investigation of the generation of a pseudo-secondary

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Investigation of the generation of a pseudo-secondary response Investigation of the generation of a pseudo-secondary response

to LPS induced by macrophage toxins to LPS induced by macrophage toxins

Kathryn Martin Remington The University of Montana

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AN INVESTIGATION OF THE GENERATION

OP A PSEUDO-SECONDARY RESPONSE TO LPS

INDUCED BY MACROPHAGE TOXINS

By

Kathryn Martin Remington

B .S ., Whitworth C ollege, 1978

Presented in p a r t ia l fu lfillm en t, o f the requirements fo r th e degree o f

Master o f Science

UNIVERSITY OF MONTANA

1981

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, ____________Chairman, Board o f Examiners

Dean, Graduate School

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ABSTRACT

Remington, Kathryn Martin, M.S., March, 1981 M icrobiology

An In v e stig a tio n o f th e Generation o f a Pseudo-Secondary Response to LPS Induced by Macrophage Toxins (79 pp.)

D irectors Jon A. Rudbach / >''

A s in g le dose o f Ilpopolysaccharide (LPS), adm inistered 21 d a f te r mice had been trea ted with the macrophage to x in s , carrageenan ( car) or m icrop articu late c r y s ta l l in e s i l i c a (MUS), generated a ty p ic a l secondary-type antibody response. The phenomenon o f pseudo-secondary responsiveness was in v estig a te d to determine a p o ss ib le mechanism for i t s gen eration . The use o f i - , K-, and

carrageenans and th o ro tra st (TT), in ad d ition to CAR and MUS, in attem pts to generate th e pseudo-secondary response, in d icated th at lysosom al d e s ta b iliz a t io n o f macrophages was a necessary r e q u is ite fo r th e generation o f the response. The ex ten t and duration o f modulation o f the r e t ic u lo e n d o th e lia l system (RES) by CAR, MUS, and TT was measured by the a b i l i t y o f mice to c lea r c o llo id a l carbon from the peripheral blood. I t was determined th at phagocytic a c t iv it y had recovered by 5 d a fter , treatm ent with macrophage to x in s and th a t i t remained normal or enhanced th e r e a fte r . Attempts to generate the pseudo-secondary response in mice which wore g e n e t ic a lly incapable o f responding to the l ip id A region o f LPS were u nsuccessfu l; thus a requirement fo r the g e n e t ic ca p a b ility to tr ig g e r a pseudo-secondary response by LPS emerged. Adm inistration o f LPS a t various in te r v a ls a f te r a primary in je c tio n o f LPS or CAR esta b lish ed , f i r s t , the k in e t ic s o f development o f true secondary responsiveness to LPS and then the k in e t ic s o f pseudo-secondary resp on siven ess. The temporal k in e t ic s o f true secondary responsiveness and pseudo-secondary resp on siven ess were id e n t ic a l . The generation o f pseudo-secondary resp on siven ess was attempted in athymlc nude m ice. F ailure to generate the response in th ese mice in d icated the T c e l l dependency o f the response. I t was p o stu la ted , then, th at pseudo-secondary resp on siven ess was the r e s u lt o f priming o f B lymphocytes by the consequences o f the.macrophage to x in s , and secondary responsiveness to LPS was generated in a normal manner.

i i


ACKNOWLEDGEMENT

I am indebted to many people, without whose h elp , I would have never

completed t h is study. Dr, Jon A, Pudbach, my th e s is ad visor, was a

good tea ch er , I thank him fo r h is expert guidance. The d irec tio n

provided by my committee members, Drs. George Card, Gary G ustafson,

and Walter H i l l , was most h e lp fu l, and I am g r a te fu l. Many o f my

fe llo w students taught me methods and tech n iq u es. I appreciate th e ir

help and fr ien d sh ip . I am g r a te fu l to my parents fo r teaching me

to s e t g o a ls fo r m yself and fo r th e ir lov in g encouragement to a tta in

those g o a ls . F in a lly , I thank my husband, David, fo r h is lo v e ,

understanding, and a ss is ta n c e . His support made i t a l l worthwhile.

I l l


TAÜLE OF CONTENTS

PageABSTRACT.................................................. ............................................................ . . . . i i

ACKNOWLEDGEMENT. ..................................................... . i ü

LIST OF TABLES..................... '.................................................................................. ..

LIST OF FIGURES..................................... . v i i i

ABBREVIATIONS............... ix

I . INTRODUCTION...................................................................... 1

H is t o r ic a l . .......................... 1

Statement o f T hesis and Approach to the Problem................. . . . 1 0

I I . MATERIALS AND METHODS............... . . 1 3

A nim als.. ................. .13

RML m ice............................................ . . . 1 3

Nude m ice............... .13

G3H/HeJ m ice........................................................................................13

Macrophage T oxins................ ............ l4

Carrageenan s . .......... 14-

M icroparticu late c r y s ta l l in e s i l i c a . . . . . .................. . . . , l 4

Thoro t r a s t ............................................ . . , . . . . , . . , , 1 4

L ip op olysacch arid e. ................................................................................. 15

C o llectio n o f serum fo r p a ssiv e

hem agglutination a ssa y s . . , . . . . . . . , . . . . . . . . . , , . . . , , , 1 5

Anti-LPS t i t e r s ............................... ................. 15

Measurement o f phagocytic a c t iv i t y by RES clearance

o f c o l lo id a l carbon...................... 17

I I I . RESULTS................. 19

I V


PageDetermination o f amount o f th o ro tra st to suppress

the RES...................... 19

Attempts to generate a pseudo-secondary response with

variou s macrophage to x in s ...................................................... . . . . 2 ^

Duration o f modulation o f RES a c t iv it y by macrophage

to x in s ........................ 32

The g e n e t ic requirements fo r generating pseudo

secondary responses! attempt to generate the

response in LPS nonresponder m ice 35

Temporal k in e t ic s fo r generating secondary

resp o n siv en ess ............... 44

K in etics o f the pseudo-secondary r e s p o n se ,. ................... .44

. Modulation o f RES a c t iv it y by CAR in athymic

nude m ice....................................... .47

Attempt to generate a pseudo-secondary response

in athymic nude m ic e . ........................ . . . . . . 5 2

IV. DISCUSSION............... 59

Macrophage to x in s and the pseudo-secondary response.................6 l

RES modulation and the pseudo-secondary response, . . . . . . . . . , 6 2

The g e n e tic requirem ents o f the pseudo-secondary

response............................... 64

The k in e t ic s fo r development o f the pseudo-secondary

resp on se............... 65

The requirement fo r T c e l l s and the pseudo-socondary

re sp o n se . ................. .66


PaceC onclusions.......................... «6?

V. SUMMARY....,............................................................................. 71

LITERATURE CITED.................................................................................. 74

V I


LIST OF TABLES

T a b l e P a g e

1, Generation o f a pseudo-secondary response with s i l i c a (mus) in white m ice......... .......................................... 25

2 , Generation o f a pseudo-secondary response with Seakem carrageenan (CAR) in white m ice..........................26

3, Generation o f a pseudo-secondary response with, i-carrageenan (i-CAR) in white m i c e , , , .................................28

4 , Generation o f a pseudo-secondary response with K-carrageenan (k-CAR) in white m i c e . , . , , , ....................... 29

5, Generation o f a pseudo-secondary response with A-carrageenan (A-CAR) in white m ice................. , . 30

6 , Attempt to generate a pseudo-secondary response with th o ro tra st (TT) in white m i c e , , , . . , , . . , , . . ..................... 31

7 , Attempt to generate a pseudo-secondary response with s i l i c a (MUFi) in LPS nonresponder mice ( i e . C3H/HeJ mice)

8 , Attempt to generate a pseudo-secondary response vrith Seakem carrageenan (CAR) in LPSl nonresponder mice ( i e . C3H/HeJ m ice)...................................................................... .43

9 , Generation o f a pseudo-secondary response with Seakem carrageenan (CAR) in the presence and absence o f T c e l l s ( i e . in athymic nude mice and th e ir normal l itte r m a te s ) .......................................... . , . 5 8

V I 1


LIÜT OF FIGURES

Figure Page

1 . RES a c t iv it y fo llow in g in je c t io n o f varying amountso f TT i . p . . . ............... 21

2 . RES a c t iv i t y fo llow in g in je c tio n o f varying amountso f TT i . v . . .......... 23

3 . Duration o f modulation o f RES a c t iv it y fo llo w in g i . v ,in je c t io n o f 10 mg o f MUS...................... . . . . . . . . . . 3 ^

Duration o f modulation o f RES a c t iv it y fo llo w in g i . p . in je c t io n o f ^,0 mg CAR. ...................... .37

5. Duration o f modulation o f RES a c t iv it y fo llo w in g i . v .in je c tio n o f 50 mg TT ................... 39

6 . Duration o f modulation o f RES a c t iv it y fo llo w in g i . v .in je c t io n o f 50 mg TT. ........................ . . 4 l

7 . The e f f e c t o f varying the tim ing o f the secondary in je c t io n o f LPS with resp ect to the primary in je c tio no f LPS............... 46

8 . The e f f e c t o f varying the tim ing o f the adm inistrationo f LPS with resp ect to the adm inistration, o f Seakem carrageenan (GAR) .............................. .49

9 . Modulation o f RES a c t iv it y by Seakem carrageenan(car) in athymic nude mice and th e ir normal l i t te r m a te s . .51

10. RES a c t iv it y fo llow in g in je c t io n o f varying amountso f Seakem carrageenan (CAR) in athymic nude m ice . . . . 5 4

11. RES a c t iv it y fo llow in g in je c t io n o f varying amounts o f Seakem carrageenan (CAR) in normal litte r m a tem ic e . ............. .56

12. The generation o f pseudo-secondary resp on siven ess . . 6 8

VI11


ABBREVIATIONS

. phagocytic index corrected fo r d iffer en ce s in body^ and organ weight

BAF B c e l l a c t iv a tin g fa c to r

B c e l l B lymphocyte

BDF B c e l l d if fe r e n t ia t io n fa c to r

Bl2f TI-2 responding memory B c e l l s

B2ÎT T-dependent responding memory B c e l l s

i-CAR io ta carrageenan

K-CAR kappa carrageenan

A-CAR lambda carrageenan

CAR Seakem carrageenan

IL-1 in te r le u k in -1

IL-2 in ter le u k in -2

i . p , in tra p er ito n ea l

i . v . intravenous

K phagocytic index

KHF k i l l e r c e l l helper fa c to r

LAF lym phocyte-activating fa c to r

LPS lip op olysacch arid e

MP m itogenic protein

MUS m icrop articu late c r y s ta l l in e s i l i c a (M in-U-Sil)

PBS phosphate buffered s a lin e

PFG plaque-form ing c e l l

RES r e t ic u lo e n d o th e lia l system

RML Rocky Mountain Laboratories

SCIF ' secondary c y to to x ic T c e l l inducing fa c to r

ix


SRBC sheep red blood c e l l s

T c e l l T lymphocyte

TCGF Ï c e l l growth fa c to r

TI T c e l l independent

TMF thymocyte m itogenic fa c to r

TSF thymocyte stim u latin g fa c to r

TT th o ro tra st

2® secondary


I . INTRODUCTION

H isto r ic a l

The production o f a n tib od ies in response to a foreign substance

i s an in te g r a l part o f an an im al's immunological d efen se . Antibody

production i s a h igh ly organized and s p e c if ic system th at can e lim i

nate invading microorganisms and requ ires the cooperation o f sev era l

c e l l ty p es.

A ntibodies are synthesized and released by a c la s s o f lymphocytes,

ca lled B lymphocytes, derived from bone marrow c e l l s . At b ir th , an

in d iv id u a l's B lymphocytes bear on th e ir su rfaces a rep erto ire o f

a n t ig e n -sp e c if ic recogn ition u n its (6 6 ), During the course o f the

anim al's l i f e , contact with an antigen w il l cause th a t a n t ig e n -sp e c if ic

B lymphocyte to d if f e r e n t ia te and to g iv e r i s e to a clone o f s p e c if ic

immunoglobulin-producing plasma c e l l s . As th e stim ulus term inates,

and antigen i s no longer p resen t, antibody sy n th es is d e c lin e s . Many

o f th e B lymphocytes become memory c e l l s which, upon subsequent

contact with the same an tigen , can sy n th esize antibody rap id ly ( l ) .

