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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 2, 2003 as doi:10.1096/fj.02-0450fje. |
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Departments of Veterinary PathoBiology and
* Pharmacology, University of Minnesota, St. Paul, Minnesota, USA; and
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
2Correspondence: Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Ave., St. Paul, MN 55108, USA. E mail: kanna001{at}tc.umn.edu
SPECIFIC AIMS
Airway smooth muscle response to contractile agonists requires elevation of intracellular calcium; in airway inflammatory diseases, smooth muscle produces an exaggerated response to contractile agonists. In the present study, we determined whether inflammatory cytokine-induced human airway smooth muscle cell hyper-responsiveness results from increased CD38 expression and cADPR-induced calcium mobilization.
PRINCIPAL FINDINGS
1. Exposure to the inflammatory cytokines TNF-
, IL-1ß for IFN-
increases CD38 expression and ADP-ribosyl cyclase activity
HASM cells arrested at Go of cell cycle were treated with 20 ng/mL of IL-1ß or TNF-, 1 IU/mL of IFN-, or 0.05% bovine serum albumin for 22 h. CD38 mRNA measured by RT-PCR revealed increased expression in cells exposed to cytokines as compared with controls (Fig. 1
). Among the cytokines tested, TNF- had greater effect on CD38 expression than IL-1ß or IFN-. Real-time PCR confirmed quantitative differences in CD38 mRNA expression between control and cytokine-treated cells and between the cytokines. CD38 protein in microsomes obtained from control and cytokine-treated HASM cells was determined by Western blot analysis, using an anti-CD38 antibody. In cytokine-treated cells, there was increased CD38 protein expression vs. controls. TNF- treatment of cells resulted in greater CD38 protein expression than the other two cytokines tested in this study. In microsomes prepared from HASM cells treated with inflammatory cytokines, activity of ADP-ribosyl cyclase, which converts ß-NAD to cADPR, was measured by monitoring the conversion of the ß-NAD analog nicotinamide guanine dinucleotide (NGD) to cGDPR using a spectrofluorometer. The specific activities of ADP-ribosyl cyclase were 
3.7-, 1.7-, and 1.9-fold higher in cells treated respectively with TNF-, IL-1ß, and IFN- than in control.
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2. The increased CD38 expression in cytokine-treated cells is associated with greater magnitude of [Ca2+]i responses to acetylcholine, bradykinin and thrombin, and greater inhibition of these responses by an antagonist of cyclic ADP-ribose
The [Ca2+]i responses to acetylcholine, bradykinin, and thrombin were measured in Fura-2-loaded HASM cells. In cells exposed to the cytokines, there was a significant augmentation of [Ca2+]i responses to all agonists compared with controls (Fig. 2
). In control and cytokine-treated cells pretreated with 8-bromo-cADPR, a cell-permeant cADPR antagonist, [Ca2+]i responses to the agonists were attenuated. The magnitude of inhibition of [Ca2+]i responses to the agonists by 8-bromo-cADPR was greater in cytokine-treated cells than in controls. TNF- treatment of HASM cells resulted in greater [Ca2+]i responses to the three agonists than the other two cytokines. There was a correlation between level of CD38 expression and magnitude of [Ca2+]i responses to agonists attributable to cADPR.
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CONCLUSIONS AND SIGNIFICANCE
The objective of this study was to determine the role of CD38-cADPR signaling in airway hyper-responsiveness. We have demonstrated that inflammatory cytokines up-regulate CD38 expression and ADP-ribosyl cyclase activity, indicating potential for increased cADPR formation in HASM cells. Cytokine treatment resulted in an exaggerated [Ca2+]i response to Ach, BK, and TH; 8Br-cADPR, a cell-permeant cADPR antagonist, attenuated this response, demonstrating a critical role for CD38-cADPR mediated calcium mobilization in airway hyper-responsiveness.
TNF- and IL-1ß are known to regulate the expression of many classes of genes, contributing to alteration of airway structure and/or airway responsiveness to agonists. In this study, we have demonstrated up-regulation of CD38 expression by inflammatory cytokines. CD38 is a type II transmembrane glycoprotein that is expressed in a variety of cell types. CD38 expression is highly regulated at multiple levels and is modulated by a variety of physiological and pathological stimuli. CD38 promoter region possesses binding sites for several transcription factors, including interferon-responsive factor 1 (IRF-1) and nuclear factor interleukin 6. Glucocorticoids, estradiol, retinoids, vitamin D3, cyclic-AMP, and cytokines such as IFN-, IL-4, and IL-7 have been shown to modulate CD38 expression. Our findings on cytokine effects on CD38 expression suggest possible control at the transcription level.
The hyper-responsiveness observed during airway inflammation is nonspecific in nature. We observed exaggerated [Ca2+]i responses to three different agonists in cytokine-treated ASM cells. The cADPR antagonist attenuated responses to all three agonists in both control and cytokine-treated cells; in cytokine-treated cells, the attenuation was much greater than in controls. These findings indicate that CD38-cADPR signaling is used by multiple agonists for calcium mobilization and that its up-regulation contributes to airway hyper-responsiveness.
Human ASM cells demonstrate hyper-reactivity to agonists that mediate their effects through G-protein-coupled receptors. How stimulation of agonist receptors leads to activation of CD38 and cADPR-mediated calcium mobilization is not clearly understood in smooth muscle cells. In other cell types, it has been demonstrated that activation of ADP-ribosyl cyclase involves G-proteins. In ASM, the effects of Ach are mediated through muscarinic receptors coupled to Gi- and Gq-type G-proteins, whereas BK and TH responses are predominantly through Gq-type G-proteins. The fact that the cADPR antagonist inhibits calcium responses to all three agonists indicates that receptors coupled to both types of G-proteins activate CD38-cADPR signaling in human ASM cells. Furthermore, it has been reported that inflammatory cytokines up-regulate the expression of G-proteins in human ASM cells. This along with the increased CD38 expression may lead to increased cADPR production and airway hyper-responsiveness.
These results provide the first evidence for a role of CD38-cyclic ADP-ribose signaling in airway smooth muscle cell hyper-responsiveness in a model of inflammation (Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0450fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0450fje 
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0.05) attenuation of calcium responses in the presence of 8Br-cADPR in control and cytokine-treated HASM cells. Data are from three different cell preparations.
B, etc.). The increased second messengers (cADPR, IP3) would result in augmented calcium release through intracellular calcium channels. Dotted arrows represent previously described findings.