- Email: [email protected]
Dissociation between proliferation and antibody formation by old mouse spleen cells in response to LPS stimulation
CELLULAR
IMMUNOLOGY
45,
295-302 (1979)
Dissociation between Old Mouse Spleen
Proliferation and Antibody Formation Cells in Response to LPS Stimulation
H. NARIUCHI Laboratory of Biological Japan, and Gerontology
Products, Research
Institute Center, Received
by
AND W. H. ADLER of Medical Science, University of Tokyo, 108 Tokyo, Baltimore City Hospitals, Baltimore, Maryland 21224 October
3, 1978
Both lipopolysaccharide (LPS)-induced proliferation and antibody formation by C57B l/6 spleen cells from old mice were studied by measuring thymidine incorporation and plaque-forming cells (PFCs) to the Z$dinitrophenyl group (DNP). There was no significant difference in the proliferative response of spleen cells from young or old mice. Anti-DNP antibody formation by spleen cells from the old mice was greatly reduced. The reduced PFC response could not explained by a shift in kinetics of the responding cells. A similar dissociation could be obtained with LPS-stimulated spleen cells from young mice by using an anti-p serum or a low concentration of hydroxyurea in the culture medium.
INTRODUCTION Because antibody formation in mice decreases with age, there has been a great deal of interest in the cellular basis of this age-related decline of the immune response. The proliferative responses of T cells to the mitogens, phytohemagglutinin (PHA) and concanavalin A (Con A), and to allogeneic cells in mixed lymphocyte cultures (MLC) have been generally shown to decrease with age (l-3). The significance, however, of these impaired T cell functions in the age-related reduction of antibody formation is still unclear. Age-related changes in B cell function are less well documented. The representation of the B cell population carrying immunoglobulin receptors does not seem to change appreciably with age in various lymphoid organs (4). The proliferative response to LPS, a B cell mitogen, of mouse spleen cells cultures does not decrease with age (5). The presence of a young-type response to LPS of cells from old mice, however, does not correlate with other B cell functions, such as the age-related decrease in the number of functional precursors of antibody-forming cells in spleen (6, 7). In this work we have studied both proliferation and antibody formation in response to LPS stimulation. We found that there is a dissociation between the proliferative response and the antibody-forming ability of spleen cells from old mice, and the possible mechanisms of the dissociation were investigated in an attempt to elucidate the B cell deficiency in the old mice. MATERIALS
AND METHODS
Mice. C57B1/6 male mice purchased from Cumberland View Farms, Clinton, Tenn., were used at 22-24 months of age. They were brought into the colony of the 295 OOOB-8749/79/080295-08$02.00/0 Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
296
NARIUCHI
AND
ADLER
Gerontology Research Center at 48-56 weeks of age and housed under standard conditions. Mice with gross pathology were excluded from these studies. The young mice used were 8 to lZweek-old C57B l/6 obtained either from the same source as the old mice or from Charles River, Mass. Cell preparation. Single cell suspensions were prepared by teasing spleens with forceps in Eagle’s minimal essential medium and passing the suspension through a 25gauge needle. LPS stimulation. For thymidine incorporation studies, 5 x lo5 spleen cells were cultured in 0.2 ml medium with 4pg LPS in Microtest II plates (Falcon No. 3040) in a humidified atmosphere of 5% CO, in air at 37°C. Five-tenths microcurie [3H]thymidine ([3H]TdR) (1.9 Ci/mmol) was added to each culture on Day 2, and the cells were harvested 17 hr later using a cell harvester (Brandel, M24 V, Rockville, Md.). The cellular material precipitated by 5% trichloroacetic acid on glass-fiber filters was dried, then solubilized with NCS in vials, and the radioactivity was determined by scintillation counting. LPS was prepared from Salmonella Minnesota R595 according to the methods of Westphal by Dr. R. Wheat, Duke University, Durham, N.C. For the antibody formation studies, 1.0 x lo6 spleen cells were cultured in 0.2 ml medium as detailed above. After variable periods of culture, from 1 to 5 days, the cells from four replicate wells were pooled, washed, and assayed for DNP PFC. Culture medium. For the thymidine incorporation studies, RPMI-1640 supplemented with 2% human serum which had been heated at 56°C for 30 min and with penicillin (100 U/ml) and streptomycin (100 &m) was used. For the antibody formation studies, spleen cells were cultured in RPMI-1640 with 20% fetal calf serum (Gibco), 5 x 10e5 M 2-mercaptoethanol, and penicillin and streptomycin. PFC assay. Cells producing antibody to DNP were assayed by a plaque technique according to the methods of Jerne and Nordin (8) as modified by Plotz et al. (9) using sheep red blood cells conjugated with DNP-alanyl-glycyl-glycyl BOC hydrazide ( 10). Reagents. Medium RPMI-1640 was purchased from Grand Island Biologicals. The fetal calf serum was obtained from Associated Biomedic System, Inc., N.Y., or Grand Island Biologicals, N.Y. Anti-p serum and anti-y,, serum were purchased from Litton Bionetics, and the specificities were confirmed by Ouchterlony analysis. Hydroxyurea was purchased from Sigma Chemicals, St. Louis, MO. RESULTS Thymidine incorporation and antibody production by spleen cells from yoing and old mice in response to LPS stimulation. Spleen cells from mice 8 weeks and 22 to