Although B lymphocytes are resp on sib le fo r the actu a l sy n th esis

and r e le a se o f a n tib o d ies , they are by no means the so le p a rtic ip a n t

in th e p rocess. Thymus-derived, or T lymphocytes, play a regu latory

ro le in humoral immunity, modulating the a c t i v i t i e s o f B c e l l s p o s i

t iv e ly or n eg a tiv e ly . . A subpopulation o f T lymphocytes, representing

approximately on e-th ird o f the p eripheral T c e l l population , bears

Lyl surface markers. Lyl c e l l s in te r a c t with antigen and aid in the


2

antibody response, thereby earning the name h elp er T c e l l s ( l 3 t 1^).

Another subpopulation o f T lymphocytes, the a m p lifier T c e l l s ,

are fu n c tio n a lly d is t in c t from helper T c e l l s and a c t to drive an tigen -

stim ulated B c e l l s to fu rth er p r o life r a tio n ( 5) .

Not a l l T c e l l s enhance the immune response. An a d d itio n a l

subpopulation o f T lymphocytes bear LyZ,3 surface an tigen s and sup

p ress B c e l l a c t iv i t y , Suppressor T c e l l s , togeth er with h elper and

a m p lifier T c e l l s , are important reg u la to rs o f antibody production

( 13. 1 4 ).

While many an tigen s require the cooperation o f T c e l l s fo r a n t i

body production, some an tigen s can stim u late the development o f an

immune response in the absence o f T c e l l s . Thymus-independent antigens

are b iochem ically d if fe r e n t from thymus-dependent an tig en s. Whereas

T-dependent antigens are u su a lly fore ign p ro te in s and ery th rocytes,

and are e a s ily degraded, T-independent an tigen s g en era lly are la rg e

polym eric m olecules with r e p e t it iv e id e n t ic a l su b u n its. T-indepen

dent an tigen s are degraded slow ly and tend to p e r s is t fo r a long tim e

in host t is s u e s (6 ) .

T-independent an tig en s, examples o f which are pneumococcal

p olysacch arid es, lip iopolysaccharides, and polymerized f la g e l l in ,

generate th e production o f an tib o d ies th at are predominately o f the

IgM c la s s . These an tigen s are o ften B c e l l m itogens and may be

non-immunological a c t iv a to r s o f the complement cascade (6) .

R ecently , i t was determined th a t the thymus-independent (Tl)

an tigen s can be divided in to two groups, based on th e ir d iffe r in g

a b i l i t y to stim u la te B c e l l s early or la t e in ontogeny and on th e ir


3

a b i l i t y to cause antibody formation in the immune d e fe c tiv e CBA/N

s tr a in o f mice (57» 72) . T rin itrophenyl B ru cella abortus (TNP-BA),

TN F-lipopolysaccharide, and TNP-polyacrylamide beads are character

i s t i c o f TI-1 an tig en s, in th a t they can stim u late antibody production

by early neonatal and CBA/N spleen c e l l s . TI-2 an tig en s, such as

TN P-Ficoll, levan , dextran, and pneumococcal polysaccharide, f a i l

to induce an antibody response in CBA/N c e l l s and cannot stim u late

neonatal c e l l s u n t i l 7 to 10 days a f te r b irth (72) .

T i t t le and Rittenburg (57) have found th a t memory B c e l l subpop

u la tio n s d i f f e r in th e ir s u s c e p t ib i l i t y to stim u lation by th ese two

c la s s e s o f TI a n tig en s. TI-1 an tigen s can cause both BlîJ (TI-2

responding) and B2b (T-dependent responding) subpopulations o f

memory E c e l l s to i n i t i a t e c e l l d iv is io n , whereas TI-2 antigens

s e le c t iv e ly stim u late BlS subpopulations.

Suppressor T c e l l s may aid in the regu la tion o f thymus indepen

dent a n tig en s. I t has been shown th at Ly2,3 c e l l s block antibody

formation by in ter feren ce with helper T c e l l a c t iv i t y (which would

suppress a response to T-dependent an tigen s) or by a c tin g on B c e l l s

and plasma c e l l s (which would suppress responses to both T-dependent

and T-independent an tigen s) ( l 4 ) .

Macrophages have been known to p lay a part in the immune response

fo r many years, but th e ir p rec ise ro le i s s t i l l not com pletely under

stood . In the early years o f immunology, macrophages were thought to

be resp o n sib le fo r both th e uptake o f antigen and fo r the production

o f a n tib o d ies ( 58) . As th e sc ien ce o f immunology matured, lymphocytes

and plasma c e l l s were shovm to be involved in the actu a l sy n th es is


4

o f a n tib o d ies , and in te r e s t in the macrophage as an e f fe c to r c e l l o f

humoral immunity subsided (58) .

In the 1960s , i t was observed th at some antibody responses

required the in te r a c tio n o f lymphocytes with e x tra c ts o f macrophages

th a t had p rev iou sly phagocytosed antigen (22, 25) . This rekindled

in te r e s t in the macrophage as an e f fe c to r c e l l in the immune response

and led to the concept o f antigen p rocessin g .

I t i s now understood th at an optim al immune response to

most an tigen s req u ires th e cooperation o f T lymphocytes, B lympho

c y te s , and macrophages. Using agents th at suppress the r e t ic u lo

en d o th e lia l system or otherw ise in a c t iv a te macrophages, various

in v e s t ig a to r s obtained evidence th at apparently showed an absolute

requirement fo r macrophages in immune resp on ses. R esu lts o f th ese

experim ents,showed th a t thymus-dependent an tigen s exh ib ited a

requirement fo r macrophages in an immune response; however, most

thymus independent an tigen s were a lso macrophage independent ( 32, 33»

55» 65 , 6 9 , 71) . R ecently, however, i t was shown th at some T c e l l -

independent an tigen s required macrophages; apparently d ir e c t B

ccll-raacrophage in te r a c tio n was required in th ese Immune responses

( t l , 32, 37).

Whereas T c e l l s and B c e l l s bore antigen recogn ition u n its on

th e ir su rfaces and resixanded to stim u lation by a s p e c if ic antigen

(4 5 , 66) , the r o le o f the macrophage in the humoral response was

n o n sp e c if ic . Macrophages, present in lymphoid and nonlymphoid t is s u e s ,

removed antigen from e x tr a c e llu la r f lu id n o n sp e c if ic a lly , Schmidtke

and Unanue (6 4 ), found th at macrophages did not d iscrim in ate between


5 .

fo re ig n and autologous p ro te in s . Furthermore, antigen uptake by

medullary macrophages was id e n t ic a l in to le r a n t, normal, and primed

animals ( 31) , Any d iscrim in ation between a n tig en ic and nonantigenic

m olecules appeared to be only with antigen-antibody Immune complexes?

macrophages had surface recep tors fo r the Fc portion o f immuno

g lo b u lin s (3 1 1 5^)t In f a c t , Humphrey and Frank ( 31) have found th at

the tim e o f lo c a liz a t io n o f antigen in macrophages o f the draining

lymph nodes corresponded to th e appearance o f c ir c u la t in g antibody

in normal ra b b its . Thus, the s p e c i f ic i t y o f the immune response l i e s

e n t ir e ly upon the lymphocytes precommitted to a s p e c if ic an tigen .

Most o f the antigen removed by macrophages was catab olized to

a nonimmunogenic form ( 3^, 6o ) , Free, c ir c u la tin g antigen was found

to be p o te n t ia lly to lero g en ic ; th erefo re an important immunological

fu nction o f t h is ca tab o lized antigen was to a id in the circumvention

o f to lera n ce (24, 34) ,

Antigen th a t escaped catabolism was "presented" by macrophages

to lymphocytes; thereby an immunogenic s ig n a l was d e liv ered . Antigen

presen tation i s not c le a r ly understood; evidence has led to severa l

models.

Macrophages re ta in a few m olecules o f an tigen , undegraded or

p a r t ia l ly degraded, on th e ir su r fa ces . M acrophage-associated antigen

was h igh ly immunogenic. When some an tigen s were adm inistered in

so lu b le form, a poor immune response was mounted. However, when the

same antigen was bound to macrophages and then adm inistered, a good

immune response fo llow ed ( 38, 59)• A lso, i t was found th at immunogeni-

c i t y was l o s t with th e removal o f an tigen from macrophage su rfaces ( 63) ,


6

This evidence suggested th at the macrophage fu n ction s by concentrating

antigen and thus promoting i t s necessary meeting with T and B lympho

c y te s .

The ro le o f the macrophage in antigen p resen ta tion , however, was

more than a mere v e h ic le fo r an tigen , Evidence suggested th at the

macrophage p lays a very a c t iv e , y et not c le a r ly defined r o le . For

example, M itchison (^O), in m acrophage-transfer experim ents, found

that antigen must be bound to v ia b le macrophages fo r induction o f

a good immune response. Furthermore, a d d itio n a l evidence was found

th at macrophages played an a c t iv e ro le in antigen p resen tation ; donor

macrophages had to be syngeneic with a r e c ip ie n t , in tra n sfer experi

ments, for good immunogenicity to be m anifested ( 20, b2 , 50) .

Hot a l l undograded anti/'cn was bound to tVic macrop!iag;c su rface .

Imjiiunogenic antigen m olecules were found a f te r surface antigens

were removed by p r o te o ly t ic enzymes or worn blocV.nd by s p e c i f i c

antibody (19, 62) , This suggested another way in which antigen

could be presented to lymphocytes by macrophages. Following the

" in ter io r iza ,tio n ” o f antigen by macrophages, un degraded antigen

m olecules were re lea sed , lead ing to the stim ulation o f lymphocytes

(62) , Such seq u estra tion o f antigen .by macrophages, followed by

slow r e le a se , would allow the immune system to be sl.imu'inted over a

long period o f time.

Subsequent to the d iscovery that processing o f antigen by

macrophages was necessary fo r some antibody responses, a h ighly

immunogenic RHA-antigen complex was iso la te d from ex tra c ts n f macro

phages th a t had been incubated with antigen (b, 23, 2? ) . macrophage


7

p rocessing o f antigen was not a b so lu te ly necessary to obtain t h is

complex, but y ie ld s were s ig n if ic a n t ly greater when antigen was taken

up by macrophages (4-, 2 ? ). Askonas and Rhodes (4) then suggested th at

macrophages did not form a s p e c if ic new inform ational RNA, but

processed the antigen in some way to make i t more immunogenic. I t

was p ostu lated by o th ers, however, that the macrophage ENA served

as a ca rr ier or an adjuvant fo r the a n tig en ic determinant and the

RNA-antigen complex, although the RNA was n o n sp ec ific , could be the

means by which inform ation e l i c i t in g antibody production was processed

( 27) . Other in v e s t ig a to r s (4?, 48) subsequently found th at RNA

from various sources (even nonmammalian sources) could complex

with antigen to form a very immunogenic "super antigen". This led

some to question whether super an tigen s operated in the in vivo

immune response ( 26) . This matter s t i l l remains a co n tro v ersia l

is s u e .

The exact ro le o f RNA in immune p rocesses has not y et been

d efin ed . I t may fu nction in immune induction when an antigen i s

introduced in to the animal or may am plify the immune response by

r ecru itin g uncommitted c e l l s rece iv in g antibody mRNA from c e l l s

responsive to a n tig en ic stim u lation ( l 7 ) .

Although many o f the d e ta i ls remain unclear, evidence has

shown th at the function o f macrophages in immune induction i s to

present antigen to T and B lymphocytes. Macrophages can a lso in f lu

ence lymphocytes by the lym pho-stim ulatory products th at they se c r e te

(61) . The sy n th es is and secre tio n o f b io lo g ic a lly a c t iv e products

by macrophages has been known fo r some tim e. The l i s t o f monokines


8

in c lu d es neutral p ro te ln ases such as coHcigenase, e la s ta s e , and

plasminogen a c t iv a to r , as w ell as lysozyme and various complement

p ro te in s ( 6 l ) .

In recent years, an in creasin g number o f lymphocyte modulating

agents have been d escribed . Various immune fu n ction s have been

ascribed to th ese fa c to r s by the in v e s t ig a to r s who have reported

them, ■ ranging from stim u latin g the p r o life r a tio n o f thymocytes

(21, 25) to su b stitu tin g fo r macrophages in the induction o f helper

T c e l l s in plaque forming c e l l (PFC) responses (20) .

A current concensus i s th a t the numerous catalogue o f lympho-

sim ulatory monokines can be reduced to a s in g le fa c to r th at has

m u ltip le fu nction s (39) . This fa c to r , c a lle d in ter le u k in 1 ( i L - l ) ,

i s produced e x c lu s iv e ly by macrophages and i s e s s e n t ia l for the

production o f another fa c to r , In terleu k in 2 (IL -2) , by T c e l l s . IL-2

mediates T c e l l p r o life r a t io n . The magnitude o f IL-2 production i s

dependent upon the quantity o f IL-1 a v a ila b le ; th ere fo re , IL-1 and

IL-2 function in a bimodal am p lifica tio n system which determines the

exten t o f T c e l l c lo n a l expansion ( 56) .

