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The Role of Nerve Growth Factor (NGF) on Mast Cells During Allergic Inflammation

The Role of Nerve Growth Factor (NGF) on Mast Cells During Allergic Inflammation. Natalie Sandoval Biol 520 March 9, 2009. Background Information. Neurotrophins (Neurotrophic Factors). Family of polypeptide growth factors

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The Role of Nerve Growth Factor (NGF) on Mast Cells During Allergic Inflammation

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  1. The Role of Nerve Growth Factor (NGF) on Mast Cells During Allergic Inflammation Natalie Sandoval Biol 520 March 9, 2009

  2. Background Information

  3. Neurotrophins(Neurotrophic Factors) • Family of polypeptide growth factors • Responsible for the survival, development and function of neurons in the peripheral and central nervous systems (CNS) Neurotrophins in the brain Deister et al 2006 www.science-autism.org

  4. Neurotrophin Family Members • Nerve Growth Factor • Brain-derived neurotrophic factor (BDNF): • Active in areas, such as the hippocampus • Activates the TrkB receptor • Neurotrophin 3 (NT-3): • Stimulates a number of neurons • Activate two of the receptors (TrkC and TrkB) • Neurotrophin 4 (NT-4): • Recently discovered and least understood • Activates the TrkB receptor Reichardt, et al 2006 Marinova et al 2007,

  5. Nerve Growth Factor (NGF) • Discovered in 1950 by Rita Levi-Montalcini • Activates TrkA receptor • 118 aminoacid polypeptide: • α,β, and γ • Mutation in gene encoding TrkA leads to a role in pain production www.als.lbl.gov/als/science/sci_archive/89receptor.html Marinova et al 2007, Thacker et al. 2007

  6. Neurotrophin Receptors • Tropomyosin-related kinase (Trk) receptor: • Ligand specific • High affinity receptor • TrkA, TrkB, TrkC • P75 neurotrophin receptor (p75NTR): • Tumour necrosis receptor superfamily • All neurotrophins bind with similar affinity • Low affinity recepter Kondyli et al 2005, Nockher et al. 2006

  7. p75NTR NGF Trk A Cytoplasm TRAF-6 Sh2 SOS Grb2 MAP kinase Pathway NIK Ras NF-kB pathway Raf NF-kB IKK IkB MEK IkB MAPK Nucleus Prosurvival genes Prodifferentation genes Neurotrophin Signaling Pathway NF-kB After Reichardt 2006, Kondyli et al 2005, Molecular Cell Biology, 5 edition

  8. NFG Interacts with Immune cells • NFG has a role outside the nervous system. • Current data, • Show that cells of the immune system express NGF • NGF can influence the developing immune response • Mast cells were identified as the first non-neuronal cell type influenced by NGF. Micera et al 2003

  9. Where do Mast Cells Come From? Immunobiology (7edition)

  10. Mast cell degranulation Mast cell intact Mast Cells (MC) & Activation • First responders to infection • Key effector cells of allergic inflammation • Contains many granules rich in histamine and heparin • Stimulated to degranulate by: • Cross-linking of Immunoglobulin E (IgE) receptors • NT-3? • NGF?? • Releases its granules and various hormonal mediators: • Histamine • Tryptase www.jralphb.co.uk/allerg.htm Bischoff et al. 2007

  11. Bidirectional Communication between NGF and MCs • Allergies: are caused by an immune response to a normally harmless substance • Immune response is damaging rather than helpful to the host • Immediate responses • NGF plasma levels are increased in times of stress (allergic reactions) • Mast cells numbers are also increased • Mast cells depend on NGF for homing, survival and differentiation • Could NGF possibly act as a chemoattractant for these immune cells? Vega et al. 2003

  12. Histamine Important protein involved in many allergic reactions The release of histamine cause allergic symptoms Itching, burning sensation, sneezing and wheezing Tryptase Secretory granule-derived serine protease contained in mast cells Marker for mast cell activation Histamine and Tryptase www.bio.davidson.edu/courses/Immunology/

  13. Allergic Diseases • Atopic Dermatitis: chronic inflammatory skin disease • Associated with elevated IgE levels, eosinophils, and mast cell hyperplasia and NGF? • Any age (early infancy) • Affects both males and females • Not contagious • Extremely itchy skin Pfaar et al. 2009 www.derm-student.com/Atopic_dermatitis.html

  14. Epidermal keratinocytes IgE antibody Fc receptor TrK A NGF ? Histamines Allergic Inflammation Itch Itch! Allergen Mast Cell After Nockher et al. 2006

  15. Hypothesis • Nerve growth factor (NGF) activates mast cells during allergic inflammation.

  16. Overview • Support Hypothesis: • Clinical Relationship between NGF and allergic diseases in mice • Molecular level of mast cell activation by NGF in mouse line • Refute Hypothesis: • No clinical relationship of NGF with allergic diseases • Mast cell activation in vivo studies of another neurotrophin

