LIPOIDS AND IMMUNOLOGICAL REACTIONS It has been recognized that the antibacterial sera ...

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LIPOIDS AND IMMUNOLOGICAL REACTIONS
I. TaE RELATION ~1O PHOSPHOLIPINS TO THE TX't'E-SPEClFIC REAC-
TIONS FO ANTIPNEUMOCOCCUS HORSE DNA RABBIT SERA
Bx FRANK L. HORSFALL, JR., M.D., nNA KENNETH GOODNER, PH.D.
(From eht Hospital of The rellefekcoR Institute/or Medical )hcraeseR
(Received for publication, June ,31 1935) D
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It has been recognized that the antibacterial sera produced by the loa
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immunization of the horse and the rabbit with Type I pneumococci, d fro
although possessing type specificity, exhibit marked differences in m h
certain immunological properties. The distinctive characteristics of ttp://ru
the two sera have interested numerous investigators, but as yet there pre
is almost no evidence to indicate what constituents of the two sera ss.o
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are responsible for the differentiating qualifies. /je
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The precipitates formed by the inter-reaction of type specific antipneumococcus rticle
antibodies and specific capsular polysaccharide can be differentiated by their -pd
cipocsorcam characteristics, which are dependent upon the species by which the f/62/4
antibody is produced. For example, the precipitate with immune horse serum /48
5
is a granular opaque cake, while that produced in a similar manner with immune /11
8
rabbit serum is a thin translucent disc. Schiemann and Casper )1( have shown 05
7
that these immune precipitates form characteristic cipocsorcim pictures which 2/4
8
can be distinguished one from the other. Zinsser and Parker )2( have demon- 5.p
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strated that antipneumococcus horse serum and homologous pneumococci, or f b
the specific soluble substance, do not bind complement, although with anti- y gu
e
pneumococcus rabbit serum complement is fixed. Avery and Tillett )3( have st o
n
shown that antipneumococcus horse serum does not passively sensitize guinea 0
6
pigs to the homologous capsular polysaccharide, while after the injection of Fe
b
antipneumococcus rabbit serum these animals can be shocked anaphylactically rua
with the specific capsular polysaccharide. Etinger-Tulczynska )4( states that ry 2
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antipneumococcus rabbit serum constantly causes the phenomenon of gnulleuQ 23
with homologous pneumococci, while antipneumococcus horse serum does so
very irregularly. In a previous communication )5( it has been shown that anti-
pneumococcus horse serum must be used in definitely limited quantities to protect
mice against large numbers of homologous pneumococci. It has also been demon-
strated that when this optimum protective quantity of horse serum is exceeded,
protective action is progressively diminished until with large amounts of serum
584
486 LIPOIDS DNA LACIGOL0'NUMMI .SglOITCAER I
protection is completely abolished. This phenomenon has been termed the
"prozone." Antipneumococcus rabbit serum, on the other hand, does not exhibit
a similar result, but protects when comparable quantities are used. In confirma-
tion of the prozone phenomenon with immune horse serum, the protocols of other
workers also demonstrate the failure of greater than optimal amounts of horse
serum to protect (Yosioka (6), Felton (7) and Sobotka and Friedlgnder (8)). It
has been repeatedly shown by Felton )9( that the immune globulin fraction of
antipneumococcus horse serum is precipitated by tenfold dilution with distilled
water. However, the immune globulin fraction of antipneumococcus rabbit
serum remains in solution after similar water dilution.
