the frequency of lps-responsive b cells to autologous and heterologous thyroglobulin

CELLULAR IMMUNOLOGY 132, 94-101 (1991)

The Frequency of LPS-Responsive B Cells to Autologous and Heterologous Thyroglobulin’

RUDOLF C. KUPPERS

The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21205

Received Jul-v IO. 1990; accepted August 26, 1990

The frequency of precursors within the mouse splenic B cell pool, reactive with mouse thyroglobulin (mTg) was estimated using a limiting dilution assay system. The mean frequency was found to be l/3900 B ceils. The results provide a minimal estimate of the frequency of mTg- reactive B cells. The frequency of mTg-reactive B cells was not influenced by the MHC locus, as both high- and low-responder strains showed similar frequencies. While the frequency of B cells reactive to human Tg was found to be similar to that reactive to mTg, only 20% of the mTg- reactive clones also cross-react with human Tg. Similarly, only 30% of huTg reactive clones were found to react with mTg. Therefore, a large proportion of Tg-reactive antibodies are restricted to self-determinants and not determinants to conserved regions of the Tg molecule. 0 1991 Academic

Press, Inc.

INTRODUCTION

The presence of self-reactive lymphocyte clones in normal individuals can be demonstrated using animal models. Autoimmune disease of both the systemic- (SLE-like) and organ-specific types can be induced experimentally using a variety of approaches. The murine experimental autoimmune thyroiditis model (EAT) is such a model. Dis- ease is induced by immunization with mouse thyroglobulin (mTg) in adjuvant (1) or with LPS (2) or can also be induced by the administration of soluble mTg at low doses over an extended period of time (3). These studies demonstrate that mTg-specific lymphocytes capable of reacting to this self antigen are present in the normal lymphocyte pool. Fundamental questions are whether individuals prone to autoimmunity might have more self-reactive cells than normal individuals, and whether these cells arise from the normal lymphocyte pool or are an aberrant population. In the former case, autoreactive cells should be found, perhaps commonly, in all individuals, whereas the latter might argue that these cells would be rare in the repertoire.

Estimates of anti-dsDNA reactive B cell precursors in mice has suggested a frequency of about 1 per 500 spleen cells (4). Interestingly, they are found at similar frequencies in normal as well as the SLE-prone MRL and NZB mouse strains. Studies in the EAT model have given some insight into the frequency of thyroid-specific cells in normal animals. Clagett and Weigle (5) reported the frequency of nonprimed lymphocytes capable of binding radiolabeled mTg as l/ 1470. The lymphocytes detected in this type

I Supported by U.S. Public Health Service Grants AR 3 I632 and AI 2 1088.

94

0008-8749/9 1 $3.00 Copyright 0 1991 by Academic Press, Inc. AU ri&tr of reproduction m any form reserved.

FREQUENCY OF B CELLS TO THYROGLOBULIN 95

of binding assay are generally considered to be B cells, supporting a lack of tolerance to Tg in the B cell population. Charriere and Salmero have also reported the frequency of thyrocyte-specific cytotoxic T cells at l/5440 (6) demonstrating the presence of T cell precursors in normal animals capable of recognizing self-antigen, though at a much lower frequency than that of B cells.

In this paper we have examined the frequency of polyclonally activated B cells which produce antibodies reactive with autologous and heterologous Tg.

METHODS AND MATERIALS

Mice. All the strains used were obtained from the Jackson Laboratories (Bar Har- bor, ME).

Limiting dilution assuyjbr antibody forming cells. The limiting dilution assay was performed as described previously (4, 7). Briefly, NH&l-treated splenic lymphocytes at appropriate dilutions were plated into 96-well flat-bottom microculture plates (NUNC, Roskilde, Denmark) in groups of 24 wells/dilution at a final volume of 200 ~1. Irradiated (2000 R) thymus and/or peritoneal exudate cells were used as feeder cells. Feeders cells were added at 2 X 105/well. The medium used was (Y MEM with nucleic acids (GIBCO, Long Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT), 2-mercaptoethanol (5 X 10e4 M), glutamine (2 mM) and penicillin/streptomycin. B cells were activated by the addition of 50 pg/ml of LPS (Escherichia coli 0 1 1 I :B4, Sigma Chemical Co., St. Louis, MO). Control wells contained feeder cells alone.

After incubation at 37”C, in 5% CO2 for 7-9 days. the culture supernatants were collected and tested for specific antibody in an ELISA assay as described below.

