mized mice was not caused only by the removal of splenic haemopoietic tissue because the splenectomized mice showed exaggerated reticulocytosis (Cox and.
Br. J. exp. Path. (1979) 60, 466
IMPAIRED REGULATION OF ERYTHROCYTE AUTOANTIBODY PRODUCTION AFTER SPLENECTOMY K. 0. COX AND J. J. FINLAY-JONES* From the Schools of Biological Sciences and Medicine*, The Flinders University of South Australia, Bedford Park, South Australia Received for publication April 2, 1979
Summary.-C3H mice were given 4 i.p. injections, each of 2 x 108 WAG rat RBC, at weekly intervals. The production of erythrocyte autoantibodies elicited by the crossreacting rat RBC was assessed using the average direct Coombs' test (DCT) score. Autoantibody production reached higher levels and persisted significantly longer in mice splenectomized 15 days before the first injection of rat RBC. This increased production of autoantibodies was not prevented by injecting each splenectomized mouse i.v. with 5 x 107 syngeneic spleen cells immediately after splenectomy. Similarly, splenectomy of mice already DCT+ significantly prolonged autoantibody production which was not prevented by injections of 108 cells prepared from the spleens removed at splenectomy. Transfer of spleen cells from mice already DCT+ to mice before the injections of rat RBC were started in the recipients caused a significant reduction in the amount of RBC autoantibodies produced. This suppression of autoantibody production was greater in unsplenectomized mice than in splenectomized mice. The results show that the spleen is involved in the regulation of these erythrocyte autoantibody responses. It is hypothesized that, in addition to the cellular component of the spleen, the splenic architecture and/or environment contributes to the regulation of autoantibody responses. MICE injected with cross-reacting rat RBC produce erythrocyte autoantibodies and develop autoimmune haemolytic anaemia (Playfair and Marshall-Clarke, 1973; Cox and Keast, 1973, 1974). Removal of the spleen before the first injection of rat RBC causes the mice to become more anaemic, to remove 51Cr-labelled syngeneic RBC from circulation faster, and to have increased amounts of RBC autoantibody in their sera (Cox and Keast, 1974). The increased anaemia in splenectomized mice was not caused only by the removal of splenic haemopoietic tissue because the splenectomized mice showed exaggerated reticulocytosis (Cox and Keast, 1974). Further, a more recent report concluded that the blood values and haemopoietic potential of splenectomized mice are not significantly different from those of unsplenectomized mice (Anosa, 1976).
These observations led to the hypothesis (Cox and Keast, 1974) now supported by experimental evidence (Naysmith and Elson, 1977; Cooke, Hutchings and Playfair, 1978; Gare and Cox, 1978; Gare and Cox, unpublished), that the spleen contains cells that can suppress production of erythrocyte autoantibodies in this model system. In view of the use of splenectomy in humans to treat patients with autoimmune haemolytic disease (reviewed by Bowdler, 1976) it was undertaken, using this experimental model, to determine whether the spleen and/or spleen cells have some unique role in regulating production of erythrocyte autoantibodies. Evidence is presented to show that autoantibody production is significantly enhanced after splenectomy and that this enhancement is not due totally to the removal of a splenic suppressor-cell population.