The distinguishing characteristics of sera produced by the immuni- D
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zation of animals of different species with the same bacterial cells are n
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presented in Table I. de
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TABLE I h
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ehT gnMsiugnitsiD scitsiretcarahC of esroH dna tibbaR Type I succoconruenpitnA ://ru
areS pre
ss.o
Typec occus I anhtoirpsneeu mo- serum Tycpoec cus I raanbtbiiptn eumo- serum rg/jem
/a
with Precipitate *SSS ...................... cake Granular Translucent csid rticle
tnemelpmoC with fixation SSS .............. + -pd
noitazitisnes evissaP of pig guinea to SSS ..... t + f/62
Neufeld's gnulduQ nonemonehp ............. 4- + /4/4
Prozone nonemonehp ........................ + 85/1
Precipitation of fraction immune by 9 volumes 180
of water distilled ......................... + 57
2
/4
8
5
* SSS indicates acetyl polysaccharide of Type I Pneumococcus. .p
d
f b
y g
Although the fraction of antipneumococcus horse serum which con- ue
tains the type specific antibodies has been extensively studied, partic- st on
0
ularly with regard to the so called immune protein, relatively little 6 F
e
b
investigation has been directed toward the lipoids of the serum. ru
a
Felton and Kauffmann (10) have shown that the sera of horses immun- ry 2
0
2
ized with Type I or Type II Pneumococcus contain, on the average, 3
27 per cent more lipoid than does normal horse serum. They also
state that the extraction of lipoids from antipneumococcus horse
serum, by means of alcohol and ether in the cold, does not alter the
protective action of the serum.
The present investigation was undertaken with the purpose of defin-
FRANK L. HORSFALL~ JR.~ AND KENNETH GOODNER 487
ing the rSle of lipoids in the immunological reactions of antipneumo-
coccus serum. It was also desired to learn what relation lipoids might
bear to the distinctive differences between antipneumococcus sera
produced by the horse and the rabbit.
The simplest means by which the significance of the lipoid fraction
of antipneumococcus serum can be studied is to compare the immuno-
logical reactions of serum before and after the extraction of lipoids.
This method has been applied to other immune sera by previous in-
vestigators. Hardy and Gardiner (11) removed lipoids from diph-
theria antitoxin by means of alcohol and ether in the cold and showed
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o
that the lipoid-free globulin became soluble in saturated NaCI solu- wn
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a
tion. They state that the antitoxic properties of the serum were d
e
d
not changed by extraction. Hartley (12) extracted lipoids by a simi- fro
m
lar method from a large number of antisera, including rabbit anti- http
horse, rabbit anti-human, horse antityphoid, and horse diphtheria ://ru
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antitoxin. He was able to demonstrate a loss of precipitating ability ress.o
in the extracted antiprotein sera, but found that diphtheria anti- rg
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toxin and antityphoid sera still gave the usual characteristic reactions. m
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rticle
EXPERIMENTAL -p
d
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Metkods of Lipoid Extraction.--Two procedures have been used for the re- 2/4
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moval of lipoids from the immune sera. These were so designed as to permit of 8
5
thorough extraction by means of lipoid solvents and quantitative recovery of /11
8
0
undenatured protein. 57
2
(A) Alcokol and Etker Extraction.--The following method, somewhat modified /4
8
5
from that used by Hartley (12), was employed. Antipnenmococcus serum was .p
d
introduced drop by drop, with stirring, into ten volumes of absolute ethyl alcohol f by g
previously cooled to -10°C. The centrifuge tubes containing the alcohol and u
e
the precipitated serum were frequently and vigorously shaken, and the tempera- st o
n
ture was constantly kept below -5°C. Mter 6 hours extraction the precipitate 06
F
was collected by rapid centrifugation at a temperature below -2°C., the super- e
b
natant liquid was decanted, and the precipitate again extracted at -10°C. with rua
a quantity of absolute alcohol equal to that first used. Mter 21 hours extraction ry 20
2
the precipitate, collected in the same manner as before, was extracted with an- 3
hydrous ether for 10 hours at -10°C., and for a second time with anhydrous ether
for 10 hours at room temperature. The precipitate was packed by centrifugation,
the supernatant liquid decanted, and the precipitate freed of the last traces of
ether by vacuum distillation. The resulting dry residue was dissolved in a
quantity of saline equal to the original serum volume.
