Nerve Growth Factor Induces a Neurokinin-1 Receptor- Mediated Airway Hyperresponsiveness in Guinea Pigs

ANNICK de VRIES, MARK C. DESSING, FERDI ENGELS, PAUL A. J. HENRICKS, and FRANS P. NIJKAMP

Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

	ABSTRACT

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Because asthmatic patients show increased nerve growth factor (NGF) serum levels, we examined the effect of NGF on airway function. Intravenously administered NGF potentiates the histamine- induced bronchoconstriction with a maximum of over 200% in anesthetized spontaneously breathing guinea pigs. Doses of 8 ng and 80 ng NGF/kg body weight induce a significant hyperresponsiveness to histamine. NGF itself does not affect airway reactivity. Airway hyperresponsiveness is observed 30 min and 3 h after NGF administration, and has disappeared after 24 h. The neurokinin-1 receptor antagonist SR 140333 completely blocks the NGF-induced hyperresponsiveness, pointing to a role for tachykinins. This is the first report showing a direct relation between peripherally administered NGF and airway hyperresponsiveness. Taking into consideration that plasma NGF levels have been shown to be elevated in asthmatic patients, our result points to an important role for NGF in the pathogenesis of asthma.

	INTRODUCTION

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Nerve growth factor (NGF) is a member of the neurotrophin family of proteins that can regulate neuronal development, maintenance, and recovery from injury. NGF has been extensively studied in relation to neurite outgrowth. However, it now becomes clear that NGF also plays a crucial role in inflammation (1). Inflammation can lead to an enhanced production and release of NGF. Inflammatory mediators, including interleukin-1 (IL-1), IL-4, IL-5, tumor necrosis factor , and interferon- have been shown to induce the release of NGF (2, 3). In addition to neurons, non-neuronal cells such as mast cells (4), fibroblasts (2), and epithelial cells (5) are able to synthesize NGF.

NGF affects immune cell activity, as it promotes inflammatory mediator release from basophils (6), mast cells (7, 8), T and B cells (9), and macrophages (12). NGF is also able to sensitize neurons and induce an enhanced production of substance P and other tachykinins (13, 14). An enhanced innervation of predominantly sensory nerves, producing substance P, can be found in the airways of transgenic mice overexpressing NGF in the airways (15). An enhanced expression of tachykinin messenger RNA (mRNA) in the nodose ganglia in a guinea pig model for asthma has been shown as well (16). Previously, we have shown a role for tachykinins in the development of airway hyperresponsiveness in the guinea pig (17, 18).

Recently, increased levels in serum NGF were found in asthmatic patients (19). Airway inflammation and hyperresponsiveness are characteristic features in asthmatic disease (20), and NGF seems to be a possible mediator in these events. Therefore, we investigated whether NGF can induce airway hyperresponsiveness and whether tachykinins are involved.

	METHODS

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Lung Function Measurement

Male Hartley guinea pigs (400 to 600 g; Harlan CPB, Zeist, The Netherlands) were anesthetized with urethane (2 g/kg body weight, intraperitoneally). Lung function was measured in spontaneously breathing guinea pigs, essentially after Amdur and Mead (21). Air flow and tidal volume were determined by cannulating and connecting the trachea via a Fleisch flow head (Meijnhart, The Netherlands) to a pneumotachograph. A pressure transducer (MP45-2; Validyne Engineering Corp., Northridge, CA) measured the transpulmonary pressure by the difference between the tracheal cannula and an esophageal cannula. In this way pulmonary resistance (RL) was determined breath by breath by dividing transpulmonary pressure by airflow at isovolumetric points. RL values were averaged per three breaths and are presented as actual value. A small polyethylene catheter (PE-50) was placed in the right jugular vein for intravenous administration of different compounds. Animal studies were approved by the Animal Care Committee of Utrecht University.

