Table of Contents Author Guidelines Submit a Manuscript
BioMed Research International
Volume 2014, Article ID 609086, 7 pages
http://dx.doi.org/10.1155/2014/609086
Research Article

Anti-Inflammatory Effects of the Nicotinergic Peptides SLURP-1 and SLURP-2 on Human Intestinal Epithelial Cells and Immunocytes

1Department of Dermatology, University of California, 134 Sprague Hall, Irvine, CA 92697, USA
2National Cancer Institute, Kiev 03022, Ukraine
3Department of Biological Chemistry, University of California, 134 Sprague Hall, Irvine, CA 92697, USA
4Institute for Immunology, University of California, 134 Sprague Hall, Irvine, CA 92697, USA

Received 18 March 2014; Accepted 17 April 2014; Published 4 May 2014

Academic Editor: Maryna Skok

Copyright © 2014 Alex I. Chernyavsky et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A search for novel and more efficient therapeutic modalities of inflammatory bowel disease (IBD) is one of the most important tasks of contemporary medicine. The anti-inflammatory action of nicotine in IBD might be therapeutic, but its toxicity due to off-target and nonreceptor effects limited its use and prompted a search for nontoxic nicotinergic drugs. We tested the hypothesis that SLURP-1 and -2—the physiological nicotinergic substances produced by the human intestinal epithelial cells (IEC) and immunocytes—can mimic the anti-inflammatory effects of nicotine. We used human CCL-241 enterocytes, CCL-248 colonocytes, CCRF-CEM T-cells, and U937 macrophages. SLURP-1 diminished the TLR9-dependent secretion of IL-8 by CCL-241, and IFNγ-induced upregulation of ICAM-1 in both IEC types. rSLURP-2 inhibited IL-1β-induced secretion of IL-6 and TLR4- and TLR9-dependent induction of CXCL10 and IL-8, respectively, in CCL-241. rSLURP-1 decreased production of TNFα by T-cells, downregulated IL-1β and IL-6 secretion by macrophages, and moderately upregulated IL-10 production by both types of immunocytes. SLURP-2 downregulated TNFα and IFNγR in T-cells and reduced IL-6 production by macrophages. Combining both SLURPs amplified their anti-inflammatory effects. Learning the pharmacology of SLURP-1 and -2 actions on enterocytes, colonocytes, T cells, and macrophages may help develop novel effective treatments of IBD.

1. Introduction

A search for novel and more efficient therapeutic modalities of inflammatory bowel disease (IBD) is one of the most important tasks of contemporary clinical and experimental medicine. Both ulcerative colitis (UC) and Crohn’s disease (CD) are epidemiologically related to smoking [14]. Most patients with UC are nonsmokers, and patients with a history of smoking usually acquire their disease after they have stopped smoking [57]. Upon cessation of smoking, patients with UC experience more severe disease progression that can be ameliorated by returning to smoking [810]. In contrast, patients with CD experience severe disease when smoking, requiring an immediate and complete cessation of any tobacco usage [3, 11]. Nicotine administration in transdermal patches or enema inhibits inflammation associated with UC [8, 1216]. Nicotine also exhibits a local therapeutic effect in CD [17], despite the fact that smoking worsens this disease. It is believed that the therapeutic effects of nicotine in IBD are mediated by the nicotinic acetylcholine (ACh) receptors (nAChRs) of gut immune cells that inhibit production of inflammatory mediators and correct specific alterations in cell cycle responses [1820]. We have previously demonstrated that nicotinic agonists abrogate PHA-dependent upregulation of TNFα and IFNγ receptors (IFNγR) in the human leukemic T-cell line CCRF-CEM (CEM) [21] and downregulate lipopolysaccharide- (LPS-) induced production of the proinflammatory cytokines IL-6 and IL-18 but upregulated IL-10 in human macrophage-like U937 cells [22]. On the other hand, recent research has conclusively demonstrated that dysregulation of intestinal epithelial cells (IEC) plays an important role in the pathogenesis of IBD [23], but the therapeutic modalities that can effectively correct function of these cells remain unknown. An important role of IEC response to nicotinic drugs in IBD has been suggested by the presence of fully developed, functional ACh axis in the intestinal epithelium, with its nicotinic arm controlling intestinal absorption, permeability, mucociliary activity, and mucin secretion, as well as IEC viability, proliferation, migration, and cohesion [2438]. Therefore, modulation of the nicotinergic anti-inflammatory pathway is considered as a novel therapeutic target for IBD [12, 3941]. Clinical trials of nicotine formulations, however, revealed severe side effects from therapeutic doses of nicotine [12, 42], which prompted a search for nontoxic nicotinergic agents that can mimic anti-inflammatory effects of nicotine in patients with IBD.

A novel paradigm of cell regulation via nAChRs has been discovered in studies of the autosomal recessive disease palmoplantar keratoderma featuring mutation of secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein- (SLURP-) 1 and impaired T-cell activity [43]. SLURP-2 expression was also discovered in the skin [44]. While various subtypes of nAChRs can be involved in the physiological regulation of cell functions by SLURPs, the biological effects of SLURP-1 are predominantly mediated by α7 nAChR and those of SLURP-2 by non-α7 nAChRs [45]. Cell function and gene expression studies [46, 47] suggested that SLURPs may play important roles in regulating both epithelial cells and immunocytes. Since nicotine has been shown to alter expression of SLURP-1 in IEC [48], we hypothesized that auto/paracrine action of SLURPs on IEC may, in part, mediate the anti-inflammatory activities of nicotine in IBD.

