Immunotoxicology 2007dll-2

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IMMUNOTOXICOLOGYIMMUNOTOXICOLOGY

BAG / SMF. Ilmu Kesehatan KulitBAG / SMF. Ilmu Kesehatan Kulit &&KelaminKelamin

FK.UNS/ RSUD.dr.Moe3wardiFK.UNS/ RSUD.dr.Moe3wardi

S!S!



ImmunotoxicologyImmunotoxicology

Study of the effects of physical andchemical agents on the immune system

Immune system is highly sensitive totoxicants – Consists of many cells undergoing rapid

proliferation and differentiation;

(eg. Bone marrow) – Immunocompetence reuires adeuate

!alance (cooperation) and functioning!etween different cellular components



Functions of the Immune ystemFunctions of the Immune ystem

!efense!efense

"omeostasis"omeostasis

u#$eillanceu#$eillance



%natomical O#gani&ation%natomical O#gani&ation

'xte#nal ec#eto#y ystems'xte#nal ec#eto#y ystems – (espi#ato#y) GI) u#ogenital(espi#ato#y) GI) u#ogenital

Inte#nal ec#eto#y ystemsInte#nal ec#eto#y ystems – Lymph systemLymph system

" *#ima#y lymphatic o#gans +thymus) fetal li$e#)*#ima#y lymphatic o#gans +thymus) fetal li$e#),one ma##o-.,one ma##o-.

" econda#y lymphatic o#gans +spleen) lympheconda#y lymphatic o#gans +spleen) lymphnodes) pe#itoneal ca$ity) lymph ducts.nodes) pe#itoneal ca$ity) lymph ducts.

– (eticuloendothelial system(eticuloendothelial system +mononuclea#+mononuclea#

phagocytes/ mac#ophages) monocytes.phagocytes/ mac#ophages) monocytes.



INN%T' IMMUNITYINN%T' IMMUNITY

*"YIC%L*"YIC%L

0%((I'(0%((I'(

1in) mucous1in) mucous

mem,#anemem,#ane

C'LLC'LL

g#anulocytes)g#anulocytes)

monocytes)monocytes)

mac#ophagesmac#ophages

C"'MIC%LC"'MIC%L

0%((I'(0%((I'(

p") lipids) en&ymesp") lipids) en&ymes

%!%*TI2' IMMUNITY%!%*TI2' IMMUNITY

"UMO(%L"UMO(%L

0 cells0 cells

anti,odiesanti,odies

C'LL M'!I%T'!C'LL M'!I%T'!

T cellsT cells

lympho1ineslympho1ines

M*M*



Cellula# Components of theCellula# Components of the

Immune ystemImmune ystem

Neut#ophils and Mac#ophagesNeut#ophils and Mac#ophages – #onspecific (innate) immunity

– Secrete !iological mediators

– $rocess and present antigen (%$) – $attern recognition receptors; &c receptors for

anti!ody coated antigens (%$)

LymphocytesLymphocytes – Specific (acuired) immunity

– Specific antigen receptors

– B cells (humoral immunity)

– ' cells (cell mediated immunity)



Cellula# Inte#actions in theCellula# Inte#actions in the

Immune ystemImmune ystem

%G

M*M*

*%

00

ensiti&edensiti&ed

00

%cti$ated%cti$ated

ThTh M*M*

T#egT#eg

ensiti&edensiti&ed

ThTh

TcytTcyt

0mem0mem

plasmaplasma

Y Y

%ntigen%ntigendest#uctiondest#uction

M*M*

+3.+3.

+3.+3.

+4.+4.

+4.+4.

+3.+3.

+3.+3.

+4.+4.

(10%) (90%)



"ematopoietic 0one Ma##o- tem Cells"ematopoietic 0one Ma##o- tem Cells

Stem cells

can differentiate

into manydifferent cell types

ow does it

decide what

to !ecome



56 "N#IRNM"N$ in whi%h it deelo's( whi%h'rimar) l)m'hoid or*an +ie ,one marrow sth)mus-

2. $KIN"S AND GR0$1 FA$RS$KIN"S AND GR0$1 FA$RS stem%ells are e2'osed to durin* this 'ro%ess

T-o ma7o# influences8

Cont#ol of tem Cell !iffe#entiationCont#ol of tem Cell !iffe#entiation



O#igin and !e$elopment ofO#igin and !e$elopment of

Immune CellsImmune Cells

temtem

cellscells

$rogenitor$rogenitor

cells cells

*ymphoid*ymphoid

+ranulocyte+ranulocyte

0ON' M%((O90ON' M%((O9

%yeloid%yeloid

%onocyte%onocyte

,-/,-/

' cell' cell01/01/

B cellB cell21/21/

0LOO!0LOO!

%$%$

TIU'TIU'

$%#$%# (31/)(31/)

eosinophileosinophil (4,/)(4,/)

!asophil!asophil (45/)(45/) mastmast

BB

committedcommitted

di$idingdi$iding

noncommittednoncommitted

di$idingdi$idingCell MediatedCell Mediated

ImmunityImmunity

"umo#al"umo#al

ImmunityImmunity

:;<:;<

thymusthymus

,u#sa,u#sa

e=ui$alente=ui$alent



Innate ImmunityInnate Immunity

Uniersal and eolutionaril) %onsered me%hanismUniersal and eolutionaril) %onsered me%hanism

o host deense a*ainst ine%tion4 5rst line oo host deense a*ainst ine%tion4 5rst line o

deense4deense4

6redates ada'tie immune res'onse6redates ada'tie immune res'onse

– Found in all multi%ellular or*anismsFound in all multi%ellular or*anisms

+ada'tie onl) in erte,rates-+ada'tie onl) in erte,rates-

– Uses re%e'tors and e7e%tors that are an%ient inUses re%e'tors and e7e%tors that are an%ient in

their linea*etheir linea*e

–

6roides 'rote%tion a*ainst a wide ariet) o6roides 'rote%tion a*ainst a wide ariet) o'atho*ens +'attern re%o*nition re%e'tors-'atho*ens +'attern re%o*nition re%e'tors-

Distin*uishes sel rom non8sel 'ere%tl)Distin*uishes sel rom non8sel 'ere%tl)

Dee%ts in innate immunit) are er) rare and almostDee%ts in innate immunit) are er) rare and almost

alwa)s lethalalwa)s lethal



%etchni6off watched the reaction to a splinter inserted into a%etchni6off watched the reaction to a splinter inserted into astarfish. emocytes (amoe!a-li6e cells) arrived and tried tostarfish. emocytes (amoe!a-li6e cells) arrived and tried to

ingest the foreign !ody; if they could not7 they walled it off.ingest the foreign !ody; if they could not7 they walled it off.

