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Am J Respir Cell Mol Biol. 2009 April; 40(4): 454–463.
Published online 2008 October 16. doi:  10.1165/rcmb.2008-0346OC
PMCID: PMC2660562

Vγ1+ T Cells and Tumor Necrosis Factor-Alpha in Ozone-Induced Airway Hyperresponsiveness

Abstract

γδ T cells regulate airway reactivity, but their role in ozone (O3)-induced airway hyperresponsiveness (AHR) is not known. Our objective was to determine the role of γδ T cells in O3-induced AHR. Different strains of mice, including those that were genetically manipulated or antibody-depleted to render them deficient in total γδ T cells or specific subsets of γδ T cells, were exposed to 2.0 ppm of O3 for 3 hours. Airway reactivity to inhaled methacholine, airway inflammation, and epithelial cell damage were monitored. Exposure of C57BL/6 mice to O3 resulted in a transient increase in airway reactivity, neutrophilia, and increased numbers of epithelial cells in the lavage fluid. TCR-δ−/− mice did not develop AHR, although they exhibited an increase in neutrophils and epithelial cells in the lavage fluid. Similarly, depletion of γδ T cells in wild-type mice suppressed O3-induced AHR without influencing airway inflammation or epithelial damage. Depletion of Vγ1+, but not of Vγ4+ T cells, reduced O3-induced AHR, and transfer of total γδ T cells or Vγ1+ T cells to TCR-δ−/− mice restored AHR. After transfer of Vγ1+ cells to TCR-δ−/− mice, restoration of AHR after O3 exposure was blocked by anti–TNF-α. However, AHR could be restored in TCR-δ−/−mice by transfer of γδ T cells from TNF-α–deficient mice, indicating that another cell type was the source of TNF-α. These results demonstrate that TNF-α and activation of Vγ1+ γδ T cells are required for the development of AHR after O3 exposure.

Keywords: ozone, airway responsiveness, γδ T cells, TNF-α

CLINICAL RELEVANCE

This article demonstrates for the first time the essential role of a unique subset of T lymphocytes in the development of ozone-induced airway hyperresponsiveness.

Ozone (O3) is a highly reactive oxidizing agent and continues to be a persistent ambient pollutant despite years of considerable effort to reduce levels in the United States (1). Toxic pulmonary effects have been demonstrated in animals and humans, and in particular in urban environments and the workplace (2). Various adverse sequelae of O3 exposure have been documented with airway hyperresponsiveness (AHR) to nonspecific stimuli, epithelial sloughing, and neutrophil accumulation in the airways. The mechanisms leading to O3-induced effects in the lung are not well understood, nor are there data linking a common mechanism resulting in AHR, neutrophil accumulation, and epithelial cell damage. The high reactivity of O3 and its low solubility in water would prevent it from passing through the lung epithelial lining fluid to act directly on the underlying epithelial cells (3). Since the lung epithelial lining fluid is composed of lipids to a large extent, it has been suggested that O3 exerts its toxic effects via oxidized lipid mediators that can act as signaling molecules (37).

Both humans and mice vary considerably in their response to O3, and genetic factors are important in dictating susceptibility to O3-induced damage (811). Inflammatory mediators likely play a major role in the pathogenesis of O3-induced AHR, lung inflammation, and injury. Perhaps linked to the genetic variability is the activity of these inflammatory mediators. Tumor necrosis factor (TNF)-α has been implicated in the pathogenesis of O3-induced lung inflammation and injury. O3 exposure enhances TNF-α release and TNF receptor expression in airway cells and tissues (12, 13). In positional cloning studies, Tnf was identified as a candidate susceptibility gene for lung inflammation induced by O3 (10). These findings are supported by the protection afforded against development of O3-induced AHR and inflammation in the absence of a TNF response (10, 1316).

In addition to TNF-α, other factors have been implicated, including interleukin (IL)-1β, whose levels increase in response to inhaled O3, and where AHR, airway neutrophilia, and structural damage can be significantly reduced when the IL-1 receptor is targeted by a receptor antagonist (17). Complement activation also plays an important role in the development of O3-induced AHR and airway neutrophilia, and in this study, the O3-iduced neutrophil response did not appear to be necessary for the O3-induced AHR (18).

