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A skin-sensitizing and thermolabile antibody in the mouse
Life Bciences Yol . 5 pp . 123-128, 1966 " Printed in Great Brita~n .
Pergamon Press Ltd.
A 3RIN-3EN3ITIZII~ AND THSRMOLABILB ANTIBODY IN THE MOU3E I .Idota and Junis M.Peixoto Department of Histology, Medical School, University of 3áo Paulo, 3áo Paulo, 3 .P ., Brazil and Division of Physiology, Instituto Oswaldo Crus, Rio de Janeiro, ®., Brazil (&eceiXed 10 June 1966 ; in final form 1 August 1966) A skin-fazing, heat labile and "mast
cell
sensitizing"
antibody was reoently found in the rat by one of us
(1, 2, 3, 4) .
The fact that the mouse stands phylogenetically close added showing
to
the that
peritoneal
recent
ability
the
mast
observation
cell
disappears after a
to
arises
of
early
histamine
from
mouse
sensitization
after
and
short time lead us to investigate the presence
experiments showing
skin-sensitizing
the rat
Prouvost-Danon et al . (5)
release
of a similar antibody in the mouse . results
of
to
and
The present paper reports the that
thermolabile
the
mouse
antibody
produces
that
is
a
here
provisionally called "mouse early antibody" . Material and Methods Animals . Female, adult
albino
mice
xeighing 18-25 g were
used
throughout . Antigens . Crydtallane bovine
serum
albumin
albumin (Ea) two tames crystallised (Mann
(B3A)
Research
and
hen
egg
laboratories,
New York, U.S .A .) were used as antigens . Method of immunization . The animals
were
subcutaneous
B3A
infection
of
Sa
or
immunized either with a adsorbed
hydroxide or xith an intraperitoneal infection 123
of
on
aluminium
either antigen
1724
TR~IOLàBILE ARTIHODY
along xith a suspension organisms .
of
Vol . 5, Ao . 18
Hordetella Dertussis vaccine, phase I
Bach animal received
and xere bled 10 days later .
a single dose of 50 ~g Ea or HSA
Technique for obtaining antiserum . Small obtained from sensitized animals by
samples
puncture
of
oP
blood
the
ophthalmic
venous plexus according to the technique described by Paaaud (6) and xere
alloxed
to
clot .
Serum
xas
were
Halpern and separated
by
centrifugation in a refrigerated centrifuge and kept at 24C . Passive cutaneous anaphylaxis (PCA) . PCA reactions by the technique of Ovary (7) . xith
an
electric
irritation of the
hair skin .
or
care txo
being
taken
intradermal
fine
short
bevel
hypodermic
needle .
afterxarde or at various times later, the intravenously xith 0.5 ml of a blue
in
saline
(0 .85 per cent NaCl)
Twenty to 30 minutes after
antigen
to
avoid
skin
xith
Seventy-two
animals
0.25 per cent
shaved
injections of
antiserum xere then made on each side of the dorsal very
produced
The backs of the mice xere
clipper, One
xere
xere
solution
containing 1 mg
infection, the
killed xith an overdóse oP ether, the akin was
hours
injected of
Evans
antigen .
animals
inverted
a
were
and
the
Yesion diameter xas measured on the inner surface of the skin with a transparent ruler. BPfect of heatin g. In order to find out whether thermolabile experiments were performed in xhich antiserum to induce PCA xas studied after
the
antibody xas
the
heating
ability
the
of
antiserum
for 30 minutes in a xater-bath kept at 564C . Results and Discussion To follox the appearance and persistence oP mouse antibody, mice xere infected xith a single dose
of
at various times afterxards and their serum antibody
early
antigen, bled detected
by
Yol . 5, No . 18 PCA reaction . that mouse
TAERYOLABILE ARTIBODY
1~25
Inspection of the results shown in Table I
early
antibody
is
a very
transient
suggest
antibody
that
appears on the 10th day folloxing immunization and by the 20th no longer detectable . to be similar
to
In this respect mouse early antibody
is
seems
rat "mast cell aeneitizing" antibody (2) and to
rabbit PCA antibody recently described by Zvaifler and Hacker (8) . T A B L S
I
Appearance and Persistence of Mouse
Early Antibody in
Sera of Immunized Animale* Mouse
Days after immunization 7
10
15
20
26
PCA (mm) 1
0~
10
12
0
0
2
0
12
10
0
0
3
0
15
15
0
0
0
6
6
0
0
5
0
8
12
0
0
6
0
15
8
0
0
* The animals were injected with 50 H3A adsorbed on aluminium hydroxide and were bled hg by the first time 7 days later . Antibody was detected by PCA using a 72 hours latent period . Each figure ie the mean of 4 animals . Other similarity
of
mouse
early
antibody
with
rat "mast cell
aeneitizing" antibody is its property of remaining for a long time in homologous skin after passive transfer .
