CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY 15, 293-300 (1980) Reduction of Circulating Immune Complexes by Calorie Restriction in (NZB x NZW) F, Mice’ S. SAFAI-KUTTI, Sloan -Kettering G. FERNANDES, Y. WANG, B. SAFAI, R. A. GOOD, AND N. K. DAYS Institute for Cancer Research, New York, New York 10021 Received August 20, 1979 Levels of circulating immune complexes (CIC) by the Raji cell radioimmunoassay were analyzed in sera of (NZB x NZW)F, (B/W) mice at four ages (3, 9, 12, and 18 months) on different levels of total food intake (Purina lab chow and two defined diets at either 20 caYday or 10 Cal/day). In all three groups of animals the CIC levels at 3 months of age were essentially normal irrespective of dietary intake and increased dramatically in sera of mice of both sexes at 6 and 9 months. The highest CIC values were recorded just prior to death at 12 months of age in sera of mice fed Purina lab chow. In contrast, significantly lower CIC values were observed at 12 months of age on the IO-Cal intake (P i 0.001). C3 and Ig deposits in the kidney lesions of the mice were strikingly less in those mice fed the IO-Cal diet than those fed either of the other two diets. This value was also significantly lower than that of the animals fed 20 cal per day of a defined diet. These findings suggest that diet may influence lifespan of the shortlived autoimmune-prone B/W mice by inhibiting the formation and deposition of immune complexes in vital organs. INTRODUCTION (NZB x NZW)F, (B/W) mice are known to be susceptible to autoimmune diseases and to die at an early age of immune complex nephritis (1,2). Fernandez et al. (3,4) have shown that the lifespan of these animals is dramatically prolonged by dietary restriction from weaning and that a low calorie intake has a profound effect on their immune function. It was also demonstrated that the deposition of y-globulin in a capillary distribution in the glomeruli of these animals was markedly inhibited by dietary caloric restriction (4). Indeed, even when restriction of food intake was initiated long after the onset of autoimmunity, e.g., at 4-5 months, lifespan was increased, severity of glomerulonephritis reduced, and deposition of Ig and complement in a capillary distribution minimized (5). Andrews et al. (6) have recently shown that the levels of circulating immune complexes (UC) in B/W mice and several other autoimmune strains are regularly elevated. It therefore appears that an association between CIC and glomerular damage exists. The purpose of the present study was to investigate the levels of CIC in sera of B/W mice at different ages on different levels of total food intake and to correlate the changes in UC with degree of renal damage. ’ Aided by grants from USPHS CA-08748, Al-11843, NS-11457, and Ag-00541, ACS-IM-185, The Molin Foundation, and the Zelda R. Weintraub Foundation. ’ To whom requests for reprints should be addressed: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, N.Y. 10021. 293 0090-1229/80/030293-08$01.00/O Copyright All rights 0 1980 by Academic Press. Inc. of reproduction in any form resewed. 294 Experimental SAFAI-KUTTI Animals MATERIALS and Diets ET AL. AND METHODS B/W mice of both sexes were maintained from weaning on three different diets as described earlier (3). In group I the animals were fed Purina lab chow ad fibitum; group II was given a defined diet and fed approximately 20 cal per day and group III was fed a diet restricted to 10 cal per day, but provided vitamins and minerals in an amount equal to that given mice of group II. The restricted diet is the one that was previously shown to at least double the lifespan of B/W mice (3). Sera of the mice of each group were studied at 3,9, and 12 months, and animals on the low caloric diet also at 18 months of age. Blood was drawn by orbital sinus puncture, allowed to clot on ice, centrifuged in a refrigerated centrifuge, aliquoted immediately, and stored at -70°C until used. Aliquots were thawed only once for each test. Raji Cell Radioimmunoassay To measure serum levels of circulating immune complexes in B/W mice, the Raji cell radioimmunoassay previously described by Theofilopoulos et al. (7) was modified as follows: Raji cells derived from Burkitt’s lymphoma were cultured in Eagle’s minimum essential medium (MEM). The cells used in the experiments were obtained 48 hr after the initiation of the culture. Before each test the viability of the cells (80-90%) was