γδ T cells represent a small population (1–5%) of T lymphocytes; however, they are found in greater numbers on mucosal and epithelial surfaces, and recent studies revealed the critical role of these cells in the protection against pathogens and tumor cells (19). In the development of allergen-induced AHR, it was apparent from studies of TCR δ chain-deficient mice, which lack γδ T cells, that γδ T cells can regulate AHR, independent of the airway inflammatory response. Moreover, specific γδ T cell subsets play important regulatory roles with different activities (20). In the allergen-induced development of lung allergic responses, the Vγ1+ subset enhances the airway response to methacholine (MCh), whereas the Vγ4+ γδ subset suppresses AHR without any influence on airway inflammation (21, 22). King and coworkers suggested that intraepithelial γδ T cells can protect the host from O3-induced lung damage by reducing the inflammatory response in the lung; the subset of γδ T cells responsible for these effects was not determined (23).

Here, we demonstrate that γδ T cells, and specifically Vγ1+ T cells, are essential to the development of O3-induced AHR and that TNF-α is an important link to this Vγ1-dependent, O3-induced AHR.

MATERIALS AND METHODS

Animals

C57BL/6 (wild-type; WT) mice, B6.129P2-Tcrdtm1Mom/J (TCR-δ chain-deficient; TCR-δ−/−) mice and B6;129P2-Tcrbtm1Mom/J (TCR β chain-deficient; TCR-β−/−) mice (background: C57BL/6 strain), were bred at National Jewish, or B6;129S-Tnftm1Gkl/J (TNF-α–deficient; TNF-α−/−) mice were purchased from Jackson Laboratory (Bar Harbor, ME); all were studied at ages of 8 to 12 weeks. All genotypes of the modified mice were confirmed by flow cytometry analysis as previously described (21). All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of National Jewish Health.

Experimental Protocol

Mice were exposed to O3 at a concentration of 2.0 ppm for 3 hours. The parameters were measured 6 to 8 hours after O3 exposure. T cell depletion was achieved after injection of 200 μg hamster anti–TCR-δ monoclonal antibodies (mAb) from clones GL3 and 403.A10 (1:1 mixture), anti-Vγ1 mAb from clone 2.11, or anti-Vγ4 mAb from clone UC3 into the tail veins of mice 3 days before O3 exposure. This dosing regimen was optimized for T cell depletion (20, 21) and monitored routinely to confirm depletion of more than 95% of splenic γδ T cells or specific subsets. Control antibody treatments were performed with the same amount of ChromePure Syrian hamster IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Throughout this article, we use the nomenclature for murine TCR-Vγ genes introduced by Heilig and Tonegawa (24).

In some experiments, mice received rat anti-mouse TNF-α (MP6-XT3: AMC 3814; Biosource, Camarillo, CA) or isotype rat IgG1 as control, just before O3 exposure. The single dose of anti-mouse TNF-α Ab was 250 μg based on previous results (25). All antibodies were suspended in 200 μl of PBS at the time of intravenous injection.

O3 Exposure

Mice were exposed to O3 at 2.0 ppm for 3 hours in stainless steel wire cages. Cages were set inside 240-liter laminar flow inhalation chambers. HEPA-filtered room air was passed through these chambers at 25 changes/hour. Room temperature was maintained at 20 to 25°C. O3 was generated by directing compressed medical-grade oxygen through an electrical discharge O3 generator (Sander Ozonizer, Model 25; Erwin Sander Elektroapparatebau GmbH, Uetze-Eltze, Germany) located upstream of the exposure chamber. The O3-air mixture was metered into the inlet air stream with mass flow controllers (Model #1359C; MKS Instruments Inc., Andover, MA). Exposure to HEPA-filtered air was done in a separate chamber with age- and treatment-matched control animals. O3 concentrations were continuously monitored at mouse nose levels within the chamber with a photometric O3 analyzer (Model 400A; Advanced Pollution Instrumentation, Inc., San Diego, CA) and recorded on a strip-chart recorder. Calibration of the O3 analyzer was performed by the Colorado Department of Public Health and Environment (Denver, CO).