RBC AUTOIMMUNITY INCREASED BY SPLENECTOMY
46'
less steel sieve, using the plunger from a syringe, MATERIALS AND METHODS into about 5 ml of BSS. The cell suspension was Mice and rats.-The source of experimental then 3-4 times through a 19-gauge animals was as described by Cox and Koh needleaspirated to break up clumps. The suspension was (1977). to 10 ml and allowed to stand for 3-5 Immunization protocol and method for splen- madeinupa centrifuge tube, on ice, for the clumps ectomy. These procedures have been described min to settle. The supernatant was transferred to by Cox and Keast (1974). Briefly, inbred C3H another centrifuge tube leaving the large clumps mice aged 7-12 weeks at the time of the first behind. The mixture was centrifuged at 500 g for injection were immunized i.p. 4 times at weekly 5 min. The supernatant was removed and the intervals, each time with 2 x 108 RBC from cells were made up to 10 ml with BSS and inbred WAG rats. In any one experiment, the washed again. Cells from all the spleens in a splenectomized mice were matched for age and group were pooled and the number of viable sex with unsplenectomized mice. Splenectomy nucleated cells was estimated in a haemoor sham splenectomy was performed either 15 cytometer using the ability to exclude Trypan days before the first injection of rat RBC or 8 blue as a criterion of viability. The cell concendays after the fourth injection of rat RBC. of a pool of spleen cells was adjusted to Coombs' tests.-Blood was collected from mice tration 108 viable nucleated cells/0-6 ml and 0 3 ml by puncture of the retro-orbital venous plexus or ml was injected i.v. into each mouse. into phosphate-buffered saline with glucose In 0-6 all where spleens from mice experiments (BSS), pH 7 4, 325 mOsm/l (Cunliffe and Cox, injected with rat RBC transferred, the 1979). The RBC were washed 3 times, each time control spleens were from were age- and sex-matched in about 100 volumes of BSS, and made up to a mice that had not been injected with rat RBC. final concentration of about 20-30%o RBC in BSS. One drop of the RBC suspension was mixed with one drop of a rabbit antiserum, RESULTS directed against mouse serum, which had been The production of erythrocyte autodiluted to give optimal reactions against various RBC suspensions ranging from weakly DCT antibodies in unsplenectomized mice (Direct Coombs' Test) positive to strongly DCT reached a maximum level 1 week after the positive (DCT+). The antiserum and the RBC were mixed on an opalescent tile and examined fourth injection of rat RBC and declined with and without a Maggie lamp, for agglutina- to low levels within 2 weeks (Fig. 1). tion after 5 min and 10 min. The results were Production of erythrocyte autoantibodies recorded after 10 min as follows: was significantly enhanced and prolonged 0=no agglutination visible with x 10 mag- in mice splenectomized 15 days before the nification 1=agglutination just visible with x 10 mag- rat RBC injections started (Fig. 1). This exacerbation and prolongation of RBC nification but not with the naked eye 2 = agglutination visible with the naked eye Rat RBC 3 = large clumps of cells visible but many free I I I cells remaining 4 = large clumps of cells but few free cells remaining ,Z; Before reactions of 1-4 were recorded it was checked that the RBC had not agglutinated E when BSS was used in place of the Coombs' serum. An average DCT score (Cooke et al., 1978) was determined from these results for each group for each day tested. a) Statistical analyses. It was recognized that the average DCT score was derived from data of ordinal-level measurement, and nonparametric tests (Kruskal-Wallis one-way analysis of Time after first injection of rat RBC weeks variance by ranks and the Mann-Whitney U FIG. 1.-Exacerbation and prolongation of tests) were used to determine whether the erythrocyte autoantibody production in differences between groups were significant mice splenectomized 15 days before injec(Siegel, 1956). P values below 0 05 were accepted tions of rat RBC. There was a minimum of as significant. 11 mice in each group. * 0 Not Intravenous injections of spleen cells. Spleens splenectomized 0 --- 0 Splenectomized were placed in BSS in Petri dishes on ice. Each 0 O Splenectomized and given normal spleen was pushed gently through a fine stainspleen cells. aJ
0
0 0
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K. 0. COX AND J. J. FINLAY-JONES
468
autoantibody production was not significantly affected by injecting each splenectomized mouse i.v. with 5 x 107 syngeneic spleen cells within 2 h of splenectomy (Fig. 1). After several weeks, autoantibody production in both groups of splenectomized mice declined (Fig. 1). It was observed that removal of spleens from mice 8 days after the final injection of rat RBC significantly prolonged the production of erythrocyte autoantibodies (Fig. 2). This prolongation of RBC autoantibody production was not significantly prevented by injecting each splenectomized mouse, within 2 h of splenectomy, with 108 spleen cells that were prepared from the removed spleens (Fig. 2). In the final series of experiments, spleen cells from mice immunized with rat RBC were transferred to unimmunized mice, both splenectomized and unsplenectomized. Control mice received spleen cells from unimmunized mice. All recipients were then injected with rat RBC and tested for the presence of RBC autoantibodies. The results (Table) show 3 main points. First, the transfer of spleen cells from mice immunized with rat RBC ("immune") to unimmunized, unsplenectomized mice before the rat RBC injections were started significantly delayed production of RBC autoantibodies. Secondly, the transfer of this suppressorcell population to splenectomized mice before the rat RBC injections started led
(D
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0
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m CD
I
.:t
0
1
2
3 4 5 6 7 8 9 Time after first injection of rat RBC weeks
10
11
FIG. 2. Prolongation of erythrocyte autoantibody production in mice splenectomized 29 days after the first injection of rat RBC. There was a minimum of 10 mice in each group. 0 * Not splenectomized 0 --- 0 Splenectomized 0 O Splenectomized and reconstituted with cells from the removed spleens.
