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Interleukin-2–induced Increased Airway Responsiveness and Lung Th2 Cytokine Expression Occur after Antigen Challenge through the Leukotriene Pathway

University of Montreal Hospital Center, Notre-Dame Hospital; and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada

Correspondence and requests for reprints should be addressed to Dr. Paolo M. Renzi, 2065 Alexandre-de-Seve, 8th Floor, Z-8905, Montreal, QC, H2L 2W5, Canada. E-mail: renzip{at}earthlink.net

	ABSTRACT

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Previous studies have shown that the allergic late airway response (LR) is dependent on the leukotriene (LT) pathway in Brown Norway (BN) rats. In this same model, interleukin-2 (IL-2) has been shown to increase allergic airway responses without increasing LT production. This study examined the relationship between the upregulation of cellular immunity with IL-2 and the LT pathway in ovalbumin-sensitized BN rats. Airway responsiveness to LTD₄ was significantly increased in BN rats pretreated with IL-2 (20,000 U twice a day for 4.5 days). Treatment with montelukast, a cysteinyl LT₁ receptor antagonist, blocked IL-2's induced increase of the LR to ovalbumin challenge. When cytokine expression was assessed either by semiquantitative polymerase chain reaction or in situ hybridization, we found that montelukast decreased the amount of IL-4 mRNA expression in the lungs while increasing the amount of interferon- mRNA expression 8 hours after challenge. These results indicate that upregulation of cellular immunity with IL-2 can increase the sensitivity of the airways to LTD₄ and that inhibition of the LT pathway will block the LR and modulate cytokine expression after antigen challenge.

Key Words: leukotriene D₄ • Brown Norway rats • interleukin-4 • interferon- • airway inflammation

	INTRODUCTION

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Asthma is the most commonly reported respiratory disease in clinical practice, affecting both children and adults (1). This disease is characterized by reversible airflow obstruction, increased bronchial responsiveness, and airway inflammation (2). It has been shown that inflammation of the airways can lead to chronic and possibly irreversible changes that affect the airway physiologic response to different stimuli (3). Within the last decades, cysteinyl leukotrienes (cys-LTs) have been shown to be potent bronchoconstrictor agents and important inflammatory mediators in the pathophysiology of both acute and chronic asthma (1). LTs can induce smooth muscle contraction, increase vascular permeability, stimulate mucous secretion, and recruit eosinophils into the lungs (4). These inflammatory mediators are more potent than acetylcholine and histamine as contractile agonists of human airways (5). Although structurally different, LTC₄, LTD₄, and LTE₄, collectively known as cys-LTs, have similar effects on the airways. Both mast cells and eosinophils can release cys-LTs when activated by a variety of factors, including IgE-dependent mechanisms (4). It has also been shown that the cys-LTs can collaborate with cytokines, namely IL-5, in the recruitment of eosinophils into the asthmatic airways (6).

Recently, new drugs aimed at blocking the actions of LTs in the airways have been developed. Several drugs have focused on the LTD₄ receptor cys-LT₁, which mediates bronchial smooth muscle contraction (7). LTC₄ and LTE₄ also bind to the cys-LT₁ receptor, but the potency of LTE₄ is lower by a factor of 10 to 100 (7). LTD₄ receptor antagonists have been shown to protect against bronchoconstriction provoked by inhaled LTD₄, exercise, cold air, platelet-activating factor, and allergen (8). Studies in patients with mild, moderate, and severe asthma have shown that the improvements in lung function by these drugs are additive to ß₂ agonists, suggesting that the effects of endogenous LTs are more than constricting bronchial smooth muscle (8). Although anti-LTs can inhibit airway smooth muscle contraction, they also seem to affect airway inflammation. Indeed, administration of montelukast, a cys-LT₁ receptor antagonist, has been shown to reduce sputum eosinophils in patients with asthma (9).