Determination of Airway Resistance and Dynamic Compliance

Airway resistance (RL) and dynamic compliance (Cdyn) were determined as a change in airway function after aerosolized methacholine (MCh) challenge as previously described (26). Mice were anesthetized with sodium pentobarbital (100 mg/kg), tracheostomized, and mechanically ventilated at a rate of 160 breaths/minute with a constant tidal volume of air (0.2 ml). After each MCh challenge, the data were continuously collected for 1 to 5 minutes and maximum values of RL and minimum values of Cdyn were taken to express changes in these functional parameters. Data are presented as percent change from baseline values recorded after challenge with saline.

Determination of Cell Numbers in Bronchoalveolar Lavage

Immediately after the assessment of AHR, lungs were lavaged via the tracheal cannula with Hanks' balanced salt solution (HBSS, 1 ml). Total leukocyte numbers were measured (Coulter Counter; Coulter Corporation, Hialeah, FL). Differential cell counts were made from cytocentrifuged preparations (Cytospin 2; Shandon Ltd., Runcorn, Cheshire, UK), stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA). As the number of macrophages, neutrophils, and epithelial cells in bronchoalveolar lavage (BAL) were good indicators of the response to O3 exposure (27), these cell types were identified by standard hematologic procedures and at least 200 cells counted under ×400 magnification in a blinded manner.

Histopathologic Study

Lungs were fixed after inflation and immersion in 10% formalin. To identify epithelial injury and airway inflammation in formalin-fixed airway tissue, sections were stained with hematoxylin/eosin.

Adoptive Transfer of γδ T Cells

Mononuclear cells were isolated from spleens of WT mice, TCR-β−/− mice, or TNF-α−/− mice. Cells were recovered by mincing the tissues and subsequently passing them through a stainless steel sieve. Cells were then washed and isolated by Histopaque-1083 (Sigma, St. Louis, MO) gradient centrifugation at 2,000 rpm for 20 minutes. For isolation of γδ T cells, the cells were applied to T cell immunocolumns (Cedarlane Laboratories Ltd., Burlington, NC) according to the manufacturer's direction. For isolation of Vγ1+ cells, the cells were incubated with biotinylated anti-Vγ1 monoclonal antibodies 2.11 (15 min, 4°C), then washed and incubated with streptavidin-conjugated magnetic beads (Miltenyi Biotec, Gladbach, Germany) for 10 minutes at 4°C. The cells were passed twice through magnetic columns to purify Vγ1+ cells, achieving a more than 95% purity.

The recovered γδ (4 × 104 or 8 × 104 cells/mouse) or Vγ1+ T cells (5 × 104 cells/mouse) were transferred intravenously into TCR-δ−/− mice 16 to 20 hours before O3 exposure. Within 30 minutes to 1 hour, intravenously transferred γδ T cells reach steady-state levels in the lung (28). Prior to and following adoptive transfer, the deficiency of surface receptors from TCR-δ−/− mice or recruitment of transferred cells into lungs were monitored by flow cytometric analysis of blood and lung tissue as previously described (21). Assays of AHR and BAL were performed 6 to 8 h after completion of the O3 exposure.

Flow Cytometry Analysis

Enriched lung cells, following preincubation with naive mouse serum in staining buffer (PBS, 2% FCS, and 0.2% sodium azide), were labeled with phycoerythrin (PE)-conjugated anti-CD69 (BD Pharmingen) and biotinylated anti-Vγ1 antibodies (clone 2.11). The suspension was then incubated with FITC-coupled streptavidin. Fluorescence intensity was compared with negative controls and cells were incubated with FITC-streptavidin alone. Results were analyzed using CellQuest software, and all analyses used a light scatter gate designated to include only small lymphocytes.

