellem A, Colantonio L, Bhakta S, Sozzani S, Mantovani A, Sinigaglia F, D'Ambrosio D. Inhibition by IL-12 and IFN-alpha of I-309 and macrophage-derived chemokine production upon TCR triggering of human Th1 cells. Eur J Immunol 2000;30:1030–1039.[CrossRef][Medline]
  19. Politz O, Kodelja V, Guillot P, Orfanos CE, Goerdt S. Pseudoexons and regulatory elements in the genomic sequence of the beta-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1. Cytokine 2000;12:120–126.[CrossRef][Medline]
  20. Farber JM. Mig and IP-10: CXC chemokines that target lymphocytes. J Leukoc Biol 1997;61:246–257.[Abstract]
  21. Flier J, Boorsma DM, van Beek PJ, Nieboer C, Stoof TJ, Willemze R, Tensen CP. Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. J Pathol 2001;194:398–405.[CrossRef][Medline]
  22. Gasperini S, Marchi M, Calzetti F, Laudanna C, Vicentini L, Olsen H, Murphy M, Liao F, Farber J, Cassatella MA. Gene expression and production of the monokine induced by IFN-gamma (MIG), IFN-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein-10 (IP-10) chemokines by human neutrophils. J Immunol 1999;162:4928–4937.[Abstract/Free Full Text]
  23. Piali L, Weber C, LaRosa G, Mackay CR, Springer TA, Clark-Lewis I, Moser B. The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig. Eur J Immunol 1998;28:961–972.[CrossRef][Medline]
  24. Fujieda S, Diaz-Sanchez D, Saxon A. Combined nasal challenge with diesel exhaust particles and allergen induces in vivo IgE isotype switching. Am J Respir Cell Mol Biol 1998;19:507–512.[Abstract/Free Full Text]
  25. Jinquan T, Jing C, Jacobi HH, Reimert CM, Millner A, Quan S, Hansen JB, Dissing S, Malling HJ, Skov PS, et al. CXCR3 expression and activation of eosinophils: role of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma. J Immunol 2000;165:1548–1556.[Abstract/Free Full Text]
  26. Fahy O, Tsicopoulos A, Hammad H, Pestel J, Tonnel AB, Wallaert B. Effects of diesel organic extracts on chemokine production by peripheral blood mononuclear cells. J Allergy Clin Immunol 1999;103:1115–1124.[CrossRef][Medline]
  27. Asselin S, Conjeaud H, Minty A, Fradelizi D, Breban M. Stable polarization of peripheral blood T cells towards type 1 or type 2 phenotype after polyclonal activation. Eur J Immunol 1998;28:532–539.[CrossRef][Medline]
  28. Imai T, Nagira M, Takagi S, Kakizaki M, Nishimura M, Wang J, Gray PW, Matsushima K, Yoshie O. Selective recruitment of CCR4-bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine. Int Immunol 1999;11:81–88.[Abstract/Free Full Text]
  29. Fahy O, Hammad H, Senechal S, Pestel J, Tonnel AB, Wallaert B, Tsicopoulos A. Synergistic effect of diesel organic extracts and allergen Der p 1 on the release of chemokines by peripheral blood mononuclear cells from allergic subjects: involvement of the map kinase pathway. Am J Respir Cell Mol Biol 2000;23:247–254.[Abstract/Free Full Text]
  30. Diaz-Sanchez D, Dotson AR, Takenaka H, Saxon A. Diesel exhaust particles induce local IgE production in vivo and alter the pattern of IgE messenger RNA isoforms. J Clin Invest 1994;94:1417–1425.