Materials

In the present study we used the precursor of NGF, murine NGF 7S (Sigma Chemical Co., St. Louis, MO and Alomone Labs, Jerusalem, Israel) in the guinea pig, as all neurotrophic factors are highly conserved in different species (22). NGF was administered intravenously at doses of 0.8, 8, or 80 ng/kg body weight. NGF was injected 30 min, 3 or 24 h before starting airway function measurement. The airway resistance was measured in the anesthetized guinea pig upon intravenous administration of increasing doses of histamine. The dose- response curve lasted 30 to 40 min. The neurokinin-1 receptor antagonist SR 140333 (23), 2 µg/kg body weight (kindly provided by Sanofi Recherche, Montpellier, France) was administered intravenously 10 min before the injection of 8 ng NGF/kg body weight. Pilot experiments revealed that 2 µg SR 140333/kg body weight completely blocked the substance P-induced decrease in blood pressure in the anesthetized guinea pig (data not shown). In control animals, instead of NGF or SR 140333, vehicles were administered; saline containing 0.01% bovine serum albumin and saline containing 1% ethanol, respectively.

Statistics

Means and standard error of the mean (SEM) were calculated. p Values were determined using an unpaired Student's t test for the comparison of two means. For multiple comparisons of single means an analysis of variance (ANOVA) followed by Bonferroni's test was used. Probability values of p < 0.05 were considered significantly different.

	RESULTS

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A strong hyperresponsiveness (an increase in contractile response) to histamine was found 30 to 70 min after injection of 8 ng NGF/kg body weight (Figure 1). The airway resistance after the highest dose of histamine (20 µg/kg body weight) increased significantly from 2.3 ± 0.3 cm H₂O/ml s¹ in the control animals to 6.0 ± 0.6 cm H₂O/ml s¹ in the NGF-treated animals (p < 0.01). NGF did not induce any change in airway resistance by itself (basal resistance 0.10 ± 0.023 cm H₂O/ml s¹ in control animals and 0.13 ± 0.013 cm H₂O/ml s¹ in NGF (8 ng/kg body weight) animals.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Induction of airway hyperresponsiveness to histamine by NGF. NGF (8 ng/kg body weight) (solid bars) or vehicle (open bars) was administered 30 min before administration of increasing doses of histamine (intravenously). Bars represent mean ± SEM (n = 5 per group). *p < 0.05 control versus NGF-treated, Student's t test.

The effect of NGF on airway responsiveness to histamine was dose-dependent (Figure 2). Administration of 0.8 ng NGF was without effect on the histamine-induced increase in airway resistance, whereas 80 ng NGF induced an airway hyperresponsiveness equal to 8 ng NGF.

View larger version (25K): [in this window] [in a new window]   Figure 2.   Effect of different doses of NGF on airway responsiveness. Saline and three different doses of NGF, 0.8 ng, 8 ng, or 80 ng NGF/kg body weight, were administered 30 min before administration of increasing doses of histamine. Only the airway resistance to the highest dose of histamine, 20 µg histamine/kg body weight, is depicted. Bars represent mean ± SEM (n = 14 for pooled controls, n = 4/5 for NGF-treated animals). *p < 0.01 control versus NGF treated, Student's t test.

A significant increase in airway responsiveness could still be found 3 h after the injection of NGF (Figure 3). Maximal values were 2.8 ± 0.6 cm H₂O/ml s¹ in saline- and 5.4 ± 0.4 cm H₂O/ ml s¹ in NGF-treated animals (p < 0.01). Histamine-induced increases in airway resistance were no longer different from control animals 24 h after the intravenous injection of NGF.

View larger version (24K): [in this window] [in a new window]   Figure 3.   Effect of a single injection of NGF on the airway responsiveness measured at different time points. A single dose of NGF (80 ng/kg body weight) was administered 30 min, 3 h, or 24 h before administration of the doses of histamine. Only the airway resistance to the highest dose of histamine, 20 µg histamine/kg body weight, is depicted. Bars represent mean ± SEM (n = 15 for pooled controls, n = 4/5 for NGF-treated animals). *p < 0.02 control versus NGF-treated, Student's t test.

The neurokinin-1 receptor antagonist SR 140333 was able to completely inhibit the NGF-induced airway hyperresponsiveness (Figure 4).

