Transcript Biol 520_NS_Presenation_March 9 - Cal State LA

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
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
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
Marinova et al 2007, Reichardt, et al 2006
Nerve Growth Factor (NGF)

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

Discovered in 1950 by Rita LeviMontalcini
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
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
Neurotrophin Signaling Pathway
p75NTR
NGF
Cytoplasm
TRAF-6
Trk A
Sh2
SOSGrb2
MAP kinase Pathway
NIK
Ras
NF-kB pathway
Raf
IKK
MEK
IkB NF-kB
IkB
NF-kB
Nucleus
Prosurvival genes
MAPK
Prodifferentation
genes
After Reichardt 2006, Kondyli et al 2005, Molecular Cell Biology, 5 edition
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
Where do Mast Cells Come From?
Immunobiology (7edition)
Mast Cells (MC) & Activation


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Mast cell degranulation
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??
Mast cell intact
Releases its granules and various
www.jralphb.co.uk/allerg.htm
hormonal mediators:
– Histamine
– Tryptase
Bischoff et al. 2007
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

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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
Histamine and Tryptase

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
www.bio.davidson.edu/courses/Immunology/
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
Allergen
Allergic Inflammation
Epidermal keratinocytes
IgE antibody
Fc receptor
TrK A
Mast Cell
NGF ?
Histamines
After Nockher et al. 2006
Hypothesis
 Nerve
growth factor (NGF) activates
mast cells during allergic inflammation.
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
Experimental Results:
Support Hypothesis
Mouse Model for Atopic Dermatitis
Parent Mice
♂ DS-Nh (Nh/Nh) X ♀ DS (+/+)
F1 Generation
♂ ♀ Mice DS-Nh (Nh/+)
Raised under conventional
conditions and after 1-2
weeks develop AD-like
dermatitis symptoms,
erythema, dry skin, etc.
Yoshioka et al. 2005
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
NGF increased in Human Patients
with Atopic Dermatitis
No Y-axis
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
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
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
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
Experimental Results:
Refute Hypothesis
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
STAGE 3
 Subjective symptoms:
– Pruritus and sleep loss
STAGE 2
STAGE 1
adserver.sante.univ-nantes.fr/Scorad.html Schulte-Herbruggen et al. 2007
No Correlation Between NGF levels
and Atopic Dermatitis
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
Schulte-Herbruggen et al. 2007
NGF Does Not Promote Growth of
Human Intestinal Mast Cells
SCF=Stem Cell Factor
NT-3=Neurotrophin-3
(A)SCF is needed to promote MC recovery.
(B)Synergistic effect of SCF with NT-3.
(C)No significant increase when NGF is added.
Lorentz et al. 2007
NT-3 Activates Mast Cells
in vivo in Mice Skin
In Vivo Study
Biopsies of back skin of
C57BL/6 mice
(A) NT-3 induces the degranulation
of skin MCs
Metz et al. 2004
Conclusion
Summary
Yoshioka et al. 2005
Fischer et al. 2005
Support Hypothesis
Groneberg et al. 2005
Toyoda et al. 2003
Lorentz et al. 2007
Schulte-Herbruggen
et al. 2007
Metz et al. 2004
Refute Hypothesis
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.
Histamine
NGF
Allergies
MCs
Tryptase
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).
Block nerve growth factor (NFG) in DS-Nh mice
Make antibody
against NGF
Inject Anti-NGF
No Anti-NGF
Marker
PBS
Untreated DS-Nh
Treated DS-Nh
Future Experiments
1 2 3 4
DS-Nh mice
DS-Nh mice
Remove
tissue
Remove
tissue
Isolate
MC
Isolate
MC
Less expression ofNGF
RT-PCR
SCORAD
RT-PCR
SCORAD
Yoshioka et al 2005
References
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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.
References
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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
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Nockher, W.N., Renz, H., (2006) Neurotrophins in allergic diseases: From neuronal growth factors
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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 Reviews 18, 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 Reviews 7, 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
Questions?