of Serum

13, 237-245 (1979)





Chemical Immunology. The Weizmann Institute of Science, Rehovot, Israel Received November 3, 1978

The absorption out of antinuclear antibody (ANA) from sera of highly ANA-positive C57BL/6 mice was obtained by different cellular and noncellular factors. A single injection of DNA, liver nuclei, thymocytes, or repeated injections of a soluble material produced by normal mouse splenocytes with (SIRS+) or without (SIRS-) previous stimulation with Con A into ANA-positive mice induced a transient decrease in ANA level, reaching minimal levels at about 14 days after treatment. A similar elimination of ANA was also achieved by in vitro treatment of positive ANA sera from mice or SLE patients with thymocytes, SIRS (+), or SIRS (-). The absorbed ANA could be released by further treatment with DNase, thus indicating that the absorption-out phenomenon involved immune-complex formation.

INTRODUCTION NZB x NZW/Fl (B/W Fl) mice serve as a model for autoimmune disease since they spontaneously develop antinuclear antibodies (ANA), LE cells, immune complex glomerulonephritis, and thereby earlier death (1). Evidence has accumulated implicating loss of suppressor T-cell activity associated with the pathogenesis of autoimmune disease in B/W Fl mice (2, 3). That implication (and the similarity of the B/W Fl autoimmune disease to human SLE) led many investigators to try and restore the T-cell activity in order to cure the disease. Improved survival of mice has been shown after treatment with multiple syngeneic young thymus grafts (4). Suppression of autoimmune hemolytic anemia has been demonstrated in B/W Fl after treatment with syngeneic young thymocytes (5). Reduction of immunoglobulin level, ANA, proteinurea, and renal pathology were induced following treatment of B/W Fl with supernatant fluid (SIRS) produced by spleen cells stimulated by Con A (6). Rich and Pierce (7) demonstrated in NZB mice that suppression of plaque-forming cells (PFC) against sheep red blood cells (SRBC) could be mediated by SIRS, suggesting that the latter activated T-suppressor cells and thereby caused suppression pf antibody formation by B cells. Krakauer er al. (6) claimed that SIRS reduced the signs of autoimmune disease in B/W Fl mice by affecting a central mechanism, namely spleen cells activated by Con A-secreting a mediator causing the activation of T-suppressor cells in the injected mice, Recently some of the physicochemical properties of SIRS were described (8- lo), although isolation of the molecule has not yet been achieved. Antinuclear antibodies are present in human autoimmune diseases, in mice ’ This work was supported in part by a grant from the Israel Cancer Association. 2 On leave from the Department of Medicine C and Unit of Clinical Immunology, Kaplan Hospital, Rehovot, Israel. 237 0090-1229/79/070237-09$01.00/O Copyright All


0 1979 by Academic of


in any

Press. Inc. form






suffering from autoimmune disease, and in strains of mice without any autoimmune manifestations. The disappearance of ANA, coinciding with the development of malignant disease was demonstrated in mice. Thus, Haran-Ghera et al. (11) found a negative relation between the appearance of lymphomas and ANA levels in SJWJ mice. Walker and Bole (12) were able to show reduction in ANA levels in B/W Fl mice developing lymphomas after treatment with cyclophosphamide. Lately Peled et al. (13) demonstrated a reduction in ANA levels durmg spontaneous or induced murine leukemogenesis. Elimination of ANA was also achieved in vitro by incubating ANA-positive sera with normal or leukemic thymocytes (13). This elimination was correlated with the appearance of DNA anti-DNA complexes. By incubating these sera with DNase, the “release” of ANA was demonstrated. The aim of the present studies was to elaborate the absorption-out phenomenon by cellular and noncellular factors and the possible mode of action involved. MATERIALS


