Modulatory Modulatory Functions of Functions of --

MSH MSH Neuropeptide in Neuropeptide in

the Immune the Immune SystemSystem

Saray FelixSaray FelixDepartment of Biological Sciences- Department of Biological Sciences-

California State University Los California State University Los AngelesAngeles

Winter 2009Winter 2009

EvolutionEvolution

ProkaryotesProkaryotes Eubacteria (true bacteria)Eubacteria (true bacteria) Archaeabacteria Archaeabacteria ProtistsProtists

EukaryotesEukaryotes FungiFungi PlantsPlants Invertebrates and Invertebrates and

ArthropodsArthropods VertebratesVertebrates

(Murphy, K. Janeway’s Immunobiology 2008)

www.bordalierinstitute.com/.../

evolutionTree.gif

Evolution of the Immune Evolution of the Immune SystemSystem

Ubiquitous Immune SystemUbiquitous Immune System Every organism is attacked by Every organism is attacked by

pathogenspathogens Response MechanismResponse Mechanism

Provides protection against Provides protection against invading pathogensinvading pathogens

Similar strategies across Similar strategies across phyla with diversity in phyla with diversity in responseresponse

biology.creighton.edu/courses/BIO432/Evolution%2005.htm (Murphy, K. Janeway’s

Immunobiology 2008)

Evolution of the Innate Evolution of the Innate Immune System Immune System

www.nature.com/.../n6/images/ni0607-547-

F1.jpg (Litman and Cooper, Nature Immunology 2007)

Fly Snail Mollusk Amphioxus Lamprey Teleost Chicken Mouse/Human

Evolution of Innate Evolution of Innate ImmunityImmunity

PhagocytosisPhagocytosis Not observed in plantsNot observed in plants Many invertebrates and Many invertebrates and

all vertebratesall vertebrates Antimicrobial PeptidesAntimicrobial Peptides

Defensins- in many Defensins- in many multicellular organisms multicellular organisms including plantsincluding plants

Differ in structural Differ in structural detaildetail

Pattern Recognition Pattern Recognition ReceptorsReceptors Toll homologs found in Toll homologs found in

many species- from many species- from plants to mammalsplants to mammals

Complement SystemComplement System Not observed in plantsNot observed in plants Many invertebrates and Many invertebrates and

vertebratesvertebrates

biology.creighton.edu/courses/BIO432/Evolution%2005.htm (Murphy, K. Janeway’s

Immunobiology 2008)

First Line of Defense: First Line of Defense: SkinSkin

Skin acts as a Skin acts as a barrier for barrier for pathogenspathogens

Epidermal cells Epidermal cells contain necessary contain necessary immune cells to immune cells to activate a response activate a response against pathogensagainst pathogens

αα-MSH and skin-MSH and skin Express and secrete Express and secrete

αα-MSH-MSH(Maaser et al., N.Y. Acad. Sci. 2006)

POMCPOMC PPro-ro-oopiopiommelanoelanoccortinortin Precursor PolypeptidePrecursor Polypeptide

Contains 241 aa Contains 241 aa residuesresidues

Undergoes post-Undergoes post-translational cleavagetranslational cleavage

ProteasesProteases Subtilisin enzymes- a Subtilisin enzymes- a

pro-hormone convertase pro-hormone convertase (PC) that belongs to the (PC) that belongs to the Serine protease groupSerine protease group

Tissue-specificTissue-specific

(Bohm et al J. Invest. Dermatology 2006)

POMC ProductionPOMC Production

It can be synthesized by:It can be synthesized by: Corticotroph cells in the anterior Corticotroph cells in the anterior

pituitary glandpituitary gland Melanotrope cells in the intermediate Melanotrope cells in the intermediate

lobe of the pituitary glandlobe of the pituitary gland Most Neurons in the arcuate nucleus or Most Neurons in the arcuate nucleus or

the hypothalamusthe hypothalamus Few neurons in the dorsomedial Few neurons in the dorsomedial

hypothalamus and in the brainstemhypothalamus and in the brainstem Melanocytes in the skinMelanocytes in the skin

