LL-37 induced cystitis and the receptor for advanced glycation end-products (RAGE) pathway

Lindsi McCoard Roundy, Wanjian Jia, Jianxing Zhang, Xiangyang Ye, Glenn D. Prestwich, Siam OottamasathienQ

Department of Medicinal Chemistry and Center for Therapeutic Biomaterials, University of Utah, Salt Lake City, USA.
Department of Pharmacotherapy, Primary Children’s Medical Center, University of Utah, Salt Lake City, USA.
Department of Surgery and Division of Pediatric Urology, Primary Children’s Medical Center, Salt Lake City, USA.

DOI: 10.4236/abb.2013.48A2001                

Abstract

To elucidate pathways in bladder inflammation, we employed our physiologically relevant LL-37 induced cystitis model. Based on inflammatory studies involving other organ systems implicating the receptor for advanced glycation end-products (RAGE), we first hypothesized that RAGE is critically involved in LL-37 induced cystitis. We further hypothesized that a common RAGE ligand high mobility group box 1 (HMGB1) is up-regulated in bladders challenged with LL-37. Finally, we hypothesized that NF-κB dependent inflammatory genes are activated in LL-37 induced cystitis. Testing our first hypothesis, C57Bl/6 mice were challenged with either saline (control) or 320 μM of LL-37 intravesically for 1 hr. After 12 or 24 hours, tissues were examined with immunohistochemistry (IHC) for RAGE, and both mRNA and protein isolation for respective qRT-PCR and Western Blot analysis. Our second hypothesis was tested by employing HMGB1 IHC. Testing our final hypothesis, qRT-PCR was performed investigating five genes: TNFα, IL-6, IL-1β, GM-CSF, COX-2. In control and LL-37 challenged tissues, IHC for RAGE revealed similar qualitative expression. Evaluation with qRT-PCR and Western Blot for RAGE revealed diminished expression at the mRNA and protein level within LL-37 challenged bladders. IHC for HMGB1 revealed a moderate qualitative increase within LL-37 challenged tissues. Finally, with the exception of TNFα, all NF-κB dependent inflammatory genes yielded substantial up-regulation. We have employed our LL-37 induced cystitis model to gain insight to wards a possible mechanistic pathway involved in bladder inflammation. This work provides data for future studies involving the inflammatory ligand HMGB1, RAGE, and receptor pathways that activate NF-κB.

Keywords

LL-37; Cathelicidin; Bladder Inflammation; Bladder Fibrosis; Spina Bifida; Myelomeningocele; Interstitial Cystitis; RAGE; HMGB1; NF-Kappa B