C57BW6 female mice, 8 months old, were obtained from the Breeding Center of the Weizmann Institute of Science. ANA. ANA was determined by indirect immunofluorescent technique on human blood smears as described by Norins and Holmes (14). A drop of the examined serum was incubated on alcohol-fixed smears for 30 min at 37°C in a humidified atmosphere. The slides were washed with PBS and dried. A drop of fluorescinated goat antimouse IgG and IgM was added to the dried slides and incubated at 37°C for 30 min. The slides were washed with PBS and dried. The slides were stained for 3 min with Evans blue 0.06% in distilled water and examined for ANA in Zeiss fluorescence microscope. The intensity of fluorescence (as an estimate of ANA level) was rated as negative (13)to four-plus ( + + + +) scale (see Fig. 1). The preparation of the smears and the examinations for ANA were done always by one of us. The numbers given in the results refer to the total count of plus (+) observed in the whole group. A soluble immune-response suppressor (SIRS). The preparation of SIRS was carried out according to the method described by Rich and Pierce (7). A suspension of spleen cells from 4- to 6-week-old female B/W FI, at a concentration of 2 x lOYm1, was incubated for 36 hr in culture medium RPM1 + 5% FCS in the presence of Con A (2 pg/ml). The cells were then centrifuged and the supernatant was absorbed on Sephadex G-50 for 1 hr in the cold to remove Con A from SIRS. This preparation was marked as SIRS (+). In the same way OSIRS (+) was prepared from spleen of 12 months old B/W Fl mice. As controls to SIRS, spleen cells from young (4-6 weeks) and from old (12 months) B/W Fl mice were incubated for 36 hr in medium lacking Con A (prepared as the above). These preparations were Animals.

named SIRS( -) and OSIRS( -), respectively. Liver cell nuclei. Nuclei were prepared from liver tissue removed

from 2- to 6-month-old B/W Fl mice (usually males). The method of the preparation was a modification of the method described by Goldblat and Bustin (15). Fresh livers were homogenized in TKM-sucrose [0.25 M sucrose, in 0.01 M Tris (pH 7.4)0.025 M KCl-0.005 M Mg Clz] solution in a Potter-Elvejehm homogenizer. The resulting suspension was overlaid on a cushion of TKM-2.5 M sucrose and cen-


FIG. 1. The fluorescent intensity rate of ANA. (a) negative 0; (b) (+): (c) (++): (e) (++++).

Cd) (+++):

trifuged for 40 min at a speed of 24,000 t-pm in a Beckman L3-50 centrifuge with head SW-27. The pellet was collected and washed three times in PBS and injected into mice in 0. l-ml concentration of 6. lo6 nuclei/ml. Thyrnocytes. Thymocytes were prepared from young l- to 2-month-old C57BW6 females. Cell suspensions were washed three times with PBS and thereafter injected ip in concentration of 5. IO’ cells/ml. DNA. Calf thymus (double-strand) DNA (Worthington) was injected ip 0.125 mg in PBS. Demonstration of complexes of DNA-anti-DNA by the use of DNase. The method used was described by Harbeck ef n/. (16). Two parallel samples of sera were taken. An equal volume of PBS containing 20 pg of DNase and 0.6 pmol MgCl, was added to the serum sample for digestion, whereas an equal volume of





PBS was added to the control sample. Both samples were mixed and incubated for 1 hr at 37°C. After an overnight incubation at 4”C, the samples were incubated at 37°C for additional 4 hr. The 0.01 ml of 1 M EDTA in distilled water was then added to all samples. To obtain a final serum dilution of 1:5, 0.4 M borate buffer (0.01 M borate, 0.15 M NaCl, pH 8.3) was added to the digested samples. To the control samples 0.4 M borate buffer containing 20 I.L~ DNase and 0.6 pmol MgCl, was added to obtain the same final serum solution. Subsequently, the determination of antibody of the digested and undigested serum samples were done in parallel, applying the immunofluorescence test on fixed blood smears. RESULTS In Vivo Absorption

out of ANA

In previous studies (13) it was shown that one injection of thymocytes into ANA highly positive C57BL/6 mice reduced their ANA incidence with 17 days and thereafter the levels of ANA reached the starting levels. In the present experiment we attempted to further investigate the influence of various agents on the level and maintenance of ANA in the injected mice. Thirty-six ANA highly positive mice were divided into four groups (9 mice in each group); one group received 0.5 ml of SIRS ip the second group received 0.125 mg of DNA, and the third group received liver-cell nuclei in a dose of 2. lo6 nuclei ip. The fourth group served as a control group. ANA level was evaluated 24 hr before the injection and thereafter every 5 days for 6 weeks. The results are summarized in Fig. 2. All 3 groups showed the same reduction in ANA levels, a reduction which was maximal between Days 12 and 16 after treatment. SIRS (+) caused a reduction of 87% in ANA level (from a total count of +23 to +3). DNA caused a reduction of 70%, (from total count of +21 to +6) and liver-cell nuclei 91% (from +22 to +2). In the control group the total count of ANA level was +24 at the beginning of the experiment and +25 after 14 days. The levels of ANA reached the initial positive level within 45 days after treatment. The Possible

Krakauer T-suppressor

Mode of Action of SIRS et al. have proposed that

SIRS acted through activation of cells (6). It seemed, therefore, of interest to test whether other

