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Mechanism of Immunological Tolerance in Cyclophosphamide-Treated Mice, Study notes of Immunology

The mechanism of immunological tolerance in mice treated with cyclophosphamide. The study found that specific tolerance lasted for up to nine months in some animals and required repeated administration of sheep red blood cells (SRBC) for maintenance. The smallest number of SRBC required for tolerance induction was 107. The document also discusses the role of antigen in inducing and maintaining tolerance in drug-treated animals. The study concludes that drug-induced immunological tolerance differs significantly from both pneumococcal polysaccharide paralysis and classical, acquired tolerance.

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Clin.
exp.
Immunol.
(1970)
6,
87-99.
ON
THE
MECHANISM
OF
IMMUNOLOGICAL
TOLERANCE
IN
CYCLOPHOSPHAMIDE-TREATED
MICE
AMIRA
MANY*
AND
R.
S.
SCHWARTZ
Clinical
Immunology
Service,
New
England
Medical
Center
Hospitals,
and
Department
of
Medicine,
Tufts
University
School
of
Medicine,
Boston,
Massachusetts
(Received
28
July
1969)
SUMMARY
The
mechanism
of
immunological
tolerance
of
sheep
red
blood
cells
(SRBC)
in
mice
treated
with
a
single
dose
of
cyclophosphamide
was
studied.
Specific
tolerance
lasted
for
as
long
as
nine
months
in
some
animals,
and
its
maintenance
required
the
repeated
administration
of
SRBC.
Anti-SRBC
antibody-forming
cells
were
significantly
reduced
in
the
tolerant
mice,
and
X-irradiated
recipients
of
their
spleens
were
specifically
tolerant
of
SRBC.
The
smallest
number
of
SRBC
required
for
the
induction
of
tolerance
was
107;
this
was
the
smallest
number
of
SRBC
that
could
elicit
antibody
synthesis
within
5
days
in
normal
mice.
Since
the
effects
of
cyclophosphamide
on
antibody-forming
cells
last
only
5
days,
it
was
concluded
that
the
mechanism
of
tolerance
induction
involved
destruction
of
antigen-
stimulated
cells.
In
support
of
this
is
the
finding
that
mice
treated
with
small
doses
of
cyclophosphamide
were
rendered
tolerant
only
when
SRBC
were
given
before
the
drug.
Drug-induced
immunological
tolerance
thus
appears
to
differ
significantly
from
both
pneumococcal
polysaccharide
paralysis
and
classical,
acquired
tolerance.
A
central
loss
of
immunocompetence
does
not
occur
in
the
former,
while
the
latter
requires
for
its
induction
the
administration
of
antigen
in
a
dose
or
form
that
does
not
stimulate
antibody
synthesis.
INTRODUCTION
Drug
induced
immunological
tolerance
is
an
antigen-specific
deletion
of
immunological
responsiveness
that
results
from
treatment
with
cytotoxic
agents.
Many
examples
of
this
phenomenon
have
been
described
(Schwartz,
1968)
since
its
discovery
(Schwartz
&
Dame-
shek,
1959),
but
the
mechanism
of
tolerance
induction
in
drug-treated
animals
remains
obscure.
The
role
of
the
chemotherapeutic
agent
in
bringing
about
tolerance
has
been
emphasized
frequently,
but
the
equally
important
function
of
antigen
in
inducing
and
Correspondence:
Dr
Robert
S.
Schwartz,
Clinical
Immunology
Service,
New
England
Medical
Center
Hospitals,
171
Harrison
Avenue,
Boston,
Massachusetts
021
11,
U.S.A.
*
Present
address:
Tel-Hashomer
Hospital,
Tel
Aviv
Medical
School.
87
pf3
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Download Mechanism of Immunological Tolerance in Cyclophosphamide-Treated Mice and more Study notes Immunology in PDF only on Docsity!

Clin. exp. Immunol.^ (1970) 6, 87-99.

