The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fahy, O.
Right arrow Articles by Tsicopoulos, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fahy, O.
Right arrow Articles by Tsicopoulos, A.
The Journal of Immunology, 2002, 168: 5912-5919.
Copyright © 2002 by The American Association of Immunologists

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 Production1

Olivier Fahy2,*, Stéphanie Sénéchal2,*, Jérôme Pène{dagger}, Arnaud Scherpereel*, Philippe Lassalle*, André-Bernard Tonnel*,{ddagger}, Hans Yssel{dagger}, Benoît Wallaert*,{ddagger} and Anne Tsicopoulos3,*,{ddagger}

* Institut National de la Santé et de la Recherche Médicale Unité 416, Institut Pasteur de Lille, Lille, France; {dagger} Institut National de la Santé et de la Recherche Médicale Unité 454, Montpellier, France; and {ddagger} Clinique des Maladies Respiratoires et Center Hospitalier Régional et Universitaire de Lille, Lille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Diesel exhausts and their associated organic compounds may be involved in the recent increase in the prevalence of allergic disorders, through their ability to favor a type 2 immune response. Type 2 T cells have been shown to be preferentially recruited by the chemokines eotaxin (CCL11), macrophage-derived chemokine (MDC, CCL22), and thymus activation-regulated chemokine (CCL17) through their interaction with CCR3 and CCR4, respectively, whereas type 1 T cells are mainly recruited by IFN-{gamma}-induced protein-10 (CXCL10) through CXCR3 binding. The aim of the study was to evaluate the effect of diesel exposure on the expression of chemokines involved in type 1 and 2 T cell recruitment. PBMC and alveolar macrophages from house dust mite allergic patients were incubated with combinations of diesel extracts and Der p 1 allergen, and chemokine production was analyzed. Diesel exposure alone decreased the constitutive IP-10 production, while it further augmented allergen-induced MDC production, resulting in a significantly increased capacity to chemoattract human Th2, but not Th1 clones. Inhibition experiments with anti-type 1 or type 2 cytokine Abs as well as cytokine mRNA kinetic evaluation showed that the chemokine variations were not dependent upon IL-4, IL-13, or IFN-{gamma} expression. In contrast, inhibition of the B7:CD28 pathway using a CTLA-4-Ig fusion protein completely inhibited diesel-dependent increase of allergen-induced MDC production. This inhibition was mainly dependent upon the CD86 pathway and to a lesser extent upon the CD80 pathway. These results suggest that the exposure to diesel exhausts and allergen may likely amplify a deleterious type 2 immune response via a differential regulation of chemokine production through the CD28 pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The prevalence of allergic diseases has strongly increased in the last 20 years, in particular in industrialized countries (1), and urban atmospheric pollution is one of the potential culprits. Exhaust from diesel-powered engines is now the main source of particles in urban air pollution (2). In particular, polyaromatic hydrocarbons (PAH)4 adsorbed on the carbon core of diesel exhaust particles (DEP) are believed to exacerbate the allergic inflammatory reaction. For example, DEP-PAH induce Ig switch toward IgE and act as an adjuvant by potentiating both total and specific IgE production by committed B cells (3, 4). DEP-PAH can also act directly on many effector cells, such as macrophages, neutrophils, mast cells, and eosinophils, by inducing their recruitment and by triggering the release of proinflammatory mediators (5, 6, 7). Interestingly, combined exposure to diesel extracts and allergen strongly potentiates the effect of each stimulus, further enhancing IgE production, and skewing the immune response toward a type 2 cytokine profile (8).

Chemokines are small chemotactic cytokines that are involved in the initiation of inflammatory reactions by specifically recruiting the actors of the inflammatory process at the site of inflammation (9). Recently, certain chemokines and their corresponding receptors have been shown to be associated with type 1- and type 2-mediated immune response (10). Indeed, whereas type 1 T cells preferentially express the chemokine receptor CXCR3 and are attracted by its ligand IFN-{gamma}-induced protein-10 (IP-10 or CXCL10) (11), type 2 T cells preferentially express CCR3 and CCR4, and are attracted respectively by eotaxin (CCL11), and by monocyte-derived chemokine (MDC or CCL22) and thymus and activation-regulated chemokine (TARC or CCL17) (12, 13, 14).

The aim of this study was therefore to evaluate the ability of diesel exhausts to favor the development of a type 2 immune response by affecting the production of chemokines involved in the attraction of type 1 and type 2 T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of diesel exhaust and extraction of DEP-PAH

DEP were obtained from a light-duty diesel-powered passenger car (Renault, Pollution Department, Boulogne-Billancourt, France), and DEP-PAH were extracted in dichloromethane (CH2Cl2) solvent. The resulting extract was then analyzed by HPLC to determine the concentration of its main organic constituents, as previously described (15). The stock solution (25 µg DEP-PAH/ml CH2Cl2) was stored at +4°C in the dark, and was diluted freshly each time in RPMI 1640 before use.

Donors

Venous blood and alveolar macrophages (AM) were collected from allergic asthmatic patients sensitive to house dust mite. All had a clinical history of asthma, and showed positive skin-prick tests toward Dermatophagoides pteronyssinus (Der.pt) allergen, positive radioallergosorbent test (RAST class 61 4), and elevated serum IgE levels (>450 IU/ml).

Bronchoalveolar lavages (BAL) were obtained from these patients after inhaled corticosteroids and {beta}2-adrenergic drugs had been discontinued for at least 1 wk. Theophyllin and cromolyn sodium were also stopped at least 1 day before the lavages. BAL were performed, as previously described (16), by instillation of saline solution into the bronchoalveolar tree under fiberoptic bronchoscopic observation. The study was approved by the ethical committee of the Centre Hospitalier Régional Universitaire de Lille (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale Lille No. 9307).