-MSH-MSH Alpha-Alpha-MMelanocyte elanocyte SStimulating timulating HHormoneormone Neuropeptide- 13 amino acid peptide with the tripeptide Neuropeptide- 13 amino acid peptide with the tripeptide

11-13 (KPV) aa containing the “active” sequence 11-13 (KPV) aa containing the “active” sequence A hormone secreted by the anterior lobe of the pituitary gland A hormone secreted by the anterior lobe of the pituitary gland

that regulates skin color in humans and other vertebrates by that regulates skin color in humans and other vertebrates by stimulating melanin synthesis in melanocytes and melanin stimulating melanin synthesis in melanocytes and melanin granule dispersal in melanophores granule dispersal in melanophores

Anti-inflammatory propertiesAnti-inflammatory properties Down-regulates pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α, Down-regulates pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α,

etc.)etc.) Up-regulates anti-inflammatory cytokines (IL-10, etc.Up-regulates anti-inflammatory cytokines (IL-10, etc.

Anti-microbial propertiesAnti-microbial properties Stimulates melanin production to generate oxygen radicalsStimulates melanin production to generate oxygen radicals Indirectly stimulates stimulates immune functionIndirectly stimulates stimulates immune function Directly inhibits pathogen proliferationDirectly inhibits pathogen proliferation

(Maaser et al., N.Y. Acad. Sci. 2006)

-MSH Receptors-MSH Receptors MMelanoelanoccortin ortin RReceptors (MCR): G-protein-linked receptors whose eceptors (MCR): G-protein-linked receptors whose

transductiontransductionoccurs via induction of adenylyl cyclase and an increase in cAMPoccurs via induction of adenylyl cyclase and an increase in cAMP MC1R- highest MC1R- highest αα-MSH affinity-MSH affinity

Key player in regulation of pigmentation in mammals and inflammation Key player in regulation of pigmentation in mammals and inflammation Detected in melanocytes, neutrophils, monocytes, dendritic cells, Detected in melanocytes, neutrophils, monocytes, dendritic cells,

endothelial cells, B-lymphocytes, glioma cells, and astrocytesendothelial cells, B-lymphocytes, glioma cells, and astrocytes MC2RMC2R

Stimulates secretion of adrenal steroidsStimulates secretion of adrenal steroids Detected in adrenal cortex, skin, murine adipocytesDetected in adrenal cortex, skin, murine adipocytes

MC3RMC3R Activation effects are related to feeding and energy homeostasisActivation effects are related to feeding and energy homeostasis Expressed in the CNS, gastro-intestinal system (stomack, duodenum, Expressed in the CNS, gastro-intestinal system (stomack, duodenum,

pancreas), and kidneyspancreas), and kidneys MC4RMC4R

Activation effects are related to feeding behavior, energy homeostasis, Activation effects are related to feeding behavior, energy homeostasis, erectile function, inflammation, and neuroprotectionerectile function, inflammation, and neuroprotection

Expressed primarily in CNS (hypothalamus, vagus nerve, basal ganglia, Expressed primarily in CNS (hypothalamus, vagus nerve, basal ganglia, hippocampus, and cerebral cortexhippocampus, and cerebral cortex

MC5RMC5R Might mediate stimulation of the production of pheromones, influencing Might mediate stimulation of the production of pheromones, influencing

sexual behaviorsexual behavior Expressed in exocrine glands (sebaceous, Harderian, lacrimal, and Expressed in exocrine glands (sebaceous, Harderian, lacrimal, and

preputial glands)preputial glands)(Lasaga et al., Peptides 2008)

MelanoCortin ReceptorsMelanoCortin Receptors

Receptors for Receptors for -MSH in the skin can be -MSH in the skin can be found on:found on: Epidermal melanocytes and keratinocytesEpidermal melanocytes and keratinocytes Endothelial cellsEndothelial cells Mast cellsMast cells Adipocytes Adipocytes

(Bohm et al. J. Invest. Dermatology 2006)

FibroblastsFibroblasts Dermal Papilla cellsDermal Papilla cells SebocytesSebocytes

