Published April 1, 1978
EFFECT OF COLCHICINE ON THE ANTIBODY R E S P O N S E
I. Enhancement of Antibody Formation in Mice*
BY P A N G
N. S H E K $ AND A L B E R T
H. C O O N S §
(From the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115)
* Supported by grant 20811 from the National Institutes of Health.
Research Fellow of the American Heart Association. Present address: Connaught Laboratories, Willowdale, Ontario M 2 N 5T8, Canada.
§ Career Investigator of the American Heart Association.
iAbbreviations used in this paper: BSS, balanced salt solution;CC, colchicine;DT, diphtheria
toxoid; HGG, human g a m m a globulin; KLH, keyhole limpet hemocyanin; LCC, lumicolchicine;
LDso, mean lethal dose; PFC, plaque-forming cells;SRBC, sheep erythrocytes; TKB, TNP-KLHbentonite; TNP, 2,4,6-trinitrophenyl;VB, vinblastine.
J. ExP. MZD. © The Rockefeller University Press • 0022-1007/78/0401-121351.00
1213
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In 1954, some experiments were carried out in this laboratory on the effect ofcolchicine
(CC) 1 on antibody formation in rabbits. At that time, we had found that antibody
formation was associated with rapid and extensive cell divisions in the plasma cell and
its immediate precursors which had just been associated with antibody synthesis (1). We
thought, therefore, that the injection of CC would inhibit antibody formation, and
Tanaka and Coons carried out some experiments to investigate this proposition. Much to
our surprise, CC enhanced the antibody response in rabbits by a factor of --- 8. The results
of these experiments are simple to relate. We administered antigen to naive or primed
rabbits with or without CC, and measured the antibody response in the serum. The
enhancing effect was found to depend on the administration of CC on the same day as
the antigen. It was ineffective when given 2 days before or 2 days after the injection of
antigen. Indeed, it had a slightly depressive effect when given 2 days after the antigen,
and an even greater effect when given on day 4 after antigen administration. But given
simultaneously with the antigen, it had an increasingly pronounced effect as the dose
was increased from 0.5 to 2 mg/kg. The latter dose killed three of the four rabbits tested,
but the survivor had an antibody titer on the 8th day of his response 50 times higher
than the control rabbits which received no CC. These facts in the rabbit were true for
both the primary and secondary antibody response. At that time, we were at a loss to
explain these findings and published them only in an abbreviated form (2, 3).
When Gershon discovered the existence of the suppressor cell in 1970 (4), it seemed
possible that the effect of CC could be explained by the elimination of some or all of the
suppressor cells which are evidently stimulated to arise during every antibody response.
It is noteworthy that in 1952, Taliaferro et al. (5) investigated the effect of Xirradiation (total body radiation with 600 or 700 rods) on hemolysin production against
sheep erythrocytes in the rabbit. They found that peak titers were reached when the
antigen was administered from 6 h to 10 min before irradiation took place. 10 years later,
Dixon and McConahey (6) also investigated the effects of whole body radiation on the
antibody response to bovine gammaglobulin in the rabbit. They found that the peak
antibody titer to a primary stimulus was about four times that of the control value and
appeared when the irradiation was carried out 2.5 days aider the injection of antigen,
although there was also a smaller elevation when the irradiation was carried out 1 day
after an antigen injection.
Published April 1, 1978
1214
ENHANCEMENT
OF THE ANTIBODY RESPONSE BY COLCHICINE
In view of our early findings with CC and the effects of X-ray and endotoxin,
we decided to test the w o r k i n g hypothesis t h a t these m a t e r i a l s killed suppressor
cells. To do this, it was necessary to c a r r y out experiments with a n i m a l s in
which cell transfers could be carried out, n a m e l y inbred mice. This paper
describes the conditions required for the effective e n h a n c e m e n t of the antibody
response by CC in mice. O u r results indicate t h a t CC is effective in p r o m o t i n g
the antibody response of mice to protein antigens, e.g. d i p h t h e r i a toxoid and
h u m a n g a m m a globulin, as well as to a hapten, 2,4,6-trinitrophenyl (TNP). For
m a x i m a l e n h a n c e m e n t , the d r u g m u s t be a d m i n i s t e r e d s i m u l t a n e o u s l y with the
antigen. In an a c c o m p a n y i n g paper, we shall present evidence which demonstrates the action of CC on suppressor cells.