Cell culture

PBMC. PBMC from 12 patients were prepared from blood collected on heparin, as previously described (17). PBMC (2 x 106 cells/well) were cultured in 12-well flat-bottom microculture plates (Nunc, Roskilde, Denmark) in complete RPMI 1640 in the presence or absence of CH2Cl2, DEP-PAH (50 ng/ml), or Der p 1 (100 ng/ml; Indoor Biotechnologies, Cardiff, U.K.). Culture supernatants were collected after 24 h, filtered through a 0.2-µm-pore-sized filter (Sartorius AG, Göttingen, Germany), aliquoted, and stored at -20°C for further chemokine and cytokine quantifications. The toxicity of the CH2Cl2 solvent and the DEP-PAH solution added to the cell culture was evaluated by a trypan blue test. The volume of CH2Cl2 or DEP-PAH solubilized in CH2Cl2 added to 1 ml medium was limited to 2 µl. No toxic effect was detected (viability >97% for all conditions) after 24 h. The addition of CH2Cl2 in cell cultures had no significant effect on chemokine production. The optimal concentrations of DEP-PAH and kinetics of stimulation were determined in preliminary experiments. Time course (2, 9, 18, 24, and 48 h) and diesel dose response (5 and 50 ng DEP-PAH/ml medium) experiments showed that the effect of DEP-PAH on Der p 1-induced MDC production was optimal at a dose of 50 ng/ml following a culture period of 24 h. At this concentration and culture time, no differences in IP-10 production, irrespective of the concentrations of DEP-PAH, were observed, and these experimental conditions were therefore selected for use in the entire study. Where indicated, neutralizing anti-IL-4, anti-IL-13, and anti-IFN-{gamma} mAbs (R&D Systems, Abingdon, U.K.) were used at a final concentration of 10 µg/ml. This concentration had been determined to bind over 90% of the corresponding specific cytokine by ELISA (data not shown). In experiments assessing the involvement of the CD28 pathway, a blocking chimeric CTLA-4-Ig was used (18) (a kind gift from J. Ellis, GlaxoSmithKline, Stevenage, U.K.) at concentrations ranging from 2.5 to 25 µg/ml, as well as blocking Abs for CD80 (clone BB1) and CD86 (clone IT2.2), both from BD Biosciences (Pont de Claix, France), at concentrations of 10 and 0.1 µg/ml, respectively, as previously described (19). The fusion protein or blocking Abs were added at the same time as allergen and/or DEP-PAH. Percentages of inhibition or amplification were calculated after subtracting the corresponding control values from the analyzed values (i.e., control value for Der p 1 and DEP-PAH for DEP-PAH + Der p 1 conditions). In some experiments, monocytes and CD4+ T cells were further purified using magnetic depletion (CD4 isolation kit and CD14 beads, respectively, from Miltenyi Biotech, Paris, France). Cell purity was over 97% in both cases.

Alveolar macrophages. BAL fluids from three patients were filtered through a sterile surgical gauze and centrifuged at 400 x g for 10 min at +4°C. After three washings, the pellet was resuspended (106 cells/well) in complete RPMI 1640, and incubated with diesel extracts and allergen under conditions similar to those described for PBMC. Percentages of macrophages, lymphocytes, neutrophils, and eosinophils were 90.1 ± 2.2, 7.5 ± 2.1, 1.7 ± 0.3, and 0.5 ± 0.2%, respectively, as assessed on Giemsa-stained cytospin preparations.

Generation of skin-derived human Th1 and Th2 clones. Human Th1 and Th2 clones were generated from cutaneous delayed-type hypersensitivity reactions, induced in healthy human volunteers after an initial sensitization and a subsequent challenge with 2,4 dinitrochlorobenzene, as previously described (20, 21). Briefly, T cell clones were analyzed for the production of IL-4, IL-5, IFN-{gamma}, and IL-10 by cytokine-specific ELISA, as described previously (22), and T cell clones with a polarized Th1 or Th2 cytokine production profile were selected and maintained in culture by weekly stimulation with feeder cells (23). Three to four days after each restimulation, the cultures were split and further expanded in medium containing 20 U/ml rIL-2. T cell clones were cultured in Yssel’s medium, supplemented with 1% AB+ human serum, and used in experiments between 10 and 12 days after the last stimulation with feeder cells.

Quantification of chemokine and cytokine levels in cell culture supernatants

Concentrations of MDC, IP-10, eotaxin, TARC (R&D Systems), IL-4, IL-13, and IFN-{gamma} (Diaclone, Besançon, France) were measured by ELISA, according to the manufacturer’s recommendations, and expressed in pg/ml. The level of sensitivity was 10 pg/ml for all chemokines, 1 pg/ml for IL-4, and 5 pg/ml for IL-13 and IFN-{gamma}. Results for chemokine concentrations were expressed as median ± interquartile (Q1 and Q3) values.

Chemokine, chemokine receptor, and cytokine mRNA expression

mRNA expression was analyzed using semiquantitative RT-PCR. After removal of the supernatants, PBMC were resuspended in TRIzol reagent (Life Technologies, Gaithersburg, MD), and total cellular RNA was extracted according to the manufacturer’s procedure. Total RNA was treated with RNase-free DNase (Boehringer Mannheim, Meylan, France) to eliminate a possible contamination by genomic DNA. RNA was then extracted and precipitated in ethanol. RNA concentrations were measured using a spectrophotometer. RNA integrity was determined by visualizing the 18S and 28S rRNA bands with ethidium bromide after electrophoresis on a 2% formaldehyde gel. RT-PCR was performed as previously described (17). PCR primers for human GAPDH, MDC, IP-10, IL-4, IL-13, IFN-{gamma}, CCR4, and CXCR3 were purchased from Eurogentec (Seraing, Belgium). The sequences of the primers were as follows: GAPDH sense, 5'-GTCTTCACCACCATGGAG-3', and antisense, 5'-CCAAAGTTGTCATGGATGACC-3'; MDC sense, 5'-TTGTCCTCGTCCTCCTTGCTGTGGC- 3', and antisense, 5'-AATCATCTTCACCCAGGGCACTCTG-3'; IP-10 sense, 5'-CGATTCTGATTTGCTGCCTTAT-3', and antisense, 5'-GACATCTCTTCTCACCCTTCTTTTT-3'; IL-4 sense, 5'-TGCCTCCAAGAACACAACTG-3', and antisense, 5'-AACGTACTCTGGTTGGCTTC-3'; IL-13 sense, 5'-GAGTGTGTTTGTCACCGTTG-3', and antisense, 5'-TACTCGTTGGCTGAGAGCTG-3'; IFN-{gamma} sense, 5'-GCAGAGCCAAATTGTCTCCT-3', and antisense, 5'-ATGCTCTTCGACCTCGAAAC-3'; CCR4 sense, 5'-TTGGACTATGCCATCCAGGC-3', and antisense, 5'-AATTCCCTCTGGAGAAACCC-3'; CXCR3 sense, 5'-CCTTCCTGCCAGCCCTCTACA-3', and antisense, 5'-CCACCACGACCACCACCAC-3'.