In this study, we analyzed the roles of SLURP-1 and -2 in the physiological regulation of the key elements of the pathobiology of IBD controlling intestinal inflammation and facilitating healing of intestinal ulcers. The results demonstrated that SLURPs can abolish expression of the IBD-related mediators of inflammation in both IEC and immunocytes. Learning the pharmacology of the SLURP-1 and -2 actions on enterocytes, colonocytes, T-cells, and macrophages may therefore help develop novel effective treatments of UC and CD.

2. Materials and Methods

2.1. Cells and Reagents

Human IEC: the small intestine enterocyte cell line CCL-241 and the colonocyte cell line CCL-248, human lymphoblastoid T-cell line CEM, and human monoblastoid tumor cell line U937 were purchased from ATCC (Manassas, VA) and grown in the respective ATCC complete growth media at 37°C in a humid, 5% CO2 incubator. To differentiate into macrophages, the U937 cells were treated with 200 nM PMA (Sigma-Aldrich Corporation, St. Louis, MO) and allowed to adhere to tissue culture plate for 3 days [49]. The full length recombinant (r)SLURP-1 and rSLURP-2 were manufactured at Virusys Corporation (Sykesville, MD), as detailed elsewhere [50]. The previously characterized anti-SLURP-1 and -2 monoclonal antibodies 336H12-1A3 and 341F10-1F12, respectively [46, 47], were from Research and Diagnostic Antibodies (North Las Vegas, NV). Normal mouse IgG (NIgG) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary mouse antibodies to human ICAM, IL-1β, IL-6, IL-10, TNFα, and IFNγ receptor (IFNγR) and ELISA kits for measuring human IL-6 and CXCL10 were purchased from R&D Systems (Minneapolis, MN). The IL-8 ELISA kit was from BD Biosciences (San Jose, CA). Both recombinant IL-1β and INFγ were from R&D Systems and both E. coli DNA and LPS from E. coli K12 strain (LPS-EK) were purchased from InvivoGen (San Diego, CA).

2.2. Quantitative Immunocytochemical Assay (QIA)

The QIA (a.k.a. in-cell western), a high throughput quantitative assay of cellular proteins, was performed in situ, as described in detail elsewhere [46], using the reagents and equipment from LI-COR Biotechnology (Lincoln, NE). The CCL-241, CCL-248, CEM, or U937 cells, 1 × 106/well of a 96-well plate, were incubated in respective growth media with or without rSLURPs for 16 h, fixed in situ, washed, permeabilized with Triton solution, incubated with the LI-COR Odyssey Blocking Buffer for 1.5 h, and then treated overnight at 4°C with a primary antibody. The cells were then washed and stained for 1 h at room temperature with a secondary antibody, and expression of the protein of interest was quantitated using the LI-COR Odyssey Imaging System. Sapphire700 (1 : 1000) was used to normalize for cell number/well.

2.3. Statistical Analysis

Results were expressed as mean ± SD, and statistical significance was determined by ANOVA with Dunnett’s posttest using the GraphPad Prism software (GraphPad Prism Software Inc., San Diego, CA). The differences were deemed significant when the calculated value was <0.05.

3. Results

3.1. Anti-Inflammatory Effects of rSLURP-1 and -2 on IEC

In in vitro experiments utilizing cultured human enterocytes and colonocytes, CCL-241 and CCL-248, respectively, we recreated an aspect of IBD pathophysiology involving the proinflammatory action of IL-1β, IFNγ, and Toll-like receptor 4- (TLR4-) and TLR9-ligands (i.e., LPS-EK and E. coli DNA, resp.) on intestinal epithelium [5153]. TLR4 and TLR9 regulate cytokine secretion, cell survival, and intestinal barrier function, and their expression on IEC is upregulated in IBD [5257]. We hypothesized that, in response to these mediators, CCL-241 and CCL-248 cells would express proinflammatory molecules eliciting mucosal homing of T-cells and recruiting other types of inflammatory cells. Exposed IEC indeed showed upregulated expression of IL-6, IL-8, CXCL10, and ICAM-1 (Figure 1).

609086.fig.001
Figure 1: Anti-inflammatory effects of rSLURP-1 and -2 on IEC. The anti-inflammatory effects of 0.01 μg/mL of rSLURP-1 (S1) and -2 (S2) on secretion of IL-6, IL-8, and CXCL10 (ELISA) and expression of ICAM-1 (QIA) by CCL-241 and CCL-248 stimulated for 16 h in a humid, 5% CO2 incubator at a cell density of 1 × 106 cells/well with 100 U/mL of IL-1β (IL-6 assay), 25 μg/mL of the TLR9 ligand E. coli DNA (IL-8), 100 ng/mL of the TLR4 ligand LPS-EK (CXCL10), or 100 U/mL of INFγ (ICAM-1) were measured as described in Materials and Methods. Some cells were exposed to S1 or S2 in the presence of 1 μg/mL of anti-SLURP-1 or -2 monoclonal antibodies (Ab). Each experiment was performed in triplicate. Asterisk = , compared to untreated cells. Pound sign = , compared to an inflammatory stimulant given alone.

