A novel molecular marker of severe asthma Sergio Bonini Professor of Medicine, Second University of Naples INMM-CNR, ARTOV, Rome se.bonini@gmail.com i@ il Modena March 3, 2009
Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma Bonini S, Lambiase A, Bonini S, Angelucci F, Magrini L, Manni L,Aloe L PNAS 1996; 93: 10955-10960 Increased NGF circulating levels in allergic diseases Increased NGF circulating levels in allergic diseases Relationship to tissue mast cells and plasma Substance P Relationship to severity of disease and bronchial hyperreactivity
NGF and NGF receptors in allergic inflammation and tissue remodelling APC Th B Fibroblasts SC Multipotent progenitors MC Eos Bonini S et al. PNAS 1996; 93: 10955 Lambiase A et al. JACI 1997; 100: 408 Solomon A et al. JACI 1998; 102: 454 Epithelial cells Pellegrini i G et al. J Cell Biol 1999; 145: 769 Lambiase A et al IOVS 1998; 39: 1272 Lambiase A et al. N Engl J Med 1998; 338: 1174 Lambiase A et al. Arch Ophthalmol l 2000; 188: 1446 Micera A et al. PNAS 2001; 98: 6162 Manni L et al. Clin Exp Rheumatol 2003; 21: 617 Micera A et al. Clin Exp Allergy 2005; 35: 650 Mi A t l E E R 2006 83 747 Micera A et al. Mol Vis 2007; 13: 981 Bracci-Laudiero L et al. J Neuroimmunol 2003; 136: 130 Micera A et al. Exp Eye Res 2006; 83: 747
Fibroblasts produce, store, release and respond to NGF
Study models Biopsies from human fibrotic lung and skinscartissues Coltures of lung, skin and eye fibroblasts In vivo effects of NGF in humans Lambiase et al, NEJM 1998; Bernabei et al, Lancet 1999; Lambiase et al, Arch Ophthalmol 2000; Micera et al PNAS 2001
Micera et al PNAS 2001; 98: 6162 67
Micera et al PNAS 2001; 98: 6162 6167
Micera et al PNAS 2001; 98: 6162 6767
Micera et al PNAS 2001; 98: 6162 67
Lambiase A, Rama P, Bonini S, Caprifoglio G, Aloe L Topical treatment with nerve growth factor for corneal neurotrophic ulcers New Engl J Med 1998; 338: 1174-1180
Epithelial cells produce, store, release and respond to NGF
NGF production by human corneal epithelial cells 1Kb + NGF TrkA p75 NTR 650 bp 500 bp 400 bp 300 bp 200 bp NGF = 368 bp TrkA = 246 bp p75 NTR = 521 bp 100 bp
NGF CORNEAL LEVELS FOLLOWING EPITHELIAL DEBRIDEMENT 4000 control * 3000 lesioned ) NGF (pg/gr) 2000 * * 1000 0 Baseline 2 12 24 48 96 168 Hours Lambiase A, IOVS 2000
Functional effects of NGF
Relative changes of p63 delta mrna in human conjunctival cells primary culture after NGF stimulation (evaluated by Real Time PCR) 1,2 1 0,8 0,6 0,4 0,2 0
Relative changes of mucins mrnas in human conjunctival cells primary culture after NGF stimulation (evaluated by Real Time PCR) control (1%FBS) NGF (10 ng/ml) NGF (100 ng/ml) 18,00 16,00 14,00 12,00 10,00 8,00 6,00 4,00 2,00 0,00 Mucin 1 Mucin 4 Mucin 5ac
NGF in Tissue Remodeling A novel function of NGF: a regulatory role in mucus production