%ammals do exactly the same thing to foreign !odies.%ammals do exactly the same thing to foreign !odies.

'lie Metchni1off8 >Fathe# of Mac#ophages?'lie Metchni1off8 >Fathe# of Mac#ophages?

5@;A No,el p#i&e5@;A No,el p#i&e

*hagocytosis4 to de$ou# *hagocytosis4 to de$ou#



8yster hemocyte %ouse macrophage

http9::itgmv2.f6.de:www:itg:dia!ate:images.html<fighttp9::www.mdsg.umd.edu:oysters:oys!lood.htm

'he similarity !etween an inverte!rate and mammalian phagocyte isstri6ing; they also use many of the same mechanisms7 including the

production of reactive oxygen species



ch#omic catgutsilicone d#oplets

%tlas of G#anulomatous !iseases Yale (osen) M6!6

'he clear (unstained) glo!ules of silicone have

!een released from a ruptured !reast implant.

'hey cannot !e ingested; at this early stage7

%$ accumulate to wall off the material; note %$

that has !ecome a multinucleated giant cell

showing the =asteroid !odies> characteristic ofthe foreign !ody reaction.

'he suture material is not digesti!le !y

macrophages (%$)7 so it has !een walled

off !y fi!ro!lasts.

Fo#eign 0ody (eactions in "umans



Innate Immunity8 FunctionsInnate Immunity8 Functions

6roides a ,arrier to 'reent the6roides a ,arrier to 'reent thes'read o ine%tions'read o ine%tion– Me%hani%al +ti*ht 9un%tions: moement-Me%hani%al +ti*ht 9un%tions: moement-

– hemi%al +att) a%ids: en;)mes: '1:hemi%al +att) a%ids: en;)mes: '1:antimi%ro,ial 'e'tides-antimi%ro,ial 'e'tides-

– Mi%ro,iolo*i%al +normal <ora-Mi%ro,iolo*i%al +normal <ora-

– Mu%osal sura%esMu%osal sura%es» Res'irator): GI: Re'rodu%tieRes'irator): GI: Re'rodu%tie

– S=in +e'ithelial %ells-S=in +e'ithelial %ells-» 0ounds: ,urns: inse%t ,ites0ounds: ,urns: inse%t ,ites




Identi5es and eliminates 'atho*ensIdenti5es and eliminates 'atho*ens

– Non8ada'tie re%o*nition s)stemsNon8ada'tie re%o*nition s)stems

– A%tiates mole%ules that tar*et theA%tiates mole%ules that tar*et themi%ro,e and aid in it>s identi5%ationmi%ro,e and aid in it>s identi5%ation

»$hese a%tors ma) ,e sura%e$hese a%tors ma) ,e sura%e

e2'ressed: released rom immunee2'ressed: released rom immune

%ells or 'resent within %ir%ulator)%ells or 'resent within %ir%ulator)s)stems)stem




Initiates an in<ammator) res'onseInitiates an in<ammator) res'onse

– Rea%tion to in9ur) or ine%tionRea%tion to in9ur) or ine%tion» $rauma to tissues or %ells$rauma to tissues or %ells

» 6resen%e o orei*n material +non8sel-6resen%e o orei*n material +non8sel-» Ine%tious a*ents +iruses: ,a%teria: un*i-Ine%tious a*ents +iruses: ,a%teria: un*i-

– Deliers e7e%tor mole%ules and immuneDeliers e7e%tor mole%ules and immune

%ells to the site o in9ur)/ine%tion%ells to the site o in9ur)/ine%tion

–om'onentsom'onents» Granulo%)tes: M6: se%reted a%torsGranulo%)tes: M6: se%reted a%tors

» Blood essels +endothelium-Blood essels +endothelium-

» 6lasma 'roteins6lasma 'roteins




6roides si*nals to alert the6roides si*nals to alert the

ada'tie immune s)stem toada'tie immune s)stem to

a%tiate an e7e%tie s'e%i5%a%tiate an e7e%tie s'e%i5%immune res'onseimmune res'onse

– U're*ulation o %o8stimulator)U're*ulation o %o8stimulator)

mole%ulesmole%ules M1 %lass II: D?@/?M1 %lass II: D?@/?

– Indu%tion o %)to=ine/%hemo=ineIndu%tion o %)to=ine/%hemo=ineres'onseres'onse (( I!8: I!8CI!8: I!8C



Innate Immunity8Innate Immunity8

Cellula# ComponentsCellula# Components

G#anulocytesG#anulocytes

– $olymorphonuclear leu6ocytes$olymorphonuclear leu6ocytes

($%#7 neutrophils)($%#7 neutrophils)

– ?osinophils?osinophils

– Basophils (!lood)Basophils (!lood)

– %ast Cells (tissues)%ast Cells (tissues)

Mononuclea# *hagocytes +('.Mononuclea# *hagocytes +('.

– %onocytes (!lood)%onocytes (!lood)

– %acrophages (tissue)%acrophages (tissue)

alle#gic and

hype#sensiti$ity #eactions

fight

pathogens

inflammation

fight pathogens

inflammation

cytotoxicity

immune #egulation



Innate Immune CellsInnate Immune Cells




Mac#ophages and G#anulocytesMac#ophages and G#anulocytes

6ha*o%)ti6ha*o%)ti

%%

're%ursor're%ursor

ss

mono,lastmono,last 'romono%)te'romono%)te monocytemonocyte

macrophagemacrophage

myelo!lastmyelo!last

,aso'hili%,aso'hili%

'rom)elo%)te'rom)elo%)te

eosinophiliceosinophilicpromyelocytepromyelocyte

neutrophilicneutrophilic

promyelocytepromyelocyte

,aso'hil,aso'hil

eosinophileosinophil

#eutrophil#eutrophil

($%#)($%#)

mastmast

cellcell

0one Ma##o-0one Ma##o- 0lood0lood TissueTissue



Neut#ophils

+*MN.

