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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 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.
