Transcript Alpha-MSH - Cal State LA - Instructional Web Server

Modulatory Functions of
-MSH Neuropeptide in
the Immune System
Saray Felix
Department of Biological Sciences- California
State University Los Angeles
Winter 2009
Evolution
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Prokaryotes
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Eubacteria (true bacteria)
Archaeabacteria
Protists
Eukaryotes
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Fungi
Plants
Invertebrates and Arthropods
Vertebrates
www.bordalierinstitute.com/.../evolutionTree.gif
(Murphy, K. Janeway’s Immunobiology 2008)
Evolution of the Immune System
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Ubiquitous Immune System
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Every organism is attacked by
pathogens
Response Mechanism
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Provides protection against
invading pathogens
Similar strategies across phyla with
diversity in response
biology.creighton.edu/courses/BIO432/Evolution%2005.htm
(Murphy, K. Janeway’s Immunobiology 2008)
Evolution of the Innate Immune
System
Fly
Snail
Mollusk Amphioxus Lamprey
www.nature.com/.../n6/images/ni0607-547-F1.jpg
Teleost
Chicken Mouse/Human
(Litman and Cooper, Nature Immunology 2007)
Evolution of Innate Immunity
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Phagocytosis
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Antimicrobial Peptides
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Defensins- in many
multicellular organisms
including plants
Differ in structural detail
Pattern Recognition
Receptors
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Not observed in plants
Many invertebrates and all
vertebrates
Toll homologs found in many
species- from plants to
mammals
Complement System
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Not observed in plants
Many invertebrates and
vertebrates
biology.creighton.edu/courses/BIO432/Evolution%2005.htm
(Murphy, K. Janeway’s Immunobiology 2008)
First Line of Defense: Skin
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Skin acts as a barrier for
pathogens
Epidermal cells contain
necessary immune cells
to activate a response
against pathogens
α-MSH and skin
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Express and secrete αMSH
(Maaser et al., N.Y. Acad. Sci. 2006)
POMC
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Pro-opiomelanocortin
Precursor Polypeptide
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Contains 241 aa residues
Undergoes posttranslational cleavage
Proteases
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Subtilisin enzymes- a prohormone convertase (PC)
that belongs to the Serine
protease group
Tissue-specific
(Bohm et al J. Invest. Dermatology 2006)
POMC Production
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It can be synthesized by:
Corticotroph cells in the anterior pituitary gland
 Melanotrope cells in the intermediate lobe of the
pituitary gland
 Most Neurons in the arcuate nucleus or the
hypothalamus
 Few neurons in the dorsomedial hypothalamus and
in the brainstem
 Melanocytes in the skin
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-MSH
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Alpha-Melanocyte Stimulating Hormone
Neuropeptide- 13 amino acid peptide with the tripeptide 11-13
(KPV) aa containing the “active” sequence
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A hormone secreted by the anterior lobe of the pituitary gland that regulates
skin color in humans and other vertebrates by stimulating melanin synthesis
in melanocytes and melanin granule dispersal in melanophores
Anti-inflammatory properties
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Down-regulates pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α, etc.)
Up-regulates anti-inflammatory cytokines (IL-10, etc.