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Deveaud, C.M., Macarak, E.J., Kucich, U., Ewalt, D.H., Abrams, W.R. and Howard, P.S. (1998) Molecular analysis of collagens in bladder fibrosis. The Journal of Urology, 160, 1518-1527. doi:10.1016/S0022-5347(01)62606-5
[2]	Metcalfe, P.D., Wang, J., Jiao, H., Huang, Y., Hori, K., Moore, R.B. and Tredget, E.E. (2010) Bladder outlet obstruction: Progression from inflammation to fibrosis. BJU International, 106, 1686-1694. doi:10.1111/j.1464-410X.2010.09445.x
[3]	Wiseman, O.J., Fowler, C.J. and Landon, D.N. (2003) The role of the human bladder lamina propria myofibroblast. BJU International, 91, 89-93. doi:10.1046/j.1464-410X.2003.03802.x
[4]	Lukban, J.C., Whitmore, K.E. and Sant, G.R. (2002) Current management of interstitial cystitis. Urologic Clinics of North America, 29, 649-660. doi:10.1016/S0094-0143(02)00055-1
[5]	Theoharides, T.C. (2007) Treatment approaches for painful bladder syndrome/interstitial cystitis. Drugs, 67, 215-235. doi:10.2165/00003495-200767020-00004
[6]	Theoharides, T.C. and Sant, G.R. (2001) New agents for the medical treatment of interstitial cystitis. Expert Opinion on Investigational Drugs, 10, 521-546. doi:10.1517/13543784.10.3.521
[7]	Toft, B.R. and Nordling, J. (2006) Recent developments of intravesical therapy of painful bladder syndrome/interstitial cystitis: A review. Current Opinion in Urology, 16, 268-272. doi:10.1097/01.mou.0000232048.81965.16
[8]	Oottamasathien, S., Jia, W., McCoard, L., Slack, S., Zhang, J., Skardal, A., Job, K., Kennedy, T.P., Dull, R.O. and Prestwich, G.D. (2011) A murine model of inflammatory bladder disease: Cathelicidin peptide induced bladder inflammation and treatment with sulfated polysaccharides. The Journal of Urology, 186, 1684-1692. doi:10.1016/j.juro.2011.03.099
[9]	Nijnik, A. and Hancock, R.E. (2009) The roles of cathelicidin LL-37 in immune defences and novel clinical applications. Current Opinion in Hematology, 16, 41-47. doi:10.1097/MOH.0b013e32831ac517
[10]	Yamasaki, K., Di Nardo, A., Bardan, A., Murakami, M., Ohtake, T., Coda, A., Dorschner, R.A., Bonnart, C., Descargues, P., Hovnanian, A., et al. (2007) Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea. Nature Medicine, 13, 975-980. doi:10.1038/nm1616
[11]	Johansson, J., Gudmundsson, G.H., Rottenberg, M.E., Berndt, K.D. and Agerberth, B. (1998) Conformationdependent antibacterial activity of the naturally occurring human peptide LL-37. The Journal of Biological Chemistry, 273, 3718-3724. doi:10.1074/jbc.273.6.3718
[12]	Hori, O., Brett, J., Slattery, T., Cao, R., Zhang, J., Chen, J.X., Nagashima, M., Lundh, E.R., Vijay, S., Nitecki, D., et al. (1995) The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system. The Journal of Biological Chemistry, 270, 25752-25761. doi:10.1074/jbc.270.43.25752
[13]	Brett, J., Schmidt, A.M., Yan, S.D., Zou, Y.S., Weidman, E., Pinsky, D., Nowygrod, R., Neeper, M., Przysiecki, C., Shaw, A., et al. (1993) Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues. American Journal of Pathology, 143, 1699-1712.
[14]	Yan, S.F., Du Yan, S., Ramasamy, R. and Schmidt, A.M. (2009) Tempering the wrath of RAGE: An emerging therapeutic strategy against diabetic complications, neurodegeneration, and inflammation. Annals of Medicine, 41, 408-422.
[15]	Englert, J.M., Hanford, L.E., Kaminski, N., Tobolewski, J.M., Tan, R.J., Fattman, C.L., Ramsgaard, L., Richards, T.J., Loutaev, I., Nawroth, P.P., et al. (2008) A role for the receptor for advanced glycation end products in idiopathic pulmonary fibrosis. American Journal of Pathology, 172, 583-591. doi:10.2353/ajpath.2008.070569
[16]	He, M., Kubo, H., Ishizawa, K., Hegab, A.E., Yamamoto, Y., Yamamoto, H. and Yamaya, M. (2007) The role of the receptor for advanced glycation end-products in lung fibrosis. American Journal of Physiology—Lung Cellular and Molecular Physiology, 293, L1427-1436. doi:10.1152/ajplung.00075.2007
