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Experimental murine amyloid. III. Amyloidosis induced with endotoxins

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Experimental Murine Amyloid. 111.
Amyloidosis Induced with Endotoxins
By WERNER
F. Bmm, M.D., JAMXS T. WILLERSON,
M.D., RICHARD
ASOFSKY,
M.D.,
JOHN
N. SHEAGREN,
M.D.,
AND
A new model for the study of experimental amyloid is described using parenteral administration of endotoxin. The
differential responses in the various
strains of mice examined suggested that
genetic factors may be involved in murine
A
SHELD~N
M. WOLFF,M.D.
amyloid formation. Many similarities
were noted between casein- and endotoxin-induced amyloidosis. It is suggested
that endotoxin may be involved in the
pathogenesis of several forms of experimental amyloidosis.
is a fibrillar glycoprotein found are reported. The immunoglobulin rein the tissues of man and animals sponses and reticuloendothelial activity
under a variety of clinical and laboratory after repeated administration of endotoxin
conditions.l Numerous procedures have were also examined. Commercially obbeen used for the induction of experimental tained and a highly purified preparation of
amyloid. These include the administration Salmonella endotoxin were studied for their
of live or killed bacteriaz4 and the repeated ability to induce amyloid. The possible
injection of various macromolecular sub- role of endotoxin in some of the models of
stances such as serum proteins,6s6 ribo- experimental amyloid induction is disnucleic acid,7Vs adjuvant?JO and ~ a s e i n . ~ J l cussed.
Recently, we reported that injection of
E. coli endotoxin induced amyloid in
MATERIALSAND METHODS
mice.12 The histologic findings and strain
Animals
differences noted simulated the amyloid
Mice were obtained from the Rodent and Rabbit
produced in mice by repeated injections
Production Section, Animal Production Unit
of casein.13
In this study, evidence is presented which (NIH). The strains included CSH/Hen, AKR,
AL/N, C57BL/6JN, Balb/c AnN, and White
suggests that endotoxins may be responsi- Swiss (G.P.). Male mice, 6 to 7 weeks old, were
ble for the amyloidosis induced by repeated used in each of the experiments. New Zealand
parenteral administration of casein. Our albino rabbits of either sex, weighing approxifindings in six different strains of mice using mately 2 Kg., were used for bioassay of endotoxin
E. coli endotoxin for induction of amyloid preparations.
MYLOID
WERNERF. BARTH,M.D.: Senior Investigator, Senior Investigator, Laboratory of Clinical InvextiArthritis and Rheumatism Branch, National In- gation, National Institute of Allergy and Infecstitute of Arthritis and Metabolic Diseams, tious Diseases, Bethesda, Maryland. SHELDON
M.
M.D.: WOLFF,M.D. : Clinical Director, National InstiBethesda, Maryland. JAMES T. WILLERSON,
Clinical Associatq Arthritis and Rheumatism tute of Allergy and Infectiaus Diseases, Bethesda,
Branch, Natiorurl I&itute
of Arthritis and
Maryland.
Metabolic Diseases, Bethesda, Maryland. RICHARD
Reprint requests should be addressed to DT.
ASOFSKY,M.D.: Head, Experimental Pathology Werner F. Barth, Head, Arthritis Division, DeSection, Laboratory .f Microbial Immunity, NG
partment of Medicine, University of Maryland
tional Institute of Allergy and Infeetiow Diseases, School of Medicine, Baltimore, Maryland 21201.
Bethesda, Maryland. JOHN N. SHEAGREN,
M.D.:
ARTHRITIS AND
RHEUMATISM, VOL. 12, No. 6 (DECEMBER,
1969)
615
616
BARTH ET AL.