Next, we sought to determine if rSLURP-1 or -2 can inhibit production of these proinflammatory molecules. rSLURP-1 significantly () diminished the TLR9-dependent secretion of IL-8 by CCL-241, but not CCL-248, and the IFNγ-induced upregulation of ICAM-1 in both types of IEC (Figure 1). rSLURP-2 inhibited the IL-1β-induced secretion of IL-6 and TLR4- and TLR9-dependent induction of CXCL10 and IL-8, respectively, in CCL-241. The specificity of these effects was demonstrated by ability of anti-SLURP antibodies to abolish the inhibitory activity of corresponding rSLURP. A mixture of both nicotinergic peptides almost completely inhibited upregulated expression of all tested inflammatory molecules in both types of IEC (Figure 1), which is in keeping with the synergistic mechanisms of their biological action [58, 59].

3.2. Anti-Inflammatory Effects of rSLURP-1 and -2 on Immunocytes

rSLURP-1 significantly () decreased production of TNFα by CEM, downregulated IL-1β and IL-6 secretion by U937 cells, and moderately upregulated IL-10 production by both types of immunocytes (Figure 2). rSLURP-2 significantly () downregulated TNFα and IFNγR in CEM and reduced IL-6 production by U937 cells (Figure 2). Combining both rSLURPs amplified their anti-inflammatory effects.

fig2
Figure 2: Anti-inflammatory effects of rSLURP-1 and -2 on immunocytes. The anti-inflammatory effects of rSLURP-1 (S1) and -2 (S2), 0.01 μg/mL, on production of proinflammatory cytokines and IL-10 by the CEM stimulated with 10 μM PHA (a) and by the differentiated U937 macrophages stimulated with 200 ng/mL LPS (b) incubated for 16 h in a humid, 5% CO2 incubator at a cell density of 1 × 106 cells/well were measured by QIA, as detailed in Materials and Methods. Each experiment was performed in triplicate. Asterisk = , compared to intact cells. Pound sign = , compared to PHA or LPS given alone.

4. Discussion

Results of the present study demonstrated for the first time that SLURP proteins can produce anti-inflammatory effects by abolishing expression of IBD-related mediators of inflammation in both IEC and immunocytes. These findings suggest that SLURPs may become prototype drugs for the treatment of IBD, because they mimic the inhibitory effect of nicotine and some noncanonical nAChR ligands on gut inflammation. Clinical use of rSLURPs should avoid nicotine-like toxicity, such as off-target and nonreceptor intracellular effects, because SLURPs are the physiological substances produced at low levels by IEC [25] and immunocytes [60] that alter cell functions by acting at nAChRs [46, 47]. Notably, quercetin—a flavonoid that exhibits its nicotinergic activity through α3, α7, and α9 nAChRs [6164]—produces an anti-inflammatory effect and ameliorates experimental IBD [65, 66].

Both α7 and non-α7 subtypes of nAChRs might mediate anti-inflammatory effects of rSLURP-1 and -2 in IEC, CEM, and U937 cells. It has been reported that activation of nAChRs inhibits secretion of IL-1β and IL-8 in IEC [67, 68]. SLURP inhibition of the production of proinflammatory cytokines in the IEC activated by TLR ligands may have important clinical implication, because compounds inhibiting the immune stimulation involving TLR ligands, especially TLR4, have been reported to be potentially useful for treatment of IBD [31]. Both nicotine and SLURP-1 bind with a high affinity to α7 nAChR [46, 69] and both upregulate local production of IL-10 (Figure 2 and [70]), which is otherwise decreased in patients with IBD [71]. T-cells also express α4 and β2 subunits [20] that could be activated by rSLURP-2. Activation of α4β2 inhibits immune reactivity [72, 73].

The differences between effects of each rSLURP protein may be due to their predominant action at distinct nAChR subtypes expressed on the cell membrane of different kinds of immunocytes [21, 22] and IEC. By RT-PCR, CCL-241 cells uniquely express α3, whereas CCL-248, α2 and α5, and both cells also express α7 and α9 nAChRs (data not shown), which is different from the colonic cell line HT29 that carries α4-made nAChR [38]. The variations of the nAChR profiles among distinct IEC types help explain regional variations of intestinal responses to smoking/nicotine [4, 70, 7476].

Previous studies indicated that SLURP-1 can potentiate the ACh action at α7 nAChR leading to modifications in functions of cutaneous epithelial cells [77] and immunocytes [78]. Since both IEC and immune cells express this nAChR subtype, the anti-inflammatory effects of SLURP-1 in the gut may result from its action on both cells types simultaneously. Additionally, since SLURP-1 has been shown to upregulate production of ACh by immunocytes [78], this endogenously produced and secreted agonist may further potentiate the α7-mediated anti-inflammatory effect of SLURP-1.