  31. Fujimaki H, Ui N, Endo T. Induction of inflammatory response of mice exposed to diesel exhaust is modulated by CD4(+) and CD8(+) T cells. Am J Respir Crit Care Med 2001;164:1867–1873.[Abstract/Free Full Text]
  32. Walters DM, Breysse PN, Wills-Karp M. Ambient urban Baltimore particulate-induced airway hyperresponsiveness and inflammation in mice. Am J Respir Crit Care Med 2001;164:1438–1443.[Abstract/Free Full Text]
  33. Nightingale JA, Maggs R, Cullinan P, Donnelly LE, Rogers DF, Kinnersley R, Fan Chung K, Barnes PJ, Ashmore M, Newman-Taylor A. Airway inflammation after controlled exposure to diesel exhaust particulates. Am J Respir Crit Care Med 2000;162:161–166.[Abstract/Free Full Text]
  34. Hashimoto K, Ishii Y, Uchida Y, Kimura T, Masuyama K, Morishima Y, Hirano K, Nomura A, Sakamoto T, Takano H, et al. Exposure to diesel exhaust exacerbates allergen-induced airway responses in guinea pigs. Am J Respir Crit Care Med 2001;164:1957–1963.[Abstract/Free Full Text]
  35. Kobayashi T. Exposure to diesel exhaust aggravates nasal allergic reaction in guinea pigs. Am J Respir Crit Care Med 2000;162:352–356.[Abstract/Free Full Text]
  36. Hashimoto S, Gon Y, Takeshita I, Matsumoto K, Jibiki I, Takizawa H, Kudoh S, Horie T. Diesel exhaust particles activate p38 MAP kinase to produce interleukin 8 and RANTES by human bronchial epithelial cells and N-acetylcysteine attenuates p38 MAP kinase activation. Am J Respir Crit Care Med 2000;161:280–285.[Abstract/Free Full Text]
  37. Boland S, Baeza-Squiban A, Fournier T, Houcine O, Gendron MC, Chevrier M, Jouvenot G, Coste A, Aubier M, Marano F. Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production. Am J Physiol 1999;276:L604–L613.
  38. Diaz-Sanchez D, Jyrala M, Ng D, Nel A, Saxon A. In vivo nasal challenge with diesel exhaust particles enhances expression of the CC chemokines rantes, MIP-1alpha, and MCP-3 in humans. Clin Immunol 2000;97:140–145.[CrossRef][Medline]
  39. Salvi SS, Nordenhall C, Blomberg A, Rudell B, Pourazar J, Kelly FJ, Wilson S, Sandstrom T, Holgate ST, Frew AJ. Acute exposure to diesel exhaust increases IL-8 and GRO-alpha production in healthy human airways. Am J Respir Crit Care Med 2000;161:550–557.[Abstract/Free Full Text]
  40. Miller MD, Krangel MS. The human cytokine I-309 is a monocyte chemoattractant. Proc Natl Acad Sci USA 1992;89:2950–2954.[Abstract/Free Full Text]
  41. Selvan RS, Zhou LJ, Krangel MS. Regulation of I-309 gene expression in human monocytes by endogenous interleukin-1. Eur J Immunol 1997;27:687–694.[Medline]
  42. Brenier-Pinchart MP, Pelloux H, Simon J, Ricard J, Bosson JL, Ambroise-Thomas P. Toxoplasma gondii induces the secretion of monocyte chemotactic protein-1 in human fibroblasts, in vitro. Mol Cell Biochem 2000;209:79–87.[CrossRef][Medline]
  43. Panina-Bordignon P, Papi A, Mariani M, Di Lucia P, Casoni G, Bellettato C, Buonsanti C, Miotto D, Mapp C, Villa A, et al. The C–C chemokine receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic asthmatics. J Clin Invest 2001;107:1357–1364.[Medline]
  44. Hieshima K, Imai T, Baba M, Shoudai K, Ishizuka K, Nakagawa T, Tsuruta J, Takeya M, Sakaki Y, Takatsuki K, et al. A novel human CC chemokine PARC that is most homologous to macrophage-inflammatory protein-1 alpha/LD78 alpha and chemotactic for T lymphocytes, but not for monocytes. J Immunol 1997;159:1140–1149.[Abstract]
  45. Reape TJ, Rayner K, Manning CD, Gee AN, Barnette MS, Burnand KG, Groot PH. Expression and cellular localization of the CC chemokines PARC and ELC in human atherosclerotic plaques. Am J Pathol 1999;154:365–374.[Abstract/Free Full Text]
  46. Goebeler M, Trautmann A, Voss A, Brocker EV, Toksoy A, Gillitzer R. Differential and sequential expression of multiple chemokines during elicitation of allergic contact hypersensitivity. Am J Pathol 2001;158:431–440.[Abstract/Free Full Text]
  47. Kodelja V, Muller C, Politz O, Hakij N, Orfanos CE, Goerdt S. Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern. J Immunol 1998;160:1411–1418.[Abstract/Free Full Text]
  48. Adema GJ, Hartgers F, Verstraten R, de Vries E, Marland G, Menon S, Foster J, Xu Y, Nooyen P, McClanahan T, et al. A dendritic-cell-derived C–C chemokine that preferentially attracts naive T cells. Nature 1997;387:713–717.[CrossRef][Medline]