$resent in !lood (@1-31/ of ABC)

#ot normally present in tissues Short lifespan - 25 hours

&unctions9

– &irst cell at the site of infection:inury

Ingest and 6ill micro!es after !actericidal

mechanisms activated (!inding to pathogen)



Mononuclea#

*hagocytes

Blood - monocytes (2-@/ ABC)

'issues - macrophages

– mature form of monocytes – found in tissues (ex.7 gastrointestinal tract7 lung7 liver7

!rain7 s6in7 spleen); reticuloendothelial system (?S)

&unctions9

– $hagocytie and 6ill after !actericidal mechanismsactivated

– $roduce cyto6ines:chemo6ines (initiate inflammation)

– Dntigen presentation (activate adaptive immunity)

– 'umor surveillance and cytotoxicity



(eticuloendothelial ystem(eticuloendothelial ystem

Consists of fixed and wanderingConsists of fixed and wanderingmacrophages located throughout the !odymacrophages located throughout the !ody

(tissues7 sinusoids7 lymph system)(tissues7 sinusoids7 lymph system) %aor locations9%aor locations9 Liver and Lung Liver and Lung

FunctionsFunctions9 anti!acterial resistance7 tumor9 anti!acterial resistance7 tumorresistance7 defense against shoc67 antigenresistance7 defense against shoc67 antigen

processing7 lipid meta!olism7 proteinprocessing7 lipid meta!olism7 proteinturnover7 iron meta!olismturnover7 iron meta!olism




Mac#ophages and G#anulocytesMac#ophages and G#anulocytes

$hagocytic$hagocytic

precursorsprecursors

mono!lastmono!last promonocytepromonocyte monocytemonocyte

macrophagemacrophage

myelo!lastmyelo!last

!asophilic!asophilic


eosinophiliceosinophilicpromyelocytepromyelocyte

neutrophilicneutrophilic


!asophil!asophil

eosinophileosinophil

#eutrophil#eutrophil

($%#)($%#)

mastmastcellcell

0one Ma##o-0one Ma##o- 0lood0lood TissueTissue



Functions of *hagocytic CellsFunctions of *hagocytic Cells

Mac#ophages and Neut#ophilsMac#ophages and Neut#ophils *ocaliation and removal of foreign su!stances*ocaliation and removal of foreign su!stances

(ie.7 pathogens) (%$ and $%#) (ie.7 pathogens) (%$ and $%#) Inflammation- response to infection or inuryInflammation- response to infection or inury

(%$ and $%#) (%$ and $%#) Initiate wound healing (%$)Initiate wound healing (%$)

Secretory functions (%$)Secretory functions (%$) Immunologic functions9 process and presentImmunologic functions9 process and present

antigen (%$)antigen (%$) 'umor cell surveillance and cytotoxicity (%$)'umor cell surveillance and cytotoxicity (%$)



Functions of *hagocytic Leu1ocytesFunctions of *hagocytic Leu1ocytes

+mono) M* and *MN.+mono) M* and *MN.

Locali&ation and (emo$al of Fo#eignLocali&ation and (emo$al of Fo#eign

u,stancesu,stances (Inflammation and wound(Inflammation and wound

healing)healing)

--ChemotaxisChemotaxis9 directed migration to site of inury;9 directed migration to site of inury;

chemical mediators (chemotactic factors)chemical mediators (chemotactic factors)

--*hagocytosis*hagocytosis9 ingestion of foreign su!stances9 ingestion of foreign su!stances

--Meta,olic dest#uctionMeta,olic dest#uction9 digestion and 6illing9 digestion and 6illing8xygen-dependent (%$87 8I7 #I)8xygen-dependent (%$87 8I7 #I)

8xygen-independent (cationic proteins78xygen-independent (cationic proteins7

lysoyme7 '#&7 porphorins lysoyme7 '#&7 porphorins

F i f t F ti f



ec#eto#y Functions ofec#eto#y Functions of

Mac#ophagesMac#ophages

Second most potent secretory cell inSecond most potent secretory cell in

body body

?nymes capa!le of degrading?nymes capa!le of degrading

extracellular matrix proteins (collagenase7extracellular matrix proteins (collagenase7

elastase)elastase)

$roducts involved in host defense (8I7$roducts involved in host defense (8I7

#I7 eicosanoids)#I7 eicosanoids)

egulatory proteins (I*-27 '#&egulatory proteins (I*-27 '#&αα7 p+?5)7 p+?5)



Mac#ophage4de#i$ed Mediato#sMac#ophage4de#i$ed Mediato#s

Binding proteins (transferrin7 fi!ronectin) Complement components $roteolytic enymes (lysoyme7 neutral protease7 acid

hydrolyases)

?nyme inhi!itors (αα55-macroglo!ulin plasmin inhi!itor)-macroglo!ulin plasmin inhi!itor) ?ndogenous pyrogen (I*-2) eactive oxygen intermediates (superoxide7 hydrogen

peroxide7 hydroxyl radical) eactive nitrogen intermediates (nitric oxide7 peroxynitrite) Bioactive lipids ($D&7 $+7 *'7 'BE) Chemotactic factors +rowth factors:cyto6ines (I*-27 '#&7 I*-@7 &+&7 CS&)

M h M di t C lM h M di t C l



(NI(NI

%cti$ated%cti$atedMac#ophageMac#ophage

'#&-'#&-αα

I*-2I*-2

$roteolytic$roteolytic

?nymes?nymes

BioactiveBioactive

*ipids*ipids(OI(OI

Mac#ophage Mediato#s Can alsoMac#ophage Mediato#s Can also

!amage "ost Tissues!amage "ost Tissues

chemo6ineschemo6ines



*#oduction of (OI

+(espi#ato#y 0u#st.

euires #DF$ oxidase #DF$ #DF$G

G

5 8B 5 8-

Superoxide dismutase

5 8- B8B

%yeloperoxidase ($%#)

B8B G C24 Chloramines

upe#oxide anion

"yd#ogen pe#oxide



(eacti$e Oxygen Inte#mediates(eacti$e Oxygen Inte#mediates

*ipid $eroxidation*ipid $eroxidation

%em!rane7 $rotein and F#D%em!rane7 $rotein and F#DFamageFamage

"BOB

OB

4

O"4



(eacti$e Nit#ogen Inte#mediates(eacti$e Nit#ogen Inte#mediates

#itric oxide and peroxynitrite#itric oxide and peroxynitrite

Nit#ic oxideNit#ic oxide- formed from l-arginine !y the- formed from l-arginine !y theenymeenyme nit#ic oxide synthase +NO.nit#ic oxide synthase +NO.