Materials
Escherichiu coli 0127:B8 and Salmonella
typhosu 0901 endotoxins (Difco Laboratories, Detroit, Mich. ) were dissolved in phosphate-buffered
saline (pH 7.4) at a concentration of 2.0 mg./ml.
and frozen at -2OOC until used. A highly purified
endotoxin derived from Salmonella enteritidis'
was also employed.14
Hammersten Casein ( Nutritional Biochemical
Company, Cleveland, Ohio) was slowly dissolved
in 0.05 M NaHC03 at a concentration of 8.5
Gm. per cent and then allowed to pervaporate
in dialysis tubing for 48 hours in the cold to a
final concentration of approximately 12 Gm. per
cent. The casein preparations were stored at 4OoC
and assayed within 1 week for pyrogenicity. The
casein was prepared in sterile glassware and
pyrogen-free reagents to a concentration of 8.5
Gm. per cent both with and without added
Merthiolate ( 1:10,000).
Induction of Amyloid
Mice were injected subcutaneously with endotoxin in alternate sites on the back, five times per
week. The administration of 500 pg. daily caused
a significant number of deaths in White Swiss
mice during the first 3 days of this schedule. When
the initial dose was decreased to 400 fig. for the
first 3 days, followed by 500 pg. daily thereafter,
mortality was negligible. This dose schedule was
used in all experiments unless otherwise indicated.
Animals injected with equal volumes of phosphatebuffered saline (0.25 ml.) served as controls.
Groups of animals were sacrificed at intervals of
1-2 weeks. Spleen tissue was obtained for histologic examination and serum for determination
of immunoglobulin levels. Frequently liver and
kidney tissues were also examined. All slides were
examined by hematoxylin and eosin stain. Virtually
all slides were also examined by the Congo red
stain. The presence of green birefringence using
polarized light on Congo red-stained sections was
considered specific for amyloid.15
Grading of Amyloid
'
A new system was devised to grade the amount
of amyloid in the spleens of mice:
Grade 1 . A definite perifollicular rim of amyloid
in one or more of the splenic follicles.
Grade 2 . More than half of all follicles in the
random section of the spleen examined show a rim
of amyloid. This grade also included those sections
*Kindly supplied by Dr. J. A. Rudbach, Rocky
Mountain Laboratory NIAID, Hamilton, Mont.
in which a small area of bridging between closely
adjacent follicles was present.
Grade 3. All splenic follicles show a rim of
amyloid with significant bridging of amyloid between neighboring follicles.
Grade 4. Extensive splenic amyloid with bridging
between almost all follicles, some distortion of the
follicular architecture, and involvement of the e n tral areas of follicles.
Grade 5. Fifty per cent or more of the spleen replaced by amyloid; splenic architecture completely
distorted.
All slides were examined by two different observers who had no knowledge of the tissue source.
The grading system used proved rapid and reproducible. Interobserver differences were never
more than a grade and occurred in less than 5
per cent of the slides examined.
Mouse Immunoglobulins
Serum immunoglobulin levels were measured by
a modification16 of the method of radial immunodiffusion17 using antisera specific for each of the
five classes of mouse immunoglobulins.l~J9A mixture of mouse myeloma sera, previously calibrated
with purified myeloma proteins, was employed as
a standard. Mouse blood was obtained by retroorbital puncture at the times indicated and sera
stored at -2OOC until used.
Reticuloendothelial (RES) Phagocytic Function
RES phagocytic activity was evaluated using a
carbon suspension (Pelikan Ink Cll-l431a,
Guenther-Wagner, New York) as the colloidal test
substance using a standard method.20 The carbon
suspension was diluted 2 parts carbon to 1 part
saline, and an amount determined which gave a
half-time of clearance ( T - %) of approximately
10 min. in control animals.21 This amount was
found to be 0.01 ml. of the suspension per gram
of mouse. Clearances were not determined serially
in the same mice, but groups of 6 to 16 individually numbered mice were studied at each point in
time. Immediately after completion of the clearance studies on any given group, the animals were
sacrificed, tissues examined histologically, and the
degree of splenic amyloid determined.