The number of amplification cycles for each product was determined to define optimal conditions for linearity and to permit semiquantitative analysis of signal strength. Twenty-five cycles were performed for amplification of GAPDH; 30 for MDC, CCR4, and CXCR3; 35 for IL-4 and IFN-{gamma}; 36 for IP-10; and 42 for IL-13. Amplification was initiated by a 1-min denaturation step at 94°C, and was then followed by 25–39 cycles at 94°C for 1 min; 55°C for 1 min for GAPDH, IL-4, IL-13, CCR4, and IFN-{gamma}; 60°C for IP-10; 65°C for MDC and CXCR3; and 72°C for 1 min, followed by 7-min extension at 72°C using a DNA thermal cycler (Mastercycler 5330; Brinkmann Instruments, Westbury, NY). Amplified PCR products were separated by gel electrophoresis in 1.5% agarose after Gelstar nucleic acid staining (FMC Bioproducts, Rockland, ME). DNA m.w. marker VI (0.15–2.1 kb) was purchased from Boehringer Mannheim. The intensity of each spot was calculated by densitometry analysis, and results were expressed as percentage of OD of each corresponding GAPDH housekeeping gene (Gel Analyst; Clara Vision, Orsay, France).

Chemotaxis assay

T cell clones were harvested and resuspended at a concentration of 106 cells/ml in complete RPMI 1640. In some experiments, CD4+ T cells purified from allergic patients were used instead of T cell clones. The chemotaxis protocol was performed with a 48-well microchemotaxis chamber (NeuroProbe, Cabin John, MD) using 5-µm-pore polycarbonate filters (Nuclepore, Pleasanton, CA) for 2 h at 37°C in 5% CO2, using supernatants from PBMC and alveolar macrophages (AM) exposed to CH2Cl2 solvent, 50 ng/ml DEP-PAH, 100 ng Der p 1, or both DEP-PAH and Der p 1. T cells that had migrated through the filter and reached the bottom well were counted under a light microscope with a magnification of 500-fold using a Thomas cell. Each condition was performed in triplicate, and at least four fields were counted for each well. The results were expressed as the subtraction of the mean number of migrating T cells with complete RPMI 1640 medium alone (negative control) from the mean number of T cell clones that migrated specifically in response to chemokines present in the culture supernatants. For positive controls, human rMDC and rIP-10 were used for Th2 and Th1 clones, respectively, at a concentration of 6 x 10-7 M (Tebu, Le Perray-en-Yvelines, France). For neutralization assays, samples were incubated for 1 h at 37°C before the chemotaxis assay with anti-MDC-neutralizing polyclonal Abs (R&D Systems) or anti-human IP-10 mAb (BD Biosciences) at a final concentration of 50 µg/ml. A chemokinesis effect was ruled out, as previously described (17). Results corresponding to the chemotaxis assays were expressed as mean ± SEM.

Statistical analysis

Statistical analysis for chemokine variations was performed using the nonparametric Wilcoxon’s paired rank test and the paired t test for chemotaxis assays. Values of p < 0.05 were regarded as statistically significant. Statistical analysis was performed using the Statview 4.11 software (Abacus Concepts, Berkeley, CA) on Macintosh.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DEP-PAH selectively potentiate Der p 1-induced MDC and decrease IP-10 release from PBMC and AM

Exposure of PBMC from asthmatic patients to 100 ng/ml purified Der p 1 induced a significant production of MDC, whereas DEP-PAH alone had no effect (Fig. 1GoA). However, the addition of DEP-PAH to Der p 1 had a potentiating effect on Der p 1-induced MDC production (+105%). Similar results were obtained with AM from allergic subjects, although the synergistic effect of DEP-PAH on Der p 1-induced MDC release (+20.5%) was weaker than that observed for PBMC (Table IGo).



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 1. Chemokine release by PBMC from allergic patients, stimulated with DEP-PAH and/or Der p 1. Production of MDC (A) and IP-10 (B) by PBMC from patients allergic to house dust mite (n = 12), stimulated with 50 ng/ml DEP-PAH and/or 100 ng/ml Der p 1 for 24 h. Control samples were stimulated with CH2Cl2, used to dissolve the DEP-PAH. Results are expressed as median ± interquartiles. *, p < 0.05 vs control; #, p < 0.01 vs control; {dagger}, p < 0.01 vs Der p 1.

 

View this table:
[in this window]
[in a new window]
  		Table I. Production of chemokines MDC, IP-10, eotaxin and TARC by alveolar macrophages from allergic patients under DEP-PAH and/or Der p 1 stimulation1

 
A spontaneous production of IP-10 was observed in supernatants from unstimulated PBMC and AM. Incubation of PBMC and AM with Der p 1 had no effect on the spontaneous level of IP-10, while incubation with DEP-PAH induced a decrease in IP-10 production as compared with the control (-24% for PBMC and -23% for AM) (Fig. 1GoB and Table IGo). For AM, the combined exposure to DEP-PAH and Der p 1 markedly decreased spontaneous IP-10 production (-28.4%), although no significant effect was observed for PBMC, most likely due to the high interindividual variability in this culture condition.

The concentrations of TARC and eotaxin in the supernatants from PBMC (data not shown) and AM (Table IGo), exposed to diesel extracts and/or Der p 1, were very low (=20 pg/ml), irrespective of the kinetics of stimulation and the concentrations of DEP-PAH and/or Der p 1. No difference in TARC and eotaxin production was observed between the different conditions of cell stimulation, suggesting that DEP-PAH acted selectively on the regulation of MDC and IP-10 release.

To better evaluate the effects of the combined DEP-PAH and allergen Der p 1 exposure on the chemokine profile, results were expressed as the ratio between MDC and IP-10 production, reflecting the balance between pro-Th2 and pro-Th1 chemokines. As shown in Table IIGo, this ratio was increased after incubation of the PBMC and AM with Der p 1 or with both diesel extracts and allergen. Exposure of AM, but not PBMC, to DEP-PAH alone, resulted in an increase of this ratio. These results suggest that chemokine production by PBMC and AM reflects a pro-Th2 pattern, as a consequence of the increased MDC production by PBMC and the decreased IP-10 production by AM.