%acrophages9 (%acrophages9 (NOIINOII) induced !y) induced !yinflammatory cyto6ines (I&#inflammatory cyto6ines (I&#γ γ 7 '#&7 '#&αα) and) and!acterially-derived products (*$S)!acterially-derived products (*$S)

ighly la!ile; oxidies nucleic acids7ighly la!ile; oxidies nucleic acids7

mem!ranes7 proteinsmem!ranes7 proteins #itric oxide reacts with superoxide anion#itric oxide reacts with superoxide anion

formingforming pe#oxynit#itepe#oxynit#ite

Dntitumor:anti!acterial activity Dntitumor:anti!acterial activity



*#oinflammato#y Cyto1ines*#oinflammato#y Cyto1ines

'umor necrosis factor -α

Interleu6in-2

Interleu6in-@

Interleu6in-2H

Chemo6ines Interferon-γ



Tumo# Nec#osis Facto#4Tumo# Nec#osis Facto#4

$roinflammatory

$rimes phagocytes to produce8I and #I

Cytotoxic

Induces apoptosis and necrosis



Immune Functions of Mac#ophagesImmune Functions of Mac#ophages

Tumo# u#$eillance

Tumo# Cytotoxicity

%ntigen p#ocessing andp#esentation



Immune Functions of Mac#ophagesImmune Functions of Mac#ophages

%ntigen p#ocessing and

p#esentation

Tumo# Cytotoxicity

Tumo# u#$eillance

M h * i fM h * i f



Mac#ophage *#ocessing ofMac#ophage *#ocessing of

%ntigens%ntigens

%acrophages function as accessory cells or%acrophages function as accessory cells or

antigen processing cellsantigen processing cells

%acrophage associated antigen is 2111x more%acrophage associated antigen is 2111x more

immunogenicimmunogenic

$rocessing of antigens involves change so that it$rocessing of antigens involves change so that it

!inds %C II (Ia) proteins; may involve!inds %C II (Ia) proteins; may involve

unfolding7 partial degradation7 selection forunfolding7 partial degradation7 selection forepitope with high affinity for %C IIepitope with high affinity for %C II

euired for '-helper cell recognition of antigenseuired for '-helper cell recognition of antigens

8ther D$C9 B cells7 epithelial cells8ther D$C9 B cells7 epithelial cells



%ntigen *#ocessing%ntigen *#ocessing

$hagocytosis of antigen$hagocytosis of antigen

$artial degradation or unfolding$artial degradation or unfolding

Binding to %C II (Ia) proteinsBinding to %C II (Ia) proteins

e-expression of processed antigene-expression of processed antigen

on cell surfaceon cell surface

$resentation to '-helper cells$resentation to '-helper cells



%ntigen *#ocessing and *#esentation%ntigen *#ocessing and *#esentation

II

Dntigen

presentation



Mac#ophage +%*C.Mac#ophage +%*C.

' cell receptor

$rocessed Dntigen

C!C!

C! Th CellC! Th Cell

M"CM"C

Class IIClass II

Cyto1inesCyto1ines

%d ti I it%d ti I it



%dapti$e Immunity8%dapti$e Immunity8

"umo#al Immunity"umo#al Immunity

%ediated !y%ediated !y 0 lymphocytes0 lymphocytes which producewhich produce

anti,odiesanti,odies oror immunoglo,ulins +Ig.immunoglo,ulins +Ig. inin

response to antigen challengeresponse to antigen challenge

%nti,odies%nti,odies9 glycoproteins; selective7 highly9 glycoproteins; selective7 highly

specific; found inspecific; found in γ−γ−glo!ulin fraction ofglo!ulin fraction of

serum (humoral!lood)serum (humoral!lood)

Fi$e ClassesFi$e Classes9 physical7 chemical and9 physical7 chemical and

antigenic differencesantigenic differences



Classes of %nti,odiesClasses of %nti,odies

IgMIgM88 primary immune response (3/); 'ype IIIprimary immune response (3/); 'ype IIIhypersensitivity reaction; immune complexes;hypersensitivity reaction; immune complexes;

B cell receptor B cell receptor

IgGIgG88 secondary immune response7 B memorysecondary immune response7 B memorycells (31/)cells (31/)

Ig%Ig%88 external secretions7 produced locally againstexternal secretions7 produced locally against!acteria and viruses (2/)!acteria and viruses (2/)

Ig'Ig'88 'ype I hypersensitivity reactions7 minute'ype I hypersensitivity reactions7 minuteamountsamounts

Ig!Ig!88 um!ilical cord !lood7 primitive recognition orum!ilical cord !lood7 primitive recognition orregulation; B cell receptor regulation; B cell receptor



%nti,ody t#uctu#e%nti,ody t#uctu#e

&a!&a!

&c&c

eavy chaineavy chain

*ight chain*ight chain

C-terminusC-terminus

#-terminus#-terminus &a!&a!

4ss4

ssssssss

papainpapain +B Fa, f#agments.+B Fa, f#agments.

pepsin + 5 Fa, f#agment.