ELISA ussuj.ji,r antibody production. The basic ELISA assay used has been described before (8). Briefly, EIA plates (Linbro, Flow Lab., McLean, VA) were coated with antigen in carbonate buffer at pH 9.8. The antigens used included mTg (2 pg/ml), huTg (2 pg/ml), and TNP-BSA (2 pg/ml). After coating (5 hr, room temperature), the plates were blocked with 1% BSA for 2 hr, and then culture supernatants were added (50-100 PI/well). The plates with supernatants were incubated overnight at 4°C. The plates were washed and 100 ~1 of an alkaline phosphatase conjugated goat anti-mouse IgG (H + L chains) (Sigma Chemical Co., St. Louis, MO) was added. After 2-3 hr, the plates were washed and substrate (p-nitrophenol phosphate, 1 mg/ ml) was added. Plates were read in an ELISA reader (MR 600, Dynatech Sci., Cam- bridge, MA) after 4 hr incubation and again after further incubation overnight at 4°C. Normally. there was little difference in the results between these time points.

Analysis qf’duta. The mean OD of the control wells, feeder cells alone, plus two SEM was used as the threshold value for positive wells. Typically, the frequency of detectable antibody producing cells to BSA (used to block the ELISA plates) was <I/ 60,000 spleen cells; therefore, no correction for these B cells has been made. The data were analyzed statistically using the minimal X2 method to give a 95% confidence level, and the X-P value (9). The precursor frequency of the cells was determined at Fo = 0.37 (10). Only experiments with P values of c.01 are presented.

To convert frequency determinations from “frequency per spleen cell” to “frequency per B cell,” values were adjusted assuming that 40% of splenic cells are B lymphocytes.

RESULTS

The goal of these experiments was to obtain an estimate of the frequency of B cells producing antibodies reactive with mTg. Two major conditions must be met before

96 RUDOLF C. KUPPERS

frequency estimates can be made using the Poisson distribution analysis, i.e. (i) cooperative/suppressive interactions within the cultures are not rate limiting, and (ii) only one cell type is limiting ( 10). That these conditions have been met is demonstrated in Fig. 1.

C57Bl/lO spleen cells were cultured as described and the supernatants assayed for antibodies reactive with TNP-BSA and mTg. The precursor frequencies of splenic cells producing detectable antibody to TNP-BSA and mTg were determined as described under Methods. The calculated X-P values (P < 0.95) indicate that the data reasonably fit a straight line arguing against cooperative/suppressive interactions in the culture system. Furthermore, the line intercepts the Y-axis at 1.0 showing that only a single cell is limiting. Therefore, the conditions for using the Poisson analysis have been met. Experiments not meeting these criteria have not been included.

In several experiments, the precursor frequency of TNP-reactive cells was determined to assess the response to a heterologous antigen. It is clear from other studies that the frequency of TNP-reactive B cells is high. The mean frequency in the experiments here was l/l9 10 (range: 478-4637) spleen cells. The range here is consistent with other reports (4, 11).

In Table 1, frequency determinations for mTg-reactive cells have been compiled, representing the results of 10 experiments with 16 individual determinations. The mean of all frequency determinations for LPS-induced, mTg specific B cells was 1 precursor per 9750 spleen cells, or l/3900 B cells. A wide range of frequencies from

Cells/Well X 10M3

FIG. 1. Frequency determination of TNP-BSA and mTg antibody producing BlO splenic cells. Spleen cells were placed in microtiter plate wells at the concentrations indicated in groups of 24, together with feeder cells and LPS. After 6 days the culture supernatant from each well was assayed in an ELISA assay for antibody to TNP-BSA or mTg. Positive wells were those whose OD were above the mean of feeder cell alone control wells plus two standard deviations. Frequency was determined at 0.37 negative wells (---). Cl, TNP-BSA; n , mTg. (- - -) represents a 95% confidence level.


TABLE I

Frequency of B Cells Reactive to MTg in High- and Low-Responder Mice”

Strain Expt No Frequency

high responders Frequency

low responders

C3H

CBA DBA/2 BL6 C3HSW BIO BIO.BR BIO BIO.BR BL6 BIO.BR Bl0 BIO.BR BIO BlO.D2

II III IV V

VI

VII

Vlll

IX

X

Mean:’ 9,656 + 5460

20.326

6.594 2.012

10.532 15.158

9.690 1.61 I

9,897

Il.478

j-356

I I .926

8.07 I

11,187

7.955

I I xl66 1153 A

9,825 i- 2380

” The frequency of mTg-reactive antibody producing cells was determining by polyclonally activating splenic B cells under limiting dilution culture conditions. Culture supernatants were tested for mTg reactivity using an ELISA assay as described under Methods and Materials.

h Frequency = l/number k SEM.

the different experiments was found in these experiments. Possible reasons for this broad range will be discussed later.