View this table:
[in this window]
[in a new window]
  		Table II. Ratio between MDC production and IP-10 production for PBMC and alveolar macrophages from allergic patients under DEP-PAH and/or Der p 1 stimulation1

 
DEP-PAH- and Der p 1-stimulated PBMC and AM supernatants exhibit a predominant Th2 chemotactic activity

To confirm the notion that stimulation of PBMC and AM by DEP-PAH and Der p 1 induces an environment that preferentially attracts Th2 cells, human Th1 and Th2 cell clones were used for chemotaxis assays. As shown in Fig. 2GoA, supernatants from PBMC incubated with DEP-PAH, Der p 1, or both compounds showed no enhanced capacity to attract Th1 clones, as compared with control supernatants. In contrast, supernatants, obtained from PBMC stimulated with both DEP-PAH and Der p 1, displayed a significant increased capacity to chemoattract Th2 clones. The addition of a neutralizing anti-MDC Ab to the supernatants before the assay reduced the Th2 cell-attracting activity of all supernatants, in particular those from DEP-PAH- and Der p 1-stimulated PBMC (70.5% inhibition vs control). Supernatants from DEP-PAH- and Der p 1-stimulated PBMC, to which a neutralizing anti-IL-8 mAb had been added, showed no inhibitory effect on their ability to induce chemotaxis of Th2 clones (data not shown). Purified CD4+ T cells isolated from allergic patients were not attracted by allergen- and/or diesel-stimulated PBMC supernatants, probably because of the low number of circulating Th2 cells (data not shown).



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 2. Capacity of PBMC from allergic patients stimulated with DEP-PAH and/or Der p 1 to induce chemotaxis in Th1 and Th2 clones. Supernatants of PBMC (A) and AM (B) from allergic patients, stimulated with CH2Cl2 in the absence or presence of 50 ng/ml DEP-PAH and/or 100 ng/ml Der p 1, were harvested after 24 h and used in an in vitro chemotaxis assay with either Th1 or Th2 clones. Where indicated, culture supernatants had been preincubated with neutralizing anti-MDC or anti-IP-10 mAb at a final concentration of 50 µg/ml. Recombinant human MDC or IP-10 at a final concentration of 6 x 10-7 M were used as positive control. Results are expressed as mean ± SEM. n = 5 and 3 for supernatants from PBMC and AM, respectively. N.S., Nonstatistically significant. *, p < 0.05.

 
Similarly, the migration of Th1 clones was not differentially affected by each of the supernatants from stimulated AM (Fig. 2GoB). In contrast, the number of migrating Th2 clones was markedly increased by supernatants of AM incubated with DEP-PAH and Der p 1. Under the latter condition, the addition of a neutralizing anti-MDC Ab had a strong inhibitory effect on Th2 clone migration (corresponding to a 63% inhibition vs control).

Modulation of chemokine and chemokine receptor mRNA expression

MDC and IP-10 mRNA production at 24 h was altered in a similar way to what was observed at the protein level. As shown in Fig. 3Go, A and B, mRNA expression for MDC did not change following exposure of PBMC to DEP-PAH alone, while MDC transcripts were enhanced following exposure to Der p 1. However, a strong potentiating effect of both stimuli was observed on the expression of MDC transcripts. In contrast, DEP-PAH induced a decrease in IP-10 mRNA expression, while exposure to Der p 1 alone or to both DEP-PAH and Der p 1 did not affect the expression of IP-10 transcripts, as compared with nonstimulated PBMC, demonstrating that DEP-PAH signaling acts on both transcriptional and translational levels of MDC and IP-10 genes. MDC mRNA expression was not detected at 0 or 2 h. MDC transcripts started to appear at 9 h, with variations that reflected the results of productions of proteins. Similarly, confirming the observation that freshly isolated PBMC spontaneously produce IP-10, mRNA for this chemokine was expressed constitutively and started to decrease 9 h after DEP-PAH exposure. No transcriptional variations were observed for the MDC and IP-10 chemokine receptors CCR4 and CXCR3, respectively, at the various time points (Fig. 3GoC).



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 3. Analysis of mRNA expression in PBMC from allergic patients stimulated with DEP-PAH and/or Der p 1. MDC and IP-10 mRNA expression was determined by semiquantitative RT-PCR (A) following stimulation of PBMC from patients allergic to house dust mite with CH2Cl2 (Control, lane C), 50 ng DEP-PAH (lane 1), 100 ng Der p 1 (lane 2), or DEP-PAH and Der p 1 (lane 3). One representative experiment of three is shown. Values of chemokine mRNA expression were determined by densitometric analysis (B) and normalized against transcripts for the GAPDH housekeeping gene. C, Kinetics of MDC, IP-10, CCR4, and CXCR3 mRNA expression was determined by semiquantitative RT-PCR analysis following culture of PBMC before, and 2, 9, and 24 h after stimulation by DEP-PAH and Der p 1. Values of OD for the chemokine and chemokine receptor mRNA are normalized against transcripts for GAPDH. D, Kinetics of IL-4 and IFN-{gamma} mRNA expression was performed and analyzed, as described above.

 
CD28, but not cytokines, mediates DEP-PAH enhancement of allergen-induced MDC production by PBMC

To identify the signaling pathway triggered by DEP-PAH, the importance of IL-4, IL-13, and IFN-{gamma} was first evaluated as a potential primary stimulus for chemokine production. We assessed in PBMC supernatants both the presence of these cytokines and the effect of neutralizing Abs. The concentrations of IL-4 and IFN-{gamma} at 2, 4, 9, 24, and 48 h remained very low (ranging from 6 to 18 pg/ml, data not shown) and showed no variation between the various culture conditions. There was no production of IL-13. To further evaluate the relationship between chemokine and cytokine expression, we monitored the kinetics of expression of mRNA coding for IL-4, IL-13, and IFN-{gamma}, before and 2, 9, and 24 h after the stimulation of the cells. As shown in Fig. 3GoD, no variation of IFN-{gamma} expression was observed following culture of the cells under the various experimental conditions. Similarly for IL-4, apart from a slight increase in the expression of this cytokine at 2 h, no variation in the expression of IL-4 transcripts was detected in cells stimulated with DEP-PAH and/or Der p 1. Expression of IL-13 mRNA was undetectable (data not shown).