-MSH and Melanin-MSH and Melanin

Stimulates Melanocytes Stimulates Melanocytes via MC1Rvia MC1R -MSH increases cAMP -MSH increases cAMP

and PKA in melanocytesand PKA in melanocytes In turn increases PKC and In turn increases PKC and

Tyrosinase mRNA Tyrosinase mRNA synthesissynthesis

PKC activates Tyrosinase PKC activates Tyrosinase in the melanosome in the melanosome (lysosome related (lysosome related organelles in melanocytes organelles in melanocytes enriched with tyrosinase enriched with tyrosinase proteins)proteins)

Tyrosine is synthesized Tyrosine is synthesized into Melanininto Melaninbric.postech.ac.kr/…/images/kcib-5-

3.jpg

MelanogenesisMelanogenesis Tyrosine, DOPA or L-Tyrosine, DOPA or L-

DOPA are Melanin DOPA are Melanin precursorsprecursors

End products are End products are Eumelanins (brown Eumelanins (brown and black pigments), and black pigments), Pheomelanins (red and Pheomelanins (red and light colored pigments) light colored pigments) and O2 radicalsand O2 radicals

MelaninsMelanins Bind metal ions and Bind metal ions and

organic cations to organic cations to efficiently scavenge for efficiently scavenge for free reactive radicalsfree reactive radicals

Synthesized by Synthesized by organisms organisms representative of all representative of all biological kingdomsbiological kingdoms(MacKintosh, J. Theoretical

Biology 2001)

Melanin StorageMelanin Storage Keratinocytes store the melanin granulesKeratinocytes store the melanin granules

Melanocytes transfer melanin granules via Melanocytes transfer melanin granules via dendritesdendrites

Invertebrate MelaninInvertebrate Melanin ChromatophoresChromatophores

Pigment containing and light-reflecting Pigment containing and light-reflecting cellscells

Melanin: negatively charged Melanin: negatively charged hydrophobic pigmentshydrophobic pigments Synthesized by the pro-Phenoloxidase Synthesized by the pro-Phenoloxidase

(pro-PO) pathway activation system (pro-PO) pathway activation system activated upon microbe infectionactivated upon microbe infection

Dopa decarboxylase (Ddc) coverts dopa to Dopa decarboxylase (Ddc) coverts dopa to dopaminedopamine

Involved in wound healing, parasite Involved in wound healing, parasite defense, cuticle hardening, and defense, cuticle hardening, and melanizationmelanization

Phenoloxidase (PO) catalyses several Phenoloxidase (PO) catalyses several steps of the eumelanin pathwaysteps of the eumelanin pathway

last component of the pro-PO activation last component of the pro-PO activation system that ultimately results in melanin system that ultimately results in melanin productionproduction

May be involved in blood clotting (activity May be involved in blood clotting (activity may strengthen protein-protein cross-links)may strengthen protein-protein cross-links)

Acts as anti-microbial via direct killingActs as anti-microbial via direct killing(Cerenius et al., Trends in Immunology 2008)

(Kan et al, J. Biological Chemistry 2008)

HypothesisHypothesis

-MSH is a melanin -MSH is a melanin activator with innate activator with innate immune functions in immune functions in vertebrates and vertebrates and invertebratesinvertebrates

Experimental Experimental FindingsFindings

POMC, the ACTH/melanocortin precursor, is POMC, the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes secreted by human epidermal keratinocytes

and melanocytes and stimulates and melanocytes and stimulates melanogenesismelanogenesis

Rousseau et al, FASEB Journal 2007Rousseau et al, FASEB Journal 2007 (A)(A) POMC and POMC and

derivative peptides derivative peptides induce induce melanogenesismelanogenesis -MSH is most -MSH is most

potent stimulator potent stimulator moleculemolecule

(B & C)(B & C) Higher Higher levels of POMC are levels of POMC are needed to stimulate needed to stimulate melanogenesismelanogenesis

(D)(D) POMC POMC stimulated pigment stimulated pigment cell dendricity in a cell dendricity in a dose-dependent dose-dependent mannermanner

85%

63%

23%

Increasing [POMC]

POMC stimulated cell pellets

Human melanocytes express Human melanocytes express functional Toll-Like Receptor 4functional Toll-Like Receptor 4