Data Analysis

One-way ANOVA was used to determine the levels of differences among all groups. Comparisons for all pairs were analyzed using Tukey-Kramer honest significant difference test, and P values for significance were set at 0.05. All data were expressed as the mean ± SEM.

RESULTS

AHR and Airway Inflammation after O3 Exposure in TCR-δ−/− Mice

WT mice exposed to filtered air showed a small dose response to inhaled MCh when RL and Cdyn were monitored. After exposure of WT mice to O3, the mice developed significant increases in RL and decreases in Cdyn to inhaled MCh in a dose-dependent fashion (Figure 1A). In contrast, exposure to O3 failed to trigger increases in RL or decreases in Cdyn in the TCR-δ−/− mice.

Figure 1.Figure 1.
Failure of TCR-δ–deficient mice to develop airway hyperresponsiveness (AHR) after O3 exposure. C57BL/6 (wild-type, WT) and TCR-δ−/−−/−) mice were exposed to 2 ppm O3 for 3 hours. (A) Airway ...

Despite the failure to alter airway reactivity after O3 inhalation, exposure to O3 elicited increases in numbers of neutrophils in the BAL of TCR-δ−/− mice similar to those observed in WT mice. After O3 exposure, the numbers of airway epithelial cells in the BAL fluid of TCR-δ−/− mice were also significantly increased after O3 exposure, similar to those in the BAL fluid of O3-exposed WT mice, indicating comparable epithelial cell damage (Figure 1B).

Examination of lung tissue revealed that O3 exposure caused airway epithelial injury and inflammatory cell infiltration in the airways of both TCR-δ−/− and WT mice (Figure 2). These changes were observed primarily in major airways but not in small airways.

Figure 2.
Histopathologic changes in WT and TCR-δ−/− mice after O3 exposure. Epithelial desquamation in major airways was identified in both WT and TCR-δ−/− mice exposed to O3. Insets represent histology of small ...

These data demonstrate that O3 exposure, while causing airway (neutrophilia) inflammation and epithelial cell damage, failed to induce AHR in the absence of γδ T cells.

Effects of Depletion of γδ T Cells on O3-Induced AHR and Airway Inflammation in WT Mice

To confirm these findings of the importance of γδ T cells in the development of O3-induced AHR and ensure that this was not an indirect consequence of genetic manipulation, we investigated the effects of depleting γδ T cells on O3-induced AHR and airway inflammation in WT mice treated with anti–TCR-δ mAb. O3 exposure caused significant increases in RL and decreases in Cdyn to inhaled MCh in WT mice. However, if WT mice were treated with anti-δ before O3 exposure, increases in airway reactivity (RL and Cdyn) to inhaled MCh failed to develop (Figure 3A). In contrast, treatment with control Ab did not affect the development of O3-induced AHR. Anti–TCR-δ mAb treatment did not affect the numbers of neutrophils or epithelial cells in BAL fluid (Figure 3B).

Figure 3.Figure 3.
Effect of depletion of γδ T cells on O3-induced AHR and airway inflammation in WT mice. Depletion was performed using an anti-δ antibody before O3 exposure. (A) Airway responsiveness to inhaled MCh. The baseline values of airway ...

These data confirmed that γδ T cells were essential for changes in airway reactivity to O3 but were not required for effects on airway inflammation response or epithelial cell damage.

Effect of Depletion of Specific γδ T Cell Subsets on O3-Induced AHR and Airway Inflammation

Both the Vγ1+ and Vγ4+ γδ T cell subsets appear to be important regulators of airway reactivity, at least in response to allergen-induced AHR (21). In light of the findings demonstrated for γδ T cells on O3-induced AHR, we next determined if a specific subset was involved. Using specific antibodies, we depleted either Vγ1+ or Vγ4+ T cells before O3 exposure of C57BL/6 mice. After depletion of Vγ1 cells, but not of Vγ4 cells, there was a significant reduction in MCh-induced airway reactivity after O3 exposure (Figure 4A). These findings identified the important role of Vγ1+ T cells in the development of O3-induced AHR. Depletion of either Vγ1+ or Vγ4+ T cells had no effects on the cell composition of BAL fluid (Figure 4B).