It is known that the antibody response to mTg is strongly influenced by genes of the major histocompatibility region. The H-2k.q.S strains are high responder haplotypes, whereas the H-2h.d haplotypes are associated with low responsiveness (12). The I-A region has been shown to be the main regulatory locus within the H-2 region (13). The strains of mice examined in Table 1 are of both the high- and low-responder haplotypes. When high and low responder haplotypes were compared, there was no significant difference in their mean precursor frequencies (Table 1). The high-responder strains had a mean frequency of 1 per 9655 spleen cell and the low responder strain I per 9825 spleen cells. This finding would argue that the H-2 region does not influence the size of the pool of LPS-activated B cell precursors to mTg.

The lack of an effect of the H-2 region on the B cell precursor pool size is further supported when B 10 congenic mouse strains are compared (Table 1, experiments VII- X). Because these animals differ at the H-2 locus, but share the same background genes, the effect of non-MHC genes on the response is eliminated. The BlO/B6 and B 10.D2 low-responder strains are of the H-2’ and H-2d haplotypes, respectively. B lO.BR is H-2k, a high responder haplotype. Since there is a wide variation in the frequency of mTg-specific B cells between experiments, the most meaningful way to examine the data is to compare results within an experiment rather than by comparing the means. In experiments VII, VIII, and X, the frequencies are essentially the same between the high- and low-responder strains within the technical limits of this type of


experiment. There is, however, a large difference between the B 1 O.BR and B 10 strains in Experiment IX, the high responder having a higher precursor frequency. Overall, however, the results from these four experiments do not consistently show a significant difference between low- and high-responder strains within the B 10 congenic mice, or when all strains were pooled. Therefore, the B cell precursor pool size does not appear to be regulated by the H-2 locus.

The response to heterologous thyroglobulin is more typical to that of a response to a foreign antigen (14-17). That cross-reactivity between mTg and heterologous Tg does exist is evidenced by serological cross-reactivity and the ability of heterologous Tg to induce EAT in mice (1, 15- 18). One might expect that since humans are phy- logenically rather remote from mice, little tolerance to human epitopes would have occurred. The frequency of B cells reactive to human determinants of Tg would be expected to be higher than that to mouse determinants. This comparison was made and the frequencies of splenic cell reactive to mTg and huTg are shown in Table 2. Clearly this expectation was not met for there appears to be little difference in the precursor frequencies to these Tgs.

It is possible, however, that most of the mTg-reactive B cells detected are directed to cross-reactive epitopes expressed on huTg. To answer this, supernatants from individual culture wells were divided and assayed simultaneously on both mTg and huTg. A two by two table was generated showing the number of clones producing mTg-specific Ab, huTg-specific antibody, or cross-reactive antibodies. The data were from cell dilutions in which the precursor frequency was around 0.5 precursors/well. This minimizes the likelihood of a well containing more than one precursor either to huTg or mTg. In Table 3, the results from three experiments were compiled. Of the 40 wells positive for mTg or huTg reactivity, 30% were specific for huTg, whereas 58% were mTg specific, and only 13% were cross-reactive. Therefore, it appears that B cell clones to the conserved determinants shared between the Tgs are not as common as B clones to more unique sequences of Tg.

DISCUSSION The results presented provide an estimate of the number of B cell precursors reactive

with mTg within the LPS-responsive, normal B cell pool in mice. This frequency is about 1 precursor per 9700 spleen cells or about l/3900 B cells.

TABLE 2

Comparison of the Frequency of Mouse Tg and Human Tg Reactive B Cells

B cell frequency” to

Experiment Strain mTg huTg

VI CBA 2,012 6,070 VII BIO 11,066 8,396

BlO.BR I 1,926 12,364 BIO.D2 7,153 6,494

Mean: 8,039 8,331

a Spleen cells were cultured under limiting dilution culture conditions for 7-9 days. The culture supematants were then tested for mTg and huTg reactivity using an ELISA assay as described under Method and Materials. Frequency = l/number.


TABLE 3

Cross Reactivity of Clones to Mouse and Human Tg’

huTg

mTg + 5 23

12 40

a Individual well supernatants. assayed against both mTg and huTg at selected cells dilutions from the experiments above, were categorized as being either mTg specific, huTg specific, or cross reactive (mTg and huTg reactive) using an ELISA assay. n = 80 wells.