  17. Experimental Results:Support Hypothesis

  18. Parent Mice ♂ DS-Nh (Nh/Nh) X ♀ DS (+/+) F1 Generation ♂ ♀ Mice DS-Nh (Nh/+) Mouse Model for Atopic Dermatitis Raised under conventional conditions and after 1-2 weeks develop AD-like dermatitis symptoms, erythema, dry skin, etc. Yoshioka et al. 2005

  19. NGF, IgE and histamine levels increased in DS-Nh mice Conventional: exposed to S. aureus SPF: free from S. aureus Levels increased only in DS-Nh mice conventionally housed. Yoshioka et al. 2005

  20. No Y-axis NGF increased in Human Patients with Atopic Dermatitis Control: 31 subjects (no history allergies) 18males, 13 females, mean age of 27 AD: 42 subjects with (AD) 26 males, 16 females, mean age of 24.9 (B): NGF levels Significant increase NGF in patients with AD in comparison to the control subjects with no history of allergies (p<0.005). Toyoda et al .2003

  21. NGF Activates Mast Cells Directly (A)Tryptase Activity with different concentrations of NGF (B) Tryptase Activity w/ or w/o NGF (C) Histamine Activity w/ or w/o NGF Black bars= with NGF White bars= without NGF Groneberg et al. 2005

  22. NGF induces TrkA receptor mRNA expression in Human Mast Cell line-1 Lane 1: No cDNA Lane 2: 1 day with NGF Lane 3: 2 days with NGF Lane 4: 4 days with NGF Lane 5: 10 days with NGF HD= Histidine decarboxylase Tryptase and TrkA mRNA levels increased in cells cultured in NGF for 1 to 10 days. Groneberg et al. 2005

  23. p75NTR Expression Increased in Lesional MCs in Atopic Dermatitis Skin N: Normal skin AD: Atopic Dermatitis (A) The neurotrophin p75 expression in found in skin cells of patients with AD. (B) p75 expression was significantly increased in MC of patients with AD. Fischer et al. 2008

  24. Experimental Results:Refute Hypothesis

  25. STAGE 3 STAGE 2 STAGE 1 SCORAD • SCORing Atopic Dermatitis: A clinical tool for assessing the severity of atopic dermatitis. • Takes into account two main criteria: • The Extent • The Intensity of the following symptoms: • Erythema, edema/papulation, oozing/crust, excoriation, lichenification and dryness • Subjective symptoms: • Pruritus and sleep loss adserver.sante.univ-nantes.fr/Scorad.html Schulte-Herbruggen et al. 2007

  26. AD: 57 patients (31 males, 26 females, mean age 33 yrs) Healthy controls: 50 volunteers (26 males, 24 females, mean age 48 yrs) NGF serum concentrations in AD patients do not differ from the control. No correlation between NGF levels and severity of AD No Correlation Between NGF levels and Atopic Dermatitis Schulte-Herbruggen et al. 2007

  27. NGF Does Not Promote Growth of Human Intestinal Mast Cells SCF=Stem Cell Factor NT-3=Neurotrophin-3 • SCF is needed to promote MC recovery. • Synergistic effect of SCF with NT-3. • No significant increase when NGF is added. Lorentz et al. 2007

  28. NT-3 Activates Mast Cells in vivo in Mice Skin In Vivo Study Biopsies of back skin of C57BL/6 mice • NT-3 induces the degranulation • of skin MCs Metz et al. 2004

  29. Conclusion

  30. Summary Yoshioka et al. 2005 NGF, histamine and IgE levels are increased in DS-Nh mice NGF levels are Increased human patients with AD Support Hypothesis Fischer et al. 2005 NGF activates MC directly (TrkA recptor) Groneberg et al. 2005 NGF p75NTR receptor expression is increased in lesional MC Toyoda et al. 2003 Lorentz et al. 2007 NGF levels are not increased in human patients with AD Refute Hypothesis NGF does not promote growth of human intestional MCs Schulte-Herbruggen et al. 2007 NT-3 activates MCs in vivo Metz et al. 2004

  31. Histamine Fc NGF MCs Allergies TrkA Tryptase Take Home Message • In allergic diseases (atopic dermatitis) NGF levels are increased. • The NGF receptors, TrkA and p75NTR are expressed on MCs. • NGF activates mast cells to release mediators during allergic inflammation.

  32. Conclusion • Based on my finding my hypothesis is supported: • Nerve growth factor activates mast cells during an allergic inflammation. • Mast cells are activated directly by NGF in HMC-1 (Groneberg et al 2005). • Mast cells are activated by SCF and NT-3 the paper was poorly written and they measure MC recovery using trypan blue (measures dead cells) (Lorentz et al 2007).

