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Resistance to tolerance induction in B cells of autoimmune mice—Abnormal expansion of low affinity IgG-producing B-cell population
CELLULAR
71, 303-314
IMMUNOLOGY
(1982)
Resistance to Tolerance Induction in B Cells of Autoimmune Mice-Abnormal Expansion of Low Affinity IgG-Producing B-Cell Population YASUKO Department
TSUNETSUGU
AND MICHIO
FUJIWARA’
of Immunology. Institute of Medical Science, University of Tokyo, Tokyo 108, Received
February
23, 1982;
Japan
accepted May 18, 1982
Several strains of mice are known to develop spontaneous autoimmune diseases like lupus erythematosus and they show various immunological abnormalities as well. Despite different genetic backgrounds, they manifest various immunological abnormalities in common, e.g., polyclonal B-cell activation (PBA) and resistance to tolerance induction. To elucidate mechanisms of the development of autoimmunity, tolerance inducibility was examined in autoimmune and normal mice using trinitrophenylated carboxymethyl cellulose (TNP-CMC) as tolerogen which is known to induce TNP-specific B-cell tolerance without the participation of T cells. NZB and MRL/Mp-lpr/lpr mice were used as autoimmune mice and C57BL/6, BALB/c, and MRL/Mp-+/+ mice as nonautoimmune mice. When TNP-CMC-injected mice were challenged with T-independent antigens, all of the mice tested were shown to be tolerant. In contrast, when TNP-CMC-injected mice were challenged with T-dependent antigen and secondary IgG responses were assessed, autoimmune mice showed rather hyperreactivity, while nonautoimmune mice showed hyporesponsiveness. Cyclophosphamide improved this defective tolerance inducibility. By the solid-phase radioimmunoassay it was revealed that average affinity of serum anti-TNP antibodies produced in TNP-CMC-injected mice was low. Such low affinity antibodies were produced in large amount in autoimmune mice. Hence, it was suggested that B-cell clones destined to produce low affinity IgG antibodies were responsible for the resistance to tolerance induction and such clones were expanding in autoimmune mice.
INTRODUCTION New Zealand mice (NZB and (NZB X NZW)F, mice) have been widely utilized as an animal model of hemolytic anemia or systemic lupus erythematosus (SLE) and have offered much information on the etiopathogenesis of autoimmune diseases (1, 2). These mice have been revealed to manifest various immunological abnormalities in the functions of immunocompetent cells: hyperactivity of B cells (6, 7) or distorted regulatory circuits (8). These abnormalities seem to be predetermined at the precursors of both T and B cells (9) and controlled by multiple genes (10, 11). Recently MRL/Mp-lpr/fpr (MRL/l) and BXSB (especially male) mice, displaying spontaneous lupus nephritis, were developed by Murphy and Roths (12) and studies using them have shed new light on the etiopathogenesis of SLE (13’ Address correspondence University of Tokyo, 4-6-1,
to M. Fujiwara, Department of Immunology, Shirokanedai, Minato-ku Tokyo 108, Japan.
Institute
of Medical
Science,
303 0008-8749/82/120303-12%02.00/O Copyright 0 1982 by Academic Press, Inc. All right8 of reproduction in any form rcscrved.
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TSUNETSUGU
AND FUJIWARA
19). These autoimmune strains of mice have different genetic backgrounds (e.g., haplotype of histocompatibility gene complex and genes participating in the development of SLE-like syndrome). Despite the difference in the genetic backgrounds, they manifest some immunological abnormalities in common. Increased polyclonal B cell activation is the most prominent feature ( 14, 16, 19) and its role in the development of murine lupus has been investigated extensively. As another immunological abnormality, the resistance to tolerance induction has been pointed out (20-26). If autoimmunity is assumed to come out from the escape of selftolerance, a clue to the pathogenesis of autoimmune diseases could be obtained by studying the mechanisms involved in defective tolerance induction. In this report, tolerance induction in B cells was studied by using trinitrophenylated carboxymethyl cellulose (TNP-CMC) as a tolerogen. It was revealed that tolerance in TNP-specific B cells was not attained when the TNP-CMC-treated autoimmune mice were immunized with a T-dependent (TD) antigen, TNP-keyhole limpet hemocyanin (KLH), and secondary anti-TNP IgG responses were assessed. The implication of the resistance to tolerance induction was discussed in relation to the development of autoimmunity. MATERIALS
AND METHODS
Mice. NZB, MRL/l, and MRL/Mp-+/+ (MRL/n) mice were maintained at our laboratory animal facilities. C57BL/6 and BALB/c mice were obtained from the Animal Breeding Unit of the Institute of Medical Science, University of Tokyo. Female mice were used at 6-10 weeks of age. Antigens. As T-independent (TI) antigens, TNP-lipopolysaccharide (TNP-LPS) and TNP2,8-Ficoll were used. The subscript indicates mean number of TNP groups per 400,000 daltons of Ficoll. TNP-LPS was donated by Dr. T. Tadakuma (Department of Microbiology, School of Medicine, Keio University). Ficoll (Pharmacia, Uppsala) was trinitrophenylated according to the method described by Inman (27). These antigens were dissolved in phosphate-buffered saline (PBS). As a TD antigen, TNP-KLH was used. KLH (Calbiochem, San Diego, Calif.) was conjugated with TNP as described by Kiefer (28). A preparation of TNP,,-KLH (29 TNP groups per 100,000 daltons of KLH) was either emulsified with complete Freund’s adjuvant (CFA) or dissolved in PBS. Tolerance induction and challenge immunization. CMC was obtained commercially (Wako Junyaku, Tokyo). TNP-CMC was made as described by Diner et al. (29) and used as a tolerogen to induce TNP-specific B-cell tolerance (Hapten density of TNP-CMC used here was 9-l 1). A group of mice was injected with 0.5 mg of TNP-CMC ip three times every other day and control group of mice was left untreated. All mice were challenged with the antigen usually 3 days after the final tolerogen injection. TNP-LPS or TNP2,*- Ficoll was injected ip at a dose of 1 or 10 pg, respectively and mice were sacrificed 4 days later. When mice were challenged with TD antigen, emulsion of TNP,,-KLH in CFA was injected SCat a dose of 100 pg and the same dose of TNP,,-KLH was boosted ip 16 days later. Mice were sacrificed 5 days later. Sheep red blood cells (SRBC) were injected simultaneously to test the specificity of the tolerance. Cyclophosphamide (Shionogi Seiyaku, Osaka) was dissolved in saline immediately before use and injected as described in the text.
