IL-4 and IL-5 mRNA expression in induced sputum of asthmatic subjects: Comparison with bronchial wash

doi:10.1016/S0091-6749(99)70497-5

Journal of Allergy and Clinical Immunology

Volume 103, Issue 2, February 1999, Pages 238-245

https://doi.org/10.1016/S0091-6749(99)70497-5 Get rights and content

Abstract

Background: The local production of T_H2 -type cytokines is thought to orchestrate the ongoing eosinophilic inflammation and contribute to the pathophysiologic features of allergic asthma. Previous studies investigating cytokine expression in asthmatic individuals have used invasive fiberoptic bronchoscopy techniques. To date, there have been no reports of cytokine mRNA expression in induced sputum as a means of quantifying local inflammatory events. Objectives: We examined whether IL-4, IL-5, and IFN-γ mRNA expression could be detected in cells from induced sputum in subjects with mild asthma and normal control subjects. In addition, we compared the profile of inflammatory cells and cytokine mRNA in sputum and bronchial wash fluid. Methods: Cells positive for IL-4, IL-5, and IFN-γ mRNA were determined by using in situ hybridization on cytospun aliquots of sputum induced by successive inhalations of hypertonic saline. Inflammatory cells were quantified by using immunologic cell surface markers and immunocytochemistry. Results: IL-4 and IL-5 mRNA were detected in the sputum of all asthmatic subjects, and the number of cells expressing these cytokines was significantly higher than that found in control subjects. Colocalization studies showed CD3-positive T cells were the major sources of IL-4 and IL-5 mRNA. Conclusions: This study demonstrates that induced sputum can be used to detect mRNA for T_H2 -type cytokines in bronchial asthma and that the increase in IL-4 and IL-5 mRNA expression is similar to that seen with more invasive techniques. The qualitative differences in inflammatory cell numbers between sputum induction and bronchial wash are consistent with their sampling of different airway compartments. (J Allergy Clin Immunol 1999;103:238-45.)

Section snippets

Subjects

Thirteen patients fulfilling the American Thoracic Society criteria for asthma,¹⁵ with documented airways reversibility to inhaled β₂ -agonists and an increased airways responsiveness to methacholine (PC₂₀ < 8 mg/mL), were recruited from the Asthma Clinic at the Montreal Chest Hospital, McGill University. These patients were classified as having mild-to-moderate asthma and had their symptoms controlled by regular use of inhaled selective β₂ -agonists. All patients were atopic on the basis of

ISH

To detect IFN-γ, IL-4, and IL-5 mRNA in cytospins of induced sputum and cells from bronchial wash fluid, radiolabeled complementary riboprobes (cRNA antisense) were used. The cDNA sequences for IFN-γ, IL-4, and IL-5 were inserted into pGEM vectors, grown in Escherichia coli , and linearized with the appropriate enzymes. Before ISH, in vitro transcription to generate sense and antisense probes was performed in the presence of ³⁵S-uridine triphosphate (UTP) and the appropriate T7 or SP6

ICC

To detect cytokine immunoreactivity recovered by sputum induction and bronchial wash, we used specific primary mAbs for IL-4 (Genzyme, Cambridge, Mass) and IL-5 (5A5; a kind gift of Dr Jan Tanvernier, Ghent, Belgium). To ascertain the phenotype of inflammatory cells, immunohistochemical markers for eosinophils (major basic protein [MBP]), T lymphocytes (CD3), B lymphocytes (CD20), neutrophils (elastase), and macrophages (CD68) were used. ICC was performed by using a modified alkaline

RESULTS

Fig 1, A and B show representative examples of APAAP immunostaining for MBP and CD3 in an asthmatic individual.