Moreover, production of MDC in PBMC stimulated with allergen and DEP-PAH was not inhibited upon addition of anti-IL-4, anti-IL-13, or anti-IFN-{gamma} Ab (Fig. 4GoA), consistent with the cytokine mRNA expression. These results show that the observed variations in chemokine production were not mediated by cytokines.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 4. Production of MDC by PBMC from allergic patients stimulated with Der p 1 with or without DEP-PAH is dependent upon the CD28, but not the cytokine pathway. A, Inhibitory effect of neutralizing mAbs against IL-4, IL-13, and IFN-{gamma} on Der p 1 (100 ng/ml) ± DEP-PAH (50 ng/ml)-induced MDC production by PBMC from patients allergic to house dust mite (n = 3) at 24 h of culture. Neutralizing Abs were added at a final concentration of 10 µg/ml. Results are expressed as percentage of inhibition ± SEM. B, Inhibitory effect of CTLA-4-Ig on Der p 1 ± DEP-PAH-induced MDC production by PBMC from patients allergic to house dust mite (n = 4) at 24 h of culture. Results are expressed as percentage of inhibition ± SEM. C, Inhibitory effect of blocking Abs against CD80 (10 µg/ml), CD86 (0.1 µg/ml), or a combination of both mAb on Der p 1 ± DEP-PAH-induced MDC production by PBMC from allergic patients to house dust mite (n = 2) at 24 h of culture. Results are expressed as percentage of inhibition ± SEM.

 
Second, the involvement of CD28 was evaluated using a CTLA-4-Ig fusion protein. This chimeric molecule blocks the CD28 costimulatory pathway by binding to CD80 and CD86. CTLA-4-Ig inhibited in a dose-dependent manner allergen-induced MDC production from PBMC, with a maximal effect at a concentration of 25 µg/ml (97.3%) (Fig. 4GoB). MDC production by PBMC, induced by both DEP-PAH and Der p 1 stimulation, was also markedly inhibited by CTLA-4-Ig at a lower concentration than for Der p 1 (86.5% inhibition at 2.5 µg/ml), suggesting that both stimuli triggered MDC production via the CD28 pathway. The inhibition of the CD28 pathway by the chimeric CTLA-4-Ig protein did not counteract the decreased IP-10 levels in DEP-PAH-stimulated PBMC (data not shown). The respective part of CD80 and CD86 in this process was evaluated by using blocking Abs. Anti-CD86 mAb inhibited Der p 1-induced MDC production (47% inhibition), whereas anti-CD80 mAb had no effect (Fig. 4GoC). When diesel extracts were coincubated with Der p 1, an inhibitory effect on MDC production was observed with both anti-CD86 mAb (75% inhibition) and CD80 mAb (10% inhibition) also to a lesser extent. These results suggest that the variations in MDC production were mainly mediated through CD86 costimulation, with an additional effect of diesel extracts on the CD80 pathway.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DEPs and associated DEP-PAH are known to affect several parameters of the inflammatory reaction (24). They are responsible for the dysregulation of parameters more specifically involved in allergy, namely IgE synthesis and the establishment of a type 2 cytokine profile (8). They activate effector cells such as eosinophils and neutrophils, and we and others have demonstrated previously that this effect is mediated, in part, by production of chemokines such as RANTES, IL-8, monocyte chemotactic protein-3, and macrophage-inflammatory protein-1{alpha} (15, 25, 26, 27). In allergic subjects, a simultaneous exposure to diesel extracts and to the specific allergen greatly potentiates the production of these chemokines (17). In agreement with differential chemokine receptor expression on the cell surface, certain chemokines have been found to preferentially recruit either type 2 (MDC, TARC, or eotaxin) or type 1 (IP-10) T lymphocytes, and thus to favor the establishment of type 2 or type 1 immune responses (10).

MDC is mainly produced by monocytes/macrophages as well as T cells, and binds selectively to CCR4, expressed on type 2 T cells and basophils (28). In the present study, we show that exposure of PBMC and AM from allergic patients to diesel extracts resulted in a strong increase of allergen-induced MDC production. When purified monocytes or CD4+ T cells from allergic patients were stimulated with allergen with or without diesel extracts, no increased production of MDC was observed (data not shown), showing that both cell types were necessary to obtain this effect. This effect was associated with an enhanced propensity of these culture supernatants to induce chemotaxis of Th2, but not Th1 clones, suggesting that diesel extracts may intensify an ongoing allergic reaction, through enhanced MDC production. This finding is in agreement with the known effects of MDC on Th2 cell recruitment (29, 30). The involvement of the latter chemokine in diesel extract- and allergen-induced recruitment of Th2 clones was furthermore confirmed by the use of a neutralizing anti-MDC Ab. It is of note that the inhibitory effect was not total, suggesting that other Th2 cell-attracting mediators, such as TARC and eotaxin, may be involved. However, in our experimental conditions, the production of these chemokines by PBMC and AM was not affected by diesel extracts and/or allergen stimulation. For the latter chemokine, these results are in agreement with a recently published study showing the absence of eotaxin induction following intranasal DEP challenge in humans (25). In contrast, for the former chemokine, human bronchial epithelial cells derived from allergic asthmatic patients have been shown to overexpress TARC with very little or no MDC expression (31). Thus, MDC and TARC may both participate in the allergic response, but originate from different cell sources.