Bioassay of Casein for Endotoxic Activity
The casein solutions were injected in rabbits for
appraisal of pyrogenicity and effect on circulating
white blood cells. Details of the housing, recording
of temaerature., m
- lotting
- of fever curves, induction
of pyrogenic tolerance, and injections are recorded
617
EXPERIMENTAL MURINE AMYLOID. 111
Amyloid in
Table 1.-Splenic
G.P. Mice
Following Administration of E. coli Endotoxin
Grade of Amyloid a
No. with Amyloidosis
5
10
15
20
injections
injections
injections
injections
( 1 week)
(2 weeks)
(3 weeks)
( 4 weeks )
Saline
E . coli Endotoxin
0/6
1/6
8/10
17/18
20/20
0/10
0/8
0/24
0.16
1.7
2.5
3.4
* Derived by adding the numerical grade assigned to the amount of amyloid in each animal and
dividing by the total number of mice in the group.
of Autoclaving Endotoxin on Amyloid Induction in G.P. Mice
Table 2.-Effect
Group
A
B
C
Endotoxin
Given Daily, pg.
No. of
Injections
500
500
14
14
14
500
Autoclaved,*
hr.
-
No. Animals with Amybid/
Total Receiving Endotoxin
15
43
'250°F.
f The single animal with splenic amyloid had a small amount with a few perifollicular foci (grade 1).
elsewhere.22 Antibody titers were measured in
serial twofold dilutions of serum using the bentonite flocculation technic.23 The number of white
blood cells in blood obtained by ear vein puncture were counted in duplicate in an electronic
counter (Model B, Coulter Electronics, Hialeah,
Fla.) after lysis of red blood cells with saponin.
Differential counts of 100 white blood cells were
performed on Wright-stained blood smears.
RESULTS
Amyloidosis in White Swiss (G.P.) Mice
With repeated administration of E. coli
endotoxin, amyloid was detected as early as
1 week after start of the injections (Table
1). Subsequently, in this mouse strain,
amyloid appeared in the spleens of almost
all animals after 10 or more injections. The
earliest deposition was perifollicular; later
the red pulp and germinal follicle were
progressively involved. The amount deposited in the spleen increased with the
longer injection schedule, and amyloidosis
was then also detected in the liver and kidneys. None of the 48 animals injected with
buffered saline alone developed amyloidosis.
When the endotoxin was autoclaved for
prolonged periods, such that pyrogenicity
in rabbits was almost completely abolished,
the ability to induce amyloid was also lost
(Table 2).'
Amyloidosis in Different Strains of Mice
Using the same injection schedule,
amyloid formation in different strains of
mice was evaluated. In some strains (AKR
and G.P. ) , amyloidosis developed early and
in virtually all animals (Table 3). I n other
strains, such as the Balb/c, C3H/Hen, and
C57BL, amyloid was of a lesser degree, appeared later, and in fewer animals.
Consistency with respect to the degree
and rapidity of development of amyloid
was noted within each of the three strains
in which several experiments were performed. In three separate experiments, the
C3H/Hen strain developed less amyloid, in
fewer animals, and after delay when compared with the G.P. mice (Table 2 and
reference 12). After 10 injections of endotoxin in G.P. mice, 8 out of 10 animals
showed amyloid in two different experi'A few rabbits became febrile when 4 mg. of
endotoxin was administered intravenously, a dose
uniformly lethal prior to autoclaving. No rabbits
developed fever following an intravenous injection
of 400 pg. of endotoxin subjected to prolonged
autoclaving.
618
BARTH ET AL.
Table 3.-Splenic
Amyloid in Different Strains of Mice Following Administration
of E. coli Endotoxin
Saline, 30 Injections
E . coli Endotoxin
Strain
10 Injections (grade)
AL/N
C57BL
4/21
Balb/c
C3H/Hen
0/10
1/11
AKR
G.P.
* 5 of
0/8
10/10
8/10
(2.1)
(1.9)
20 Injections (grade)
0/3
l0/15
9/9
2/11
(1.5)
(2.1)
9/9
10/10
(4.2)
(4.3)
30 Injections (grade)
-
0/10
9/9
9/11
7/10
9/9
11/11
*
0/8
(3.2)
(1.8)
(1.6)
(4.2)
(4.5)
0/10
0/8
0/10
0/10
these mice were sacrificed after 10 injections of saline.
ments; the same grade of amyloid was
found on both occasions (Tables 1 and 2).