Anti-microbial properties
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Stimulates melanin production to generate oxygen radicals
Indirectly stimulates stimulates immune function
Directly inhibits pathogen proliferation
(Maaser et al., N.Y. Acad. Sci. 2006)
-MSH Receptors
Melanocortin Receptors (MCR): G-protein-linked receptors whose transduction
occurs via induction of adenylyl cyclase and an increase in cAMP
 MC1R- highest α-MSH affinity
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MC2R
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Activation effects are related to feeding and energy homeostasis
Expressed in the CNS, gastro-intestinal system (stomack, duodenum, pancreas), and kidneys
MC4R
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Stimulates secretion of adrenal steroids
Detected in adrenal cortex, skin, murine adipocytes
MC3R
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Key player in regulation of pigmentation in mammals and inflammation
Detected in melanocytes, neutrophils, monocytes, dendritic cells, endothelial cells, Blymphocytes, glioma cells, and astrocytes
Activation effects are related to feeding behavior, energy homeostasis, erectile function,
inflammation, and neuroprotection
Expressed primarily in CNS (hypothalamus, vagus nerve, basal ganglia, hippocampus, and
cerebral cortex
MC5R
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Might mediate stimulation of the production of pheromones, influencing sexual behavior
Expressed in exocrine glands (sebaceous, Harderian, lacrimal, and preputial glands)
(Lasaga et al., Peptides 2008)
MelanoCortin Receptors
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Receptors for -MSH in the skin can be found on:
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Epidermal melanocytes and keratinocytes
Endothelial cells
 Fibroblasts
Mast cells
 Dermal Papilla cells
 Sebocytes
Adipocytes
(Bohm et al. J. Invest. Dermatology 2006)
-MSH and Melanin
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Stimulates Melanocytes via
MC1R
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bric.postech.ac.kr/…/images/kcib-5-3.jpg
-MSH increases cAMP and
PKA in melanocytes
In turn increases PKC and
Tyrosinase mRNA synthesis
PKC activates Tyrosinase in the
melanosome (lysosome related
organelles in melanocytes
enriched with tyrosinase
proteins)
Tyrosine is synthesized into
Melanin
Melanogenesis
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Tyrosine, DOPA or L-DOPA
are Melanin precursors
End products are
Eumelanins (brown and
black pigments),
Pheomelanins (red and light
colored pigments) and O2
radicals
Melanins
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Bind metal ions and organic
cations to efficiently scavenge
for free reactive radicals
Synthesized by organisms
representative of all biological
kingdoms
(MacKintosh, J. Theoretical Biology 2001)
Melanin Storage
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Keratinocytes store the melanin granules
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Melanocytes transfer melanin granules via dendrites
Invertebrate Melanin
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Chromatophores
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Pigment containing and light-reflecting cells
Melanin: negatively charged hydrophobic
pigments
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Synthesized by the pro-Phenoloxidase (pro-PO)
pathway activation system activated upon
microbe infection
Dopa decarboxylase (Ddc) coverts dopa to
dopamine
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Phenoloxidase (PO) catalyses several steps of
the eumelanin pathway
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Involved in wound healing, parasite defense,
cuticle hardening, and melanization
last component of the pro-PO activation system
that ultimately results in melanin production
May be involved in blood clotting (activity may
strengthen protein-protein cross-links)
Acts as anti-microbial via direct killing
(Kan et al, J. Biological Chemistry 2008)
(Cerenius et al., Trends in Immunology 2008)
Hypothesis
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-MSH is a melanin activator
with innate immune functions in
vertebrates and invertebrates
Experimental Findings
POMC, the ACTH/melanocortin precursor, is secreted by
human epidermal keratinocytes and melanocytes and
stimulates melanogenesis
Rousseau et al, FASEB Journal 2007
85%
Increasing [POMC]
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63%
23%
POMC stimulated
cell pellets
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(A) POMC and
derivative peptides
induce melanogenesis
 -MSH is most
potent stimulator
molecule
(B & C) Higher levels
of POMC are needed
to stimulate
melanogenesis
(D) POMC stimulated
pigment cell dendricity
in a dose-dependent
manner
Human melanocytes express functional Toll-Like
Receptor 4
Ahn et al., Experimental Dermatology 2008
Lane 1: G3PDH
Lane 2: TLR4
Lane 3: CD14
Lane 4: MyD88
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RT-PCR analysis
demonstrate that
human
melanocytes
express TLR4,
CD14, and
MyD88 mRNA
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LPS treatment
induced
upregulation of
TLR4 and
MyD88 mRNA
in human
melanocytes
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LPS stimulated
melanin granule
production resulting
in increased melanin
pigmentation
Microbial induced
melanogenesis may
be activated via
TLR4
Activation of MC4R reduces the inflammatory response
and prevents apoptosis induced by LPS and IFN-γ
Caruso et al., Endocrinology 2007
Lane 1: blank
Lane 2: HP
Lane 3: Astrocytes
Lane 4: Liver
Lane 1: HP
Lane 2: Astrocytes
Lane 3: Liver
Lane 4: (-) control
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RT-PCR determined that
MC4R RNA but not
MC3R RNA is detected
in astrocytes
Western blotting detected
MC4R protein in
astrocytes
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-MSH attenuated the stimulatory
effects of LPS/IFN-γ on iNOS
mRNA and protein expression
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MC4R is involved in the antiinflammatory effect of -MSH in
astroglial cells
Antimicrobial effects of -MSH peptides
Cutuli et al., J. Leukocyte Biology 2000
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Fig. 1
Fig. 2
(1) -MSH peptides, at
various concentrations,
inhibited S. aureus colony
formation
 (2) -MSH peptides had
inhibitory effects on C.