Figure 4.Figure 4.Figure 4.Figure 4.
Effect of depletion of Vγ1+ or Vγ4+ T cells on O3-induced AHR and airway inflammation in WT mice. Depletions were performed with specific antibodies before O3 exposure. (A) RL and Cdyn. The baseline airway function values ...

Numbers and Activation of Vγ1+ T Cells in the Lung after O3 Exposure

To determine if the numbers and/or activation of Vγ1+ T cells were altered after O3 exposure, we analyzed lung cells for Vγ1, CD3, and CD69 expression, an activation-associated marker, after O3 or filtered air exposure. As shown in Figure 4C, the total number of Vγ1+ T cells in the lungs was not altered after O3 exposure. However, the percentage of CD69+ Vγ1+ T cells was increased in the lungs after O3 exposure (Figure 4D).

Restoration of O3-Induced AHR by Adoptive Transfer of γδ or Vγ1+ T Cells

In parallel to the depletion experiments, we confirmed the role of γδ T cells in adoptive transfer experiments in TCR-δ−/− recipient mice. As described above, TCR-δ−/− mice did not develop AHR after exposure to O3 (Figure 5A). Adoptive transfer of (total) γδ T cells from TCR-β−/− mice into TCR-δ−/− mice before O3 exposure reconstituted O3-induced increases in RL and decreases in Cdyn with higher numbers of transferred cells leading to greater changes in airway reactivity. Adoptive transfer of γδ T cells also did result in an increase in numbers of epithelial cells, without influencing the numbers of neutrophils and macrophages in BAL fluid (Figure 5B). Adoptive transfer of Vγ1+ γδ T cells (5 × 104 cells/mouse) into TCR-δ−/− also reconstituted the increases in RL and decreases in Cdyn after O3 exposure (Figure 5C). O3-exposed TCR-δ−/− and WT mice had a similar BAL cell composition, including numbers of neutrophils and epithelial cells, and transfer of Vγ1+ γδ T cells into TCR-δ−/− mice had little effect other than modestly increasing numbers of epithelial cells (data not shown).

Figure 5.Figure 5.Figure 5.Figure 5.
Adoptive transfer of total γδ T cells or Vγ1 γδ T cells restores AHR in δ−/− mice after O3 exposure. (A) The baseline values of airway function were comparable among experimental groups; ...

On histopathologic examination, O3 caused epithelial injury in the airways of both WT and TCR-δ−/− mice. The extent of the epithelial injury in major airways was the same after O3 exposure of TCR-δ−/− mice, regardless of whether they received total Vγ1+ T cells (Figure 5D), γδ T cells, or no T cells at all. No apparent changes were observed in small airways.

Inhibitory Effect of Anti–TNF-α on Vγ1+ T Cell–Dependent O3-Induced AHR

Given the reconstitution of AHR in TCR-δ−/− mice that received Vγ1+ T cells and the potential role of TNF-α in the overall response to O3 (13), we evaluated the consequences of neutralizing TNF-α using antibody administered after adoptive transfer of Vγ1+ T cells into TCR-δ−/− mice. Transfer of Vγ1+ cells to TCR-δ−/− mice restored O3-induced increases in RL and decreases in Cdyn (Figure 6A). Treatment with anti–TNF-α mAb just before O3 exposure but after Vγ1+ T cell transfer completely suppressed development of AHR in the Vγ1-recipient TCR-δ−/− mice (Figure 6A). However, treatment with anti–TNF-α did not have any effect on BAL cell composition, including numbers of epithelial cells (Figure 6B) or airway damage (data not shown).

Figure 6.Figure 6.
Effect of anti–TNF-α on O3-induced AHR and airway inflammation in δ−/− mice recipients of transferred Vγ1+ γδ T cells. Anti–TNF-α was injected just before initiation ...