determined by the trypan blue dye exclusion test. Preparation of Aggregated Mouse IgG Chromatographically purified mouse IgG (Cappel Laboratories, Cochranville, Pa. 19330) was dissolved in phosphate-buffered saline (PBS, pH 7.3) and then centrifuged at 40,OOOg for 20 min to obtain an aggregate-free solution. The supernatant was removed and incubated in a waterbath at 63°C for 30 min. The solution containing the aggregates was then centrifuged a second time as above and the upper one-third of the supernatant was separated frozen in small aliquots at -70°C in a Revco freezer until used. Just before each analysis the solution was thawed at room temperature. Preparation of lz51 Rabbit anti-mouse IgG The IgG fraction of a rabbit anti-mouse IgG containing antibody (both heavy and light chains) (Cappel Laboratories) was labeled with lz51 (New England Nuclear, Boston, Mass.) by the chloramine T method as described by McConahey and Dixon (8). The specific activity of the lzsI rabbit anti-mouse IgG (4.5 mg/ml) was 0.2 &i/mg protein. The standard curve for the Raji cell radioimmunoassay was obtained as follows: 10 ~1 of different dilutions of aggregated mouse y-globulin (AMG) was preincubated with 15 ~1 of normal human serum (diluted 1:4, 1:6, or 1: 10) as a source of complement. It was found that the ideal standard curve was obtained when normal human serum was diluted with normal saline at 1:6. Incubation was carried out at 37°C for 30 min. Next 4.0 x lo6 Raji cells in 50 ~1 MEM were added and the mixtures incubated at 37°C for 45 min. The cells were then washed three times with 1 ml of MEM (at 900g for 5 min) in a refrigerated centrifuge. After the final wash, 100 ~1 of lz51 rabbit anti-mouse IgG was added to each CALORIES AND IMMUNE COMPLEXES 295 tube. The cells were then washed three times again as above and the radioactivity of the pellet was determined in an autogamma scintillation spectrometer (Packard autogamma scintillation spectrometer). All tests were run in duplicate. For the assay of CIC in mouse sera, the sera from B/W mice were diluted 1:6 with normal saline and 25 ~1 of this mixture was incubated with 50 ~1 of Raji cells and the test carried out as described above. Standard statistical methods were used for the calculation of mean values, standard deviation (SD), and standard error of the mean (SE). Unless otherwise stated, mean values 2 SE are reported. Immunofluorescence Studies Kidney sections embedded in optimum cutting temperature (OCT) compound Tissue-Tek II, (Lab-Tek products, Division of Miles Laboratory, Naples, Ill.) were frozen in a cryostat and stored at -70°C. The frozen kidney sections were cut into 6 pm sections in a cryostat (- 15 to -2O”C), placed on glass slides, and stained with fluorescein-conjugated antisera (antimouse IgG, IgM, IgA, or C3) (Cappel Laboratories) according to standard procedures (9). These antisera were previously tested for their specificity by immunodiffusion and immunoelectrophoresis and diluted 1: 10 to 1:20 in PBS. Grading of Immunojluorescence Staining An arbitrary grading system was adopted with scores of 0 (none), 1 (lowest), 2, 3, and 4 (highest), depending on the observed intensity of the immunofluorescence staining. In addition to the intensity of the immunofluorescence, the number of glomeruli involved were also counted. Each series of slides was examined at least at two different times. Most often, scores of the two examinations proved to be identical, but in the event of discrepancy, the grades obtained were averaged. Statistical Methods The logarithmic transformation was used on the CIC values to stabilize the variances. Analysis of variance methods were used to compare across sex, age, and diets. The Neuman-Keuls tests for multiple comparisons were carried out at each age level to compare across the three diets (10). RESULTS Serum Levels of CIC in B/W Mice on Different Diets The standard curve obtained in our laboratory for the Raji cell radioimmunoassay for mouse serum is presented in Fig. 1. As shown, this curve resembles closely the standard curve for Raji cell radioimmunoassay obtained in our laboratory with human serum. Prior to each experiment, a standard curve was set up. Figure 2 shows the changes of CIC levels with age of male mice fed three different diets. At the age of 3 months no difference was observed between the levels of CIC in mice of the three dietary groups. The mean 2 standard error CIC concentration for mice on standard