Mucus and Allergic Diseases Mucusisanaqueoussolutionoflipidsandproteins coating the mucosal surfaces and representing an important barrier against pathogens Mucin (MUC) glycoproteins gy represent the major (10-50% wt/wt) component of mucus, and the major determinant of its visco-elastic properties p In humans, MUC are encoded by at least 20 genes whose protein backbone transcripts are secreted by epithelial cells and then extensively glycosilated In the airways, MUC 5AC (preferentially secreted by goblet cells) and MUC 5B (preferentially secreted by submucosal glands) are the most represented mucins, increasing in pathological states
Regulation of mucus production Mechanisms Differentiation of epithelial cells into mucus producing cells (mucus cells hyperplasia and/or metaplasia) Hypersecretion (mucin genes overexpression, goblet cell degranulation, etc.) Regulatory factors Bacteria; Pollutants and chemicals; Biological products; Cytokine (IL-1β IL-4,IL-6,, IL-9, IL-13, TNFα, IFNγ); Growth Factors (EGF/EGF-R)
NGF LEVELS IN PLASMA AND TEARS OF VKC PATIENTS 9000 7000 control VKC 5000 3000 NGF pg/ml 1000 300 200 100 0 Plasma Tear
Study models Transgenic mice overexpressing NGF at the level of Clara cells Effects of NGF on human epithelial cell coltures Epithelial ldifferentiation Mucin gene expression Mucin protein content and release by goblet cells
Hyperinnervation of the Airways in Transgenic Mice Overexpressing Nerve Growth Factor Gary W. Hoyle, Regina M. Graham, Jeffrey B. Finkelstein, Kim-Phoung Thi Nguyen, David Gozal and Mitchell Freidman Am J Resp Cell Mol Biol 1998; 18: 149-157
Goblet cells in gut of NGF transgenic mice
Modulation of mucus production in a transgenic mouse model of asthma Groups Mice Treatment 1 (PBS/NGF-tg) NGF-tg PBS/OVA 2 (OVA/NGF-tg) NGF-tg OVA/OVA 3 (wt) C57/BL6 (wt) PBS/PBS 4 (OVA) C57/BL6 (wt) OVA/OVA 5 (PBS/NGF-tg) NGF-tg PBS 6 (OVA/NGF-tg) NGF-tg OVA Bonini S. et al, J Allergy Clini Immunol 2006 ;117:S250 Bonini Se, Lambiase A, Renz H et al. in preparation
Mucins expression in NGF transgenic mice A target gen ne expression 40 30 20 10 0-10 MUC1 MUC4 MUC5ac Rest analysis: NGF-tg mice vs Wt D target gene expression 7 6 5 4 3 2 1 0 MUC1 MUC4 MUC5ac Rest analysis: OVA/NGF-tg mice vs. OVA M MUC5ac B C target gen ne expression 7 6 5 4 3 2 1 0 MUC1 MUC4 MUC5ac Rest analysis; OVA mice vs Wt 7 target gene expression 6 5 4 3 2 1 0 MUC1 MUC4 MUC5ac Rest analysis: OVA/NGF-tg mice vs PBS/NGF-tg mice MU UC5ac, X600 F muc5ac, OD E 80 60 40 20 0 PBS OVA PBS OVA NGF-TG WT Sensitization/Challenge OVA- NGF-tg NGF-tg Isotype
NGF promotes epithelial cell differentiation to goblet cells
Epithelium Mucus metaplasia and mucin gene expression Frossard N. et al. Eur J Pharmacol 2004; 500: 453-65
Collaborations Stefano Bonini, Alessandra Micera, Alessandro Lambiase Eye Clinic, University Campus Biomedicus Rome, Italy Megon Bresciani, Marie France Lalibertè, François Lalibertè, CHU Arnaud de Villeneuve, CHU Arnaud de Villeneuve, France Claudia Gramiccioni, University of Foggia, Institute of Respiratory Diseases, Italy Harald Renz, Dept. of Clinical Chemistry and Molecular Diagnostics, Philipps-University Marburg, Germany Francesca Levi-Schaffer, Dept. of Pharmacology, School of Pharmacy, Hadassah Med. School, Hebrew Univ., Jerusalem, Israel Abraham Solomon Joseph Frucht Pery Jacob Pe er Abraham Solomon, Joseph Frucht Pery, Jacob Pe er Dept. of Ophthalmology, Hadassah Univ. Hosp., Jerusalem, Israel