The mechanism of action leading to increased MDC production was then investigated. The first hypothesis tested was the involvement of the cytokine pathway. MDC production in monocytes is known to be induced by IL-4 and IL-13 and inhibited by IFN-{gamma} (32). The production of MDC by T lymphocytes is also up-regulated by IL-4, IL-5, and IL-6, and inversely correlates with levels of IFN-{gamma} (33). Surprisingly, no modification was observed in the production levels of IL-4, IL-13, and IFN-{gamma} by PBMC from allergic patients, which remained very low in all conditions of stimulation. The more sensitive technique of mRNA amplification was then used to better evaluate possible modulations of cytokine expression. Variations in IL-4, IL-13, and IFN-{gamma} mRNA expression did not appear to correlate with the variations in MDC production and mRNA expression, as one could have expected in view of the studies cited above. The relatively high initial level of IL-4 mRNA expression is likely to be due to the allergic status of the donors, as well as to nonspecific activation, since no differences were observed between various stimulation conditions. Furthermore, neutralizing mAb against IL-4 and IL-13 did not affect MDC production by PBMC stimulated with Der p 1 in the presence or absence of DEP-PAH, ruling out a role for type 2 cytokines in DEP-PAH-dependent MDC production, and suggesting the involvement of other signaling pathways. Besides cytokines, other stimuli such as those mediated by costimulatory molecules have been shown to regulate chemokine production, and in particular the IL-4-independent CD28-mediated pathway has been implicated in chemokine production. In mice, Herold et al. (34) have demonstrated an inhibition of macrophage-inflammatory protein-1{alpha} production by Ag-stimulated T cells from CD28-deficient, but not from IL-4 knockout mice. Moreover, DEP have been shown to induce and enhance the expression of mRNA coding for CD80, a natural ligand of CD28, in nasal lavage cells (24). Finally, CD28 activation is involved in Th2 cell development (35) and particularly in allergic inflammation (36). Therefore, the potentiating effect of DEP-PAH on Der p 1-induced MDC release might result from an increase of CD28-mediated costimulatory signals, since in both PBMC and AM cultures T cells were present, allowing the costimulation through this pathway. This hypothesis was tested by using a CTLA-4-Ig fusion protein known to inhibit B7 (CD80/CD86)-CD28 interaction (18). Allergen-induced MDC production by PBMC was markedly inhibited by CTLA-4-Ig fusion protein, as was the potentiation of MDC production in the presence of diesel extracts, suggesting that both stimuli acted through the CD28 pathway. More specifically, Der p 1 was shown to act through CD86 costimulation, while the potentiating effect of diesel extracts was linked to CD80 costimulation. These results are in agreement with a previous study showing that IL-5 production by allergen-stimulated T cell lines from allergic patients was CD86, but not CD80 dependent (19). The present study provides a novel insight into the mechanisms of CD28-induced Th2 development, showing the involvement of the Th2 cell-attracting chemokine MDC. Moreover, it shows that diesel pollutants can activate the CD28 pathway and enhance the recruitment of Th2 cells through selective chemokine production.

Concomitantly, the pro-Th1 chemokine IP-10 was also evaluated and appeared to be differentially regulated by DEP-PAH. IP-10 is produced by monocytes/macrophages and T cells and is constitutively expressed by human keratinocytes (37) and epithelial cells (38). This chemokine has been suggested to have a potential role in the maintenance of protective, type 1-dominated responses in nonatopic subjects (39). In this study, we show that human PBMC also produce IP-10 constitutively. Exposure of PBMC and AM from allergic asthmatic patients to diesel extracts alone induced a decrease of the constitutive production of IP-10. Cell depletion experiments showed that this effect was linked to a direct effect on T cells (data not shown). The signaling pathway that governs the decrease of IP-10 remains undetermined. The production of IP-10 is known to be regulated by cytokines, and in particular, is increased by IFN-{gamma} (38). However, in the present work, the role of cytokines is unlikely, since no variation in IFN-{gamma} protein and mRNA expression could be demonstrated. The inhibition of the CD28 pathway by the chimeric CTLA-4-Ig protein also did not modify the inhibitory effect observed on IP-10 levels. Taken together, these data suggest that diesel extract exposure alone may indirectly favor a type 2-dominated response by decreasing the release of the pro-Th1 chemokine IP-10.

For both PBMC and AM, the resulting balance between MDC and IP-10 in the presence of DEP-PAH and Der p 1 was markedly in favor of a pro-type 2 T cell response, as confirmed by their selective chemoattractant ability on Th2 clones. Although the number of samples for AM from asthmatic subjects was limited because of ethical constraints, and therefore did not allow statistical analysis, the results obtained showed a clear decrease in IP-10 production. Variations in MDC production by AM were not as prominent as those in PBMC, which might be due to the constitutive production of MDC by macrophages, as opposed to its induced production by monocytes (30). As AM are in direct and early contact with diesel exhaust pollution, our results suggest that diesel extract exposure can orientate the chemokine balance in the lung toward a pro-type 2 T cell response, even in the absence of specific allergen. The early dysregulation in the production of chemokines associated with the balance of type 1/type 2 T cells may provide an entry in the amplifying loop between the production of these chemokines and the recruitment of type 2 T cells, and thus participate in the exacerbation of the allergic reaction and in turn to the severity of the symptoms. In conclusion, diesel exhaust pollution, via the CD28 pathway, appears to regulate production of selective chemokines, leading in combination with the relevant allergen, to the exacerbation of the type 2 immune response.


   Acknowledgments
 
We thank Dr. P. Gosset for critical review of this work. We thank the staff of Renault Lardy for providing DEP-PAH and for HPLC analysis, Dr. Jonathan Ellis (GlaxoSmithKline) for providing the fusion protein CTLA-4-Ig, and the team of the Pneumology Department of the Calmette Hospital of Lille for allergic patient recruitment and for providing BAL samples.


   Footnotes
 
1 This work was supported by a grant from Agence de l’Environnement et de la Maîtrise de l’Energie, PRIMEQUAL-PREDIT (No. 97034 from Ministère de l’Environnement), and Comité National contre les Maladies Respiratoires et la Tuberculose. Back

2 O.F. and S.S. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Anne Tsicopoulos, Institut National de la Santé et de la Recherche Médicale Unité 416, Institut Pasteur de Lille, 1 rue du Professeur Calmette, Boite Postale 245, 59 019 Lille, France. E-mail address: anne.tsicopoulos{at}pasteur-lille.fr Back

4 Abbreviations used in this paper: PAH, polyaromatic hydrocarbon; AM, alveolar macrophage; BAL, bronchoalveolar lavage; DEP, diesel exhaust particle; IP-10, IFN-{gamma}-induced protein-10; MDC, macrophage-derived chemokine; TARC, thymus activation-regulated chemokine. Back

