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Regulation of TNF-- and IFN--induced CXCL10 expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease¹

ELIZABETH L. HARDAKER^*, ALICIA M. BACON^*, KAREY CARLSON^*, AMY K. ROSHAK^*, JAMES J. FOLEY^*, DULCIE B. SCHMIDT^*, PETER T. BUCKLEY^*, MEGHAN COMEGYS^*, REYNOLD A. PANETTIERI, JR., HENRY M. SARAU^* and KRISTEN E. BELMONTE^*,2

^* GlaxoSmithKline, Respiratory and Inflammation, Centre for Excellence in Drug Discovery, King of Prussia, Pennsylvania, USA; and
University of Pennsylvania Medical Center, Pulmonary, Allergy and Critical Care Division, Philadelphia, Pennsylvania, USA

²Correspondence: UW2532, Respiratory and Inflammation, Centre for Excellence in Drug Discovery, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, PA 19406, USA. E-mail: Kristen.E.Belmonte{at}GSK.com

SPECIFIC AIMS

The chemokine CXCL10 (interferon--inducible protein-10, IP-10) and its receptor, CXCR3, have been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). While a variety of inflammatory cells produce CXCL10 in response to antigens, irritants, and cytokines, we hypothesized that the airway smooth muscle (hASM) in human COPD patients and primary hASM cells in culture secrete CXCL10, which promotes inflammatory responses in COPD.

PRINCIPAL FINDINGS

1. CXCL10 is expressed by the airway smooth muscle in the lungs of patients with COPD
Lung tissue was obtained at autopsy from patients diagnosed with COPD. ELISA assays performed on whole lung homogenates demonstrated that CXCL10 levels were elevated eightfold in the COPD patients compared with those obtained from unaffected subjects (Fig. 1 A). Immunohistochemistry performed on bronchial sections revealed increased expression of CXCL10 in the airways of COPD subjects. Significant CXCL10 expression was detected in the airway smooth muscle (Fig. 1B ). These observations suggest that CXCL10 is elevated in the airways of COPD subjects and that the airway smooth muscle may actively participate in secreting CXCL10.

View larger version (121K): [in this window] [in a new window]   Figure 1. A) Measurement of CXCL10 protein (pg/mL) in homogenates of human lung obtain from patients at autopsy was measured by ELISA. Each point represents a measurement from an individual patient that was measured in triplicate. B) Expression of CXCL10 protein in sections of human lung as detected by immunohistochemistry using a monoclonal antibody against human CXCL10 (brown) and counterstained with hematoxylin (purple). SM, smooth muscle; L, airway lumen (n=3 for normals, n=5 for COPD).

2. Cytokines associated with COPD have a synergistic effect on the expression of CXCL10 by primary cultured human airway smooth muscle cells
In light of the novel finding that airway smooth muscle readily expresses CXCL10 in the lungs of patients with COPD, we examined whether IFN- and TNF-, cytokines implicated in the pathogenesis of COPD, stimulated CXCL10 expression in primary cultured hASM. CXCL10 protein expression was modestly induced by IFN- and TNF- (each at 0.01–10 ng/mL), and synergistically induced expression was observed when both cytokines were combined. Significant levels of CXCL10 protein (516.0±215.3 ng/mL) were measured when the cells were treated simultaneously with 0.1 ng/mL of TNF- and IFN-, whereas little or no CXCL10 protein was detectable at this concentration when added individually. When 10 ng/mL of IFN- and TNF- were added concurrently, highly elevated concentrations of CXCL10 protein were produced by the hASM after just 5 h of treatment (7155±2363 pg/mL). In contrast, no measurable CXCL10 protein was present until at least 8 h when treated with TNF- alone and not until 18 h when treated with IFN- alone. The synergistic effect of IFN- and TNF- on expression of CXCL10 protein is important, since elevated levels of both cytokines are found in the lungs of patients with COPD.

3. Human airway smooth muscle does not express CXCR3
Since cultured hASM secrete CXCL10 protein, we tested whether the hASM cells also expressed CXCR3, the receptor for CXCL10. IFN- and/or TNF- had little effect on steady-state expression levels of CXCR3 by cultured hASM; no receptor activation was noted in calcium mobilization assays nor was any receptor binding observed in radioligand binding studies designed to detect the presence of functional CXCR3 receptors on hASM. These data are consistent with published reports that CXCR3 may be expressed preferentially on activated T lymphocytes.

