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

During Allergic Inflammation

Natalie SandovalBiol 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)

Deister et al 2006 www.science-autism.org

Neurotrophins in the brain

Neurotrophin Family Members Nerve Growth FactorNerve 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,

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

Thacker et al. 2007

www.als.lbl.gov/als/science/sci_archive/89receptor.html

Marinova et al 2007,

Neurotrophin Receptors

Kondyli et al 2005, Nockher et al. 2006

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

After Reichardt 2006,

NF-kB

Kondyli et al 2005,

Neurotrophin Signaling Pathway

Trk A

IKK

NF-kBIkB

Prosurvival genes

p75NTR

TRAF-6

NIK

IkB

Sh2

Grb2SOS

Ras

Raf

MEK

MAPK

Prodifferentation genes

Cytoplasm

Nucleus

NGF

MAP kinase Pathway

NF-kB pathway

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

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

Bischoff et al. 2007

www.jralphb.co.uk/allerg.htm

Mast cell degranulation

Mast cell intact

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

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

www.derm-student.com/Atopic_dermatitis.htmlPfaar et al. 2009

Allergic Inflammation

After Nockher et al. 2006

Itch

Itch!

Fc receptor

IgE antibody

Histamines

TrK AMast Cell

Allergen

Epidermal keratinocytes

NGF ?

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

Yoshioka et al. 2005

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.

NGF, IgE and histamine levels increased in DS-Nh mice

Yoshioka et al. 2005

Conventional:Conventional: exposed to S. aureus

SPF:SPF: free from S. aureus

Levels increased only in DS-Nh mice conventionally housed.

NGF increased in Human Patients with Atopic Dermatitis

Toyoda et al .2003

ControlControl: 31 subjects (no history allergies)18males, 13 females, mean age of 27

AD:AD: 42 subjects with (AD)26 males, 16 females, mean age of 24.9

(B):B): NGF levels

Significant increase NGF in patients with AD in comparison to the control subjects with no history of allergies (p<0.005).

No Y-axis

NGF Activates Mast Cells Directly

Groneberg et al. 2005

((A)A) Tryptase Activity with different concentrations of NGF

(B)(B) Tryptase Activity w/ or w/o NGF(C)(C) Histamine Activity w/ or w/o

NGF

Black bars= with NGFWhite bars= without NGF

NGF induces TrkA receptor mRNA expression in Human Mast Cell line-1

Groneberg et al. 2005

Lane 1:Lane 1: No cDNALane 2:Lane 2: 1 day with NGFLane 3:Lane 3: 2 days with NGFLane 4:Lane 4: 4 days with NGFLane 5:Lane 5: 10 days with NGF

Tryptase and TrkA mRNA levels increased in cells cultured in NGF for 1 to 10 days.

HD= Histidine decarboxylase

p75NTR Expression Increased in Lesional MCs in Atopic Dermatitis Skin

Fischer et al. 2008

N: Normal skinAD: 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.

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/crustoozing/crust,, excoriation, lichenification and dryness

Subjective symptoms:– Pruritus and sleep loss

Schulte-Herbruggen et al. 2007adserver.sante.univ-nantes.fr/Scorad.html

STAGE 1

STAGE 2

STAGE 3

No Correlation Between NGF levels and Atopic Dermatitis

Schulte-Herbruggen et al. 2007

NGF serum concentrations in AD patients do not differ from the control.

No correlation between NGF levels and severity of AD

AD: 57 patients(31 males, 26 females, mean age 33 yrs)

Healthy controls: 50 volunteers(26 males, 24 females, mean age 48 yrs)

NGF Does Not Promote Growth of Human Intestinal Mast Cells

Lorentz et al. 2007

(A)SCF is needed to promote MC recovery.(B)Synergistic effect of SCF with NT-3.(C)No significant increase when NGF is added.

SCF=Stem Cell Factor

NT-3=Neurotrophin-3

NT-3 Activates Mast Cells in vivo in Mice Skin

Metz et al. 2004

(A) NT-3 induces the degranulation of skin MCs

In Vivo Study

Biopsies of back skin of C57BL/6 mice

Conclusion

Summary

Support Hypothesis

Refute Hypothesis

Yoshioka et al. 2005

Fischer et al. 2005

Toyoda et al. 2003

Groneberg et al. 2005

Metz et al. 2004

Schulte-Herbruggen et al. 2007

Lorentz et al. 2007

NGF, histamine and IgE levels are increased in DS-Nh mice NGF levels are Increased human patients with AD

NGF activates MC directly (TrkA recptor)

NGF p75NTR receptor expression is

increased in lesional MC

NGF levels are not increased in human

patients with ADNGF does not promote growth of human intestional MCs

NT-3 activates MCs in vivo

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.

NGF

Histamine

TryptaseTrkA

MCs

Fc

Allergies

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).

Future Experiments

Yoshioka et al 2005

Block nerve growth factor (NFG) in DS-Nh mice

1 2 3 4

Mar

ker

PB

SU

ntre

ated

DS

-Nh

Tre

ated

DS-

Nh

Less expression ofNGF

Remove tissue

Inject Anti-NGF No Anti-NGF

Make antibody against NGF

Remove tissue

IsolateMC

IsolateMC

RT-PCR

DS-Nh mice DS-Nh mice

SCORADSCORAD

RT-PCR

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.

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 analgesia 105, 839-847. Nockher, W.N., Renz, H., (2006) Neurotrophins in allergic diseases: From neuronal growth factors

to intercellular signaling molecules. Journal of Clinical Immunology 117, 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 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?