5. Conclusions

Both rSLURP-1 and -2 inhibit production of inflammatory mediators in human enterocytes, colonocytes, T-cells, and macrophages. Combining both rSLURP proteins amplifies the anti-inflammatory effects. The anti-inflammatory effects of nontoxic nAChR ligands such as SLURPs may therefore ameliorate disease in CD and UC patients. Identification of the predominant types of nAChRs mediating anti-inflammatory effects of each SLURP protein on IEC and immunocytes should help elucidate the intracellular signaling pathways.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment

This work was supported, in part, by internal funds from University of California-Irvine School of Medicine.

References

  1. R. J. Motley, J. Rhodes, S. Kay, and T. J. Morris, “Late presentation of ulcerative colitis in ex-smokers,” International Journal of Colorectal Disease, vol. 3, no. 3, pp. 171–175, 1988. View at Google Scholar · View at Scopus
  2. I. Koutroubakis, O. N. Manousos, S. G. M. Meuwissen, and A. S. Pena, “Environmental risk factors in inflammatory bowel disease,” Hepato-Gastroenterology, vol. 43, no. 8, pp. 381–393, 1996. View at Google Scholar · View at Scopus
  3. D. T. Rubin and S. B. Hanauer, “Smoking and inflammatory bowel disease,” European Journal of Gastroenterology and Hepatology, vol. 12, no. 8, pp. 855–862, 2000. View at Google Scholar · View at Scopus
  4. R. Eliakim, F. Karmeli, P. Cohen, S. N. Heyman, and D. Rachmilewitz, “Dual effect of chronic nicotine administration: augmentation of jejunitis and amelioration of colitis induced by iodoacetamide in rats,” International Journal of Colorectal Disease, vol. 16, no. 1, pp. 14–21, 2001. View at Publisher · View at Google Scholar · View at Scopus
  5. A. D. Harries, A. Baird, and J. Rhodes, “Non-smoking: a feature of ulcerative colitis,” British Medical Journal, vol. 284, no. 6317, p. 706, 1982. View at Google Scholar · View at Scopus
  6. R. F. Logan, M. Edmond, K. Somerville, and M. J. S. Lanman, “Smoking and ulcerative colitis,” British Medical Journal, vol. 288, no. 6419, pp. 751–753, 1984. View at Google Scholar · View at Scopus
  7. R. J. Motley, J. Rhodes, and G. A. Ford, “Time relationships between cessation of smoking and onset of ulcerative colitis,” Digestion, vol. 37, no. 2, pp. 125–127, 1987. View at Google Scholar · View at Scopus
  8. H. de Castella, “Non-smoking: a feature of ulcerative colitis,” British Medical Journal, vol. 284, no. 6330, p. 1706, 1982. View at Google Scholar · View at Scopus
  9. J. Birtwistle and K. Hall, “Does nicotine have beneficial effects in the treatment of certain diseases?” British Journal of Nursing, vol. 5, no. 19, pp. 1195–1202, 1996. View at Google Scholar · View at Scopus
  10. J. M. Wolf and B. A. Lashner, “Inflammatory bowel disease: sorting out the treatment options,” Cleveland Clinic Journal of Medicine, vol. 69, no. 8, pp. 621–631, 2002. View at Google Scholar · View at Scopus
  11. R. J. Hilsden, D. C. Hodgins, A. Timmer, and L. R. Sutherland, “Helping patients with Crohn's disease quit smoking,” American Journal of Gastroenterology, vol. 95, no. 2, pp. 352–358, 2000. View at Publisher · View at Google Scholar · View at Scopus
  12. G. A. Thomas, J. Rhodes, and J. R. Ingram, “Mechanisms of disease: nicotine—a review of its actions in the context of gastrointestinal disease,” Nature Clinical Practice Gastroenterology & Hepatology, vol. 2, no. 11, pp. 536–544, 2005. View at Google Scholar
  13. B. Coulie, M. Camilleri, A. E. Bharucha, W. J. Sandborn, and D. Burton, “Colonic motility in chronic ulcerative proctosigmoiditis and the effects of nicotine on colonic motility in patients and healthy subjects,” Alimentary Pharmacology and Therapeutics, vol. 15, no. 5, pp. 653–663, 2001. View at Publisher · View at Google Scholar · View at Scopus
  14. J. McGrath, J. W. McDonald, and J. K. Macdonald, “Transdermal nicotine for induction of remission in ulcerative colitis,” Cochrane Database of Systematic Reviews, no. 4, Article ID CD004722, 2004. View at Google Scholar · View at Scopus
  15. R. D. Pullan, J. Rhodes, S. Ganesh et al., “Transdermal nicotine for active ulcerative colitis,” New England Journal of Medicine, vol. 330, no. 12, pp. 811–815, 1994. View at Publisher · View at Google Scholar · View at Scopus
  16. J. T. Green, G. A. O. Thomas, J. Rhodes et al., “Pharmacokinetics of nicotinic carbomer enemas: a new treatment modality for ulcerative colitis,” Clinical Pharmacology and Therapeutics, vol. 61, no. 3, pp. 340–348, 1997. View at Publisher · View at Google Scholar · View at Scopus