Interchain disulfide !onds sta!ilie domain structureInterchain disulfide !onds sta!ilie domain structure

which is tied to Ig function; differences in Igwhich is tied to Ig function; differences in Ig

molecular weight due to differences in heavy chainsmolecular weight due to differences in heavy chains

Jaria!le regionJaria!le regionantigen !inding siteantigen !inding site

Cellular !inding siteCellular !inding site



%nti,ody t#uctu#e%nti,ody t#uctu#e

IgMIgM99 pentamer pentamer

Ig%Ig%99 monomeric; !ecomes multimeric inmonomeric; !ecomes multimeric inendothelium; acuires glycoprotein that protectsendothelium; acuires glycoprotein that protects

it against digestionit against digestion

Y Y Y Y Y

Dinetics of %nti,odDinetics of %nti,ody



Dinetics of %nti,odyDinetics of %nti,ody

*#oduction*#oduction

*#ima#y immune #esponse*#ima#y immune #esponse99 IgMIgM

econda#y immune #esponseeconda#y immune #esponse99 IgMIgM andand IgGIgG

– Shorter lagShorter lag

– igher levels of specific Ig+ producedigher levels of specific Ig+ produced – Steady state level persists longer Steady state level persists longer

– Ig+ predominatesIg+ predominates

– Kuantitative difference !etween primary andKuantitative difference !etween primary and

secondary immune response due to increase in thesecondary immune response due to increase in thenum!er of potentially reactive B cellsnum!er of potentially reactive B cells

– %d7u$ants%d7u$ants9 Ig+ produced after primary response;9 Ig+ produced after primary response;secondary Ig+ response sustainedsecondary Ig+ response sustained



Dinetics of %nti,ody *#oductionDinetics of %nti,ody *#oduction



0 Lymphocyte !iffe#entiation0 Lymphocyte !iffe#entiation

!one!one

marrowmarrow$re-B$re-B

cellcell

BursaBursa

euivalenteuivalent B cellB cell

%ntigen%ntigen

' cell' cell--

processedprocessed

antigenantigenSensitied B cellSensitied B cell

$lasma cell$lasma cell %emory cell%emory cell

YY %ntigen%ntigen

Y

Y Y

Y Y

Y Y

LysisLysis

Y Y %G%G

complementcomplement

5;<5;<

@;<@;<

M*M*

Y Y

Y Y Y Y



Immune (esponse8 0 CellsImmune (esponse8 0 Cells

%nti,ody%nti,odyt#igge#ingt#igge#ing

ClonalClonal*#olife#ation*#olife#ation

Matu#ationEMatu#ationE!iffe#entiation!iffe#entiation

%ntigen%ntigen

smallsmall

B cellB cell

SmallSmall

B cellB cell

*arge*argeB cellsB cells

*arge*arge

B cellsB cells

lympho,lastlympho,last

lympho,lastlympho,last

00

memo#ymemo#y

00

0000

00

memo#ymemo#y

00

00

00

Y Y Y Y

U U

U U

UU

Y Y

Y Y

UU

Y Y

UU Y Y

UU

Y Y

U U

Y Y Y Y

U U



Monoclonal %nti,odiesMonoclonal %nti,odies

'echniue developed !y %iller and Lohler (20H1);'echniue developed !y %iller and Lohler (20H1);revolutionied immunology; involves developmentrevolutionied immunology; involves development

of single anti!ody secreting immortalied cellof single anti!ody secreting immortalied cell

– Inect mice with antigen (5 x)Inect mice with antigen (5 x)

– Dfter 5 wee6s7 collect spleen cells (B cells); Dfter 5 wee6s7 collect spleen cells (B cells);

+$'G7 IgG7 +-+$'G7 IgG7 +-

– &use with mouse myeloma (+$'-7 Ig-7 +G) in $?+&use with mouse myeloma (+$'-7 Ig-7 +G) in $?+and D' (hypoxanthine7 aminopertin7 thymidine) mediumand D' (hypoxanthine7 aminopertin7 thymidine) medium

– Collect and clone hy!ridoma cells (+$'G7 IgG7 +G)Collect and clone hy!ridoma cells (+$'G7 IgG7 +G)

– Select for clones that produce specific anti!odySelect for clones that produce specific anti!ody




Monospecific8Monospecific8 normally anti!odies extremelynormally anti!odies extremely

polymorphic even when directed against singlepolymorphic even when directed against single

antigen; normal antisera heterogeneity7 therefore noantigen; normal antisera heterogeneity7 therefore no

two antisera identical two antisera identical

(ep#oduci,le(ep#oduci,le9 i9 identical anti!ody more specificdentical anti!ody more specific

and relia!leand relia!le

Unlimited =uantitiesUnlimited =uantities99 permanent cell line7 growspermanent cell line7 grows

indefinitely and produces very large amounts ofindefinitely and produces very large amounts ofanti!ody.anti!ody.

%ntigen need not ,e pu#e o# cha#acte#i&ed%ntigen need not ,e pu#e o# cha#acte#i&ed 99anti!ody reacts with only one determinantanti!ody reacts with only one determinant




*otential Use*otential Use everse !iological effects of certain agentseverse !iological effects of certain agents

(digitalis7 morphine7 6epone7 histamine)(digitalis7 morphine7 6epone7 histamine)

Frug scavengers9 low levels drugs andFrug scavengers9 low levels drugs and

chemicals (dioxin)chemicals (dioxin)

Frug targeting9 cancer treatmentFrug targeting9 cancer treatment

'umor targeting'umor targeting

Identification and purification of proteinsIdentification and purification of proteins



%ntigens%ntigens

Su!stance recognied as =foreign>Su!stance recognied as =foreign>

apten9 foreign su!stance that !inds anti!ody;apten9 foreign su!stance that !inds anti!ody;does not elicit immune responsedoes not elicit immune response

Immunogen9 foreign su!stance that !inds antigenImmunogen9 foreign su!stance that !inds antigenand elicits immune responseand elicits immune response

Carrier9 large proteinCarrier9 large protein apten G Carrier Immunogenapten G Carrier Immunogen

?pitope9 part of antigen that is recognied !y?pitope9 part of antigen that is recognied !yanti!odyanti!ody

Immunodominant site9 epitopes that are moreImmunodominant site9 epitopes that are morehighly charged or more accessi!lehighly charged or more accessi!le



Types of %ntigensTypes of %ntigens

T4independent antigensT4independent antigens – Complex car!ohydratesComplex car!ohydrates

– Fo not reuire processingFo not reuire processing

– Can directly interact with B cellsCan directly interact with B cells – #o memory#o memory

T4dependent antigensT4dependent antigens

– euire macrophages or other D$Ceuire macrophages or other D$C – euire '-helper cellseuire '-helper cells