Received for publication December 13, 2001. Accepted for publication March 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sly, R. M.. 1999. Changing prevalence of allergic rhinitis and asthma. Ann. Allergy Asthma Immunol. 82:233.[Medline]
  2. United Nations. 1994. Air pollution in the world’s megacities. United Nations Environment Programme 36:5.
  3. Fujieda, S., D. Diaz-Sanchez, A. Saxon. 1998. Combined nasal challenge with diesel exhaust particles and allergen induces in vivo IgE isotype switching. Am. J. Respir. Cell Mol. Biol. 19:507.[Abstract/Free Full Text]
  4. Takenaka, H., K. Zhang, D. Diaz-Sanchez, A. Tsien, A. Saxon. 1995. Enhanced human IgE production results from exposure to the aromatic hydrocarbons from diesel exhaust: direct effects on B-cell IgE production. J. Allergy Clin. Immunol. 95:103.[Medline]
  5. Kanemitsu, H., S. Nagasawa, M. Sagai, Y. Mori. 1998. Complement activation by diesel exhaust particles (DEP). Biol. Pharm. Bull. 21:129.[Medline]
  6. Saneyoshi, K., O. Nohara, T. Imai, F. Shiraishi, H. Moriyama, H. Fujimaki. 1997. IL-4 and IL-6 production of bone marrow-derived mast cells is enhanced by treatment with environmental pollutants. Int. Arch. Allergy Immunol. 114:237.[Medline]
  7. Terada, N., K. Maesako, K. Hiruma, N. Hamano, G. Houki, A. Konno, T. Ikeda, M. Sai. 1997. Diesel exhaust particulates enhance eosinophil adhesion to nasal epithelial cells and cause degranulation. Int. Arch. Allergy Immunol. 114:167.[Medline]
  8. Diaz-Sanchez, D., A. Tsien, J. Fleming, A. Saxon. 1997. 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. 158:2406.[Abstract]
  9. Teran, L. M., D. E. Davies. 1996. The chemokines: their potential role in allergic inflammation. Clin. Exp. Allergy 26:1005.[Medline]
  10. Homey, B., A. Zlotnik. 1999. Chemokines in allergy. Curr. Opin. Immunol. 11:626.[Medline]
  11. Bonecchi, R., G. Bianchi, P. P. Bordignon, D. D’Ambrosio, R. Lang, A. Borsatti, S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129.[Abstract/Free Full Text]
  12. Sallusto, F., C. R. Mackay, A. Lanzavecchia. 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005.[Abstract/Free Full Text]
  13. Siveke, J. T., A. Hamann. 1998. T helper 1 and T helper 2 cells respond differentially to chemokines. J. Immunol. 160:550.[Abstract/Free Full Text]
  14. Zingoni, A., H. Soto, J. A. Hedrick, A. Stoppacciaro, C. T. Storlazzi, F. Sinigaglia, D. D’Ambrosio, A. O’Garra, D. Robinson, M. Rocchi, et al 1998. The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells. J. Immunol. 161:547.[Abstract/Free Full Text]
  15. Fahy, O., A. Tsicopoulos, H. Hammad, J. Pestel, A. B. Tonnel, B. Wallaert. 1999. Effects of diesel organic extracts on chemokine production by peripheral blood mononuclear cells. J. Allergy Clin. Immunol. 103:1115.[Medline]
  16. Tonnel, A. B., M. Joseph, P. Gosset, E. Fournier, A. Capron. 1983. Stimulation of alveolar macrophages in asthmatic patients after local provocation test. Lancet 1:1406.[Medline]
  17. Fahy, O., H. Hammad, S. Senechal, J. Pestel, A. B. Tonnel, B. Wallaert, A. Tsicopoulos. 2000. 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. 23:247.[Abstract/Free Full Text]
  18. Hidi, R., V. Riches, M. Al-Ali, W. W. Cruikshank, D. M. Center, S. T. Holgate, R. Djukanovic. 2000. Role of B7-CD28/CTLA-4 costimulation and NF-{kappa}B in allergen-induced T cell chemotaxis by IL-16 and RANTES. J. Immunol. 164:412.[Abstract/Free Full Text]
  19. Larche, M., S. J. Till, B. M. Haselden, J. North, J. Barkans, C. J. Corrigan, A. B. Kay, D. S. Robinson. 1998. Costimulation through CD86 is involved in airway antigen-presenting cell and T cell responses to allergen in atopic asthmatics. J. Immunol. 161:6375.[Abstract/Free Full Text]
  20. Yssel, H., J. E. De Vries, M. Koken, W. Van Blitterswijk, H. Spits. 1984. Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones. J. Immunol. Methods 72:219.[Medline]
  21. Lecart, S., V. Boulay, N. Raison-Peyron, J. Bousquet, L. Meunier, H. Yssel, J. Pene. 2001. Phenotypic characterization of human CD4+ regulatory T cells obtained from cutaneous dinitrochlorobenzene-induced delayed type hypersensitivity reactions. J. Invest. Dermatol. 117:318.[Medline]
  22. Pene, J., A. Desroches, L. Paradis, B. Lebel, M. Farce, C. F. Nicodemus, H. Yssel, J. Bousquet. 1998. Immunotherapy with Fel d 1 peptides decreases IL-4 release by peripheral blood T cells of patients allergic to cats. J. Allergy Clin. Immunol. 102:571.[Medline]
  23. Spits, H., H. Ijssel, C. Terhorst, J. E. de Vries. 1982. Establishment of human T lymphocyte clones highly cytotoxic for an EBV-transformed B cell line in serum-free medium: isolation of clones that differ in phenotype and specificity. J. Immunol. 128:95.[Medline]
  24. Nel, A. E., D. Diaz-Sanchez, D. Ng, T. Hiura, A. Saxon. 1998. Enhancement of allergic inflammation by the interaction between diesel exhaust particles and the immune system. J. Allergy Clin. Immunol. 102:539.[Medline]
  25. Diaz-Sanchez, D., M. Jyrala, D. Ng, A. Nel, A. Saxon. 2000. In vivo nasal challenge with diesel exhaust particles enhances expression of the CC chemokines RANTES, MIP-1{alpha}, and MCP-3 in humans. Clin. Immunol. 97:140.[Medline]
  26. Hashimoto, S., Y. Gon, I. Takeshita, K. Matsumoto, I. Jibiki, H. Takizawa, S. Kudoh, T. Horie. 2000. 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. 161:280.[Abstract/Free Full Text]
  27. Boland, S., A. Baeza-Squiban, T. Fournier, O. Houcine, M. C. Gendron, M. Chevrier, G. Jouvenot, A. Coste, M. Aubier, F. Marano. 1999. Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production. Am. J. Physiol. 276:L604.[Abstract/Free Full Text]
  28. Chvatchko, Y., A. J. Hoogewerf, A. Meyer, S. Alouani, P. Juillard, R. Buser, F. Conquet, A. E. Proudfoot, T. N. Wells, C. A. Power. 2000. A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock. J. Exp. Med. 191:1755.[Abstract/Free Full Text]
  29. Imai, T., M. Nagira, S. Takagi, M. Kakizaki, M. Nishimura, J. Wang, P. W. Gray, K. Matsushima, O. Yoshie. 1999. 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. 11:81.[Abstract/Free Full Text]
  30. Andrew, D. P., M. S. Chang, J. McNinch, S. T. Wathen, M. Rihanek, J. Tseng, J. P. Spellberg, III C. G. Elias. 1998. STCP-1 (MDC) CC chemokine acts specifically on chronically activated Th2 lymphocytes and is produced by monocytes on stimulation with Th2 cytokines IL-4 and IL-13. J. Immunol. 161:5027.[Abstract/Free Full Text]
  31. Sekiya, T., M. Miyamasu, M. Imanishi, H. Yamada, T. Nakajima, M. Yamaguchi, T. Fujisawa, R. Pawankar, Y. Sano, K. Ohta, et al 2000. Inducible expression of a Th2-type CC chemokine thymus- and activation-regulated chemokine by human bronchial epithelial cells. J. Immunol. 165:2205.[Abstract/Free Full Text]
  32. Bonecchi, R., S. Sozzani, J. T. Stine, W. Luini, G. D’Amico, P. Allavena, D. Chantry, A. Mantovani. 1998. Divergent effects of interleukin-4 and interferon-{gamma} on macrophage-derived chemokine production: an amplification circuit of polarized T helper 2 responses. Blood 92:2668.[Abstract/Free Full Text]
  33. Galli, G., D. Chantry, F. Annunziato, P. Romagnani, L. Cosmi, E. Lazzeri, R. Manetti, E. Maggi, P. W. Gray, S. Romagnani. 2000. Macrophage-derived chemokine production by activated human T cells in vitro and in vivo: preferential association with the production of type 2 cytokines. Eur. J. Immunol. 30:204.[Medline]
  34. Herold, K. C., J. Lu, I. Rulifson, V. Vezys, D. Taub, M. J. Grusby, J. A. Bluestone. 1997. Regulation of C-C chemokine production by murine T cells by CD28/B7 costimulation. J. Immunol. 159:4150.[Abstract]
  35. King, C. L., R. J. Stupi, N. Craighead, C. H. June, G. Thyphronitis. 1995. CD28 activation promotes Th2 subset differentiation by human CD4+ cells. Eur. J. Immunol. 25:587.[Medline]
  36. Burr, J. S., S. L. Kimzey, D. R. Randolph, J. M. Green. 2001. CD28 and CTLA4 coordinately regulate airway inflammatory cell recruitment and T-helper cell differentiation after inhaled allergen. Am. J. Respir. Cell Mol. Biol. 24:563.[Abstract/Free Full Text]
  37. Boorsma, D. M., J. Flier, S. Sampat, C. Ottevanger, P. de Haan, L. Hooft, R. Willemze, C. P. Tensen, T. J. Stoof. 1998. Chemokine IP-10 expression in cultured human keratinocytes. Arch. Dermatol. Res. 290:335.[Medline]
  38. Sauty, A., M. Dziejman, R. A. Taha, A. S. Iarossi, K. Neote, E. A. Garcia-Zepeda, Q. Hamid, A. D. Luster. 1999. The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells. J. Immunol. 162:3549.[Abstract/Free Full Text]
  39. Gangur, V., F. E. Simons, K. T. HayGlass. 1999. IP-10 mediated reinforcement of human type 1 cytokine synthesis to environmental allergens among non-atopic subjects. Int. Arch. Allergy Immunol. 118:387.[Medline]



