Lecture Evolution of Immunity.pdf - School of Life Sciences

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Lecture Evolution of Immunity.pdf - School of Life Sciences

EVOLUTION OF IMMUNITY


RELEVANCEEconomicimportanceEcologicalimportance


RELEVANCEUseful & ethically acceptablemodels to study immunity & diseaseLive in microbe-rich and/orcontaminated environments – the‘living lab’Novel pathways and molecules:potential applicationsLong evolutionary history:defences honed by time


DISEASES/ABNORMALITIES IN ANIMALSDermo diseaseSPX pustulesShell diseaseBlack band diseaseShell thickeningFibropapillomasSkin papillomasBlack sea urchin plagueBacterial ulcers & fungal infectionPhocine distempervirus


SOME FACTORS FACILITATINGINCREASE IN DISEASEGlobal transfer of pathogensAquacultureEnvironmental changeEnvironmentaldegradation


Aquaculture problems………• Stock animals suffer stress• Growth and flesh qualitybecome impaired• Disease epidemics occurPROFIT IS REDUCED


Million metric tons producedValue million US$Worldwide growth of shrimp farming21000ValueProduction8000600014000200001970 1975 1980 1985 1990 1995 20000Production (million tonnes)


VERTEBRATESECHINODERMS/ASCIDIANS>95% speciesBody cavityCoelomatesancestryAcoelomates/PseudocoelomatesMulti-celledancestryDiploblastsARTHROPODSANNELIDSMOLLUSCSROTIFERANSNEMATODESFLATWORMSCNIDARIANSSPONGESHuge diversity in:FormBiologyDistribution/habitatsWould you expectimmune processes to bethe same?No – huge diversity ofcells & moleculesYes – common frameworkexists throughtout phyla


TRENDS IN THE EVOLUTION OF IMMUNITY• All animals can• phagocytose• perform cytotoxicity/graft rejection responses• execute clotting (those with ‘blood’)• Majority synthesize non-specific antimicrobial factors, AMPs• Majority have developed dedicated cells for immunologicalpurposes• Greatest complexity in mammals• All invertebrates lack Ig


EVOLUTION OF IMMUNITY – DRIVING FORCES• Increased longevity• Risk of radiationdamage• Endothermy• Viviparity• Parasite/pathogenselection pressure


INVERTEBRATE IMMUNITY: EARLY IDEASMetchnikoff1880s1970sPhagocytic theoryof immunityRatcliffe• Adaptive component present, but hiddenCooper• Enhanced response could be induced• Complement & induced primitive antibodies present• Agglutinins equivalent to Ab in terms of binding, inducibility• True immunity only in coelomates• Opsonins ‘evidence’ of adaptive immunityFUNCTIONAL SIMILARITIES DO NOT DEMONSTRATEHOMOLOGY


INVERTEBRATE IMMUNITY: LATER DEVELOPMENTS1980s-onwardsBayne Smith RaftosLectins provide biochemicaldiversity for recognition of nonselfcarbohydrate moieties(Bayne)Protease cascades representprimitive forms of complement(Smith)Biochemical relationship of certain immune proteins to Ig Superfamilye.g. FREPS, hemolin (Adema, Faye)Inducible proteins (e.g. cecropins) confer adaptability oninvertebrates (Boman, Hultmark, Hoffmann)Gene homologues to complement in invertebrates (Smith, Raftos)


SUMMARY - PORIFERAEXTERIOR BARRIERSRecognition:Communication:Chemicaldominant overphysicalRecognition processesgood – lectins andaggregation factorspurified. Recentevidence of TLRReaction:No specificmolecules identifiedPhagocytosis occurs for both feeding and defenceCytotoxic reactions strong and production of soluble, toxic effecterswidespread throughout phylum


SUMMARY - CNIDARIAEXTERIOR BARRIERSRecognition:Chemical dominantover physicalFew details known ofrecognition processesCommunication:Reaction:Observations madeof cell movement towound sites - nospecific moleculesidentifiedPhagocytosis dominant, cytotoxic reactions good, encapsulation recorded.Evidence for ROS generation, production of soluble, toxic effecterswidespread


Invertebrate blood cellsDiversity of cell types increases as phylabecome more ‘advanced’


MOLLUSCS: Recognition and TLR pathwaysElements ofpathway identifiedin oystersMontagnani et al (2004)FEBS Letters, 561 75-82Escoubas et al (1999)FEBS Letters, 453 293-298Members of IgSFidentified insnails:Fibrinogenrelatedproteins(FREPS)