We have been interested in the relationship between LTs and cell-mediated immunity. We have previously reported in the Brown Norway (BN) rat that the late airway response (LR) can be inhibited by a cys-LT₁ receptor antagonist (10). Interestingly, pretreatment of sensitized BN rats with interleukin (IL)-2, a T cell growth factor, causes an increase in the number of inflammatory cells present in the lung lavage fluid (11) and an increased airway response to ovalbumin (OA) (11). Similar effects have also been shown in guinea pigs pretreated with IL-2 (12). The inflammatory reaction induced by IL-2 administration is similar to the immunologic response to viral infections such as human T-lymphotrophic virus type I (13) and respiratory synctial virus (14). Human T-lymphotrophic virus type I infection causes T lymphocyte activation and proliferation, whereas respiratory synctial virus infection leads to increased airway hyperresponsiveness and increased cellular inflammation, namely neutrophils, eosinophils, and T lymphocytes (15). We have thus employed IL-2 pretreatment of BN rats to assess the mechanism by which upregulation of cellular immunity affects the airway response to antigen. Because the LR can be inhibited in BN rats by a cys-LT receptor antagonist, we have previously assessed whether pretreatment with IL-2 caused an increase in LT production. We found that the amounts of bile LTs after antigen challenge of IL-2–pretreated rats did not differ with that of control animals (16). These findings led us to study (1) whether IL-2 affects the reactivity of the airways to LTs, and (2) whether antagonism of the cys-LT₁ receptor with montelukast will affect the effects of IL-2 on the LR and cytokine mRNA production. We show here that upregulation of the immune response with IL-2 increases the sensitivity of the airways to LTs. We also report that montelukast modulates cytokine mRNA production after antigen challenge.

	METHODS

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Animals, Sensitization, and Measurement of Lung Resistance
Sixty-two highly inbred male BN (SSN strain) rats that were 7 to 8 weeks old were obtained from Harlan Sprague-Dawley Inc. (Walkerville, MD) and were sensitized on Day 1 by a subcutaneous injection of 1 ml of saline containing 1 mg of OA and 200 mg of aluminum hydroxide (Sigma Chemicals, St. Louis, MO). Rats were anesthetized with urethane (1 g/kg intraperitoneally). Lung resistance (R_L) was measured (10).

Experimental Protocol
For the assessment of LTD₄ responsiveness, 22 rats were given either 0.2 ml of saline or 20,000 units of human recombinant IL-2 diluted in 0.2 ml of saline subcutaneously twice a day for 4.5 days from the 9th to the 14th day after sensitization. On Day 14, rats were challenged with incremental doses of LTD₄ (0.05 to 1,000 ng/ml [17]) (Cayman Chemical Co., Ann Arbor, MI) intratracheally in 50 µl until a doubling in R_L occurred.

In addition, two control groups and two experimental groups consisted of 20 sensitized rats each given 0.2 ml of saline or 20,000 units of human recombinant IL-2, respectively, subcutaneously twice a day for 4.5 days as described previously here. All rats were challenged with an aerosol of saline (control) or OA (experimental) (1 mg/ml) for 5 minutes. Before and 2 hours after the latter challenge, rats received an intravenous injection of 0.36 ml of saline or montelukast (MK-0476, 0.5 mg/kg; Merck Frosst, Montreal, Quebec, Canada). R_L was measured at baseline and at 5, 10, 15, 20, and 30 minutes after challenge and subsequently every 15 minutes for a period of 8 hours.

Eight hours after OA challenge, the lungs were lavaged through the tracheal tube by five instillations and immediate retrieval of 5 ml of saline at room temperature, and inflammatory cells were counted (11).

RNA Preparation, Reverse Transcription, Semiquantitative Polymerase Chain Reaction, and In Situ Hybridization
TRIzol reagent (GIBCO BRL, Montreal, QC, Canada) was used to isolate total RNA from frozen lung biopsies. Reverse transcription was performed on 5 µg of total RNA with Moloney murine leukemia virus reverse transcriptase (GIBCO BRL) in the presence of RNasine (Pharmacia, Montreal, Quebec, Canada). For semiquantitative experiments, the reaction mixture contained 5 µCi/ml of [³²P]dATP as a tracer. Specific primers were used to amplify selected cytokine messages (18). To confirm the differences in Th1/Th2 cytokine expression obtained by semiquantitative polymerase chain reaction (SQ-PCR), we performed in situ hybridization for IL-4 and interferon (IFN)- on lung biopsies (18).