-bY 3 a



2 20 ‘s E

= IO 8 days



FIG. 2. Effect of SIRS (+), N-DNA, and liver nuclei LCN on ANA level. C57BL/6 mice aged 12 months highly positive for ANA were injected either with SIRS, N-DNA, or LCN, and the ANA level was determined at different time intervals after the injection.





preparations of splenocyte supernatants (not activated by Con A) would act similarly. We, therefore, compared the effect of SIRS and other preparations of splenocyte supematants not stimulated with Con A [SIRS (-) on ANA levels]. In a similar way, preparations of supematants from cultures of spleen cells from old mice with (OSIRS+) and without (OSIRS-) Con A-stimulation were used. It seemed of interest to test the action of OSIRS from B/W Fl mice on ANA levels since old NZB mice were known to lack T-suppressor function (2). Forty C57BIJ6 mice selected as highly positive ANA were divided into four groups. Each group consisting of 10 mice, received three injections (1.5 ml) total of the different preparations at a 2-week interval (0.5 ml ip priming a total of 1.5 ml). Group A-IO mice were treated with SIRS(+); Group B-with SIRS(-); Group C with OSIRS (-); Group D with OSIRS (-). ANA levels were estimated 24 hr before injection and thereafter once a week for 8 weeks, the last test being done 6 weeks after termination of the treatment. The results summarized in Fig. 3 indicate there was a significant decrease in ANA level upon injection of the different SIRS preparations. In three groups (A, B, C) the reduction was maximal (87.5%, 91.5%, 92.3%, respectively) 2 weeks after injection. In the group treated with OSIRS (-) direct correlation between time of injection and maximal decrease in ANA level could not be demonstrated. Six weeks after termination of the treatment the level of ANA reached the starting point in all groups. The same duration of time was required for ANA levels to come back to normal after a single injection. It seems, therefore, that repeated injections of SIRS could not result in the prolonged decreased of ANA levels. The Induction of Complexes following ANA Absorption out in Vivo Recent studies have proposed that the reduction of ANA levels after thymocyte injection was due to the formation of immune complexes probably of DNA-anti-DNA type (13). In order to further establish the existence of such complexes sera from mice rendered ANA negative following treatments described in the two previous experiments were incubated with DNase in order to release the ANA out of its complexed form. Sera from three groups of mice treated with SIRS (+>, DNA, or liver-cell nuclei (as described in the first experiment), and sera from each group of the four groups treated with the various SIRS preparations have been pooled and then divided into three samples for each of the groups. Two


OSIRS(~) OSIRS I- 1 SIRS t-c) SIRS (-)


FIG. level.

3. Effect

of repeated












on ANA











After DNase

Before DNase

I 2 3 4 5 6 7 8 9

+ 8 + 8 + 0 0 0 e 3

+ + + f3 + 0 + e I3 4

I3 I9 0 e + I3 +++ 8 0 4






After DNase + ++ t + + + ++++ e + 12



Before DNase

After DNase

0 + + 0 0 0 + 0 + 4

+ ++ + 0 +

t ++ + it+ 12

count( +)

samples from each group were treated with DNase. The third was treated with PBS and termed as control. The results are summarized in Tables 1 and 2. Sera taken from mice of the first experiment show maximal release of ANA activity, after treatment with DNase, in the mice treated previously with LCN and DNA. The pools of sera from the experiment showed increase of ANA activity after treatment with DNase, the sample termed as control showed no change in ANA level.

IN LEVEL Initial ANA level (before treatment)




ANA level

In viva

effect on ANA level


ANA level after DNase

after in vitro incubation

ANA level after DNase

Pools of sera






from mice treated with SIRS (+)

+++ +++

+ +

++++ ++

++ ++++

++++ ++++

Pools of sera from mice treated with SIRS(-)

++++ ++++ ++++

+ + +

++ +++ ++

+ +++ +++

++ ++ ++

Pools of sem from mice treated with OSIRS( +)

+++ +++ +++

+ + +

++++ ++ ++

++ + +++

++ +++ +++

Pools of sera from mice treated with OSIRSt-J

+++ f-4-f +++

+ + +

+++ +++ ++

+ + ++

++ ++ ++

’ Each pool of sera has been divided into three samples: The tirst two give the experimental results and are controls of each other. The third sample (in the in vitro experiments) has been incubated with PBS and serves as control.