albicans viability at
somewhat higher
concentrations
 -MSH peptides have
anti-microbial effects on
gram-positive (S.a.)
bacteria and fungus (C.a.)
Anti-microbial action of melanocortin peptides and
identification of a novel X-Pro-D/L-Val sequence in Grampositive and Gram-negative bacteria
Charnley et al., Peptide 2008
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Control
peptide: AAA
KPV peptide
APV peptide
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-MSH peptide 11-13
containing the LysineProline-Valine sequence
as well as the AlanineProline-Valine sequence
decreased microbe
viability in a dosedependent manner
-MSH peptide sequence
1-13 and 11-13 possess
anti-microbial effects
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There was no significant
differences between the
peptides
-MSH reduces the internalization of S. aureus and downregulates HSP 70, integrins and cytokine expression in
human keratinocyte cell lines
Donnarumma et al., Experimental Dermatology 2004
TNF-α
2hr
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24hr
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IL-8
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ICAM-1
Ctrl S.a. (S.a + α-MSH)
Ctrl
PA
PA + α-MSH
LTA
LTA + α-MSH
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S. aureus, protein A (PA), and
lipoteichoic acid (LTA) induced
the release of TNF-α, IL-8, and
ICAM-1 from human
keratinocytes
-MSH pre-incubated human
keratinocytes strongly downregulated TNF-α, IL-8, and
ICAM-1 release
-MSH lowers proinflammatory molecules in LTA
and PA stimulated keratinocytes
-MSH has protective roles at
the cutaneous level by reducing
infection and inflammatory
process
The immunomodulating neuropeptide -MSH
suppresses LPS-stimulated TLR4 with IRAK-M in
macrophages
Taylor, J. Neuroimmunology 2005
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APC spleen cells incubated with LPScontaminated ovalbumin enhanced
IFN-γ production
APC cells incubated with S.a.
ovalbumin were limited in stimulating a
strong IFN-γ production
LPS-stimulated APC cells pre-incubated
with -MSH could not enhance IFN-γ
production
S.a-stimulated APC cells pre-incubated
with -MSH did not change nor
enhaced the IFN-γ production
-MSH is limited to antagonizing a
TLR4-associated response in APC
Innate immunity in insects: surface-associated dopa
decarboxylase-dependent pathways regulate phagocytosis,
nodulation and melanization in medfly haemocytes
Sideri et al., Immunology 2007
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(b and c) Nodulation (aggregates) and
melanization is evident after 10+ mins
of bacterial infection in medfly
haemocytes
(d- g) No aggregates and reduced
melanization resulted in haemocytes preincubated with Ddc inhibitors
(benserazide, carbidopa, anti-Ddc, or
anti-proPO)
(h and i) haemocytes pre-incubated
with anti-proPO and then with either
dopa or dopamine failed to induce
melanization but formed small nodules
PO and Ddc activities are key regulatory
molecules of haemocyte nodulation and
melanization in presence of bacteria
0 min E.coli
infection
10 min
E.coli
60 min
E.coli
AntiDdc
benserazide
carbidopa
Anti-proPO
Anti- proPO + dopa
Anti- proPO +
dopamine
Conclusions
-MSH is a melanin activator with innate immune functions in
vertebrates and invertebrates
Author
Key Findings
Hypothesis
Supported
Critique
Rousseau et al.