Even though the EAT response is strongly influenced by the MHC, the results here indicate no difference in the frequency of Tg-reactive B cells between high- and low- responder strains. This would argue that the regulatory effects of the MHC do not influence the size of the autoreactive B cell population. There also appears to be little difference in the frequency of anti-DNA-producing B cells between different strains (4). It is more likely that the MHC is important in regulating the T cell response to self-antigens which then control B cell responses through helper/suppressor cell interactions. We cannot, however, exclude an influence of the Ig-gene loci on autoreac- tivity ( 19).

It was noted earlier, that the range varied considerably between strains of mice and between experiments. There are several possible reasons for this wide range. First, the actual frequency of mTg-reactive B cells may differ significantly from animal to animal. Second, there may be genetically controlled differences between mouse strains. Third, there may be experimental variation in not only the detection of antibody-producing cells. but also in cloning efficiency, i.e., the number of cells which actually grow and give rise to a measurable clone. Because of these problems, the frequency estimates made here must be taken as minimal estimates for not all Tg-specific B cells are likely detected nor LPS responsive and, therefore, would not be detected in this assay system.

In most mouse strains, between one-third and one-tenth of B cells will grow in response to LPS (20). In these experiments the range of cloning efficiencies were estimated between one-fourth and one-thirtieth, depending on the experiment and the strains examined. Using an approximate mean value of one-tenth, or 10% for the cloning efficiency in these experiments, the absolute frequency for mTg-reactive cells would be about l/390 B cells. However, the validity of factoring in the cloning efficiency has certain inherent problems.

LPS-responsive cells may not be representative of the complete B cell repertoire. It has been suggested that LPS-responsive cells appear to be newly released B cells which may not have undergone selection (2 1, 22). They may not, therefore, include cells of the functionally mature immune system. While the repertoire of the LPS-sensitive population appears stochastic when the V-gene usage frequency is compared to the complexity of the various V-gene families, it may not completely parallel that of the mature B cell population (23-28). The LPS-responsive population might be thought of as expressing a potential repertoire which has not been expanded and from which autoreactive B cells might be selected.


In addition, the origin of autoreactive antibodies is also unclear. Studies of the autoimmune lupus mouse strains MRL, NZB, and NZB/W, suggest that B cells which produce these autoantibodies come from a unique B cell subset, a subset expressing the CD5 cell surface marker (29, 30). Many or all of the autoreactive Abs show mul- tireactivity to common antigens in addition to autoantigens. These observations suggest that at least a portion of the autoimmune B cell population may not be derived from the larger B cell pool, but from a maturationally distinct population.

Clearly Tg is a self-antigen and can induce thyroiditis; however, antibodies appear to play little or no pathological role in the typical murine EAT lesion (2, 8, 15, 18, 3 l-34), although they are an important component in spontaneous autoimmune thyroiditis in the OS chicken (35). In humans, there is no clear role for anti-Tg autoantibodies in lymphocytic thyroiditis. No evidence showing that high-affinity antibodies may be important in disease induction or pathology has been presented. Current evidence that B cells can also act as antigen-presenting cells might directly implicate them, and not necessarily antibody in the disease process (36). Therefore, the impor- tance of mTg-reactive B cells cannot be eliminated.

In conclusion, this work demonstrates that in mice, B cells capable of producing antibodies to autologous Tg occur at a high frequency in LPS-responsive B cell population. A high proportion of these B cells show high specificity for mouse Tg with little cross-reactivity to Tg from unrelated species suggesting that conserved epitopes are not seen, or do not exist. These results are consistent with those of Souroujon et al. (37) who reported that autoreactive antibodies occur at frequencies comparable to or higher than those of foreign antigens. Our results do not support any form of active selection against or tolerance of B cell clones reactive with Tg. Clearly, we cannot say whether the antibodies produced by the clones detected in the limiting dilution assay have a pathological role; however, it would be difficult to explain how any form of clonal deletion/anergy would be selective only for antibodies having a pathological impact. The immune system is clearly very adaptive and, certainly, mechanisms exist which selectively prevent overwhelming autoreactive responses. This control likely resides with the T cell. Clearly, we need to see if autoreactive T cells likewise occur at high frequencies.