The AL/N strain was examined twice
because of the high mortality noted in the
first experiment. Twenty-five out of 35
mice died in the first 2 weeks of administration of E. coli endotoxin. Most deaths occurred during the first 3 days. There was
equally high mortality in a second experiment using 35 more mice; hence there are
no data available after 2 weeks' treatment.
Virtually none of the C3H/Hen mice and
very few of the G.P., Balb/c, or AKR mice
died on this injection schedule. Higher
mortality than was observed in these 4
strains was seen in the C57BL strain. Consequently, in this strain, on this schedule,
the later time points could not be studied.
None of the saline-treated animals, in any
strain, developed amyloidosis.
purified S. enteritidb endotoxin was
studied. Three of 4 mice that received 15 injections of 0.5 mg./day of this preparation
for 3 weeks developed splenic amyloid.
Immunoglobulin Responses Following Administration of Endotoxin
The immunoglobulin responses in G.P.
and C3H/Hen mice following repeated administration of endotoxin are shown in Figs.
1-4. In the G.P. mice, 7s-yl levels were only
slightly increased with high doses of endotoxin but not increased with the lower
doses (Fig. 1 ) . In this strain, 7S-y2,, levels
were decreased but other immunoglobulins
unchanged after 5 weeks' treatment with
0.005 mg. of endotoxin (Fig. 2 ) . At the
higher doses, there was an early and
sustained increase in levels of 7 S - y ~and
~
yM and a decrease in levels of yA (Figs. 1
and 2). The immunoglobulin responses in
this strain contrasted with the C3H mice
which showed a marked increase in 7Syl
levels after high doses of endotoxin for 6
weeks but little increase with the smaller
doses (Fig. 3). The changes in 7S-yzB, yM,
and y,, noted in the G.P. mice were not
found in the C3H mouse (Fig. 4 ) .
Production of Amyloid by S . typhosa and
Highly Purified S . enteritidis Endotoxins
Twenty-one of 25 G.P. mice examined
after 15 injections (3 weeks) of S . typhosa
endotoxin showed splenic amyloidosis. The
limited study with this endotoxin suggested
that it was qualitatively similar to E . coli
RES Phagocytic Function During Adminendotoxin in the type of histological lesions
istration of Endotoxin
induced. In order to determine whether
Table 4 shows serial changes in the rate
amyloidosis induced by the commercial
of
clearance of colloidal carbon in G.P. and
products was not secondary to some other
contaminating nonendotoxin material," the
*Aerobic and anaerobic cultures of the comeffect of repeated injections of a highly mercial Difco endotoxin were negative.
6 19
EXPERIMENTAL MURINE AMYLOID. 111
G.l?
MICE WITH
AMYLOIDOSIS
16.0
-
12.0
-
0
1
CONTROL ENDOTOXIN ENDOTOXIN ENDOTOXIN ENDOTOXIN
0.005mg
0.5mg
0.5mg
0.5mg
( 2 wks.)
(4 wks.)
(5 wks.)
(5 wks.)
Fig. 1.-The 7S-yl immunoglobulin response following administration of E. coli
endotoxin to White Swiss (G.P.) mice. Each point represents the level in an individual
mouse. Mean levels are indicated by the horizontal bars.
-
G.P.
<
6.0-
UJ
5.0 -
-P
5
-J
0
0
0
2 4.0.
A
0
0
COO0
ooo
0"
0
0
v
0
0
00
00.
-co-
080
+O
CONTROL
ti
ENDOTOXIN
0.005mg
( 5wks.)
naoa
0
e.
M
0
.+
* so
A
??
AA
W
0 " :
&?A
!:
-
i
a A a4 4 - 5
ENDOTOXIN
0.5 mg
(2wks )
ENDOTOXIN
0.5mg
(4w k s . )
ENDOTOX IN
0.5mg
(5wks.)
