Transcript Mast Cells are Major Target of Neuronal Substance P to Induce

Substance P Mediate Activation of Mast cell
by Allowing Nerve-mast Cell interaction
California State University, Los Angeles
Bio 520: Advanced Immunology Seminar
Abby Yilma: 2/2/09
Background
•
•
•
•
•
•
Neurotransmitters
Substance P
Receptor molecule
Mast cells
Adhesion molecule
Nerve-mast cell interaction
Neurotransmitter
• Chemicals that transmit nerve cell
impulse from one cell to another cell
– Can be neuron to neuron or neuron
to other cells such as immune cells
Neurotransmitter
in side vesicles
Vesicles fuse and release their
contents into the synapse
• The release of chemical is driven by
– Arrival of action potential at the
synapse
• Effect of a neurotransmitter usually
depends on
– Chemical properties of the receptors
Synaptic
cleft
Bind to a receptor on the
postsynaptic membrane
• Stimulatory or inhibitory
www.nature.com/.../fig_tab/nm0907-1018_F1.html
Class of Neurotransmitter Molecules
Molecules
Effects
Acetycholine (Ach)
Both stimulatory & inhibitory
Monoamine group (dopamine)
Stimulatory effects
Amino acid group (Gamma
aminobutyric acid)
Inhibitory effects
Peptide (Endorphaline, Enkephline, Inhibitory, Inhibitory, stimulatory
and Substance P)
Substance P (SP)
• Discovered by Von Euler and Gaddum in 1931
– Extracts of horse brain and intestine contained a
hypotensive and spasmogenic factor
– Later found to be protein
• Isolated and characterized by Leeman’s group in
1971
– Bovine hypothalamus
O’Connor et al., 2004
Structure of SP
• Polypeptide synthesized in
the ribosome and then
cleaved into undecapeptide
(11 aa)
– SP: Arg-Pro-Lys-Pro-Gln-GlnPhe-Phe-Gly-Leu-Met-NH2
• Belongs to tachykinin family
– Share common carboxyterminal sequence
– Essential for receptor
interaction and activation
O’Connor et al., 2004
Where is SP Found?
• Widely distributed in the sensory neurons (afferent
neuron)
– Physical stimulation such as touch, vision and hearing
– Chemical simulation such as capsaicin
• Active component of chilly peppers & produce sensation of burning
• Native to the gastrointestinal, respiratory tracts and
genitourinary tract
• Effect mediated by specific receptor called neurokinin-1
receptor (NK-1R)
– Expressed in nervous cells and immune cells
O’Connor et al., 2004 & Bulut et al., 2008
Neurokinin-1 Receptor (NK-1R)
•Glycoprotein (407 aa, 46 kDa)
•7 α-helical transmembrane loops
•Many serine and threonin residue
at cytoplasmic carboxyl terminal
• Interaction via the third cytoplasmic loop
• Cytoplasmic carboxyl terminus becomes phosphorylated
– Cause desensitization of the receptor in response of repeated
application of agonist
O’Connor et al., 2004
SP’s Role in Regulating Body
Functions
• In the central nerve system
• Participates in regulating neuronal survival and degeneration
• In the spinal cord
• Participates in neurotransmission of pain
• Immune cells (?) mast cells?
• Proinflammtory mediator
•
•
Induces inflammation
Contributes to chronicity of inflammatory response
O’Connor et al., 2004
Mast cells
• First describe by Paul Ehrlich in
1878
• A resident of several types of
tissues
Human skin mast cell grown on human
skin fibroblast feeder layers
– Contains many granules
• Visible by light microscope
– Rich in histamine
• Involves in local immune response
• Acts as neurotransmitter
– Located in close proximity to nerves
• Skin, intestinal mucosa
www.aad.org/education/students/mastcellsfig1.htm
Ito et al., 2006
Activation of Mast Cells
Degranulating
Mast cells
• Direct injury
– Physical or chemical
• Cross-linking of immunoglobin (IgE)
– Through Fc epsilon RI (FcєRI)
• Complement protein
– Produced in response to evasion
• Substance P (?)
http://instructional1.calstatela.edu/eporter/BIOL520/
Molecules Released By Mast Cells into the
Extracellular Environment
• Preformed mediators from the granules
– Histamine
– Heparin (anticoagulant)
– Serine (phosphorylated by kinase during cell singling)
• Newly formed lipid mediators from cell membrane
– Prostaglandia D2 (inhibitor of platelet aggregation)
– Leukotrien C4 ( act on inflammatory response)
• Cytokines
– Critical for development and function of innate and adaptive immunity
– Example: IL-3, TNFa, and GM-CSF
• GM-CSF stimulates stems cells to produce granulocytes and monocytes
• Molecules that can influence neuronal activity upon interaction with
nerve cells (?)