ON THE MECHANISM OF IMMUNOLOGICAL

TOLERANCE IN CYCLOPHOSPHAMIDE-TREATED MICE

AMIRA MANY* AND R. S. SCHWARTZ

Clinical Immunology Service, New^ England Medical Center^ Hospitals,

and Department of Medicine, Tufts University School of Medicine,

Boston, Massachusetts

(Received 28 July 1969)

SUMMARY

The mechanism ofimmunological tolerance of sheep red blood cells (SRBC) in mice

treated with a single dose of cyclophosphamide was studied. Specific tolerance

lasted for as long as nine months in some animals, and its maintenance required

the repeated administration of SRBC. Anti-SRBC antibody-forming cells were

significantly reduced in the tolerant mice, and X-irradiated recipients of their

spleens were specifically tolerant of SRBC. The smallest number of SRBC required

for the induction^ of^ tolerance^ was 107; this was the smallest number of SRBC that

could elicit (^) antibody synthesis within (^5) days in normal (^) mice. Since the effects of

cyclophosphamide on antibody-forming cells last only 5 days, it was concluded

that the mechanism of tolerance induction involved destruction of antigen- stimulated cells. In support of this is the finding that mice treated with small doses of cyclophosphamide were rendered tolerant only when SRBC were given before the drug. Drug-induced immunological tolerance thus appears to differ significantly from both^ pneumococcal polysaccharide paralysis and classical, acquired tolerance. A central loss of (^) immunocompetence does not occur in the former, while the latter requires for its induction (^) the administration of antigen in a dose or form that does not stimulate antibody synthesis.

INTRODUCTION

Drug induced^ immunological tolerance is an antigen-specific deletion of immunological responsiveness that results from treatment with (^) cytotoxic agents. Many examples of this

phenomenon have been described (Schwartz, 1968) since its discovery (Schwartz & Dame-

shek, 1959), but the mechanism of tolerance induction in drug-treated animals remains

obscure. The role of the chemotherapeutic agent in bringing about tolerance has been emphasized frequently, but the (^) equally important function of antigen in inducing and

Correspondence: Dr^ Robert S. Schwartz, Clinical Immunology Service, New England Medical Center Hospitals, 171 Harrison Avenue, Boston, Massachusetts 021 11, U.S.A.

  • (^) Present address: Tel-Hashomer Hospital, Tel Aviv Medical School.

87

88 Amira Many and R. S. Schwartz

maintaining tolerance in drug-treated animals is not well^ appreciated.^ In^ this paper^ we will present evidence that in drug-induced immunological tolerance there^ is, as in^ acquired immunologic tolerance, a central loss of^ specific^ immunological^ reactivity. However,^ the induction of tolerance in^ cyclophosphamide-treated^ mice^ requires^ an^ amount^ of^ antigen that can stimulate antibody synthesis in normal^ animals.

MATERIALS AND METHODS

Animals

C57BL/6J mice, aged 8-10 weeks, were housed in plastic cages and had free access to water and a standard mouse diet (Purina).

Antigens

Sheep red blood cells (SRBC) (Colorado^ Serum^ Co.)^ and^ horse^ red^ blood cells^ (HRBC) (a gift of the Massachusetts Public^ Health Biological^ Laboratories)^ were^ washed three times in 0 15 M^ saline and^ resuspended^ to^ the^ desired^ concentration^ in 0^ 15 M^ saline.^ Unless stated

otherwise, the^ antigenic challenge^ was^5 x^108 cells^ (0.1 ml^ of^ a^ suspension with^ a^ haematocrit

of 20 volumes^ °/%) injected intraperitoneally.

Cyclophosphamide (Cytoxan®,^ Mean^ Johnson^ Laboratories)

The drug was dissolved in 0-08 M saline and administered intraperitoneally in^ concentra- tions of from 1-5 to 3 0 mg/ml, depending upon the experimental design.

Spleen cell suspension

Spleens were excised and^ minced with^ scissors,^ and the^ fragments^ were^ gently^ pressed

through tantalum^ gauze into^ a^ small volume of chilled^ Ringer's^ solution.^ Aseptic^ technique

was used throughout.

X-Irradiation

Mice were exposed in lucite containers to 850 r^ using a^ Westinghouse deep therapy^ unit

(250 kV, 15 mA, target distance 50 cm) at a^ rate^ of^46 r/min.

Measurement of the immune response

Two methods were^ used:^ (a) The^ haemolytic plaque^ assay^ (Jerne^ &^ Nordin,^ 1963).