laboratory chow was 21 2 2 &ml. The corresponding means for animals on 20 and 10 cal of the defined diet were 37 2 6 and 20 2 5 pg/ml, respectively. All three groups of animals studied at 9 months of age had elevated values for CIC, and no statistical difference between the mean values was observed. Thereafter, however, the mice on the diet containing only 10 cal per 2% SAFAI-KUTTI ET AL. 9x cm 6x 3x 4 AGGREGATED 40 200 400 1200 MOUSE IgG, Hg/ml FIG. 1. Standard curve established for Raji cell radioimmunoassay. day showed progressively decreasing values for serum CIC up to the age of 18 months. The highest mean CIC (1217 + 420 pg/ml) was recorded in animals on lab chow diet at the age of 12 months and at this point the mean CIC in the group of mice on 10 cal (90 + 17 &ml) was significantly lower (P < 0.001) than that of mice fed lab chow. The mean value was also significantly lower (P < 0.01) than that for animals on 20 cal of the defined diet (518 -+ 179 &ml). At the age of 18 months this difference between 10 cal and the few survivors on the other two diets 20 Calorie IO Calorie --NORMAL IO I 2 4 I 6 I 8 IO 12 14 I6 16 I 18 I 20 Age (months) FIG. 2. Changes of levels of circulating immune complexes with age of male mice fed three different diets. CALORIES AND IMMUNE 297 COMPLEXES became still more apparent (P < 0.001). No statistical differences were observed between CIC levels of mice at any age on lab chow and the 20-cal defined diet. The median and range of the CIC values and the mean and standard deviation of the logarithm of the CIC values for each diet, age, and sex are given in Table 1. Neumal-Keuls multiple comparison tests were based on the mean of the logarithm of the CIC values. Figure 3 compares CIC levels obtained in sera of female mice on the three different diets. At the age of 3 months, all mice on the three different diets had comparable serum levels of CIC (16 kg/ml). Highly elevated levels for CIC were recorded in sera of animals on all three diets at the age of 9 months as had been TABLE LEVELS Age (months) Male 3-4 9 12 OF CIRCULATING IMMUNE 1 COMPLEXES ON DIFFERENT IN SERA DIETS OF MALE AND CIC Diet (‘.M n Standard 20 10 10 10 10 Standard 20 10 Standard 20 10 B/W FEMALE MICE Log CIC Range Mean SD 33 24 16-35 16-80 16-64 3.00 3.48 3.30 0.30 0.50 0.45 10 10 10 392 2% 208 144-6400 176-2240 108-720 6.31 5.97 5.48 1.21 0.82 0.64 10 10 10 840 256 84 304-4800 96- 1760 24- 196 6.75 5.74 4.30fl.b 0.83 1.04 0.70 16-112 3.74 0.79 56- 1440 RX-960 16-56 6&td 5.89d 3.35”,’ 1.12 0.62 0.51 2.77 2.77 2.77 - Median 17 6 40 Standard 20 10 7 7 480 400 21 Standard 20 10 10 10 10 16 16 16 9 Standard 20 10 10 10 10 680 432 280 196- 1920 224-2720 144-960 6.44 6.31 5.71 0.78 0.87 0.60 12 Standard 20 10 10 12 10 632 344 102 196-3200 40-4000 32- 176 6.53 5.79 4.46”,b 0.98 1.40 0.61 15 10 J 16 16- 160 3.65 1.11 15 15-18 15-18 19 Female 3-4 10 9 n Significantly lower than standard diet, b Significantly lower than 2C-cal diet, P CSignificantly lower than 20-cal diet, P d No. of animals used represents a pool 16 16 16 < 0.001, Neuman-Keuls test. 0.01. 0.001. from three different experimental groups. P < i 298 SAFAI-KUTTI 101 2 ET AL. 4 IO & FIG. 3. Changes of levels of circulating different diets. 4 14 I5 12 kil:“+hs~ immune complexes with age of female mice fed three observed for the male mice but no statistical difference between the groups was noted. At the age of 12 months, the mean value for CIC of mice on 10 cal per day (100 + 16 pg/ml) had declined considerably and was significantly lower (P < 0.001) than the mean for animals on standard laboratory chow (1044 ? 323 pg/ml), and that of the mice on 20 cal (767 2 331 &ml) (see Table 1). All female mice on lab chow and 20 cal died shortly after 12 months and CIC levels of the surviving female mice on the IO-Cal diet dropped further to 64 2 23 &ml at I5 months of age. Glomerular Deposition of Immunoglobulins and Complement in BIW Mice These results are summarized in Table 2. Kidney sections of mice on high caloric intake (17 of 22) showed extensive granular deposits of IgG and C3 (3 -4t) in the glomerular tufts. In contrast, the deposition of IgG and C3 of mice on the restricted calorie diet (lo- 15 animals) was minimal and confined primarily to the mesangium. Kidney sections of 17/26 mice given 20 cal per day demonstrated 2-3t deposition of IgG and C3 in a capillary distribution. DISCUSSION In the present report we confirm the findings of Andrews