Detection and characterization of OX40 ligand expression in human airway smooth muscle cells: A possible role in asthma?

doi:10.1016/j.jaci.2003.12.311

Journal of Allergy and Clinical Immunology

Volume 113, Issue 4, April 2004, Pages 683-689

https://doi.org/10.1016/j.jaci.2003.12.311 Get rights and content

Abstract

Background

The airway smooth muscle (ASM) cell, originally thought of as a passive structural cell, is now well recognized as an active participant in the pathologic events that occur during persistent asthma. Cell-surface molecules play an important role in the development of an immune response. A number of cell-surface molecules are expressed on ASM cells, and these might contribute to the inflammatory reaction.

Objective

The purpose of this study was to determine whether OX40 ligand (OX40L), a molecule known to be involved in T-cell activation, was present on the ASM cell surface.

Methods

We used real-time RT-PCR to detect mRNA expression and flow cytometry, ELISA, and immunoprecipitation to detect the presence of cell-surface protein on ASM cells isolated from asthmatic and nonasthmatic individuals. ELISAs and Western blotting were used to determine the functional outcomes of engagement of OX40L.

Results

OX40L was present on both asthmatic and nonasthmatic ASM cells. Engagement of OX40L with recombinant OX40:Fc resulted in a significantly greater increase in release of IL-6 from ASM cells of asthmatic patients than from ASM cells of nonasthmatic patients (P<.01). Ligation of OX40L resulted in a rapid translocation of protein kinase C β₂ to the cell membrane.

Conclusion

Because the receptor for OX40L, OX40, is expressed on CD4⁺ T cells within 48 hours of stimulation through the T-cell receptor, elucidation of the cross-talk between OX40 and OX40L could be very important in understanding the interaction of cells present in the inflamed airways of an asthmatic patient.

Section snippets

Chemicals

The following compounds were obtained from the following sources: Dulbecco modified Eagle medium (DMEM), PBS, penicillin, streptomycin, amphotericin B, and trypan blue were obtained from Invitrogen (Heidelberg, Australia); EDTA was purchased from Ajax (Auburn, Australia); FBS was obtained from Commonwealth Serum Laboratories (Melbourne, Australia); and BSA was acquired from Sigma (St Louis, Mo).

Antibodies and recombinant proteins

FITC-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc, West Grove,

RT-PCR to detect OX40L mRNA expression

Expression of OX40L was normalized to the level of 18S rRNA in each reaction. OX40L mRNA was expressed equally in cells from both nonasthmatic and asthmatic patients (data not shown).

OX40 and OX40L presence on ASM cells measured by means of ELISA

Both nonasthmatic and asthmatic ASM cells express OX40L, measured with 5A8, on the cell surface (Fig 1, A). The results are expressed as a percentage of absorbance (450 nm) of cells alone, which was set at 100%. The absorbance in the presence of 5A8 was significantly greater than the IgG₁ isotype control for both

Discussion

OX40L is a 34-kd protein expressed on the surface of activated B lymphocytes,¹⁷ activated dendritic cells,⁴ microglial cells,¹⁸ and vascular endothelial cells.¹⁹ This, to our knowledge, is the first report of the expression of OX40L on the surface of human ASM cells. In this study we have demonstrated that stimulation of OX40L with rOX40:Fc mediates a signal transduction pathway involving PKC β₂ and release of IL-6. These observations support the importance of interactions between ASM cells and

Acknowledgements

We acknowledge the collaborative effort of the cardiopulmonary transplant team and the pathologists at St Vincent's hospital, Sydney, and the thoracic physicians and pathologists at Royal Prince Alfred Hospital, Concord Repatriation Hospital, and Strathfield Private Hospital and Rhodes Pathology, Sydney. We also acknowledge the contribution of Dr Gregory King at the Woolcock Institute of Medical Research for supplying the asthmatic biopsy specimens.

References (39)

J.K. Burgess et al.
Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex: characterization of the epitopes
Blood
(1998)
C. Hicks et al.
Quantitation of platelet derived growth factor receptors on cells from human arterial segments
J Immunol Methods
(1994)
A.I. Chen et al.
Ox40-ligand has a critical costimulatory role in dendritic cell:T cell interactions
Immunity
(1999)
M.C. Faux et al.
More on target with protein phosphorylation—conferring specificity by location
Trends Biochem Sci
(1996)
Y. Gokmenpolar et al.
Mapping of a molecular determinant for protein kinase C beta(Ii) isozyme function
J Biol Chem
(1998)
J. Bousquet et al.
Airways remodelling in asthma: no doubt, no more?
Int Arch Allergy Immunol
(1995)
K. Murata et al.
Impairment of antigen-presenting cell function in mice lacking expression of OX40 ligand
J Exp Med
(2000)
S. Flynn et al.
CD4 T cell cytosine differentiation: the B cell activation molecule, OX40 ligand, instructs CD4 T cells to express interleukin 4 and upregulates expression of the chemokine receptor, Blr-1
J Exp Med
(1998)
Y. Ohshima et al.
Expression and function of OX40 ligand on human dendritic cells
J Immunol
(1997)
A.D. Watts et al.
A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in “reverse signalling.”
EMBO J
(1999)

A.L. Lazaar et al.