Data Analysis
The concentration of LTD₄ required to double R_L (EC₂₀₀R_L) was obtained by linear interpolation between the two concentrations bounding the point at which R_L reached 200% of the control value. Comparisons of airway responsiveness to LTD₄ between groups were performed with log-transformed data and were analyzed using unpaired, nonparametric Mann-Whitney tests. The LR was calculated as the area under the curve of R_L above the baseline value over the 3- to 8-hour period after challenge (10). Data were analyzed using a Kruskal-Wallis nonparametric analysis of variance test followed by Dunn's multiple comparisons test; p values were considered significant when less than 0.05.

	RESULTS

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Pretreatment with IL-2 Increases Airway Responsiveness to LTD₄
To determine the relationship between LTs and upregulation of cell-mediated immunity, rats were pretreated with either saline or IL-2 for 4.5 days and after general anesthesia, and endotracheal intubation was challenged on the 14th day after sensitization with exponentially increasing doses of LTD₄ to measure airway responsiveness. Rats pretreated with IL-2 showed an increased airway responsiveness to LTD₄ (Figure 1) . The mean dose of LTD₄ that caused a doubling in resistance was 88.2 ± 38.4 ng/ml (n = 10; one rat died during the procedure) for IL-2-treated rats. Rats pretreated with saline required a higher dose of LTD₄ to double R_L (665.7 ± 47.6 ng/ml, n = 11, p < 0.05).

View larger version (11K): [in this window] [in a new window]   Figure 1. Effect of IL-2 on airway responsiveness to LTD4. Rats (n = 21) received either saline or IL-2 subcutaneously (20,000 U twice a day for 4.5 days pre-LTD4 challenge) and were then challenged with increasing doses of LTD4 (0.05, 0.5, 5.0, 50, 500, 1,000 ng/ml) until baseline RL doubled. EC200RL was calculated as the amount of LTD4 necessary to double RL. *p < 0.05 between IL-2–treated and saline-treated control rats.

View larger version (13K): [in this window] [in a new window]   Figure 2. Effect of IL-2 alone or IL-2 and montelukast on the LR to OA. OA-sensitized rats (n = 35) were given either IL-2 (20,000 U twice a day) or saline for 4.5 days starting on Day 9. On the 14th day, they received either montelukast (0.5 mg/kg) or saline intravenously before and 2 hours after OA challenge. RL was measured for 8 hours after OA challenge, and the LR was calculated as the area under the curve for RL values obtained from 4 to 8 hours after OA challenge. *p < 0.05 between IL-2–treated animals that received saline intravenously and IL-2–treated animals that received montelukast intravenously before and 2 hours after OA challenge.

View larger version (42K): [in this window] [in a new window]   Figure 3. Effect of IL-2 pretreatment or IL-2 and montelukast on BAL inflammatory cells after OA challenge. BALF (n = 35) was recovered 8 hours after OA or saline challenge for cell differential analysis. *p < 0.05 between OA-challenged IL-2–pretreated rats and saline-pretreated and saline-challenged rats.

View larger version (57K): [in this window] [in a new window]   Figure 4. Effect of IL-2 pretreatment with or without montelukast on Th1 (IFN-, C)/Th2 (IL-4, A; IL-5, B) total lung cytokine mRNA expression after OA challenge. Lungs (n = 30) were fixed in liquid nitrogen 8 hours after challenge for SQ-PCR analysis. *p < 0.05 between IL-2–pretreated rats that received either saline or montelukast intravenously before and 2 hours after OA challenge.