Thus
injected with antiserum
early
reaction could still Table II .
be
containing
mouse
induced 10 days later
when as
mice
were
antibody a is
This fact ie in contrast with the reeulta of
shown
PCA in
Munoz and
126
THEEIQOLABILE ANTIBODY
Anaeker
(9)
xho
xorking
xith
antibody
Yol . 5, No . 18 present
hyperimmunized mice observed th at mouse antibody rapidly from the passively sensitized skin . T A $ L B
in
serum
disappears
Other
of
very
property
that
I I
Persistence of Mouse Early Antibody in Homologous Skin * Days betxeen antibody and antigen infection 3
~
5
6
7
8
9
l0
3
3
PCA 2 4
3
4
4
4
4
4
3
3
4
4
0
4
3
0
3
4
_
~
_
_
3
2
O
~
3
3
3
2
i
4
3
3
2
2
1
1
~
s Animals in~eeted xith 0.025 ml mouse antiserum obtained 10 days after a single injection of fa plus ~ertuesie . Mach figure represents a different mouse . React onia are graded according to their diameter : 0 = no reaction ; 1 = ~F mm ; 2 = ~- 6 mm ; 3 = 6-10 mm ; ~ > 10 mm ; - = not done . distinguish mouse early antibody from antibodies present in of hyperimmuni:ed mice is its thermolability . Table III heating
of
antisera
completely destroys the ability
containing of
these
As it is mouse
sera
serum
shoxn
in
early antibody to
induce a PCA
reaction 72 hours later. Hox heating affects the antibody molecule is not knoxn .
It may denature the entire molecule, or change i t s
ability to combine xith antigen or its capacity to become fined to the tissues . Mouse immunoglobulins have been xell studied by Fahey et al .(10, 11, 12) xho shoxed that mice produce four major classes of immunoglobulins . TKO major classes of 7 3 ~ -globulin, i.e .,the
Yol . 5, No . 18
1727
THERIIOLABILE ANTIBODY
7 S ~1-globulin and 7 S ~ 2-globulin as xell as 19 S macroglobulins and
~lA (~32A)-globulina . Furthermore the 7 S ~2-globulina
been
shorn
to
include
i~2b-globulina .
txo
subclasses, the
All five immunoglobulina
have
~ 2a-globulina
have
and
antibody activity
but only mouse ~ 1-globulina xere found capable of sensitizing the mouse for
PCA (13) .
Mouse
immunoglobulin
detected in serum or peritoneal fluid animals .
fractions
taken
from
have
been
hyperimmuaiaed
Such fluids probably do not contain mouse early antibody
as no heat labile or akin-sensitizing antibody xas described among their
immunoglobulin
characterize the
fractions .
Preliminary
attempts
~`-globulin fraction responsible
early antibody activity have not been euceaeful serum antibody concentration
for due
the
mouse
the
to
Taken together the present
to lox
results
suggest that mouse early antibody may be very similar to rat "mast cell eensitizing° antibody .
Hoxever they
T A B L E
may
not
be
identical
I I I
Effect of~Heating (56PC) on Ability of Atouse Bnrly Antibody to Induce PCA* Antiserum Control serum
Heated serum
anti-sa 1
15
0
Anti-Ba 2
18
0
3
10
0
12
0
Anti-Ba
Anti-SSA
* Animals injected xith 0 .010 ml mouse antiserum obtained 10 days after a single infection of Ba plus B:pertuasie . PCA reactions induced 72 hours after sen~a i~z~ion . Bach figure is the mean of 3 animals .