to reduced levels of autoantibodies, but not to the same extent as in unsplenectomized mice. Thirdly, removal of the spleen, followed by injection of normal spleen cells i.v., exacerbated and prolonged erythrocyte autoantibody production. DISCUSSION
Mice injected with cross-reacting rat RBC produce RBC autoantibodies (Playfair and Marshall-Clarke, 1973; Cox and Keast, 1973, 1974) and develop cells that suppress autoantibody production (Naysmith and Elson, 1977; Cooke et al., 1978;
TABLE. Suppression of Erythrocyte Autoantibody Production in Splenectomized and in Unsplenectomized Mice Receiving Suppressor Cells before the Injections with Rat RBC Average direct Coombs' test score/ number of mice DCT+ (Time after first rat RBC injection)* No. of mice 9 9 12 12
Spleen cells Splenectomy transferred No Normal No Immunet Yes Normal Yes Immune
A
26 days 1.00/6 0/0 1.83/11 0.42/2
32 days 0 67/3 0/0 1-33/8 0.17/1
39 days 0 33/3 0/0 1 00/6 0/0
* The mice were given 4 i.p. injections, each of 2 x 108 rat RBC, at weekly intervals. t The donor mice were given 8 i.p. injections each of 2 x 108 rat RBC. The first 6 injections were at weekly intervals starting at the age of 6-8 weeks. The 7th injection was 6 months later and the 8th injection 1 month after the 7th injection. The spleens were taken from the mice 5 weeks after the 8th injection of rat RBC.
RBC AUTOIMMUNITY INCREASED BY SPLENECTOMY
Gare and Cox, 1978; Gare and Cox, unpublished). The experiments presented were designed to test whether splenectomy impaired the induction of suppressor cells, and/or the expression of their activity. The results show that splenectomy of inice before the rat RBC injections significantly impaired the ability of the mice to regulate production of erythrocyte autoantibodies. This is shown indirectly by the prolongation and exacerbation of RBC autoantibody production in splenectomized mice (Fig. 1). In mice with intact spleens, sutppressor cells for autoantibody production have been shown to be present in the spleen after 2 injections of rat RBC and after various injections of rat RBC for up to 6 months later (Cooke et al., 1978; Gare and Cox, unpublished). The possibilitv that the increased average DCT score in splenectomized mice was due to an inability to remove autoantibody-coated RBC from circulation is discounted by the observation that splenectomized mice remove 5ICr-labelled syngeneic cells from circulation faster than unsplenectomized mice (Cox and Keast, 1974). The increased autoantibody production was not totally due to removal of suppressor cells or precursor suppressor cells because reconstitution of the splenectomized mice with 5 x 107 viable nucleated spleen cells did not restore suppressor activity (Figs 1 and 2). This suggests that the cellular component of the spleen is not solelyT responsible for effective regulation of RBC autoimmunity by the spleen. Thus it appears likely that splenic architecture and/or environment contribute to the induction and/or expression of suppressorcell activity for autoimmune responses. From other studies of cells regulating responses to foreign antigens it has been postulated that suppressors are produced in the spleen (Romball and Weigle, 1977; Sy et al., 1977) and that the splenic environment contributes to the expression of suppressor-cell function (Wu and Lance, 1974). The fact that autoantibody production in both groups of splenectomized mice declined after several weeks
469