Fig. 2.-Serum levels of 7 s - y ~ S~ -~~ ZIgA,
B , and IgM in G.P. mice after repeated
injections of E. coli endotoxin. Each point represents the level in an individual mouse.
Mean levels are indicated by the horizontal bars.
CSH/Hen mice. Acceleration of the clearance rate was seen at the end of 1 week in
the G.P. mice. By the end of the second
week, the clearance rate had returned to
control levels, where it remained despite
progressively increasing amounts of amy-
loid in the spleen and liver. Similar alterations in the rate of carbon clearance were
demonstrated in the C3H/Hen mice, but
over a more extended period of time. Acceleration was seen at 3 weeks in this strain
with return to control levels at 6 weeks.
620
BARTH ET AL,
C3"
MICE WITH
AMYLOIDOSIS
0
0
'6
0
'/10
'10
18
00
28.0
0
00
24.0
0
T
20.0
'SY,
(mg/ml)
16.0
0
0
12.0
0
0
8.0
4.c
4
0
-
0
0
0
0
0
0
0
0
1
~
:NDOTOXIN ENDOTOXll :NDOTOX II :NDOTOXIN
0.005mg
0.00Smg
0.5 mg
0.5mg
(3wkr.)
(6 wkr.)
(6 wkr.1
(3 wkr.)
CONTROL
Fig. 3.-The
7S-yl immunoglobulin response to various doses of E. coli endotoxin
in C3H mice. Each point represents the level in an individual mouse. Mean levels are
indicated by the horizontal bars.
.*o
0
O
h
==
A*& A A
0
ENDOTOX IN
0.005mg
(3wks. 1
** .
- 0= *
=
*-.
8
+
0
ENDOTOX I N
0.005mg
(6 wks.)
I
1
ENDOTOX IN
0.5 mg
(3w k s . )
ENDOTOXIN
0.5 mg
(6wks.)
Fig. 4.-Responses
of 7S-y2*, ~ S - YIgA,
~ ~ and
,
IgM to repeated administration of
E. coli endotoxin in the C3H mouse. Each point represents the level in an individual
mouse. Mean levels are indicated by the horizontal bars.
621
EXPERIMENTAL MURINE AMYLOID. 111
of Clearance of Colloidal Carbon at Various Times During the Development
of Endotoxin-Induced Amyloidosis in Two Strains of Mice
Table 4.-Rates
Half-Time of Clearance (TM) Performed at:
Mouse Group 0
Baseline
Control
G.P.
Endotoxin $
treated G.P.
Control
C3H/Hen
Endotoxin $
treated C3H/Hen
10.5+0.9t 13.1+1.6
10.5 f 0.9
2 Weeks
3 Weeks
4 Weeks
12.1+0.9
10.4f0.8
12.1f0.6
1 Week
5.2
+ 0.4
10.9
+ 1.3
+ 0.6
8.8 + 0.6
8.8
+ 1.0 13.2 -I- 1.0
10.4 + 1.7
4.2 + 0.3 Q
6 Weeks
11.3
+ 1.5
8.2 + 1.0
9.3
Each group consisted of a minimum of 6 animals.
(min.) f S. E.
$ E. coli endotoxin in dose of 0.5 mg. subcutaneously five times per week.
$ Significantly different from control: p
0.01 ( t test).
O
t Mean T 95
<
Table 5 . F e b r i l e Responses in Rabbits Following Intravenous Administration
of Casein and E. coli Endotoxin
Agent Administered
0
NaHCO,
8.5 Gm. % casein
0.85 Gm. % casein
8.5 Gm. % casein
E. coli endotoxin
E. coli endotoxins
8.5 Gm. % casein 11
Amount,
ml.
1.0
1.0
1.o
1.0 ml. daily for 6 days
0.5 pg.
0.5 p g .
1.0 ml.
