IL-1 receptors mediate persistent, but not acute, airway hyperreactivity to ozone in guinea pigs

Abstract

Ozone exposure in the lab and environment causes airway hyperreactivity lasting at least 3 days in humans and animals. In guinea pigs 1 day after ozone exposure, airway hyperreactivity is mediated by eosinophils that block neuronal M(2) muscarinic receptor function, thus increasing acetylcholine release from airway parasympathetic nerves. However, mechanisms of ozone-induced airway hyperreactivity change over time, so that depleting eosinophils 3 days after ozone makes airway hyperreactivity worse rather than better. Ozone exposure increases IL-1beta in bone marrow, which may contribute to acute and chronic airway hyperreactivity. To test whether IL-1beta mediates ozone-induced airway hyperreactivity 1 and 3 days after ozone exposure, guinea pigs were pretreated with an IL-1 receptor antagonist (anakinra, 30 mg/kg, intraperitoneally) 30 minutes before exposure to filtered air or to ozone (2 ppm, 4 h). One or three days after exposure, airway reactivity was measured in anesthetized guinea pigs. The IL-1 receptor antagonist prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone exposure. Ozone-induced airway hyperreactivity was vagally mediated, since bronchoconstriction induced by intravenous acetylcholine was not changed by ozone. The IL-1 receptor antagonist selectively prevented ozone-induced reduction of eosinophils around nerves and prevented ozone-induced deposition of extracellular eosinophil major basic protein in airways. These data demonstrate that IL-1 mediates ozone-induced airway hyperreactivity at 3 days, but not 1 day, after ozone exposure. Furthermore, preventing hyperreactivity was accompanied by decreased eosinophil major basic protein deposition within the lung, suggesting that IL-1 affects eosinophil activation 3 days after ozone exposure.

Figure 1.

IL-1β was present in bone marrow of control guinea pigs (open bar) and was slightly increased 1 day after ozone exposure (shaded bar). At 3 days after ozone exposure (solid bar), IL-1β levels in bone marrow were almost twice as high as in controls. IL-1β concentrations were normalized to total protein. *P = 0.057 from control. Data are means ± SE, n = 5.

Figure 2.

Blocking IL-1 receptors does not prevent airway hyperreactivity 1 day after ozone exposure (A) but does prevent airway hyperreactivity 3 days after exposure (C). In anesthetized guinea pigs, electrical stimulation of both vagus nerves causes frequency-dependent bronchoconstriction (measured as an increase in pulmonary inflation pressure) in air-exposed guinea pigs (open circles) that was significantly potentiated (A) 1 and (B) 3 days after ozone exposure (solid circles). Pretreatment with the IL-1 receptor antagonist prevented airway hyperreactivity 3 days after ozone exposure (C, solid squares), but not 1 day after exposure (A, solid squares). The IL-1 receptor antagonist did not affect air-exposed controls (C, open squares). Vehicle treatment did not change vagally mediated bronchoconstriction in either ozone- or air-exposed animals (B, dashed lines next to respective controls). *The entire frequency response after ozone exposure is significantly different from that of air-exposed guinea pigs. ^‡The entire frequency response in the presence of IL-1 receptor antagonist is significantly different from ozone-exposed guinea pigs. Data are expressed as means ± SE, n = 3–7.

Figure 3.

Blocking IL-1 receptors increases airway smooth muscle contraction to intravenous acetylcholine. Acetylcholine-induced bronchoconstriction was measured in vagotomized guinea pigs as an increase in pulmonary inflation pressure. (A) One and (B) three days after ozone exposure, smooth muscle contraction slightly increased (solid circles) compared with air-exposed animals (open circles). (C) The IL-1 receptor antagonist slightly increased smooth reactivity in all groups. Vehicle treatment did not change acetylcholine-induced bronchoconstriction in either air- or ozone-exposed animals (B, dashed lines next to respective controls). ^‡The entire frequency response in the presence of IL-1 receptor antagonist is significantly different from that of ozone-exposed guinea pigs. Data are expressed as means ± SE, n = 4–7.

Figure 4.

Bradycardia in response to (A and B) electrical stimulation of both vagus nerves, or to (C and D) intravenous acetylcholine in vagotomized guinea pigs, was measured as a fall in heart rate in beats per minute. Neither ozone (A–D, solid circles) nor the IL-1 receptor antagonist (A–D, solid squares) affected vagally or acetylcholine induced bradycardia. Vehicle treatment did not change bradycardia in response to vagal stimulation or acetylcholine in either air- or ozone-exposed animals (B and D, dashed lines next to respective controls). Data are expressed as means ± SE, n = 4–6.

Figure 5.

(A) Inflammatory cells in bronchoalveolar lavage were not increased 1 day after ozone exposure, with the exception of neutrophils. (B) However, 3 days after ozone exposure, all inflammatory cells were significantly increased in bronchoalveolar lavage. Treatment with IL-1 receptor antagonist did not change inflammatory cells in bronchoalveolar lavage in controls or at either time point after ozone exposure. *Significantly different from air-exposed controls. Data are expressed as means ± SE, n = 4–7.

Figure 6.

Circulating inflammatory cells in peripheral blood were not changed by ozone or by the IL-1 receptor antagonist at (A) 1 or (B) 3 days after ozone exposure, with the exception of lymphocytes, which were significantly inhibited by the IL-1 receptor antagonist 3 days after ozone exposure (B). Data are expressed as means ± SE, n = 3–6.

Figure 7.

Eosinophils are present in airways and around nerves in control and ozone-exposed animals. Nerves in guinea pig lungs were stained black with antibody to PGP9.5, and eosinophils were stained red with chromotrope 2R (asterisks) (A–D) and quantified in six to eight different airways per guinea pig (E and F). Even in air-exposed control animals there are some eosinophils around airway nerves (A; open bars in E and F), which are decreased by ozone (C; solid bars in E and F). The ozone-induced decrease in total lung eosinophils was not affected by the IL-1 receptor antagonist (E), but the IL-1 receptor antagonist did prevent ozone induced eosinophil loss around nerves (F). Data in E and F are expressed as means ± SE, n = 4–6. *Significantly different from air-exposed animals treated with the IL-1 receptor antagonist.

Figure 8.

Blocking IL-1 receptors prevented extracellular deposition of eosinophil major basic protein (MBP) (labeled with an antibody to MBP) within airways 3 days after ozone exposure. After staining, slides were coded and analysis performed blinded, and intact eosinophils (as identified by solid spheres of MBP staining in excess of 8 μm in diameter) were excluded. Airways of air-exposed control guinea pigs do contain some extracellular MBP (A, and open bar in E), which was not significantly changed 3 days after ozone exposure (C, and solid bar in E). IL-1 receptor antagonist significantly decreased deposition of extracellular MBP (D, and dark gray bar in E). (E) Data are expressed as mean fluorescence intensity per area of the airway in μm². Data are means ± SE, n = 4–6. ^‡Significantly different from ozone-exposed animals. Scale bar for A–D, 50 μm.

Figure 9.

Airway hyperreactivity 1 day after ozone exposure is mediated by resident eosinophils (Eos_R) that release MBP and inhibit M₂ muscarinic receptor function on parasympathetic nerves. The mechanisms of hyperreactivity 3 days after ozone exposure have not been determined; however, it is possible that hyperreactivity is mediated by substance P induced by nerve growth factor (NGF) released from eosinophils and that the IL-1 receptor antagonist (IL-1RA) is blocking this pathway. Alternatively, the IL-1 receptor antagonist may protect a new, beneficial population of eosinophils (Eos_N).