Ahn et al., Experimental Ahn et al., Experimental Dermatology 2008Dermatology 2008

RT-PCR RT-PCR analysis analysis demonstrate demonstrate that human that human melanocytes melanocytes express TLR4, express TLR4, CD14, and CD14, and MyD88 mRNAMyD88 mRNA

LPS treatment LPS treatment induced induced upregulation upregulation of TLR4 and of TLR4 and MyD88 mRNA MyD88 mRNA in human in human melanocytesmelanocytes

LPS stimulated LPS stimulated melanin granule melanin granule production resulting production resulting in increased melanin in increased melanin pigmentationpigmentation

Microbial induced Microbial induced melanogenesis may melanogenesis may be activated via TLR4 be activated via TLR4

Lane 1: G3PDHLane 2: TLR4Lane 3: CD14Lane 4: MyD88

Activation of MC4R reduces the Activation of MC4R reduces the inflammatory response and prevents inflammatory response and prevents apoptosis induced by LPS and IFN-apoptosis induced by LPS and IFN-γγCaruso et al., Endocrinology 2007Caruso et al., Endocrinology 2007

RT-PCR determined RT-PCR determined that MC4R RNA but that MC4R RNA but not MC3R RNA is not MC3R RNA is detected in astrocytesdetected in astrocytes

Western blotting Western blotting detected MC4R detected MC4R protein in astrocytesprotein in astrocytes

-MSH attenuated the -MSH attenuated the stimulatory effects of stimulatory effects of LPS/IFN-LPS/IFN-γγ on iNOS mRNA on iNOS mRNA

and protein expressionand protein expression MC4R is involved in the anti-MC4R is involved in the anti-

inflammatory effect of inflammatory effect of -MSH -MSH

in astroglial cellsin astroglial cells

Lane 1: blankLane 2: HPLane 3: AstrocytesLane 4: Liver

Lane 1: HPLane 2: AstrocytesLane 3: LiverLane 4: (-) control

Antimicrobial effects of Antimicrobial effects of -MSH -MSH peptidespeptides

Cutuli et al., J. Leukocyte Cutuli et al., J. Leukocyte Biology 2000Biology 2000

(1)(1) -MSH peptides, -MSH peptides, at various at various concentrations, concentrations, inhibited S. inhibited S. aureusaureus colony formationcolony formation

(2)(2) -MSH peptides -MSH peptides had inhibitory effects had inhibitory effects on C. on C. albicansalbicans viability at somewhat viability at somewhat higher concentrationshigher concentrations

-MSH peptides have -MSH peptides have anti-microbial effects anti-microbial effects on gram-positive on gram-positive (S.a.) bacteria and (S.a.) bacteria and fungus (C.a.) fungus (C.a.)

Fig. 1

Fig. 2

Anti-microbial action of melanocortin Anti-microbial action of melanocortin peptides and identification of a novel X-Pro-peptides and identification of a novel X-Pro-

D/L-Val sequence in Gram-positive and D/L-Val sequence in Gram-positive and Gram-negative bacteriaGram-negative bacteria

Charnley et al., Peptide 2008Charnley et al., Peptide 2008 -MSH peptide 11-13 -MSH peptide 11-13

containing the Lysine-containing the Lysine-Proline-Valine Proline-Valine sequence as well as sequence as well as the Alanine-Proline-the Alanine-Proline-Valine sequence Valine sequence decreased microbe decreased microbe viability in a dose-viability in a dose-dependent manner dependent manner

-MSH peptide -MSH peptide sequence 1-13 and sequence 1-13 and 11-13 possess anti-11-13 possess anti-microbial effectsmicrobial effects There was no There was no

significant differences significant differences between the peptidesbetween the peptides

Control peptide: AAA

KPV peptide

APV peptide

-MSH reduces the internalization of S. -MSH reduces the internalization of S. aureusaureus and down-regulates HSP 70, and down-regulates HSP 70,

integrins and cytokine expression in human integrins and cytokine expression in human keratinocyte cell lineskeratinocyte cell lines