Number of
Rabbits
Mean F.I.,f
cm.2
3
3
3
4
4
4
3
3.7
36.3
16.6
17.0
43.5
28.3
21.0
Mean 3 Hr. Temp.,*
C
0.13
1.63
0.40
0.18
1.95
1.28
0.33
* All agents given I.V.
t F.I. = 5 hr. fever index, cm.2 Fever index is area under a fever curve
in which 1 degree and
1hour equals 1 inch.
$ 3 hr. temp. degrees C above baseline 3 hours after agent injected.
$ Rabbits had received 1 ml., 8.5 Gm. %, casein daily (I.V.) for 7 days prior to challenge with
endotoxin.
I I Rabbits had received 7 daily I.V. injections of 3 pg. E. coli endotoxin.
Amyloid did not develop in these C3H
mice but was present in virtually all of the
G.P. mice after 10 injections (Table 1).
“‘Endotoxic” Properties of Casein
The febrile responses of rabbits to the
intravenous injection of casein are presented in Table 5. Every sample of casein
tested was pyrogenic. The febrile responses
to 8.5 Gm. per cent of casein were higher
than to 0.85 Gm. per cent of casein. All
rabbits which received a single injection of
the higher dose of casein had biphasic
fever curves typical of those seen after
administration of endotoxin. The daily administration of casein resulted in pyrogenic
refractoriness ( “tolerance”) to subsequent
challenge with casein characterized by a
lower febrile response and a monophasic
fever curve. Cross tolerance developed between E. coli endotoxin and casein; E.
coli-tolerant rabbits showed diminished responses to casein, and casein-tolerant rabbits had similarly decreased febrile
responses to E. coli endotoxin. Results of
studies shown in Table 5 were derived
with casein solutions prepared within 1-2
days of testing. Similar experiments were
622
BAR=
Table 6.-Effect
Time, hr.
0
0.5
2
4
6
24
ET AL.
of Casein on Blood Leukocyte Count in Rabbits'
WBC
Granulocytes t
9425
6200
4600
6375
8500
8325
3420
2873
1628
5252
6130
4391
Mononuclears t
6005
3327
2172
1123
2370
3934
'Rabbits received 1 ml. of 8.5 Cm. % casein I.V. at 0 time. The results are the mean values of 4
rabbits.
t Granulocytes =polymorphonuclear leukocytes and basophiles.
Mononuclears = lymphocytes and monocytes.
performed with stock casein solutions, and
the results obtained were essentially identical to those in Table 5.
An aliquot of a casein solution was autoclaved at 250°F for 2 hours and tested €or
pyrogenicity. The material was pyrogenic
prior to autoclaving but not afterward.
Such a change is characteristic of endotoxins .24
Further evidence that the casein was
contaminated with endotoxin is presented
in Table 6. Casein induced a prompt leukopenia, followed by a granulocytosis. Such
results are similar to those reported following the administration of bacterial endotoxins to rabbits.25
One week following the intravenous administration of 1 ml. of casein, all 4 rabbits
tested showed low titers of circulating antibody to either the somatic antigens of E.
coli or S. typhosa. Antibodies detectable by
the bentonite flocculation test are rarely
present prior to the administration of endotoxic polysaccharides.26Such cross-reacting
antibodies as appear after administration of
endotoxin are regarded as nonspecific and
do not serve as an indicator of the type
of endotoxin present in casein.
different microorganisms as well as a highly
purified endotoxin to induce amyloid suggests that amyloid induction may be a
general property of endotoxins and not due
to some unidentified contaminants in the
commercial preparations.
Amyloid was initially deposited in the
spleen and only later in the liver and kidney. There were progressively increasing
amounts of amyloid in these organs after
longer periods of endotoxin administration.
