Human eosinophils release matrix metalloproteinase-9 on stimulation with TNF-α,☆☆,

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Abstract

Background: The eosinophil is a prominent cell in allergic lung inflammation and is exposed to a range of cytokines, including TNF-α, at the site of allergen challenge. Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) produced by inflammatory cells are thought to play a crucial role in interstitial matrix turnover and tissue remodeling in acute and chronic lung diseases. In addition, protein kinase C is known to be important in MMP-9 expression and secretion. Objective: We investigated the regulation of eosinophil-derived MMP-9 and TIMP proteins by TNF-α. Methods: Using RT-PCR and gelatin zymography, we investigated the ability of human eosinophils to produce and secrete active MMP-9 on stimulation with TNF-α. We also studied the production of TIMP-1 and TIMP-2 in eosinophils by using Western blotting. Results: The gelatinolytic activity of MMP-9 in unstimulated eosinophils was low, but it increased by 95% after TNF-α stimulation. This increase was regulated at both the transcriptional and translational levels. The transcription inhibitor actinomycin D, the nuclear factor κB (NFκB) inhibitor N-CBZ-Leu-Leu-Leu-AL, the protein synthesis inhibitor cycloheximide, and the protein kinase C inhibitor H7 significantly decreased MMP-9 activity in TNF-α–treated cells. TIMP-1 and TIMP-2 gene expression and protein production varied significantly among different cell donors. Conclusion: Eosinophils, on stimulation with TNF-α, may play a major role in asthmatic airway remodeling through increased MMP-9 production at the inflammatory site. (J Allergy Clin Immunol 1999;104:983-90.)

Section snippets

Isolation of peripheral blood eosinophils

Peripheral blood eosinophils were isolated from atopic asthmatic volunteers who had given their informed consent, as described previously.20, 21 Briefly, heparinized blood (100 mL) was mixed with 20 mL of dextran (6%; Fluka, Buchs, Switzerland) in RPMI-1640 medium (Bio Whittaker, Walkersville, Md). Erythrocytes were then sedimented for 30 minutes at room temperature. The plasma was layered on a Ficoll (Pharmacia, Quebec, Canada) cushion (15 mL) and centrifuged for 25 minutes at 1000g at room

RT-PCR

Total RNA was isolated from 2 × 106 eosinophils by using the Quiagen RNeasy kit (Ontario, Canada). The average amount of RNA obtained from 2 × 106 eosinophils was 300 ng. One third of the RNA was reverse transcribed by using superscript II reverse transcriptase (Gibco BRL, Ontario, Canada) and random hexamers (50A 260 U; Boehringer Mannheim GmbH, Mannheim, Germany) as primers. Thereafter, PCR was performed in 20-μL reactions by using the following primers (25 μmol/L) for MMP-9

MMP-9 and TIMP expression in human eosinophils

Transcriptional expression of MMP-9, as well as that of TIMP-1 and TIMP-2, was studied in human peripheral blood eosinophils by means of RT-PCR. MMP-9 and TIMP-2 gene expression could be detected in both resting and TNF-α–stimulated eosinophils (Fig 1, A ), whereas TIMP-1 gene expression appeared to vary between different subjects (data not shown).

. A, A representative RT-PCR experiment of resting and TNF-α–stimulated eosinophils. Cells were incubated for 24 hours at 37°C in the presence or

DISCUSSION

The recent description of the capacity of eosinophils to release MMP-9 has opened new vistas toward our understanding of the process of airway remodeling in asthma.25 Our novel data revealed that human eosinophil–derived MMP-9 expression and secretion is upregulated on TNF-α stimulation. Gelatin zymography provided an excellent method to show MMP-9 activity in these cells. NFκB was previously shown to be involved in MMP-9 activation in fibroblasts,26 transformed cells,27, 28 and renal cells.29

Acknowledgements

We thank Jolanta Sawicka for technical help, and Dr Harissios Vliagoftis and Dr Greg Sawicki for useful and ongoing discussions.

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    Supported by the Medical Research Council of Canada, the Alberta Heritage Foundation for Medical Research, and the Glaxo-Wellcome Heritage Award, University of Alberta. A. S. was also supported by Hoffman-La Roche, ESCF. R. M. and M. D. are Alberta Heritage Senior Medical Scholars.

    ☆☆

    Reprint requests: Redwan Moqbel, PhD, FRCPath, Pulmonary Research Group, University of Alberta, 5-74 HMRC, Edmonton, Alberta T6G 2S2, Canada.

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