Materials and Methods
Male or female BALB/c mice, 8- to 16-wk-old, were obtained from The Jackson
Laboratory, Bar Harbor, Maine; from Charles River Breeding Laboratories, Wilmington, Mass.;
or from the West Seneca Laboratory, Health Research Inc., West Seneca, N. Y. Animals were
maintained in cages with free access to laboratory mouse chow and acidified, chlorinated water.
Antigens and Immunization. Purified diphtheria toxoid (DT) and human gamma globulin
(HGG) were supplied by the Massachusetts Department of Public Health, Division of Biologic
Laboratories, Boston, Mass. The hapten-carrier conjugate, trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH), was prepared from 2,4,6-trinitrobonzene sulfonic acid (Eastman Kodak Co.,
Rochester, N. Y.) and KLH (Calbiochem, San Diego, Calif.) according to the procedure described
by Rittenberg and Amkraut (12). The conjugate used in the present study had a ratio of 762 tool of
TNP/mol of KLH. In some experiments, TNP-KLH was absorbed on bentonite particles by the
method of Gallily and Garvey (13) for the purpose of immunization. All mice were immunized
intraperitoneally. The doses of antigens used for immunization are specified in the text. CC
(Sigma Chemical Co., St. Louis, Mo.) in physiological saline was administered intraperitoneally
to appropriate groups of animals at doses ranging from 0.25 to 1.5 mg/kg body weight, depending
upon the experimental protocol.
Passive Hemagglutination. Mice were bled from the ophthalmic venous plexus and the sara
obtained were inactivated at 56°C for 30 min. Circulating antibody levels were measured by the
hemagglutination of sheep erythrocytes, (SRBC; Colorado Serum Co., Denver, Colo.) covalently
coupled to the protein antigen by bis-diazobenzidine according to the method of Stavitsky and
Arquilla (14). For the detection of hapten-specific antibodies, the indicator SRBC were coupled to
Animals.
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Fourfold enhancement of diphtheria antitoxin in guinea pigs was described by
Greenberg and Fleming (7) when pertussis vaccine was injected subcutaneously at the
same time. Johnson et al. (8) described the enhancement of antibedy formation to protein
antigens by the injection of endotoxin from bacterial cell wall.
In 1963, White (9) published a review entitled ~Factors affecting the antibody response"
in which he described some unpublished data (Farthing and White, 1959) which
demonstrated the enhancing effect of CC (1 mg/kg) on both the primary and secondary
response of guinea pigs to diphtheria toxoid. They reported about a 10-fold difference in
both primary and secondary responses when CC was administered 6 h before or 6 h after
the antigen.
In 1976, Bash, Singer, and Waksman (10) reported that cyclophosphamide (20-50 rag/
kg) given on the day of antigen injection to donor rats abrogated the suppressive effect on
recipients of the T-cell fraction of rat spleen cells. Finally, Burchiel and Melmon (11)
reported that the mitotic inhibitors, CC, cytosine arabinoside, and hydroxyurea, all
produced dose-dependent augmentation of the antibody response in cultures of murine
spleen cells.
These findings clearly predict the existence of a suppressor cell since both X-ray and
CC were known to kill dividing cells. The effect of endotoxin is not so clear, but perhaps
it interferes with cell division at a critical moment.
Published April 1, 1978
PANG
N.
S H E K A N D ALBERT
H . COONS
1215
Results
Primary Antibody Response. Mice injected intraperitoneally with 100 ~g of
TNP-KLH-bentonite (TKB) on day 0 gave a peak hapten-specific IgM PFC
response on days 6 and 7 (Fig. 1 A). The simultaneous administration of CC (1
mg/kg body weight) with 100 ~g of TKB to animals in the experimental group
increased the IgM PFC response by about twofold. The hapten-specific IgC PFC
response of CC-treated animals was also greater than that of control animals
(Fig. 1 B). Significant elevation in the circulating anti-TNP antibody titers was
observed in immunized animals treated with CC (Fig. 2). The enhanced
antibody level appeared as early as 7 days after immunization, and the
difference in antibody titer between the two groups lasted for at least 14 days.