REFERENCES

I. Rose, N. R., Twarog, F. J., and Crowle, A. J., J. Immune/. 106, 698, 1971. 2. Esquivel, P. S., Rose, N. R., and Kong, Y. M.. J. Exp. Med. 145, 1250, 1977. 3. ElRehewy, M., Kong, Y. M., Giraldo, A. A., and Rose, N. R., Eur. J. Immunol. 11, 146, 1981. 4. Pisetsky, D. S., and Caster, S. A., Cell. Immunul. 72, 294, 1982. 5. Clagett. J. A., and Weigle, W. O., J. Exp. Med. 139, 643, 1974. 6. Charreire, J., and Salamero, J., Tissue ilntigens 23, 72, 1984. 7. Lefkovits, I., and Waldmann, H., “Limiting Dilutjon Analysis ofCellsin the Immune System.” Cambridge

Univ. Press, Cambridge, 1979. 8. Kuppers, R. C., Suiter, T., Gleichmann, E., and Rose, N. R., Eur. J. Immunol. 18, 161, 1988. 9. Taswell, C., J. Immunol. 126, 1614, 1981.

10. Lefkovits, I., and Waldmann, H., Immunol. Toduy 5, 265, 1984. 11. Andersson, J., Coutinho, A., and Melchers, F., J. Exp. Med. 145, 1520, 1977. 12. Vladutiu, A., and Rose, N. R., Cell Immunol. 17, 106-I 13, 1975. 13. Beisel, K. W., David. C. S., Giraldo, A. A., Kong, Y. M., and Rose, N. R.. Immunogenetics 15, 427,

1982. 14. Tomazic, V., and Rose, N. R., Immunology 30, 63, 1976. 15. Romball, C. G., and Weigle, W. O., J. Immunol. 106, 698, 1984.


16. Simon, L. L., Krco. C. J., David, C. S., and Kong, Y. M., Cell Immunol. 94, 243, 1985. 17. Champion, B. R., Hutchings, P.. Davies, S., Marshall-Clarke, S., Cooke, A., and Roitt, I. M., Immunology

58, 51, 1986. 18. Simon, L. L., Justen, J. M.. Giraldo, A. A., Krco, C. J., and Kong, Y. M., Clin. Immun. Immunopathol

39, 345, 1986. 19. Juy, D.. Primi. D., Sanchez, P., and Cazenave, P.-A., Eur. J. Immunol. 12, 24, 1982. 20. Levy. M., Eur. J. Immunol. 14, 864, 1984. 21. Primi, D., and Cazenave, P. A., J. Exp. Med. 159, 1253, 1984. 22. DeFreitas, A. A., and Coutinho. A., J. Exp. Med. 154, 994, 1981. 23. Primi, D.. Mami. F., LeGuern. C., and Cazenave, P.-A., J. Exp. Med. 156, 181, 1982. 24. Manser, T., Huang. S-Y.. and Gefter. M. L.. Science 226, 1283, 1984. 25. Dildrop, R., Krawinkel. U.. Writer. E., and Rajewsky, K., Eur. J. Immunol. 15, 1154, 1985. 26. Monestier, M.. Manheimer-Lory, A., Bellon, B.. Painter, C., Dang, H., Talal, N., et ul. J. C/in. Invest.

78. 753. 1986. 27. Hartman. A. B.. Mallett, C. P., Srinivasppa. J., Prabhakar. B. S.. Notkins. A. L., and Smith-Gill. S. J..

Mol. Immunol. 26, 359, 1989. 28. Bona. C., Kasturi. K. N.. Mayer. R., and Fidanza, V., Autoimmunrfy 2, 39, 1988. 29. Herzenberg. L. A.. Stall. A. M., Lalor, P. C.. Sidman, C., Moore, W. A.. Parks, D. R., and Herzenberg,

L. A., Immunol. Rev 93, 81. 1986. 30. Casali, P., and Notkins, A. L., Immunol. Toduy 10, 364. 1989. 31. Okayasu, I., Clin. Immunoi. Immunopathol. 36, 101, 1985. 32. Braley-Mullen. H., Johnson M., Sharp, G. C., and Kyriakos, M.. Ccl/ Immunol. 93, 132, 1985. 33. Claggett. J. A.. Wilson. C. B., and Weigle, W. O., J. Exp. Med. 140, 1439, 1974. 34. Vladutiu. A.. and Rose. N. R., J. Immunol. 106, I 139, 197 I, 35. Jarosrewski, J., Sundick. R. S.. and Rose. N. R., Clin. Immunol. Immunopzthol. 10, 95, 1978. 36. Hutchings, P.. Rayner, D. C.. Champion. B. R.. Marshall-Clarke, S., Macatonia, S., Roitt, I., and Cooke,

A.. 6w .J. Immunol. 17, 393, 1987. 37. Souroujon. M.. White-Scharf M. E., Andre-Schwartz. J.. Gefter, M. L.. and Schwartz, R. S.. J. Immunol.

140,4173. 1988.

the frequency of lps-responsive b cells to autologous and heterologous thyroglobulin

Documents