The many monokines, now determined to be the s in g le fa c to r ,

in ter le u k in 1, included the lym phocyte-activating fa c to r (LAF) or

m itogenic protein (MP), as i t i s a lso ca lle d (21, 25) . LAF stim

u lated the p r o life r a t io n o f thymocytes and enhanced th e ir responses

to l e c t in s (21, 25) . Also included in the l i s t o f macrophage

products th at comprised IL-1 are the B c e l l a c t iv a tin g fa c to r (BAF),

which encouraged th e d if fe r e n t ia t io n o f B c e l l s to plasma c e l l s

( 12, 70) , B c e l l d if fe r e n t ia t io n fa c to r (BDF), and T c e l l rep lacing


9

fa c to r s ( 39) .

Rather than a s in g le fa c to r , IL-2 was once thought to be many

products o f macrophage and T c e l l in te r a c tio n . Included in t h is l i s t

were thymocyte stim u la tin g fa c to r (TSF), thymocyte m itogenic fa c to r

(TMF), T c e l l growth fa c to r (TOGF), costim u lator, k i l l e r c e l l helper

fa c to r (KHF), and secondary cy to to x ic T c e l l inducing fa c to r (SCIF)

(39).

C h a ra cter istic s o f each o f th e in ter le u k in s are known, ILr-1

i s produced e x c lu s iv e ly by m acroph^es, and i t s a c t iv it y i s n e ith er

H-2 nor sp e c ie s r e s tr ic te d , IL-1 i s a com paratively sm all m olecule,

being 12,000 to 18,000 d a lton s, and does not p o ssess the a b i l i t y

to maintain in v itr o long term cu ltu res o f c y to to x ic T c e l l s (39) .

A la rg er m olecule (30,000 to 35.000 d a lto n s), IL-2

required both macrophages and T c e l l s fo r i t s production. The

a c t iv it y o f IL-2 i s H-2 u n restr ic ted , and i t can promote and maintain

in v itr o long term cu ltu res o f cy to to x ic T c e l l s (39) . O bviously, the

in te r le u k in s in flu en ce the immune response, and th e ir r o le i s

becoming in crea s in g ly c lea rer .

Much inform ation o f the r o le o f macrophages in humoral immunity

has been gained s in ce the tim e they were thought to sy n th es ise

cintibody; s t i l l th e ir p r e c ise fu nction in immunity remains to be

d efin ed . Macrophages w i l l continue, to be an in te r e s t in g and contro

v e r s ia l to p ic o f research .


10

statem ent o f T hesis and AT:proach to the Problem

S p e c if ic macrophage to x in s have been used as t o o ls to impair

the r e t ic u lo e n d o th e lia l system (RES) fo r in v e s t ig a tin g the ro le o f

macrophages in generating antibody responses. R ecently, in the

course o f one such study wherein carrageenan (CAR) and m icroparticu

la te c r y s ta l l in e s i l i c a (FiUS) were employed as macrophage to x in s , à

unique phenomenon was observed. While running con tro ls fo r a study

on the e f f e c t s o f the s i l i c a and carrageenan on doses o f antigen

th at were priming mice fo r a secondary antibody response, Becker

and Rudbach (7 , 9} observed th a t one dose o f lip op o lysacch arid e (IP S ),

given to mice exposed to macrophage to x in s 21 days p rev iou sly , e l i c i t e d

a ty p ic a l secondary type resi^onse. This "pseudo-secondary" response

was m anifested by an increased production o f t o t a l antibody and by

maintenance o f the high antibody t i t e r , th e c h a r a c te r is t ic s o f a

secondary resi)onse to a T c e l l independent an tigen .

The purpose o f the present study was to explore the mechanism(s)

by which the pseudo-secondary response was generated . Aspects o f

the pseudo-secondary response th a t were studied included attem pts

to generate th e response with macrophage to x in s o ther than KU? and

CAR, examination o f RES modulation by the various macrophage to x in s ,

an in v e s t ig a t io n o f the g e n e t ic requirem ents fo r generating the

pseudo-secondary response, an in v e s t ig a tio n o f the temporal k in e t ic s

fo r developing the pseudo-secondary response, and the dependency o f

the pseudo-secondary response on T c e l l s .

In th e in v e s t ig a t io n s o f Becker and Rudbach (7 , 9 ) , Seakem


1 1 .

carra."oenan, a natural mixture o f K- and A- car ra. n on an s , wan uaed.

The present study examined the oapa.hility o f and A-cartageenans,

as w ell as thorium d ioxide (th o ro tra st) to f';onnrate pseudo-secondary

resp on siven ess. F ir s t , however, prelim inary work wan required to

e s ta b lish proper dosaye royimeno fo r th o ro tra st (TT). This was

necessary because the carrayeonans and MU" exerted th e ir to x ic e f f e c t s

by d e s ta b iliz in g lysosom es and causiny subsequent a u to ly s is o f

macrophaye s j TT was thought to suppress RES function by a p h y sio lo g ica l

blockade o f macropha,yes (68) . Once the data had been generated, then

an attempt was made to co rre la te the mechanisms by which RÉO suppression

was achieved with the ca p a b ility o f the various macrophage to x in s

to generate a pseudo-secondary response.

The exten t and duration o f Rit" inodula,tion by MUf, CAR, and TT

a lso were examined, with sp e c ia l consideration given to a study o f

the s ta te o f the systems a t the tim e which the tr ig g er in g dose o f

LP£i was adm inistered in the pseudo-secondary response.

The use o f mice th at did not resTOud to LPS allowed in v e st ig a tio n

o f the g en e tic requirements o f the pseudo-secondary response. The

capabl11ty o f macrophage to x in s to "prime" th ese mice fo r secondary

resp on siven ess was exam.ined and compared id th the g en e tic in a b i l i t y

o f th e mice to mount a secondary anti-LPS rr?r.ponsc.

The k in e t ic s o f true secondary res.ponsi veness wore esta b lish ed

and were compared to the k in e t ic requirements o f pseudo-secondary

resjx in sivcness.

F in a lly , an attempt was made to generate Die pseudc-sccondary

response in athym ic, nude mice. Prelim inary work was necessary to


12

e s ta b lish an adequate experim ental system; CAR p o te n tia te s the

en d otox ic ity o f LPS in normal mice and t h is p o ten tia tio n was

increased in nude mice. Once the system was e s ta b lish ed , the gener

ation o f pseudo-secondary responsiveness was attempted in nude mice.

The ca p a b ility o f nude mice to generate pseudo-secondary resp on sive

ness in the absence o f T c e l l s was compared with th e ir ca p a b ility

to mount a true secondary response without the aid o f T c o l l s .

A ,strong co rre la tio n between true secondary responsiveness

and pseudo-secondary responsiveness was observed. I t was postu lated

th at the macrophage to x in s , fUf, and the carragcenans, were resp on sib le

fo r priming mice fo r a secondary anti-LPP response.


I I . MATERIALS AÏÏD FKCRODS

Animals, ( i ) RHL m ire, Male Pwiss-W elstor derived mice

were obtained from the Rooky Mountain Laboratories (RML)» Hamilton,

MT, RKL mice arc derived from the U.1IIÎÎ colony and have retained ■

nearly the same amount and type o f g en e tic va r ia tio n found in natural

murine populations (4 6 ). They wore allowed standard lab chows and

water sd lib itu m . Mice used fo r experiments were 4 to 8 weeks o ld .

( i i ) fude m ice. C ongenitally athymic nude mice were obtained

from our colony at the U n iversity o f Montana, The o r ig in a l stock

were aquircd from the Rocky Mountain L aboratories, Hamilton, MT,

Nude o ffsp r in g were the product o f crossin g heterozygous fem ales

with nude m ales. The heterozygous fem ales were obtained by crossing

N.NIH Swiss-W cbster derived fem ales with nude males.

A ll mice were housed in cages containing s t e r i l iz e d wood

ch ips as l i t t e r . Mice were maintained on autoclaved food (Nayne

Lab lilox S te r i l iz a b le , Chic3.go, 1L) ari l i bi tum.

M ice■received water contain ing 10 mg o x y te tra cy c li ne (Poche

Labor a tori or, N utlcy, N J )/] ,ite r . This seemc'' to improve th e ir

general hea’Ith , nude mice were a lso given ?1? mg metre’■ id azolc

(Fls-gyl, fo a r lc and Co,, Columbus, G :l)/li te r o f water for l.d days

fo llow in g wcn.nlng.

Six to cig !it wocl- old mice o f Lotli c -x es were usx:d for in vu r;ligation s,

( l i l ) C3t*/li?J m ice, Male C^h/Hed mi ee were purchased from

Jackson Laboratories (bar Harbor, Mlj) and were maintained on standard

lab chow and water nA l i bi turn. Mice wore allovred to a cc lim a tize

13


fo r one week a f te r t^v;ir a r r iv a l before boin,''; use^ For in v e s t ig a t io n s ,

&%pcrimonts wore performed on 6 to 8 week old mice,

kacrophaee to x in s , (i.) Ca.rra.'^nonans. Calcium carragoenan (Soakem

9)1 K-carrareonan (P.ECII 5862) , X-carrageenan (PKCd 5863) , and

i-carrogeenan (PJ'JfJH 586 1-) wore obtained from karine C ollo ids

(S p r in g fie ld , N j), The carragcenans were suspended in warm ( 36c)

phosphago buffered s a lin e (PbS) (O .I5 M ilaCl, 0,0033 ii 1% , pH 7 ,2 )

to the desired concentration and were used w ithin 2b h o f preparation,

The appropriate suspensions were brought to 37C before in tra p er ito n ea l

in je c t io n ,

( l i ) r ii.cropartid ila te c r y s ta l l in e s i l i c a . S i l ic a p a r t ic le s

(M in-U -Sil, W hittaker, Clark and D an iels, I n c ., Hew York, NY) l e s s

than 5 nm in s i s e were subjected to fu rth er fra c tio n a tio n according

to the method described by Pecker and budbach (7 , 9 ) .

B r ie f ly , $0 g o f s i l i c a (H in -U -fil) were suspended in ';{){) ml

o f d i s t i l l e d , deionized water and were subjected to u ltra so n ic

v ib ra tio n s (branson, Danbury, Cl’) a t room temperature fo r "}0 s . The

suspension was d ilu ted with an equal volume (590 ml.) o f d i s t i l l e d ,

deion ized water, pieced in a l l graduated cy lin d er and allowed to

s e t t l e a t room temperature fo r 2b h. Fraction I I I , corresponding

to the 590-759 ml %)ortion from the top , was removed and washed

tw ice with d i s t i l l e d , deion ized water. I t was allowed to d?'v in

a warm (60C) oven fo r b-8 h. Prior to ini.ravenous in jec l.lo ti, i t

was resuspended to the desired concentration in warm (379) FI:?.

( i i i ) T h orotrgst, s ta b il iz e d thorium d ioxide (T iiorotrast,

Testagar and Co., I n c , , D e tro it, Ml), con ta ini.ng ?J\-zO/. tiiorium


15

dioxide by volume (z 60 mf;/ml), 25X aqueous d extrin , and 0 , 1 5 --

m ethylparasept, was adm inistered in traven ou sly . Appropriate

d ilu t io n s were made with FBS,

Lipopolynaccharicln. L ipopolysaccharide (LF?) was prepared from

E scherich ia c o l l 0113 (Braude s tra in ) as a phnnol-water ex tra ct

by the method described by Rudbach, e t a l . (5 2 ). A 1 m^/ml stock

so lu tio n was made by d isso lv in g the lyojihi li'^îed lipopolysaccharido

in PBS. This so lu tio n was dispensed in to v ia ls and frosen a t -f.OC

u n til use, Appropriate d ilu t io n s o f the stock so lu tio n were made

immediately before use.

C o lle c t ion o f serum for passive hem arrintination as.sayr.. Nice

were an esth etized with ether (ill. R. fqulbb end Bons, New York, NY)

and exsanguinated by an a x il la r y in c is io n . The whole blood was

allowed to c lo t a t room temperature fo r approximately 1 h and nt

Ac overn ight. The serum was separated from the c lo t by ce n tr i

fugation at 1,000 X g fo r 10 min. I t was dispensed in to v ia ,ls ,

heated a.t 5 iC for 30 min and stored a t -ROC.

Anti-LTTI t i t e r s . Antibodies spnci Tic for LIB were determined

by p a ssiv e hem agglutination a ssa y s . Bboep rod blood c e l l s (CRLC),

coated with LPC from k. coJ_i 011.3 wer,. used as in d ica to r c e l l s .