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305
Assay for anti-TNP plaque-forming cells (PFC). Numbers of anti-TNP PFC in the spleen were determined by the technique of Cunningham and Szenberg (30) using TNP-horse red blood cells (TNP-HRBC) as target. In the indirect (IgG) PFC assay, rabbit anti-mouse immunoglobulin was added for the development of plaques. The lot of serum used here was preliminarily checked to have little blocking effect on the appearance of IgM PFC. Numbers of indirect PFC were obtained by subtracting those of direct PFC. Serum anti-TNP antibodies. Levels of serum antibodies were measured by the solid-phase radioimmunoassay according to the method of Tsu and Herzenberg (3 1). Briefly, using 96-well microtiter plate (Tomy Seiko, Tokyo) wells were coated with the antigen by adding 50 ~1 of 100 pg/ml of TNP-bovine serum albumin (TNP-BSA) (either TNPS,,-BSA or TNP,,-BSA) for 1 hr at room temperature. After wells were washed three times with PBS containing 1% BSA and 0.1% NaN3, 40 ~1 of adequately diluted test or standard sera was added to the coated wells and incubated for 1 hr. Then, wells were washed similarly and 40 ~1 of ‘*‘I-labeled purified rabbit anti-mouse immunoglobulin was incubated for another 1 hr. Finally, wells were thoroughly washed, dried, and cut into plastic tubes. Radioactivity of each well was counted with the Autogamma Scintillation Counter (Packard Instrument, Downers Glove, Ill.). In each assay ‘*‘I-counts bound to wells were converted to units by comparison with the binding curve obtained from the standard anti-TNP serum. In this study antibody titer in the standard serum was arbitrarily determined as 100 units. The ratio of antibody units bound to TNP5,,-BSA per those bound to TNPs7BSA was represented as the average affinity of anti-TNP antibodies as validated by Herzenberg et al. (32). Statistical analysis. Results of PFC assay were expressed as geometric mean $ standard error (SE). Levels of serum antibodies were shown as arithmetic mean f standard deviation (SD). The percentage response was calculated as follows:
% Response =
mean PFC numbers or serum titers of tolerant mice x 100. mean PFC numbers or serum titers of immune control mice RESULTS
Tolerance Induction in the Primary IgM Anti-TNP
Response
NZB and C57BL/6 mice were tolerized with TNP-CMC as described under Materials and Methods and challenged with 10 pg of TNP,,,-Ficoll on Day 3 of the final TNP-CMC injection. Anti-TNP PFC in the spleen were assayed 4 days later. The representative results are shown in Fig. 1. Both strains of mice were deeply rendered tolerant; the percentage response was 0.8% in NZB and 0.3% in C57BL/6 mice, respectively. The tolerance was specific since there was no significant difference in anti-SRBC response between tolerized and control mice (Fig. 1, dotted column). In the following experiments no significant difference was observed in anti-SRBC PFC response between tolerized and control group of mice. Therefore, the results were omitted from the figure and tables. As tolerance inducibility was reported to change with the age of mice (20-21, 23), the same tolerizing schedule was carried out in 6- to 8-month-old NZB mice. The percentage response in these mice was 4.3%.
306
TSUNETSUGU
AND FUJIWARA
PFC/SPLEEN
(x109
15-
10: : : :
5-
~
:.
: : : : . :.
i
3
::.::; j:.:. .:.:.: .. .. .. :::::: :.:.:. .:.:.: :::::: .A.. ;:::: :::.:. .:.:.: 55.. :.:.:. _..... .:.:.: . ..A :.:i’. .:.:.: ::g ... . .. i’.:.: :.:.:. .:.:.: .,.... :.:.:’ .:.:.: :::::: ... ..
TOLERIZEO
NZB
CONTROL
(2M)
TOLERIZEO
CONTROL
C57Bt/6Q~,
TCtERi
NZB(6-EM)
FIG. 1. NZB and C57BL/6 mice were tolerized with TNP-CMC at the age indicated in parentheses. Three days later 10 pg of TNP*,s-Ficoll and 2 X 10’ of SRBC were injected and direct PFC assay was done; 0, anti-TNP PFC; q , anti-SRBC PFC.
The results of the challenge immunization with TNP-LPS were similar to those with TNP,,,-Ficoll; the percentage response was 0.6% in NZB and 0.8% in C57BL/ 6 mice, respectively (Table 1). When the challenge was postponed until Day 11 or 20 after the TNP-CMC injection, percentage response became slightly higher. But again both strains of mice were tolerized almost to the same degree, 3.2-6.0s response in NZB and 1.0-2.7s response in C57BL/6 mice. Thus, TNP-CMC induced stable tolerance, irrespective of strain and age of mice, to the subsequent challenge immunization with TI antigen. Tolerance Induction
in the Secondary IgG Anti-TNP
Response
In the following experiments, it was examined whether TNP-CMC induced tolerance in the secondary anti-TNP response by a TD antigen or not. As a preliminary experiment, C57BL/6 mice were injected with TNP-CMC three times every other day and primed with TNP,,-KLH in CFA 3 days after the final tolerogen injection. The mice were boosted 16 days later and splenic IgG anti-TNP PFC were enumerated. The percentage response was 4.8%. When the priming was postponed until 3 or 6 weeks later, the responsiveness recovered to 8.0 or 16%, respectively.
DEFECTIVE
B-CELL TOLERANCE TABLE
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MICE
1
Tolerance Induction in the Primary IgM Anti-TNP
Response’
Direct PFC/spleen’ (% response) Day ofb challenge 3
TNP-CMC + + -
Antigen
NZB
TNP-LPS
427 72,500 1,400 171,000
TNP-Ficoll
11
+ -
TNP-LPS
20
+ + -
TNP-LPS
z : 5 r
C57BL/6
1.37 (0.6) 1.16 1.32 (0.8) 1.06
327 46,000 364 135,000
4,760 5 1.41 (5.3) 90,200 $ 1.15 2,420 40,500 5,310 168,000
TNP-Ficoll
5 r 2 5
z 5 5 3
1.14 (0.8) 1.20 1.19 (0.3) 1.16
1,150 5 1.50 (2.7) 41,900 5 1.28
1.41 (6.0) 1.21 1.36 (3.2) 1.05
331 33,900 2,600 180,000
5 5 5 5
1.61 (1.0) 1.38 1.52 (1.4) 1.12
a NZB and C57BL/6 mice were injected with TNP-CMC as described under Materials and Methods. TNP-CMC-injected and control untreated mice were immunized with either TNP-LPS or TNP-Ficoll and numbers of direct anti-TNP PFC were enumerated 4 days later. Each group consists of five mice. b The days are indicated when TNP-LPS or TNP-Ficoll was immunized after the final TNP-CMC injection. ’ Geometric means of PFC numbers are shown with standard error.
Next, tolerance inducibility in this experimental system was examined in autoimmune mice, comparing with normal strains of mice. NZB, C57BL/6, and BALB/c mice were tolerized and immunized as described above. The results obtained are summarized in Table 2. A remarkable difference was observed between normal and autoimmune mice, that was, while C57BL/6 and BALB/c mice were rendered tolerant (4.8 and 1.3% response, respectively), TNP-CMC-injected NZB mice showed augmented response ( 132%) as compared with immune control mice. TABLE
2
Tolerance Induction in the Secondary IgG Anti-TNP Strain NZB
C57BL/6
BALB/c
Number of mice
TNP-CMC
Response”
Indirect PFC/spleen
% Response 132
(14) (13)
+ -
389,000 5 1.50 295,000 2 1.24
(12)
+ -
31,000 F 1.53 653,000 F 1.20
4.8
(11) (10) (10)
+ -
6,780 5 1.94 504,000 5 1.25
1.3
a Tolerized and untreated mice were immunized with TNP-KLH Indirect anti-TNP PFC were enumerated 5 days later.
in CFA and boosted 16 days later.
TSUNETSUGU
AND FUJIWARA
TABLE
3
Affinities of Antibodies in Tolerized and Immune Control Mice” Serum IgG antibodies* (Unit) TNPCMC
Indirect PFC/spleen
NZB
+ -
577,000 5 1.81 442,000 $ 1.22
C57BLJ6
+ -
12,200 5 2.50 833,000 5 1.37
BALB/c
+ -
1,610 5 2.41 608,000 5 1.20
Strain
(W TNP,-BSA
Affinity (l/h)
24.6 f 7.96 233.9 + 63.0
62.5 + 19.3 154.8 + 37.3
0.40 + 0.06 1.44 + 0.23
(1.5)
2.9 + 0.93 168.9 + 42.0
11.2 f 2.88 179.9 + 33.4
0.29 f 0.05 0.91 + 0.11
(0.3)
2.1 f 185.9 f
8.7 f 5.94 200.6 + 27.9
0.53 f 0.24 0.97 f 0.12
response) (131)
TNP,-BSA
0.68 4.69
’ Mice were tolerized and immunized with TNP-KLH as usually. At the time of sacrifice blood was collected and serum anti-TNP antibodies were estimated. Each group consists of five mice. * In the solid-phase radioimmunoassay, both TNPs,,-BSA (TNP,-BSA) and TNPs,-BSA (TNP,,-BSA) were used as the antigens. Average affinities of antibodies were expressed with the ratio of the binding (see Materials and Methods).