The administration of CC to animals immunized with HGG also significantly
(P < .001) enhanced the serum antibody titer at 1 wk, but not 4 days, after
immunization (Fig. 3). DT was found to be a relatively poor antigen for the
induction of a primary response in mice even when CC was given.
Secondary Antibody Response. The immunization of mice with two injections of DT (20 Lf per injection) 20 days apart, gave a consistent response in
terms of the mean circulating antibody titer I0 days after the second injection of
antigen (Fig. 4). When colchicine was also administered to similarly immunized
animals at the time of priming (group II), at the time of challenge (group III), or
at both times (group IV), the subsequent antibody response was increased by
about 15-fold. The kinetics of the secondary response in mice which received
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T N P as described by Rittenberg and P r a t t {15). Antibody titers were expressed as the reciprocal of
t h e highest serum dilution which gave positive h e m a g g l u t i n a t i n g reactions.
Preparation of Cell Suspensions and Hemolytic Plaque Assay. Mice were sacrificed by cervical
dislocation. The spleens were harvested and gently teased with sharp forceps in balanced salt
solution (BSS; 16). The dispersed spleen cells were filtered through stainless steel screens and
were washed three times in BSS by centrifugation at 1,000 rpm for 10 min each time. The washed
spleen cells were assayed for antibody-forming cells by a modified method (16) of the J e r n e
hemolytic plaque technique (17). Indicator SRBC were prepared according to the method of Kapp
and I n g r a h a m (18) for HGG-SRBC and of Rittenberg and P r a t t (15) for TNP-SRBC. Briefly, the
assay of plaque-forming cells was performed as follows. Glass culture tubes (10 × 75 ram)
containing 0.3 ml of 0.7% agarose (L'Industrie Biologique Francaise S. A., Gennevilliers, France)
in BSS, 50 Izl of HGG-SRBC (7.5%) and 20 ~l of SRBC-absorbed 0.5% bovine serum albumin
solution were preincubated in a 41-42°C water-bath. 50 Izl of a spleen cell suspension containing
105-10 ~ viable nucleated cells were added to each tube. The mixture was gently but thoroughly
mixed and was then spread on a microscope glass slide previously coated with 0.1% agarose. After
the agarose had solidified, the slides were inverted, placed on plexiglass trays and incubated at
37°C in a humidified chamber. After a~ incubation period of 1.5-2.0 h, freshly reconstituted
guinea pig complement (Pel-Freez Farms, Inc., Rogers, Ark.), at 1:15 dilution, was flooded u n d e r
the slides. After an additional 1.5 h incubation, the n u m b e r of plaque-forming cells (PFC) on each
slide was enumerated under a low-power microscope. For the development of IgG PFC, a
previously determined optimal dilution of a polyvalent rabbit anti-mouse IgG a n t i s e r u m (kindly
supplied by Dr. Carl W. Pierce of the Jewish Hospital of St. Louis, St. Louis, Mo.) was incorporated in the agarose mixture. The n u m b e r of IgG PFC was calculated from the difference
between the n u m b e r of IgM PFC and the total n u m b e r of PFC developed with the anti-IgG
antiserum.
Preparation of Lumicolchicine. Lumicolchicine was prepared by the irradiation of a CC
solution in a quartz cuvet placed at 12 inches from a UV lamp (Osram High Pressure Mercury
Arc, HBO 200; Osram, Munich, W. Germany) for 30 min. The successful conversion of CC to predominantly beta- and gamma-lumicolchicines was monitored by the appearance of two isosbestic
points at 255 and 305 nm in t h e i r UV spectra, and by a decrease of absorbancy at 350 nm (19).
