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1981
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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Date : MAR 9 3981_________
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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
Approved by;
, ____________Chairman, Board o f Examiners
Dean, Graduate School
I lM iLDate
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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
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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
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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
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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
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PaceC onclusions.......................... «6?
V. SUMMARY....,............................................................................. 71
LITERATURE CITED.................................................................................. 74
V I
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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
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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
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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
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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
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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
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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
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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
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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
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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) ,
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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
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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
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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
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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 .
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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
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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
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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.
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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
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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
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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.
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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
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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;
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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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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
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21
|olut
§ceI-SoX
OlO
8
r
J— L
§ IîrilVA ^ NV3W
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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
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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
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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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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. Table 1
Generation o f a pseudo-secondary response with s i l i c a (MUS) in w hite m ice.
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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.
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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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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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^
DAY 0 DAY 0 DAY 21 DAY 25
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 .
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GROUP TPEATKE-NT®- |0 b AB TTTERd
DAY 0 DAY 0 DAY 21 DAY 25
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
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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
DAY 0 DAY 0 DAY 21 DAY 25
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.
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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
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, 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 .
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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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
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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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
37
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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 A; Control mice
Line B: T est mice
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
39
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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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4 l
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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
DAY 0 DAY 0 DAY 21 DAY 25
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 .
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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).
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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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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 A; Control mice
Line Bs Tost mice
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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V
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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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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6
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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
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m
3X
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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
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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
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sdnpy w uodyy
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00
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3o
X
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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
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i \ / \
o»
ui
v>v_3o
X
S 9 n |p ^ yo UDdyy
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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.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
CD■ DOQ .C
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C/)Wo"3O
8
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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<
DAY 0 DAY 0 DAY 21 DAY 26
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
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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
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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.
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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.
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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
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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
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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
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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
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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
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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
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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-
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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 .
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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
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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
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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,
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7 +
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