In Vitro Induction




of Complexes

The possible formation of immune complexes (DNA-anti-DNA)


treatment with SIRS was also tested in vitro. Highly positive ANA sera were incubated for an hour at 37°C with the different SIRS preparations or PBS as control. The levels of ANA were recorded in each of the experimental groups. The absorbed sera were incubated with DNase and ANA levels were tested again. The results summarized in Table 2 showed remarkable decrease in ANA levels in all the tested groups after treatment with SIRS. Further incubation with DNase caused release of ANA into serum except in the group which has been treated with OSIRS(-) (group D). ANA Absorption from Sera of SLE Patients After demonstrating the in vivo and in vitro “absorption out” of ANA from sera of mice it seemed of interest to test ANA absorption from positive sera of SLE patients. Thus 13 highly positive ANA sera of patients with SLE were divided into four groups. Sera from group A were incubated for 60 min at 37°C with SIRS (+), sera from group B with SIRS(-), and sera from group C with thymocytes. A fourth group served as control and was incubated for 60 min at 37°C with PBS. The results shown in Table 3 indicate a marked decrease in the ANA level in all the groups tested when compared to the initial levels which are shown on the left column under the heading control. After incubation with DNase the control group showed no change, whereas, the other groups showed a remarkable release of ANA. DISCUSSION The aim of the present studies was to define means by which high levels of ANA in the sera of mice could be diminished or eliminated. The testing of different agents acting on the immune system or refractory to the immune system could indicate their possible mode of action. The main contribution of the present investigation was to demonstrate that certain cellular and noncellular agents could similarly absorb the circulating ANA, thereby inducing DNA-anti-DNA complexes detected by DNase treatment. The fact that different agents, cells, pure nucleic acids, nuclei, and SIRS, had similar effects on the elimination of ANA from sera would suggest that absorption rather than central humoral factor was involved in this phenomenon. A noncellular soluble immune-response suppressor, supematant of young spleen cells stimulated with Con A and shown to have a suppressor effect of the primary response to SRBC (7), prevented the manifestation of autoimmunity observed in B/W Fl mice. Krakauer et al. (6) proposed that the effect of SIRS was related to augmentation of the T-suppressor cells which progressively declined in adult B/W Fl mice (17). The loss of T-suppressor cells in old B/W Fl mice was therefore suggested to be the main cause of autoimmunity (2, 17). In another recent work (18) it was shown that reduced T-suppressor-cell activity was involved in autoimmune disease of B/W Fl mice. The involvement of T-suppressor cells in autoimmunity is still a controversial issue. The work of Roubinian et al. (18) claims that removal of thymus or spleen from male B/W Fl mice resulted in earlier death which suggests that regulatory influ-





ences in this sex exerted a truly suppressive effect on disease expression. By contrast, thymectomy in female B/W Fl mice resulted in significantly prolonged survival, suggesting that the thymus exerts a deleterious rather than beneficial effect on disease expression in this sex. The formulation supports the concept that the predominant regulatory effect in female B/W Fl mice is helper rather than suppressor. The fact that SIRS, as well as native DNA and liver-cell nuclei, affected the transient elimination of ANA in a similar way might suggest another mechanism of action of SIRS rather than activation of T-suppressor cells. Thus, SIRS might contain, in addition to factors stimulating T-suppressor cells, also nuclear constituents which could absorb ANA upon injection into ANA-positive mice. The fact that supernatants of unstimulated spleen cells and of old B/W Fl mice, which are believed to lack T-suppressive effect, were found in the present studies to reduce ANA levels in mice might speak for other factors responsible for ANA diminution. The results would suggest that SIRS acts as an absorbent of ANA rather than as a mediator for activation of T-suppressor cells. The discontinuation of SIRS treatment resulted in a gradual increase of ANA level. The necessity to administer repeated injections of spleen supernatants in order to maintain maximal decrease of ANA for a long period might be attributed to the requirement of continuous absorption of newly formed ANA from the serum rather than to sole activity on a central mechanism. The interest to implicate the present studies which were performed in mice to human systemic lupus erythematosus (SLE) is obvious. The pathogenesis of this disease is believed to be linked to the loss of suppressor-T-cell activity, thus releasing B cells to form antibodies against tissue and sera antigens. Lately, Horowitz et al. (19) showed a reduction in T-cell activity in serum from patients



Treatment SIRS( +) Patient I 2 3 4 5 6 7 8 9 10 11 12 13 Total

( +)

ANA after absorption ++ + + ++++ f + 0 ++ 9 e 0 8 e 12




of sera with: SIRS(-)