γδ T Cells Are Not the Source of TNF-α

To determine if γδ T cells were themselves the source of TNF-α, we examined the consequences of reconstituting TCR-δ−/− mice with γδ T cells from TNF-α−/− mice. Adoptive transfer of γδ T cells from TNF-α−/− or WT mice into TCR-δ−/− mice before O3 exposure reconstituted O3-induced increases in RL and decreases in Cdyn (Figure 7A), suggesting that TNF-α−/− was derived from another cell type activated by O3, possibly damaged epithelium. BAL cell composition was similar after transfer of γδ T cells from either TNF-α−/− or WT mice (Figure 7B).

Figure 7.Figure 7.
TNF-α–deficient γδ T cells reconstitute O3-induced AHR in γ−/− mice. TNF-α–sufficient and TNF-α–deficient γδ T cells were injected intravenously into ...

DISCUSSION

Acute exposure of susceptible mice to O3 leads to the rapid development of AHR, airway neutrophilia, increased numbers of epithelial cells in BAL fluid, and histologic evidence of epithelial sloughing. These changes tend to peak at 6 to 12 hours, with resolution over the ensuing 24 to 48 hours (17). Since subacute O3 exposure (0.3 ppm O3 for 48–72 h) was shown to induce airway inflammation but not AHR (13), the protocol followed here, exposure to 2 ppm O3 for 3 hours is similar to that used by others and one in which a genetic susceptibility locus linked to TNF-α has been delineated (10, 13, 29). One of the primary responses to O3, the development of AHR in response to the nonspecific bronchoconstrictor MCh was virtually eliminated in mice genetically deficient in γδ T cells. This finding was confirmed in WT mice treated with a γδ T cell–depleting antibody. Thus, it appeared that γδ cells were essential to the development of O3-induced AHR, and this was confirmed in experiments in which transfer of γδ T cells restored the AHR response in TCR-δ−/− mice.

Although AHR, airway neutrophilia, and epithelial sloughing all result from acute O3 exposure, only airway function and not the neutrophilia or epithelial damage primarily in major airways was affected by the absence or depletion of the γδ T cells, suggesting a dissociation in the pathways leading to these events. A similar dissociation may be observed in individuals with asthma, in whom asthma control (AHR) may be difficult to achieve, and acute exacerbations remain despite significant improvement in airway eosinophilia or other biomarkers after inhaled corticosteroid therapy (30). We also previously showed that after complement depletion or treatment with an IL-1 receptor antagonist, AHR, airway neutrophilia, and epithelial damage were suppressed in parallel, but one could dissociate a requirement for airway neutrophilia in the development of O3-induced AHR (17). Koohsari and colleagues showed that the severity of airway epithelial injury after chlorine gas exposure is greater in TCR-δ−/− mice, but that the inflammatory response and the change in airway responsiveness to MCh were reduced (31). These findings also demonstrate the dissociation of AHR and airway epithelial injury. The dissociation of γδ T cell effects on development of AHR and airway inflammation is very characteristic of the effects of this population of T cells in the response to allergen challenge of sensitized mice, in which AHR regulation appeared to be independent of airway eosinophilia (20, 32).

Another parallel between O3-induced and allergen-induced AHR is in the role of Vγ1+ T cells. Under both conditions, Vγ1+ γδ T cells appear essential to the development of AHR (21). Whereas Vγ4+ γδ T cells down-regulated allergen-induced AHR, their role in O3-induced AHR remains uncertain. In WT mice, depletion of Vγ4+ γδ T cells was without effect on O3-induced AHR whereas depletion of Vγ1+ γδ T cells abrogated AHR. Further, supporting the role of this γδ T cell subset, activation of Vγ1+ γδ T cells in the lungs of O3-exposed mice, identified by CD69 expression, was increased and in reconstitution experiments transfer of Vγ1+ γδ T cells into TCR-δ−/− mice restored O3-induced AHR. The location of Vγ1+ γδ T cells has been studied in the mouse lung, and they are found in the alveoli, the lamina propria/smooth muscle layers, and around blood vessels, including locations within the blood vessel wall (33). Dziedzic and White reported on the response to O3 in mice lacking T cells (34). They suggested that the site of the interactions may be on epithelial cells that line the alveolar duct and the alveoli. Taken together, the interaction of Vγ1+ γδ T cells and damaged alveolar epithelial cells may be critical to the development of AHR after O3 exposure.