Donnarumma et al., Experimental Donnarumma et al., Experimental Dermatology 2004Dermatology 2004 S. S. aureusaureus, protein A (PA), , protein A (PA),

and lipoteichoic acid (LTA) and lipoteichoic acid (LTA) induced the release of induced the release of TNF-TNF-αα, IL-8, and ICAM-1 , IL-8, and ICAM-1 from human keratinocytesfrom human keratinocytes

-MSH pre-incubated -MSH pre-incubated human keratinocytes human keratinocytes strongly down-regulated strongly down-regulated TNF-TNF-αα, IL-8, and ICAM-1 , IL-8, and ICAM-1 releaserelease

-MSH lowers pro--MSH lowers pro-inflammatory molecules in inflammatory molecules in LTA and PA stimulated LTA and PA stimulated keratinocyteskeratinocytes

-MSH has protective -MSH has protective roles at the cutaneous roles at the cutaneous level by reducing infection level by reducing infection and inflammatory process and inflammatory process Ctrl S.a. (S.a + α-

MSH)

TNF-α

IL-8

ICAM-1

2hr

CtrlPAPA + α-MSHLTALTA + α-MSH

24hr

The immunomodulating neuropeptide The immunomodulating neuropeptide -MSH suppresses LPS-stimulated -MSH suppresses LPS-stimulated TLR4 with IRAK-M in macrophagesTLR4 with IRAK-M in macrophagesTaylor, J. Neuroimmunology 2005Taylor, J. Neuroimmunology 2005

APC spleen cells incubated with APC spleen cells incubated with LPS-contaminated ovalbumin LPS-contaminated ovalbumin enhanced IFN-enhanced IFN-γγ production production

APC cells incubated with S.a. APC cells incubated with S.a. ovalbumin were limited in ovalbumin were limited in stimulating a strong IFN-stimulating a strong IFN-γγ productionproduction

LPS-stimulated APC cells pre-LPS-stimulated APC cells pre-incubated with incubated with -MSH could not -MSH could not enhance IFN-enhance IFN-γγ production production

S.a-stimulated APC cells pre-S.a-stimulated APC cells pre-incubated with incubated with -MSH did not -MSH did not change nor enhaced the IFN-change nor enhaced the IFN-γγ production production

-MSH is limited to antagonizing -MSH is limited to antagonizing a TLR4-associated response in a TLR4-associated response in APCAPC

Innate immunity in insects: surface-Innate immunity in insects: surface-associated dopa decarboxylase-dependent associated dopa decarboxylase-dependent

pathways regulate phagocytosis, nodulation pathways regulate phagocytosis, nodulation and melanization in medfly haemocytesand melanization in medfly haemocytes

Sideri et al., Immunology 2007Sideri et al., Immunology 2007 (b and c)(b and c) Nodulation Nodulation

(aggregates) and melanization is (aggregates) and melanization is evident after 10+ mins of bacterial evident after 10+ mins of bacterial infection in medfly haemocytesinfection in medfly haemocytes

(d- g)(d- g) No aggregates and reduced No aggregates and reduced melanization resulted in melanization resulted in haemocytes pre-incubated with haemocytes pre-incubated with Ddc inhibitors (benserazide, Ddc inhibitors (benserazide, carbidopa, anti-Ddc, or anti-proPO)carbidopa, anti-Ddc, or anti-proPO)

(h and i)(h and i) haemocytes pre- haemocytes pre-incubated with anti-proPO and incubated with anti-proPO and then with either dopa or dopamine then with either dopa or dopamine failed to induce melanization but failed to induce melanization but formed small nodulesformed small nodules

PO and Ddc activities are key PO and Ddc activities are key regulatory molecules of haemocyte regulatory molecules of haemocyte nodulation and melanization in nodulation and melanization in presence of bacteriapresence of bacteria

0 min E.coli infection

10 min E.coli

60 min E.coli

benserazide

carbidopa

Anti-Ddc

Anti-proPO

Anti- pro-PO + dopa

Anti- pro-PO + dopamine

ConclusionsConclusions

-MSH is a melanin activator with innate immune -MSH is a melanin activator with innate immune functions in vertebrates and invertebratesfunctions in vertebrates and invertebrates

AuthorAuthor Key FindingsKey Findings

HypothesHypothesis is

SupporteSupportedd

CritiqueCritique

Rousseau et al.