[17]	Queisser, M.A., Kouri, F.M., Konigshoff, M., Wygrecka, M., Schubert, U., Eickelberg, O. and Preissner, K.T. (2008) Loss of RAGE in pulmonary fibrosis: Molecular relations to functional changes in pulmonary cell types. American Journal of Respiratory Cell and Molecular Biology, 39, 337-345. doi:10.1165/rcmb.2007-0244OC
[18]	Baldwin, A.S., Jr. (1996) The NF-kappa B and I kappa B proteins: New discoveries and insights. American Journal of Respiratory Cell and Molecular Biology, 14, 649-683. doi:10.1146/annurev.immunol.14.1.649
[19]	Barnes, P.J. and Adcock, I.M. (1997) NF-kappa B: A pivotal role in asthma and a new target for therapy. Trends in Pharmacological Sciences, 18, 46-50. doi:10.1016/S0165-6147(97)89796-9
[20]	Neurath, M.F., Pettersson, S., Meyer zum Buschenfelde, K.H. and Strober, W. (1996) Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice. Nature Medicine, 2, 998-1004. doi:10.1038/nm0996-998
[21]	Schmidt, A.M., Hori, O., Brett, J., Yan, S.D., Wautier, J.L. and Stern, D. (1994) Cellular receptors for advanced glycation end products. Implications for induction of oxidant stress and cellular dysfunction in the pathogenesis of vascular lesions. Arteriosclerosis, Thrombosis, and Vascular Biology, 14, 1521-1528. doi:10.1161/01.ATV.14.10.1521
[22]	Stancovski, I. and Baltimore, D. (1997) NF-kappaB activation: The I kappaB kinase revealed? Cell, 91, 299-302. doi:10.1016/S0092-8674(00)80413-4
[23]	Wang, X.C., Saban, R., Kaysen, J.H., Saban, M.R., Allen, P.L., Benes, E.N. and Hammond, T.G. (2000) Nuclear factor kappa B mediates lipopolysaccharide-induced inflammation in the urinary bladder. The Journal of Urology, 163, 993-998. doi:10.1016/S0022-5347(05)67870-6
[24]	Woronicz, J.D., Gao, X., Cao, Z., Rothe, M. and Goeddel, D.V. (1997) IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK. Science, 278, 866-869. doi:10.1126/science.278.5339.866
[25]	Foell, D., Wittkowski, H. and Roth, J. (2007) Mechanisms of disease: A “DAMP” view of inflammatory arthritis. Nature Clinical Practice Rheumatology, 3, 382-390. doi:10.1038/ncprheum0531
[26]	Schmidt, A.M., Yan, S.D., Wautier, J.L. and Stern, D. (1999) Activation of receptor for advanced glycation end products: A mechanism for chronic vascular dysfunction in diabetic vasculopathy and atherosclerosis. Circulation Research, 84, 489-497. doi:10.1161/01.RES.84.5.489
[27]	See, W.A., Zhang, G., Chen, F., Cao, Y., Langenstroer, P. and Sandlow, J. (2009) Bacille-Calmette Guerin induces caspase-independent cell death in urothelial carcinoma cells together with release of the necrosis-associated chemokine high molecular group box protein 1. BJU International, 103, 1714-1720. doi:10.1111/j.1464-410X.2008.08274.x
[28]	Javaherian, K., Liu, J.F. and Wang, J.C. (1978) Nonhistone proteins HMG1 and HMG2 change the DNA helical structure. Science, 199, 1345-1346. doi:10.1126/science.628842
[29]	Wang, H., Bloom, O., Zhang, M., Vishnubhakat, J.M., Ombrellino, M., Che, J., Frazier, A., Yang, H., Ivanova, S., Borovikova, L., et al. (1999) HMG-1 as a late mediator of endotoxin lethality in mice. Science, 285, 248-251. doi:10.1126/science.285.5425.248
[30]	Bianchi, M.E., Beltrame, M. and Paonessa, G. (1989) Specific recognition of cruciform DNA by nuclear protein HMG1. Science, 243, 1056-1059. doi:10.1126/science.2922595
[31]	Lotze, M.T. and Tracey, K.J. (2005) High-mobility group box 1 protein (HMGB1): Nuclear weapon in the immune arsenal. Nature Reviews Immunology, 5, 331-342. doi:10.1038/nri1594
[32]	Stros, M., Ozaki, T., Bacikova, A., Kageyama, H. and Nakagawara, A. (2002) HMGB1 and HMGB2 cell-specifically down-regulate the p53-and p73-dependent sequence-specific transactivation from the human Bax gene promoter. The Journal of Biological Chemistry, 277, 7157-7164. doi:10.1074/jbc.M110233200
[33]	Merenmies, J., Pihlaskari, R., Laitinen, J., Wartiovaara, J. and Rauvala, H. (1991) 30-kDa heparin-binding protein of brain (amphoterin) involved in neurite outgrowth. Amino acid sequence and localization in the filopodia of the advancing plasma membrane. The Journal of Biological Chemistry, 266, 16722-16729.