REACTIONPHAGOCYTOSISDominant response in filter-feeders(> 85% Mytilus haemocytesphagocytic) lower rates ingastropodsCYTOTOXICITYObserved in Mytilus and CrassostreaCrassostrea: Active cytotoxic molecules comprise a complex ofinactive units, which polymerize to carry out cell lysisMechanism appears similar to that of vertebrate cytotoxic cells(Hubert et al; 1996, Eur. J. Biochem. 240, 302-306; 1997, Biochim. Biophys. Acta1361, 29-41)


SUMMARY - MOLLUSCAEXTERIOR BARRIERSRecognitionCommunicationReactionEffective physical &chemical barriersRecognition & signallingcomponents identifiedExperiments suggestgood communication,identity of molecules inquestionCellular and soluble effecters strong, particularly phagocytosis.Enzymes, AMPs, ROS widespread and highly active, especially inbivalves.


CRUSTACEAN CELL FUNCTIONS: GRANULARCELLSNon-phagocyticParticipate in capsule formation andmelanisationLabile: degranulate in vitroDegranulation – key processReleases:• Opsonins• Cell adhesion proteins• Microbicidal proteins• Cytotoxic factors• Oxyradical scavengers• Signalling molecules


RECOGNITION, REACTION, COMMUNICATIONβ-1,3 glucansPeptidoglycanLive bacteriaProPO Cascade in crustaceansProphenoloxidase(inactive)Inactive serineprotease(ProppA)ClottingPhenoloxidase(active)EncapsulationMelanin(browncolour)Active serineprotease(ppA)PhenoliccompoundsQuinonesInhibitors:proteinase inhibitors, α-2-macroglobulinKILLING


Limulus haemolymphObserved in 1950s that exposure of Limulus haemolymph toGram-negative bacteria produced solid, gel-like clotReaction also produced with low-levels of bacterial endotoxins(pyrogens)Limulus-amoebocyte lysatetest developed and approvedby FDA in 1970sUsed to detect bacterialtoxins in injectable drugs,irrigation fluids andsurgical tubing


HAEMOLYMPH COAGULATION CASCADEEndotoxin (LPS)β-1,3 glucanFactor CFactor C aFactor BFactor B aFactor G aFactor GProclotting enzymeClotting enzymeCoagulogen(soluble)Coagulin(insoluble)


Phagocytosis levels low. ProPO system triggers numerousand diverse soluble effecters, including antimicrobialpeptides, opsonins, ROS, adhesion molecules, agglutininsidentifiedSUMMARY - CRUSTACEANSEXTERIOR BARRIERSRecognitionCommunicationPhysical: very strongDegranulationand activation ofProPO systemdominatesimmune responseReaction


SUMMARY - DEUTEROSTOMESEXTERIOR BARRIERSRecognitionCommunicationReactionPhysical: variableLectins, Toll-likereceptorsComplementcomponentsidentifiedAll cellular and soluble effecters of the innatesystem identified


COMPLEMENT COMPONENTS IN DEUTEROSTOMESMolecular fragmentsof C6 and C7identified in seaurchins, also inhibitoryFactors H and IFujita et al (2004) Mol.Immunol. 41, 103-111Shah et al (2003) Dev. Comp.Immunol. 27, 529-538


WHY MIGHT Ig ONLY BE FOUND INVERTEBRATES?• Long life span?• Bigger genetic base?• ‘Higher’?• Do not undergo metamorphosis?• Only vertebrates get cancer?• Warm-blooded?• Selection pressure from parasites/pathogens?• Ig present in invertebrates but not yet identified?


Flajnik (2004) Nature, 430, 157-158


SUMMARY - VERTEBRATESEXTERIOR BARRIERSRecognition:Communication:Physical: Scales, skin,feathers, mucusChemical: Toxins, antibacterialcompoundsToll receptorsLectinsAntibodiesMHC (Majorhistocompatibilitycomplex)Reaction:CytokinesComplementPhagocytosis, cytotoxicity, encapsulation, enzymes, AMPs,toxic metabolites, antibodies, complement


ENVIRONMENTAL STRESS AND THE HOST


MICROBEHOSTENVIRONMENT


STRESS FACTORS - ECOTOURISMDisturbance - less time spent resting or feedingEnergetic costs - more energy spent in avoidance behaviourDisease/infection - increased contact increasesrisk in both directionsZoonoses: Diseases spread from animals to humans undernatural conditionse.g. seal finger/pox, TB, influenza, conjunctivitis, brucellosis