– euire maor histocompati!ility antigenseuire maor histocompati!ility antigens

– %ostly proteins%ostly proteins



%nti,ody4%ntigen Inte#actions%nti,ody4%ntigen Inte#actions

Binding of antigen to anti!odyBinding of antigen to anti!ody

8ccurs in varia!le region of anti!ody8ccurs in varia!le region of anti!ody

moleculemolecule InstantaneousInstantaneous

?xothermic?xothermic

%ay form complexes%ay form complexes Cytotoxicity mediated !y complementCytotoxicity mediated !y complement

(lysis)(lysis)



Cell Mediated ImmunityCell Mediated Immunity

%ediated !y%ediated !y T lymphocytesT lymphocytes which releasewhich releasesolu!le mediators (solu!le mediators (lympho1ineslympho1ines))

Important in host defense against viruses7Important in host defense against viruses7

certain !acteria7 fungi7 transplant reection andcertain !acteria7 fungi7 transplant reection andtumor surveillance; 'ype IJ (delayed type)tumor surveillance; 'ype IJ (delayed type)hypersensitivity (F') reactionshypersensitivity (F') reactions

T Cells8T Cells8 derived from precursor cells in !onederived from precursor cells in !onemarrow; mature in thymus; !ecome educatedmarrow; mature in thymus; !ecome educated



Types of T CellsTypes of T Cells

(egulato#y T cells(egulato#y T cells

– T4helpe# cellsT4helpe# cells88 'M (CFM) antigens ('h27 'h5)'M (CFM) antigens ('h27 'h5)

– T4#egulato#y +supp#esso# cells.8T4#egulato#y +supp#esso# cells.8 surfacesurface

CFMG:CF5G; intracellular CFMG:CF5G; intracellular &ox$, (naturally&ox$, (naturallyoccuring 'reg)occuring 'reg)

Cytotoxic +1ille#. T cellsCytotoxic +1ille#. T cells88 CFH antigensCFH antigens

Identified !y surface mar6ers (CFM7 CFH7 CF5)Identified !y surface mar6ers (CFM7 CFH7 CF5)

(monoclonal anti!odies)(monoclonal anti!odies) Aor6 together to modulate immune responseAor6 together to modulate immune response

Dctions mediated !y Dctions mediated !y cyto1inescyto1ines



TT"5"5 cells8cells8 ' helper inflammatory cells' helper inflammatory cells" Dctivate macrophages Dctivate macrophages

" Involved in cell-mediated allergies7 e.g. poison ivyInvolved in cell-mediated allergies7 e.g. poison ivy" $romote reection of transplanted tissue$romote reection of transplanted tissue

" Stimulated !y cell-!ound antigen7 and secreteStimulated !y cell-!ound antigen7 and secrete


T4helpe# cellsT4helpe# cells

TT"B"B cells8cells8 Stimulate B cells to producetimulate B cells to produce

anti!odiesanti!odies



T Cell Lympho1inesT Cell Lympho1ines

%acrophage activity factor (I&#%acrophage activity factor (I&#γ)γ)

%acrophage inhi!itory factor (%I&)%acrophage inhi!itory factor (%I&)

Interferon (I&#Interferon (I&#αα and I&#and I&#ββ)) *ymphotoxic factor *ymphotoxic factor

Interleu6in 5 (I*-57' cell growthInterleu6in 5 (I*-57' cell growth

factor)factor) Interleu6in , (I*-,7 B cell growthInterleu6in , (I*-,7 B cell growth

factor)factor)



T Lymphocyte !iffe#entiationT Lymphocyte !iffe#entiation

0one0one

ma##o-ma##o-

CytotoxicCytotoxic


M*M* educationeducation

0 cell0 cell+4.+4. +4.+4.

Y Y

+3.+3.

+3.+3.

+4.+4.

+3.+3.+3.+3.

cyto1inescyto1ines

$D

*#e4T

TcytTcyt

M*M* ThTh

T#egT#eg

ensiti&ed

Th

Thymus



T Cell 'ducationT Cell 'ducation

*earning to distinguish !etween self and non-self

Controlled !y %C (self mar6er) proteins

Selection of su!populations of immature ' cellsthrough their interactions with %C proteinsdisplayed to them in thymus

Immature ' cells recognie %C alone; as they

mature7 recognie %CGantigen %aturation dependent genetic alteration in '

cell receptor

Nneducated ' cells are destroyed in thymus



%ntigen (ecognition ,y T Cells%ntigen (ecognition ,y T Cells

Specific ' cell receptor Specific ' cell receptor

' cells are' cells are M"C #est#ictedM"C #est#icted; only recognie; only recognie

antigen and %C proteinantigen and %C protein

'-helper cells recognie processed antigen'-helper cells recognie processed antigen

and %C II (self)and %C II (self)

Cytotoxic ' cells recognie processedCytotoxic ' cells recognie processed

antigen and %C I (non-self or altered self)antigen and %C I (non-self or altered self)

Ma7o# "istocompati,ility ComplexMa7o# "istocompati,ility Complex



Ma7o# "istocompati,ility ComplexMa7o# "istocompati,ility Complex

+M"C.+M"C.

*arge cluster of immune response genes; code for*arge cluster of immune response genes; code forproteins that direct ' cell responses (mouse chrom @;proteins that direct ' cell responses (mouse chrom @;human chrom 23); %C I and %C IIhuman chrom 23); %C I and %C II

Dllow immune system to distinguish self and non-self; to Dllow immune system to distinguish self and non-self; todestroy non-self or altered self (virally infected cellsdestroy non-self or altered self (virally infected cellsand tumor cells)and tumor cells)

?xtreme polymorphism9 millions of alleles or variants;?xtreme polymorphism9 millions of alleles or variants;control individual responsiveness to antigen; inheritcontrol individual responsiveness to antigen; inherit

haplotypeshaplotypes?very individual uniue; %C mar6ers genetically?very individual uniue; %C mar6ers genetically

determined; the closer two individual are genetically7determined; the closer two individual are genetically7the closer their haplotypethe closer their haplotype

Ma7o# "istocompati,ilityMa7o# "istocompati,ility



Ma7o# "istocompati,ilityMa7o# "istocompati,ility

ComplexComplex

Class I M"C8 expressed on all somatic

cells; classic =transplantation antigens>

Class II M"C8 expressed on immune cells

(B7'7 macrophages7 thymus epithelium);

immune associated (I D) antigens; important in

immune regulation7 cell-cell communication

Class III M"C8 complement



%cti$ation of T Cells%cti$ation of T Cells

Mac#ophageMac#ophage

%ntigen%ntigen

T4helpe# T4helpe#

C C

T cellT cell

#ecepto# #ecepto#

p#ocessedp#ocessed

antigen antigen M"C IIM"C II

timulato#y cyto1inestimulato#y cyto1ines

00 M* M* T T

IL4:IL4: IL4BIL4B IFNIFNCytotoxic lympho1inesCytotoxic lympho1ines

Y Y C C

T41ille# T41ille#

alte#edalte#edself self

epitopeepitope

CFMGCFHG

*otential 'ffects of Toxicants on*otential 'ffects of Toxicants on




the Immune ystemthe Immune ystem

Immune upp#essionImmune upp#ession

– Fecreased resistance to infection7 impaired

surveillance; increased incidence cancer; delayed or

a!errant wound healing – +eneral immune suppressants

Pharmaceuticals9 corticosteroids (macrophage inhi!itors;

lympholytic); methotrexate7 cyclophosphamide (cytotoxic

agents; !one marrow suppressants)

Environmental/industrial chemicals (solvents7 $D7 !enene7

pesticides7 heavy metals7 some air pollutants)

Agents of abuse (caa!inoids7 ethanol7 to!acco)

Physical agents (NJ7 ioniing radiation)






Immune 'nhancementImmune 'nhancement

– %utoimmunity9 immune response against hosts own

tissues and cells; autoanti!odies produced (D#D);

oints7 connective tissue most sensitive; failure oforganism to recognie its own cells as =self>; leads to

inappropriate immune response against self antigens

+enetic factors

Chemicals:drugs-lupuslupus9 genetic predisposition vs drug induced (hydralaine7

procainamide); increase 'h or decreased 'reg:s cells

-acute liver failure9 alterations in self antigens; tienilic acid7

triglitione






Immune 'nhancementImmune 'nhancement – "ype#sensiti$ityEalle#gic #eactions9 most common toxic

manifestations of chemicals; undesira!le reactions produced !ynormal immune system; reuire presensitiation; most common inlung (asthma-dust; pneumonitis- inorganic chem.; F'-

!eryllium)7 s6in (contact dermatis) and +I trac6; drugs:chemicalscan act as haptens !y conugating to proteins or cells; !ecomeimmunogens/ ex7 penicillin allergy; poison ivy

– Ch#onic InflammationCh#onic Inflammation9 a!errant wound repair; foreign !odyreaction; granuloma

– Gene#al Immune timulation8 nonspecific stimulationmacrophages and:or lymphocytes; ex7 levamisole7 copolymer7estrogen7 somatotropin

" i i i ( i" iti it ( ti



"ype#sensiti$ity (eactions"ype#sensiti$ity (eactions

Type IType I9 immediate (anaphylactic) reaction (within 2-,1 min);9 immediate (anaphylactic) reaction (within 2-,1 min);

can involve s6in (urticaria7 ecema)7 eyes (conunctivitis)7can involve s6in (urticaria7 ecema)7 eyes (conunctivitis)7nasopharynx (rhinitis)7 !ronchopulmonary tissue (asthma) ornasopharynx (rhinitis)7 !ronchopulmonary tissue (asthma) or+I (gastroenteritis)+I (gastroenteritis)

$rimary exposure to antigen9 !inding of Ig? to mast cells$rimary exposure to antigen9 !inding of Ig? to mast cells

Secondary exposure9 cross !ridging of antigen to Ig?7 capping7Secondary exposure9 cross !ridging of antigen to Ig?7 capping7CaGG influx7 release of vasoactive autacoids (ex.7 histamine7CaGG influx7 release of vasoactive autacoids (ex.7 histamine7*'7 $D&)*'7 $D&)

esponse9 Inhi!ition of vascular smooth muscle; !ronchioleesponse9 Inhi!ition of vascular smooth muscle; !ronchioleconstriction7 hay fever7 uticaria7 acute anaphylaxisconstriction7 hay fever7 uticaria7 acute anaphylaxis

?xamples9 latex7 peanuts7 isocyanates7 co!alt7 nic6el?xamples9 latex7 peanuts7 isocyanates7 co!alt7 nic6el

M t C ll M di tM t C ll M di t



Mast Cell Mediato#sMast Cell Mediato#s

istamine (increased vascularistamine (increased vascularpermea!ility7 constrict smooth muscles)permea!ility7 constrict smooth muscles)

Serotonin (Increase vascularSerotonin (Increase vascularpermea!ility)permea!ility)

?osinophil chemotactic factor ?osinophil chemotactic factor

SS-D (*'B) (constrict smooth muscles;SS-D (*'B) (constrict smooth muscles;

increased vascular permea!ility)increased vascular permea!ility) $latelet activating factor (platelet$latelet activating factor (platelet

aggregation)aggregation)

" iti it ( ti" iti it ( ti




Type IIType II9 Cytotoxic hypersensitivity; anti!ody9 Cytotoxic hypersensitivity; anti!ody

dependent (Ig+ and Ig%)dependent (Ig+ and Ig%)

Binding of anti!ody to antigenBinding of anti!ody to antigenComplement fixationComplement fixation

Dctivation of complement Dctivation of complement

*ysis of antigen:anti!ody complex !y complement*ysis of antigen:anti!ody complex !y complement

$hagocytosis and degradation !y macrophages$hagocytosis and degradation !y macrophages

?xamples9 !lood transfusions7 organ transplants;?xamples9 !lood transfusions7 organ transplants;hemolytic anemia; h disease; &armerOs lung-hemolytic anemia; h disease; &armerOs lung-

!iological dusts (hypersensitivity pneumonitis)!iological dusts (hypersensitivity pneumonitis)





Type IIIType III9 Intermediate reaction (@-5M hr)9 Intermediate reaction (@-5M hr)

Immune complex hypersensitivity; (D!-Dg complexes)Immune complex hypersensitivity; (D!-Dg complexes)