. Representative examples of radiolabeled ISH and APAAP ICC of cells recovered by induced sputum. Red cytoplasmic staining in panels A and B indicates immunolocalization of MBP- and CD3-positive cells, respectively, in an asthmatic subject (arrows) . Inserts at a higher magnification show the typical morphology of eosinophils and lymphocytes, respectively. C demonstrates the paucity of

DISCUSSION

In this study we have investigated the inflammatory characteristics of induced sputum in asthmatic subjects and have demonstrated the feasibility of detecting cytokine mRNA in inflammatory cells recovered from induced sputum with ISH. Our results in asthmatic subjects indicate that there are an increased number of eosinophils and IL-4 and IL-5 mRNA–positive cells in the cellular component of induced sputum compared with that of control subjects. By using immunohisto-chemistry, we also confirmed

Acknowledgements

We thank the Glaxo Institute for Molecular Biology, Geneva, Switzerland and Dr Colin Sanderson for their generous gifts of human cDNA for the human IL-4 and IL-5. We also thank Ms Elsa Schotman, Ms Zivart Yasruel, and the nurses at the Montreal Chest Institute for their technical assistance, and Ms Linda Karpowicz for her help in the preparation of this manuscript.

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The present study was undertaken to study the effect of the nonsteroidal anti-inflammatory drug indomethacin on Th1 and Th2 immune responses. For this study, mice were immunized by s.c. injection of ovalbumin (OVA) emulsified with complete Freund's adjuvant into the base of the tail (day 0). Varying doses of indomethacin were orally administrated daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon-γ as an indicator of Th1 responses and anti-OVA IgG1 and interleukin-10 as that of Th2 responses were measured. The results showed that treatment with indomethacin was followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. Indomethacin inhibited both Th1 and Th2 responses, although the nonsteroidal anti-inflammatory drug suppressed the former more effectively than the latter. Administration of indomethacin resulted in suppression of antigen (OVA)-induced arthritis that was associated with inhibition of anti-OVA IgG2a but not IgG1 production. These results suggest that nonsteroidal anti-inflammatory drugs may downregulate Th1 and, to a lesser extent, Th2 immune responses. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association
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^☆: Supported by the J. T. Costello Memorial Research Fund, the Montreal Chest Research Institute, and Inspiraplex. Dr Hamid is a recipient of a Chercheur-Boursier award from the Fonds de la Recherche en Sante du Quebec. Dr Eleanor Minshall is a recipient of a Medical Research Council/Canadian Lung Association Fellowship.

^☆☆: Reprint requests: Qutayba Hamid, MD, PhD, Meakins-Christie Laboratories, McGill University, 3626 St Urbain St, Montreal, Quebec, Canada H2X 2P2.

^★: 0091-6749/99 $8.00 + 0 1/1/94552

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IL-4 and IL-5 mRNA expression in induced sputum of asthmatic subjects: Comparison with bronchial wash☆,☆☆,★

Abstract

Section snippets

Subjects

ISH

ICC

RESULTS

DISCUSSION

Acknowledgements

J Allergy Clin Immunol

Blood

Chest

Blood

Chest

J Allergy Clin Immunol

J Allergy Clin Immunol

Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma

Am Rev Respir Dis

IL-4 is an essential factor for the IgE synthesis induced in vitro by human T cell clones and their supernatants

J Immunol

Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma

N Engl J Med

Phenotype of cells expressing mRNA for TH2-type (interleukin 4 and interleukin 5) and TH1-type (interleukin 2 and interferon gamma) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects

Am J Respir Cell Mol Biol

Comparison of samples collected by sputum induction and bronchoscopy from asthmatic and healthy subjects

Am J Respir Crit Care Med

Cellular characteristics of sputum from patients with asthma and chronic bronchitis

Thorax

Use of induced sputum cell counts to investigate airway inflammation in asthma

Thorax

Spontaneous and induced sputum to measure indices of airway inflammation in asthma

Am J Respir Crit Care Med

Dissociation between airway inflammation and airway hyperresponsiveness in allergic asthma

Am J Respir Crit Care Med

Cytokine expression in normal, atopic, and asthmatic subjects using the combination of sputum induction and the polymerase chain reaction

Thorax

Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements

Am J Respir Crit Care Med