  17. J. R. Ingram, J. Rhodes, B. K. Evans, and G. A. Thomas, “Nicotine enemas for active Crohn's colitis: an open pilot study,” Gastroenterology Research and Practice, vol. 2008, Article ID 237185, 6 pages, 2008. View at Publisher · View at Google Scholar
  18. A. Bai, Y. Guo, and N. Lu, “The effect of the cholinergic anti-inflammatory pathway on experimental colitis,” Scandinavian Journal of Immunology, vol. 66, no. 5, pp. 538–545, 2007. View at Publisher · View at Google Scholar · View at Scopus
  19. M. C. Aldhous, R. J. Prescott, S. Roberts, K. Samuel, M. Waterfall, and J. Satsangi, “Does nicotine influence cytokine profile and subsequent cell cycling/apoptotic responses in inflammatory bowel disease?” Inflammatory Bowel Diseases, vol. 14, no. 11, pp. 1469–1482, 2008. View at Publisher · View at Google Scholar · View at Scopus
  20. J. Qian, V. Galitovskiy, A. I. Chernyavsky, S. Marchenko, and S. A. Grando, “Plasticity of the murine spleen T-cell cholinergic receptors and their role in in vitro differentiation of nave CD4 T cells toward the Th1, Th2 and Th17 lineages,” Genes and Immunity, vol. 12, no. 3, pp. 222–230, 2011. View at Publisher · View at Google Scholar · View at Scopus
  21. A. I. Chernyavsky, J. Arredondo, V. Galitovskiy, J. Qian, and S. A. Grando, “Structure and function of the nicotinic arm of acetylcholine regulatory axis in human leukemic T cells,” International Journal of Immunopathology and Pharmacology, vol. 22, no. 2, pp. 461–472, 2009. View at Google Scholar · View at Scopus
  22. A. I. Chernyavsky, J. Arredondo, M. Skok, and S. A. Grando, “Auto/paracrine control of inflammatory cytokines by acetylcholine in macrophage-like U937 cells through nicotinic receptors,” International Immunopharmacology, vol. 10, no. 3, pp. 308–315, 2010. View at Publisher · View at Google Scholar · View at Scopus
  23. P. Henderson, J. E. Van Limbergen, J. Schwarze, and D. C. Wilson, “Function of the intestinal epithelium and its dysregulation in inflammatory bowel disease,” Inflammatory Bowel Diseases, vol. 17, no. 1, pp. 382–395, 2011. View at Publisher · View at Google Scholar · View at Scopus
  24. T. W. Zimmerman and H. J. Binder, “Effect of tetrodotoxin on cholinergic agonist-mediated colonic electrolyte transport,” The American Journal of Physiology, vol. 244, no. 4, pp. G386–G391, 1983. View at Google Scholar · View at Scopus
  25. A. Pettersson, S. Nordlander, G. Nylund, A. Khorram-Manesh, S. Nordgren, and D. S. Delbro, “Expression of the endogenous, nicotinic acetylcholine receptor ligand, SLURP-1, in human colon cancer,” Autonomic and Autacoid Pharmacology, vol. 28, no. 4, pp. 109–116, 2008. View at Publisher · View at Google Scholar · View at Scopus
  26. C. L. Green, W. Ho, K. A. Sharkey, and D. M. McKay, “Dextran sodium sulfate-induced colitis reveals nicotinic modulation of ion transport via iNOS-derived NO,” American Journal of Physiology-Gastrointestinal and Liver Physiology, vol. 287, no. 3, pp. G706–G714, 2004. View at Publisher · View at Google Scholar · View at Scopus
  27. B. Sayer, J. Lu, C. Green, J. D. Söderholm, M. Akhtar, and D. M. McKay, “Dextran sodium sulphate-induced colitis perturbs muscarinic cholinergic control of colonic epithelial ion transport,” British Journal of Pharmacology, vol. 135, no. 7, pp. 1794–1800, 2002. View at Publisher · View at Google Scholar · View at Scopus
  28. M. Jönsson, Ö. Norrgård, and S. Forsgren, “Presence of a marked nonneuronal cholinergic system in human colon: study of normal colon and colon in ulcerative colitis,” Inflammatory Bowel Diseases, vol. 13, no. 11, pp. 1347–1356, 2007. View at Publisher · View at Google Scholar · View at Scopus
  29. P. L. Wei, L. J. Kuo, M. T. Huang et al., “Nicotine enhances colon cancer cell migration by induction of fibronectin,” Annals of Surgical Oncology, vol. 18, no. 6, pp. 1782–1790, 2011. View at Publisher · View at Google Scholar · View at Scopus
  30. O. Lundgren, M. Jodal, M. Jansson, A. T. Ryberg, and L. Svensson, “Intestinal epithelial stem/progenitor cells are controlled by mucosal afferent nerves,” PLoS ONE, vol. 6, no. 2, Article ID e16295, 2011. View at Publisher · View at Google Scholar · View at Scopus
  31. J. Wei and J. Feng, “Signaling pathways associated with inflammatory bowel disease,” Recent Patents on Inflammation and Allergy Drug Discovery, vol. 4, no. 2, pp. 105–117, 2010. View at Publisher · View at Google Scholar · View at Scopus