View larger version (36K): [in this window] [in a new window]   Figure 5. Effect of IL-2 pretreatment with or without montelukast on IL-4 (A) and IFN- (B) mRNA-positive cells after OA challenge. Lungs (n = 32) were recovered in PBS–sucrose 8 hours after OA challenge for in situ hybridization. *p < 0.05 between IL-2–pretreated rats that received either saline intravenously or montelukast intravenously 2 hours after OA challenge.

	DISCUSSION

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In this study, we pretreated rats with IL-2 to upregulate their cellular immunity. IL-2 is a T cell growth factor that is released by T lymphocytes in response to antigen presentation and leads to lymphocyte proliferation and cytokine production (16). IL-2 also stimulates T lymphocytes from subjects with asthma to release mediators that increase eosinophil proliferation and survival (19). In previous studies performed on the BN rat, we showed that IL-2 pretreatment caused an increase in the number of inflammatory cells present in the BALF (11) and an increased airway response to OA (11). We also found that IL-2 pretreatment increased the airway response to OA without affecting the in vivo production of cys-LTs before or after OA challenge (16). Because the LR in BN rats and in humans is related to LTs (10, 20), we hypothesized that IL-2 may be modulating its effects on the LR by enhancing the sensitivity of the airways to LTs. To test this hypothesis, we measured airway responsiveness to LTD₄ in BN rats pretreated with IL-2 or saline. Our results show that rats pretreated with IL-2 require a significantly lower mean dose of LTD₄ to double baseline R_L when compared with control subjects (p < 0.05). We conclude that upregulation of immune function with IL-2 increases the airway responsiveness to LTs. The precise mechanism by which IL-2 alters the responsiveness to LTD₄ is unclear. We have previously found a significant increase in BAL eosinophils after pretreatment with IL-2 compared with untreated control animals that were challenged (11) or unchallenged with antigen (OA) (16). In the experiments reported here, eosinophils were also increased in IL-2–pretreated rats after OA challenge when compared with control subjects. An increase in eosinophils in the airways may lead to the release of more mediators, such as eosinophil cationic protein (19), that have the potential to affect the airway responsiveness to LTs.

Airway smooth muscle cells have been shown to express a higher amount of cytokine receptor mRNA and surface protein in the atopic asthmatic-sensitized state (21). IL-2 upregulates the production of cytokines that can increase surface receptors (22). It is possible that IL-2 increased the expression of the cys-LT₁ receptors on the surface of airway smooth muscle cells and thus their sensitivity to LTD₄. IL-2 may also be exerting an effect at different points along the signal transduction pathway of the cys-LT₁ receptor. Activation of these receptors, which are coupled to G proteins, induces calcium release that results in airway smooth muscle contraction (23). Nabata and colleagues have shown that vascular smooth muscle cells preincubated with IL-2 had an increase in intracellular Ca²⁺ and in DNA synthesis when exposed to angiotensin II (24). Therefore, it is also possible that IL-2 may potentiate the effects of LTD₄ on the airways by increasing the activity of the signal transduction pathway.

To assess whether the cys-LTs were involved in the increased LR after pretreatment with IL-2, we administered the cys-LT₁ receptor antagonist montelukast at the time of OA challenge in rats pretreated with IL-2. The IL-2–induced LR was completely inhibited by montelukast. These results show that LTs are not only involved in the LR but also in the increased airway response to OA after pretreatment with IL-2. We have previously found that IL-2 does not increase bile LT production either before or after OA challenge of BN rats (16). It is therefore likely that IL-2's effects on the LR occur through either an increase in cys-LT₁ receptor expression or an increase in the sensitivity of the receptor to LTs as described previously here.