Assuming that B cells of NZB mice showed higher threshold for the tolerance induction, these mice were injected with larger amounts of the tolerogen. It was still difficult to induce tolerance; when 2.0 mg of TNP-CMC was injected three times every other day, the percentage response was 104% and when three times 0.5 mg of TNP-CMC injection as usual was repeated for 3 weeks, the responsiveness reduced to 34%, but not to the level of normal mice (less than 10% response). In another experiment, sera were collected at the time of PFC assay to measure the levels of serum antibodies by solid-phase radioimmunoassay and their average affinities were estimated (Table 3). In the PFC assay, NZB mice showed 131% response, in contrast with C57BL/6 and BALB/c mice which were rendered unresponsive (1.5 and 0.3% response, respectively). Estimation of anti-TNP antibodies gave rise to somewhat different results from the PFC assay. Levels of anti-TNP antibodies in tolerized NZB mice never exceeded those of control mice; percentage response in NZB mice was 40% in this experiment (62.5/154.8). Average affinity of antibodies of tolerized group was lower than those of immune control group in all strains tested. The fact seems to indicate that the augmented response observed in tolerized NZB mice was due to the expansion of B-cell clones differentiating to produce antibodies with low affinities. Tolerance inducibility in MRL/l mice was examined similarly, comparing with MRL/n mice. Like NZB, MRL/l mice showed enhanced percentage PFC response (174%), while MRL/n mice showed 9.0% response (Table 4). The average affinities of antibodies of both strains were lower than those of immune control group. Induction of TNP-CMC
Tolerance in Primed NZB Mice
From the previous results, it was suggested that tolerance by the TNP-CMC was selectively attained in B-cell population capable of producing higher affinity antiTNP antibodies. In the next experiment it was examined whether TNP-specific
DEFECTIVE
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TABLE Tolerance
Induction
in the Secondary
4
IgG
Anti-TNP
Response Serum
Strain MRL/l
MRL/n
TNPCMC
Indirect PFC/spleen
+
1,570,OOO
-
901,000
+
77,900
-
869,000
0 See footnotes
to Table
(% response)
z 1.92 z 1.74 r
1.71 5 1.67
3. Each group
TNP,-BSA
(174)
+ 61.0
108.0
k 42.1
5.82 f 168.7
in MRL
IgG
Mice”
antibodies
(Unit) Affinity (l/W
TNP,-BSA
107.8
(9.0)
consists
309
MICE
1.98
1 78.0
153.3
f 83.3
0.59 f 0.06
96.6 f 38.1
1.05 + 0.13
20.2 3~ 8.23
0.33 2 0.06
143.8 f 55.7
1.01 10.12
of five mice.
clones with higher affinity receptors can be tolerized or not. NZB and C57BL/6 mice were primed with TNP,,-KLH and 3 weeks later TNP-CMC was injected as before. Mice were boosted 3 days after the final TNP-CMC injection. Most of C57BL/6 mice died probably due to anaphylactic shock shortly after the TNPCMC injection. On the other hand, NZB mice survived and became tolerized to the degree of 3% response (Table 5). Efect of Cyclophosphamide
(Cy) on the Tolerance Induction
Cy has been used to intensify the tolerance induction (34) or to treat murine lupus (35). Moreover, the drug seems to depress the polyclonal activation of B cells (our data to be published). The following experiment examined if Cy affected tolerance induction in B cells of NZB mice. NZB mice were injected with 150 mg of Cy per kg of body weight of mouse (mg/kg body wt) 1 day before TNP-CMC injection. The augmented response (338%) was depressed to 34.5% by the preinjection of Cy (Table 6). In another experiment 20 mg/kg body wt of Cy was injected weekly, starting from 1 month of age for 10 weeks. Before the last Cy injection NZB mice were given TNP-CMC and 2 days after the last Cy injection challenged with TNPz9KLH in the usual way. Serum antibodies were measured by the binding of antisera to TNPS,-BSA. It was shown that Cy suppressed the antibody production in TNP-
TABLE Tolerance TNP-CMC
+ -
5
Induction
in Primed
Indirect
PFC/spleen
11,100 402,000
NZB
Mice” % Response
r 1.35 5 1.23
0 Mice were primed with TNP-KLH in CFA 3 weeks previously, 3 days later. Each group consists of five mice.
2.7
injected
with TNP-CMC,
and boosted
310
TSUNETSUGU
AND TABLE
Effect
CY
+ -
TNP-CMC
of Cy on the Tolerance Indirect
6 Induction
by TNP-CMC”
PFC/spleen
+
153,000
-
443,000
+
991,000
-
293,000
’ Cy (150 mg/kg body wt) was injected and assayed in the usual way.
FUJIWARA
(% response)
5 2.21 5 1.22
(34.5)
5 1.21 5 1.51
(338)
ip 24 hr before
Affinity 0.21 f 0.00 0.88 + 0.18 0.44 k 0.04 0.76 f 0.08
tolerance
induction.
Mice
were
challenged
CMC-injected NZB mice from 47 to 3.7% response and from 53 to 19.5% response, when assayed at 3 weeks and 6 weeks after the challenge, respectively (Fig. 2). DISCUSSION NZB, (NZB X NZW)F1, MRL/l, and male BXSB mice spontaneously develop lupus-like syndrome and have been widely utilized to elucidate mechanisms involved 3weeks
NZB
BALB/c
-
NZB
6weeks
BALB/c
FIG. 2. Effect of Cy on the tolerance induction. Mice were treated with 20 mg/kg body weight of Cy weekly from 1 month of age. Between the ninth and tenth Cy treatment, TNP-CMC was injected. Mice were immunized and assayed in the usual way. Total amounts of serum anti-TNP antibodies were Cy-treated group. The ordinate measured at 3 and 6 weeks after the booster immunization; 0, no Cy; shows the percentage of serum antibody titers of tolerized mice to immune control mice (56 response).