Published April 1, 1978
1216
ENHANCEMENT OF THE ANTIBODY RESPONSE BY COLCHICINE
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Fro. 1. Effect of CC on the kinetics of the TNP-spocific primary PFC response. BALB/c
mice were immunized intraperitoneally with 100 ~ g of TKB (©) or with 100/~g of TKB and
CC (1 mg/kg, Q); their spleens were harvested for PFC assay at various times after
immunization. Each point represents the mean -+ SE of the response of 4-16 animals. SC,
spleen cells.
two injections of DT and CC showed that the enhanced response occurred as
early as 5 days after the second immunization (Fig. 5). The difference in
antibody titers between CC-treated animals and control animals lasted for more
than 15 days. The effective CC dose for maximal enhancement was between 1.01.5 mg/kg body weight (Fig. 6). The drug was found to be lethal to injected
animals at doses above 1.5 mg/kg and the mean lethal dose (LDso) of CC for
BALB/c mice was 2.1 mg]kg (Fig. 7). Since a relatively large amount of purified
DT would have been needed for the sensitization of indicator SRBC in a
hemolytic plaque assay, the number of DT-specific antibody-forming cells was
not enumerated. Instead, the effect of CC on the PFC response to another
protein antigen (HGG), and to a hapten (TNP) was studied.
Mice previously primed with HGG and challenged 10 days later with the
same antigen gave a relatively low HGG-specific IgG PFC response (Fig. 8 A).
On the other hand, similarly immunized animals which were also given CC
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w0
Published April 1, 1978
PANG
N.
SHEK
AND
ANTI-TNP
8
ALBERT
PRIMARY
H.
COONS
1217
RESPONSE
< 0.002
< 0.003
I0
14
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t-.
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0
$4
t~
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x
o
2
DAYS
FIG. 2. CC-induced enhancement of serum anti-TNP hemagglutinating antibody levels.
Mice were injected intraperitoneallywith 100 ~g of T K B ([=-'~or with 100 ~g of T K B and
C C (1 mg/kg, ~ ) . Animals were bled on days 7, 10, and 14 after the injection.Each bar
represents the mean titer -+SE of 6-7 animals.
showed an IgG PFC response which was about three times as large. Since very
few, if any IgM PFC were detected by this system (20), we could only express
our HGG-specific PFC response by the number of indirect plaques obtained.
The increase in the number of antibody-forming cells was accompanied by
elevated circulating antibody levels which persisted for a longer period of time
(Fig. 8 B). In the case of the secondary PFC response to the TNP hapten, the
administration of CC to experimental animals at the time of priming enhanced
both the IgM and the IgG response (Fig. 9). The IgM PFC response of the CCtreated animals was twice as large (Fig. 9 A), and the IgC PFC response was
about four times as large as that of the control animals (Fig. 9 B).
Relationship between the Time of Administration of CC and the Enhancement of the Antibody Response. Different groups of animals were given CC at
times before (day -1), during (day 0), or after (day + 1 and day +2) the injection
of TNP-KLH. Animals in the control groups were injected with the antigen
alone. The primed animals were challenged with TNP-KLH on day 14. 3 days
after the second injection, the spleens of the immunized animals were assayed
for their secondary hapten-specific PFC response. Maximal enhancement of the
PFC response was observed when CC was administered to animals on the same
day as the injection of antigen (Fig. 10 A). Although minimal enhancement was
observed when the drug was given to animals 1 day before immunization, no
elevation in the PFC response was noted when the drug was administered to
animals 1 or 2 days after immunization. Similar results were observed in the
primary response; the drug was most effective when given to animals on the
same day as antigen injection (Fig. 10 B).
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7
Published April 1, 1978
1218
ENHANCEMENT
OF THE ANTIBODY RESPONSE BY C O L C H I C I N E
ANTI-HGG
PRIMARY
n.s.
RESPONSE
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(11)
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OT.I.CC
SECONDARY
RESPONSE
TITER
IO
t5
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I
DT
< 0-001
DT
(111]
DT
DT+CC
(IV)
DT.FCC
DT+CC
I
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<o.oo,
< 0.001
FIG. 4. CC-mediated enhancement of the secondary antibody response of mice immunized
with DT. Control animals in group I were primed with 20 Lf of D T on day -20 and
challenged with a similar dose of antigen on day 0. Animals in the experimental groups
were immunized in a similar fashion except that CC, at a dose of I mg/kg body weight, was
also administered at the time of priming (group If),at the time of challenge (group III),or
at both times (group IV). All animals were bled 10 days after the second injection. Each bar
represents the mean titer ± SE of 8 animals.