(Fig. 1) may be an indication of some additional suppressor mechanism that is utilized in times of deficient splenic function. Splenectomy of mice 8 days after the final injection of rat RBC, at a time when the animals contain splenic suppressors that can significantly delay erythrocyte autoantibody production on transfer to unimmunized mice (Cooke et al., 1978; Gare and Cox, 1978; Gare and Cox, unpublished), impaired the ability of the mice to reduce autoantibody production (Fig. 2). This was shown to occur irrespective of whether the mice were reconstituted with 5 x 107 cells from the removed spleens. The level of reconstitution is about 5 times greater than the minimum number of cells that has been shown to effect significant suppression in other experiments (Gare and Cox, unpublished). The observation suggests that the spleen contributes to the expression of suppressor cells for continuing autoimmune responses. In the final series of experiments spleen cells shown to contain suppressor cells were tested for their ability to delay autoantibody production in splenectomized mice that were injected with suppressor cells before the first injection of rat RBC. The results show that the suppressor cells did cause a significant level of suppression of autoantibody production in splenectomized mice although they were not as effective as in unsplenectomized mice (Table). Thus it appears that the role of the splenic environment and/or architecture in the expression of suppressor activity for autoimmune responses is/are not as important if the suppressors are present before, rather than after, the autoimmunity has started. The implications of these results for the treatment of patients with autoimmune haemolytic disease are not clear. On the one hand there is good evidence that splenectomy often improves the health of a patient with a severe haemolytic process (Bowdler, 1976). On the other, splenectomy does not invariably improve the health of a patient and in some cases the patient
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may be in worse condition after splenectomy (Bowdler, 1976). It is possible that in this latter group suppressor cells are removed by splenectomy. This work was financed by grants from the National Health and Medical Research Council of Australia and from The Flinders University Research Committee. REFERENCES ANOSA, V. 0. (1976) Post Splenectomy Blood Values, Marrow Cytology, Erythrocyte Life Span and Sequestration in Mice. Am. J. Physiol., 231, 1254. BOWDLER, A. J. (1976) The Role of the Spleen and Splenectomy in Autoimmune Haemolytic Disease. Sem. Hematol., 13, 335. COOKE, A., HUTCHINGS, P. R. & PLAYFAIR, J. H. L. (1978) Suppressor T-cells in Experimental Autoimmune Haemolytic Anaemia. Nature, 273, 154. Cox, K. 0. & KEAST, D. (1973) Erythrocyte Autoantibodies Induced in Mice Immunized with Rat Erythrocytes. Immunology, 25, 531. Cox, K. 0. & KEAST, D. (1974) Autoimmune Haemolytic Anaemia Induced in Mice Immunized with Rat Erythrocytes. Clin. exp. Immunol., 17, 319.
Cox, K. 0. & KOH, L. Y. (1977) Disappearance of IgG2B Autoantibodies Associated with Recovery from Anaemia. Clin. exp. Immunol., 27, 560. CUNLIFFE, D. A. & Cox, K. 0. (1979) Effects of Bromelain and Pronase on Erythrocyte Membranes. Molec. Immunol,, 16, 427. GARE, N. F. & Cox, K. 0. (1978) Erythrocyte Autoantibody Production Diminished by Autoantibody-induced Suppressor Cells. Proc. Aust. Soc. Med. Res., 11, 40. NAYSMITH, J. D. & ELSON, C. J. (1977) Autosuppression of an Autoantibody Response to Erythrocytes in Mice. Allergol. et Immunopath., 5, 480. PLAYFAIR, J. H. L. & MARSHALL-CLARKE, S. (1973). Induction of Red Cell Auto-antibodies in Normal Mice. Nature (New Biol.), 243, 213. ROMBALL, C. G. & WEIGLE, W. 0. (1977) Splenic Role in the Regulation of Immune Responses. Cell Immunol., 34, 376. SIEGEL, S. (1956) Nonparametric Statistics for the Behavioural Sciences. New York: McGraw-Hill. SY, M. S., MILLER, S. D., KOWACH, H. B. & CLAMAN, H. N. (1977) A Splenic Requirement for the Generation of Suppressor T-cells. J. Immunol., 119, 2095. Wu, C. Y. & LANCE, E. M. (1974) Immunoregulation by Spleen-seeking Thymocytes. II. Role in the, Response to Sheep Erythrocytes. Cell. Immunol., 13, 1.