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in the development of the autoimmune diseases. These autoimmune mice have different genetic backgrounds, and genes responsible for the development of the diseases seem to be different from strain to strain. These mice manifest various immunological abnormalities and in spite of different genetic backgrounds they share some immunological abnormalities in common. The most outstanding common characteristic is PBA, which is suspected to play an important role in the development of autoimmunity. Genetic studies, so far, have suggested that genes responsible for PBA dissociated from those for the development of autoimmune diseases (10, 11, 36). But the possibility still remains that PBA is coded by a part of multiple genes responsible for the development of autoimmunity. The problem should be elucidated by further investigations. Another noticeable abnormality observed in autoimmune mice is the resistance to tolerance induction to a TD antigen (11, 20-25) or hapten (26). Recently induction of immunological tolerance to human serum albumin (HSA) was studied in both autoimmune and nonautoimmune mice in our laboratory. It was revealed that T cells of autoimmune mice were refractory to a tolerizing regimen of HSA which induced immunological tolerance in normal mice. The resistant nature was suggested to be inherent to T-cell precursors in the bone marrow (our data, to be published). B-cell tolerance in autoimmune mice has also been studied but the results remain controversial. Some reported that autoimmune mice were much more resistant to tolerance induction than normal mice (26), while others reported that tolerance inducibility was not different between autoimmune and normal mice (37) or the difference was subtle (38). The conclusion drawn might be influenced by various experimental conditions such as age of animal, dose or form of tolerogen, type or timing of challenge immunization, and assay methods of immune responses. In this report TNP-CMC was used as a B-cell tolerogen, because it was known to induce strong hapten-specific B-cell tolerance in normal mice without the participation of T cells (29) which was also confirmed in our preliminary experiments. Various strains of mice were injected with TNP-CMC and immunized either with TI antigen (TNP-LPS or TNP-Ficoll) or TD antigen (TNP-KLH). It was revealed that all the mice treated were rendered tolerant when challenged with TI antigen. The duration of tolerance was also examined considering the possibility that tolerance might recover rapidly in autoimmune mice. There was no significant difference between NZB and C57BL/6 mice as to the degree of tolerance inducibility in the TI antigenic challenge at least within 3 weeks after the tolerogen injection. On the other hand, when challenge immunization was made with TNP-KLH and the secondary IgG antibody response was assessed, only autoimmune mice showed marked resistance to tolerance induction. These results could be explained at least partly by the existence of different subpopulations among TNP-specific B-cell clones; one population is responding to TI antigen and the other is responding to TD antigen. It may be reasonable to assume the existence of B-cell subpopulations or subsets classified by many parameters (39). The subpopulations or subsets might overlap with each other or consist of a part of B-cell compartments with different maturational stages. Cell populations responsible for the resistance to tolerance induction appear to be precursors of cells producing an IgG class of anti-TNP antibodies, especially those producing low affinity antibodies. Such a population might preferentially increase
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in autoimmune mice. Thus, as shown in the experiments (Tables 3 and 4) the average affinity of anti-TNP antibodies detected in TNP-CMC-injected NZB or MRL/l mice was low as compared with that of immune control mice. The fact is reminiscent of the cell selection theory proposed by Siskind and Benacerraf (40). According to this theory, cells with high affinity receptors should be more susceptible to tolerance induction. In this respect we examined tolerance inducibility in primed NZB mice. Precursors with high affinity receptors were expected to expand by the preimmunization with TNP-KLH in CFA (40). It was demonstrated that TNP-specific tolerance was attained in primed NZB mice to a similar degree to that in nonprimed normal mice (Table 5). This observation shows that B cells with high affinity receptors of autoimmune mice could be tolerized as those of nonautoimmune mice. The notion supports again the expansion of B cell clones with low affinity receptors in autoimmune mice, which are hardly tolerized. The causes of the expansion of such clones are as yet poorly understood. The most important factor might be genetic influence which is expressed as polyclonal B-cell activation (14, 16, 19). We assumed that treatment of autoimmune mice with Cy might give us some useful information, because Cy depressed PBA without affecting responsiveness of T- and B-cell function in autoimmune mice (our observations to be published). Abnormal resistance to tolerance induction in NZB mice was considerably improved by the treatment with Cy. This result might imply that tolerance induction in NZB mice became easier when PBA was suppressed by Cy. It should be noted, however, that other action of Cy might be operating in tolerance induction. Of particular interest, in the autoimmune mice the isotype of polyclonally produced immunoglobulins (16) or anti-DNA antibodies (46-48) is known to switch from IgM to IgG with the age of mice. Cy can prevent the isotype switch of autoantibodies which might be relevant to the treatment of autoimmune diseases (35 and our observation). Participation of suppressor cells should be excluded when we claim B-cell tolerance. There are a number of reports showing suppressor cells in tolerance induction (41-44). Suppressor cells were reported to decrease with the age of NZB mice, that might make the mice more resistant to tolerance induction (6, 7). Suppressors might be influenced by various experimental conditions as described previously (45) and tolerance is inducible without the participation of suppressor cells (29). Although we cannot entirely rule out the participation of suppressor T cells in the whole event, we have got results showing T cells from tolerized mice did not inhibit the response of primed B cells (data not shown). In conclusion autoimmune mice showed resistance to tolerance induction by TNP-CMC when immunized with TNP-KLH and they produce anti-TNP antibodies with lower affinities. Hence, it was suggested that B-cell clones destined to produce low affinity IgG antibodies were hard to be tolerized and such clones were expanding in autoimmune mice. Further elucidation of causes of the appearance of such clones will be expected to clarify the mechanisms of the resistance to tolerance induction and also the development of autoimmune disease. Although we could not explain the direct cause of PBA here, the development of the new methods such as combining molecular biology and immunology will help us to solve the problem in the near future.
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313
ACKNOWLEDGMENTS We are greatly indebted to Dr. Takehiko Uchiyama (Department of Microbiology, School of Medicine, Kitasato University) for providing generous amounts of TNP-CMC and for his helpful suggestion. We wish to acknowledge the helpful advice and stimulating discussions of Professor Kyoichi Kano (Department of Microbiology, State University of New York at Buffalo) and Dr. Kyoko Hayakawa (Department of Immunology, University of Tokyo). We also wish to thank Ms. Ai Kariyone for her technical assistance.
REFERENCES 1. Howie, J. B., and Helyer, B. J., Advan. Immunol. 9, 215, 1968. 2. Talal, N., and Steinberg, A. D., Current Topics Microbial. Immunol. 64, 19, 1914. 3. Moutsopoulos, H. M., Boehm-Truitt, M., Kassan, S. S., and Chused, T. M., J. Immunol. 119, 1639, 1971. 4. Manny, N. S., Datta, S. K., and Schwartz, R. S., J. Immunol. 122, 1220, 1979. 5. Cohen, P. L., and Ziff, M., J. Immunol. 119, 1534, 1977. 6. Krakauer, R. S., Waldmann, T. A., and Strober, W., J. Exp. Med. 144, 662, 1976. 7. Klasen, L. W., Krakauer, R. S., and Steinberg, A. D., J. Immunol. 119, 830, 1977. 8. Cantor, H., McVay-Bondreau, L., Hugenherger, J., Naidorf, K., Shen, F. W., and Gershon, R. K., J. Exp. Med. 147, 1116, 1978. 9. Morton, J. I., and Siegel, B. V., Proc. Nat. Acad. Sci. USA 71, 2162, 1974. 10. Raveche, E. S., Novotny, E. A., Hansen, C. T., Tojo, J. H., and Steinberg, A. D., J. Exp. Med. 153, 1187, 1981. 11. Theofilopoulos, A. N., and Dixon, F. J., Immunol. Rev. 55, 179, 1981. 12. Murphy, E. D., and Roths, J. B., In “Genetic Control of Autoimmune Disease” (N. Rose, P. Bigazzi, and N. L. Warner, Eds.), p. 207. Elsevier/North Holland, New York, 1978. 13. Andrews, B. S., Eisenberg, R. A., Theofilopoulos, A. N., Izui, S., Wilson, C. B., McConahey, P. J., Murphy, E. D., Roths, J. B., and Dixon, F. J., J. Exp. Med. 148, 1198, 1978. 14. Izui, S., McConahey, P. J., and Dixon, F. J., J. Immunol. 121, 2213, 1978. 15. Eisenberg, R. A., Theofilopoulos, A. N., Andrews, B. S., Peters, C. J., Thor, L., and Dixon, F. J., J. Immunol. 122, 2272, 1919. 16. Theofilopoulos, A. N., Shawler, D. L., Eisenberg, R. A., and Dixon, F. J., J. Exp. Med. 151, 446, 1980. 17. Eisenberg, R. A., Izui, S., McConahey, P. J., Hang, L., Peters, C. J., Theofilopoulos, A. N., and Dixon, F. J., J. Immunol. 125, 1032, 1980. 18. Izui, S., Kelley, V. E., McConahey, P. J., and Dixon, F. J., J. Exp. Med. 152, 1645, 1980. 19. Theofilopoulos, A. N., McConahey, P. J., Izui, S., Eisenberg, A., Pereira, A. B., and Creghton, W. D., C/in. Immunol. Immunopathol. 15, 258, 1980. 20. Staples, P. J., and Talal, N., J. Exp. Med. 129, 123, 1969. 21. Staples, P. J., Steinberg, A. D., and Talal, N., J. Exp. Med. 131, 1223, 1970. 22. Playfair, J. H. L., Immunology 21, 1037, 1971. 23. Fujiwara, M., Kariyone, A., and Kimura, M., J. Clin. Lab. Immunol. 3, 185, 1979. 24. Taurog, J. D., Smathers, P. A., and Steinberg, A. D., J. Immunol. 125, 485, 1980. 25. Laskin, C. A., Taurog, J. D., Smathers, P. A., and Steinberg, A. D., J. Immunol. 127, 1743, 1981. 26. Goldings, E. A., Cohen, P. L., McFadden, S. F., Ziff, M., and Vitetta, E. S., J. Exp. Med. 152, 730, 1980. 27. Inman, J. K., J. Immunol. 114, 704, 1975. 28. Kiefer, H., In “Immunological Methods” (I. Lefkovits, and B. Pernis, Eds.), p. 145. Academic Press, New York, 1979. 29. Diner, U. E., Kunimoto, D., and Diener, E., J. Immunol. 122, 1886, 1979. 30. Cunningham, A. J., and Szenberg, A., Immunology 14, 599, 1968. 3 1. Tsu, T. T., and Herzenberg, L. A., In “Selected methods in cellular immunology” (B. B. Mishell, and S. M. Shiigi, Eds.), p. 373. W. H. Freeman, San Francisco, 1980. 32. Herzenberg, L. A., Black, S. J., Tokuhisa, T., and Herzenberg, L. A., J. Exp. Med. 151, 1071, 1980.
