Effects of wortmannin on airways inflammation induced by allergen in actively sensitised Brown Norway rats

doi:10.1016/S0014-2999(01)01515-1

European Journal of Pharmacology

Volume 433, Issues 2–3, 21 December 2001, Pages 217-223

https://doi.org/10.1016/S0014-2999(01)01515-1 Get rights and content

Abstract

We have investigated the effect of wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase, on the immediate-type allergic response and the late phase pulmonary inflammation induced by allergen challenge in the ovalbumin-sensitised Brown Norway rat. Intratracheal (i.t.) instillation of ovalbumin induced dose-related bronchoconstrictor responses. Administration of wortmannin (1, 10 or 100 μg kg⁻¹ i.t., 1 h prior to challenge) induced a marked and dose-dependent inhibition of ovalbumin-induced bronchospasm (ED₅₀ ca. 5 μg kg⁻¹ i.t.). At similar doses, wortmannin also suppressed the bronchoconstrictor responses to 5-hydroxytryptamine and methacholine but the degree of blockade of these spasmogens (1.4–1.9-fold) was less than that of ovalbumin (>20-fold). Wortmannin, given intratracheally 1 h prior to allergen challenge, also suppressed the increases in bronchoalveolar lavage fluid leukocyte numbers and eosinophil peroxidase activity measured 24 h post challenge. However, relatively high doses were necessary (ED₅₀ ca. 100 μg kg⁻¹ i.t.). The potency of wortmannin was increased when dosed 1 h prior to and 24 h after allergen challenge and the readout was 48 h after challenge (ED₅₀ 3–5 μg kg⁻¹ i.t.). Thus, wortmannin is a potent inhibitor of the bronchoconstrictor response induced by allergen in the airways of actively sensitised Brown Norway rats. Inhibition of phosphatidylinositol-3-kinase, an obligatory step in mast cell activation in response to allergen, is the presumed mechanism of action. The fact that similar doses of wortmannin do not suppress the late response to allergen suggests a minimal role for the mast cell in generating the late response to allergen in this model. The striking increase in potency to inhibit the late response when dosed 1 h prior to and 24 h after allergen challenge with the readout taken at 48 h may represent an effect of wortmannin to suppress the migration of leukocytes.

Introduction

Mast cells play a major role in allergy and inflammation Wasserman, 1983, Galli, 1997, Metcalfe et al., 1997, Bakharevski and Ryan, 1999, Holt et al., 1999. Pivotal to the effector function of mast cells is their activation through the interaction of a multivalent antigen (allergen) with its specific immunoglobulin E (IgE) antibody bound to its high affinity receptor, FcεRI. Signalling through FcεRI is initiated by cross-linking of allergen bound to IgE which leads to mast cell degranulation and the release of mediators such as histamine, 5-hydroxytryptamine and proteases, the production of leukotrienes and prostaglandins and the release and synthesis of chemokines and cytokines Church et al., 1997, Metcalfe et al., 1997. The evidence is compelling that mast cells coordinate and facilitate the development of allergic inflammation Wasserman, 1983, Galli, 1997, Metcalfe et al., 1997.

Phosphatidylinositol-3-kinase is associated with, and activated by, a number of protein tyrosine kinases and plays an important role in signalling pathways initiated by several growth factor receptors Vlahos, 1995, Shepherd et al., 1996, Cardenas et al., 1998. Phosphatidylinositol-3-kinase is also activated in mast cells following cross-linking of multivalent allergen bound to IgE and is involved in the signal transduction pathway responsible for degranulation and the release of bioactive mediators Yano et al., 1993, Marquardt et al., 1996, Pendl et al., 1997, Bhattacharyya et al., 1998. Phosphatidylinositol-3-kinase is also involved in chemotaxis and the oxidative burst in leukocytes in response to a variety of stimuli Arcaro and Wymann, 1993, Niggli and Keller, 1997, Lennartz, 1999, Rameh and Cantley, 1999, Miike et al., 2000.

An essential factor implicating phosphatidylinositol-3-kinase in an obligatory role in inflammatory cell function is that the responses can be abolished by low concentrations of wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase Ui et al., 1995, Cardenas et al., 1998. We report here the effect of wortmannin on the immediate-type allergic response and the late phase pulmonary inflammation induced by allergen challenge in an animal model of allergic asthma, the ovalbumin-sensitised Brown Norway rat.