  49. Pardo A, Smith KM, Abrams J, Coffman R, Bustos M, McClanahan TK, Grein J, Murphy EE, Zlotnik A, Selman M. CCL18/DC-CK-1/PARC up-regulation in hypersensitivity pneumonitis. J Leukoc Biol 2001;70:610–616.[Abstract/Free Full Text]
  50. Tokuriki A, Seo N, Ito T, Kumakiri M, Takigawa M, Tokura Y. Dominant expression of CXCR3 is associated with induced expression of IP-10 at hapten-challenged sites of murine contact hypersensitivity: a possible role for interferon-gamma-producing CD8(+) T cells in IP-10 expression. J Dermatol Sci 2002;28:234–241.[CrossRef][Medline]
  51. Lauw FN, Simpson AJ, Prins JM, van Deventer SJ, Chaowagul W, White NJ, van der Poll T. The CXC chemokines gamma interferon (IFN-gamma)-inducible protein 10 and monokine induced by IFN-gamma are released during severe melioidosis. Infect Immun 2000;68:3888–3893.[Abstract/Free Full Text]
  52. Kraft M, Riedel S, Maaser C, Kucharzik T, Steinbuechel A, Domschke W, Luegering N. IFN-gamma synergizes with TNF-alpha but not with viable H. pylori in up-regulating CXC chemokine secretion in gastric epithelial cells. Clin Exp Immunol 2001;126:474–481.[CrossRef][Medline]
  53. Flier J, Boorsma DM, Bruynzeel DP, Van Beek PJ, Stoof TJ, Scheper RJ, Willemze R, Tensen CP. The CXCR3 activating chemokines IP-10, Mig, and IP-9 are expressed in allergic but not in irritant patch test reactions. J Invest Dermatol 1999;113:574–578.[CrossRef][Medline]
  54. Brice GT, Graber NL, Hoffman SL, Doolan DL. Expression of the chemokine MIG is a sensitive and predictive marker for antigen-specific, genetically restricted IFN-gamma production and IFN-gamma-secreting cells. J Immunol Methods 2001;257:55–69.[CrossRef][Medline]
  55. Sallusto F, Lenig D, Mackay CR, Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med 1998;187:875–883.[Abstract/Free Full Text]
  56. Nanki T, Lipsky PE. Lack of correlation between chemokine receptor and T(h)1/T(h)2 cytokine expression by individual memory T cells. Int Immunol 2000;12:1659–1667.[Abstract/Free Full Text]
  57. Sallusto F, Kremmer E, Palermo B, Hoy A, Ponath P, Qin S, Forster R, Lipp M, Lanzavecchia A. Switch in chemokine receptor expression upon TCR stimulation reveals novel homing potential for recently activated T cells. Eur J Immunol 1999;29:2037–2045.[CrossRef][Medline]
  58. D'Ambrosio D, Iellem A, Bonecchi R, Mazzeo D, Sozzani S, Mantovani A, Sinigaglia F. Selective up-regulation of chemokine receptors CCR4 and CCR8 upon activation of polarized human type 2 Th cells. J Immunol 1998;161:5111–5115.[Abstract/Free Full Text]
  59. Power CA, Meyer A, Nemeth K, Bacon KB, Hoogewerf AJ, Proudfoot AE, Wells TN. Molecular cloning and functional expression of a novel CC chemokine receptor cDNA from a human basophilic cell line. J Biol Chem 1995;270:19495–19500.[Abstract/Free Full Text]
  60. Kim CH, Kunkel EJ, Boisvert J, Johnston B, Campbell JJ, Genovese MC, Greenberg HB, Butcher EC. Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with extralymphoid tissue homing potential. J Clin Invest 2001;107:595–601.[Medline]
  61. Sato K, Kawasaki H, Nagayama H, Enomoto M, Morimoto C, Tadokoro K, Juji T, Takahashi T. Chemokine receptor expressions and responsiveness of cord blood T cells. J Immunol 2001;166:1659–1666.[Abstract/Free Full Text]
  62. Rabin RL, Park MK, Liao F, Swofford R, Stephany D, Farber JM. Chemokine receptor responses on T cells are achieved through regulation of both receptor expression and signaling. J Immunol 1999;162:3840–3850.[Abstract/Free Full Text]
  63. Andrew DP, Ruffing N, Kim CH, Miao W, Heath H, Li Y, Murphy K, Campbell JJ, Butcher EC, Wu L. C–C chemokine receptor 4 expression defines a major subset of circulating nonintestinal memory T cells of both Th1 and Th2 potential. J Immunol 2001;166:103–111.[Abstract/Free Full Text]
  64. D'Amico G, Frascaroli G, Bianchi G, Transidico P, Doni A, Vecchi A, Sozzani S, Allavena P, Mantovani A. Uncoupling of inflammatory chemokine receptors by IL-10: generation of functional decoys. Nat Immunol 2000;1:387–391.[CrossRef][Medline]



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