Differences were detected in the ease of
induction and amount of amyloid deposited
in the spleen among the six strains of mice
examined. For example, AKR mice had
more splenic amyloid after 10 injections
than did C3H mice after 30 injections. This
strain difference resembled that seen in a
previous study of spontaneous amyloid in
aged mice in which amyloid was more extensive and frequent in A strain mice than
in C3H mice.27
Strain differences were also observed in
the immunoglobulin responses to repeated
administration of endotoxin. Whereas C3H
mice had a marked increase in 7S-yl levels
with little increase in the other immunoglobulins, G.P. mice had increases in 7S-y1,
7S-yZB, and yy levels. Both the serum
DISCUSSION
7S-yl levels and the total immunoglobulin
This study demonstrates that the re- levels were lower in the G.P. than in the
peated administration of commercially C3H mice, however. It was noted that
available E. coli endotoxin rapidly and con- serum yA levels fell during treatment in
sistently induces amyloidosis in mice. The the G.P. mice. Possible mechanisms for a
ability of endotoxins derived from several fall in levels of some of the immunoglob-
EXPERIMENTAL MURINE AMYLOID. III
ulins with administration of endotoxin are
increased immunoglobulin catabolism or
some reciprocal regulation of the levels
among the various immunoglobulin classes.
It is apparent that amyloidosis was induced
in some mice with little or no increase in
serum immunoglobulin levels, and that two
strains of mice manifested different immunoglobulin responses following the same
antigenic stimulus.
Many of the features of amyloidosis induced by endotoxin simulated those noted
following administration of casein. Sites of
deposition and order of appearance in the
various organs in the two models were
similar. As with amyloid induced by
casein,13strain differences were present and
there was no direct relationship between
amyloidosis and an increase in serum immunoglobulin levels. Accelerated RES
phagocytosis of carbon followed by a return to normal has been described with
amyloidosis induced by c a ~ e i n ~and
~-*
was
~
duplicated in the present studies by administration of endotoxin. The impairment in
ribosome function during the early phase
of amyloid induction using casein30 has
also been produced by repeated injections
of endotoxin.3l A significant difference in
the two methods of amyloid induction is
the relative dose of inducing agent necessary. Forty to fifty milligrams of casein administered daily for several weeks are often
necessary to produce amy10id.l~In contrast,
a total dose of 0.125 mg. of E. coli endotoxin given over a 5 week period12 or 2.3
mg. given in 1 week as in this study have
induced amyloid.
In view of the many similarities between
these two models of induction of amyloid,
it was postulated that endotoxin contamination might account for casein-induced
amyloidosis. The presence of endotoxin in
casein was shown by the following: (1)A
typical biphasic febrile response in rabbits
was observed following administration of
casein; ( 2 ) a dose response relationship be-
623
tween amount of casein administered and
febrile reactivity was found; ( 3 ) febrile
tolerance with casein and cross tolerance
between both casein and endotoxin was
produced. Other evidence of the presence
of endotoxin in casein was: (4)heating in
the autoclave destroyed the pyrogenicity
of casein; (5) the hematological responses
in rabbits after casein administration were
the same as noted after endotoxin; and ( 6 )
antibodies to the somatic polysaccharides
of two different Enterobacteraceae were
detected after administration of casein.
Febrile responses in mice following repeated administration of casein were
studied over 40 years ago, and the results
were consistent with contamination by endotoxin.6
Every lot of casein we examined showed
endotoxin activity in the rabbit, but quantitation has not been possible. The type of
endotoxin( s ) present in casein is unknown.
It is probably not E. coli endotoxin since
the strain responses to E. coli endotoxin
and casein were not the same in the different strains of mice. The C57BL strain
developed little amyloid after E. coli endotoxin, but had the highest incidence in response to casein.13 Conversely, AKR mice
developed amyloidosis as readily as did
G.P. mice following administration of endotoxin but not after administration of
casein.13 Recently spores of Bacillus cereus
have been found as contaminants in the
type of casein we used.32 Possibly endotoxin from these organisms is responsible
for the amyloid reaction induced by casein.