  32. Y. Sun, B. Fihn, M. Jodal, and H. Sjövall, “Effects of nicotinic receptor blockade on the colonic mucosal response to luminal bile acids in anaesthetized rats,” Acta Physiologica Scandinavica, vol. 178, no. 3, pp. 251–260, 2003. View at Publisher · View at Google Scholar · View at Scopus
  33. G. M. Roomans, V. Vanthanouvong, A. Dragomir, I. Kozlova, and R. Wróblewski, “Effects of nicotine on intestinal and respiratory epithelium,” Journal of Submicroscopic Cytology and Pathology, vol. 34, no. 4, pp. 381–388, 2002. View at Google Scholar · View at Scopus
  34. I. Kozlova, A. Dragomir, V. Vanthanouvong, and G. M. Roomans, “Effects of nicotine on intestinal epithelial cells in vivo and in vitro: an X-ray microanalytical study,” Journal of Submicroscopic Cytology and Pathology, vol. 32, no. 1, pp. 97–102, 2000. View at Google Scholar · View at Scopus
  35. I. A. Finnie, B. J. Campbell, B. A. Taylor et al., “Stimulation of colonic mucin synthesis by corticosteroids and nicotine,” Clinical Science, vol. 91, no. 3, pp. 359–364, 1996. View at Google Scholar · View at Scopus
  36. A. Cervin, S. Lindberg, U. Mercke, and R. Uddman, “Neuropeptide Y in the rabbit maxillary sinus modulates cholinergic acceleration of mucociliary activity,” Acta Oto-Laryngologica, vol. 112, no. 5, pp. 872–881, 1992. View at Google Scholar · View at Scopus
  37. F. J. Zijlstra, E. D. Srivastava, M. Rhodes et al., “EicosanoidsEffect of nicotine on rectal mucus and mucosal eicosanoids,” Gut, vol. 35, no. 2, pp. 247–251, 1994. View at Google Scholar · View at Scopus
  38. A. E. Summers, C. J. Whelan, and M. E. Parsons, “Nicotinic acetylcholine receptor subunits and receptor activity in the epithelial cell line HT29,” Life Sciences, vol. 72, no. 18-19, pp. 2091–2094, 2003. View at Publisher · View at Google Scholar · View at Scopus
  39. M. Bencherif, P. M. Lippiello, R. Lucas, and M. B. Marrero, “Alpha7 nicotinic receptors as novel therapeutic targets for inflammation-based diseases,” Cellular and Molecular Life Sciences, vol. 68, no. 6, pp. 931–949, 2011. View at Publisher · View at Google Scholar · View at Scopus
  40. J. Ghia, P. Blennerhassett, R. T. El-Sharkawy, and S. M. Collins, “The protective effect of the vagus nerve in a murine model of chronic relapsing colitis,” American Journal of Physiology-Gastrointestinal and Liver Physiology, vol. 293, no. 4, pp. G711–G718, 2007. View at Publisher · View at Google Scholar · View at Scopus
  41. J. E. Ghia, P. Blennerhassett, H. Kumar-Ondiveeran, E. F. Verdu, and S. M. Collins, “The Vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model,” Gastroenterology, vol. 131, no. 4, pp. 1122–1130, 2006. View at Publisher · View at Google Scholar · View at Scopus
  42. S. Nikfar, S. Ehteshami-Ashar, R. Rahimi, and M. Abdollahi, “Systematic review and meta-analysis of the efficacy and tolerability of nicotine preparations in active ulcerative colitis,” Clinical Therapeutics, vol. 32, no. 14, pp. 2304–2315, 2010. View at Publisher · View at Google Scholar · View at Scopus
  43. J.-W. Tjiu, P.-J. Lin, W.-H. Wu et al., “SLURP1 mutation-impaired T-cell activation in a family with mal de Meleda,” British Journal of Dermatology, vol. 164, no. 1, pp. 47–53, 2011. View at Publisher · View at Google Scholar · View at Scopus
  44. H. Tsuji, K. Okamoto, Y. Matsuzaka, H. Iizuka, G. Tamiya, and H. Inoko, “SLURP-2, a novel member of the human Ly-6 superfamily that is up-regulated in psoriasis vulgaris,” Genomics, vol. 81, no. 1, pp. 26–33, 2003. View at Publisher · View at Google Scholar · View at Scopus
  45. S. A. Grando, “Basic and clinical aspects of non-neuronal acetylcholine: biological and clinical significance of non-canonical ligands of epithelial nicotinic acetylcholine receptors,” Journal of Pharmacological Sciences, vol. 106, no. 2, pp. 174–179, 2008. View at Publisher · View at Google Scholar · View at Scopus
  46. J. Arredondo, A. I. Chernyavsky, R. J. Webber, and S. A. Grando, “Biological effects of SLURP-1 on human keratinocytes,” Journal of Investigative Dermatology, vol. 125, no. 6, pp. 1236–1241, 2005. View at Publisher · View at Google Scholar · View at Scopus
  47. J. Arredondo, A. I. Chernyavsky, D. L. Jolkovsky, R. J. Webber, and S. A. Grando, “SLURP-2: a novel cholinergic signaling peptide in human mucocutaneous epithelium,” Journal of Cellular Physiology, vol. 208, no. 1, pp. 238–245, 2006. View at Publisher · View at Google Scholar · View at Scopus
