Abstract
Objective
Fibroblasts are sentinel cells that could serve as intermediaries in the immune reaction in the inflammatory process. In this work, we investigate the action of the muscarinic agonist carbachol (CARB) on the expression and function of nitric oxide synthase (NOS) and cyclooxygenase (COX) in fibroblasts under normal or inflammatory conditions.
Methods
The normal fibroblast cell line, 3T3, from NIH swiss mouse embryo, was used. The inflammatory milieu was mimicked with lipopolysaccharide (LPS) (10 ng/ml) plus interferon gamma (IFNγ) (0.5 ng/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) production were measured by Griess reagent and radioimmunoassay, respectively. NOS, COX, and nuclear transcription factor kappa B (NF-κB) were studied by Western blot.
Results
CARB increased NO synthesis by 57 ± 5%, while a 150 ± 10% increase in NO liberation was triggered by LPS plus IFNγ treatment. CARB added to LPS plus IFNγ potentiated NO synthesis by 227 ± 19%. CARB also upregulated NOS1 protein expression via NF-κB activation. In addition CARB and LPS plus IFNγ stimulated PGE2 synthesis by 72 ± 9 and 42 ± 4%, respectively, while CARB added to LPS plus IFNγ treated cells produced a synergism in PGE2 liberation (130 ± 12%) via COX-2.
Conclusion
Activation of muscarinic acetylcholine receptors can mimic mild inflammatory conditions or can deepen pre-existing inflammation, establishing a fine-tuned set-up on fibroblasts that in turn could be alerting the immune system.
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References
Stuehr J, Marletta MA. Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma. J Immunol. 1987;139:518–25.
Geller DA, Nussler AK, Di Silvio M, Lowenstein CJ, Shapiro RA, Wang SC, et al. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci USA. 1993;90:522–6.
O’Brien WJ, Heimann T, Tsao LS, Seet BT, McFadden G, Taylor JL. Regulation of nitric oxide synthase 2 in rabbit corneal cells. Invest Ophthal Vis Sci. 2001;42:713-9.
Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature. 2000;406:282–7.
Frey EA, Miller DS, Jahr TG, Sundan A, Bazil V, Espevik T, et al. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med. 1992;176:1665–71.
Jones E, Adcock I, Ahmed B, Punchard N. Modulation of LPS stimulated NF-kappa B mediated nitric oxide production by PKCε and JAK2 in RAW macrophages. J Inflamm. 2007;4:1–9.
Davidson JM. Inflammation: wound repair. New York: Garland Press; 1992.
Butcher EC, Williams M, Youngman K, Rott L, Briskin M. Lymphocyte trafficking and regional immunity. Adv Immunol. 1999;72:209–53.
Buckley CD, Pilling D, Lord JM, Akbar AN, Scheel-Toellner D, Salmon M. Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation. Trends Immunol. 2001;22:199–204.
Buchli R, Ndoye A, Rodriguez JG, Zia S, Webber RJ, Grando SA. Human skin fibroblasts express m2, m4, and m5 subtypes of muscarinic acetylcholine receptors. J Cell Biochem. 1999;74:264–77.
Español A, de la Torre E, Sales ME. Parasympathetic modulation of local acute inflammation in murine submandibular glands. Inflammation. 2003;27:97–106.
Chen TR. In situ detection of micoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res. 1997;104:255–62.
Granger D, Hidds J, Perfect J, Durack D. Metabolic fate of l-arginine in relation to microbiostatic capability of murine macrophages. Clin Invest. 1990;85:264–73.
Fiszman G, Cattaneo V, de la Torre E, Español A, Colombo L, Sacerdote de Lustig E, et al. Muscarinic receptors autoantibodies purified from mammary adenocarcinoma bearing mice sera stimulate tumor progression. Int Immunopharma. 2006;6:1323–30.
Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem. 1976;72:248–54.
Español AJ, de la Torre E, Fisman GL, Sales ME. Role of non-neuronal cholinergic system in breast cancer progression. Life Sci. 2007;80:2281–5.
Español AJ, Sales ME. Different muscarinic receptors are involved in the proliferation of murine mammary adenocarcinoma cell lines. Int J Mol Med. 2004;13:311–9.
Granstrom E, Kindhal H. Radioimmunoassay of prostaglandins and thomboxanes research. New York, Raven Press. 1978;5:119–210.
Goren N, Cuenca J, Martín-Sanz P, Boscá L. Attenuation of NF-kB signaling in rat cardiomyocytes at birth restricts the induction of inflammatory genes. Cardiovascular Res. 2004;64:289–97.
Bulseco DA, Poluha W, Schonhoff CM, Daou MC, Condon PJ, Ross AH. Cell-cycle arrest in TrkA-expressing NIH3T3 cells involves nitric oxide synthase. J Cell Biochem. 2001;8:193–204.
Noga O, Hanf G, Schäper C, O’Connor A, Kunkel G. The influence of inhalative corticosteroids on circulating nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in allergic asthmatics. Clin Exp Allergy. 2001;31:1906–12.