I or s e n s i t i . s s t i o n , 1 mg o f LIB wa ; dirx'.elved in i ml o f fi,1. Î- lO/j.

b u f fe r (pn 7,R) and then placed in a b o i l i n g ’■■.ater la th f o r f ) h,

B'Ctir'itizod BBEC were prepn.'-ed by combin1 n;, 0 .?5 ml o f paci'''d,

wa: hod B IBG (Colorado Beruir Co., b'nvc'r, CO), '"'.O ml o f Fj B, and

1 .0 ml o f the b o i l ed LFB. This mixture was in oui latr-d at fo r

30 min w i th - freq u en t inlxin; , The c e l l : w're t'H;n vrac’ "d 1>t'î ce in.


16

cold PBS and f in a l ly resuspended in m icro titer d ilu en t ( l^ normal

rab b it serum (in a c tiv a ted ($60 fo r ^0 min) and adsorbed with SRBC)

in PBS) to a concentration o f 0.5"^. The s e n s it iz e d SRBC were used

Immediately a f te r preparation.

Normal SRJ3C were prepared by resuspending packed, washed SRBG,

in m ic ro titer d ilu e n t, to a concentration o f 0 . 5/».

To each w ell o f a m icro titer p la te (T iter tek , Linbro Chemical

Co., I n c ., New Haven, GT), 0 .05 ml o f d ilu en t was added. Then,

0 .0 5 ml o f t e s t serum was added to th e f i r s t w ell and s e r ia l two

fo ld d ilu t io n s o f th e serum were made with 0 .05 ml d ilu to r s (Cooke

Engineering Co., Alexandria, VA). In a l l ca ses, d u p lica te t e s t s

o f each serum were performed, Next, 0 ,05 ml o f the se n s it iz e d PRBC

was added to w e lls 1-11 and 0 ,05 ml o f normal PRBC was added to

the 12th w e ll o f each row. Each p la te was g en tly a g ita ted , covered,

and placed in a humid chamber. Hemagglutination was recordéd a f te r

incubation fo r 2 h at room temperature (2Ü-22C) and again a f te r

overn ight incubation a t li-C. A p o s it iv e hem agglutination t i t e r was

read as the rec ip ro ca l o f th at d ilu tio n o f serum th at did not g iv e

a button con tro l p a ttern .

Hemagglutination t i t e r s are expressed as values o f x , derived

from th e equation x = lo g 2 (HD/2), where HD was the rec ip ro ca l o f

the h ig h est d ilu tio n o f scrum th at produced homa^'glutination o f

the s e n s it iz e d PBBG. Thus, the t i t e r i s the tube number o f the

endpoint when the f i r s t w e ll contained a l / 4 d ilu tio n o f antiserum .

Sera th a t gave no hem agglutination a t the low est d ilu tio n were

a r b itr a r ily assigned a t i t e r o f 0, i e . , a d ilu t io n o f l / 2 . T iter s


17

were expressed as the geom etric mean o f the d u p lica te t e s t s .

Measurement o f phagocytic a c t iv it y hy FES clearance o f c o l lo id a l

carbon. Phagocytic a c t iv it y was measured by the a b i l i t y o f normal

and macrophage to x in -tr ea te d mice to c lea r carbon p a r t ic le s from

th e peripheral b lood. C o llo id a l carbon (Pelikan C ll/l^ ^ la , Koh-

I-Noor, Bloomsbury, NJ) was s ta b iliz e d with \% g e la t in (Baker

Chemical Co., P h illip sb u rg , Nj) and was in je c ted in travenously as

a volume o f 0,1 ml th a t contained l6 ,9 mg o f carbon. At 5 min

and 15 min a f te r the in je c t io n o f carbon, mice were an esth etized

with ether (lü, R. Squibb and Sons, New York, NY) and 0 ,02 ml o f

blood was removed from the r e tr o -o r b ita l p lexus in heparinized

c a p illa ry tubes (C ap illary micro hem atocrit 73810, Kimble, Toledo,

o h ). Blood was ly sed in 2 ,0 ml o f 0,1% NapCO (J . T. Baker Chemical

Co,, P h illip sb u rg , N j). O ptical d e n s it ie s o f the lysed blood

samples were measured a t 650 nra in a spectrophotom eter (Beckman DU-2,

Beckman Instrum ents, I n c ., Palo A lto , CA). A fter the 15 min b leed ing,

the mice were sa c r if ic e d and the wet w eights o f the l iv e r s and sp leen s

wore determined.

The phagocytic index ( k) was determined fo r each mouse by the

fo llow in g formula;

K = logj f) OD at 5 ^In - log^ CD a i 15 min

10 min

The phagocytic index corrected fo r d iffe re n c e s in body and

organ weight (o<) was determined fo r each mouse by the fo llow in g

formula;


18

o (= V / k x ( l i v e r wel/rht 4- sp leen w eight) t o t a l body w eight


I I I . nUÜULTS

Determination o f amount o f th o ro tra st to suijprens the RES.

E xtensive work by o th er in v e s t ig a to r s had e s ta b lish ed the standard

dosages fo r CAR and MUS ( 7 ,9 ) . Only lim ited rep orts in older

l it e r a tu r e had described the use o f IT to suppress the RES in mice;

th ere fo re , prelim inary experiments sought to determine e f fe c t iv e

doses fo r TT. A ltera tion o f RES a c t iv it y was determined by comparing,

with normal mice, the a b i l i t y o f t e s t mice to c lea r carbon p a r t ic le s

from the p erip h eral blood 24 h a f te r th e f in a l in je c t io n o f TT.

F ig . 1 shows th e e f f e c t o f varying amounts o f TT, adm inistered l . p . ,

on the clearance o f c o l lo id a l carbon. S ig n ific a n t RES suppression

occurred with 250 mg doses o f TT. However, even m u ltip le l . p .

In jec tio n s o f I 50 mg TT on consecutive days did not a lte r RES

a c t iv i t y .

The in je c t io n o f varying amounts o f TT i . v . a lso in fluenced

phagocytic a c t iv i t y , as shown in F ig . 2. Doses o f 50 mg and 100 mg

TT i . v , were comparable in suppressing RES fu n ctio n . Again, the

phagocytic a c t iv it y o f mice th at were given m ultip le in je c t io n s

o f 50 mg TT i . v , on su ccess iv e days was s im ila r to th ose mice

th a t received PBS. With consideration g iven to the en d otox ic ity

p o ten tia tin g e f f e c t s o f thorium d ioxide (8 ) , 50 mg i . v , o f TT was

the chosen dose to be used. This was a compromise between an

amount th a t would suppress e f f e c t iv e ly the RES a c t iv i t y , but would

not p o te n tia te , too g r e a t ly , en d o to x ic ity .

19


F ig , 1 . RES a c t iv it y fo llo w in g in je c t io n o f varying amounts

o f Tr i , p , RES a c t iv it y was measured by th e a b i l i t y o f t e s t

mice to c lea r carbon p a r t ic le s from th e p erip h era l blood.

Mice were te s te d 2^ h a f te r the f in a l in je c t io n o f TT, Each

value rep resen ts the ar ith m etic mean o f two m ice.

Symbols*.

Open bars: S in g le In je c t io n o f TT

V er tica l crosshatches; TT given on two con secu tive days

H orizontal crosshatches: TT given on f iv e con secu tive days

20


21

|olut

§ceI-SoX

OlO

8

r

J— L

§ IîrilVA ^ NV3W


F ig . 2 . RKS a c t iv i t y fo llo w in g in je c t io n o f varying amounts

o f TT i . v . RES a c t iv i t y was measured by th e a b i l i t y o f t e s t

mice to c le a r carbon p a r t ic le s from, th e p erip h era l blood,

Kice were te s te d 24 h a f te r th e f in a l in je c t io n o f TT. Each

value rep resen ts the ar ith m etic mean o f two m ice.

Symbols:

Open bars: E’in g le in je c t io n o f TT.

V ertica l crosshatches: TT given on two con secu tive days

22


23

[

[

[I I I I

'Ùno 6

C< O 00T T Q

1

s

s

oin

mCN

mt

gO

f

3 n iV A j Q N V iW


24

Attempts to venerate a Tiseudo-secondary rasponse with various

macrouhage to x in s . I t had been shown p rev iou sly by Becker and

Rudbach (7 , 9) th a t a s in g le dose o f LPS, given to mice exposed to

MUS or CAR 21 days e a r l ie r , e l i c i t e d a ty p ic a l secondary type

response. This "piseudo-secondary" response could not be generated

in the absence o f the macrophage to x in s ( l e . when PBS was adm inistered

in p la ce o f MUS or CAR); th erefo re , i t became o f in te r e s t to determine

the r o le the macrophage to x in s played in the generation o f the

pseudo-secondary response. The use o f macrophage to x in s th at would

suppress the RES by a mechanism other than th at employed by KUS and

CAR w a s .f e lt to be an important step in the in v e s t ig a tio n o f the

r o le o f the macrophage to x in in the pseudo-secondary response.

In an attempt to r e -e s ta b lish the system, groups o f 5 mice

were trea ted with MUS or CAR on day 0 and then were given one or

two in je c t io n s o f LPS, S in g le doses o f LPS were given e ith e r a t

. 6 h a f te r treatm ent with macrophage tox in or on day 21. Mice,

rece iv in g two in je c t io n s o f LPS were in jec ted 6 h a f te r treatm ent

with macrophage to x in s and again on day 21. Control mice were

given PBS on day 0 and LPS a t the tim es designated . Sera were

c o lle c te d on day 25.

Tables 1 and 2 show that mice given MUS or CAR on day 0 and

a s in g le dose o f LPS 21 days la t e r do indeed generate a pseudo- •

secondary antibody response.(groups A). The magnitude o f th is

response was comparable to a tr u e . secondary response ( i e , a response

to primary and secondary in je c t io n s o f LPS) (groups E and F ) , The

pseudo-secondary response o f groups A con trast sharply with the


CD■ D

OQ.C

gQ.

■DCD

C/)

o '3O

8

. Table 1

Generation o f a pseudo-secondary response with s i l i c a (MUS) in w hite m ice.

CD

3.3"CD

CD■DOQ.Cao3

■DO

CDQ.

■DCD

C/)C /)

GROUP tbsatî-:e?:t^ b 20 c AB TITSFt'

DAY 0 DAY 0 DAY 21 DAY 29

A MU? NONE LP? 10B PE? NONE LP? 5.5G MU? LP? ■ NONE 5D PE? LP? NONE 6E MU? LP? ■ LP? 10.5F PB? LP? LP? 11

6 h a f te r MU? or PE?n o mg KÜP i . v .^0.1 ug LF? i . v .n .O u g L F P i .p .

Antibody t i t e r s were obtained ,by p a ss iv e hem agglutination t e s t s . Each value rep resen ts the antibody t i t e r o f sera pooled from f iv e m ice. T ite r s are expressed as values o f x , derived from th e equation, x = lo g 2 ( - 1 /2 ) , where HE was th e rec ip ro ca l o f the h ig h est d ilu tio n o f sera th a t produced hem agglutination.

VA

CD■ D

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C/)

o"3O

8

ci'

33"CD

CD■DOQ .CaO3

■DO

CDQ .

Table 2

G eneration o f a pssudo-seconclary response with Geakem carrageenan (CAR) in white mice,

GROUP TREATMEUT f b pOC AS TITER

BAY 0 BAY C DAY 21 DAY 2<

A CAP NOYE ' LP? 10.5 ■B PE? RONE LP? 7 .5C CAR LP? NONE 8 .5D PB? LP? NONE 5E CAR •LP? LP? 11? PE? LP? LP? 10 .5G GAR NCR3 NONE 2 .5R PR? RCaE' ■ NONE 0

roON

CL 5.0 mg GAR i . p .■g C .l ug LFS i . v . 6 h a f te r CAR or PE?.

1 .0 ug IP? i .p . Antibody t i t e r s were obtained by p assive hem agglutination t e s t s . Each value rep resen ts th e antibody

t i t e r o f sera pooled from 5 m ice. T ite r s are expressed as va lu es o f x , derived from th e equation,X = log? (H D/z), where >!D was th e rec ip ro ca l o f the h ig h est d ilu tio n o f sera th a t produced hem agglu tination ,

■ 27

response o f con tro l mice th at recoivet! PB? on clay 0 and the s in g le

in je c t io n o f LP? on day 21 (groups B ) .