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DAY 4
DAY 7
FIO. 3. Effect of C C on the circulating anti-HGG antibody titers. Mice were injected
intraperitoneally with 100 ~g of H G G (F-'3) or with 100 /~g of H G G and C C (1 mg/kg,
l).
Each bar represents the m e a n titer ± SE of 6 animals bled on day 4, and of 13
animals bled on day 7. n.s., not significant.
Published April 1, 1978
PANG
N.
SHEK
AND
ALBERT
H.
1219
COONS
15
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0
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DT
D,z
g
0
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10
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15
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20
I
25
30
DAYS AFTER 2rid I N J E C T I O N
FIG. 5. Effect of CC on the kinetics of the secondary response to DT. Control a n i m a l s (Q)
were given two intraporitoneal injections of 20 Lf of DT 20 days apart. Animals in the
experimental group (@) were similarly immunized except t h a t CC, at a dose of 1.5 mg/kg,
was also administered at the time of antigen injection. Each point represents the m e a n
titer _+ SE of 7-8 animals.
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FIG. 6. Titration curve of the effect of different doses of CC on antibody formation.
Control mice were given two intraperitoneal injections of 20 Lf of DT 20 days apart.
Animals in the experimental groups were similarly immunized except t h a t CC, at various
doses as indicated in the abscissa, was also given. All a n i m a l s were bled 10 days after the
second injection, and t h e i r serum anti-DT h e m a g g l u t i n a t i n g antibody titers were determined. Each point represents the m e a n titer _+ SE of 5 animals.
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I
5
Published April 1, 1978
1220
ENHANCEMENT OF THE ANTIBODY RESPONSE BY COLCHICINE
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BODY W E I G H T
Comparison of the Effect of Two Different Anti-Mitotic Drugs on the Antibody
Response. Vinblastine (VB), another anti-mitotic drug, was tested for its
effectiveness in enhancing the antibody response. Three groups of animals were
similarly immunized with TKB. Animals in group II also received CC (1 mg/kg)
and those in group III were injected with a similar dose of VB on the day of
immunization. 6 days later, the spleens of the treated animals were assayed for
their hapten-specific PFC response. Results shown in Table I indicate that VB
was as effective as CC in enhancing the anti-TNP PFC response.
Effect of Lumicolchicine on the Antibody Response. Lumicolchicine (LCC),
the structural isomer of CC, was tested for its effectiveness to enhance antibody
formation. Mice in the control group were injected intraperitoneally with 100
ttg of TKB on day 0. Two other groups of animals were immunized in a similar
manner, except that CC was also administered to I group and LCC to the other.
The dose given in each case was I mg/kg body weight. The hapten-specific PFC
response was assayed on day 6. Results shown in Table II indicate that whereas
CC enhanced the TNP-specific PFC response by more than 100%, the administration of LCC to immunized animals had no enhancing effect.
Discussion
Results of experiments performed in mice and reported in the present paper
confirm the original observations by Tanaka and Coons (2) that CC is effective
in enhancing the antibody response. Apparently, there is no species difference
in terms of the capacity of CC to promote antibody formation since the drug
works well in rabbits (2, 3), guinea pigs (9), hamsters (21), and in mice as
presently reported.
Previous reports on CC-induced enhancement expressed the enhancing effect
only in terms of the serum antibody level, and the magnitude of the maximal
enhancement varied from 3- to 50-fold (2, 9, 21, 22). In the present study with
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FIG. 7. Determination of the LDso of CC in mice. BALB/c mice (10 animals/group) were
injected intraperitoneally with various doses of CC ranging from 0 to 3.0 mg/kg body
weight'. Each point represents the percent of animals which survived for at least 10 days
after CC injection.
Published April 1, 1978
PANG
400
N.
SHEK
AND
ALBERT
H.
1221
COONS
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DAYS
AFTER
2nd
;
;
;
,0
INJECTION
Fro. 8. Enhancement of the secondary antibody response to HGG by CC. Mice in the
control group (©) wez:egiven 2 intraperitoneal injections of 100 ~g of HGG 14 days apart.