'Indirect' plaques (presumably caused^ by^ 7S^ antibodies)^ were^ assayed by^ the method of

Dresser & Wortis (1965), which was modified by incubating the^ agar plates for^1 hr^ at^ 40C

after the addition of rabbit anti-mouse^ IgG. (b) Serum^ haemagglutinins were^ estimated^ in

microtitre plates (Sever, 1962). In^ all^ cases, the^ initial dilution of^ serum was^ 1:20. Titres^ are

expressed as^ the^ number of^ the^ last^ well^ in the^ plate^ that showed^ an^ agglutination^ pattern.

RESULTS

Immunological tolerance^ of SRBC^ in^ cyclophosphamide^ treated^ mice

Twenty-five adult^ mice^ were^ injected^ once^ with^ cyclophosphamide,^150 mg/kg,^ and,

within several minutes, they received 5 x^108 SRBC.^ Every 5 days thereafter^ they were^ given

Amira Many and R. S. Schwartz

5 x 108 SRBC. Another group of twenty mice^ was treated^ in the same way^ except^ for the injection of cyclophosphamide. Mice of both^ groups were bled^ every^5 days, just^ before injection of the SRBC.^ The^ experiment^ was^ continued^ for^ about 9^ months.^ The^ agglutinin titres found in^ the^ drug-treated^ animals^ were^ always^ less^ than^ those^ of^ the^ control^ mice (Fig. 1). During the first 4 months of the experiment, responses to SRBC by the cyclo-

phosphamide-treated mice were severely depressed; thereafter, the titres of anti-SRBC

agglutinins slowly increased,^ but^ never^ reached^ the^ levels found in^ the^ control^ mice. Each^ of

the mice in the control group responded to the antigen; the fraction of cyclophosphamide- treated mice forming antibodies to the SRBC rose gradually (Fig. 2), but never reached

100%. Recovery^ of^ responsiveness^ (as^ defined by^ the^ presence^ of^ any^ anti-SRBC antibodies^ in

serum) was linear during the first 5 months; thereafter, a^ small, constant^ fraction^ of the

experimental animals failed to form agglutinins.

14-

12

10 X

@ 8 _ c, 0

o 0

a-A - 0 0 20 4 60 8 10

FIG. 3. Specificity of drug-induced immunological tolerance. The experiment was carried out as in Fig. 1; beginning on day 5 control and experimental animals were given HRB3C every 5 days. (^) 0r, Control; (^) *, experimental; *, response to sheep red blood cells.

Tolerance specificity was determined by challenging the cyclophosphamide-treated and control mice with a second antigen, HRBC. Five days after the first injection of SRBC, the mice were challenged with 5 x 108 HRBC. This was repeated every 5 days. Fig. 3 shows that the cyclophosphamide-treated mice did not differ from those of the control group with respect to their responsiveness to HRBC.

The central loss of^ immunocompetence Tolerance was induced in mice by the method just described, and 20 days later, or 5^ days after the fourth injection of SRBC, the number of plaque forming cells^ (PFC)^ in^ their spleens was enumerated. Two^ groups^ of mice^ were^ studied: in one, the^ initial dose^ of SRBC

was 5 x 108; in the other it was 5 x 109. Antibody-forming cells were found in considerably

reduced numbers in the drug-treated animals (Fig. 4). The^ numbers^ of mice are small, but

it seems that an initial dose of 5 x i09 SRBC, although no more immunogenic in normal mice

than 5 x 108 SRBC, was more tolerogenic in cyclophosphamide-treated mice.

90

Immunological (^) tolerance in mice 91

Mice were rendered tolerant of SRBC in the manner described, and 24 hr after the third

injection of SRBC (or 16 days after treatment with cyclophosphamide) they were killed and

suspensions of their spleen cells were injected into irradiated mice. The (^) recipients were (^) given

850 r total body irradiation and, within 4 hr, 5 x 107 spleen cells intravenously from either

100,000 _ 5 x10SRBC (^5) x ISRBC Direct Indirect^ Direct Indirect plaques plaques^ plaques^ plaques

10,

a-

1,

FIG. 4. PFC in (^) normal and drug treated mice. All groups of (^) animals received injections of SRBC every (^5) days, and splenic PFC were estimated 5 days after (^) the fourth dose of SRBC. Open columns, controls; (^) hatched columns, drug treated mice. Eight animals (^) per group.