314 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.
TSUNETSUGU
AND
FUJIWARA
Dokken, J., Weksler, M. E., and Siskind, G. W., Cell. Immunol. 55, 66, 1980. Howard, J. G., and Shand, F. L., Immunol. Rev. 43, 43, 1979. Chia, D., Levy, L., Barnett, E. V., and Carnes, W. H., Immunology 43, 57, 1981. Hirose, S., Maruyama, N., Ohta, K., and Shirai, T., J. Immunol. 125, 610, 1980. Purves, E. C., and Playfair, J. H. L., Clin. Exp. Immunol. 15, 113, 1973. Pike, B. L., Layton, J. E., Rose, N. R., and Nossal, G. J. V., Cell. Immunol. 63, 385, 1981. Kincade, S. W., Adv. Immunol. 31, 177, 1981. Siskind, G. W., and Benacerraf, B., Adv. Immunol. 10, 1, 1969. Kolsch, E., Stumpf, R., and Weber, G., Trunsplant. Rev. 26, 56, 1975. Nachtigal, D., Zan-Bar, I., and Feldman, M., Transplant. Rev. 26, 87, 1975. Weigle, W. O., Sieckmann, D. G., Doyle, M. V., and Chiller, J. M., Transplant. Rev. 26, 186, 1975. Scott, D. W., Cell. Immunol. 37, 327, 1978. Fujiwara, M., and Kariyone, A., Immunology 34, 51, 1978. Papoian, R., Pillarisetty, R., and Talal, N., Immunology 32, 75, 1977. Steward, M. W., and Hay, F. C., Clin. Exp. Immunol. 26, 363, 1976. Sawada, S., and Talal, N., J. Immunol. 122, 2309, 1979.
71, 303-314
IMMUNOLOGY
(1982)
Resistance to Tolerance Induction in B Cells of Autoimmune Mice-Abnormal Expansion of Low Affinity IgG-Producing B-Cell Population YASUKO Department
TSUNETSUGU
AND MICHIO
FUJIWARA’
of Immunology. Institute of Medical Science, University of Tokyo, Tokyo 108, Received
February
23, 1982;
Japan
accepted May 18, 1982
Several strains of mice are known to develop spontaneous autoimmune diseases like lupus erythematosus and they show various immunological abnormalities as well. Despite different genetic backgrounds, they manifest various immunological abnormalities in common, e.g., polyclonal B-cell activation (PBA) and resistance to tolerance induction. To elucidate mechanisms of the development of autoimmunity, tolerance inducibility was examined in autoimmune and normal mice using trinitrophenylated carboxymethyl cellulose (TNP-CMC) as tolerogen which is known to induce TNP-specific B-cell tolerance without the participation of T cells. NZB and MRL/Mp-lpr/lpr mice were used as autoimmune mice and C57BL/6, BALB/c, and MRL/Mp-+/+ mice as nonautoimmune mice. When TNP-CMC-injected mice were challenged with T-independent antigens, all of the mice tested were shown to be tolerant. In contrast, when TNP-CMC-injected mice were challenged with T-dependent antigen and secondary IgG responses were assessed, autoimmune mice showed rather hyperreactivity, while nonautoimmune mice showed hyporesponsiveness. Cyclophosphamide improved this defective tolerance inducibility. By the solid-phase radioimmunoassay it was revealed that average affinity of serum anti-TNP antibodies produced in TNP-CMC-injected mice was low. Such low affinity antibodies were produced in large amount in autoimmune mice. Hence, it was suggested that B-cell clones destined to produce low affinity IgG antibodies were responsible for the resistance to tolerance induction and such clones were expanding in autoimmune mice.
INTRODUCTION New Zealand mice (NZB and (NZB X NZW)F, mice) have been widely utilized as an animal model of hemolytic anemia or systemic lupus erythematosus (SLE) and have offered much information on the etiopathogenesis of autoimmune diseases (1, 2). These mice have been revealed to manifest various immunological abnormalities in the functions of immunocompetent cells: hyperactivity of B cells (6, 7) or distorted regulatory circuits (8). These abnormalities seem to be predetermined at the precursors of both T and B cells (9) and controlled by multiple genes (10, 11). Recently MRL/Mp-lpr/fpr (MRL/l) and BXSB (especially male) mice, displaying spontaneous lupus nephritis, were developed by Murphy and Roths (12) and studies using them have shed new light on the etiopathogenesis of SLE (13’ Address correspondence University of Tokyo, 4-6-1,
to M. Fujiwara, Department of Immunology, Shirokanedai, Minato-ku Tokyo 108, Japan.
Institute
of Medical
Science,
303 0008-8749/82/120303-12%02.00/O Copyright 0 1982 by Academic Press, Inc. All right8 of reproduction in any form rcscrved.
304
TSUNETSUGU
AND FUJIWARA
19). These autoimmune strains of mice have different genetic backgrounds (e.g., haplotype of histocompatibility gene complex and genes participating in the development of SLE-like syndrome). Despite the difference in the genetic backgrounds, they manifest some immunological abnormalities in common. Increased polyclonal B cell activation is the most prominent feature ( 14, 16, 19) and its role in the development of murine lupus has been investigated extensively. As another immunological abnormality, the resistance to tolerance induction has been pointed out (20-26). If autoimmunity is assumed to come out from the escape of selftolerance, a clue to the pathogenesis of autoimmune diseases could be obtained by studying the mechanisms involved in defective tolerance induction. In this report, tolerance induction in B cells was studied by using trinitrophenylated carboxymethyl cellulose (TNP-CMC) as a tolerogen. It was revealed that tolerance in TNP-specific B cells was not attained when the TNP-CMC-treated autoimmune mice were immunized with a T-dependent (TD) antigen, TNP-keyhole limpet hemocyanin (KLH), and secondary anti-TNP IgG responses were assessed. The implication of the resistance to tolerance induction was discussed in relation to the development of autoimmunity. MATERIALS
AND METHODS
Mice. NZB, MRL/l, and MRL/Mp-+/+ (MRL/n) mice were maintained at our laboratory animal facilities. C57BL/6 and BALB/c mice were obtained from the Animal Breeding Unit of the Institute of Medical Science, University of Tokyo. Female mice were used at 6-10 weeks of age. Antigens. As T-independent (TI) antigens, TNP-lipopolysaccharide (TNP-LPS) and TNP2,8-Ficoll were used. The subscript indicates mean number of TNP groups per 400,000 daltons of Ficoll. TNP-LPS was donated by Dr. T. Tadakuma (Department of Microbiology, School of Medicine, Keio University). Ficoll (Pharmacia, Uppsala) was trinitrophenylated according to the method described by Inman (27). These antigens were dissolved in phosphate-buffered saline (PBS). As a TD antigen, TNP-KLH was used. KLH (Calbiochem, San Diego, Calif.) was conjugated with TNP as described by Kiefer (28). A preparation of TNP,,-KLH (29 TNP groups per 100,000 daltons of KLH) was either emulsified with complete Freund’s adjuvant (CFA) or dissolved in PBS. Tolerance induction and challenge immunization. CMC was obtained commercially (Wako Junyaku, Tokyo). TNP-CMC was made as described by Diner et al. (29) and used as a tolerogen to induce TNP-specific B-cell tolerance (Hapten density of TNP-CMC used here was 9-l 1). A group of mice was injected with 0.5 mg of TNP-CMC ip three times every other day and control group of mice was left untreated. All mice were challenged with the antigen usually 3 days after the final tolerogen injection. TNP-LPS or TNP2,*- Ficoll was injected ip at a dose of 1 or 10 pg, respectively and mice were sacrificed 4 days later. When mice were challenged with TD antigen, emulsion of TNP,,-KLH in CFA was injected SCat a dose of 100 pg and the same dose of TNP,,-KLH was boosted ip 16 days later. Mice were sacrificed 5 days later. Sheep red blood cells (SRBC) were injected simultaneously to test the specificity of the tolerance. Cyclophosphamide (Shionogi Seiyaku, Osaka) was dissolved in saline immediately before use and injected as described in the text.