Animals in the experimental group (e) were similarly immunized except CC, at a dose of 1
mg/kg, was also administered at the time of antigen injection. At various times after the
second injection, the animals were bled for the determination of serum hemagglutinating
anti-HGG antibody titers (B) and their spleens were harvested for the assessment of their
PFC response (A). Each point represents the mean response +- SE of 4-10 animals. SC,
spleen cells.
mice, o u r d a t a indicate t h a t CC-induced e n h a n c e m e n t of t h e c i r c u l a t i n g
a n t i b o d y t i t e r v a r i e d f r o m as low as 3-fold (in t h e p r i m a r y r e s p o n s e to H G G ,
Fig. 3) to as h i g h as a b o u t 15-fold (in the s e c o n d a r y response to DT, Fig. 4) m o r e
t h a n t h a t of the control group. In addition to e v a l u a t i n g the s e r u m a n t i b o d y
titer, this s t u d y also e x a m i n e d t h e cellular aspect t h r o u g h t h e a s s e s s m e n t of
the P F C response. T h e d a t a indicate t h a t the n u m b e r of a n t i b o d y - f o r m i n g cells
in the spleens of C C - t r e a t e d a n i m a l s w a s significantly increased. F o r e x a m p l e ,
the hapten-specific p r i m a r y P F C response w a s doubled (Fig. 1) and the
s e c o n d a r y I g G PFC response w a s e n h a n c e d by a t least fivefold (Fig. 9). In t h e
case of the secondary P F C response to H G G , the response of C C - t r e a t e d a n i m a l s
w a s at least t h r e e t i m e s as large as t h a t of control a n i m a l s (Fig. 8 A). This
Downloaded from on October 1, 2016
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Published April 1, 1978
1222
ENHANCEMENT
OF T H E
ANTIBODY RESPONSE
BY C O L C H I C I N E
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6
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4
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b.
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u. 400
200
DAYS
(After
challenge
with
antigen)
FIG. 9. Effect of CC on the secondary TNP-specific PFC response. Mice in the control
group (©) were given 2 intraperitoneal injections of 100 ~g of TNP-KLH 14 days apart.
Animals in the experimental group (Q) were similarly immunized except CC, at a dose of 1
mg/kg, was also given at the time of priming. On each day between 2 and 6 days after
challenge, spleens of the immunized animals were harvested for the assay of their haptenspecific PFC response. Each point represents the mean response _+ SE of five animals. SC,
spleen cells.
increase in the PFC response w a s a c c o m p a n i e d by an e l e v a t i o n of the s e r u m
antibody titer (Fig. 8 B). T h e s e results implied that t h e a d m i n i s t r a t i o n of CC at
the t i m e of antigenic p e n e t r a t i o n affected certain cellular e v e n t s leading to an
increase in the n u m b e r of antibody-forming cells w h i c h in turn increased the
circulating antibody level.
For effective e n h a n c e m e n t of t h e antibody r e s p o n s e by CC, there are at least
t w o critical factors w h i c h h a v e to be observed; (a) t h e dose of CC used, and (b)
t h e t i m e of its administration.
W i t h i n t h e dose r a n g e that is not lethal for mice, m a x i m a l e n h a n c e m e n t m a y
be obtained by the injection of 1.0-1.5 m g CC/kg body w e i g h t (Fig. 6). An
increasingly pronounced e n h a n c i n g effect w a s also observed in t h e rabbit w h e n
the dose of CC w a s increased from 0.5 to 2.0 m g / k g (2). It is of interest to note
that despite the t r e m e n d o u s difference in susceptibility to t h e lethal effect of CC
b e t w e e n mice (LDso 2.1 mg/kg; Fig. 7) and h a m s t e r s (LD~o 300 mg/kg; Ref. 22)
the optimal dose of CC for t h e e n h a n c e m e n t of antibody formation lies w i t h i n
the s a m e range of about 1.0-1.5 mg/kg.
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600
Published April 1, 1978
PANG
N. S H E K
AND
ALBERT
SECONDARY TNP-SPECIFIC
H. C O O N S
1223
PFC RESPONSE
A
.........
....
x-2OO
......