View larger version (21K): [in this window] [in a new window]   Figure 4.   Effect of the neurokinin-1 receptor antagonist SR 140333 on the NGF-induced airway hyperresponsiveness. Vehicle or SR 140333 was administered intravenously 10 min before saline or NGF. Saline or NGF (8 ng/kg body weight) was administered 30 min before administration of increasing doses of histamine. Vehicle-saline (open bars); SR 140333-saline (cross-hatched bars); vehicle-NGF (solid bars); SR 140333-NGF (double cross-hatched bars). Bars represent mean ± SEM (n = 4/5 per group). *p < 0.05 vehicle-NGF versus SR 140333-NGF and versus vehicle-saline, ANOVA followed by Bonferroni's test.

	DISCUSSION

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Asthmatic patients show a highly elevated level of serum NGF (19). This is the first study showing a rather acute increase of airway responsiveness to histamine after a single, peripheral injection of NGF. Therefore, we postulate a role for NGF in the development of asthma. The induction of airway hyperresponsiveness by NGF was dose-dependent as 8 ng and 80 ng NGF/kg body weight caused a significant increase in airway responsiveness, whereas no effect was found using the lower dose of 0.8 ng NGF. NGF showed an increase on airway responsiveness within 30 min to 3 h after the administration, whereas no increase could be determined 24 h after a single injection of 80 ng NGF.

NGF is able to augment neurogenic inflammation (24), and this could be one of the mechanisms via which airway hyperresponsiveness was induced. It is known that NGF changes the properties of sensory nerve endings by inducing a very fast accumulation of second messenger in synaptosomes (25), by sensitizing the nerve terminal (24), or by altering neuropeptide levels in sensory nerves (14). Previous studies, in our and other groups, have shown a role for tachykinins in IL-5 (17), 13-hydroxyoctadecadienoic acid (18), ozone (26), and citric acid (27) induced airway hyperresponsiveness in animal models. Moreover, tachykinin levels are elevated in asthmatics (28), and protective effects of neurokinin receptor antagonists have been reported (29). In this study we show that the NGF-induced airway hyperresponsiveness is mediated via the neurokinin-1 receptor. The preferred ligand for the neurokinin-1 receptor is substance P; however, there is also a relative high affinity for neurokinin A (28).

Two receptors are known for NGF: the low-affinity receptor p75 neurotrophin receptor and the high-affinity tyrosine kinase receptor A (TrkA). Upon contact NGF binds to the TrkA receptor and in a tyrosine kinase-dependent manner phosphorylates key transduction-related proteins or ion channels, thereby sensitizing the peripheral sensory nerve ending (25, 30). Alternatively, the NGF-TrkA complex is internalized and retrogradely transported to the nucleus, where the preprotachykinin mRNA levels (precursor for substance P and neurokinin A) and protein levels (substance P and neurokinin A) are altered (14, 31). The p75 low-affinity NGF receptor is also shown to be responsible for an upregulation in tachykinin content in sensory nerves (32). Any of these mechanisms could account for an increased release of substance P, presumably from the sensory nerve endings, and thereby induction of airway hyperresponsiveness. The NGF-induced neuronal sensitization could also be indirect, via the release of sensitizing mediators from TrkA expressing inflammatory cells, e.g., mast cells and monocytes (7, 24, 30).

In conclusion, this study shows a clear role for the neurokine NGF in developing airway hyperresponsiveness to histamine. Asthmatic patients show an elevated level of serum NGF (19). This suggests a possible relationship between the high NGF serum levels and the pathological situation in asthmatic patients. In the guinea pig 8 to 80 ng NGF/kg body weight (approximately 25 ml blood in a guinea pig weighing 500 g) induced an airway hyperresponsiveness (Figure 2). The dose range in which NGF caused an airway hyperresponsiveness was comparable to the serum levels of NGF found in asthmatic patients: 150 pg NGF/ml serum (19). In several animal models for pain, neutralizing antibodies against NGF have been proven to attenuate the pathological changes caused by inflammation or NGF (1, 33, 34) and even the increased levels of substance P (35). Thus, NGF might be a new target for therapy in asthma.

	Footnotes

Correspondence and requests for reprints should be addressed to Annick de Vries, Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands. E-mail: A.deVries{at}pharm.uu.nl

(Received in original form August 13, 1998 and in revised form December 14, 1998).

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