DEFECTIVE
B-CELL TOLERANCE
IN AUTOIMMUNE
MICE
305
Assay for anti-TNP plaque-forming cells (PFC). Numbers of anti-TNP PFC in the spleen were determined by the technique of Cunningham and Szenberg (30) using TNP-horse red blood cells (TNP-HRBC) as target. In the indirect (IgG) PFC assay, rabbit anti-mouse immunoglobulin was added for the development of plaques. The lot of serum used here was preliminarily checked to have little blocking effect on the appearance of IgM PFC. Numbers of indirect PFC were obtained by subtracting those of direct PFC. Serum anti-TNP antibodies. Levels of serum antibodies were measured by the solid-phase radioimmunoassay according to the method of Tsu and Herzenberg (3 1). Briefly, using 96-well microtiter plate (Tomy Seiko, Tokyo) wells were coated with the antigen by adding 50 ~1 of 100 pg/ml of TNP-bovine serum albumin (TNP-BSA) (either TNPS,,-BSA or TNP,,-BSA) for 1 hr at room temperature. After wells were washed three times with PBS containing 1% BSA and 0.1% NaN3, 40 ~1 of adequately diluted test or standard sera was added to the coated wells and incubated for 1 hr. Then, wells were washed similarly and 40 ~1 of ‘*‘I-labeled purified rabbit anti-mouse immunoglobulin was incubated for another 1 hr. Finally, wells were thoroughly washed, dried, and cut into plastic tubes. Radioactivity of each well was counted with the Autogamma Scintillation Counter (Packard Instrument, Downers Glove, Ill.). In each assay ‘*‘I-counts bound to wells were converted to units by comparison with the binding curve obtained from the standard anti-TNP serum. In this study antibody titer in the standard serum was arbitrarily determined as 100 units. The ratio of antibody units bound to TNP5,,-BSA per those bound to TNPs7BSA was represented as the average affinity of anti-TNP antibodies as validated by Herzenberg et al. (32). Statistical analysis. Results of PFC assay were expressed as geometric mean $ standard error (SE). Levels of serum antibodies were shown as arithmetic mean f standard deviation (SD). The percentage response was calculated as follows:
% Response =
mean PFC numbers or serum titers of tolerant mice x 100. mean PFC numbers or serum titers of immune control mice RESULTS
Tolerance Induction in the Primary IgM Anti-TNP
Response
NZB and C57BL/6 mice were tolerized with TNP-CMC as described under Materials and Methods and challenged with 10 pg of TNP,,,-Ficoll on Day 3 of the final TNP-CMC injection. Anti-TNP PFC in the spleen were assayed 4 days later. The representative results are shown in Fig. 1. Both strains of mice were deeply rendered tolerant; the percentage response was 0.8% in NZB and 0.3% in C57BL/6 mice, respectively. The tolerance was specific since there was no significant difference in anti-SRBC response between tolerized and control mice (Fig. 1, dotted column). In the following experiments no significant difference was observed in anti-SRBC PFC response between tolerized and control group of mice. Therefore, the results were omitted from the figure and tables. As tolerance inducibility was reported to change with the age of mice (20-21, 23), the same tolerizing schedule was carried out in 6- to 8-month-old NZB mice. The percentage response in these mice was 4.3%.
306
TSUNETSUGU
AND FUJIWARA
PFC/SPLEEN
(x109
15-
10: : : :
5-
~
:.
: : : : . :.
i
3
::.::; j:.:. .:.:.: .. .. .. :::::: :.:.:. .:.:.: :::::: .A.. ;:::: :::.:. .:.:.: 55.. :.:.:. _..... .:.:.: . ..A :.:i’. .:.:.: ::g ... . .. i’.:.: :.:.:. .:.:.: .,.... :.:.:’ .:.:.: :::::: ... ..
TOLERIZEO
NZB
CONTROL
(2M)
TOLERIZEO
CONTROL
C57Bt/6Q~,
TCtERi
NZB(6-EM)
FIG. 1. NZB and C57BL/6 mice were tolerized with TNP-CMC at the age indicated in parentheses. Three days later 10 pg of TNP*,s-Ficoll and 2 X 10’ of SRBC were injected and direct PFC assay was done; 0, anti-TNP PFC; q , anti-SRBC PFC.
The results of the challenge immunization with TNP-LPS were similar to those with TNP,,,-Ficoll; the percentage response was 0.6% in NZB and 0.8% in C57BL/ 6 mice, respectively (Table 1). When the challenge was postponed until Day 11 or 20 after the TNP-CMC injection, percentage response became slightly higher. But again both strains of mice were tolerized almost to the same degree, 3.2-6.0s response in NZB and 1.0-2.7s response in C57BL/6 mice. Thus, TNP-CMC induced stable tolerance, irrespective of strain and age of mice, to the subsequent challenge immunization with TI antigen. Tolerance Induction
in the Secondary IgG Anti-TNP
Response
In the following experiments, it was examined whether TNP-CMC induced tolerance in the secondary anti-TNP response by a TD antigen or not. As a preliminary experiment, C57BL/6 mice were injected with TNP-CMC three times every other day and primed with TNP,,-KLH in CFA 3 days after the final tolerogen injection. The mice were boosted 16 days later and splenic IgG anti-TNP PFC were enumerated. The percentage response was 4.8%. When the priming was postponed until 3 or 6 weeks later, the responsiveness recovered to 8.0 or 16%, respectively.