POMC stimulates melanogenesis; α-MSH is a
potent melanin stimulator molecule
Yes
N/A
Various cell lines were used
under differing treatments
Ahn et al.
Melanin granule production resulted in LPSstimulted melanocytes
Yes
Yes
Melanin production was not
well discussed
Caruso et al.
α-MSH inhibits pro-inflammatory effects (LPS
induced) in astrocytes
N/A
Yes
No future experiments stated
Cutuli et al.
α-MSH directly inhibited S.a. colony formation
and C.a. tube formation
N/A
Yes
One cell lineage used at end
of experiments
Charnley et al.
-MSH peptides 11-13 possess anti-microbial
properties
N/A
Yes
No cell lineages used (only
bacterial cells)
Donnarumma
et al.
MSH down-regulated pro-inflammatory
cytokines in human keratinocytes
N/A
Yes
None
Taylor et al.
MSH inhibited TLR4 activity on proinflmmatory cytokines
N/A
Yes
None
Sideri et al.
PO and Ddc are key for melanin production
during microbe invasion
No
No
Melanin as antimicrobial was
not tested thoroughly
Follow-Up Experiments
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Melanocytes and/or keratinocytes lacking all melanin pre-cursors
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Infect -MSH pretreated cells with S. aureus
Test for microbe inhibition/killing
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BALB/c (immuno-deficient) mice
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treated and untreated with varying concentrations of -MSH
Injected with S. aureus or PBS (control)
Tested for immune response and/or microbe inhibition
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Examines role of melanin granule/production in microbe host defense
Investigates whether -MSH anti-microbial properties enhance immune
response
BALB/c mice with POMC-KO genes
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Treated and untreated with -MSH at various concentrations
Injected with S. aureus or control
Testes for immune response and/or microbe killing
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Analyses whether -MSH treatment is effective drug therapy
Take Home Message
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-MSH can be processed (from POMC) in many
tissues and various cells
-MSH neuropeptide is an immunomodulator in the
innate immune response
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Down-regulates pro-inflammatory cytokines via upregulation of anti-inflammatory cytokines
Anti-microbial properties can be direct (kills microbe) or
indirect (enhances immune response)
-MSH activates melanogenesis in skin cells
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Melanin granules may have anti-microbial properties in
vertebrates
Melanin synthesis is key molecule in invertebrate host defense
against invading pathogens
References
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Ahn, J.H., Park, T.J., Jin, S.H., Kang, H.J. (2008) Human melanocytes express functional Toll-like receptor 4. Experimental
Dermatology 17, 412-417
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Bohm, M., Luger, T.A., Tobin, D.J., Garcia-Borron, J.C. (2006) Melanocortin Receptor Ligands: New Horizons for Skin Biology
and Clinical Dermatology. Journal of Investigative Dermatology 126, 1966-1975
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Caruso, C., Durand, D., Schioth, H.B., Rey, R., Seilicovich, A., Lasaga, M. (2007) Melanocortin 4 Receptors Reduces the
Inflammatory Response and Prevents Apoptosis Induced by Lipopolysaccharide and Interferon- in Astrocytes. Endocrinology 148,
4918-4926
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Cerenius, L., Lee, B.L., Soderhall, K. (2008) The proPO system: pros and cons for its role in invertebrate immunity. Trends in
Immunology 29, 263-271
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Charnley, M., Moir, A.J.G., Douglas, C.W.I., Haycock, J.W., (2008) Anti-microbial action of melanocortin peptides and
identification of a novel X-Pro-D/L-Val sequence in Gram-positive and Gram-negative bacteria. Peptides 29, 1004-1009