  33. Marker PBS Untreated DS-Nh Treated DS-Nh DS-Nh mice Make antibody against NGF DS-Nh mice No Anti-NGF Inject Anti-NGF 1 2 3 4 Remove tissue Remove tissue Isolate MC Isolate MC Less expression ofNGF RT-PCR RT-PCR SCORAD SCORAD Future Experiments Block nerve growth factor (NFG) in DS-Nh mice Yoshioka et al 2005

  34. References • Fischer, T.C., Lauensteinw, H.D., Serowkaz, F., Pilznerw, C., Groneberg, D.A. and Welker, P. (2008) Pan-neurotrophin receptor p75NTR expression is strongly induced in lesional atopic mast cells. Clinical and Experimental Allergy 38, 1168–1173. • Groneberg, D.A., Serowka, F., Peckenschneider, Artuc, M., Gruzkau, A., Fischer, A., Henz, B.M., and Welker, P. (2005) Gene expressionand regulation of nerve growth factor in atopic dermatitis mast cells and the human mast cell line-1. Journal of Neuroimmunology 161, 87- 92. • Kondyli, M., Varakis, J., and Assimakopoulou, M. (2005) Expression of p75NTR and Trk neurotrophin receptors in the enteric nervous system of human adults. Anatomical Science International 80, 223-228. • Lorentz, A., Hoppe, J., Worthmann, H., Gebhardt, T., Hesse, U., Bienenstock, J., and Bischoff. S.C. (2007) Neurotrophin-3, but not nervegrowth factor, promotes survival of human intestinal mast cells. Neurogastroenterol Motil 19, 301-308. • Marinova, T., Philipov, S. and Aloe, L. (2006) Nerve Growth Factor Immunoreactivity of Mast Cells in Acute Involuted Human Thymus. Inflammation 30, 38-42. • Metz, M., Botchkarev, V.A., Botchkareva, N.V., Welker, P., Tobin, D.J., Knop, J., Maurer, M. and Paus, R. (2004) Neurotrophin-3 regulates mast cell functions in neonatal mouse skin. Experimental Dermatology 13, 273-281. • Pfaar, O., Raap, U., Holz, M., Hormann, K., and Klimek, L. (2009) Pathophysiology of itching and sneezing in allergic rhinitis. Swiss Med Weekly 139, 35-40. • Schulte-Herbruggen, O., Folster-Holst, R., Von Elstermann, M., Augustin, M., and Hellweg, R. (2007) Clinical Relevance of Nerve Growth Factor Serum Levels in Patients with Atopic Dermatitis and Psoriasis. Int Arch Allergy Immunology 144, 211-216. • Toyoda, M., Nakamura, M., Makino, T., and Morohashi. (2003) Localization and content of nerve growth factor in peripheral blood eosinophils of atopic dermatitis patients. Clinical and Experimental Allergy 33, 950-955. • Yoshioka, T., Hikita, I., Asakawa, M., Hirasawa, T., Deguchi, M., Matsutani, T., Oku. H., Horikawa, T., and Arimura, A. (2006) Spontaneous scratching behavior in DS-Nh mice as a possible model for pruritus in atopic dermatitis. Immunology 118, 293-301.

  35. References • Deister, C. and Schmidt, C.E. (2006) Optimizing neurotrophic factor combinations for neurite outgrowth. J. Neural Eng.3, 172-179 • Reichardt, L.F. (2006) Neurotrophin-regulated signaling pathways. Philosophical Transactions of the Royal Society 361, 1545-1564. • Thacker et al. (2007) Pathophysiology of Peripheral Neuropathic Pain: Immune • Cells and Molecules. Anesthesia and analgesia105, 839-847. • Nockher, W.N., Renz, H., (2006) Neurotrophins in allergic diseases: From neuronal growth factors to intercellular signaling molecules. Journal of Clinical Immunology117, 583-588. • Lodish, et al., Molecular Cell Biology, 5 edition • Micera, A. et al. (2003) Nerve growth factor and tissue repair remodeling: trkA and p75NTR, two receptors one fate. Cytokine & Growth Factor Reviews18, 245–256. • Janeway, et al., 7 edition 2008, Garland Publishing. • Bischoff, S.C. (2007) Role of mast cells in allergic and non-allergic immune response: comparison of human and murine data. Nature Reviews7, 93-103. • Vega, J.A., Garcia-Suarez, O., Hannestad, J., Perez-Perez, M., and Germana, A. (2003) Neurotrophins and the Immune system. J. Anat.203, 1-19. • www.bio.davidson.edu/courses/Immunology/ • www.jralphb.co.uk/allerg.htm • www.als.lbl.gov/als/science/sci_archive/89receptor.html • www.science-autism.org • www.derm-student.com/Atopic_dermatitis.html

  36. Questions?

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