DEFECTIVE
B-CELL TOLERANCE TABLE
IN AUTOIMMUNE
307
MICE
1
Tolerance Induction in the Primary IgM Anti-TNP
Response’
Direct PFC/spleen’ (% response) Day ofb challenge 3
TNP-CMC + + -
Antigen
NZB
TNP-LPS
427 72,500 1,400 171,000
TNP-Ficoll
11
+ -
TNP-LPS
20
+ + -
TNP-LPS
z : 5 r
C57BL/6
1.37 (0.6) 1.16 1.32 (0.8) 1.06
327 46,000 364 135,000
4,760 5 1.41 (5.3) 90,200 $ 1.15 2,420 40,500 5,310 168,000
TNP-Ficoll
5 r 2 5
z 5 5 3
1.14 (0.8) 1.20 1.19 (0.3) 1.16
1,150 5 1.50 (2.7) 41,900 5 1.28
1.41 (6.0) 1.21 1.36 (3.2) 1.05
331 33,900 2,600 180,000
5 5 5 5
1.61 (1.0) 1.38 1.52 (1.4) 1.12
a NZB and C57BL/6 mice were injected with TNP-CMC as described under Materials and Methods. TNP-CMC-injected and control untreated mice were immunized with either TNP-LPS or TNP-Ficoll and numbers of direct anti-TNP PFC were enumerated 4 days later. Each group consists of five mice. b The days are indicated when TNP-LPS or TNP-Ficoll was immunized after the final TNP-CMC injection. ’ Geometric means of PFC numbers are shown with standard error.
Next, tolerance inducibility in this experimental system was examined in autoimmune mice, comparing with normal strains of mice. NZB, C57BL/6, and BALB/c mice were tolerized and immunized as described above. The results obtained are summarized in Table 2. A remarkable difference was observed between normal and autoimmune mice, that was, while C57BL/6 and BALB/c mice were rendered tolerant (4.8 and 1.3% response, respectively), TNP-CMC-injected NZB mice showed augmented response ( 132%) as compared with immune control mice. TABLE
2
Tolerance Induction in the Secondary IgG Anti-TNP Strain NZB
C57BL/6
BALB/c
Number of mice
TNP-CMC
Response”
Indirect PFC/spleen
% Response 132
(14) (13)
+ -
389,000 5 1.50 295,000 2 1.24
(12)
+ -
31,000 F 1.53 653,000 F 1.20
4.8
(11) (10) (10)
+ -
6,780 5 1.94 504,000 5 1.25
1.3
a Tolerized and untreated mice were immunized with TNP-KLH Indirect anti-TNP PFC were enumerated 5 days later.
in CFA and boosted 16 days later.
TSUNETSUGU
AND FUJIWARA
TABLE
3
Affinities of Antibodies in Tolerized and Immune Control Mice” Serum IgG antibodies* (Unit) TNPCMC
Indirect PFC/spleen
NZB
+ -
577,000 5 1.81 442,000 $ 1.22
C57BLJ6
+ -
12,200 5 2.50 833,000 5 1.37
BALB/c
+ -
1,610 5 2.41 608,000 5 1.20
Strain
(W TNP,-BSA
Affinity (l/h)
24.6 f 7.96 233.9 + 63.0
62.5 + 19.3 154.8 + 37.3
0.40 + 0.06 1.44 + 0.23
(1.5)
2.9 + 0.93 168.9 + 42.0
11.2 f 2.88 179.9 + 33.4
0.29 f 0.05 0.91 + 0.11
(0.3)
2.1 f 185.9 f
8.7 f 5.94 200.6 + 27.9
0.53 f 0.24 0.97 f 0.12
response) (131)
TNP,-BSA
0.68 4.69
’ Mice were tolerized and immunized with TNP-KLH as usually. At the time of sacrifice blood was collected and serum anti-TNP antibodies were estimated. Each group consists of five mice. * In the solid-phase radioimmunoassay, both TNPs,,-BSA (TNP,-BSA) and TNPs,-BSA (TNP,,-BSA) were used as the antigens. Average affinities of antibodies were expressed with the ratio of the binding (see Materials and Methods).
Assuming that B cells of NZB mice showed higher threshold for the tolerance induction, these mice were injected with larger amounts of the tolerogen. It was still difficult to induce tolerance; when 2.0 mg of TNP-CMC was injected three times every other day, the percentage response was 104% and when three times 0.5 mg of TNP-CMC injection as usual was repeated for 3 weeks, the responsiveness reduced to 34%, but not to the level of normal mice (less than 10% response). In another experiment, sera were collected at the time of PFC assay to measure the levels of serum antibodies by solid-phase radioimmunoassay and their average affinities were estimated (Table 3). In the PFC assay, NZB mice showed 131% response, in contrast with C57BL/6 and BALB/c mice which were rendered unresponsive (1.5 and 0.3% response, respectively). Estimation of anti-TNP antibodies gave rise to somewhat different results from the PFC assay. Levels of anti-TNP antibodies in tolerized NZB mice never exceeded those of control mice; percentage response in NZB mice was 40% in this experiment (62.5/154.8). Average affinity of antibodies of tolerized group was lower than those of immune control group in all strains tested. The fact seems to indicate that the augmented response observed in tolerized NZB mice was due to the expansion of B-cell clones differentiating to produce antibodies with low affinities. Tolerance inducibility in MRL/l mice was examined similarly, comparing with MRL/n mice. Like NZB, MRL/l mice showed enhanced percentage PFC response (174%), while MRL/n mice showed 9.0% response (Table 4). The average affinities of antibodies of both strains were lower than those of immune control group. Induction of TNP-CMC
Tolerance in Primed NZB Mice
From the previous results, it was suggested that tolerance by the TNP-CMC was selectively attained in B-cell population capable of producing higher affinity antiTNP antibodies. In the next experiment it was examined whether TNP-specific
DEFECTIVE
B-CELL
TOLERANCE
IN AUTOIMMUNE
TABLE Tolerance
Induction
in the Secondary
4
IgG
Anti-TNP
Response Serum
Strain MRL/l
MRL/n
TNPCMC
Indirect PFC/spleen
+
1,570,OOO
-
901,000
+
77,900
-
869,000
0 See footnotes
to Table
(% response)
z 1.92 z 1.74 r
1.71 5 1.67
3. Each group
TNP,-BSA
(174)
+ 61.0
108.0
k 42.1
5.82 f 168.7
in MRL
IgG
Mice”
antibodies
(Unit) Affinity (l/W
TNP,-BSA
107.8
(9.0)
consists
309
MICE
1.98
1 78.0
153.3
f 83.3
0.59 f 0.06
96.6 f 38.1
1.05 + 0.13
20.2 3~ 8.23
0.33 2 0.06
143.8 f 55.7
1.01 10.12
of five mice.
clones with higher affinity receptors can be tolerized or not. NZB and C57BL/6 mice were primed with TNP,,-KLH and 3 weeks later TNP-CMC was injected as before. Mice were boosted 3 days after the final TNP-CMC injection. Most of C57BL/6 mice died probably due to anaphylactic shock shortly after the TNPCMC injection. On the other hand, NZB mice survived and became tolerized to the degree of 3% response (Table 5). Efect of Cyclophosphamide
(Cy) on the Tolerance Induction
Cy has been used to intensify the tolerance induction (34) or to treat murine lupus (35). Moreover, the drug seems to depress the polyclonal activation of B cells (our data to be published). The following experiment examined if Cy affected tolerance induction in B cells of NZB mice. NZB mice were injected with 150 mg of Cy per kg of body weight of mouse (mg/kg body wt) 1 day before TNP-CMC injection. The augmented response (338%) was depressed to 34.5% by the preinjection of Cy (Table 6). In another experiment 20 mg/kg body wt of Cy was injected weekly, starting from 1 month of age for 10 weeks. Before the last Cy injection NZB mice were given TNP-CMC and 2 days after the last Cy injection challenged with TNPz9KLH in the usual way. Serum antibodies were measured by the binding of antisera to TNPS,-BSA. It was shown that Cy suppressed the antibody production in TNP-
TABLE Tolerance TNP-CMC
+ -
5
Induction
in Primed
Indirect
PFC/spleen
11,100 402,000
NZB
Mice” % Response
r 1.35 5 1.23
0 Mice were primed with TNP-KLH in CFA 3 weeks previously, 3 days later. Each group consists of five mice.
2.7
injected
with TNP-CMC,
and boosted
310
TSUNETSUGU
AND TABLE
Effect
CY
+ -
TNP-CMC
of Cy on the Tolerance Indirect
6 Induction
by TNP-CMC”
PFC/spleen
+
153,000
-
443,000
+
991,000
-
293,000
’ Cy (150 mg/kg body wt) was injected and assayed in the usual way.