4. CXCL10 expression by human airway smooth muscle is differentially regulated by TNF- and IFN-
Although the precise mechanism by which TNF- and IFN- synergize to induce CXCL10 expression remains unknown, evidence suggests that NF-B activation and its binding to a site within the CXCL10 promoter may mediate cytokine-induced CXCL10 expression. We next investigated whether CXCL10 expression by IFN- and TNF- in hASM cells was dependent on NF-B activation. CXCL10 expression in hASM appeared to be transcriptionally regulated by TNF-, since it significantly increased steady-state levels of CXCL10 mRNA. IFN- increased accumulation of CXCL10 mRNA, but to a lesser degree than that obtained with TNF-. Together, TNF- and IFN- synergistically increased CXCL10 mRNA levels. The TNF--regulated increase in CXCL10 expression in hASM cells appears to be dependent on NF-B, since stimulation with TNF- resulted in NF-B activation that lasted for 24 h. A novel salicylanilide analog that blocks phosphorylation of IB (and thus binding of NF-B) blocked TNF--stimulated CXCL10 production (Fig. 2 A). In contrast, IFN- stimulation of hASM resulted in only a small increase of NF-B activation; the salicylanilide analog inhibitor had little effect on IFN--stimulated CXCL10 production (Fig. 2B ), indicating that NF-B is not the primary transcription factor activated by IFN-. More likely, IFN- activates receptors coupled to STAT-1 (signal transducer and transcriptional activator), which binds at the ISRE (interferon-stimulated response element) enhancer site within the CXCL10 promoter. When IFN- and TNF- were added simultaneously, NF-B was activated earlier (at t = 0.25 h) and was sustained for up to 24 h. Despite IFN- not directly activating the NF-B signaling pathway, the presence of both cytokines enhanced NF-B activation. Surprisingly, although the salicylanilide analog did not inhibit IFN--induced CXCL10 expression, the salicylanilide analog did inhibit CXCL10 production when cells were stimulated by IFN- and TNF- (Fig. 2C ). Taken together, these data suggest that while IFN- and TNF- may work individually through disparate signaling pathways to stimulate CXCL10 secretion, together these cytokines appear to enhance transcription of the CXCL10 gene in an NF-B-dependent manner.

View larger version (24K): [in this window] [in a new window]   Figure 2. A) Effect of the salicylanilide analog NF-B inhibitor on TNF- or IFN--stimulated CXCL10 production in primary cultured hASM cells stimulated for 24 h with TNF- (10 ng/ml) in the presence or absence of the salicylanilide analog (0.03–10 µM). B) Primary cultures of hASM were stimulated for 24 h with IFN- (10 ng/ml) in the presence of the salicylanilide analog (0.03–10 µM). C) Primary cultures of hASM were stimulated for 24 h with IFN- and TNF- (each at 10 ng/ml) in the presence of the salicylanilide analog (0.03–10 µM). Supernatants were tested for the presence of CXCL10 by ELISA. Data represents the mean ± SEM of triplicate determinations from 2–3 different human donors, ***P < 0.001.

CONCLUSIONS AND SIGNIFICANCE

In summary, the present study demonstrates that elevated levels of CXCL10 exist in the airways of patients with COPD. Although it is evident that a variety of cell types, especially inflammatory cells, secrete CXCL10, we now show that the airway smooth muscle can serve as a source of CXCL10 in inflamed airways. IFN- and TNF-, cytokines associated with the pathogenesis of COPD, alone and in combination, induce CXCL10 secretion. We demonstrated that NF-B plays a significant role in the regulation of CXCL10 expression by TNF-, a minor role in IFN--induced CXCL10 expression, but a pivotal role for the synergistic effect of TNF- and IFN- on CXCL10 expression in airway smooth muscle. Collectively, our data for the first time suggest that hASM can be a source of CXCL10, which may play an important role in the pathogenesis of COPD and could serve as a target for pharmacological intervention in the treatment of this condition.

View larger version (69K): [in this window] [in a new window]   Figure 3. Summary of CXCL10 expression in COPD. A) In COPD, damage to the airway initiates the release of a cascade of inflammatory mediators, including INF- and TNF-. B) These mediators are chemotactic for inflammatory cells such as lymphocytes, neutrophils, and macrophages. These cells are capable of producing additional IFN- and TNF- when activated, as well as CXCL10 (C). Increased concentrations of TNF- and IFN- in lung tissue result in enhanced induction of the CXCL10 gene (D) by the airway smooth muscle (E), leading to a synergistic effect on CXCL10 protein production in the airway (F).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0170fje

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