  48. A. Pettersson, G. Nylund, A. Khorram-Manesh, S. Nordgren, and D. S. Delbro, “Nicotine induced modulation of SLURP-1 expression in human colon cancer cells,” Autonomic Neuroscience: Basic and Clinical, vol. 148, no. 1-2, pp. 97–100, 2009. View at Publisher · View at Google Scholar · View at Scopus
  49. K. Nilsson, K. Forsbeck, M. Gidlund et al., “Surface characteristics of the U-937 human histiocytic lymphoma cell line: specific changes during inducible morphologic and functional differentiation in vitro,” Hamatologie und Bluttransfusion, vol. 26, pp. 215–221, 1981. View at Google Scholar · View at Scopus
  50. A. I. Chernyavsky, J. Arredondo, D. J. Putney, J. S. Marsh, and S. A. Grando, “Potential role for epithelial nicotinic receptors in tobacco related oral and lung cancers,” Journal of Stomatological Investigation, vol. 2, no. 1, pp. 5–14, 2008. View at Google Scholar
  51. S. Dionne, F. M. Ruemmele, and E. G. Seidman, “Immunopathogenesis of inflammatory bowel disease: role of cytokines and immune cell-enterocyte interactions,” Nestle Nutrition Workshop Series. Clinical & Performance Programme, vol. 2, pp. 41–61, 1999. View at Google Scholar · View at Scopus
  52. E. Cario, “Toll-like receptors in inflammatory bowel diseases: a decade later,” Inflammatory Bowel Diseases, vol. 16, no. 9, pp. 1583–1597, 2010. View at Publisher · View at Google Scholar · View at Scopus
  53. S. C. Gribar, R. J. Anand, C. P. Sodhi, and D. J. Hackam, “The role of epithelial Toll-like receptor signaling in the pathogenesis of intestinal inflammation,” Journal of Leukocyte Biology, vol. 83, no. 3, pp. 493–498, 2008. View at Publisher · View at Google Scholar · View at Scopus
  54. M. Akhtar, J. L. Watson, A. Nazli, and D. M. McKay, “Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-kappaB-independent pathway,” The FASEB Journal, vol. 17, no. 10, pp. 1319–1321, 2003. View at Google Scholar · View at Scopus
  55. P. S. Bridger, M. Mohr, I. Stamm et al., “Primary bovine colonic cells: a model to study strain-specific responses to Escherichia coli,” Veterinary Immunology and Immunopathology, vol. 137, no. 1-2, pp. 54–63, 2010. View at Google Scholar
  56. C. Sodhi, R. Levy, R. Gill et al., “DNA attenuates enterocyte Toll-like receptor 4-mediated intestinal mucosal injury after remote trauma,” American Journal of Physiology-Gastrointestinal and Liver Physiology, vol. 300, no. 5, pp. G862–G873, 2011. View at Publisher · View at Google Scholar · View at Scopus
  57. A. Levin and O. Shibolet, “Toll-like receptors in inflammatory bowel disease-stepping into uncharted territory,” World Journal of Gastroenterology, vol. 14, no. 33, pp. 5149–5153, 2008. View at Publisher · View at Google Scholar · View at Scopus
  58. A. I. Chernyavsky, M. Kalantari-Dehaghi, C. Phillips, S. Marchenko, and S. A. Grando, “Novel cholinergic peptides SLURP-1 and -2 regulate epithelialization of cutaneous and oral wounds,” Wound Repair and Regeneration, vol. 20, no. 1, pp. 103–113, 2012. View at Publisher · View at Google Scholar · View at Scopus
  59. A. I. Chernyavsky, S. Marchenko, C. Phillips, and S. A. Grando, “Auto/paracrine nicotinergic peptides participate in cutaneous stress response to wounding,” Dermato-Endocrinology, vol. 4, no. 3, pp. 324–330, 2012. View at Publisher · View at Google Scholar
  60. Y. Moriwaki, K. Yoshikawa, H. Fukuda, Y. X. Fujii, H. Misawa, and K. Kawashima, “Immune system expression of SLURP-1 and SLURP-2, two endogenous nicotinic acetylcholine receptor ligands,” Life Sciences, vol. 80, no. 24-25, pp. 2365–2368, 2007. View at Publisher · View at Google Scholar · View at Scopus
  61. B. H. Lee, S. Choi, T. Shin et al., “Quercetin enhances human α7 nicotinic acetylcholine receptor-mediated ion current through interactions with Ca2+ binding sites,” Molecules and Cells, vol. 30, no. 3, pp. 245–253, 2010. View at Publisher · View at Google Scholar · View at Scopus
  62. B. H. Lee, S. H. Choi, T. J. Shin et al., “Effects of quercetin on α9α10 nicotinic acetylcholine receptor-mediated ion currents,” European Journal of Pharmacology, vol. 650, no. 1, pp. 79–85, 2011. View at Publisher · View at Google Scholar · View at Scopus
  63. B. H. Lee, S. H. Hwang, S. H. Choi et al., “Quercetin inhibits α3β4 nicotinic acetylcholine receptor-mediated ion currents expressed in Xenopus oocytes,” Korean Journal of Physiology and Pharmacology, vol. 15, no. 1, pp. 17–22, 2011. View at Publisher · View at Google Scholar · View at Scopus