Once the .system was r e -e s ta b lish e d , the use o f macrophage

to x in s o ther than KU? or CAR were employed in attem pts to generate

pseudo-secondary resp on ses. Various isom ers o f carrageenan e x is t

which suppress the RE? in a manner s im ila r to CAR ( ie , by lysosom al

d e s ta b il iz a t io n ) , i - , K~, and A -carrageenans were given to groups

o f 5 mice on day 0 . LP? was adm inistered on day 21, I t can be seen

in Tables 3? and 5 th a t, l ik e CAR, the i , k", and A isom ers o f

carrageenan would a lso e l i c i t pseudo-secondary responses (groups A ).

Those responses a lso mirrored true secondary responses in magnitude

(groups E and F ) , Cont o i mice, rece iv in g PB? on day 0 and LP? on

day 21 (groups B) responded in a primary fash ion , thus in d ica tin g

th a t the secondary resp on siven ess o f groups A was generated by the

i - i K -, and A-carrageenans,

The carrageenan8 and MU? suppressed the RE? by lysosom al

d e s ta b iliz a t io n and concomitant c y to to x ic ity (3 , 15)• To determine

i f th e mechanism o f RE? suppression by macrophage to x in s played a

part in the generation o f the pseudo-secondary, resjjonse, TT was

■ employed, TT i s a macrophage to x in that suppressed phagocytic

a c t iv i t y by RE? blockade; no c y to to x ic ity was observed (68) . Table

6 demonstrated th a t mice given TT on day Ü and LP? on day 21 do not

gen erate a pseudo-secondary response (group A ). The anti-LP? response

o f th ese mice was e s s e n t ia l ly th e same as the mice th at received

PB? on day 0 and LP? on day 21 (group b ) . Mice th a t were given

PB? or TT and two in je c t io n s o f LP? (groups E and F) generated


CD■ DOQ .C

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o

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Generation

Table 3

o f a pseudo-secondary response with i-carrageenan (i-CAR) in w hite m ice.

GROUP TPSATHEHT®- f b 2# c AE TITER^


A i-CAR NONE LP? 9E PE? NONE LP? 7 .5C i-CAR LP? NONE kD PB? LP? NONE 5

. E i-CAR LP? LP? 11F PE? LP? LP? 10 .5G i-CAR NONE NOME 1 .5H PE? NONE NONE 0

roco

■D Si 5 .0 ïïig i-CAR i . p . 0 .1 ug LP? i . v . 6 h a f te r i-CAR or PB? 1 .0 ug LP? i . p . Antibody t i t e r s were obtained by p a ssiv e hem agglutination t e s t s . Each value rep resen ts th e 'antibody

t i t e r o f sera pooled from f iv e m ice. T ite r s are expressed as v lau es o f x , derived from th e equation,X = log2 (HD/z), where HD was the reciprocal of the highest dilution of sera that producedhem agglutination .

CD■ D

OQ .C

gQ .

73 Table 4CD

w' G eneration o f a pseudo-secondary response with K-carrageenan (K-CAE) in w hite m ice.

o

8

(O '

3.3"CD

CD ■DO Q.Cao

:S bcr C

GROUP TPEATKE-NT®- |0 b AB TTTERd


A K-GAR NOITB IPS 11B FES NONE IPS 5.5G K-GAR IPS NONE 6D PBS IPS NONE 6E K-CAR LPS . IPS 10.5F PBS IPS •IPS ■ 11

tsj\o

5 .0 ng k-CAB i . p ,0 .1 ’jg LFS i . v , 6 h a f te r K-C/iR or FES1 .0 ug LPS i .p .

^ Antibody t i t e r s were obtained by p a ssiv e hem agglutination t e s t s . Each value rep resen ts the antibodyK- t i t e r o f sera pooled from 5 m ice. T ite r s are expressed as va lu es o f x , derived from the equation ,g X = log^ (HD/2), where HD was th e rec ip ro ca l o f the h ig h est d ilu tio n o f sera th a t produced hemagglu-^ t in a t io n .CD

IC/)C/)

CD■ D

OQ .C

gQ .

■DCD

(/)o"3O

8

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CD■DOQ .CaO3

■DO

CDQ .

CD

C/)C /)

Table 5

G eneration o f a pseudo-secondary response with A-carrageenan (A-CAR) In white m ice.

GROUP TREATMENTS-- io b 2* ° AE TIT3RÜ


A A-car RONS LPS 8B FES NONE LPS 5 .5G A-car LPS NONE 7D PES LPS NONE ■ 6E PBS LPS LPS 11 y

I 5 5 .0 mg A-CAH i .p .o 0 .1 ug LPS i . v . 6 h a f t e r A-CAR or PBS^ ° 1 .0 ug LPS i . p .

Antibody t i t e r s were obtained by p a ssiv e hem agglutination t e s t s . Each value rep resen ts the antibody t i t e r o f sera pooled from 5 m ice. T ite r s are expressed as va lu es o f x , derived from th e equation, x = lo g , (HD/2), where HD was th e rec ip ro ca l o f the h ig h est d ilu tio n o f sera th a t produced hem agglutination.

CD■ D

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C/)C/)

°' Table 6O

8

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CD■DOQ .CaO3"OO

CDQ .

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Attempt to gen erate a pseudo-secondary response with th o ro tra st (TT) in w hite m ice.

GROUP TR ATMEHT b 2 c AB TlTEpd

DAY 0 DAY 0 DAY 21 DAY 2S

A TT NONE IPS 5E P3S ÎÎCNE IPS 6 .5C TT IPS NONE 5D PBS IPS NONE 4 .5E TT IPS IPS 10F PES ■ IPS IPS. 9 .5'j TT NONE NONE 0u PES NONE NONE 2

w

a

ÇD C

, 50 mg TT i . v ,0.1 ug LFf. i . v . 6 h, a f te r TT or PES1 ,0 ug IPS i . p .Antibody t i t e r s were obtained by p a ssiv e hem agglutination t e s t s , üach value rep resen ts the antibody t i t e r o f sera pooled from 3 m ice. T ite r s are expressed as va lu es o f x , derived from the equation,X =» log? (H l/z ) , where HD was the rec ip ro ca l o f the h ig h est d ilu tion - o f sera th a t producedhem agglutination .

32

tru e secondary resp on ses. Thus, i t appeared th at the lysosom al

d e s ta b il iz a t io n o f macrophages was e s s e n t ia l fo r the generation o f

a pseudo-secondary response.

Duration o f modulation o f RE? a c t iv it y by mgcrouhapie to x in s .

The use o f various macrophage to x in s in attem pts to generate pseudo

secondary responses had in d icated th a t lysosom al d e s ta b iliz a t io n and

the concomitant c y to to x ic ity o f macrophages was a requirement fo r

th e generation o f a pseudo-secondary response. JJarlier work by

Becker and Rudbach (7 , 9 )t however, had shown that by 4 or 5 days

a f te r treatm ent with MUS or CAR, RES function had returned to normal.

Therefore, RE? a c t iv it y was follow ed fo r 21 days a f te r treatment with

macrophage to x in s in order to determine i f RE? function was s t i l l

normal a t the tim e the e l i c i t in g dose o f LP? was g iven , or i f

phagocytic a c t iv i t y had again become suppressed, thereby allow ing

a bolus o f antigen to stim u late the antibody producing c e l l s . The

macrophage to x in s , MU?, CAR, and TT were used to con trast the func

t io n a l s ta te o f the RE? a t various tim es a fte r macrophage tox in

ad m in istration with the means by which RE? modulation was achieved

( c e l l c y to to x ic ity v s . RE? blockade). I t was assumed th at the

?eakem carrageenan would produce r e s u lt s s im ila r to the Isomers o f

carrageenan; th ere fo re , the la t t e r were omitLed in t h is study. Groups

o f th ree mice were g iven macrophage to x in s on day Ü. Phagocytic a c t i

v ity was a ssessed by the a b i l i t y o f mice to c lea r carbon p a r t ic le s

from th e p erip h eral b lood . Control mice received PB? on day 0 , F ig . 3

shows th e RE? a c t iv i t y o f mice a f te r rece iv in g KU?. By day 3#

p hagocytic fu n ction had returned to normal, and by day 4 and th e r e a fte r .


F ig . 3» Duration o f inodii I a t ion o f Hulf' a c t iv i t y fo llow in g i . v .

in jec tio n o f 10 o f KUS. RES a c t iv ity was measured by the

a b i l i t y o f t e s t mice to c le a r carbon p a r t ic le s from the

peripheral blood, Kice were given s i l i c a at time 0 and were

te s ted on the days in d ica ted . Control mice received PIS a t

time 0, Each value represents the ar ithm etic mean o f three mice.

Symbols:

Line A: Control mice

Line Test mice

33


to

00 <!

/

\

I -I 1

a

et

00

- tO

Q

Pi

oo

. J


35

phagocytic fu nction was even enhanced. Thus, on day 21, the day

th e tr ig g e r in g dose o f LPS fo r the pseudo-secondary response was

g iven , phagocytic a c t iv it y was g rea ter than normal. S im ilar ly ,

in F ig , 4 , i t i s seen th a t on day 4 , fo llo w in g treatm ent with CAR,

RES fu nction had returned to normal. T hereafter an enhancement

o f phagocytic a c t iv i t y was observed.

Treatment o f mice with TT suppressed RES function fo r 50 h

(F ig , 5 ) « F ig , 6 shows th at a f te r day 2 phagocytic function had

recovered and was even enhanced. Thus, th e s ta te o f modulation

o f th e RES was s im ila r fo r the 21 days fo llow in g the adm inistration

o f CAR, MUS or TT, although the to x in s a ffec te d modulation o f the

RES by d iffe r e n t mechanisms.

The /renetic requirements fo r generating rsnndo-secondary

responses; attempt to generate the response in IPS nonrespondor

m ice, ■ C3H/HeJ mice can mount a primary response to LPS, but lack

th e g e n e t ic ca p a b ility to produce a secondary response (53)•

Experiments wore designed to t e s t whether C^H/UeJ mice had the

g e n e tic ca p a b ility to generate a pseudo-secondary response.

Groups o f 3 mice were given MUS or CAR on day 0 and LPS on

day 21. Control mice received PBS on day 0 and LPS a t the appro

p r ia te tim es. Sera,was c o lle c te d on day 25. Table 7 shows the

antibody responses o f mice th a t received MUS on day 0 , Mice th at

were g iven MUS on day 0 and LPS on day 21 (group A) did not generate

a pseudo-secondary response. The t i t e r o f th a t group was s im ila r

to the primary responses in con tro l groups (groups B, D, and e) ,

Likew ise, the data in Table 8 demonstrate th a t mice th a t were given


F ig , 4 . Duration o f modulation o f RE3 a c t iv i t y fo llo w in g l . p .

in je c t io n s o f $ .0 mg CAR. RES a c t iv i t y was measured by th e

a b i l i t y o f t e s t mice to c le a r carbon p a r t ic le s from the

p erip h eral b lood . Mice were g iven CAR a t tim e 0 and were te s te d

on th e days In d ica ted , Control mice rece iv ed PBS a t tim e 0 ,

Each value rep resen ts th e a r ith m etic mean o f 3 mice.

Symbols:

Line A; Control mice

Line B; T est mice

36


37

r

\

\

. 09

\

Isan |o^youoa^


F ig , 5» Duration o f modulation o f RES a c t iv i t y fo llo w in g i . v .

in je c t io n o f 50 mg TT. RES a c t iv i t y was measured by th e

a b i l i t y o f t e s t mice to c le a r carbon p a r t ic le s from the p e r i

pheral b lood. Mice were g iven TT a t tim e 0 and were te s te d a t

th e tim es in d ic a te d . Control mice received PBS a t tim e 0 , Each

value rep resen ts the a r ith m etic mean o f 2 or 3 mice.

Symbols:


Line B: T est mice

38


39

iû

\

t 1 I

§o J U J L

o

o«

3O

X

o«

- s ®

*®0,lDA>o uoay /


F ig , 6 , Duration o f modulation o f RE? a c t iv i t y fo llo w in g i . v .

in je c t io n o f 50 mg TT. RE? a c t iv i t y was measured by the

a b i l i t y o f t e s t mice to c le a r carbon p a r t ic le s from the p e r i

pheral b lood. Mice were g iven TT a t tim e 0 and were te s te d a t

the tim es in d ic a te d . Control mice rece ived PB? at tim e 0 , Each

value rep resen ts the a r ith m etic mean o f 2 or 3 m ice.

Symbols:

Line A: Control mice

Line B: T est mice


4 l

fiû

Ii s

d

_

—

d

MOo

sonfoyvysuoav^


CD"OOQ .C

gQ .

"OCD

C/)

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8

ci'

33"CD

CD"OOQ .CaO3

"OO

CDQ .