128
Yol . 5, No . 18
TBEEIIOLABILE ANTIBODY
since observation of mouse maat cella after PCA reactions have not revealed the characteristic mast cell disruption observed tissues after PCA .
Further experimental
xork
is
in
rat
needed on this
point . Acknowledgements The Authors Oreenford, Division,
Middlesex,
are
grateful
&igland
Purl River, N. T.,
to
(ilaao Iatioratories Ltd.,
and
to
U.S .A .
for
Lederle a
laboratories
generous
gift
of
pertuasis vaccine . References 1 . I .Mota, Fed .Proc . ~, 559 (1963) . 2 . I .Mota, Life Sciences l, 465 (1963) . 3. I .Mota, Life Sciences i, 917 (1963) . 4 . I .Mota, Immunology ~, 681 (1961F) . 5 . A.Provoust-Danon, M.Silva Lima
and
M.Queiroz Javierre,
Life
Sciences ~, 289 (1966) . 6. B.N .Halpern and A .Pacaud, C.R .Soc .Biol. 1~4~, 1465 (1951) . 7 . z. Ovary, J .Immunol . 81, 355 (1958) . 8 . N.Zvaifler and E.L .Hecker, Fed .Proc . 2~, 250 (1965) . 9 . J .Munoz and R.L .Anacker, J.Immuno]. . ~, 640 (1959) . 10 . J .L .Fahey,
J.Wunderlich
and
R.Mishell,
J.~cD .Msd . ~, 223
(1964) . 11 . J .L .Fahey, J .Wunderlich
and
R .Mishell,
J .~cp .Med . ~, 243
(1964) . 12 . Z .Ovary, W .F .Barth and J.L .Fahey, J.Immunol . ~4, 410 (1965) . 13 . R .S .Nussenzweig, C .Merryman and B.Benacerraf, 315 (1964) .
J~Bxp .Med . ~,
Pergamon Press Ltd.
A 3RIN-3EN3ITIZII~ AND THSRMOLABILB ANTIBODY IN THE MOU3E I .Idota and Junis M.Peixoto Department of Histology, Medical School, University of 3áo Paulo, 3áo Paulo, 3 .P ., Brazil and Division of Physiology, Instituto Oswaldo Crus, Rio de Janeiro, ®., Brazil (&eceiXed 10 June 1966 ; in final form 1 August 1966) A skin-fazing, heat labile and "mast
cell
sensitizing"
antibody was reoently found in the rat by one of us
(1, 2, 3, 4) .
The fact that the mouse stands phylogenetically close added showing
to
the that
peritoneal
recent
ability
the
mast
observation
cell
disappears after a
to
arises
of
early
histamine
from
mouse
sensitization
after
and
short time lead us to investigate the presence
experiments showing
skin-sensitizing
the rat
Prouvost-Danon et al . (5)
release
of a similar antibody in the mouse . results
of
to
and
The present paper reports the that
thermolabile
the
mouse
antibody
produces
that
is
a
here
provisionally called "mouse early antibody" . Material and Methods Animals . Female, adult
albino
mice
xeighing 18-25 g were
used
throughout . Antigens . Crydtallane bovine
serum
albumin
albumin (Ea) two tames crystallised (Mann
(B3A)
Research
and
hen
egg
laboratories,
New York, U.S .A .) were used as antigens . Method of immunization . The animals
were
subcutaneous
B3A
infection
of
Sa
or
immunized either with a adsorbed
hydroxide or xith an intraperitoneal infection 123
of
on
aluminium
either antigen
1724
TR~IOLàBILE ARTIHODY
along xith a suspension organisms .
of
Vol . 5, Ao . 18
Hordetella Dertussis vaccine, phase I
Bach animal received
and xere bled 10 days later .
a single dose of 50 ~g Ea or HSA
Technique for obtaining antiserum . Small obtained from sensitized animals by
samples
puncture
of
oP
blood
the
ophthalmic
venous plexus according to the technique described by Paaaud (6) and xere
alloxed
to
clot .