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Cutuli, M., Cristiani, S., Lipton, J.M., Catania, A., (2000) Antimicrobial effects of -MSH peptides. Journal of Leukocyte Biology
67, 233-239
Donnarumma, G., Paoletti, I., Buommino, E., Tufano, M.A., Baroni, A. (2004) -MSH reduces the internalization of
Staphylococcus aureus and down-regulates HSP 70, integrins and cytokine expression in human keratinocyte cell lines. Experimental
Dermatology 13, 748-754
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Hillyer, J.F., Christensen, B.M. (2005) Mosquito Phenoloxidase and Defensin Colocalize in Melanization Innate Immune
Responses. Journal of Histochemistry and Cytochemistry 53, 689-698
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Kan, H., Kim, C., Kwon, H., Park, J., Roh, K., Lee, H., Park, B., Zhang, R., Zhang, J., Soderhall, K., Ha, N., Lee, B.L. (2008)
Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect. Journal of Biological Chemistry 283, 25316-25323
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Lasaga, M., Debeljuk, L., Durand, D., Scimonelli, T.N., Caruso, C. (2008) Role of -melanocyte stimulating hormone and
melanocortin 4 receptor in brain inflammation. Peptides 29, 1825-1835
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Litman, G.W., Cooper, M.D. (2007) Why study the evolution of immunity? Nature Immunology 8, 547-548
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Maaser, C., Kannengiesser, K., Kucharzik, T. (2006) Role of the Melanocortin System in Inflammation. Annals New York Academy
of Science 1072, 123-134
References Cont’d
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Mackintosh, J.A. (2001) The Antimicrobial Properties of Melanocytes, Melanosomes, and Melanin and the Evolution of Black Skin.
Journal of Theoretical Biology 211, 101-113
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Nosanchuk, J.D., Casadevall, A. (2003) The contribution of melanin to microbial pathogenesis. Cellular Microbiology 5, 203-223
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Popovich, P.G., Longbrake, E.E. (2008) Can the immune system be harnessed to repair the CNS? Nature Reviews Neuroscience 9.
481-493
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Rosas, A.L., MacGill, R.S., Nosanchuk, J.D., Kozel, T.R., Casadevall, A. (2002) Activation of the Alternative Complement Pathway
by Fungal Melanins. Clinical and Diagnostic Laboratory Immunology 9, 144-148
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Rousseau, K., Kauser, S., Pritchard, L.E., Warhurst, A., Oliver, R.L., Slominski, A., Wei, E.T., Thody, A.J., Tobin, D.J., White, A.
(2007) Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and
melanocytes and stimulates melanogenesis. FASEB Journal 21, 1844-1856

Sideri, M., Tsakas, S., Markoutsa, E., Lampropoulou, M., Marmaras, V.J. (2008) Innate immunity in insects: surface-associated dopa
decarboxylase-dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes. Immunology 123,
528- 537
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Scholzen, T.E., Sunderkotter, C., Kalden, D.H., Brzoska, T., Fastrich, M., Fisbeck, T., Armstrong, C.A., Ansel, J.C., Luger, T.A.
(2003) Alpha-Melanocyte Stimulating Hormone Prevents Lipopolysaccharide-Induced Vasculitis by Down-Regulating Endothelial
Cell Adhesion Molecule Expression. Endocrinology 144, 360-370

Tang, H., Kambris, Z., Lemaitre, B., Hashimoto, C. (2006) Two Proteases Defining a Melanization Cascade in the Immune System
of Drosophila. Journal of Biological Chemistry 281, 28097-28104
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Tang, H., Kambris, Z., Lemaitre, B., Hashimoto, C. (2008) A Serpin that Regulates Immune Melanization in the Respiratory System
of Drosophila. Developmental Cell 15, 617-626
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Taylor, A.W. (2005) The immunomodulating neuropeptide alpha-melanocyte-stimlating hormone (alpha-MSH) suppresses LPSstimulated TLR4 with IRAK-M in macrophages. Journal of Neuroimmunology 162, 43-50
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www.copewithcytokines.org
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www.wikipedia.com
The End…
Questions???