After DNase

+ + + + + ++++ + + +++ + ++++ + + 21




I3 f3 0 9 8 ++ + + ++ +++ 9 + ++ 12

Thymocytes After DNase

+++ + ++ 8 + ++ + ++ ++ + ++++ e + 20


I9 e + e’ + 0 + + + 0’ + 8

Control After DNase


e’ e’ t++ + ++ +++t + e’ I5

(PBS) After

DNase +++ ++ +++ ++++ +++t +++ +++ +++ +++ +++ +++ ++ 0 36

+++ ++ +++ +++ +++ +++ ++ +++ +++ +t +++ ++ + 33

: Thirteen samples of human ANA highly positive (as shown in the control column) divided into four groups. Three were incubated with one of each of the following agents: SIRS (+), SIRS (-), and thymocytes, and the fourth with PBS.




with SLE and restoration of that activity by incubating the cells with thymosin or cultured thymic epithelium. There is also a hormonal influence on the development of the disease, thus the female to male ratio is 8: 1. The disease occurs in women in the childbearing age and it tends to be aggravated during or after pregnancy. For obvious reasons we could not do in viva experiments in patients with SLE, but as can be seen in Table 3, a short incubation of human ANApositive sera from SLE patients with SIRS and SIRS (->, as well as thymocytes reduced the ANA levels. Such results raised the possibility that SIRS might contain nuclear constituents which could neutralize the activity of the antibodies to nuclear material and thereby form immune complexes such as DNA-anti-DNA. Furthermore, we could release ANA activity after incubating the sera with DNase (Fig. 3). There is an ongoing study in our laboratory which tries to correlate the level of ANA to degree and intensity of disease. If there was evidence that decrease of ANA level could be related with amelioration of disease, it might open a new attitude to treatment of SLE by offering “absorption out” treatment. ACKNOWLEDGMENT We are indebted to Professors Nechama Haran-Ghera and Zvi Bentwich for theiradvice discussions.

and fruitful

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Howie, J. B., and Helger, B. J., Adv. Immunol. 9, 215, I%& Steinberg, A. D., Arth. Rheum. 17, 11, 1974. Steinberg, A. D., J. Zmmunol. 113, 1, 1974. Kysela, S., and Steinberg, A. D., Clin. Zmmunoi. Zmmunopathol. 2, 133, 1973. Gershwin, E. M., and Steinberg, A. D., Clin. immunol. Zmmunopathol. 4, 38, 1975. Krakauer, R. S., Strober, W., Reppeon, D. L., and Waldman, T. A., Science 196, 56, 1977. Rich, R. R., and Pierce, C. W., J. Immunol. 112, 1360, 1974. Reinstem, J. L., and Steinberg, A. D., J. Zmmunol. 119, 217, 1977. Pierce, C. W., Tadakuma, T., Kuhner, A. L., and David, J. R., In “Mitogens and Immunology” (J. J. Oppenheim and D. L. Rosenstreich, Eds.), pp. 583-595, Academic Press, New York, 1976. Tadakuma, T., Kuhner, A., David, J., and Pierce, L., Fed. Proc. 34, 838, 1975. Haran-Ghera, N., Ben-Yaakov, hi., Peled, A., and Bentwich, Z., J. Nat. Cancer Znsr. 50, 1227, 1973. Walker, S. E., and Bole, G. G., Clin. Exp. Zmmunol. 24, 210, 1976. Peled, A., Brahmi, S., Haran-Ghera, N., and Bentwich, Z., J. C/in. Lab. Immunol., in press. Norms, L. C., and Holmes, M. C., J. Zmmunol. 93, 148, 1964. Goldblatt, D., and Bustin, M., Biochemistry 14, 1689, 1975. Harbeck, R. J., Bardone, E. J., Kohler, P. F., and Carr, R. I., J. C/in. fnvesr.52, 789, 1973. TaJal, N., Transplant. Rev. 31, 15, 1976. Roubinian, J. R., Papoian, R., and Talal, N., J. Zmmunol. 118, 1524, 1977. Horowitz, S., Borcherding, W., Moorthy, A. V., Chesney, R., Schulte-Wesserman, H., Hong. R., and Goldstein, A., Science 197, 999, 1977.

Absorption of serum antinuclear antibodies.

CLINICAL IMMUNOLOGY AND Absorption IMMUNOPATHOLOGY of Serum 13, 237-245 (1979) Antinuclear Antibodies’ DAVIDGELTNER~ AND ALPHAPELED Departmen...
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