Serum
xas
were
Halpern and separated
by
centrifugation in a refrigerated centrifuge and kept at 24C . Passive cutaneous anaphylaxis (PCA) . PCA reactions by the technique of Ovary (7) . xith
an
electric
irritation of the
hair skin .
or
care txo
being
taken
intradermal
fine
short
bevel
hypodermic
needle .
afterxarde or at various times later, the intravenously xith 0.5 ml of a blue
in
saline
(0 .85 per cent NaCl)
Twenty to 30 minutes after
antigen
to
avoid
skin
xith
Seventy-two
animals
0.25 per cent
shaved
injections of
antiserum xere then made on each side of the dorsal very
produced
The backs of the mice xere
clipper, One
xere
xere
solution
containing 1 mg
infection, the
killed xith an overdóse oP ether, the akin was
hours
injected of
Evans
antigen .
animals
inverted
a
were
and
the
Yesion diameter xas measured on the inner surface of the skin with a transparent ruler. BPfect of heatin g. In order to find out whether thermolabile experiments were performed in xhich antiserum to induce PCA xas studied after
the
antibody xas
the
heating
ability
the
of
antiserum
for 30 minutes in a xater-bath kept at 564C . Results and Discussion To follox the appearance and persistence oP mouse antibody, mice xere infected xith a single dose
of
at various times afterxards and their serum antibody
early
antigen, bled detected
by
Yol . 5, No . 18 PCA reaction . that mouse
TAERYOLABILE ARTIBODY
1~25
Inspection of the results shown in Table I
early
antibody
is
a very
transient
suggest
antibody
that
appears on the 10th day folloxing immunization and by the 20th no longer detectable . to be similar
to
In this respect mouse early antibody
is
seems
rat "mast cell aeneitizing" antibody (2) and to
rabbit PCA antibody recently described by Zvaifler and Hacker (8) . T A B L S
I
Appearance and Persistence of Mouse
Early Antibody in
Sera of Immunized Animale* Mouse
Days after immunization 7
10
15
20
26
PCA (mm) 1
0~
10
12
0
0
2
0
12
10
0
0
3
0
15
15
0
0
0
6
6
0
0
5
0
8
12
0
0
6
0
15
8
0
0
* The animals were injected with 50 H3A adsorbed on aluminium hydroxide and were bled hg by the first time 7 days later . Antibody was detected by PCA using a 72 hours latent period . Each figure ie the mean of 4 animals . Other similarity
of
mouse
early
antibody
with
rat "mast cell
aeneitizing" antibody is its property of remaining for a long time in homologous skin after passive transfer .
Thus
injected with antiserum
early
reaction could still Table II .
be
containing
mouse
induced 10 days later
when as
mice
were
antibody a is
This fact ie in contrast with the reeulta of
shown
PCA in
Munoz and
126
THEEIQOLABILE ANTIBODY
Anaeker
(9)
xho
xorking
xith
antibody
Yol . 5, No . 18 present
hyperimmunized mice observed th at mouse antibody rapidly from the passively sensitized skin . T A $ L B
in
serum
disappears
Other
of
very
property
that
I I
Persistence of Mouse Early Antibody in Homologous Skin * Days betxeen antibody and antigen infection 3
~
5
6
7
8
9
l0
3
3
PCA 2 4
3
4
4
4
4
4
3
3
4
4
0
4
3
0
3
4
_
~
_
_
3
2
O
~
3
3
3
2
i
4
3
3
2
2
1
1
~
s Animals in~eeted xith 0.025 ml mouse antiserum obtained 10 days after a single injection of fa plus ~ertuesie . Mach figure represents a different mouse . React onia are graded according to their diameter : 0 = no reaction ; 1 = ~F mm ; 2 = ~- 6 mm ; 3 = 6-10 mm ; ~ > 10 mm ; - = not done . distinguish mouse early antibody from antibodies present in of hyperimmuni:ed mice is its thermolability . Table III heating
of
antisera
completely destroys the ability
containing of
these
As it is mouse
sera
serum
shoxn
in
early antibody to
induce a PCA
reaction 72 hours later. Hox heating affects the antibody molecule is not knoxn .