Eglen RM. Muscarinic receptor subtypes in neuronal and non-neuronal cholinergic function. Auton Autacoid Pharmacol. 2006;26:219–33.
Wessler I, Kilbinger H, Bittinger F, Unger R, Kirkpatrick CJ. The non-neuronal cholinergic system in humans: expression, function and pathophysiology. Life Sci. 2003;72:2055–61.
de la Torre E, Genaro AM, Ribeiro ML, Pagotto R, Pignataro OP, Sales ME. Proliferative actions of muscarinic receptors expressed in macrophages derived from normal and tumor bearing mice. Biochim Biophys Acta. 2008;782:82–9.
Sterin-Borda L, Ganzinelli S, Berra A, Borda E. Novel insight into the mechanisms evolved in the regulation of the m1 muscarinic receptor, iNOS and nNOS mRNA levels. Neuropharmacology. 2003;45:260–9.
Molina-Holgado E, Khorchid A, Liu H, Almazan G. Regulation of muscarinic receptor function in developing oligodendrocytes by agonist exposure. Br J Pharmacol. 2003;13:47–56.
Carbone DL, Moreno JA, Tjalkens RB. Nuclear factor kappa-B mediates selective induction of neuronal nitric oxide synthase in astrocytes during low-level inflammatory stimulation with MPTP. Brain Res. 2008;1217:1–9.
Guizzetti M, Bordi F, Dieguez-Acuña FJ, Vitalone A, Madia F, Woods JS, et al. Nuclear factor kappaB activation by muscarinic receptors in astroglial cells: effect of ethanol. Neuroscience. 2003;120:941–50.
Pausawasdi N, Ramamoorthy S, Crofford LJ, Askari FK, Todisco A. Regulation and function of COX-2 gene expression in isolated gastric parietal cells. Am J Physiol Gastrointest Liver Physiol. 2002;282:G1069–78.
Fraser CC. G protein-coupled receptor connectivity to NF-kappaB in inflammation and cancer. Int Rev Immunol. 2008;27:320–50.
Dumont I, Peri KG, Hardy P, Hou X, Martinez-Bermudez AK, Molotchnikoff S, et al. PGE2, via EP3 receptors, regulates brain nitric oxide synthase in the perinatal period. Am J Physiol. 1998;275:R1812–21.
Razani-Boroujerdi S, Behl M, Hahn F, Pena-Philippides JC, Hutt J, Sopori ML. Role of muscarinic receptors in the regulation of immune inflammatory response. J Neuroimmunol. 2008;194:82–8.
Salvemini D. Regulation of cyclooxygenase enzymes by nitric oxide. Cell Mol Life Sci. 1997;53:576–82.
Davel L, D′Agostino A, Español A, Jasnis MA, Lauría de Cidre L, de Lustig ES et al. Nitric oxide synthase-cyclooxygenase interactions are involved in tumor cell angiogenesis and migration. J Biol Reg Homeost Agent. 2002;16:181–9.
Dolan S, Kelly JG, Huan M, Nolan AM. Transient up-regulation of spinal cyclooxygenase-2 and neuronal nitric oxide synthase following surgical inflammation. Anesthesiology. 2003;98:170–80.
Bonizzi G, Karin M. The two NF-kB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 2004;25:280–9.
Tran K, Merika M, Thanos D. Distinct functional properties of IkBa and IkBb. Mol Cell Biol. 1997;17:5386–99.
Beg AA, Finco TS, Nantermet PV, Baldwin AS Jr. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of IkBa: a mechanism for NF-kB activation. Mol Cell Biol. 1993;13:3301–10.
Gough DJ, Levy DE, Johnstone RW, Clarke CJ. IFNγ signaling—does it mean JAK–STAT? Cytokine Growth Factor Rev 2008;19:383–94.
Boivin MA, Roy PK, Bradley A, Kennedy JC, Rihani T, Ma TY. Mechanism of interferon-gamma-induced increase in T84 intestinal epithelial junction. J Interferon Cytokine Res. 2009;29:45–54.
Schliebs R, Heidel K, Apelt J, Gniezdzinska M, Kirazov L, Szutowicz A. Interaction of interleukin-1β with muscarinic acetylcholine receptor-mediated signaling cascade in cholinergically differentiated SH-SY5Y cells. Brain Res. 2006;1122:78–85.
Acknowledgments
The authors want to thank Mrs. María E. Castro and Ana I. Casella for their excellent technical assistance. Alejandro Español, Nora Goren, María L. Ribeiro, and María E. Sales are established investigators from the National Research Council (CONICET). Authors want especially to thank to the University of Buenos Aires for the grant UBACYT MO04, M064 and to the National Agency for the Promotion of Science and Technology (ANPCyT) for the grant PICT 2006-485 that supported this research.
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Español, A.J., Goren, N., Ribeiro, M.L. et al. Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells. Inflamm. Res. 59, 227–238 (2010). https://doi.org/10.1007/s00011-009-0097-4
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DOI: https://doi.org/10.1007/s00011-009-0097-4