FUJIWARA
(% response)
5 2.21 5 1.22
(34.5)
5 1.21 5 1.51
(338)
ip 24 hr before
Affinity 0.21 f 0.00 0.88 + 0.18 0.44 k 0.04 0.76 f 0.08
tolerance
induction.
Mice
were
challenged
CMC-injected NZB mice from 47 to 3.7% response and from 53 to 19.5% response, when assayed at 3 weeks and 6 weeks after the challenge, respectively (Fig. 2). DISCUSSION NZB, (NZB X NZW)F1, MRL/l, and male BXSB mice spontaneously develop lupus-like syndrome and have been widely utilized to elucidate mechanisms involved 3weeks
NZB
BALB/c
-
NZB
6weeks
BALB/c
FIG. 2. Effect of Cy on the tolerance induction. Mice were treated with 20 mg/kg body weight of Cy weekly from 1 month of age. Between the ninth and tenth Cy treatment, TNP-CMC was injected. Mice were immunized and assayed in the usual way. Total amounts of serum anti-TNP antibodies were Cy-treated group. The ordinate measured at 3 and 6 weeks after the booster immunization; 0, no Cy; shows the percentage of serum antibody titers of tolerized mice to immune control mice (56 response).
DEFECTIVE
B-CELL TOLERANCE
IN AUTOIMMUNE
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in the development of the autoimmune diseases. These autoimmune mice have different genetic backgrounds, and genes responsible for the development of the diseases seem to be different from strain to strain. These mice manifest various immunological abnormalities and in spite of different genetic backgrounds they share some immunological abnormalities in common. The most outstanding common characteristic is PBA, which is suspected to play an important role in the development of autoimmunity. Genetic studies, so far, have suggested that genes responsible for PBA dissociated from those for the development of autoimmune diseases (10, 11, 36). But the possibility still remains that PBA is coded by a part of multiple genes responsible for the development of autoimmunity. The problem should be elucidated by further investigations. Another noticeable abnormality observed in autoimmune mice is the resistance to tolerance induction to a TD antigen (11, 20-25) or hapten (26). Recently induction of immunological tolerance to human serum albumin (HSA) was studied in both autoimmune and nonautoimmune mice in our laboratory. It was revealed that T cells of autoimmune mice were refractory to a tolerizing regimen of HSA which induced immunological tolerance in normal mice. The resistant nature was suggested to be inherent to T-cell precursors in the bone marrow (our data, to be published). B-cell tolerance in autoimmune mice has also been studied but the results remain controversial. Some reported that autoimmune mice were much more resistant to tolerance induction than normal mice (26), while others reported that tolerance inducibility was not different between autoimmune and normal mice (37) or the difference was subtle (38). The conclusion drawn might be influenced by various experimental conditions such as age of animal, dose or form of tolerogen, type or timing of challenge immunization, and assay methods of immune responses. In this report TNP-CMC was used as a B-cell tolerogen, because it was known to induce strong hapten-specific B-cell tolerance in normal mice without the participation of T cells (29) which was also confirmed in our preliminary experiments. Various strains of mice were injected with TNP-CMC and immunized either with TI antigen (TNP-LPS or TNP-Ficoll) or TD antigen (TNP-KLH). It was revealed that all the mice treated were rendered tolerant when challenged with TI antigen. The duration of tolerance was also examined considering the possibility that tolerance might recover rapidly in autoimmune mice. There was no significant difference between NZB and C57BL/6 mice as to the degree of tolerance inducibility in the TI antigenic challenge at least within 3 weeks after the tolerogen injection. On the other hand, when challenge immunization was made with TNP-KLH and the secondary IgG antibody response was assessed, only autoimmune mice showed marked resistance to tolerance induction. These results could be explained at least partly by the existence of different subpopulations among TNP-specific B-cell clones; one population is responding to TI antigen and the other is responding to TD antigen. It may be reasonable to assume the existence of B-cell subpopulations or subsets classified by many parameters (39). The subpopulations or subsets might overlap with each other or consist of a part of B-cell compartments with different maturational stages. Cell populations responsible for the resistance to tolerance induction appear to be precursors of cells producing an IgG class of anti-TNP antibodies, especially those producing low affinity antibodies. Such a population might preferentially increase
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in autoimmune mice. Thus, as shown in the experiments (Tables 3 and 4) the average affinity of anti-TNP antibodies detected in TNP-CMC-injected NZB or MRL/l mice was low as compared with that of immune control mice. The fact is reminiscent of the cell selection theory proposed by Siskind and Benacerraf (40). According to this theory, cells with high affinity receptors should be more susceptible to tolerance induction. In this respect we examined tolerance inducibility in primed NZB mice. Precursors with high affinity receptors were expected to expand by the preimmunization with TNP-KLH in CFA (40). It was demonstrated that TNP-specific tolerance was attained in primed NZB mice to a similar degree to that in nonprimed normal mice (Table 5). This observation shows that B cells with high affinity receptors of autoimmune mice could be tolerized as those of nonautoimmune mice. The notion supports again the expansion of B cell clones with low affinity receptors in autoimmune mice, which are hardly tolerized. The causes of the expansion of such clones are as yet poorly understood. The most important factor might be genetic influence which is expressed as polyclonal B-cell activation (14, 16, 19). We assumed that treatment of autoimmune mice with Cy might give us some useful information, because Cy depressed PBA without affecting responsiveness of T- and B-cell function in autoimmune mice (our observations to be published). Abnormal resistance to tolerance induction in NZB mice was considerably improved by the treatment with Cy. This result might imply that tolerance induction in NZB mice became easier when PBA was suppressed by Cy. It should be noted, however, that other action of Cy might be operating in tolerance induction. Of particular interest, in the autoimmune mice the isotype of polyclonally produced immunoglobulins (16) or anti-DNA antibodies (46-48) is known to switch from IgM to IgG with the age of mice. Cy can prevent the isotype switch of autoantibodies which might be relevant to the treatment of autoimmune diseases (35 and our observation). Participation of suppressor cells should be excluded when we claim B-cell tolerance. There are a number of reports showing suppressor cells in tolerance induction (41-44). Suppressor cells were reported to decrease with the age of NZB mice, that might make the mice more resistant to tolerance induction (6, 7). Suppressors might be influenced by various experimental conditions as described previously (45) and tolerance is inducible without the participation of suppressor cells (29). Although we cannot entirely rule out the participation of suppressor T cells in the whole event, we have got results showing T cells from tolerized mice did not inhibit the response of primed B cells (data not shown). In conclusion autoimmune mice showed resistance to tolerance induction by TNP-CMC when immunized with TNP-KLH and they produce anti-TNP antibodies with lower affinities. Hence, it was suggested that B-cell clones destined to produce low affinity IgG antibodies were hard to be tolerized and such clones were expanding in autoimmune mice. Further elucidation of causes of the appearance of such clones will be expected to clarify the mechanisms of the resistance to tolerance induction and also the development of autoimmune disease. Although we could not explain the direct cause of PBA here, the development of the new methods such as combining molecular biology and immunology will help us to solve the problem in the near future.
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ACKNOWLEDGMENTS We are greatly indebted to Dr. Takehiko Uchiyama (Department of Microbiology, School of Medicine, Kitasato University) for providing generous amounts of TNP-CMC and for his helpful suggestion. We wish to acknowledge the helpful advice and stimulating discussions of Professor Kyoichi Kano (Department of Microbiology, State University of New York at Buffalo) and Dr. Kyoko Hayakawa (Department of Immunology, University of Tokyo). We also wish to thank Ms. Ai Kariyone for her technical assistance.
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