et al. (6) and present an adaptation of the Raji cell radioimmunoassay technique of Theofilopoulos et al. (7) for analyzing CIC in sera of mice. Our findings reveal as did theirs that in sera TABLE HISTOLOGICAL STUDIES 2 BY IMMUNOFLUORESCENCE WITH THREE OF KIDNEY DIFFERENT TISSUE FROM MICE DIETS IgG and C3 Diet Total no. of animals 3-4t Lab chow IO cal 20 cal 22 24 26 17 None None 2-3t 5 6 20 O-It None 18 6 CALORIES AND IMMUNE COMPLEXES 299 of B/W mice fed laboratory chow ad libitum, the CIC become elevated by 6 months of age and rise progressively thereafter. This increase in CIC is associated with the deposition of IgG and C3 in a capillary distribution in the glomeruli. We extended our studies to investigate the influence of calorie restriction on circulating immune complex levels. In the present study serum CIC levels and their association with glomerular deposition of IgG and C3 were analyzed whenever possible at four different ages (3, 9, 12, and 18 months) of B/W mice fed on three different diets (lab chow, defined diet at either 20 or 10 Cal/day). In all three groups of animals, the CIC levels at 3 months of age were essentially normal irrespective of dietary intake. Thereafter an increase in serum CIC levels occurred in all animals. At the age of 9 months, the serum CIC levels were dramatically increased in mice of both sexes of each dietary group. The highest serum CIC values were recorded in the animals fed standard lab chow at 12 months of age, i.e., just prior to death. In contrast, significantly lower CIC values were observed in animals at 12 months of age on the lo-cal intake. Male mice at 18 months of age fed 10 cal per day showed a further decrease in CIC values while the few surviving mice fed lab chow or a defined diet with higher calorie intake showed persistent high levels of CIC. Female mice did not survive to this age. No differences of CIC in mice fed lab chow or 20 cal per day was evident at any age. It is of some interest that C3 and Ig deposits in the kidney lesions of the mice on lab chow ad libitum were apparently greater than those of the group fed the defined diet at a level of 20 cal per day. The basis for this difference is not apparent since the CIC levels were not significantly different in the two groups. The mice fed calorie-restricted diet had strikingly less deposition of immunoglobulin and C3 in the capillaries of the glomeruli than mice of either of the other two groups. Nonetheless, deposition of small amounts of Ig could be demonstrated but it was confined to the mesangium. These findings appear to link the influence of calorie restriction on length of life and development of severe kidney disease in B/W mice to an influence on CIC. In prior work we (3, 4) and others (11) have found that anti-DNA antibody production in B/W mice is diminished by calorie restriction. By contrast, the amount of virus in the tissues is not diminished by calorie restriction (1 I). Further, our previous studies showed that dietary manipulations inhibit thymic involution and the decline of certain immunologic functions with age in B/W mice (4). It is clear from the work of Andrews et al. (6) that at least some of this decline is due to autoimmunity directed toward lymphoid cells. The present observations were especially challenging in that calorie restriction influences the levels of CIC most strikingly or entirely after the animals have reached 9 months of age. Just why this should be is not immediately apparent and deserves further analysis. Even though the levels of CIC were similarly elevated at 6 and 9 months in all dietary groups, the fall of CIC levels in the calorierestricted animals was associated with striking inhibition of deposition of apparent immune complexes in the glomeruli. Our prior studies (3, 5) established that glomerular lesions are minimal in the diet-restricted animals at 12-14 months. These histopathologic and immunologic analyses taken together with observations recorded herein make it seem likely that either calorie restriction influences the nature of immune complexes or that renal lesions and immune complex deposits SAFAI-KUTTI 300 once developed in these mice are reversible. to clarify this issue. ET AL. Further investigation will be needed REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Talal, N., Transplant. Rev. 31, 240, 1976. Lambert, P. H., and Dixon, F. J., J. Exp. Med. 127, 507, 1968. 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