I
-I
0
I
I
I
2
I
2
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B
0
DAY of CC ADMINISTRATION
(in relotion to
day of Ist immunizotion)
FIG. I0. Effectof varying the time of C C administration on the enhancement of the P F C
response. Groups of mice (fiveanimals per group) were given CC, at a dose of i mg/kg, on
various days (as indicated on the abscissa)before, during, and afterthe immunization with
100/~g of T N P - K L H on day 0. Control animals received the same dose of antigen but no C C
was given. 14 days after the first injection, all animals were challenged with 100 ~g of
T N P - K L H . The spleens of the injectedanimals were harvested and assayed for their TNPspecific P F C response 3 days after the second injection. Each point represents the mean
response _ SE of the CC-treated animals (e) or of the control animals (©). SC, spleen cells.
The timing of the injection of CC seems to be critical. For the maximal
enhancing effect, the drug has to be administered simultaneously with or on
the same day as the antigen (Fig. 10). Only minimal enhancement of the PFC
response was observed when CC was given 1 day before the injection of antigen.
Effective enhancement had been reported in the guinea pig when CC was given
as early as 6 h before antigen injection (9). On the other hand, administration of
the drug 1 or 2 days after immunization was ineffective (Fig. 10). CC was also
found to be incapable of enhancing the antibody response in rabbits when it was
given 2 days before or 2 days after the injection of antigen (2). The failure of CC
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-I
Published April 1, 1978
1224
ENHANCEMENT
OF THE A N T I B O D Y RESPONSE
BY C O L C H I C I N E
TABLE I
Enhancement of the Primary Hapten-Specific PFC Response by CC and VB
Day 0
Group
Number
of mice
Day 6
TNP-specific IgM PFC
Treatment
Per 106 SC
I
II
III
12
11
12
TKB*
TKB + CC§
TKB + VBII
125 ± 30
263 ± 74
248 ± 57
P*
Per spleen
Mean +-SE
19,500 ± 3,010
<0.001
39,420 ± 6,630
<0.002
38,290 ± 5,850
P
<0.002
<0.002
* P values in comparison to group I.
i00 ~g T K B injected i.p.
§ C C administered at 1 mg/kg body weight, i.p.
IIV B sulfate administered at 1 mg/kg body weight, i.p.
SC, spleen cells.
Day 0
Group
Number
of mice
Day 6
TNP-specific IgM PFC
Treatment
Per 106 SC
I
II
III
6
6
6
TKB$
TKB + CC§
TKB + LCCII
170 ± 28
370 -+ 32
160 -+ 41
P*
Per spleen
Mean +-SE
28,700 ± 5,090
<0.002
64,820 + 6,950
n.s.
29,640 ± 7,910
P
<0.003
n.s.
* P values in comparison to group I.
* 100 ~g TKB injected i.p.
§ CC administered at 1 mg]kg body weight, i.p.
II LCC administered at 1 mg/kg body weight, i.p.
n.s., not significant; SC, spleen cells.
to e n h a n c e the antibody response w h e n it was administered as e a r l y as 1 day
after i m m u n i z a t i o n suggests t h a t certain critical r e g u l a t o r y cellular events
m u s t have t a k e n place w i t h i n 24 h after contact with antigen. Indeed, E a r d l e y
and Sercarz (23, 24) recently d e m o n s t r a t e d in their e l e g a n t studies t h a t
r e g u l a t o r y suppressor cells appeared as e a r l y as 24 h after p r i m i n g with
antigen, and t h a t cell divisions were required for the expression of suppressive
activities. T a k e n together, these observations strongly support our hypothesis
t h a t CC m a y act on the e a r l y dividing suppressor cell or its precursors.
The route of CC a d m i n i s t r a t i o n does not seem to be important. In this study,
we injected CC and the a n t i g e n separately into the peritoneal cavity. However,
effective e n h a n c e m e n t had also been observed w h e n CC and the a n t i g e n were
injected via different routes. For example, CC h a d been a d m i n i s t e r e d subcutaneously to rabbits i m m u n i z e d in the foot-pad (2), and i n t r a p o r i t o n e a l l y to
h a m s t e r s i m m u n i z e d i n t r a v e n o u s l y (21, 22); in both cases, antibody formation
was increased.