TABLE 1. Specific, adoptive tolerance of SRBC in (^) irradiated recipients of spleen cells from mice with (^) drug-induced tolerance of SRBC

Repopulation by spleen cells (^) from: Days after irradiation (^) Normal mice Tolerant mice

Anti-SRBC (^) Anti-HRBC Anti-SRBC Anti-HRBC

(^10) 2-6±3-0 (^0) 0-3±0-8 1-0+2- (^15 5) 3±1-2 70+1-7 0-3+0-8 (^) 6-8±1- (^20) 11-0±3-0 11-0±3-0 0-8±1-5 10-8+1- 25 12-3+1-0 (^) 1-0±1-

tolerant or normal (^) mice. Five days later all receipients were (^) injected with a mixture of 5 x (^108) SRBC+5x 108 HRBC. These (^) antigens were given every 5 days (^) thereafter. The

results, listed in Table 1, demonstrate that the specific tolerance of SRBC induced in

cyclophosphamide-treated mice could be transferred to irradiated recipients.

Immunological tolerance in mice 93

Fig. 5 shows that the prolonged tolerance in cyclophosphamide-treated mice was^ not due to persistence of^ non-specific drug injury.^ Recovery from cyclophosphamide by^ 120 hr in the absence of additional antigen was demonstrated by studying serially the immune

responses of mice given antigen either simultaneously with or 120 hr after the drug (Fig. 5).

10 to_

8;

0~~~~~~~~

6 I

Days before or otter cyclophospharnide FIG. 7. Sensitization or tolerance induction in mice given SRBC before or after cyclophos- phamide. Horizontal stippled bar shows the standard deviation of anti-SRBC titres of mice not given the drug. Eight to ten mice per group.

The differences in the number of splenic PFC formed by these two groups is significant at each point studied. This indicates that although cyclophosphamide is of undoubted im- portance in making possible the induction of immunological tolerance, antigen must be present if the drug is to exert an immunologically specific effect.

Requirement for an immunogenic stimulus The smallest number of SRBC needed for the induction of immunological tolerance in

cyclophosphamide-treated mice was determined in groups of animals given 102, i04, 106,

107 or 108 SRBC, together with 150 mg/kg cyclophosphamide. Control animals received no drug. Every 5 days thereafter all mice received 5 x 108 SRBC. Tolerance developed only in those groups of drug-treated mice that received an initial dose of antigen capable of provoking an immune response within five days in normal mice (Fig. 6). The smallest tolero- genic dose of antigen was 107 SRBC. A 'low zone' of tolerance induction was not detected.

Inhibition of antigen-selected cells SRBC were injected at different times before or after a single dose (150 mg/kg) of cyclo- phosphamide. Every 5 days after the first dose of antigen^ the various groups of mice received 5 x 108 SRBC. Haemagglutinin titres were measured on day 20. Tolerance was induced when antigen was given any time from -72 to + 24 hr before or after the drug (Fig. 7), but it was not possible to induce tolerance when antigen was given 48 hr or more after, or 96 hr or more before cyclophosphamide.

Amira Many and R. S. Schwartz

06

0 4

2 0

0~~~~~~~

0 25 50 75 100 125 150 Dose of cyclophosphamide (mg/kg) FIG. 8. Sensitization or tolerance induction in mice given different doses of cyclophosphamide before or after 5 x 108 SRBC. Horizontal stippled bar shows the standard deviation of anti- SRBC titres of mice not given the drug. Six to eight mice per group. *, Cyclophosphamide on day 0; o, cyclophosphamide on day + 1.

2 5 x 10' SRBC

10

8

4

2

0

0 10 20 30 40 50 Days FIG. 9. Tolerance or sensitization in mice given cyclophosphamide, 75 mg/kg, either^ simultan- eously with (@) or 1 day after (A) 5 x^108 SRBC.^ o, No^ cyclophosphamide.

94

96 Amira Many^ and R. S.^ Schwartz

Specificity of the maintenance^ antigen^ was determined^ in^ a group^ of mice that was^ given HRBC instead^ of SRBC^ every^5 days after^ an inducing^ injection of SRBC. The^ results (Fig. 12) show that only mice maintained with SRBC were persistently tolerant^ of^ that antigen.