A part of the results was presented at the meeting of the British Pharmacological Society held in July 1999 (Tigani et al., 1999).

Section snippets

Animals

Male Brown Norway rats weighing 200–300 g were supplied by Biological Research Laboratories (Füllinsdorf, Switzerland). They were kept at an ambient temperature of 22±2 °C under a 12-h normal phase light–dark cycle and fed on NAFAG pellets supplied by Nahr und Futtermittel, Gossau, Switzerland. Drinking water was freely available. All experiments were carried out with the approval of the Veterinary Authority of the City of Basel (Kantonales Veterinaeramt, Basel-Stadt).

Sensitisation procedure

Ovalbumin (20 μg ml⁻¹) was

Acute bronchoconstrictor responses to intratracheal administration of ovalbumin in actively sensitised Brown Norway rats: effect of pretreatment with wortmannin

Intratracheal (i.t.) instillation of ovalbumin (3–60 mg kg⁻¹) induced dose-related bronchoconstrictor responses, which peaked at 5 min and resolved within 15 min (Fig. 1). Pretreatment with wortmannin (1, 10 or 100 μg kg⁻¹ i.t., 1 h prior to challenge) induced a dose-dependent inhibition of the bronchospasm induced by ovalbumin (60 mg kg⁻¹). The ED₅₀ was approximately 5 μg kg⁻¹ (Fig. 2). By interpolation from the ovalbumin dose–response relationship, the degree of blockade by wortmannin (10 μg

Discussion

Our results demonstrate potent inhibition by wortmannin of the acute early bronchoconstrictor response to ovalbumin in actively sensitised Brown Norway rats. At similar doses, wortmannin also inhibited the bronchoconstrictor responses to 5-hydroxytryptamine and methacholine. However, the degree of inhibition of these spasmogens (1.4–1.9-fold following the 10 μg kg⁻¹ dose of wortmannin) was markedly less than that of ovalbumin (>20-fold). Thus, nonselective suppression of bronchial contractility

References (31)

M.E. Cardenas et al.
Signal transduction cascades as targets for therapeutic intervention by natural products
Trends Biotechnol.
(1998)
T. Du et al.
Effect of nedocromil sodium on allergen-induced airway responses and changes in the quantity of airway smooth muscle in rats
J. Allergy Clin. Immunol.
(1996)
J.T. Hom et al.
Antigen induced recruitment of eosinophils: importance of CD4+ T cells, IL-5 and mast cells
Clin. Immunol. Immunopathol.
(1994)
M.R. Lennartz
Phospholipases and phagocytosis: the role of phospholipid-derived second messengers in phagocytosis
Int. J. Biochem. Cell Biol.
(1999)
V. Niggli et al.
The phosphatidylinositol 3-kinase inhibitor wortmannin markedly reduces chemotactic peptide-induced locomotion and increases in cytoskeletal actin in human neutrophils
Eur. J. Pharmacol.
(1997)
L.E. Rameh et al.
The role of phosphoinositide3-kinase lipid products in cell function
J. Biol. Chem.
(1999)
M. Ui et al.
Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase
Trends Biotechnol.
(1995)
S.I. Wasserman
Mediators of immediate hypersensitivity
J. Allergy Clin. Immunol.
(1983)
H. Yano et al.
Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells
J. Biol. Chem.
(1993)
A. Arcaro et al.
Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses
Biochem. J.
(1993)

O. Bakharevski et al.

Mast cells as a target in the treatment of rheumatoid arthritis

Inflammopharmacology

(1999)

N. Beckmann et al.

Pulmonary oedema induced by allergen challenge in the rat: non-invasive assessment by magnetic resonance imaging

Magn. Reson. Med.

(2001)

S.B. Bhattacharyya et al.

Activated T lymphocytes induce degranulation and cytokine production by human mast cells following cell-to-cell contact

J. Leukocyte Biol.

(1998)

M.K. Church et al.

Human mast cells and basophils

R.P. Eady et al.

Nedocromil sodium and sodium cromoglycate: pharmacology and putative modes of action

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