"OCD

C/)C /)

Attempt tc generate a pseudo-secondary response with s i l i c a (MUS)

in IPS nonresponder mice ( i e . CJll/'AeJ m ice),

GROUP TREATPEMT AE TITERÜ


A KUO NONE ■ IPS ^.5S PIS NONE IPS 4.5c FES NONE NONE 0D PBS IPS NONE 2E FES IPS . IPS 4.5

atccl

ro

8.0 ng r-;us i . v .0.1 Ug LrS i . v ,1 .0 ug LFS i . p .Antibody t i t e r s were obtained by p a ssiv e h enagglu tination t e s t s . Each value rep resen ts the. antibod2/ t i t e r o f sera pooled fro% 3 ^ ic e . T ite r s are expressed as va lu es o f x , derived fron th e equation , x = log? (HD/z), where HD was the rec ip ro ca l o f the h ig h est d ilu tio n o f sera th a t produced hem agglutination .

CD■ D

OQ .C

gQ .

■DCD

C/)C/)

Table 8

Attempt to generate a pseudo-secondary response with Seakem carrageenan (C/H)

in LPS nonresponder mice ( i e . in C3H/HeJ m ice).

CD

33"CD

CD■DOQ .Cao3"Oo

CDQ .

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C/)C/)

GROUP TREATKITIT 1© b 2® . AB TIT3RÜ

DAY 0 DAY 0 DAY 2i DAY 25

A CAR NONE' LPS ^ .5B PBS NONE LPS 4 .5G CAR NONE NONE 0D PBS NONE NONE 0E CAR NONE CAR 0F PBS LFS NONE' 4 .5G PBS LPS LFS . 4

abcd

5 .00 .1 ug LFS i . v ,1 ,0 ug LFS i .p .Antibody t i t e r s were obtained by p a ssiv e her.agglutination t e s t s . Each value rep resen ts the antibody t i t e r o f sera pooled fron 2 or 3 n ic e . T ite r s are expressed as va lu es o f x , derived fron the equation, x = log^ (K L/z), where HD was the rec ip ro ca l o f th e h ig h est d ilu tio n o f sera th a t produced h en a cg lu tin a tio n .

44

CAR on day 0 and LPS, on day 21 (group A), produced anti-LPS t i t e r s

th a t were comparable to the primary t i t e r s o f con tro l groups (groups

B, F, and G ).

These data in d ica ted th a t i f mice did not p ossess the g e n e tic

c a p a b ility o f ex h ib itin g secondary responsiveness to LPS, they

could not generate pseudo-secondary responsiveness to LPS. There

fo r e , th e g e n e t ic requirem ents fo r a pseudo-secondary response mimiced

th o se requirem ents fo r a true secondary response.

Temporal k in e t ic s fo r ^eneratinR secondary resp on siven ess.

Before the k in e t ic s fo r generating pseudo-secondary responsiveness

could be examined, i t was necessary to r e -e s ta b lish the temporal

requirem ents fo r generating a true secondary response. A fter t h is

was e s ta b lish ed , k in e t ic comparisons o f pseudo-secondary responsiveness

with tru e secondary responsiveness could be made.

Groups o f th ree mice were given primary in je c t io n s o f LPS on

day 0 and secondary in je c t io n s o f LPS on varying days a f te r the

primary in je c t io n s . Sera were c o lle c te d 4 days a f te r th e f in a l

in je c t io n s o f LPS.

Data from F ig . 7 show th at a second in je c t io n o f LPf. would not

generate a secondary response u n t i l approximately the 9th day a f te r

th e primary in je c t io n . T hereafter, anti-LPS t i t e r s increased u n t il

the end o f the experim ent, 21 days a f te r the primary in je c t io n .

K in etics o f the pseudo-secondary response. Once the k in e t ic s

fo r generating a tru e secondary response were e s ta b lish e d , the

k in e t ic s o f th e pseudo-secondary response could be examined. These

data might in d ic a te a s im ila r ity in the mechanisms by which the


F ig , 7 , The e f f e c t o f varying th e tirrlng o f the secondary

in je c t io n o f LPS with resp ec t to th e primary in je c t io n o f

LPS. 1 ,0 ug LPS was g iven i , p , on day 0 , A second, 1 ,0 ug

in je c t io n was adm inistered i , p , on th e days in d ic a te d . Con

t r o l mice received only a s in g le 1 ,0 ug dose o f LFS on day 0 ,

N ice were h ied 4 days a f te r th e f in a l in je c t io n o f LPS,

Antibody t i t e r s were obtained by p a ss iv e hem agglutination

t e s t s . Each value rep resen ts the antibody t i t e r o f sera

pooled from th ree m ice. T ite r s are expressed as va lu es o f

X, derived from the equation , x = logg (HD/z), where HD was

th e rec ip ro ca l o f the h ig h est d ilu t io n o f sera th a t produced

hem agglutination ,

Symbols:


Line Bs Tost mice


w«rV*

%

XoÛ


V

1

47

two responses were generated . Therefore, groups o f four mice were . '

given CAR on day 0 , IPS was adm inistered a t th ree day in te r v a ls

a f te r th e In je c t io n o f th e macrophage tox in and sera were c o lle c te d

4 days a f te r th e in je c t io n o f EPS, F ig , 8 shows th a t a pseudo

secondary response could not be mounted before approximately the , • -

9th day a f te r treatm ent with CAR, Pealc secondary anti-LPS t i t e r s

were seen on day 12 and th e r e a fte r . I t was concluded th at the

k in e t ic s o f a pseudo-secondary response generated with CAR were

s im ila r to th e k in e t ic s o f a true secondary response. Therefore,

th e manner by which th e pseudo-secondary response was generated

required tim ing th a t was s im ila r to th a t required by the mechanism

generating tru e secondary resp on siven ess.

Modulation o f RES a c t iv it y by CAR in athymic nude mic e . Before

th e generation o f pseudo-secondary responsiveness could be attempted

in nude mice, i t was necessary to determine i f RES suppression

could be achieved with the standard doses o f macrophage to x in s ,

MUS was found to be to x ic to the nude mice a t doses th a t suppressed

RES fu n ction ; th erefo re , MUS was om itted in the fo llow in g in v estig a ti<

Groups o f mice were given the standard dose o f CAR a t tim e 0 ,

Phagocytic a c t iv i t y was a ssessed by th e a b i l i t y o f mice to c le a r

c o l lo id a l carbon from, th e p eripheral blood u n t i l 72 h a f te r th e

ad m in istration o f CAR, The phagocytic fu nction o f mice treated

w ith GAR was compared with th e phagocytic function o f both nude

mice th a t rece iv ed PBS a t tim e 0 and normal lit te r m a te mice th a t

received CAR o r PBS a t tim e 0 , On F ig , 9 i t can be seen th a t the

RES fu n ction o f th e nude mice th a t received CAR was i n i t i a l l y suppresi


F ig , 8 , The e f f e c t o f varying th e tim ing o f th e aO m inistration

o f LPS with resp ect to th e aclm inistratlon o f Seakem carrageenaB

( car) , car was g iven on day 0 and LPS was adm inistered on th e

days in d ica ted . Mice were b led 4 days a f t e r th e in je c t io n o f

LPS, Antibody t i t e r s were obtained by p a ss iv e hem agglutination

t e s t s . Each value rep resen ts the antibody t i t e r o f sera pooled

from four m ice, T ite r s are expressed as va lu es o f x , derived

from the equation , x = log^ (HD/2), where HD was the r ec ip r o c a l

o f the h ig h est d ilu t io n o f sera th a t produced hem agglutination .

Symbols;

Line A; PBS day 0; 0 ,1 ug LPS day 0

Line Bs PBS day 0; 1 ,0 ug LPS day 21

Line G: PBS day 0; 0 ,1 ug IPS day 0; 1 ,0 ug LPS,day 21

Line D; 5 .0 mg CAR day 0; 1 ,0 ug LPS on the days in d ica ted


V

in

c0*»ok«cÊ5«n0.m»

0

6

■*®»u q y


J'lc. 9. Modulation o f Rno a c t iv i t y by :.',oai.oni ccu’ray.oonan (CAR)

in athymic nudo mice and th e ir normal l i t t e r mat os . a c t i v i t y

was measured by the a b i l i t y o f mice to c lear carbon p a r t i c l e s

from the peripheral blood, Mice were c;iven 5.0 mv CAR i . p . a t

time 0 and wore te s ted at the tim es in d ica ted . Control mice

received PBS at time 0 . iJach value rep resen ts the a r ith m etic

mean o f two mice.

Symbols:

Line A: Nudo control mice

Line L: Normal control mice

Line G: Normal t e s t mice

Line D: Nudo te s t mice

50

R eproduced with permission of the copyright owner. Further reproduction prohibited without permission.

m

3X


52

to a le v e l below th at suppression obtained in CAR-treated euthymie

m ice. By 70 h a f te r treatm ent with CAR, the phagocytic a c t iv ity o f

th e nude mice had returned to normal.

From th ese data i t was determined th at the standard dose o f

CAR could suppress RK? function ip nude mice to a degree s im ila r

to th e suppression achieved with CAR in normal mice. Therefore,

5 mg CAR was used in attem pts to generate a pseudo-secondary response

in nude mice,

CAR p o te n tia te s the en d otox ic ity o f LPS (8 ) , and th is standard

dose proved to be to x ic to the nude mice in the long-term experiments

th a t were required fo r determining pseudo-secondary resp on siven ess,

Experiments were then designed to determine whether or not a dose o f

CAR could be found th at would s t i l l produce e f fe c t iv e RES suppression,

but be l e s s to x ic fo r the nude m ice. Nude mice were given varying

amounts o f CAR a t tim e 0 . RES a c t iv ity was measured a t the tim es

in d ica ted by th e a b i l i t y o f mice to c lea r carbon i ia r t lc le s from

th e p erip h eral blood. F ig . 10 and 11 show the RES function o f

nude mice and th e ir normal litte r m a te s for 72 h a f te r treatment

with CAR, In both nude and in litte r m a te con tro ls W,0 mg CAR was

the low est dose that would s ig n if ic a n t ly suppress phagocytic a c t iv i t y .

Therefore, t h is was .chosen to be the standard dose o f CAR in the

fo llo w in g experim ents with nude mice.

Attempt to generate a pseudo-secondary rcspoTis" In alhynilc

nude m ice. The requirement fo r T c e l l s in the pseudo-secondary

response was a ssessed by the use o f co n g en ita lly athymic nude

m ice, LP8 i s a T c e l l independent antigen and can e l i c i t good


P ig , 10, RE? a c t iv i t y fo llo w in g in je c t io n o f varying amounts

o f ?eakem carrageenan (CAR) in athymic nude m ice. Mice were

given varying amounts o f CAR a t tim e 0 and RE? a c t iv it y was

measured a t the tim es in d ic a te d . Control mice received PB? ai

tim e 0 , BE? fu nction was te s te d by th e a b i l i t y o f mice to

c le a r carbon p a r t ic le s from the p erip h era l b lood . Each value

rep resen ts th e ar ith m etic mean o f two m ice.

C.Symbols !

Line Ai 1 ,0 mg CAR i .p .

Line Bi 2 .0 mg CAR i . p .

Line C; 3 ,0 mg CAR i ,p .

Line Ds 4 .0 mg CAR i ,p .

Line Ei Control mice

53


!\ i /

sdnpy w uodyy

S

:

00

I /)

3o

X

%


F ig , 11, RES a c t iv i t y fo llo w in g in je c t io n o f varying amounts

o f Seakem carrageenan (CAR) in normal l it te r m a te m ice. Mice

were given varying amounts o f CAR a t tim e 0 and RES a c t iv i t y

was measured a t th e tim es in d ic a te d . Control mice rece iv ed

PBS a t tim e 0 , RES fu n ctio n was te s te d by th e a b i l i t y o f

mice to c lea r carbon p a r t ic le s from th e p erip h era l b lood.

Each value rep resen ts th e ar ith m etic mean o f two m ice.

Symbols:

Line A: 1 ,0 mg CAR i , p .

Line B: 2 ,0 mg CAR i .p .

Line C: 3 ,0 mg CAR i ,p .

Line Di 4 ,0 mg CAR i , p .

Line Ei Control mice

55


i \ / \

o»

ui

v>v_3o

X

S 9 n |p ^ yo UDdyy


57

antibody responses in the absence o f Ï c e l l s . Athymic nude mice

were used to determine i f a pseudo-secondary response could a lso

be generated in the absence o f T c e l l s .