Stress ?Mental,emotional orphysical strain/tension…..Biologically – “…stage produced by environmental or otherfactor that extends adaptive responses of an animal beyondthe normal range, or which disturbs normal functioning to suchan extent that chances of survival are significantly reduced….”(Brett, 1958)


CASE STUDY – THE ‘SEA EMPRESS’ OIL SPILL‘Sea Empress’ grounded Feb 1996> 70 000 t crude oil spilt, > 200 kmcoastline contaminatedExtensive mortalities observed****


PHAGOCYTIC ACTIVITY IN HAEMOCYTES –MYTILUS EDULIS FIELD DATAIngestion of zymosan mg -1 protein3020100*** *** ***Feb 96 Mar 96 Apr 96 May 96 Oct 96 Dec 96 Mar 97 Jun 97 Oct 97 Dec 97 Mar 98Oxwich OTenby TDaleDAngleA


EXTRACELLULAR SUPEROXIDE GENERATION- PHENANTHRENE EXPOSURE0.50.4Change in OD mg -1 protein0.30.20.10Day 0 0 50 100 200 400 0 50 100 200 400*CMDay 7 Day 14Phenanthrene (µg l -1 )100% mortalityof C. eduleCerastodermaMytilus


PHAGOCYTIC ACTIVITY - PHENANTHRENE EXPOSURE90Percentage phagocytosis6030* *Day 0 0 50 100 200 400 0 50 100 200 400CMDay 7Phenanthrene (µg l -1 )Day 14100% mortalityof C. eduleCerastodermaMytilus


IMMUNE FUNCTIONS IN MYTILUSFIELD SITESEngland, Wales, Sweden,Italy, SpainResults highly variableLAB STUDIESNO DIFFERENCEPhagocytosis Cd, Cu Cd, phenanthrene TBT, oil, fluoranthenephenanthreneRespiratory burstBaPfluorantheneCdCuEnzymesCd, fluoranthene,FluoranthenePCBs PCBs PCBs


DEVELOPMENT OF TOOLS TO ASSESS IMMUNE STATUSMonoclonal antibodies:positive for specificimmune cell moleculescDNA probes:e.g. for antibacterial peptidesMGD-1 defensinM K A A F V L L V V G L C I M T D V A T A G F G C P N N1 CCA AAT ATG AAA GCA GCA TTC GTC TTG TTG GTG GTT GGT TTG TGC ATC ATG ACG GAC GTG GCG ACT GCT GGG TTT GGC TGT CCA AAC AATY A C H Q H C K S I R G Y C G G Y C A G W F R L R C T C Y R91 TAT GCC TGT CAT CAA CAC TGT AAG TCA ATT CGA GGA TAC TGT GGG GGA TAC TGT GCA GGG TGG TTC AGA TTG AGG TGC ACA TGC TAT AGAC G G R R D D V E D I F D I Y D N V A V E R F ***181 TGC GGC GGG AGA AGG GAC GAT GTG GAG GAT ATT TTT GAT ATC TAT GAT AAT GTA GCA GTA GAA CGT TTC TGA ATC TAG AAG ATA TTG GTT271 TAA AAA CAA AGA GGA TGA AAA TGG TAT CAT CTC TAT TAA CAG GCG TTA TCT TAC TGA TAC TTA ACA TAT ATT TAA TAA TTC TTT TTG GTT361 TTG AAT GTA TTC TCT CCA GTT CTA AAC ATG TTT GAA AAA ATA TAA AAT AAA CCC TTA AAA AAA AAA AAA AAA AA


EFFECT OF THE OIL-DERIVED PAHPHENANTHRENE ON PEPTIDE EXPRESSION INHAEMOCYTESPeptide cDNA probesMGD-1Mytilin BMyticin A18S rRNA0 50 200 400phenanthrene (ppb)Unpublished data, collaboration with Philippe Roch (Drim Laboratory)


MARINE MAMMAL IMMUNOTOXICOLOGYPROBLEMSAvailability, access, expenseShort turn-around time forassays essential (< 24 h)Research infrequent, studiestend to be long-termStandard mammlian immunology markers/techniques showno cross-reactivityExtrapolated functions of immune cells may not be correctEthical considerations


PHOCINE DISTEMPER IN GREY AND HARBOURSEALS1988: 18 000 dead seals inNorthern Europe, ca.13% grey seals(Halichoerus grypus)2002: 21 000 mortalities,ca. 4% confirmed asgrey seals.Symptoms: similar to caninedistemper.Death often due to secondarybacterial infections.Causative agent - morbillivirusSpecies sympatricFound in same areas,same haul-out sites