(B) Alcokol, Petroleum Etker and Etker Extraction.--Antipneumococcus serum
488 LIPOIDS AND IM~gUNOLOGICAL REACTIONS. I
treated identical manner a was in to that in described Method A, until the second
extraction with absolute alcohol had been completed. The precipitate was then
extracted with low boiling point (40-60°C.) petroleum ether for 42 hours at
-5°C. The precipitate was packed by centrifugation and extracted with an-
hydrous ether exactly as in Method .A
The precipitates recovered after these extractive procedures form
perfectly clear solutions in 0.9 per cent saline. In physical appear-
ance these solutions do not differ materially from the original sera.
It will be observed that certain definite temperatures were main-
tained at the various stages of the extractive methods. These are
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of importance for two reasons: (a) If the temperature of the solvents w
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is allowed to exceed certain limits the proteins are rapidly denatured, ad
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d
and (b) if the temperature is maintained below a certain point the fro
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extraction of lipoids is considerably retarded. h
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seitreporP of Type Extraction Serum after Horse I Antipneumococcus of ://rup
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Lipoids ss.o
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Type I antipneumococcus horse serum which had been extracted /jem
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with alcohol and ether in the cold by Method A was studied by means rticle
of the usual in vitro and in vivo tests to determine what changes, if -p
d
f/6
any, had been produced in the immunological characteristics of the 2
/4
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serum as a result of the extraction of lipoids. Quantitative deter- 8
5
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minations before and after extraction showed a loss of 92 per cent of 18
0
5
serum lipoids. In confirmation of the early work of Hoppe-Seyler 72
/4
(13), and that of numerous others, it was found that ether extraction 85
.p
d
alone removed only a relatively small proportion of total lipoids from f b
immune serum. It was also found that ether extraction, or alcohol y gu
e
and ether extraction, of dried serum was inadequate and removed only st o
n
0
a moderate proportion of total serum lipoids. The results of agglu- 6 F
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tination and precipitation tests with immune horse serum before and bru
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after the extraction of lipoids are presented in Table II. ry 2
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2
3
Although the unextracted immune horse serum, at a dilution of 1:80, agglu-
tinated a suspension of Type I pneumococci, no visible agglutination could be
detected when extracted serum was mixed with either living or heat-killed ho-
pneumococci. mologous At a dilution of 1:4,000,000 the capsular poly- specific
saccharide forms a precipitate defirdte with the untreated serum. However, the
extracted serum does not form any visible precipitate with the capsular poly-
saccharide in the concentrations used.
FRANK L. HORS~ALL, JR., AND KENNETH GOODNER 489
The results presented in Table II indicate that the lipoids of anti-
pneumococcus horse serum are essential to the visible manifestations
of the reactions of type specific agglutination and precipitation. This
interpretation of the results of the in vitro tests is tenable if it is pos-
sible to show that some specific immune property, as, for example,
protective action, remains unaltered in the extracted serum, since it
TABLE II
noitanitulggA of Type I ,iccocomuenP dna noitatipicerP witk Capsular Poly-
edirakccas yb Type I succocomuenpitnA esroH Serum, erofeb dna after -cartxE
of tion sdiopiL D
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Type I antlpneumococcus ad
horse serum ed
Serum dihtion extraction Before Lot 4 extraction (Method A) Lot After 4 (A.E.) from http://ru
p
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noitanitulggA fo I Type Pneumococcus 0012::11 ++++++++* ' -- t ss.org
o4:1 +++ - /jem
o 6018::11 + + + /article
-p
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f/6
polysac- Acetyl charide noitulid 2/4/4
8
5
noifatipicer with Type I raluspac -casylop 000,01:1 ++++ -t /118
edifahc 000,04:1 ++++ 05
7
000,004:1 + + + + - 2/48
000,000,4:1 ++ 5.p
d
f b
* sgnidaeR after 2 hours at .C°73 and 81 hours at .C°0 y g
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t No ecnedive in although precipitation, or agglutination visible of these tubes est o
there saw a slight .ecnecselapo n
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is obvious that the antibody might have been destroyed completely b
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by the extractive procedure. The results of protection tests in mice ry 2
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with immune horse serum, before and after the extraction of lipoids, 23
are shown in Table III.