This article has been cited by other articles:


Home page
 International Journal of ToxicologyHome page
L. A. Burns-Naas, K. L. Hastings, G. S. Ladics, S. L. Makris, G. A. Parker, and M. P. Holsapple
What's So Special about the Developing Immune System?
International Journal of Toxicology, March 1, 2008; 27(2): 223 - 254.
[Abstract] [Full Text] [PDF]

Home page
 Hum Exp ToxicolHome page
P E Schwarze, J Ovrevik, M Lag, M Refsnes, P Nafstad, R B Hetland, and E Dybing
Particulate matter properties and health effects: consistency of epidemiological and toxicological studies
Human and Experimental Toxicology, October 1, 2006; 25(10): 559 - 579.
[Abstract] [PDF]

Home page
 J. Immunol.Home page
P. d. Nadai, A.-S. Charbonnier, C. Chenivesse, S. Senechal, C. Fournier, J. Gilet, H. Vorng, Y. Chang, P. Gosset, B. Wallaert, et al.
Involvement of CCL18 in Allergic Asthma
J. Immunol., May 15, 2006; 176(10): 6286 - 6293.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
C.-M. Kang, A.-S. Jang, M.-H. Ahn, J.-A. Shin, J.-H. Kim, Y.-S. Choi, T.-Y. Rhim, and C.-S. Park
Interleukin-25 and Interleukin-13 Production by Alveolar Macrophages in Response to Particles
Am. J. Respir. Cell Mol. Biol., September 1, 2005; 33(3): 290 - 296.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. D. Finkelman, M. Yang, T. Orekhova, E. Clyne, J. Bernstein, M. Whitekus, D. Diaz-Sanchez, and S. C. Morris
Diesel Exhaust Particles Suppress In Vivo IFN-{gamma} Production by Inhibiting Cytokine Effects on NK and NKT Cells
J. Immunol., March 15, 2004; 172(6): 3808 - 3813.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
J. T. Souto, J. C. Aliberti, A. P. Campanelli, M. C. Livonesi, C. M.L. Maffei, B. R. Ferreira, L. R. Travassos, R. Martinez, M. A. Rossi, and J. S. Silva
Chemokine Production and Leukocyte Recruitment to the Lungs of Paracoccidioides brasiliensis-Infected Mice Is Modulated by Interferon-{gamma}
Am. J. Pathol., August 1, 2003; 163(2): 583 - 590.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. Senechal, P. de Nadai, N. Ralainirina, A. Scherpereel, H. Vorng, P. Lassalle, A.-B. Tonnel, A. Tsicopoulos, and B. Wallaert
Effect of Diesel on Chemokines and Chemokine Receptors Involved in Helper T Cell Type 1/Type 2 Recruitment in Patients with Asthma
Am. J. Respir. Crit. Care Med., July 15, 2003; 168(2): 215 - 221.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
K. S. Harrod, R. J. Jaramillo, C. L. Rosenberger, S.-Z. Wang, J. A. Berger, J. D. McDonald, and M. D. Reed
Increased Susceptibility to RSV Infection by Exposure to Inhaled Diesel Engine Emissions
Am. J. Respir. Cell Mol. Biol., April 1, 2003; 28(4): 451 - 463.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fahy, O.
Right arrow Articles by Tsicopoulos, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fahy, O.
Right arrow Articles by Tsicopoulos, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS