- 1 WTAL QE W@AOJW ONLY

(1)

I M U M M * T a Y U 1 D O M R FKTORS P R M X K I D WHEN 3ENSITWB) LYMPHOCYTES ARE STlMUlATTD WITH ANTIGEN

- 1 WTAL QE W@AOJW ONLY

- mvBEx~h.pXEb

+*

(2)

(3)

(4)

(5)

(6)

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S m W O F T H E T H E S I S

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PI& OF-EXPERIMENT=

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3 1

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1. EXPERImNTS USING GUINEA-PIGS

.

,

.

1. ~xperiments using hinea-pi'l lynphosytes

., obtained byDextra" sedimentation of : ,

. .

* * .

peripheral blood.

.

,

. . . ' . . . . .

12

. . .

.

Materials and llethods,. 11

. .

rr: E * ~ I M ~ " + S using quinea-pig 1,""phocyfes obtained. by washing cells from the, .

. . .

.

-. perironea1 cavity. 45

: . .

. . . .

,

.

materials and nechoa?. 45

, " . _' . .

~ e m l t s .

^. . . . .

. 48 I . Emerinents guinea-pig l y n p h ~ s ~ t e g

~beained by cc triiugarian a i peripheral '\

. - , .

. . . . : .

blood oa ficoll-~ypaque mixture. 55

.

.,,

.

,

.

^, _(il~xperinents an guinea-pig skin-reactive

.

far;tbr'"~ing the mrbroax system.

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(7)

.

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. ^, .

.

. . . .

. .

m. actors a f f e c t i n g lympFecyte t r a n s f o r m t i o n .

. . --

i n vitro.,

. . .

^I ^{7 6 .}

& r e r i a l a a n d Methods 77

. . . .

. .

.

i e s n ~ t s

.

:

. . .

⁸⁹

1 .

% D i s c u s s i o n

. . . ^: . . .

92 _.

.

_,

V.

.

The proaubea - i w s r i g a r i a n by g u r n e a - p i g p e r i p h e r a l blood ^Ofi n t l m a t o r y factors

. . .

1 p P M ~ Y f e s S t i m u l a t e d w i t h PPD.

. . .

10P

.

naterials and nerhcas

. . ' . . .

lb)

. . . . . .

“ .

~ e s b i t a

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^_Discuss~on

. . .

114

.

;.

, 1 .

B. EXPERIHENTI I4 MAN

* . . =. ' experiments

^oninflm;rory fa&-

...

produced by human p e r i p h e r a l . blood fmphod

c y t e s s t i m u l a t e d with PPD.

. . .

118

, .

. .

H a t e r i a l s and Methods

. . .

6 llB

. . , .

r r . prperinients ~ a l r i ~ out after t h e develdpnent .

'

. , . .

. . . , of the g u i n e a - p i g s y s k for t h e prodvcrion . . ^and^assayof s k i n - r e a c t i v e f a c t o r u s i n g

. . . .

.: . . ,

p e ~ i p l i e r a l blood lyolphocytes. 1 3 0 '

.

,

-

l i l The d & e l o p e n r of t h e modified beu?syre '

. . _. .

Wrgratiop Test.

. . .

:

. . .

133

. H a t e r i a l s and Methods

. . . ... ,

ⁱ

.

114

. ' . -

" .

.

Resylts

. . .

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(8)

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MQraLion Test.

. . . . . . . .

(9)

1 I:

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^..

'; -me t u b e r c u l i n r e a c t i o n i a thought spe:ifie i m e ' .' -rellpoo<a by a e n a i t i s e d l g q h o e y t e a to the a n t i g e n . It i s 7 h a r b c " t e r i s e d

.

, ^"h i s r o l p g l s a l l y by infi1trafil.n with mononuclear ie&ryteii: ;

I n e a p e r i m n t 6 , d e a c r i b e d i n t h i s t h e s i s , mncnuclear IeuLocylea

.

''

.

*

fro.peripherai blood of t u b e r e u l i n - s e n s i ~ v e w i n e a p i g s . end k i e r i n g of g r e a t e r t h a n 90%; s-li lymphoiytes, w e r e p t i m u l a r e d

&

I . .

,

. .

in s e r u ~ f r e e

miuv ria

c h i t u b e r c y ~ t i . a n l i g e i . PPD,

&

3 hays.

, .

. ,

.

^'" .eonc&r&rcd =el;-free s u p e r n a k r a fmm t h e s e c v l t u r e s produced an ,

. .

-infla-ror, r e a c t i o n when i n j e c t e d i n r r a d y m l l y i n t o u#.+dnized

. .

guinaa-pigs. Supmstsnfa frnm c u l t u r e s w i t h o u t -'tip" did oof ,

-

, -

.

^, ^' ^pradvcesn i n f l i o a a t o r y ' r e a p o n a e . The r e a c t i o n w a s eharacferimed by _.

. .

,

. . . . .

. . .

. . . : .. . .

.. .

.

me

s u p a m s t - t s poasesaing inflan.mfory a c t i v i t y contained a f a c t o r ' i n h i b i t i n 8 L$e'rai$retibn ? f p e r i t o n e a l aucrophagaa from uniannmiled . ^, gui"?-pigs.

.

m

experiments i n man, it was demonstrated b a r p e r i p h e t a l blood 1e"kocyt.s,.cmoisring of 1 0 % lymphocyte? and 90% polynolphs from 1 , , r u d e r c u l i o - e e n s i t i v e donors, were i n h i b i t e d i o t h e i r -=menis b y rho

. .

dl.

.

'

. .

t d e r c ~ l i n - ~ ~ g a t i v e donors, WBB blooked b y 1

un

a c t i n w c i n D. In,

. .

~smp~imafmt~ from P P P s t i m d a t e d serun-mncainiqg c u l t v r e s of nononuclear.

-~ ^. ^' ^. ^.

c e l l s Erom i n d i v i d u a l s s e n s i t i v e t s . t d e r c u l i n ; t h e r e occurred factor ' - '

. ^. . ^.

^capableof i n h i b i t i n g the mwm,nt of ,polymorphe.

mi.

factor pas nor ' '

.

. prodmeed by c e l l s f m n t u b e r c u l i n n e g a t i v e donors and vsa not d e m ' a r r a b l e .' i n the absence of serum. Amaver, s a r m f r e e c u l t f i l l of p e r i p h e r a l '

.

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(10)

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blood mrmrauciear.ceiib rr, t u b e r c u ~ i ~ - ~ ~ ~ i t i ; e d o m e ?hen s t i & ~ a t e d '

.

e.

with PPD +leased a splvble factor produciog,:ryfhelba and induration

. .

,

.

when i n j e & b d inrradarnally i n t o ~inrmunired guin.spigs.. This reaction,

. .

^' ^I

.

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v a maximal at 4-6 hours. meb appea?=d ra be a earrela~ipn between antigen-apeclfic release of. this inflmmtory factor w d the ability

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a£ aupernatants.to ,enhance d g r a r i m nf polymorphs.

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(11)

EUWARD J m m

(1749-1823)

From

a p o r t d l

by J. k Smith, 1801

(12)

- 9 ' . . .

. Xli\RY EARGE,

X

~ w d b r i . in thi. palill. \%.as ino-

culmed rvitlr naiolour niallrr in Ihc,ynr l i & .

4

f l l < ~ *

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. ,_a_,~ ~ R m c c Pa paliB calour roo11 nly,rsrrd B o u t llic

e

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.

- ! . . _pll.li

s h L

:Ile nixtter ~ w r ittkricd. a , d lirrrd i t l ~ l l ~ n t l ~ e r

t

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^,

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^{' '} cstcn~vcly, but dicda!ray in r rc>v?ayrhitlxoar prodnciny

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any variolour .lymplomr'. Sltc $.r Lncc I,ren rcpedcdly

.

.'

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,

.cmployeycd a n nurL lo Snna!l+or plicnlr, lrilllout espe-

.

' -

. .

r e i n g a n y ill conk9~cncrr. Thirwomen bad i l k Cow

'

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I.osls,l>cn Ihc livcd in ihc rcrrk~ or r Furmcr ill dlir prrinr _,.,

. . .

, .

. .

.,

..

,

.

tl~irly.onc yems berorc.

. ; .

b

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.

I . .

a i h . a & ~ ~ t b ~ t ~ . ~ O I O U C .noct,wtirn *ha Gnrm ir , I C . ~ C ~ L. 0j.n _{, . ,.}

. . .

1,. h a l i ~xcisa lhnammmtimm the rxn$o *t,icl, ;his apP~jfi rprnd*~

.

:

.

, . ^, thandm it pmdu.~. tlr Snrll Par. I n d r d ilbmdmrs alinofl a r.itcrion by

.

. . W I ~ I C % W ~ C = ~ a ~ t ~ ~ ~ l > ~ ~ i ~ t l ~ f i l ~ ~ i ~ r ~ n ; ~ ~ W ~ I I ~ C ~ C ~ C ~ V C J ~ J ~ O ~ . 1,ktm, '

. >

a% Ba.hanp, which mdubclhrsugb lilr. had k m p d ~ r r d in the .Bian, or

. . . -

^_I-'

. . ,

.

dirpotlian 10 *aim, i n I c rrii.1. nr ~ h r k i n : mi it ir rmsrk.~lc tm. '

'

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^'whnl>crthi. chnwc has hrm~rUc0ctl the Small h r or dw Cow POX. (la% .

.

- ' ' ~ ~ ~ ~ i r ~ ~ ~ ~ i ~ ~ ~ ~ r ~ ~ ~ ~ ~ ~ ~ ~ i ~ ~ ~ ~ ~ i ~ n ~ ~ m i ~ ~ i ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ i ~ . ~ ~ ~ ~ ~

.ariob4 mat,", ,

'

. '', CASE

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" F I C ~ 2. A page IF- Jenner's "an exquiry into m a causes and ' '

.

, .

,

- .

effects 05 the va,ioibe 'vacchee. (1798) describing a delayed hypersen-

. ..

. ,

.

^gitivityreaotion .to the Aaaiwx xi- Jsnner's. c o m n t * u w n the '

.

reac(iq. .

. .

(13)

4s" Me

a l t i g e n

-isulin

^{i e}

;"jested

^lntot h e s k i n of

an

3 :

.. .

.

a n i o l a ~ or ian s e n s i t i s * t o that antigen by preyioas':dntac< w i t h the-

, .

t v b e r c ~ e ba<iJlus, a delayed h y p e r ~ e n n i t i v i t ~ reaction t o Me tuber- - 4 ,

..

^,

. ^. ,

^. ^r

d i n m y occur.. his i s ~ h a ~ ~ c i ~ r z s r d

w

t h e g r a a u a d e v e ~ o p n e ; ~ t

. .

of ,

\, . .

a duf1 rsd ;aired area Ular i s f i r m and'mdurated, aheariw ^afeera

. .

^,

.

. .

f e w hours b u i not r e a c h i n g i t s .

y*im . . ,

size u b t g 24 or 48 hours. '

. .

*,

. . ..

~ u b s e q u e n t i y the r e a c t i d " f a d e s over the next f w day$ and w t r e l y

. .

' ' diaapp.arsi' The e 1 u o l d a t i o n . o ~ th r loems b; which t h i s r e a c t i o n accurg

. .

.. . .

\

-

conprises the scope of thi.s th~lesis.

. 0

:

,

_{. .} . . _{. .} . .

. ^,

' lNTIMOUCTlON ,

Prohahly'the f i r s t d e s c r i p t i o n of a delayed. hypersensitivity

.

^' .

.'

.

r e a c t i o n appeared i n 1798 i n ~ e d e r ' s "A" enquiiy i n p 'the

. .

^'

. '

.

-

_{. .}

causes and

_.

srfects bf t n e v ? r i o ~ a e ~ a c c i n a e - . =one. it i s vorthy o f .

-

.

mentiin: bur i n ' a m t i o n , ~knner's i n t e r p r e t a t i o n o; t h e phenomenon ,

.

' , must.be considered rernarknble 'lPlw5es 1 6 21.

. .

sire the s u g g e s t i o n i s of an ind",-.nq change t h a t occurs.

i n the Day after expornre to infectitin, enabling i t tb reject i n an '

.

, a c e l e r a t e a mnwr, mtterto vhioh it has hesols& s e n a i t i s e d . ware

6:~

^,. ^{a l s o}

^.

^that

^.

chinqe i s G*ed to the i"flaona)tO* response of the

ksaela 'dv

_$thin_{t h e}s k i n and a t t e n t i o n i s d r d n to *e usefulness of the reaction.

:

.

, as a a i a g r a s t i c test for inmunit$. ,

.

' b, a ' ,

.

process a n t i c i p a f i n g even some d a r n i d e a s concerning i e mture.

. '

.

. .

' .I

. .

^, Indiea, ' i r v a e nor u n t i l Is 0 M a t a a i i i l a r p h e n o m ~ n . . r a a , /

. ' ^P

h e s c r i h & by RDbert M a h l ~ i - s 31 i n h i s i n v e s t i g a t i a n s into t u b e r -

* .

^su1osis.

.

^. ^,

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.

^,

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.

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(14)

(15)

.-..

. r t a healthy guiB&-pig be Inoculated with a pure e u l t i v a t i o . o r t u b e r c i + a c i l l i . t h e i n o c u l a t i o n b i n d

'.

g e n e r a l l y becomes sealed, and seems to h e a l up during t h e next few aa9s. It is o n l v ~ i n the course of from r t o n t o f o u r t e e n days t h a t a hard nodule i s formed, .which s i s t s u n t i l .eon ape"., fommg an "lceratrnb the death of t h e animal, b u r . t h e oase i s s p t W h l ~ h per-

.

very d i f f e r e n t .rr.an already tuberculous animal be

i n o c u l a t e d . me m o s t surrable anlmls for thl. ~ ~ ~ t i - nent are t h o s e that nave already been s u c c e s s f u l l y , i n o c v l s t e d four $0 s i x we% p r e v i o u s l y . I" t h e Case , of such an a n l n a l a l s o t h e emall i n o c u l a t i o n wound b e c o m r sealed at f i r s t , b u t no nodule i s farmed, a , ' pesu1i.r change taking place at t h e pol"* Of mo.u1atron.

Already, on fhe f i r s t or second day, t h e spot b e m s hard and dark-co1oureti; and UlLI 15 not sonfined to t h e p o i n t of m o c u l a t i o " , h u t s p r e a d s arovnd t o a diemeter Of 0 : 5 t o I o e n t i n e t r e . During t h e nent Few days it becomes lnore and more c l e a r that the epidermis thus changed i s ' n e c r o t i c . F i F l l y it i s bhrarn o f f . and a flat u l c e r a t e d surface remains, which q e n e r a n v h e a l s . . 4"icLly and completely, w i t h o u t c a r l y i n g i n f e c t i o n to.

t h e neighbourlng l y w h a t i c glands. Th". the i n o c u l a t e d t u b e r c l e b a c i l l i act q v i t e d i f f e m t b on the skii o f a healthy guinea-pig and o n e m t of. rvbercv1ous *ne.

But this remarkable a c t i o n does n o t helong e x c l v e i v e l y to l i v i n g tubercle b a c i l l i , bur .la0 i n t h e same d e g r e e to dead ones, "herher k i l l e d by low renperarwes of long durarron. which I flrsr t r i e d , or by b o i l i n g h e a t , or by c e r t a i n chermcals." '

,

.

Subsequently. t h i s r e a c t i o n became Xnam as t h e m s h phenomenon.

m c h , h i n s e l f followed t h i s work up with the devel?pinent of a b a c t e r r a l extract w i t h the.sme,actiulry, whish he called old m b e r c u l i n ll(och, 1891). T h i s c o n s i s t e d of a g l y c e n n broth culkvre of human-type,. ' ' w c 0 b a c b r i a t u b e r o d l o s i = , g r a m For 6-8 weeks and t h e n c o n c e n r r r t e d by

'h

-

b o r l i n g , f i n a l i y being freed from b a c i l l i by f i l t r a t i o n .

.

. ^.

K O C ~ a l s o d e s c r i b e d t h e s y s t e m i c t u b e r e v l i n r e a c t i o n , which m y f a l l o w t h e a d n i n i ~ t r a t i o n of Ulbercv1in t 0 , s e n s i t i v e i n d i v i d u a l s by

;:. . .

syntemis,

. . "

subcuraneous

.

or even i n t r a d e r - l

*

routes:- xis f e a t u r e s depend, upon t h e dose aQminrrtered and ? h e d e g r e e of s e n s i t i v i t y , i n i t s mildest

.

^,

fohn d e i n g n c h a r a c e e r i ~ e a by fever, kl:rhxng and general rnalais? and when.

.

^s-

^.

.

, . .

¹

(16)

.

\ ⁶

.

^mostsevere draavoing hypotension, h w t h e m i a and .

.

vearneas ynich may

.

' , resulk i n death.

. ..

. .

1

^me^cutaneoust u b e r c u l i n r & s r i o n was f u r t h e r i n v e s r i g a t e a by

E p s t e i n (1891) a5d von p i r w t (19071 using

4-

cont?ct k c h n i q u e which

_{. .} -

&are h i s n-, and by Mantou. (19101 who>ntrddused t h e n o r s t a n d a r d rechniquL oi inrrasuranemus t l s r i n g . me c q n j v n c r i v a l reacqron test

.

^'

was a l s o inPzoavsea a r t h i s t i n e

.

( c a ~ m e t t e , 19071. m e s e L r e a s t i o p s - were a l l enplayed i n t h e d i a ~ n o s i s of t W r c u l o s i s .

/.;?

'

I t was von PirqueL dnd *Chick (1903) who f i r s t reca &>&the

3,:. .

: c u b e x c u ~ l n r e a s k i a n as an = n e r g i d phenomenon analogous ;o oher recent- .

. . .

l y d e s c r i b e d ilanunological r e a c t i o n s .*Ch as anaphylaxis or'.Ule nrfhus

_it" . ....

S k i d Reaction. :*_

-

.

^~ovever,i n 1921. zlnsser shoved t h a t t u b c r o u l i n s e n s i r l v i h

. .

d i f f e r e d fro. o t h e r re..gnLred f o m s of a l l e r g y i n t h a t it could not.

ed p n s s i v e l y u ~ i n g ~ s p m .

.

Furthemre, i n 1925 mnsrer and Muelex suggested t h a t "these substances upon which ( b a c t s r i - a11 a l l e r g y depends m y passes= p r p t e a e i v e functions d i f f e r e n t fion and ,

.

based on a d i f f e r e n t lneshanisai from those possrssed b y

Indeed, as was later p o i n t e d but both.!" aniny.1 experiments and c l i n r c a l experience. t h e r e appearq m be a close p a r a l l e l between host r e s ~ s r a n o e and the p r e v a i l i n g level of ruberealin r e n s i t r v i t y (Wilson

?r a l . . 1940; Heinbeck, 1936, 1949; mlaniels et 01.. 1948;' Bloah. 19551

.'

c e r t a i n evidence subseqvently appeared m denanstrate a dieaoci-

\ .

a t i ~ n between r u g e r c u l i n e e n s i t i v i e y and z m n i t y l w ~ l s o n e t al.. 1940;

. .

' R a f f e l , 1950; Rich, 1951: Crrrule. 1958).' However,, as WacLaness (19671 ' h a s p o i n t e d out, i n

.

s r i t i p Of t h i s eviaenoe, one oanmt imply, from

m e -abser;ce of s k i n xe..tiuity, t h a t i m n o l o g i c a l r e a c t i v i t y at the

Q

(17)

'I

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,.

.

7

c e l l u l a r l w e l i s a b r e m . =he i n t e r r e l a t i o n s h i p of i m u n a ~ o g i c a i res-

. .

p n n e and a n t i g e n a e n r i r i v i t y , *iCh "my d e t e r n i n e a c t u a l r e s i s t a n c e ,

,

^$0disease. makes t h e a n a l y s i s o r h u n e meshanims ext=me,y d i f f > ~ " l * .

The h i s t o l o g i c a l s h a i a s t e r i s t i c S of *uberculin and a n a p h y l a c t i c

.

tvpe o f s k i n r e a c t i o n s -re clearlyidi.rin9uished by Dienen A d tfallory (1932, 19361. whereas i n those r e a c t i o n s mediated by antibody, t h e r e ' - w a s a r a p i a y %eveloping oeama of the s k i n qulckiy foilawed by a n

,

inte-e p o l m o r p h o ? ~ ~ l e a r leukocyte I P ~ I i n f i l t i q t i o n reaching i t s , h e i g h t i n 1-6 hr;. i n t h e e a r l i e s r a ralldest t u b e r c u l i n r e a c t i o n s , the c e l l u l a r i n f i l t r o < i O n war at a l l tiLl.osL,prsly m m n u c i e a r . m t h e i r enperhents, reactions began a t 4 to 6 hr and were maximal a t 24-48 u,. ~a.1~ on l p p h o c y t e s were seen w i t h i n s m a l l v e s s e l s , m v l b l y venu1es; h e r they were seen i n perivinu1ar C"fZ. o f t e n s e v e r a l "el11 '

.

d-ep. oed- was neyer ~ r ~ l u n g , 1 e a d i n g . t o t h e cancludion fhaa c e l l u l a r .

.

. a o c m u l i t i o n d must be respoisible for a % g n i f i c a n t partpf t h e induration

'

. , ' p a l p a b l e a t t h e tubetsqlin reaction s i t e s . Polymorphonuclear leuxocyte

infiltr.bio; w :seen only "hen there was necrosis: ^'

- 5 .

^{- I}

m asrcmena, -Free 119341 i d h i s s t u d i e s of the t v b e r s u l i n , ' r e a c t i o n was able to c o r r e l a t e the degree of polymorphonuclear response

with t h e s e v e r i t y of t h e resL'mnse: i n ~ r t i c u l a r , u i t h t h e extent of t h e ' ' n e c r o s i s . T h e c e l l u l a r -date uae round to c o n s i s t of mmrcytes "ihh

.

f r e q u e n t ~ymphocytes i n severe r e ~ t i o n s , up

tb

2ar e o s i n o p h i ~ s .

~ p l l and ~ i n d e 119511 and cell (195Bl a l s o e s s e n t i a l l y -"finned t h e previous f i n d i n g s , I n a d d i e i o n noring t h e i n i t i a l i n f i l - a t i o n to con-

) :

,

. .

E i s t of nxlnocyres and histiocytes'xitb a few lmphocytes and a'Wnall but d e f i n i t e p r o p a r t i o n of granulocytes. ,

I

wesslCn 119521 i p v e e i g a t e d the h i s t o l o g y of t h e r e a c t i o n i n

_- ..

~ t U b e T ~ 1 0 " ~ Tabbits. Lle fo""d,tm: a f t e r 3 'hr considerable i n f i l t r a t i o n

.

-

(18)

8

was spread over the dermis, polygv,rph. seing intersperse$ With lynphw wees. only after 24 hr did

knonuLgr

sell tiper predaninaee.

.

. .

In M 6 3 . .Bon$!hton and Spector re-examined the histology of the ^'

;

.,

tuberculin reastron in the guinea-pig. he^ found that in the first hr there ocdurred a s m l l or nade~are infla&arory meizrration of predominantly polynbrphonuslear leukocytes from the uereels. W 5-6 h r . .polymarphon"cle.r. were no longer seen in the perivascu1ar areas, where

/

oananv~lears now predominated. They were, however, visible in the inter- vascvlar spaces, rnto whrch they had apparently moved. irt 8 hr the

.

^>

.

. migration Of leukocyte., predom-nantly polymorph.nucle.rs from the

"esse15 began again, the area being heavily ~nfilirared with granu1o-

t

cyres as we11 as being o e d e m t h . But b i 2 4 h r emigration of cells was subsiding, polymorphonvCleara were lnoving away fmm vessels into the tissue. and periuasC"iar ce11.so11ections vere da.in.tea by nanonuolear sell. that had let* the aircylatron with the granulooytes but which had ,

.

^becomeinnobilized aroma the vessels. These eIlMorb considered the tuberculin reaction to have a biph%sis partern, With% minor peak at 3-4 hr (probably a "on-specific reaction to tuber"ulin1 and a major

.

~ peak at 8-12 hr. due to speoifio sensitisation.

.

The passive transfir of delayed nipersensirivity

experiaerd$3

by ~ a r ~ ~ s t e r d e r and chase in 1941 confi'med the importance 6 mnonuslear

. .

cells in the rvberculin r e a c t i o n , and chase 119451 was able to rrmsfer 't~erculin-sens~tivity m normal guinea-pigs w i n g peritoneal exudate .

..

cells frn. sensitined animals. Such cell suspensions consist PrimrilY

3.

.

of macrophages and lyrphhwtee. In these expeliments, skin reactions ',

bacane positiw follwing 'intravenous administration Of the o;lls, with- in 20-36. hr.

c

'

(19)

-Subsquenfly, passive t r a n s f e r of tvbercvlin s e n s i t i v i t y with pvre lymphocytes vaa demonstrated by Kourilsky e t al'.. (1952) snd Uessleo

.

' ' (1952).

But i f the dependence-of the tuberculin teaciian upon s p e c i f i c - a l l y sensirieed , d l s , probably lymphocytes, was thu. demnsrrate'd, the

. .

m a n s by which these c e i i a produced t h e infismmamry teepmse "as e r i l l

. . \ .

m s e l r l e d .

, \

In 1932 Rich snd.leuis had shkm char =he c e l l s tha< :auld -aormelly migrate out fmm explanted fragments of spleen o r hrne marrmw

i n the presence of tuberculin, weresinhihired and k i l l e d vhen the ex-

@

p l a n t s rere from tuberculin-aensitieEd snim1s. These cells "ere' t e n t s

-

tive1y i d e n t i f i e d ae a mixture of m o c y r e s o r macmphapa and p a y - ' m o r p p n u e ~ e a r leukocytes. mus t h e tuberculin reectian c d d consist

,

.

of sn immunologically speciff6 necrL%sis Of se"silive'ce11s vl& a s e u n d a v i n f l ~ f o l y r e a p m e .

. ..

,,

A nunher pf p*qs were published c0nffrd"g 8ich and Leuie'

.

' , original findin* (Beillpsn 6 Seiberf, 1946; Favour, 1947; F a b r i r i o , 1952; w a k s m , 1953; ~ a n g a r d s a et al.. 1955) v h i l a r others were not

,

able t o confirn'then (Baldddge 6 Kligmm. 19,51; Harki.& James. 1953). ^'

wd;eman

. w d ' & t o l f e y (1958) i n agreement v l f h t h e ' l e t f e r s o u p

.

of authors were s b l e to d e m n s r r e t e a superior s e l l s u r v i v a l of macro-

.

phages from seneieised guinea-pigs ~xpoead La tuberculin. Even mre ,

important mas me apparent p r o l i f e r a t i o n of inre-diate mrmmvclear c e l l s i n these cultures. There e p p a r e d t o t h e avthore to be a arphc- logical progression f m n small lymphocgte. to these e a l l s , . v h i e h ~ & r e ' per,ddaae aepative, hut took "p o e u t r a l red stain and had e s e w t y

b i u p h + l i c cyraplaaa Eilled with deck granules or vacuoles. mtosea

. .

(20)

. ^.

. , , , 10

. .

'

were not seen i n the c u l t u r e s but t h e f i n d i n g s q v i t e d e f i n i t e l y argued

.

asaB.t a p u r e 1 y . d e s t r u c t i v e r o l e fo; t u b e r c u l i n m t i s s u e c u l t u r e .

1 . There foliDvea the demnstration

dy

~ ~ ~11961, 1962) t h a t t ~ i ~ a

.

^' ^',

.--

t h e small,lymPhocyts from hW." p e r i p h e r a l blood can transforpl mrpho- r ^, ~ o g i s a l ~ y under q e i n f l u e n c e phytohaemagqlurinin exkracted from

' ^.

^,

' . '

the red kidney bean, P ~ W O I U ~ vulgaris. The c e l l ; become I s w e , strong-.

. . .

IY b a s b p h l l i c and b l a s t - l l k e with prominent n u c l e o l i and subsequently

- ,

divide. meoe' f i n d i n g s were confirmed by n a r ~ h a l l a n d ' w b e r t r 11963).

. .,'

, , ~ c h i e k and.P?lbinovitz (1963) aid ~ $ ~ d ~ h l ; ~ i ~ ~ ~ i i ~ ~ ef a l . (1963). . .

.

This l e d Peamarn et a1. '1196$)

'&

examine t h e efSect of tuber- c u l i n vpon lynphocykea fro. t d r ~ u l i n - s e n s i t i v e i n d i v i d u a l s i n t i s s u e - ,

.

c u l t u r e . They found a $id.lar m r p h o l o g i s a l r r a n s f o m t i o n resul+ng

. .

, , i n cell di"i.i.", where..

ceiill

from Ulbercu1in negative donors did nor ^'

\ divid.=. These ~ h s e r v a L i o n s suggested t+,e an i m n a l a g i c ~ l l y s p e c i f i c ,

recognition o f p n r i s e n

wan

made by lymphocytes of s s m i t i s e a i i ; l a i v i d u ~ , '

--

i n

mis

p h e n m h n was also reported by omer workers lcowling

et a l . . 1963; Schrek, 19b3; Marshall 6 Roberts, 1951).

.

' , Thus ly;nphocytes Of a eensitise.3 i n d i v i d u a l or animal are s t i m - latea by t h e presence of s p e c i f i c antigen to d c o m e l a r g e a c t i v e p r o t e i n ²

I

^' s y n t h e s i e i n g b l a s t . c e l l s 1Torreli et a l . , 1966; .sof:ky P Oppenheim.

1966) a h d . i t t h e r e f o r e becomes p o s r i b l ~ that the tuberculin reactron it-

.

, s e l f might.be linked to some p:odusr of the lyaphasyfe. These s e l l s , s p e c i f i c a l l y S e n s i t i s e d t o tuberculin and capable of w e m e n * through ' mhYt i s s v e s of the hody 1Darms. 19591 Marcheei.6 W a r n , 1964; Owans.

, -

1966) light‘ respond fo the presence of that a n t i g e n with the prodvction of s u b s t a n c e s e l i c i t i n g an inflanlarory response, of which the butansous - , t u b e r c u l i n r e a c t i o n i s bur one n a n i k s t a t i o n .

. . .

(21)

3

1 1 ( .

i '

. "

^.

.

- Mhers had omsidered *his W s n i b i l i w a t ah e a r l i e r period.

.

'

' z i n s a e r and Taniya 11926). ~ l l t e ' a n u e'c a l . 11938). 2$rrsre and

' ,

P u t r e f u g e s I19521 had attempted V, shar, by t h e incubation of nenai-

,

t i s e d s e l l s with antigen &

%.

t h e presence of substances which '

.

w o u l d ' s i u s e a reaction o f ihe t u b e r s v l f n type upon intrade-1 i n j e c t i o n .

.

. .

.

-

' i n t o n o m i a n ~ o a l s . wareve=, the r e s u l t s of meae e x p e r i m n t a xire nemr emvincing.

S i n i l a r l y , w&ksnan and m a 1 t s y 11&81 had i n d a t e d s e n s i t i s e o p e r i t o n e a l exudate c e l l s and l m h node o l l a w i t h . m t i g e n f o r up to . ,

.

. .

₂₄_{h r}

and Men injes(ed me resuspended Cells i n s v l t v r e f l u i d i n t o t i e '

s k i n 0, do-I a n i m l s without ptodvcing an inila-tory r e a m * except : VPM one occasiod.' They used = o n c e n t r a t e d oll aljspenaiona in s a l i n e

''

. .

and p l a s m and suspected t h a t ,st of m e c e l l s we, dauaqma

dr

k i l l e d

- - ^..

. . ^, ,

by the of the ino;lharion.period.

.

-

Hwever, i n 1959 aohanovsky reportad t h a t , after incubating c e l l a

. . .

_from_rabbi? s e n s l t i s e d to BCD w i h d i l u t e t u b e r c u l i n , the c e l l - f m e s u p e r n a t a n t s provoked an inn=-tory response men i n j e c t e d i n t r a d e r -

r

n a l l y into normal aninullg. he i n f l m t r o n appeared withln 2 4 hr an-d

I .. .

conera1 s u p e r n a t a n t s did not produce such a reaccidn. ne t e s t e d p e r i - .pher*l bled Fell*. -spleen F e l l s . " d , c e l l ~ frmn cisterns c h y l i .

.. .;

, , Thee r e s v l t s were repeated i n 1960 by Johm-ky, u s i n g s e m i - -

.

- t i s e a guineh-pig p e r i t o n e a l emdate c e l l s c u l t u r e d vi+h t u b c r s u l i n a r , '

. . .

d ~ p h t h h i i a t o u r i d

at

a c e l l F.centration of 1.25

-

^7.5x ~ Q ~ ~ ~' I' I S I, . ~

:

£0. periodn of from 2 to 20 hr. ngain.the & n a t a n t f l u i d s i n -

( - .

.

: ^I

j e c t e d i n t r a o u t m e o u s l y and the r e a c t i o n s w a s u r e d a t 24 uul 4 8 h b i y p

~ h b s Y t e cul*ures fran a n i m l s ' a e n s i t i v a tubbrcvlin nore f r e q u e n t l y ,

.

. of e l i c i t i n g an infl-tory response,

7

.. . . t ,

(22)

(23)

- . . . -

-

. .

³ ^,

. ' when incubated with t h a t a n t i g w , thanirhe&;ubaled with d i p h t h e r i i foroid as a control. nweuer,the s p e c i f i c i t y of the reap,onae was n o t always demonsrrable. .me difference be&n the eqntrol\end experi- m e q a l groups was e a i d to h a w been mast marked when those e n i i l s v i t h

. .

&e m0.t i n t e n s e and.thoee with negative reactions were eonpared. . , ' In 1960, voisio end T o d l e f , invesrigsting thei&difiesfions of- e a p i l ~ e r y p e r m e a b ~ l i w i n i r m n o l o g i c a l reaeribns using e?-s blue dye, -. had been able'co d e m s t r e t e a delayed insreaae i n p e m a b i l i t y b e g i o -

ning at 4-8 h r after the iatrade-l i n j e c t i o n of tuberculin info

,

segaitieed guinea-pigs. We h m g e i n p e r m a b i l i t y reached i t s peek at

,

24 hy (see plgmre 4).

9 A possible nediaror o r the change w a s the l y ~ h node p e m a b i l i t y factor (LNPQ d e s e d m d by Willovghby et a1. (1963) i n e x t r a c t s of lgmph npde c e l l s f y w r u b e r e u l i n J b e n e i r ~ w iuinea-pip. % i s material, which

i

'

. .

wm diatingviihed from hieemmine, serotonin, bradykinio, k a l l i k r e i n and

.

globulin p e w a b i l i t y Factor by p h a m e o l o g i c a l means, produced a signi-

b

ficimt'increaee i n vasculat p&-abi?iw a f f e r ' i n f r a d e d i n j e c t i o n

. . . .

'

.

i n t o n o m l a i d e . mere wae w *associated emigration o f le&ocyree ' '

.

,

.

. heginning with and f o l l n r e d within en e a r l y i n f i l r r a t i c a b f polgmorphonucle8r i e d o c y t e s 6 h r by ,a callul$r response , c m e i s f i n g of almst'

- .

,

. . .

e n t i r e l y h o n u c l e e r c e l l s . l h e a e cells p e r s i s t e d For several days (noughton h Spector, 1963).

-

'

ixtraets of rhe skin-bearing rvbercvlin reactiona,aearmstrefed a:.

.

rise i n eoncentration of LNPF i n p a r a e l w i t h the deuelwmeof .of t h e lesion, waning as the reaction subsided. amever, n o d a s well a s s e m i - rised lymph node c e l l s were f o k d t o urnrain t h e f a c t o r LNPP w d b o t h '

.

ralsased i t i n t o the -dim upon incubation with PPD. In addition, a ,

(24)

-

- .

- (69611, (9 ZD 'd1Od

. .

-1e as yocarX + 'omom

- "%

I . , . . z z ~ i ,

, 16961) las?a&'

we asyen , ^>' a

i '97-9 xoT ' 4 p , & n s r ) o a a rr ^,arxsx m , s u a s

.

169611 19 91 ' 6 1 d ( 3 M I 01%

,re xe *=?a + .mom F ~r . 1 ~ l G 3 ~ 0 ~ / 6 n d r ~ a d a IL -

-

I?~ZI:OTXI m a . ~ m . s u a i 16961 '896Tl

.

, IV 91 'AT06

' 1

' la apvowna

.

,

+

'oum

.

01-9 xI ~ru/hw-z.o~ras zz 'oTxT ?aa,3wstras

-

r

-

189671 UOoTB ^0". ^??emma ¹⁹^s7^'ouom ^I ^- ^x01 ^lr=76nsz)^odd ^{' s z .}

^Lorrz'r

^3m.suas

a, D l d - W T " 3

,

e z q a n v a a e = + ~ ? ~ u ~ lq) a u * ~ w 20 amnxeN uoy7 -=ad-

uo?%=zluaouas 191 u o n e r i TTaJ l o s d h ~

. .

^weautle~nuri?~ am> -uaouaj

-,sar

-

_Onp, " o y l e a lo -m=

. .

^rTaa ^.

-

_UT_3.3_-ue3u03 ^"T

*mab ^{a q 1}

.

I- . ~ . .

. -

- -.

. .

.

B X ~ J ~ Z anpoem urns -

. .

/

. . .

- . '

Y a',mz

Q

.

^,

(25)

. .

preparation of minced rar l i v e r incubaced with rat serumhad similar properties upon iolradermal i n j e c t i o n m lNPF (Specror s Willoughby,

1964).

.

_,

.

, Ihua, t h i s material may not b a ' i m o s p e e i f i e b u t s i q l y be s o a

.

'substance e x t r a c t a b l e fzoa l m h node -11s and o t h e t l i v i n g

6isska.

^.

.

LNPP i s s a i d n o t Lo be destroyed by p r o r e a l y t i e and to hsve a

. .

.

molecular weim't >100,000 (Schild & Willpughby, 1967) and these authors ^' ,

* -

.haw. raised. th. p b s s i b i i i t y that the a c t i v e principle O F LNPF map be

.

?

ribonvcleie a c t d ( M A ) o r ne o f d t s dsgladarian plraducts. Wi* was

.

.. ^.

^.

9 .

^,^. ^sham'to^havei n f l a m t o r y braperties i i n d l a r t o LNPF.

. .

me presenve of

.

LNPF a t the s i r e of a tuberculin reaction may thus& the r e s u l t a f

- .

f e l l i n f i l t r a t i o n and nor df s p e c i f i c t e l e a s e of a m;diabr'of the res- pons*;

.

, . ,,

.

,

A n u h e r of avrhors have r6cently described soluble factors.de-

.

. .

.

' aved From l m h e c y t e evlrvres w d abl* to cause akin i n f l m h o n . me

'.

esa?nrial fearurea of thebe ?re ivm~vlrieed i n Table A.

In the e.tpe.1-nls of Bemet6 and Bloom (1968) tha intrade-1 i n j e c t i b . of Che concentrated svpernstBlls f m m stimul.ted culiures .

.

= N e d skin r e a c t i o n s which q p e a r e d in 3-5 h r , were medmel at 8-12 hr, and dissppearedlby

i o

^hr. ^msaareairions f o n s i s t s d of moderate indur-

a t i o n and erythema w i t h a diamster, of 11.8 f 0.63 m v t d l e t cdntrol ^'

. . . . .

supematanre pmduced smaller eryrhemarovs reactions of 6.9 t 0.98 m

*

. .

^?I1

.

guinea-pigs t o r u l e dui p s s i b l e reactions due t o foreign his*

incompatibility antigens. ' .

.

, .

u i e r o l o g i c a l e m d n a t i o n of the r e a c t i o n s

=baed(

at four hours, an emdare coneisring yearly e n t i r e l y of m ~ n m v c l e e r c e l l s . l a t e r , , naitrophile eppeared.so that by 14-16 h r exudate of $rodmacely

* f i ' , 4

. .

.

I

(26)

(27)

' 17

. .

^*^,^* ^.

.

equal ovmbera of manonuclear c e l l s w d neurrophila w a s p r i e n t i n t h e

1

dermis and subeutaneaua tissue.

. . . .

^,

Duma& er nl. (1969) used 125~-labelled hu&" serum e l b a n t o

. .

early

changes in pemesbilify iRfecLiDo

. .

.

. ^. .

" .

: . .

-

l a f i o n o f radioactive l a b e l t h a t control s y p e r n a t w t ~ did. Huch of t h i s

, . .

accumulerion occurred w i t h i n t h e f i r s t hour. A. more delayed component

. . .. .

^wassbovn by .measurement of t h e i"durarY0" produced (see Pig&=* 5;.

, .

.

,

.

This revea1ed.a d i f f e r e n t i a l i n c r e a s e i n skin thickneks between 6 and 10 , h r after i n r r a d e m a l i n j e c t i o n , a t which rime t h e r e was e c e l l u l a r i n f i l - /

. .

t r a r i q i o r the e * l d t e s t s i r e s with mononuclear and po1yhorphm81esr

. .

, c e l l e . , x r was n o r e i t h a t l y d G c y r e eonieates'dild not qroduee such

&

--

.

infl-;tory responsa, sugg&ling t h a t acute c e l l death was nor respon- - . -6 s i b l e f a r the e f f e c t .

-

ma superharanra examined by ~ r e j c i e t a i . (1969) €05 i n f i e m a -

.. .

, ' . tory p r o p e r f i e s , "am derived from r a b b i t lymph node c e l l c u l t u r e s Bnd

.

,, were r e s t e d by i n t r a d e m a 1 i n j e c t i o n i n normal g ~ I n e a - ~ i g e . Chanses in.

. . . .

' vascular p e m e a b l l i c y were assessed by the 'use of Evan. blue dye: con-.

. .

: . . r r o l ' s u p e r n a t a n t s &om culturee 'incubated wirhovt anrigen produced a n

. .

.

^. immediate erythema with en increase i n vi;cu1er permeability rnajlmel ar

.

;

-.

30 minutes and r ~ b s e q u e n t l y fading.

-

A s l i g h t inii-&ry i n f i l t r a t i o n

7

^,

,

.fp.ly~omhon~.~e.. O ~ . C U . ~ ~ . In c ~ ~ ~S ~ P ~ . ~ ~ . . s t . .

.

^.

stimulated c u l t u r e s ; n i t i s s l y prodused an .rea of u m s i d e r a b l e p d ~ o r , ' '

peaking a t 30 min and subsequently fading. m i x e was no chadge iq vao-

- .

enla= permeability at r h i a time. A small area of cenrral'eryrhqma then ^''

' i . .

. .

occurred, gradually k l a r g i n g , yithgen aasodated increase in p e r m e a b i l i ~ , : ' ,

:

I .I

. .

1 ' ,

. .

<

. .

,

-. . . . ( .

.

: .

^-

- . . I , . -

(28)

(29)

.

,

. . .

^..

. . .

^,

^. .. . ' .

. '.

. . . . . . . . . . . - . . '

¹⁹

_,

.. .

_.

. ^. ^.

'

.

. r e p ~ ~ s i n p the pellor in 4-5 hr. &.our 12 h r l a r e r rhe eryrheaa was f o l l a r s d by indur.afi.n and at Z b hr there yaa a cono*dersble r.ast,on' '

. o

, : ( ^,F t h thk hpkrroseopls apie.rsn.. of *,delayed hy~"rsenslll"tty reaceion.

,C" .,.

. .

' . m i h ~ ~ t o ~ d g i ~ ~ ~ f i n d i n s at 12 and.%& hr : ~ h m d an inflrntory i n f i l - ' . /

.

^.

. , . .

^of& i y m o r p ~ o n s c ~ e e ~

^I

ina(goEonuclkr ~ ~ 1 1 ~ '

cse

p e u r e 6 ) .

- .

,. .

'

,

. ^' ^{, '} rne.re&~r. or the clme+-mra . .

..

o t ~ e i e e a, weiser ( 1 1 6 9 ) s-n-

.

^'

,''.<mred rh.: s"p.i.rm'rs f r o n - ~ s * s c i n u l a r ~ d .en.ltLsed Iywhphoeyreg

. . . ^. .

^.e1ieit.a ^.k* raactt."s, whereas e"pem.l*nia' ran .cultures of nqrmal '

;

,

... . .' ' .

.. . - _{, .} _.. .

^'

_. _.

^were19mp~h'.cytes supernatants

.

tnsubpted "tth or rrom sensrrised ~ y l n p h ~ s y r e s "irhour

.

entige

. i

were cultufed ^' mu*,, viehour less aerive;

SD.

ss OI ,

.

, saoe i n t e r e s t w a s

.

the n o " a p a c < f t c i n f l m y i . r y , ecrlkry of aupernat.oia de.rived from SensLtiue br noma1 ?laClp:*ee in."bsted;lch PPO (see

,

. . . _.

_,

-. - - ~ l g v r e I ) . k e ~ c t i o n a m ali caqes appeared ac about 4 hr and bagen I; ,

.

. ^.

fade eradually after L6 hr.

alsopisring

^by^48-72^{hr. '}me histology

"

'

oq"enese

Lac=,ons

ys ~OCexaminad. .

. . .

,

. . . - . . ^, ^.

^{p i e r}e t a l . (1969) i<</sriiated in ~ b n s x d e r a b l e detatl the s i n

. . . .

. . ,

R e a c t i M F S C ~ O * ( 5 W ) releaeed by a n t i g e n - a r i m u l a l e d . 1 y ~ p h h h y Y ~ ~ . In

. . .

:

.

^Chi⁼ ^hen.3sthe in,ection of pupernatants from

the

se!sirls.d lymph.node.

..

. .

_"*

.. .

eella and peritonea, e x u d e f e l y n p h o q t r s infvbared W i t h PPD produced an ,

* .

. .

L

^{. .}

inrramqteq ,reesrion'dfh eryrhemahn( tndurrtion reachin& its peak I"

, _,. 3-6 hr.,

- .

k e r e was said to ba no svch ectiusti in aupeAarmts from "on-

..

' .ensitive lymphocyte cultures incubaced "itb ,anrigen. nareuer: tnese

' .

rm.Ate

vere

"@liven. *Ithaugh there appears A to b% s d e f i n l f e dlfierence. .

. . .

.

. . .~

. .

'

-

^- ^*ween control and srl.ul..ed :"lture., here, aa in prernous . I p e r s , rhe <

I .

.

,

. .

,

..d"pp.s&l;

unef*."liafed supernacanrs-also Sf times *.,;her marked -

. .

'

-..

i n f l a m f o r y r e s p m s e s . Marerial from unfragti~nnred perironeal exudates

.-;.

, . ^rodburtfiaa.p;rifonee~ maorephp8e eulrures p r ~ v c e d ~nf;ema~o;;-l

: ^:.

. ' I ,

' .

^,'

^. ^. ^.

_ I . I

.

. .

_.

. ^" ^.

_.

:'.

.

^,

" .

,

. ^. ^.

(30)

20

' .

,

CULTURE SUPERNATANTS Lymphocyfe I

MacrOphane

"

-.

, . 6 1 ⁴⁰ 1 ^;,;

^- ^a

-.- a.

Q F 20

s

B 0

k

.

. .

< ^.

.

~ r m m .7. me =.kin reactions lm21 i n n h a l 'guinea-piga e l i c i t e d

Cross-hatches histogrug indibate su@rnatants fmrculturee incubated with the antigen. (HeisB and Weirk, 19691. .* -,

_{. *}

'

. . .

^'.

.

. .~

~

_{. .}

.

,

. . .

. . . .

.

r

, .

^' ^I ^.

"

_ .-

r - - - .

/-

(31)

reacr$ons

.

. b u t here t h e d i f f e r e n c e s between s t i m u l a t e d and e o n e r q l super-

m n*hnts were much l e s s s r r i k i n g .

:' N i e r o s c o p i c a l l y a t 6 h r t h e r e occurred a mired poiymorphmuclear- w o o n u e l e a r leukocyte i n f i l t r a t i o n i n the deeper p a r t s of t h e dermis,

.

. 8

t h e c e l l s being r n a i n l y ~ p e r i v s o e u l ~ r , There were e i t h e r Lqual n m b e r s of b o t h c e l l types o r a predominance of'$olymorphqn"clear leukocytes. A t . 24 h r t h e ' i n f i l f r a a i o n was reduced b u r s i m i l a r i n composition (see F i g u r e 81.

.

me pmduecian of inflammarorl supernatanla vas prevented by t h e preeence o f actinomyein or puramycin. t h e l a t t e r reducing p r o r e i n gym-

-

. .

, t h e s i s t o l e s s than 5% of p r e v i o u s l e v e l s . I n a d d i t i o n , pep;in des-

troyed the inflammatory a v r i v i r y o f t h e supematants w h i l s t r i b a n u e l e e s e

. ^.

or deorribonue1es4e had n o , e f f e e t . F r a c l i o n a r i o n of rhc s u p e r n a t a n t s ' on Sephadex 0-200 produced a peak c o n t a i n i n g n o e l a f t h e inflammafon,

. ..

,

.

a c t i v i t y w i t h a moleevlar weighr'approxinately-that of serul. albumin.

. .-..

, n I

, P

^-A number of o t h e r s u b s t a n c e s are d e t e c t a b l e i n t h e s u p e r n a t a n t s , o b t a i n e d f r o . qrimulared s e n a i r i s e d lymphocytes. The fallo%iing have', been idenr<fied v i r h some p r e c i s i o n by t h e i r a b i l i t y t o madify the

in

v i t r o a c t i v i t y or behevfoui,,oP s u s c e p t i b l e i n d i c a t o r e e 1 l r : ; m g r a r i o n .

-

I n h i b i t o r y A c t o r (HIP) vhich i n h i b i z ; Ehe move-nnqf norm@ unsensi- , '

' I

a s e d maeraphages (david ef a l . , 1964; BInnett S ~ l o o n , ' 1 9 6 6 ) , Chew-

. .

taerfe Factor which induces t h e d i r e c t i o n a l movemenl o f n o m a 1 unsensi-.

r i s e d nacrophsgea along a concenrr;tion g r a d i e n t 'of t h e m a t e r i e l (ward

'

er a l . , 19.69). Lymphatoxin (LT)-which damages and k i l l s e v s c e p t i b l e . target. c e l l s

ina

"an-specific manner (Granger e; a l . , 1969). The p r e s e n c e o f such substances i n a c t i v e svpernatents has been cdnsidered

.

.?

(32)

(33)

. . . ^. . . ^.

an'% vitrn c o r r e l a t e of M e d e l a y e d h y p e = s e n a i r i v i t y r e a c t i o n ITilrrence. 19681.

'

Whec f a c t o r s h& been d e s c r i b e d whose s e p a r a t b c h a r a c t e r i s t i c s ,

.

have been l e s s well worked our, e.g., b l a s t a g e n i c f a c t o r s thee s t i m u l a t e

.

n o m a 1 lymphocytes td t r a n s f o r . a n d &vide IKasakwa & I w e n s t e i n , ,1965;

Bennett. s Bloom; 1968; k n a e et a l . , 19691 va~entiae 6 mwrence, 19691:

or a fasmr that may i n h i b i t t h e mvemenr of rm-1 p e r ~ h e r a l blood P o l m r p h o n u s l e a r leukocytes I w r g , 1967. 1969).

mere i s thus p o s $ i b i ~ i r y t h a t t h e s k i n re.ctive or ' in€lmy:ory f a s t o r i s a i w l y a mixture of these f a c t o r s . A solnp1ere anme=

y

^mscne c e s e a r i 1 y a w a i t th: y i o n of mre <"fO"&

ti-" dins me physisoahemisill characteristi.. .f SW. as to HIS, c h e m t a ~ t i c f a a t o r O X LT. But it is.!mst like19 that these o t h e r f a c t a r s . a r e important i n the development of the tuberculin reas-

4

tron, s i n c e t h c i r presence appears t o correlate so "ell with the a b i l i t y to develop a delayed h y p e r s e n s i d v i r y s k i d reaction.

mu. ovnande et al: 119691 were a b l e t o show t h & , i n t h e i r

.

, e x p e ~ i m n t s . t h e supernatants fm s t i m u l a t e d lynphocyte cultures, as we11 1% prnducins an infla-tory response v m i n j e c t i o n inn, also had t h a a b i l i t y to $timulate rnrrnal' l m h o c y t e a to synt?e:ize m a , 1.e.. '

,, .

had

m i m g e n i c - a c t i v i t y , were sytoroxis to muse f i b r o b l a s t s a n d . i n h i w t e d t h e movement or 'normal p e r i t o n e a l exudate macrophag&:

Bennett and8100. 1196e) f r a a t i o n a t e d their ~ m p h o c y r e c ~ i t u - supernatants, after concentration by g e l f i l t r a h i o n , on sephaaex

1 i .

co1unns. They found a peak co*esponding t o an average molecular weight

.

, of a ~ ~ r o x i n a t e l y 67.000. warked i n h i b i t i o n of macmphage migration,was. ' fauna i n ' t h i s f r a c t i o n . d e r i v e d from stirnulared l m h o c y t e m i t u r e s . ~t

.

^'

'

.

^I ^'

.

^-

(34)

-;

: :. r ^,, ^, ^!

. .

vas.this n a r e r i a i t h a t produced infl-tow r e a g t i o n s won i n t r a d e m a 1 , injecaio". d e s e ' r e s u l t s u o ~ l d he a g a i n s t t h e sw being an anrmoay'or ,

I.\

antigen/&tibnay cowlex..

. .

. -

"=ise and we~ser 119691 s i n r l a r l y fracrionared t h e = culture s u p e r n a t a n t s , "sing sepkadex G-200 and.fauna Mar a fraction, c a r r e s - ponding to M e ?lutim peak of bsvine y:globulln and part of a

. .

L o a d pe& by t?e ~ o l m , ddnrained m s t

or

Me s y t o t o x i c a c t i v i t y , as i n d i c a t e d by ~e i n h i b l t i o n of g r n r t h of muse r e e l l s

.

'*"d a l s o inhibited macrophage nugra<ion and produced Ski" r e a c t i o n s

*'

"PO" i n t r a a e m l ' i n j e c t i o n .

Tt i s t h e r e t o r e pertinent t o examine t h e a c t i v e supe'matants in- ,

,.

^{j y a a}

tgtest

^*ors?., f o r t h e presence o f n I ~ . o r other of Me possible a

--

i n v i t r o $ o r r e l a t e s of delayed,hypersenslri"it~~

R f a r t h e r consideration i s whether the c e l l s a c t u a l l y ' p m d u c i n g

-

t h e n e d i a t o r e . a r e those lymphocytes t h a t t r a n s f o m t o blasts and siib-

...'

s e q u e n t ~ y divide. ~ n d e e d , it may be that t h e p m c e s s of t r a n s f o r n a r i c n

.

is not inseparably linked t o the prpauotion of m d i a r o r s .

:

aeonere m a s1o.m 11967) .anakrejci

. ,

(1969) were able to ahov . ^*

.

th%t "IF and SRF a< d e l e c t a b l e withit! 6 h r of incubation o f s e n s i t i v e

.

ly*hhosyres vith.a"kigen, bur t h i s doeb not a- a g a i n s t t h e =ell.

. , '

producing the nedi.+.Ore going on k. f u l l b l a s t t r a n r ; f o m t i o n . Tndeed, , , *

' i t i s ""fortunate, considering tiie l a r g e c e l l a d intigen p a n c e n t r a t i o n s

p

, .

.

"Bed i n t h e production qf

snr

t h a t i n none o f e experiments so f a r s o n s i d e r e d was l m h o s y t e t r m s f o m e i o n .crud1

l

^assessed.

^mis

^covld

I

^,be considered p a r t i c u l a r l y r e l e v a n t m the l i g h t of M e n m r o u s reports .

.

~ p p a r e n t l y denonstrating s p e c i f i c s e n s i r t e e d cell damage by r v b e r c u l i n

; cvide SUPPPI. especially.nay t h i s be inpornant where oultures are c a r p e d

-

\

- 1 WTAL QE W@AOJW ONLY