It will be observed that after the of extraction lipoids from the mice serum,
protected were against fatal with infection Type I .succoconruenP The protec-
tive titers of the before serum and in identical, were after extraction that 50.0 .cc
amounts in both instances protected against 01 9- .cc of culture.
490 SDIOP1L DNA LACIGOLONUMMI .SNOITCAER I
It is evident, therefore, that although the extraction of lipoids
caused a loss of type specific agglutinating and precipitating proper-
ties, the capacity of the extracted serum to protect mice against in-
fection remained entirely unaltered.
Perhaps the most significant peculiarity of antipneumococcus horse
serum is the prozone phenomenon, as evidenced by the failure of
greater than optimum amounts of serum to protect mice against fatal
infection with homologous pneumococcl. It is important to the def-
inition of the significance of the changes produced in horse serum
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TABLE III wn
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noitcetorP Tests with Type I Antitmeumococcus Horse Serum before dna after d
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Extraction of Lipoids fro
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Type I antlpneumococcus horse serum Virulence control ttp://ru
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Type I pneumo- re
culture coccus Before Lot extraction 4 (Method After A) extraction Lot 4 (A.E.) No mures ss.org
0.~0 cc, 0.0S cc. 0.20 cc. 0.05 co. /jem
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~-01 S s s s m rticle-p
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10-8 S s s s f/6
2
01 ~ S s S s m /4/4
~-01 S s s s 85
6-01 S S D44 /11
8
r-01 D64 05
7
8-01 D 64 2/4
8
5
.p
S ~ survival. df b
D = death at indicated number of hours. y g
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after the extraction of lipoids, to determine what effect their absence n 0
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might have upon this protective prozone. In Table IV the results Fe
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of a protection test designed to illustrate the prozone are given. a
ry 2
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2
The optimum protective quantity of antipneumococcus horse serum was not 3
changed by the extraction of lipoids by Method A, as is evidenced by the uniform
protection resulting when 1.0 cc. of either untreated or extracted serum was in-
jected with 0.05 cc. of culture. The prozone phenomenon is as readily demon-
strable in the extracted as in the unextracted serum, since in both instances with
greater than optimal amounts of serum the protective action rapidly decreases
until with 0.4 cc. of serum protection is completely lost.
I~RANK .L HORSFALL, JR., AND KENNETH OOOI)NEI~ 491
From the results of protection tests shown in Table IV, it is appar-
ent that after extraction of lipoids from antipneumococcus horse
serum the prozone phenomenon was still demonstrable as manifested
by the failure of larger amounts of the extracted serum to protect
mice against fatal infection with homologous pneumococci. Under
the conditions of this experiment it may be stated that the extraction
of lipoids from immune horse serum does not alter the prozoning
action of the serum when used in excess.
The results of the preceding experiments may be summarized by
stating that although the removal of lipoids from antipneumococcus
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horse serumr esulted in a loss of agglutinating and precipitating prop- wn
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TABLE IV d fro
noitcetorP Tests witk Increasing Amounts of eht Same Lot of Type I Antipneumo- m h
succoc Horse Serum erofeb dna enozorP after nonemonehP Extraction of Lipoids, Illustrating eht ttp://rup
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I Type antipneumococcus horse serum rg
tnuomA fo muresitna /jem
Before Lot extraction 4 (Method After A) extraction Lot 4 (A.E.) /article
6. -pd
0.4 D 22 D 32 D22 D22 f/62
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0.2 D 24 D 24 D 22 S /4
8
0.1 S S S S 5/1
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0.05 S S S S 80
5
7
2
All mice received 0.05 cc. of Type I pneumococcus culture, of virulence such /48
5
that 01 -8 cc. killed mice in 48 hours. .p
d
f b
erties in vitro, the protective action of the extracted serum in vivo y gu
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remained quantitatively unchanged. The clue to the nature of these st o
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paradoxical results was found in studies of the mechanism of protec- 6
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tion in mice which had been injected with culture and extracted bru
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serum. Although in vitro no agglutination had been visible, it was ry 2
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found that, when the extracted serum was injected together with 23
pneumococci into the peritoneum of the mouse, agglutinatioonc curred
in vivo in a manner comparable to that in controls in which unex-
tracted serum had been used.