Bulut et al., 2008
Molecules from Mast Cells That Act on
Nerve Cells
• Tryptase
– Granule-derived serine proteinase
– Used as marker for mast cell activation
• Histamine
– Released as a neurotransmitter
– Regulating physiological function in the gut
• Binds to proteinase- activated receptors 1 and 2
Ito et al., 2006
Nerve-mast Cell Interaction
• Is a true chemical synapsis formed during the
communication?
Ito et al., 2006 & Furuno et al., 2005
Adhesion Molecules Common to
Nerves and Mast cells
• In 2003, Ito and colleagues
discovered
– Synaptic cell adhesion molecules
(SynCAM) or spermatogenic
immunoglobulin superfamily
(SgIGSF)
• In later work showed
– SgIGSF/SynCAM is localized on
both side of most synapses
– Functions as a homophilic or
heterophilic adhesion molecule
• Depending the binding partners avialable
– Can span the synaptic cleft
– Drives synapse assembly
Ito et al., 2006 and Furuno et al., 2005
Hypothesis
• Mast cell activation requires not only a direct cell to cell
interaction between nerve fibers and mast cells but also
substance P, which is the main transmitter of nerve fibers
Research Articles
• In support of the hypothesis
– Proximity between nerve and mast cells
– Importance of MC/NC crosstalk in MC-driven inflammatory skin
responses
– Mast cells activation in the presence of Adhesion molecule
• Refuting the hypothesis
– Receptor-mediated SP activation of mast cells event
– Activation of mast cells by substance P in vitro without nerve
cells
In support of the hypothesis
• Importance of MC/SN crosstalk in MC-driven
inflammatory skin responses
– Mice were anesthetized
– Surgically removed the sensory nerves from the ear skin
• Employed histochemical/immunohistochemical detection technique
using SN marker proteins (SP)
– To test the importance of nerve function, denervated and control
skin sites were treated with capsaicin and vehicle (control)
• Capsaicin induces strong cutaneous neurogenic inflammation in
normal skin
• Measure increase in ear thickness at different time points using
caliper
Denervated Ear Skin Fail to Develop Inflammatory
Reaction in Response to Capsaicin
Control
d = dermis
dp = dermal papilla
e = epidermis
sg = sebaceous gland
p < 0.05 ; *** p < 0.005
n=3
Nerve fiber
presented at
physiological site
Suggests the importance of MS/SN crosstalk
Denervation results in loss of functional skin nerves
Siebenhaar et al., 2008
To examine the Proximity of Mast to
Nerve cell
• Mast cell distribution and proximity with nerve elements
in urinary bladder and stomach
– Normal tissue (+/+) from mouse bladder and stomach
– Performed histochemical/Immunohistochemistry
• For mast cells (stained with toluidine blue)
• For nerve cells detection (neuron-specific enolase (NSE) polyclonal
antibody)
– Examined the submucosa layers in both tissues
The Bladder and Stomach Contained Nerve Cells
in Higher Proximity to Mast Cells
Mast cell = black overhead
Blood vessels = black arrow
Nerve elements = white
overhead
Blood vessels play a
role in virtually every
medical condition
The distribution of the
mast cell and its
contact with nerve
element is varied in
both tissues
Andrea et al., 2005
• The role of SP on mast cells activation in vivo study
– Female mice aged 8 weeks maintained for 1 week
– Psychological stress (PS) and foot shock stress (FS) were
administered
– Killed the mice by decapitation
– Analyze the skin portion using histochemistry /immunohistochemistry
• Counted degranulated mast cells outside of the cell membrane and
calculated the ratio of the degranulated mast cell to all mast cells
• Determined SP-positive nerve fibers
Degranulated Mast Cells and SP-positive
Nerve Fibers Increased with Stressed Mice
Control
After stress was administered , the
ratio of degranulated of mast cells
increased significantly following FS
and PS
After FS and PS was
administered, the number of SPpositive nerve fibers per/skin area
was increased significantly
SP-positive nerve fibers were
detected in the upper and mid
dermis
*** p < 0.001
Substance P-positive nerve fibers in the skin
Kawana et al., 2006
• Examine whether SgiGSF adhesion molecule involves
in Mast cell degranulation
– 8 to 10-week-old mice
– Remove the mesentery portion
• Surrounds part of small intestine
– Stimulated mesentery part with capsaicin and electric pulse
• The electric pulse applied at their nerve root
– Employed histochemical/immunohistochemistry
• Stained with alcian blue and nuclear fast red
– Counted degranulated mast cells
Capsaicin Caused Mesenteric mast cell
Degranulation
Degranulated stage = overheads arrow
Arrows = stable stage