How Vγ1+ γδ T cells exert their effects on airway tone is not defined at present. Possibilities include direct interactions with airway smooth muscle or indirectly through release of mediators that themselves affect airway tone. One candidate is TNF-α. TNF-α has been implicated in O3-induced AHR (13) and the genetic link with a locus on mouse chromosome 17 close to the Tnfa gene in defining susceptibility to O3-induced inflammation and lung injury supports this concept (10). Further, TNF receptor–mediated signaling through NF-κB and MAPK/AP-1 were shown to be essential for O3-induced pulmonary toxicity (35). In the studies reported here, the reconstitution of AHR in TCR-δ−/− mice by Vγ1+ γδ T cells was completely prevented by administration of anti–TNF-α Ab before O3 exposure. Administration of anti–TNF-α Ab, although preventing AHR, did not prevent lung inflammation and injury in response to O3. These seemingly disparate results (10) need further evaluation, but are in agreement with the findings of Shore and coworkers (14), who demonstrated that TNF receptor deficiency prevented O3-induced AHR but not neutrophil accumulation in the airways. Since Vγ1+ γδ T cells only impacted AHR, one possibility is that O3 exposure induced TNF-α release, possibly from damaged airway epithelium, which in turn leads to the activation of Vγ1+ γδ T cells. We previously showed that TNF-α may activate γδ T cells leading to AHR (36) and that TNF-α regulates allergen-induced AHR through γδ T cells, specifically through the TNF-αR2 receptor (p75) (32). Since the cell transfer and anti–TNF-α administration were before O3 exposure, this effect of TNF-α on the activation of Vγ1+ T cells remains a possibility. Less likely is that the transferred Vγ1+ γδ T cells were themselves the source of TNF-α leading to the changes in AHR. Indeed, in this study, adoptive transfer of γδ T cells from TNF-α–deficient mice into TCR-δ−/− mice fully reconstituted the development of AHR, indicating that they were not a primary source of TNF-α. Other possibilities now being pursued are interactions between γδ T cells and dendritic cells, since γδ T cells can induce the maturation of dendritic cells and dendritic cells are known to produce TNF-α (19, 37).

IL-17 is a potent proinflammatory cytokine and its role in the pathogenesis of diseases including asthma has been intensively investigated. Pichavant and colleagues showed that IL-17 from NKT cells may play a key role in O3-induced but not in allergen-induced AHR (38). γδ T cells were also shown to be a source of IL-17; however, as Roark and coworkers demonstrated in a collagen-induced arthritis model, Vγ4+ γδ T cells but not Vγ1+ γδ T cells are more likely a source of IL-17 (39). The role of IL-17 and its interaction with TNF-α and γδ T cell requires further investigation.

In summary, these data identify an essential role for TNF-α and Vγ1+ γδ T cells in O3-induced AHR, and clearly dissociate alterations in airway function from other consequences of O3 exposure, airway neutrophilia and epithelial cell damage. Ozone and other air pollutants are thought to increase the risk of morbidity and acute exacerbations of asthma (40). The observation that a small subset of γδ T cells is critical to the development of altered airway function after O3 exposure highlights their functional capacity in response to a common inhaled environmental toxin. Further investigation into the role of γδ T cells in the development AHR and their manipulation may lead to novel approaches for controlling O3-related asthma exacerbations.

Acknowledgments

The author thank L. N. Cunningham and D. Nabighian (National Jewish Health, Denver, CO) for their assistance.

Notes

This study was supported by NIH grants HL-36577 and HL-61005 (to E.G.), and by EPA grant R825702. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NHLBI or the NIH.

Originally Published in Press as DOI: 10.1165/rcmb.2008-0346OC on October 16, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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