  64. Y. L. Shih, H. Liu, C. Chen et al., “Combination treatment with luteolin and quercetin enhances antiproliferative effects in nicotine-treated MDA-MB-231 cells by down-regulating nicotinic acetylcholine receptors,” Journal of Agricultural and Food Chemistry, vol. 58, no. 1, pp. 235–241, 2010. View at Publisher · View at Google Scholar · View at Scopus
  65. M. Comalada, D. Camuesco, S. Sierra et al., “In vivo quercitrin anti-inflammatory effect involves release of quercetin, which inhibits inflammation through down-regulation of the NF-κB pathway,” European Journal of Immunology, vol. 35, no. 2, pp. 584–592, 2005. View at Publisher · View at Google Scholar · View at Scopus
  66. H. H. Kim, H. Kong, B. Choi et al., “Metabolic and pharmacological properties of rutin, a dietary quercetin glycoside, for treatment of inflammatory bowel disease,” Pharmaceutical Research, vol. 22, no. 9, pp. 1499–1509, 2005. View at Publisher · View at Google Scholar · View at Scopus
  67. J. P. Van Dijk, G. S. Madretsma, Z. J. Keuskamp, and F. J. Zijlstra, “Nicotine inhibits cytokine synthesis by mouse colonic mucosa,” European Journal of Pharmacology, vol. 278, no. 1, pp. R11–R12, 1995. View at Publisher · View at Google Scholar · View at Scopus
  68. T. Spoettl, C. Paetzel, H. Herfarth et al., “(E)-metanicotine hemigalactarate (TC-2403-12) inhibits IL-8 production in cells of the inflamed mucosa,” International Journal of Colorectal Disease, vol. 22, no. 3, pp. 303–312, 2007. View at Publisher · View at Google Scholar · View at Scopus
  69. W. J. de Jonge and L. Ulloa, “The alpha7 nicotinic acetylcholine receptor as a pharmacological target for inflammation,” British Journal of Pharmacology, vol. 151, no. 7, pp. 915–929, 2007. View at Publisher · View at Google Scholar · View at Scopus
  70. R. Eliakim and F. Karmeli, “Divergent effects of nicotine administration on cytokine levels in rat small bowel mucosa, colonic mucosa, and blood,” Israel Medical Association Journal, vol. 5, no. 3, pp. 178–180, 2003. View at Google Scholar · View at Scopus
  71. T. Werner and D. Haller, “Intestinal epithelial cell signalling and chronic inflammation: from the proteome to specific molecular mechanisms,” Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis, vol. 622, no. 1-2, pp. 42–57, 2007. View at Publisher · View at Google Scholar · View at Scopus
  72. K. Matsunaga, T. W. Klein, H. Friedman, and Y. Yamamoto, “Involvement of nicotinic acetylcholine receptors in suppression of antimicrobial activity and cytokine responses of alveolar macrophages to Legionella pneumophila infection by nicotine,” Journal of Immunology, vol. 167, no. 11, pp. 6518–6524, 2001. View at Google Scholar · View at Scopus
  73. E. P. van der Zanden, S. A. Snoek, S. E. Heinsbroek et al., “Vagus nerve activity augments intestinal macrophage phagocytosis via nicotinic acetylcholine receptor α4β2,” Gastroenterology, vol. 137, no. 3, pp. 1029–1039, 2009. View at Publisher · View at Google Scholar · View at Scopus
  74. I. Murakami, Y. Hamada, S. Yamane, H. Fujino, S. Horie, and T. Murayama, “Nicotine-induced neurogenic relaxation in the mouse colon: changes with dextran sodium sulfate-induced colitis,” Journal of Pharmacological Sciences, vol. 109, no. 1, pp. 128–138, 2009. View at Publisher · View at Google Scholar · View at Scopus
  75. R. Eliakim, X. F. Qiu, and M. W. Babyatsky, “Chronic nicotine administration differentially alters jejunal and colonic inflammation in interleukin-10 deficient mice,” European Journal of Gastroenterology and Hepatology, vol. 14, no. 6, pp. 607–614, 2002. View at Publisher · View at Google Scholar · View at Scopus
  76. A. Karban and R. Eliakim, “Effect of smoking on inflammatory bowel disease: is it disease or organ specific?” World Journal of Gastroenterology, vol. 13, no. 15, pp. 2150–2152, 2007. View at Google Scholar · View at Scopus
  77. F. Chimienti, R. C. Hogg, L. Plantard et al., “Identification of SLURP-1 as an epidermal neuromodulator explains the clinical phenotype of Mal de Meleda,” Human Molecular Genetics, vol. 12, no. 22, pp. 3017–3024, 2003. View at Publisher · View at Google Scholar · View at Scopus
  78. T. Fujii, K. Horiguchi, H. Sunaga et al., “SLURP-1, an endogenous alpha7 nicotinic acetylcholine receptor allosteric ligand, is expressed in CD205(+) dendritic cells in human tonsils and potentiates lymphocytic cholinergic activity,” Journal of Neuroimmunology, vol. 267, no. 1-2, pp. 43–49, 2014. View at Publisher · View at Google Scholar