This doesn’t rule out the possibility that capsaicin might directly
activate mast cells, so the stimulated mesenteric nerve at their
root
1/3 of mast cells were degranulated after
capsaicin , which suggest that capsaicin
caused degranulation
Ito et al., 2007
SgIGSF Mediated Attachment of Mast Cell to
Neuron
Electric simulation will induce mast cells degranulation
through releasing neurotransmitters from nerves
* p < 0.05
n=7
>1/3 degranulated
Both group failed to activate mast cells
Mi wh = lack SgIGSF
mivit = express less
SgIGSF
SP = nerve
impulse
transmitted
Degranulation
restored
Ito et al., 2007
Refuting the Hypothesis
• Mast cells dependent on NK-1R to develop relationship
with nerve cells
– 10-to-12 week-old mice lack NK1-R (NK1-R-/-) and mast cell with no
expression of NK1-R (NK1-R BMR kit)
– Removed tissue from bladder mucosa
– Employed histochemistry/immunohistochemistry
• For mast cells stained with toluidine blue
• For nerve cells detection (neuron-specific enolase (NSE) polyclonal
antibody)
– Determined mast cells distribution
– Assessed the contact between mast and nerve cells
NK1-R Expression Affect the Contact Between
Mast and Nerve Cells
Mast cell = black overhead
Blood vessels = black arrow
Nerve elements = white overhead
Suggests the contact between mast and nerve
cells is mediated through NK1-R
NK1-/- BMR kit = mast cell with no expression of NK1-R
Ercan et al., 2006
• Substance P activates mast cells without nerve cells in
Vitro
– Culture human peripheral blood-derived CD34+ (HuMC) and
LAD2 mast cells overnight
• HuMC is primary cell line and LAD2 isolated from tissue
– Degranulation assay
• Cells were sensitized overnight with myeloma (cancer cells) IgE
• Cell were preincubated overnight with IgE and then stimulated with
anti-IgE and SP
– FcєRI mediated activation
• Measured β-hexosaminidase and cytokines release into
supernatant
LAD2 Stimulated with SP Released
More β-hexosaminidase
Potent activators of LAD2
SP activated LAD2 to degranulate
Kulka et al., 2007
SP Activated Mast cells to Produce Large
Cytokines
HuMC cells
N = 3; p< 0.01
SP stimulated HuMC cells to produce
small quantities of TNF GM-CSF and IL-3
LAD2 cells
SP induced LAD2 cells to produce large
quntities of TNFα, GM-CSF and IL-3
Suggests that SP can activate mast cells without nerve cells
Kulka et al., 2007
• Substance P fail to activate mast cells in human intestine
– Human mast cells isolated from normal intestinal tissue were cultured
for 1-2 weeks
– Mast cells were challenged for 1 h with different Neuropeptide, including
SP, mAB29C6, and ionomycin
• mAB29C6 and ionomycin induces IgE receptor crosslinking used as positive
controls
– Measured histamine and sulphidoleukotriene (sLT) released in cell-free
supernatants using radioimmunoassay
SP Fail to Activate Mast cells Significantly
** p < 0.001; n = 2
Positive controls
Positive controls
Control
Control
Not only SP but also other neuropeptide fail to activate mast cells
Bischoff et al., 2004
Summary: Supporting the Hypothesis
• Siebenhaar et al., 2008
– Importance of MC/SN cross talk
to develop inflammatory
reaction
• Andrea et al., 2005
– Nerve cells are in higher
proximity to mast cells
Capsaicin
• Kawana et al., 2006
– Activation of mast cells by SP
and mast-nerve cells interaction
• Ito et al., 2007
– Importance of adhesion
molecules for mast-nerve cells
interaction
Degranulation
Summary: Refuting the Hypothesis
• Ercan et al., 2006
– Receptor-mediated SP activation
of human mast cells event
– Questions the direct interaction
• Kulka et al., 2007
– Substance P activates mast cell
in vitro without nerve cell
– Arguing nerve cells are not
required for mast activation
NK1-R
• Bischoff et al., 2004
– Failure of SP to activate mast
cells
– Suggests that SP is not required
for mast cells activation
Capsaicin
Degranulation
X
X
Follow up Experiment
• Using human model to characterize the presence of
SgIGSF expression on other immune cells
Inflammation response in reddish
Mast cells
SP
Nerves
SgIGSF
Basophile
Capsaicin treated
skin tissue
Machrophage
www-biology.ucsd.edu/news/article_030603.html
Conclusion
• The different results presented from different research
group on SP’s role and MC/SN crosstalk on mast cell
activation suggests further investigation in this area,
specially using human model
• The conflicting results can place mast cells at the border
of potential critical host-pathogen interactions
Study on Proinflammatory Effects of SP
in Immune Cells
• Involves observation of abnormal level of SP, SP-nerve fibers, NK1R in disease tissue
– Knockout experiment in animal model and coculture appraoch
O’Connor et al., 2004