POMC stimulates melanogenesis; α-MSH is a potent melanin stimulator molecule

YesN/A

Various cell lines were used under differing treatments

Ahn et al. Melanin granule production resulted in LPS-stimulted melanocytes

YesYes

Melanin production was not well discussed

Caruso et al.

α-MSH inhibits pro-inflammatory effects (LPS induced) in astrocytes

N/AYes

No future experiments stated

Cutuli et al.

α-MSH directly inhibited S.a. colony formation and C.a. tube formation

N/AYes

One cell lineage used at end of experiments

Charnley et al.

-MSH peptides 11-13 possess anti-microbial properties

N/AYes

No cell lineages used (only bacterial cells)

Donnarumma et al.

MSH down-regulated pro-inflammatory cytokines in human keratinocytes

N/AYes

None

Taylor et al.

MSH inhibited TLR4 activity on pro-inflmmatory cytokines

N/AYes

None

Sideri et al. PO and Ddc are key for melanin production during microbe invasion

NoNo

Melanin as antimicrobial was not tested thoroughly

Follow-Up ExperimentsFollow-Up Experiments Melanocytes and/or keratinocytes lacking all melanin pre-cursorsMelanocytes and/or keratinocytes lacking all melanin pre-cursors

Infect Infect -MSH pretreated cells with S. aureus-MSH pretreated cells with S. aureus Test for microbe inhibition/killingTest for microbe inhibition/killing

Examines role of melanin granule/production in microbe host defense Examines role of melanin granule/production in microbe host defense

BALB/c (immuno-deficient) miceBALB/c (immuno-deficient) mice treated and untreated with varying concentrations of treated and untreated with varying concentrations of -MSH-MSH Injected with S. Injected with S. aureusaureus or PBS (control) or PBS (control) Tested for immune response and/or microbe inhibitionTested for immune response and/or microbe inhibition

Investigates whether Investigates whether -MSH anti-microbial properties enhance immune response-MSH anti-microbial properties enhance immune response BALB/c mice with POMC-KO genesBALB/c mice with POMC-KO genes

Treated and untreated with Treated and untreated with -MSH at various concentrations -MSH at various concentrations Injected with S. aureus or controlInjected with S. aureus or control Testes for immune response and/or microbe killingTestes for immune response and/or microbe killing

Analyses whether Analyses whether -MSH treatment is effective drug therapy-MSH treatment is effective drug therapy

Take Home MessageTake Home Message -MSH can be processed (from POMC) in -MSH can be processed (from POMC) in

many tissues and various cellsmany tissues and various cells -MSH neuropeptide is an immunomodulator -MSH neuropeptide is an immunomodulator

in the innate immune responsein the innate immune response Down-regulates pro-inflammatory cytokines via Down-regulates pro-inflammatory cytokines via

up-regulation of anti-inflammatory cytokinesup-regulation of anti-inflammatory cytokines Anti-microbial properties can be direct (kills Anti-microbial properties can be direct (kills

microbe) or indirect (enhances immune response)microbe) or indirect (enhances immune response) -MSH activates melanogenesis in skin cells-MSH activates melanogenesis in skin cells

Melanin granules may have anti-microbial Melanin granules may have anti-microbial properties in vertebratesproperties in vertebrates

Melanin synthesis is key molecule in invertebrate Melanin synthesis is key molecule in invertebrate host defense against invading pathogenshost defense against invading pathogens

ReferencesReferences 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

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

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

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

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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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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Litman, G.W., Cooper, M.D. (2007) Why study the evolution of immunity? Nature Immunology 8, 547-548

Maaser, C., Kannengiesser, K., Kucharzik, T. (2006) Role of the Melanocortin System in Inflammation. Annals New York Academy of Science 1072, 123-134

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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

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

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

Taylor, A.W. (2005) The immunomodulating neuropeptide alpha-melanocyte-stimlating hormone (alpha-MSH) suppresses LPS-

stimulated TLR4 with IRAK-M in macrophages. Journal of Neuroimmunology 162, 43-50

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