18 1

15

12-

6-

5 x 106 SRBC I l 0 20 40 60 80 Days FIG. 1 1. All mice were given 5 x^108 SRBC on day 0. The^ 'maintenance'^ group (A)^ received SRBC every 5 days thereafter during the entire^ experiment.^ Antigen^ was withheld^ from the other two groups from day 0 to day 30; it was then given every^5 days.^ Eight^ to ten^ mice per group. a, No maintenance; *,^ no^ cyclophosphamide.

DISCUSSION

The results of^ these experiments confirm^ those^ of Frisch^ &^ Davies^ (1965)^ and^ Aisenberg

(1967), and their important observation that mice treated with a^ single dose of^ cyclophos-

phamide can be rendered tolerant of SRBC has been extended in^ several ways.

It is apparent from these studies that the immunosuppressive effects of^ cyclophosphamide

are contingent upon two factors: cytotoxicity of the^ drug and selection of cells^ by^ antigen.

By itself, the drug causes a transient, non-specific depression of the^ immune^ response

(Aisenberg & Davis, 1968) and^ immunological^ recovery^ is^ complete^ within 120 hr^ or^ less,

depending upon the dose^ given.^ When^ administered^ together^ with^ antigen,^ the^ drug^ sup-

presses the primary immune response and, in^ addition, immunological^ memory^ is^ inhibited

(Butler & Coons, 1964). However, when^ antigen is^ given^ to^ cyclophosphamide-treated^ mice

at frequent intervals, specific tolerance^ develops.

Several observations may clarify our understanding of^ drug-induced immunological

tolerance. The first is that in cyclophosphamide-treated mice there^ is^ a^ central loss of

specific immunological responsiveness, as^ shown^ by^ the marked^ reduction^ in the number of

antibody-forming cells in their lymphoid tissue and^ by^ the^ 'transfer' of^ drug-induced^ toler-

Immunological tolerance in (^) mice

ance to heavily irradiated mice. The tolerance in these mice thus differs (^) significantly (^) from the paralysis induced by pneumococcal (^) polysaccharide, in which (^) antibody-forming cells are present but their product-antibody-is apparently absorbed (^) by persisting antigen (Howard et (^) al., 1969).

Tolerance in cyclophosphamide-treated mice is thus similar to classical, acquired immuno-

logical tolerance. But the resemblance soon ends, for our data show that an immunogenic

stimulus is required for the induction of tolerance in drug-treated animals. The dictum

that tolerogens must be immunogens (Benacerraf, 1969) is thus upheld. However, a crucial

difference between acquired and drug-induced tolerance is that in the former the tolerogen

8 f

7-

6 -

5 Ti

(^2) --1 T

004

10 20 30 40 50 60 Days FIG. 12. Specificity (^) of the maintenance antigen. Tolerance persisted only in mice given SRBC every 5 days. *, No (^) maintenance; o, HRBC maintenance; *, SRBC maintenance.

must be given in a dose or (^) form that does not stimulate antibody synthesis (Dresser (^) &

Mitchison, 1968), whereas in the latter the tolerogen must be given in a dose that can elicit

an immune response in animals not given the drug. Furthermore, unlike conventional

tolerance, a 'low zone' of tolerance was not identified in cyclophosphamide-treated mice.

Our (^) explanation of these observations rests on the unproven (^) assumption that differentiat- ing, proliferating, antigen-selected (^) cells are more sensitive to the cytotoxic effects of cyclo-

phosphamide than resting lymphocytes. The ability to induce tolerance by injecting SRBC

24 hr after a large dose of the drug may be due to unrepaired sublethal damage in antigen

sensitive cells (DeWys & (^) Kight, 1969), which, on stimulation by antigen, finally die. (^) What is more important is that smaller doses of the drug are (^) effective only after the antigenic

Immunological tolerance in mice 99

SCHWARTZ, R.S. (1968) Immunosuppressive drug therapy.^ Human^ Transplantation, p.^ 440. Grune &^ Stratton, New York. SCHWARTZ, R.S. & DAMESHEK, W. (1959) Drug-induced immunologic tolerance. Nature (Lond.), 183, 1682. SCHWARTZ, R.S. & DAMESHEK, W. (1963) The role of antigen dosage in drug-induced immunologic tolerance. J. Immunol. 90, 703. SEVER, J.L. (1962) Application of^ a^ microtechnique^ to viral^ serological^ investigations.^ J.^ Immunol.^ 88, 320.