&ormation of immune complexes; mostly Ig%; circulate in&ormation of immune complexes; mostly Ig%; circulate in!lood; attach to vessel walls (esp. 6idney); complement!lood; attach to vessel walls (esp. 6idney); complementfixation7 phagocyte and platelet accumulation; increasedfixation7 phagocyte and platelet accumulation; increasedvessel permea!ility7 edema7 decreased !lood flow7 releasevessel permea!ility7 edema7 decreased !lood flow7 releaseof hydrolytic enymes and vasoactive su!stances; vesselof hydrolytic enymes and vasoactive su!stances; vesselrupture7 hemorrhagerupture7 hemorrhage

?xamples9 arthus s6in reaction-local; serum sic6ness-?xamples9 arthus s6in reaction-local; serum sic6ness-systemic7 protracted anaphylaxis7 occupational diseasessystemic7 protracted anaphylaxis7 occupational diseases





Type I2Type I29 Felayed 'ype ypersensitivity (F')9 Felayed 'ype ypersensitivity (F')eactions (MH-35 hr)eactions (MH-35 hr)

%ediated !y macrophages (%$) and cytotoxic ' cells%ediated !y macrophages (%$) and cytotoxic ' cells('cyt)('cyt)

%acrophage present processed antigen to ' cells;%acrophage present processed antigen to ' cells;activation of 'cyt; %$ and 'cyt release cytotoxicactivation of 'cyt; %$ and 'cyt release cytotoxicmediators (CC7 I*-57 I&#mediators (CC7 I*-57 I&#γ γ 7 '#&7 '#&αα););

?xamples9 'u!erculin type allergy- other allergies of?xamples9 'u!erculin type allergy- other allergies of

infections; autoimmune diseases; allergic contactinfections; autoimmune diseases; allergic contactdermatitis (acids7 resins7 preservatives7 cosmetics7dermatitis (acids7 resins7 preservatives7 cosmetics7!eryllium7 chromium7 nic6el)- agents act as small!eryllium7 chromium7 nic6el)- agents act as small%A haptens; form complete antigens !y !inding to%A haptens; form complete antigens !y !inding todermal proteins.dermal proteins.

*#oceedu#es fo# %ssessing*#oceedu#es fo# %ssessing



*#oceedu#es fo# %ssessingg

ImmunotoxicologyImmunotoxicology

Immunopathology – +ood for assessing general immune alterations

-ematology profile9 leu6ocyte counts7 differentials

-Clinical chemistry9 Ig levels

-istology9 B%7 nodes7 spleen7 thymus

-8rgan weights9 spleen7 thymus

"ost (esistance %ssays

– potentially useful as initial screen to measureimmunocompetence; rationale- immuno-suppressed hosts are more suscepti!le toinfections or to cancer

"ost (esistance %ssays"ost (esistance %ssays




Tumo# Cell ChallengeTumo# Cell Challenge

– %easure resistance to tumor cell graft

following toxicant exposure

– Inect tumor cell suspension into flan6;

palpate wee6ly for tumor development;

measure latency and volume

– ?xamples9 *ewis lung carcinoma (F?S);%CD sarcoma ($C$); polyoma virus ('CFF7

B$7 urethane)





Infectious %gentsInfectious %gents – ange of effects may !e o!served9 mild

infection7 severe infection7 death

– Inection of infectious agent can lead toactivation of macrophages and ' cells whichdecrease severity of disease or to generalimmune suppression and increased severity of

disease – %onitor effects of toxicant on freuency and

latency of infection:mortality and effects onspecific target organs

'xamples of Chemicals and !#ugs that'xamples of Chemicals and !#ugs that



p gp g

%lte# "ost (esistance to Infections%lte# "ost (esistance to Infections

2i#uses2i#uses

– SJ ($CB)SJ ($CB)

– Influena (F'')Influena (F'')

0acte#ia0acte#ia

– ?-coli (lead7 cadmium)?-coli (lead7 cadmium)

– Salmonella (lead7 'CFF7 $CB)Salmonella (lead7 'CFF7 $CB)

– *ysteria ('CFF7 F?S7 cyclophosphamide)*ysteria ('CFF7 F?S7 cyclophosphamide)

*a#asites*a#asites

– 'richinella ($CB)'richinella ($CB)

%ssessment of *hagocytic Cell%ssessment of *hagocytic Cell



g y

FunctioningFunctioning

Fifferential countsFifferential counts

%easurement of functional activity%easurement of functional activity

(chemotaxis7 phagocytosis7(chemotaxis7 phagocytosis7cytotoxicity-!acteria7 tumor cells)cytotoxicity-!acteria7 tumor cells)

*ysosomal enyme activity*ysosomal enyme activity

Clearance capcity ?S (colloidalClearance capcity ?S (colloidal

car!on7 25-I-triolein)car!on7 25-I-triolein)

% t f " l I it% t f " l I it



%ssessment of "umo#al Immunity%ssessment of "umo#al Immunity

%easurement of anti!ody titers (immunodiffusion7%easurement of anti!ody titers (immunodiffusion7

hemagglutination7 ID7 ?*ISD)hemagglutination7 ID7 ?*ISD)

%easurement of anti!ody synthesis (plaue%easurement of anti!ody synthesis (plaue

forming assays)forming assays) %easurement of B cell surface receptors (?DC%easurement of B cell surface receptors (?DC

rosettes)rosettes)

?numeration of B cells (%a!)?numeration of B cells (%a!) %easurement of B cell proliferation (polyclonal%easurement of B cell proliferation (polyclonal

activators-*$S7 $A%)activators-*$S7 $A%)

%ssessment of Cell Mediated%ssessment of Cell Mediated



ImmunityImmunity

?numeration of ' cells (%a!)?numeration of ' cells (%a!)

%easurement of ' cell proliferation in response%easurement of ' cell proliferation in response

to mitogens ($D7 Con D)to mitogens ($D7 Con D)

Dssessment of F' response9 measure s6in Dssessment of F' response9 measure s6inresponse to sensitier (oxasalone7 FC#B)response to sensitier (oxasalone7 FC#B)

+raft vs ost reaction+raft vs ost reaction

' cell cytotoxicity- tumor cells' cell cytotoxicity- tumor cells *ympho6ine production*ympho6ine production

%ixed lymphocyte reaction%ixed lymphocyte reaction

Documents

Immunotoxicology 2007dll-2