Nude mice and th e ir normal lit te r m a te s were trea ted with CAR

on day 0 . One or two In je c t io n s o f LP? were then g iv en , S in g le doses

o f LPS were g iven e ith e r a t 6 h a f te r treatm ent with CAR or on

day 21. Mice rece iv in g two in je c t io n s o f LPS were in jec ted a t

6 h a f te r treatm ent with CAR and again on day 21. Control mice

were g iven PBS on day 0 and LPS a t th e ,t im es d esignated . Sera

were harvested on day 2$. T o x ic ity problems were encountered

in t h is experim ent, in s p ite o f the reduced CAR dosage. By the

tim e sera were harvested on day 25i t o x ic it y deaths had reduced

many groups to one or two mice. Therefore, any conclusions

drawn from t h is data are tenuous. However, from Table 9 i t can

be seen th at a pseudo-secondary response could not be mounted

in th e absence o f T c e l l s (group A). Mice with normal T c e l l s

could generate a pseudo-secondary response (group C); th erefore

th e presence o f T c e l l s appeared to be a requirement fo r the

feneration o f a pseudo-secondary response. Nude mice exh ib ited

normal primary (compare groups E and F) and secondary (compare

groups G and l ) resp on ses. Therefore, with only a minimal amount

o f data upon which to base t h is con clu sion , priming fo r a pseudo

secondary response appeared to have a T c e l l requirem ent. However,

T c e l l s were not required fo r generation o f a true secondary response.


CD■ DOQ .C

gQ .

■DCD

C/)Wo"3O

8

c i '

33"CD

CD■ D

OQ .CaO3

■DO

CDQ .

■DCD

Table 9

Generation o f a pseudo-secondary response with Seakem carrageenan (CAF.) in th e presence and absence o f T c e l l s ( i e , in athymic nude mice and th e ir normal l i t t e r m a te s ) .

GROUP MOUSE TREATMEHT 20 c A3 TITER<


A nu/nu CAP NONE IPS 3 .5B nu/nu PBS NONE LPS 3C nu/+ GAR IPS 8D nu/+ PBS ■ NONE LPS 4

S nu/nu PBS IPS NONE 2■ F nu/+ PBS IPS NONE 1

G nu/nu PBS IPS IPS 7 .5H nu/+ CAR IPS IPS e.5I nu/+ PBS IPS IPS 6

J nu/+ CAR NONE NONE 0

Vr*CO

i. ^ 4 ,0 mg CAR i . p ,° 0 ,1 ug IPS i . v ,

p 1 ,0 ug LFS i . p . Antibody t i t e r s were obtained by p a ssiv e hem agglutination t e s t s . Each value rep resen ts th e antibody

t i t e r o f sera pooled from th ree m ice. T ite r s are expressed as va lu es o f x , derived from the equation , x = lo g 2 (HD/z), where HD was the rec ip ro ca l o f the h ig h est d ilu t io n o f sera th a t produced hem agglutination .

IV . DISCUSSION

During the course o f a recent study o f the r o le o f macrophages

in antibody responses, the phenomenon o f pseudo-secondary responsive

n ess was d iscovered (7 , 9)» I t was observed that one dose, o f LFS,

given to mice th a t were trea ted with macrophage to x in s 21 d p rev iou sly ,

e l i c i t e d a ty p ic a l secondary type response. The present in v e s t ig a tio n

revealed a strong s im ila r ity between true secondary responsiveness

and th e s in g le antigen dose-pseudo-secondary response. In f a c t , i t

appeared th a t th e macrophage to x in s , CAR and MUS, primed animals

fo r a secondary anti-LPS response.

The mechanism o f pseudo-secondary responsiveness was in v e s t i

gated in the fo llo w in g manner. F ir s t , the mechanism by which

various macrophage to x in s e l i c i t e d RES suppression was correlated

with th e ir c a p a b ility to generate the pseudo-secondary response.

I t was determined th a t only th ose macrophage to x in s th a t produced

lysosom al d e s ta b iliz a t io n and subsequent c y to to x ic ity could generate

pseudo-secondary resp on siven ess. Therefore, the consequences

o f the lysosom al d e s ta b iliz a t io n and c e l l death— e ith e r the re lea se

o f fa c to r s upon macrophage a u to ly s is or fa c to rs involved in the

subsequent regeneration o f the macrophage population—appeared to

be resp o n sib le fo r priming a c t i v i t i e s .

Next, the fu n c tio n a l s ta te o f th e RES was observed, fo llow in g

treatm ent with various macrophage to x in s , u n t i l day 21. ; t h is was

the tim e at which th e e l i c i t i n g dose o f LPS was adm inistered. I t

was seen th a t by day 5 a f te r treatm ent with macrophage to x in ,

59


60

RES fu nction was normal and th a t normal or enhanced phagocytic

a c t iv i t y continued u n t i l day 21, Thus, macrophage function was

a t a normal or an enhanced le v e l a t the tim e the pseudo-secondary

response was tr ig g e re d . These fu n ctio n a l macrophages could have

p a rtic ip a ted in the generation o f the immunological responsiveness

th a t was observed.

F in a lly , the g e n e t ic and k in e t ic requirem ents, as w ell as

th e T c e l l dependency, o f a tru e secondary response.w ere compared

to those requirem ents fo r a pseudo-secondary response. The g en e tic

and k in e t ic requirem ents fo r pseudo-secondary responsiveness

appeared to be id e n t ic a l to those requirements fo r true secondary

resp on siven ess to LPS, However, one d ifferen ce was discovered

in th a t th e true secondary response to LPS could bo generated

in th e absence o f fu n c tio n a l T c e lls ', whereas pseudo-secondary

resp on siven ess could n o t. I t must be remembered, however, th at

th e data from th e attem pt to generate pseudo-seondary responsive

n ess in nude mice were tenuous. I f fu rth er in v e s t ig a t io n s can

e s ta b lish a dosage regimen o f macrophage to x in s th a t w i l l not

p o te n tia te en d o to x ic ity to too great an exten t in nude mice, more

v a lid experim ental data can be obtained on the r o le o f T c e l l s

in the pseudo-secondary response. However, i f the above data,

which in d ica ted th a t pseudo-secondary responsiveness was T c e l l

dependent, were v a lid , then perhaps the priming reaction in th e

pseudo-secondary responses was dependent on T c e l l s . This in te r

p reta tio n would change the focu s o f the e f f e c t o f the macrophage

to x in s away from th e la t t e r , tr ig g e r in g events in the secondary


61

response to a T c e l l dependent priming event.

The generation o f pseudo-secondary resp on siven ess appeared

to occur by a mechanism s im ila r to th a t generating true secondary

resp o n siv en ess. The r o le o f the macrophage to x in s in the pseudo-

secondary phenomenon may have been to generate a population o f

memoiy c e l l s th a t primed the mice fo r a s p e c if ic secondary response,

upon subsequent contact with LPS,

Macropha/re to x in s and the pseudo-secondary response. The

use o f the variou se isom ers o f carrageenan, MUS and TT in attem pts

to generate pseudo-secondary responsiveness revealed th at the

mechanism by which RES suppression was achieved was an important

fa c to r in the induction o f the response, TT suppresses phagocytic

fu nction by RES blockade (68) , No c e l l d estru ction was observed

a f te r TT treatm ent, and recovery o f phagocytic a c t iv it y was not

dependent on th e generation o f a new c e l l population (68) , Conversely,

. macrophage c y to to x ic ity was the means by which CAR and HUS produced

RES suppression . Once i^hagocytosed, GAR and MU8 were resp on sib le

fo r ly so so m a l-d e s ta b iliz a tio n , which led to rupture o f the lysosom es

with the subsequent r e le a se o f d ig e s t iv e enzymes and eventual c e l l

death (3, 1 5 ) • Pseudo-secondary responsiveness was observed with

MUS and th e various isom ers o f carrageenan; however, a pseudo

secondary response could not be mounted in the presence o f TT, There

fo r e , th e a u to ly s is o f macrophages and/or th e ir regeneration

appeared to be a r e q u is ite fo r the generation o f pseudo-secondary

resp o n siv en ess. Merely suppression o f RE8 function was not s u f f ic ie n t

to prime fo r a pseudo-secondary response.


62

RES modulation and the •psexiclo-seconclary resp on se. Examination

o f RES fu nction fo r 21 d in mice th a t had received CAR, MUS, or

TT, had determined th a t a t approximately 5 d fo llow in g adm inistration

o f th e macrophage to x in s , the suppressed phagocytic a c t iv it y had

returned to normal; in f a c t , i t was even enhanced, Although the

recovery from RES suppression by CAR, MUS, and TT appeared to

be eq^ually com plete, i t was apparent th at the mechanisms o f

recovery could not have been id e n t ic a l . The macrophages present

a t day 21 in mice th at had been given TT were those th at had been

suppressed on day 0 ; they had recovered th e ir normal phagocytic

fu nction (68) , Mice th a t were given CAR or MUS were depleted

o f th e ir macrophage populations (3, 1 5 ) , Therefore, the normal

phagocytic a c t iv it y seen a f te r day 5 was the r e s u lt o f a new

population o f macrophages. The enhanced phagocytic a c t iv it y

th a t was seen fo llow in g RES recovery in mice treated with MUS,

CAR or TT was probably due to a rebound e f f e c t .

I t had been p ostu lated p rev iou sly th a t pseudo-secondary

resp on siven ess was the r e s u lt o f ex cess iv e stim u lation o f progenitor

antibody-form ing c e l l s by a bolus o f antigen that had not been

sequestered in the macrophage-impaired mice (? ) . The present

r e s u lt s showed th at macrophage function was normal a t the time

th e e l i c i t i n g dose o f EPS was adm inistered. Therefore, antigen

seq u estra tio n would have occurred norm ally, and the e a r lie r p o stu la te

was not th e means by which the pseudo-secondary response was generated,

As mentioned^ e a r l ie r , d estru ction o f macrophages may have

been required fo r generation o f pseudo-secondary resp on siven ess.


63

The r e le a se o f fa c to r s from th e ruptured macrophage lysosom es or

from the macrophages them selves could have served to prime B

lymphocytes fo r à secondary anti-LPS response; th ese might have

su b stitu te d fo r the LPS m olecule. The immunodeterminant group o f

antigen alone apparently can stim u late unprimed. B lymphocytes

to produce a primary antibody response and to s e n s it iz e the

animal fo r a secondary anti-LPS response (6 4 ). However, tr ig g er in g

o f a secondary response required both the a n tig en ic s ig n a l and

a second, m itogenic s ig n a l; the la t t e r i s present in LPfi as the

l ip id A portion o f the m olecule (6 4 ), The macrophage-derived

fa c to r s may have been s u f f ic ie n t to su b s titu te fo r the LPS m olecule

in th e priming o f B c e l l s , so th at when LPf was adm inistered on

day 21, the p rev iou sly s e n s it iz e d B lymphocytes mounted a secondary

response.

I t i s u n lik e ly th a t the macrophage to x in s them selves primed

the B lymphocytes. CAR aJid LPS:U113 do share come stru ctu ra l

s im i la r i t ie s , in th a t both contain g a la c to se u n its ( 36, 4 o ), and

i t could be p ostu lated th at t h is stru ctu ra l s im ila r ity would

allow CAR to s u b s titu te fo r LPS in priming fu n ctio n s. However,

the pseudo-secondary response i s a lso observed with KUS, a m aterial

th a t bears no s tru c tu r a l resemblance to LPf,. Therefore, i t i s

more reasonable th a t th e consequences o f MUC and CAR adm inistration ,

rather than th e macrophage to x in s them selves, were resp on sib le '

fo r .priming fo r a secondary anti-L P f response,

. Another asp ect th at must be considered i s the population

o f new macrophages th a t arose a f te r macrophage d ep letion by CAR


6 !'

or MUS, This population was young and capable o f optim al processing

o f a n tigen . T herefore, when LPS was introduced in to the system,

i t could be processed very e f f ic ie n t ly and presented to the antibody-

forming c e l l s . These young macrophages a lso may have been capable .

o f in creased production o f IL-1 (56) . The resu lta n t p r o life r a tio n

o f T c e l l c lon es may have aided macrophage derived fa c to r s in

th e priming o f B lymphocytes.

The g en e tic requirem ents o f the pseudo-seconrlary response.

The pseudo-secondary response could not be generated in the LPS

nonresponder, C3M/KcJ m ice. C3H/HeJ mice have a d efect in a

single) autosomal dominant gene ( 53) th a t i s m anifested by an

in a b i l i t y o f th e ir c e l l s , or the lack o f a membrane comi^onent on

th e ir c e l l s , to rea ct with the l ip id A portion o f the LFf molecule

( 53, 67) . Therefore, th ese mice could not respond to th e second

s ig n a l o f LPS and could not mount a secondary response to LPS.