STANDARD MAMMLIAN IMMUNOLOGY MARKERS –LITTLE/NO CROSS-REACTIVITYImmune molecules/functions identified or used so far:Immunoglobulins: IgG, IgM, IgA - sealsCytokines: Interleukin-2 – beluga whales, sealsMHC: - sealionsROS:PhagocytosisNatural killer cell activityLymphocyte mitogenicresponseBeluga whales, seals,bottlenose dolphinMonoclonal antibodies: Homologs of lymphocyte receptors CD2,19, 21, 45 - Cetaceans


Immunotoxicology in grey and harbour sealsBlood cellsseparated intoPMN andmononuclearcellsSpecies Age / Sex NHarbour seal Adult males 4Adult females 2Juvenile males 1Juvenile females 3Grey seal Adult males 2Incubation ofAdult females 2cells with PCBJuvenile males 4mixture: 3h andJuvenile females 224hAssays:Phagocytosis: Fluorescent-labelled E.coliNK cell cytotoxicity: Fluorescent-labelled YAC-1target cells {Respiratory burst – ROS generationHammond et al (2004); Aquat.Toxicol., 74, 126-38


Phagocytosis1.41.23 h 24 h1.00.8Harbourseals0.61.20 3 30Concentration of PCBs ng/ml0 3 30Concentration of PCBs ng/ml1.11.00.9Greyseals0.80 3 30Concentration of PCBs ng/ml0 3 30Concentration of PCBs ng/ml


24 h0 3 30Concentration of PCBs ng/mlGreyseals2.502.001.501.000.50Respiratory burst6.005.004.003.002.000 3 30Concentration of PCBs ng/mlProportional change in respiratory burstProportional change in respiratory burstHarbourseals24 h


1.51.00.50.0-0.5Cytotoxicity1.51.00.50.0-0.53 24Time (h)3 24Time (h)Proportional change in cytotoxicityProportional change in cytotoxicityHarbour seals Grey sealsEffects independent of PCB exposure


References:Wootton, E.C., Dyrynda, E.A. & Ratcliffe, N.A. (2003) Bivalve immunity: comparisonsbetween the marine mussel (Mytilus edulis), the edible cockle (Cerastoderma edule)and the razor-shell (Ensis siliqua). Fish & Shellfish ImmunologyDyrynda, E. A., Law, R. J., Dyrynda, P. E. J., Kelly, C. A., Pipe, R. K. and Ratcliffe,N. A. (2000) Changes in immunocompetence of natural mussel (Mytilus edulis)populations following a major oil spill (Sea Empress, Wales UK). Marine Ecology-Progress Series 206, 155-170.Iwanaga, S. (2002). The molecular basis of innate immunity in the horseshoe crab.Current Opinion in Immunology 14, 87-95.Relf, J. M., Chisholm, J. R. S., Kemp, G. D. and Smith, V. J. (1999). Purification andcharacterization of a cysteine-rich 11.5-kDa antibacterial protein from the granularhaemocytes of the shore crab, Carcinus maenas. European Journal of Biochemistry,264, 350-357.Valerie J. Smith, Janet H. Brown and Chris Hauton (2003) Immunostimulation incrustaceans: does it really protect against infection?, Fish & Shellfish Immunology,15, 71-90


IMMUNOLOGY MOL2.2Assignment Feb 2006The assignment will comprise 30% of the unit mark and should be handed into the School OfficeDeadline: Noon, Thursday 16th MarchLate submissions will incur a penalty of 5% per dayPurpose of assignment: To explore and assess scientific papers inimmunologyYou are being asked to write a critique of a scientific publication within thejournal Developmental & Comparative Immunology (full text accessavailable from HWU library website, via ScienceDirect or from the linklisting electronic journal access).You should locate an article published during 2005 (i.e. volume 29) on anytopic that interests you. The article must be a minimum of two full printedpages. Specify your article at the start of your assignment, citing theauthor(s), title and pages. In order to minimize paper usage, do notattach a hard-copy of the article with your finished assessment.


Your critique should be a maximum of 1500 words and address the followingpoints.1. What are the central questions or hypotheses addressed by the articleand what is the background information on this topic?2. What scientific approaches/methods were taken to answer thequestion/hypothesis?3. Briefly summarize the results obtained and consider whether theyanswered the questions posed at the beginning of the article.4. Explain whether or not you found the article convincing and highlightanything you found that you consider particularly important, novel orinteresting about the article.5. If researching the topic yourself, e.g. as part of a PhD project, are thereany ways in which the work could have been improved?

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