The mechanism of amyloid formation
following administration of endotoxin is unknown, but our results suggest that immunoglobulins are not directly involved in
its pathogenesis. That genetic factors may
play a role is suggested by the different responses in the strains examined. Variations
in leukocyte responses, resistance, and
lethality following the administration of endotoxin have previously been observed in
624
BARTH ET AL.
diflerent strains of
It has been
Endotoxin may be implicated in other
suggested that chronic stimulation of cells forms of experimental amyloidosis. It is
in the reticuloendothelial system is involved ubiquitous and difficult to inactivate within the pathogenesis of amy1oidosis.l The out such vigorous procedures as prolonged
ability of endotoxin to induce amyloidosis autoclaving at high temperature^.^^ The
supports this hypothesis, since endotoxin literature contains many instances in which
rapidly localizes to these cells following contamination with endotoxin was later
parenteral a d m i n i s t r a t i ~ n ~and
~ - ~stimu~
shown to be responsible for a variety of
lates RES phagocytic function as ~ e l l . ~ biological
~ 9 ~ ~ effects.25 Materials other than
Chronic stimulation of lymphocytes is ap- casein that have been used to induce
parently inadequate, since prolonged ad- amyloid in animals include such diverse
ministration of phytohemagglutinin did not substances as live or killed bacterial
induce amyloid.3l
organisms, serum proteins, adjuvant, and
Studies thus far do not permit distinction ribonucleic acid. It is possible that endobetween endotoxin, a fraction of endotoxin,
toxin could be involved in these models.
or an endogenous product released followEndotoxin may be of pathogenetic iming endotoxin administration as a cause of
portance
in some forms of human amyloid,
amyloid formation. It is of interest that
particularly
those associated with chronic
loss of pyrogenicity by prolonged autoinfections.
The
persistent presence of endoclaving abolished the amyloidogenic properties of endotoxin. Efforts are underway toxin in chronically infected tissues in man
in our laboratory to identify the amyloido- may be analogous to the murine model degenic principal in endotoxin and to study scribed here.
its subcellular localization.
ACKNOWLEDGMENTS
The authors thank Mrs. Kay Gordon, Mr. S. B.
Ward, and Mr. J. B. Edelin for their expert tech-
nical assistance. We would also like to thank Dr.
John L. Decker and Dr. Leon Sokoloff (NIH) for
their advice and encouragement in the course of
these studies.
SUMMARYAND CONCLUSIONS
The repeated administration of commercially available E. co2i endotoxin to mice rapidly
and consistently induced amyloidosis. S. typhosa endotoxin and a highly purified S. mtrn’tidhs
endotoxin were also able to induce formation of amyloid. The development of amyloidosis
was related to the mouse strain used and in some mice was not accompanied by an increase
in serum immunoglobulin levels. Injections of endotoxin initially produced an acceleration in
reticuloendothelial clearance of carbon, whereas prolonged administration was associated with
a return to control levels.
Large amounts of endotoxin were found to be present in casein. There were many similarities
in the amyloidosis induced by administration of endotoxin and casein. It is suggested that
endotoxin may be responsible for the amyloidosis induced by repeated parenteral administration of casein, and that endotoxin may be implicated in other forms of experimental amyloidosis.
SUMMARIOIN INTERLINGUA
Le administration repetite de commercialmente disponibile endotoxina de Escherichia
coli a muses resultava in le rapide e consistente induction d e amyloidosis. Endotoxina
de Salmonella typhosa e u n altemente purificate endotoxina de Salmonella enteritidis
etiam se monstrava capace de inducer le formation de amyloide. Le disveloppamento
de amyloidosis esseva relationate con le racia murin usate, e in certe muses ill0 non
esseva accompaniate de un augment0 in l e nivellos sera1 de irnmunoglobulina. Injectiones de endotoxina produceva initialmente u n acceleration in le clearance reticulo-
625
EXPERIMENTAL MURINE AMYLOID. 111
endothelial de carbon, durante que le administration prolongate esseva associate con
un retorno a1 nivellos de controlo.
Grande quantitates de endotoxina esseva retrovate in caseina. Multe similitudes
esseva notate inter le amyloidosis inducite per le administration de endotoxina e ill0
inducite per caseina. Es suggestionate q u e endotoxina es possibilemente responsabile
pro le amyloidosis inducite per repetite administrationes parenteral de caseina e q u e
endotoxina ha possibilemente un rolo in le genese de altere formas de amyloidosis
experimental.
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