The fa c t th at C3'V*^eJ mice could not generate a pseudo-secondary

response to LPS suggested th at the g en e tic requirements for pseudo

secondary resp on siven ess 'e r e the same as those fo r true seccndary

resp on siven ess. Thus, mice must p ossess the g en e tic ca p a b ility to

respond to the second, m itogenic s ig n a l o f LPf in order for pseudo-

secondary resp on siven ess to be m anifested. This, ar;ain, supported

the h yp oth esis th at the e f f e c t s o f CAR and MUS were exerted at

the l e v e l o f priming the B lymphocytes fo r a secondary anti-LPS

response; in a l l p r o b a b ility , tr ig g e r in g o f a secondary response

occurred in a normal manner. In both ca ses, when the tr ig g er in g

dose o f LPS was adm inistered on day 21, the sonsi Lized lymphocytes


65

were stim ulated by the l ip id A portion o f the m olecule to secondary

resp o n siv en ess. Thus, upon th e ir f i r s t exposure to LPP, mice

trea ted with MUS or CAR reacted as i f they had been p reviously

s e n s it iz e d to the LPS immunodeterminants.

The k in e t ic s fo r development o f the pseudo-secondary response.

I t was e s ta b lish ed th a t th e k in e t ic s fo r development o f pseudo-

secondary resp on siven ess follow ed th ose fo r generation o f true

secondary resp on siven ess. The present study confirmed e a r lie r

work (51) th a t had determined th at a 10 to I4 d in te r v a l between

primary and secondary in je c t io n s o f LPS was required fo r generation

o f a secondary response. L ikew ise, pseudo-secondary responsiveness

required at l e a s t a 10 d in te r v a l between adm inistration o f the

macrophage to x in and in je c t io n o f LPS. I t appeared th a t the

pseudo-secondary response was generated by a mechanism that required

the same tim ing as th at mechanism producing a true secondary

response. Again, i t seemed reasonable to p o stu la te th a t the

pseudo-secondary response was tr iggered in the same manner as a

tru e secondary response; the d ifferen ce in the two responses

occurred a t th e le v e l o f priming.

An a lte r n a tiv e h yp oth esis could be suggested . The required

in te r v a l between macrophage tox in -ad m in istra tion and in je c tio n Of

LP5 was 10 d, RES fu nction was shown to have recovered from

suppression by MUS or CAR by day 5 fo llow in g treatm ent with macrophage

to x in . An a d d itio n a l 5 d may have been required fo r t h is young

population o f macrophages to have matured to the l e v e l that

they could p rocess and present antigen e f f e c t iv e ly . Once the


66

antigen handling c a p a b il i t ie s had been esta b lia h ed , th ese young

macrophages may have been ab le to process and to present antigen

much more e f f i c i e n t ly than could an o ld er population o f macrophages.

The r e s u lt o f in creased antigen processing may have been m anifested

by a heightened antibody response' to the s in g le dose o f LPS.

However, o ther r e s u lt s o f t h is study le n t the most c r e d ib il i ty

to an hyp oth esis whereby treatm ent with macrophage to x in s primed

th e B lymphocytes and the s in g le dose o f antigen triggered secondary

resp on siven ess in a normal manner.

The requirement fo r T c e l l s and the paeudo-secondary response.

The p o te n tia tio n o f en d o to x ic ity by CAR (8) was a problem in the

experim ents with nude m ice. Amounts o f CAR th at would suppress

RES fu nction but would not p o ten tia te en d otox ic ity to too

grea t an ex ten t in the short term experim ents, s t i l l did p o ten tia te

en d o to x ic ity too much fo r long.term experim ents. A n on -p oten tia tin g ,

and y e t RES-suppressing, dose o f CAR was not found. Therefore,

th e l e t h a l i t y o f th e combined treatm ent reduced the numbers o f

mice in many o f the experim ental groups to the point th a t the

data were not s t a t i s t i c a l l y sound. Any conclusions made from

th ese data were tenuous and should be subjected to v a lid a tio n

when an acceptab le experim ental system can bo designed.

The data th a t were obtained did su ggest, however, that a

pseudo-secondary response could not be generated in the absence

o f T c e l l s . This fin d in g i s contrasted to true secondary respon

s iv e n e s s , which could be generated in the absence o f T c e l l s . I f

true secondary and pseudo-secondary responses are id e n t ic a l at


6 7

th e le v e l o f LPS tr ig g e r in g (th e second s ig n a l ) , then the T c e l l

dependency o f the pseudo-secondary response occurred a t the le v e l

o f prim ing. Assuming th a t IL-1 production by young macrophages was

Important in priming B lymphocytes, then the lack o f a pseudo

secondary response in nude mice would not be su rp r is in g . The IL-1

resp on sive IL-2 producing c e l l s are lack ing or nonfunctional in

nude mice ( 56) . T herefore, th e in te r le u k in s would not function

in th e nude mice, and the mice could not have been primed, by

treatm ent with CAR, fo r pseudo-secondary responsiveness to LPS.

A sim ple T c e l l dependency o f the macrophage-derived fa c to rs

involved in priming a lso would have resu lted in the abrogation o f

the pseudo-secondary response in nude m ice. Thus, a T c e l l dependent

fa c to r th a t.co u ld prime B lymphocytes fo r a secondary anti-LPS

response would not have been ab le to operate in the athymic nude

m ice. A lso , the in te r le u k in s , which are resp on sib le fo r the

in creased number o f T c e l l s th a t might aid B lymphocytes, could

not fu nction in the nude mice, '

C onclusions. The treatm ent o f mice with the macrophage to x in s ,

CAR, or IlUfi, 21 d p rior to th e in je c tio n o f LPS resu lted in a

ty p ic a l secondary-type antibody response. The above r e s u lts

have led to the p o stu la tio n o f the fo llow in g mechanism(s) fo r the

gen eration o f t h is response (se e F ig . 1 2 ). Macrophages that were

trea ted with CAR or MU5 underwent lysosom al d e s ta b iliz a t io n and

subsequent a u to ly s is . During a u to ly s is , fa c to r s were released

from the macrophages th a t were capable o f s ig n a llin g B lymphocytes

to d i f f e r e n t ia te in to memory c e l l s . These macrophage-derived fa c to r s


FIG, 12, THE GEMERATION OF PSEUDO-SECONDARY RESPONSIVENESS

2° RESPONSE Antibody-forming (plasma

SMALL LYMPHOCYTES (Memory c e l l s )

s up er s B edan tlgeîrs^

YOUNG MACROPHAGE;

progenitor cells

otho fa ctors? _ X ^ L ?

CAR or MUS-TREATEDmacrophages

young macrophages

68


6 ?

could have functioned in a manner s im ila r to the immunodeterminant

group on the LPS m olecule; they provided the necessary s ig n a l

to B lymphocytes. When LPS was adm inistered on day 21, the primed

B lymphocytes were ab le to respond in a secondary fash ion , as i f

they had encountered LPS p rev io u sly , when in f a c t , t h is was th e ir

f i r s t exposure to the an tigen . As the macrophage population

recovered from d ep letio n by CAR or KUS, young, healthy macrophages

may a lso have contributed to the pseudo-secondary resp on siven ess.

In terleu k in production by young macrophages could have caused

the p r o life r a t io n o f c lon es o f am p lifier T c e l l s which, in turn,

may have driven B lymphocytes to fu rth er p r o life r a t io n . Operating

a lte r n a t iv e ly to or in conjunction with the above rea ctio n s, young

macrophages may have processed and presented antigen more e f f e c t iv e ly ,

thus causing a heightened antibody resiJonse, This could explain why

a s in g le dose o f LPS can, indeed, generate a secondary-type antibody

response.

The data generated in t h is study have shown th at the mechanism

by which the pseudo-secondary response was induced operated a t the

le v e l o f priming. The tr ig g er in g a c t i v i t i e s required fo r pseudo

secondary resp on siven ess appeared to be id e n t ic a l to those required for

a true secondary response. Therefore, the early theory that suggested

a mechanism whereby progen itor antibody-form ing c o l l s wore e x c e ss iv e ly

stim ulated by a bolus o f antigen th a t had not been sequestered in the

macrophage-impaired mice (9 ) was proved to be in error, and in te r e s t

was focused a t th e priming stop .

By cen terin g th e in v e s t ig a t io n o f the mechanism o f pseudo-


7 0

secondary resp on siven ess a t th e priming s tep , many q u estion s have

a r ise n . Do macrophages contain substances th at could rep lace the

LP£) immunodeterminant group in s e n s it iz in g lymphocytes? What, i f

any, r o le does the d e s ta b iliz a t io n o f macrophages play in generating

t h is sub stan ce(s)? Do in te r le u k in s play a part in generating the

pseudo-secondary response? W ill In ter leu k in s prime mice for a

pseudo-secondary response? Answers to th ese q u estion s w il l r e su lt in

a c le a re r understanding o f the mechanism by which pseudo-secondary

responsiveness i s generated .


V. SUMMARY

Previous work had esta b lish ed that a ty p ic a l secondary type

response could be generated by a s in g le dose o f LPS th a t had

been adm inistered to mice treated with the macrophage to x in s , GAR

or MUS, 21 d p rev iou sly (? , 9)» The mechanism o f t h is phenomenon

was in v estig a te d and i t was determined th at the requirements

fo r pseudo-secondary resp on siven ess p a ra lle led those fo r true

secondary resp on siven ess. The requirement fo r T c e l l s was the

only exception 5.pseudo-secondary responsiveness required the

presence o f T c e l l s and a true secondary response could be generated

in th e ir absence. I t was postu lated that the pseudo-secondary

response was generated in a manner s im ila r to the generation o f a

tru e secondary response. The d iffer en ce s between the two responses

probably occurred a t th e le v e l o f priming. Priming for true secondary

responsiveness was achieved by. previous contact with the molecule

o f LPS, but pseudo-secondary priming was dependent upon lysosom al

d e s ta b il iz a t io n o f macrophages by CAR or MUS,

Attempts to generate pseudo-secondary responsiveness with

i - , K -, and A -carrageenans were s u c c e ss fu l. However, attem pts

to generate the phenomenon with TT, a macrophage tox in that suppressed

the RES by p h y s io lo g ic a l blockade ( 68) , rather than by lysosom al

d e s ta b iliz a t io n and a u to ly s is o f macrophages, f a i le d . From th is i t

was determined th a t lysosom al d e s ta b iliz a t io n and c y to to x ic ity o f

macrophages was .a requirement fo r the generation o f the response.

Phagocytic a c t iv i t y was follow ed fo r 21 d a f te r treatment


7 2

with CAR, MUS, and TT by determining the a b i l i t y o f mice to c lea r

c o l lo id a l carbon from the peripheral b lood. I t was found th at RES

fu n ction had returned to normal by 5 d a f te r treatm ent with the

macrophage to x in s . Normal or enhanced phagocytic a c t iv it y continued

u n t i l day 21, T herefore, macrophages were fu n c tio n a lly a t a

normal or an enhanced le v e l a t the time the pseudo-secondary response

was generated ,

C3H/HeJ m ice, mice th a t lack the g en e tic ca p a b ility to mount

secondary resp on siven ess to LPS, were a lso unable to generate a

pseudo-secondary response to LPS, This in d ica ted th at the g en etic

requirem ents fo r pseudo-secondary responsiveness p a ra lle led those

fo r tru e secondary resp on siven ess.

L ikew ise, the k in e t ic s o f the pseudo-secondary response

fo llow ed those o f true secondary resp on siven ess, A second in je c tio n

o f LPS would not e l i c i t a secondary response u n t i l a t le a s t 10 d

a f te r th e primary in je c t io n o f LPS, At l e a s t 10 d were a lso required

between treatm ent with macrophage to x in s and the in je c tio n o f LPS

fo r th e generation o f pseudo-secondary resp on siven ess.

R esu lts based on r e la t iv e ly meager data suggested th a t, u n like

true secondary resp on siven ess, pseudo-secondary responsiveness

required the presence o f Ï c e l l s .

A mechanism fo r pseudo-secondary resp on siven ess was postu lated

th at a ttr ib u ted priming fo r the pseudo-secondary response to the

consequences o f the macrophage to x in s , Substances capable o f

li lymphocyte priming were released from macrophages treated with

MUS or CAR. Thus, a population o f s e n s it iz e d B lymphocytes were


73

a v a ila b le when LPS was adm inistered on day 21, These c e l l s responded

to th e antigen in a secondary fa sh io n , mounting a secondary response

to th e ir f i r s t encounter with LPS,


7 +

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investigation of the generation of a pseudo-secondary

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