Inasmuch as extracted horse serum protected mice against fatal
infection equally as well as untreated horse serum, it seemed not
492 LIPOIDS AND IMMUI~OLOGICAL REACTIONS. I
impossible that upon injection into mice the extracted serum might
have been restored to a state similar to that existing before the extrac-
tion of lipoids. This hypothesis predicates the presence of the nec-
essary lipoid substances within the mouse peritoneum, and also the
ability of the extracted serum to become reassociated with these
lipoids. If both these assumptions are correct, the extracted serum
should, after injection, regain its type specific agglutinating and pre-
cipitating capacities when withdrawn from the peritoneum and re-
tested in vitro.
To test this hypothesis, Type I antipneumococcus horse serum from D
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which the lipoids had been extracted was injected intraperitoneally n
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into mice, and after 30 minutes the peritoneal fluid was withdrawn by de
d
means of a capillary pipette. This fluid was found to be capable of fro
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agglutinating Type I pneumococci, and of precipitating with the http
specific capsular polysaccharide, although prior to injection the ex- ://ru
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tracted serum showed no visible evidence of these reactions. ress.o
This experiment indicates that although the extraction of lipoids rg
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from antipneumococcus horse serum causes the loss of certain in vitro /a
reactions, this change is not irreversible, and that under certain cir- rticle
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cumstances it is possible to restore the type specific agglutinating and df/6
2
precipitating properties to the extracted serum. /4
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8
With the purpose of determining what effect the extraction of 5/1
1
lipoids from antipneumococcus horse serum had upon certain other 80
5
7
distinctive qualifies of the unextracted serum, the extracted serum 2/4
8
5
was tested for capacity to fix complement, and also for the precipita- .p
d
bility of the immune globulin fraction upon water dilution. f by g
u
It was found that the immune horse serum, after extraction of e
st o
lipoids, did not fix complement with homologous pneumococci, or n
0
6
with the specific capsular polysaccharide, a property likewise charac- F
e
b
teristic of unextracted serum. rua
On tenfold dilution with distilled water, it was evident that the ry 2
0
2
3
solubility of the globulin fraction of the extracted horse serum had
been markedly changed, for almost no precipitate formed.
When 0.5 cc. of extracted serum was diluted with 4.5 cc. of distilled water in
a microtip centrifuge tube, allowed to stand in the ice box overnight, and the
solution centrifuged for ~ hour, the packed precipitate amounted to but 0.001 .cc
When 5.0 .cc of unextracted horse antipneumococcus serum was carried through
FRANK L. HORSFALL, JR., AND KENNETH GOODNER 493
the identical procedure, the packed precipitate equalled 0.06 cc., or 60 times the
volume of the precipitate from extracted serum.