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TABLE II
Effect of CC and LCC on the Primary Hapten-Specific PFC Response
Published April 1, 1978
P A N G N. S H E K AND ALBERT H . COONS
1225
Summary
Colchicine (CC) enhances the antibody response in mice to protein antigens,
like diphtheria toxoid and human gamma globulin, as well as to the 2,4,6trinitrophenyl hapten. Maximal enhancement was observed when CC was
administered to animals on the same day as the injection of antigen. The
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If the enhancement of the antibody response by CC is dependent on the antimitotic action of the drug, then any anti-mitotic drug, e.g. VB, should also be
capable of mediating the enhancement. Indeed, when VB was given to animals
at the time of antigen injection, it was found to be as effective as CC in elevating
the antibody response (Table I).
These observations with both CC and VB strongly suggest that there is a
connection between the anti-mitotic action of the drug and its enhancement of
antibody formation. The dose of CC (1-1.5 mg/kg) that we used to enhance the
antibody response has been shown to be effective in inhibiting mitosis in vivo
(25, 26). If the mitosis-blocking property is crucial for the enhancing effect of
CC, deprivation of its anti-mitotic capacity should render it inactive in this
regard. Indeed, we were able to show that the conversion of CC to its non-antimitotic structural isomer, LCC, abolished the enhancing effect (Table II). Thus,
it seems justifiable to say that the anti-mitotic action of CC is an essential
element for successful enhancement of the antibody response. The critical
timing in the administration of CC necessary for effective enhancement, a
feature of the effect of X-irradiation, indicates that these agents are most likely
acting upon rapidly dividing cells. It is noteworthy that X-irradiation which
also predominantly causes damage to dividing cells (27) also enhances the
antibody response in vivo (28).
In contrast to the enhancing effect reported here, the mitosis-blocking
capacity of CC has been employed to suppress the antibody response in rats by
the administration of the drug a few days after immunization, but within
several hours before the treated animals were sacrificed for the PFC assay (26).
The effective suppressing dose (1.3 mg/kg) of CC used lies within our enhancing
dose range (1.0-1.5 mg/kg). Thus, approximately the same dose of CC administered to animals may mediate either enhancement or suppression of antibody
formation depending upon the timing of drug administration in relation to the
injection of antigen. The suppressive effect of CC, observed when the drug is
injected in the later phase of the antibody response, has been suggested to be
the result of the mitotic blocking action of CC on cellular divisions of the
antibody-forming cell and its precursors (26). On the other hand, we postulate
that the enhancing effect of CC, observed when it is injected at the same time
as antigen, is the consequence of its interruption of mitosis that prevents the
generation of antigen-stimulated, early dividing suppressor cells.
It is clear, from the general effect of such anti-mitotic agents in rabbits, mice,
guinea pigs, and hamsters on both the primary and secondary antibody
responses to several protein antigens, that the early development of suppressor
cells usually, if not always, accompanies antigenic stimulation. The accompanying paper presents evidence that CC does indeed prevent the development of
suppressor cells.
Published April 1, 1978
1226
ENHANCEMENT
OF THE ANTIBODY RESPONSE BY COLCHICINE
optimal dose of CC was in the range of 1.0-1.5 mg/kg body weight. The
enhanced antibody formation was evident from elevated circulating antibody
titers and from an increased n u m b e r of antibody plaque-forming cells (PFC) of
the spleen. The circulating antibody titer of CC-treated animals was higher
t h a n t h a t of control animals by a factor of about 3-7 in the primary response,
and by a factor of at least 15 in the secondary response. In terms of the number
of antibody forming cells, CC enhanced the primary PFC response by ~100%,
and the secondary PFC response by as high as fivefold. The enhancing effect of
CC seemed to be related to its mitosis-blocking capacity since (a) vinblastine,
another antimitotic drug, was found to be as effective as CC and (b) lumicolchicine, the non-anti-mitotic structural isomer of CC, was ineffective in potentiating antibody responses. The critical timing in the administration of CC on the
same day as antigen suggests t h a t most likely, the mitotic poison was acting on
antigen-stimulated early dividing suppressor cells.
The authors wish to thank Daniel Cargill for his excellent technical assistance.
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21.
SHEK A N D ALBERT H . COONS