Studies on the mechanisms of the LR have linked its pathophysiology to several factors such as immunoglobulin E production, Th2 cytokine expression, and inflammatory cell influx (25). However, the precise mechanism by which a LR occurs remains to be determined. We studied BAL inflammatory cells and the expression of Th1 and Th2 cytokine mRNA in the lungs of rats to determine whether any of these factors were affected by inhibition of the cys-LT₁ pathway with montelukast. We found no statistically significant difference in neutrophils, macrophages, and lymphocytes in the BALF between control and experimental groups of animals. Animals challenged with OA and pretreated with IL-2 showed a significant increase in BAL eosinophils compared with the group of animals unchallenged and receiving saline (p < 0.05). The former group of animals also showed a LR, whereas the latter did not. These data support results from previous studies indicating that eosinophils play an important role in mediating a LR (26). Although there was a decrease in the number of BAL eosinophils in the group of animals that were pretreated with IL-2 and received montelukast, this difference was not statistically significant. Montelukast administration inhibited the IL-2–mediated LR without affecting the total number of cells or differential in the BALF 8 hours after OA challenge. These results suggest that airway inflammation may be present without any impact on airway tone (if LT activity is inhibited).

We assessed cytokine mRNA expression in the lung by using SQ-PCR and found that rats pretreated with IL-2 had a significant increase in IL-4 and IL-5 mRNA (Th2) and lower IFN- mRNA (Th1) expression after OA challenge when compared with animals given IL-2 and receiving montelukast at the time of challenge (p < 0.05). The change in the balance between Th1 and Th2 cytokine expression was also confirmed by in situ hybridization. In situ hybridization tests revealed that cells expressing IL-4 mRNA were more predominant and cells expressing IFN- mRNA were less predominant after pretreatment with IL-2 and OA challenge. Interestingly, these changes are similar to those described previously in the BN rat or in humans when an LR occurs (27). The addition of montelukast at the time of OA challenge leads to a predominance of cells expressing the Th1 cytokine IFN- and less cells expressing IL-4 mRNA as described in rats that do not develop a LR (28). These data demonstrate that montelukast can reverse the effects of IL-2 and OA challenge on Th1-Th2 cytokine mRNA expression.

Many studies have shown an important role of cytokines in asthma (29). IL-4 can lead to selective recruitment of inflammatory cells such as mast cells and eosinophils as well as inducing the production of IgE from B cells (30). IL-5 is involved in recruiting and activating eosinophils, cells that release proteins or mediators with the potential of causing several changes that are found in asthma such as bronchoconstriction, inflammation, and epithelial cell desquamation (27). Th2 cytokines have been shown to be increased in the lungs of atopic individuals with asthma (31). IFN- is a Th1 cytokine that may inhibit certain effects of Th2 cytokines (21). The role that cys-LTs play in modulating the balance between Th1 and Th2 cytokines has not been previously assessed. A recent study by Hasday and associates showed that patients with asthma with high cys-LTs production in the BALF 24 hours after segmental ragweed challenge also had increased IL-5, IL-6, and tumor necrosis factor- production compared with low cys-LTs producers (32). This indicates that cys-LTs production may have a direct relationship with cytokine production. Moreover, it has recently been established that the cys-LT₁ receptor is not only expressed on the smooth muscle cells of the airways but that there is also a high expression of the receptor on peripheral blood lymphocytes (23), suggesting that cys-LTs have an effect on T lymphocyte function. The results presented here suggest that cys-LTs are involved in mediating cytokine production after OA challenge as montelukast changed the profile of Th1 and Th2 mRNA expression. The mechanism by which the LT pathway affects cytokine mRNA production has not been explored.

We have confirmed previous results in rats and in guinea pigs that upregulation of cellular immunity with IL-2 increases the airway response to antigen. IL-2 increases the inflammatory response that is present around the airway before OA challenge. We have previously reported that IL-2 did not increase LT production after OA challenge (16). The results reported here clearly demonstrate that the effects of IL-2 are through an increase in the sensitivity of the airways to cys-LTs. In addition, we have described a link between LTs and Th1/Th2 cytokine production. Further studies are required to understand this relationship fully.

	Acknowledgments

 
This work was generously supported by a University Research Grant from Merck Frosst (Montreal, Quebec, Canada) and a CIHR Canada Grant (MDP# 53101).

	FOOTNOTES

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

Received in original form September 5, 2001; accepted in final form February 28, 2002

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