These results tend to show that the solubility of the immune glob-
ulin of antipneumococcus horse serum is considerably modified by
the lipoid constituents of the serum. Although a large proportion of
the immune globulin of untreated serum is precipitated by tenfold
dilution with water, almost none is precipitated by this means after
the removal of lipoids. Furthermore, the protective antibody of the
extracted serum remains almost quantitatively in the supernatant
D
liquid after tenfold water dilution. ow
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d
Absorption of evitcetorP Antibody from Extracted Type I e
d
succocomuenpitnA Horse Serum from
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The results of the preceding experiments have demonstrated that ://ru
the extraction of lipoids from Type I antlpneumococcus horse serum pre
results in an apparent loss of in vitro activity, as evidenced by the ss.o
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lack of specific precipitation and agglutination. However, since the /jem
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extracted serum protected mice as well as did unextracted serum, rticle
it became of interest to determine whether any binding of antibody -p
d
by the pneumococcus or its capsular polysaccharide occurred in vitro f/62
/4
even in the absence of the physical changes which usually denote /48
5
/1
antigen-antibody union. 1
8
0
5
7
2
In studying this phase of the problem, two separate types of absorption ex- /48
5
periments were performed. In the first, 4.0 cc. of antipneumococcus horse serum, .p
d
after the extraction of lipoids, was diluted with 6.0 cc. of saline and mixed with f by g
the living pneumococci centrifuged from 052 cc. of an 81 hour broth culture. ue
After incubation at 37°C. for 2 hours, and 81 hours in the ice chest, the mixture st o
n
was centrifuged for 1 hour. The supernatant fluid was again absorbed in a 06
F
similar way and finally was passed through a Berkefeld V filter. The second eb
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method differed from the first in that in this instance absorption was carried out a
with bacterial cells which had been heat-killed, and subsequently washed four ry 20
2
separate times with 60 cc. of saline. After each washing the pneumococci were 3
recovered by centrifugation for 1 hour. 2.0 cc. of extracted serum were mixed
with organisms prepared in this way from 051 cc. of broth culture of Type I
Pneumococcus, the mixture was incubated for 2 hours at 37°C. and kept in the ice
chest overnight. The supernatant fluid was pipetted off after centrifugation for
1 hour and again absorbed in a similar way. In both methods an equal volume
of unextracted serum was treated in an identical manner.
494
LIPOIDS AND IMMUNOLOGICAL REACTIONS. I
Following absorption with pneumococci by the above two methods,
protection tests were carried out with both the extracted and the
unextracted sera. The results of these experiments are presented in
Table V.
The results of protection tests with extracted horse serum, which
had been absorbed with pneumococci, are quite different from the
results of tests with unextracted serum similarly absorbed (Table V).
With unextracted serum, absorption removed all demonstrable pro-
tective antibody even when repeatedly washed heat-killed organisms
were used. With extracted serum, however, an appreciable amount
D
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TABLE V ad
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evitarapmoC Results of Protection Tests with Whole and Extracted Type I Anti- d fro
succocomuenp Serum, Horse after Absorption with Type I iccocomuenP m
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Type I succocomuenpltna serum horse 2.0( ).cc ://ru
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erutlucSUpneu- CcI oType conr extraction Be4 foLot re Lot (Method extraction After 4 ).E.A( A) erofeB Lot extraction 4 extraction After (Method A) Lot 4 ).E.A( ss.org/je
m
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Unabsorbed Li~lg Wash~ with Absorbed Type ~pneumococci dngI Washed rticle-pd
f/6
2
/4
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10-2 SSSD SSSS 85
l0 s SSSD SSSS DD DD DDDD DDDD /11
4-01 SS DD DD DDDD DDSS 805
SOl DDD DDD 72
DDSS DDDS /4
01 ~ DDD DDD DSSS SSSS 85
.p
7-01 DD DD DS SS df b
y g
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of protective antibody remained in an effective form in solution. st o
n
This is true even though the serum had been absorbed with a large 06
F
quantity of living pneumococci. It is apparent that if the antibody eb
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were bound in vitro by the Pneumococcus, it would be thrown out of ary 2
solution together with the Pneumococcus by centrifugation and 02
3
Berkefeld filtration. In the unextracted serum this occurred, and
protective action could no longer be demonstrated. In the serum
from which lipoids had been extracted, however, sufficient antibody
remained in solution to protect all mice against 10 -e cc. of culture,
and to protect some mice against as much as 10-* cc. These results