Asthmatic airway biopsy specimens are more likely to express the IL-4 alternative splice variant IL-4δ2☆,☆☆,★

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Abstract

Background: The human IL4 gene, has been shown to express the alternatively spliced messenger (m)RNA IL-4δ2. IL-4δ2 is missing the entire sequence from exon 2 and has been identified as an IL-4 receptor antagonist. Objective: We sought to distinguish IL-4 and IL-4δ2 mRNA in respiratory tract tissue for the first time. Methods: A novel competitive PCR assay was established with primers designed on either side of the alternative splice junction of the IL4 gene, allowing the simultaneous quantitation of both IL-4 and IL-4δ2 mRNA from one reaction. Results: IL-4 and IL-4δ2 were differentially expressed in 4 nasal polyps. No difference was seen in endobronchial biopsy specimens for IL-4 mRNA expression between control subjects (median, 2.8 × 102 copies/μg RNA; range, 0-3.7 × 103 copies/μg RNA) and asthmatic subjects (median, 1.4 × 102 copies/μg RNA; range, 0-4.7 × 102 copies/μg RNA). However, significantly more asthmatic subjects (6 of 9) than control subjects (1 of 7) expressed IL-4δ2 (P = .036). Expression of IL-4 variants was unaffected by atopic status. Conclusions: Given that IL-4δ2 is an IL-4 receptor antagonist, these results indicate that it is crucial to be able to distinguish IL-4δ2 from IL-4 when assessing IL4 gene expression. Increased expression of IL-4δ2 in stable asthmatic subjects suggests that the balance of IL-4 and IL-4δ2 may modulate asthmatic inflammation. (J Allergy Clin Immunol 1999;104:978-82.)

Section snippets

Samples

PHA-stimulated PBMCs were prepared as described previously.9 Aliquots of 3 × 106 cells were stimulated with 3 μg/mL PHA (Sigma-Aldrich, Castle Hill, NSW, Australia) for 24 hours and then collected by centrifugation, snap frozen in liquid nitrogen, and stored at –80°C.

Freshly resected nasal polyps from 4 atopic patients were obtained. They were dissected and snap frozen in liquid nitrogen before storing at –80°C.

Endobronchial biopsy specimens were obtained from 9 asthmatic volunteers with

IL-4 PCR specificity

RT-PCR of PHA-stimulated PBMC RNA yielded PCR products of 339 and 291 bp, which are consistent with being derived from IL-4 and IL-4δ2 mRNA, respectively. Restriction enzyme analysis was conducted on PAGE-purified and reamplified PCR products (data not shown). As predicted, putative IL-4δ2 PCR products were cleaved by Pst I (exon 3) and Pvu II (exon 3) but not by Bpu AI (exon 1-2 splice junction) or Hin dII (exon 2). IL-4 PCR products were cleaved by all 4 restriction enzymes, producing

DISCUSSION

In both nasal polyps and airway biopsy specimens we found that IL-4 and IL-4δ2 were variably expressed, with IL-4δ2 levels usually somewhat lower than those for IL-4. If we had used an assay that did not distinguish between IL-4 and IL-4δ2 for these samples, we would have concluded incorrectly that asthmatic subjects expressed more IL-4 compared with control subjects. Given that IL-4δ2 is an IL-4 receptor antagonist,6, 7 these results indicate that it is crucial to be able to distinguish IL-4δ2

Acknowledgements

We thank Ros Bish, RN; Dr Frank Thien; and Dr Xun Li for their clinical assistance.

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    ☆

    Supported by Glaxo Wellcome Australia and the National Health and Medical Research Council, Australia.

    ☆☆

    Reprint requests: E. Haydn Walters, MD, FRCP, FRACP, Department of Respiratory Medicine, Alfred Hospital, Prahran, Melbourne, Victoria, 3181, Australia.

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