T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis

J Exp Med

(1994)

P.R. Johnson et al.

Heparin and PGE2 inhibit DNA synthesis in human airway smooth muscle cells in culture

Am J Physiol Lung Cell Mol Physiol

(1995)

S. Carlin et al.

Protein kinase C isoforms in human airway smooth muscle cells: activation of PKC-zeta during proliferation

Am J Physiol Lung Cell Mol Physiol

(1999)

W. Durand-Arczynska et al.

Caldesmon, calponin and alpha-smooth muscle actin expression in subcultured smooth muscle cells from human airways

Histochem J

(1993)

J.K. Burgess et al.

Expression of connective tissue growth factor in asthmatic airway smooth muscle cells

Am J Respir Crit Care Med

(2003)

M.B. Sukkar et al.

GM-CSF production from human airway smooth muscle cells is potentiated by human serum

Mediators Inflamm

(2000)

M. Triantafilou et al.

Identification of Echovirus 1 and coxsackievirus A9 receptor molecules via a novel flow cytometric quantification method

Cytometry

(2001)

E. Baba et al.

Functional CD4 T cells after intercellular molecular transfer of 0X40 ligand

J Immunol

(2001)

A.L. Lazaar et al.

CD40-mediated signal transduction in human airway smooth muscle

J Immunol

(1998)

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OX40L has been detected on the surface of antigen presenting cells (APCs) 1–3 days after antigen encounter (Croft, 2003). It was thought previously that OX40L expression was limited to professional APCs such as activated B-cells, dendritic cells and macrophages, however studies have revealed that it is also expressed on NK and mast cells (Stuber et al., 1995; Ohshima et al., 1997; Weinberg et al., 1999; Kashiwakura et al., 2004; Zingoni et al., 2004) as well as structural cells such as smooth muscle cells and vascular endothelial cells in the presence of inflammatory cytokines (Burgess et al., 2004; Imura et al., 1996). These findings illustrate how the OX40-OX40L interaction is not confined to specific tissues or organs but that its effects have the potential to be widespread throughout the body.
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^☆

Supplementary data associated with this article can be found at doi:10.1016/j.jaci.2003.12.311.

Supported by an Australian Research Council SPIRT grant and Johnson & Johnson Research Pty Ltd. J. K. Burgess is supported by a National Health and Medical Research Council Peter Doherty Fellowship no. 165722.

^∗: Drs Black and Hunt contributed equally to this work.

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Mechanisms of allergyDetection and characterization of OX40 ligand expression in human airway smooth muscle cells: A possible role in asthma?☆

Abstract

Background

Objective

Methods

Results

Conclusion

Section snippets

Chemicals

Antibodies and recombinant proteins

RT-PCR to detect OX40L mRNA expression

OX40 and OX40L presence on ASM cells measured by means of ELISA

Discussion

Acknowledgements

Blood

J Immunol Methods

Immunity

Trends Biochem Sci

J Biol Chem

Airways remodelling in asthma: no doubt, no more?

Int Arch Allergy Immunol

Impairment of antigen-presenting cell function in mice lacking expression of OX40 ligand

J Exp Med

CD4 T cell cytosine differentiation: the B cell activation molecule, OX40 ligand, instructs CD4 T cells to express interleukin 4 and upregulates expression of the chemokine receptor, Blr-1

J Exp Med

Expression and function of OX40 ligand on human dendritic cells

J Immunol

A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in “reverse signalling.”

EMBO J

T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis

J Exp Med

Heparin and PGE2 inhibit DNA synthesis in human airway smooth muscle cells in culture

Am J Physiol Lung Cell Mol Physiol

Protein kinase C isoforms in human airway smooth muscle cells: activation of PKC-zeta during proliferation

Am J Physiol Lung Cell Mol Physiol

Caldesmon, calponin and alpha-smooth muscle actin expression in subcultured smooth muscle cells from human airways

Histochem J

Expression of connective tissue growth factor in asthmatic airway smooth muscle cells

Am J Respir Crit Care Med

GM-CSF production from human airway smooth muscle cells is potentiated by human serum

Mediators Inflamm

Identification of Echovirus 1 and coxsackievirus A9 receptor molecules via a novel flow cytometric quantification method

Cytometry

Functional CD4 T cells after intercellular molecular transfer of 0X40 ligand

J Immunol

CD40-mediated signal transduction in human airway smooth muscle

J Immunol

Mechanisms of allergy
Detection and characterization of OX40 ligand expression in human airway smooth muscle cells: A possible role in asthma?☆