[34]	Mullins, G.E., Sunden-Cullberg, J., Johansson, A.S., Rouhiainen, A., Erlandsson-Harris, H., Yang, H., Tracey, K.J., Rauvala, H., Palmblad, J., Andersson, J., et al. (2004) Activation of human umbilical vein endothelial cells leads to relocation and release of high-mobility group box chromosomal protein 1. Scandinavian Journal of Immunology, 60, 566-573. doi:10.1111/j.0300-9475.2004.01518.x
[35]	Rauvala, H. and Pihlaskari, R. (1987) Isolation and some characteristics of an adhesive factor of brain that enhances neurite outgrowth in central neurons. The Journal of Biological Chemistry, 262, 16625-16635.
[36]	Rouhiainen, A., Kuja-Panula, J., Wilkman, E., Pakkanen, J., Stenfors, J., Tuominen, R.K., Lepantalo, M., Carpen, O., Parkkinen, J. and Rauvala, H. (2004) Regulation of monocyte migration by amphoterin (HMGB1). Blood, 104, 1174-1182. doi:10.1182/blood-2003-10-3536
[37]	Scaffidi, P., Misteli, T. and Bianchi, M.E. (2002) Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature, 418, 191-195.
[38]	Bonaldi, T., Talamo, F., Scaffidi, P., Ferrera, D., Porto, A., Bachi, A., Rubartelli, A., Agresti, A. and Bianchi, M.E. (2003) Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. The EMBO Journal, 22, 5551-5560. doi:10.1093/emboj/cdg516
[39]	Semino, C., Angelini, G., Poggi, A. and Rubartelli, A. (2005) NK/iDC interaction results in IL-18 secretion by DCs at the synaptic cleft followed by NK cell activation and release of the DC maturation factor HMGB1. Blood, 106, 609-616. doi:10.1182/blood-2004-10-3906
[40]	Oottamasathien, S., Jia, W., Roundy, L.M., Zhang, J., Wang, L., Ye, X., Hill, A.C., Savage, J., Lee, W.Y., Hannon, A.M., et al. (2013) Physiologic relevance of LL-37 induced bladder inflammation and mast cells. The Journal of Urology, S0022-5347(13)00014-1.
[41]	Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta CT) method. Methods, 25, 402-408. doi:10.1006/meth.2001.1262
[42]	Chromek, M., Slamova, Z., Bergman, P., Kovacs, L., Podracka, L., Ehren, I., Hokfelt, T., Gudmundsson, G.H., Gallo, R.L., Agerberth, B., et al. (2006) The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection. Nature Medicine, 12, 636-641. doi:10.1038/nm1407
[43]	Park, J.S., Gamboni-Robertson, F., He, Q., Svetkauskaite, D., Kim, J.Y., Strassheim, D., Sohn, J.W., Yamada, S., Maruyama, I., Banerjee, A., et al. (2006) High mobility group box 1 protein interacts with multiple toll-like receptors. American Journal of Physiology Cell Physiology, 290, C917-C924. doi:10.1152/ajpcell.00401.2005
[44]	Park, J.S., Svetkauskaite, D., He, Q., Kim, J.Y., Strassheim, D., Ishizaka, A. and Abraham, E. (2004) Involvement of toll-like receptors 2 and 4 in cellular activetion by high mobility group box 1 protein. The Journal of Biological Chemistry, 279, 7370-7377. doi:10.1074/jbc.M306793200
[45]	Yu, M., Wang, H., Ding, A., Golenbock, D.T., Latz, E., Czura, C.J., Fenton, M.J., Tracey, K.J. and Yang, H. (2006) HMGB1 signals through toll-like receptor (TLR) 4 and TLR2. Shock, 26, 174-179. doi:10.1097/01.shk.0000225404.51320.82
[46]	Huttunen, H.J. and Rauvala, H. (2004) Amphoterin as an extracellular regulator of cell motility: From discovery to disease. Journal of Internal Medicine, 255, 351-366. doi:10.1111/j.1365-2796.2003.01301.x
[47]	Song, J. and Abraham, S.N. (2008) Innate and adaptive immune responses in the urinary tract. European Journal of Clinical Investigation, 38, 21-28. doi:10.1111/j.1365-2362.2008.02005.x
[48]	Weichhart, T., Haidinger, M., Horl, W.H. and Saemann, M.D. (2008) Current concepts of molecular defence mechanisms operative during urinary tract infection. European Journal of Clinical Investigation, 38, 29-38. doi:10.1111/j.1365-2362.2008.02006.x
[49]	Zasloff, M. (2007) Antimicrobial peptides, innate immunity, and the normally sterile urinary tract. Journal of the American Society of Nephrology, 18, 2810-2816. doi:10.1681/ASN.2007050611
[50]	Leemans, J.C., Butter, L.M., Pulskens, W.P., Teske, G.J., Claessen, N., van der Poll, T. and Florquin, S. (2009) The role of toll-like receptor 2 in inflammation and fibrosis during progressive renal injury. PLoS One, 4, e5704. doi:10.1371/journal.pone.0005704