It may denature the entire molecule, or change i t s
ability to combine xith antigen or its capacity to become fined to the tissues . Mouse immunoglobulins have been xell studied by Fahey et al .(10, 11, 12) xho shoxed that mice produce four major classes of immunoglobulins . TKO major classes of 7 3 ~ -globulin, i.e .,the
Yol . 5, No . 18
1727
THERIIOLABILE ANTIBODY
7 S ~1-globulin and 7 S ~ 2-globulin as xell as 19 S macroglobulins and
~lA (~32A)-globulina . Furthermore the 7 S ~2-globulina
been
shorn
to
include
i~2b-globulina .
txo
subclasses, the
All five immunoglobulina
have
~ 2a-globulina
have
and
antibody activity
but only mouse ~ 1-globulina xere found capable of sensitizing the mouse for
PCA (13) .
Mouse
immunoglobulin
detected in serum or peritoneal fluid animals .
fractions
taken
from
have
been
hyperimmuaiaed
Such fluids probably do not contain mouse early antibody
as no heat labile or akin-sensitizing antibody xas described among their
immunoglobulin
characterize the
fractions .
Preliminary
attempts
~`-globulin fraction responsible
early antibody activity have not been euceaeful serum antibody concentration
for due
the
mouse
the
to
Taken together the present
to lox
results
suggest that mouse early antibody may be very similar to rat "mast cell eensitizing° antibody .
Hoxever they
T A B L E
may
not
be
identical
I I I
Effect of~Heating (56PC) on Ability of Atouse Bnrly Antibody to Induce PCA* Antiserum Control serum
Heated serum
anti-sa 1
15
0
Anti-Ba 2
18
0
3
10
0
12
0
Anti-Ba
Anti-SSA
* Animals injected xith 0 .010 ml mouse antiserum obtained 10 days after a single infection of Ba plus B:pertuasie . PCA reactions induced 72 hours after sen~a i~z~ion . Bach figure is the mean of 3 animals .
128
Yol . 5, No . 18
TBEEIIOLABILE ANTIBODY
since observation of mouse maat cella after PCA reactions have not revealed the characteristic mast cell disruption observed tissues after PCA .
Further experimental
xork
is
in
rat
needed on this
point . Acknowledgements The Authors Oreenford, Division,
Middlesex,
are
grateful
&igland
Purl River, N. T.,
to
(ilaao Iatioratories Ltd.,
and
to
U.S .A .
for
Lederle a
laboratories
generous
gift
of
pertuasis vaccine . References 1 . I .Mota, Fed .Proc . ~, 559 (1963) . 2 . I .Mota, Life Sciences l, 465 (1963) . 3. I .Mota, Life Sciences i, 917 (1963) . 4 . I .Mota, Immunology ~, 681 (1961F) . 5 . A.Provoust-Danon, M.Silva Lima
and
M.Queiroz Javierre,
Life
Sciences ~, 289 (1966) . 6. B.N .Halpern and A .Pacaud, C.R .Soc .Biol. 1~4~, 1465 (1951) . 7 . z. Ovary, J .Immunol . 81, 355 (1958) . 8 . N.Zvaifler and E.L .Hecker, Fed .Proc . 2~, 250 (1965) . 9 . J .Munoz and R.L .Anacker, J.Immuno]. . ~, 640 (1959) . 10 . J .L .Fahey,
J.Wunderlich
and
R.Mishell,
J.~cD .Msd . ~, 223
(1964) . 11 . J .L .Fahey, J .Wunderlich
and
R .Mishell,
J .~cp .Med . ~, 243
(1964) . 12 . Z .Ovary, W .F .Barth and J.L .Fahey, J.Immunol . ~4, 410 (1965) . 13 . R .S .Nussenzweig, C .Merryman and B.Benacerraf, 315 (1964) .
J~Bxp .Med . ~,