24 months of age were cultured with 4 pg LPS for 3 days. Both the proliferative response and the antibody formation response were assayed using the cells from individual mice by measuring thymidine incorporation and the number of PFCs to DNP. The results shown inTable 1 are from two representative experiments carried out separately on different occasions. In each experiment spleen cells from three individual young mice were cultured and the results were combined. The data in Table 1 demonstrate no significant differences in the proliferative responses of spleen cells from either the young or the old mice. The responses of the spleen cells from some of the old mice actually exceeded those from the young mice. However,
LPS RESPONSE
297
IN OLD MOUSE SPLEEN CELLS TABLE
1
Dissociation between LPS-Induced Thymidine Incorporation and PFC Response to DNP in Aged Mice”,” Thymidine incorporation Mouse No. 1 2 3 4 5 6 7 8 9 10’
Background 2882 5917 4518 2500 7103 6511 8300 3724 2269 2387
2 448 2 778 f 902 T 69 4 228 2 2561 + 1540 -t 357 % 74 + 515
(cpm) Stimulated
104020 70865 78857 157145 113053 82232 95144 65069 149065 76252
k -t + t 2 -c 5 + t r
34927 8234 14126 3734 4499 2435 4842 4726 7764 5655
PFCiculture 301 120 124 232 70 1302 1252 248 225 1068
2 + k 2 + it k + k 2
48 11 36 77 34 31 23 2 10 107
o Twenty-two- to twenty-four-month-old C57B1/6. b One x lo6 spleen cells were cultured with 4 pg of LPS for 3 days. Results for old mice are expressed as average values of triplicate assays and standard deviations. Results for young mice are the averages of six individual mice. c Eight-week-old C57B1/6.
the anti-DNP antibody-forming cell number was greatly reduced in most of the cell cultures from the old mouse spleens, with usually only about one-fifth to one-tenth the number of PFCs seen in cultures of the young cells. The background numbers of PFCs in the unstimulated cultures were from 70 to 150 for cells from both young and old mouse spleens. However, since it was possible that the spleen cells from the old mice have an LPS response with different kinetics, the next series of experiments was designed to determine if this could explain the differences in the PFC response which had been obtained. Kinetics
of LPS-induced anti-DNPproduction
by cellsfrom young and old mice.
Using different concentrations of LPS for initial stimulation, and assaying for DNP PFC number from Day 1 to Day 5 after initiation of the culture, spleen cells from both young and old mice reached their peak responses at Day 3 and declined thereafter. Results of a representative experiment are shown in Fig. 1. The PFC responses of the old mouse spleen cells were much lower throughout the culture period, with the peak value occurring at Day 3. There were evident differences in the responses of the cells from the young and old animals to different concentrations of LPS. When a low concentration of LPS was used, the cells from the old animals would not develop any PFCs, while the cells from the young mice responded well. Effects of hydroxyurea on cellular thymidine incorporation and antibody formation. Spleen cells from young C57B l/6 mice were cultured with 4 pg LPS for 3
days at various densities in the presence of various concentrations of hydroxyurea. Results are shown in Fig. 2. Concentrations of hydroxyurea lower than 0.5 x 1O-4 M had no inhibitory effect on thymidine incorporation, while the PFC response to DNP was suppressed. When lop4 M or higher concentrations of hydroxyurea were added to the culture medium, both the thymidine incorporation and the PFC responses were suppressed. Therefore, the hydroxyurea had a greater effect on the
298
NARIUCHI
AND ADLER
antibody formation, and in the presence of low concentrations of hydroxyurea the cells from the young mice behave in the same fashion as do the cells from old animals, in that they will incorporate thymidine in response to LPS stimulation but will not form anti-DNP PFCs. Effects of anti-immunoglobulin antiserum on the LPS response of spleen cells. Spleen cells from young C57B1/6 mice were cultured with 4 pg LPS in the presence of anti-p antibody. The proliferative response again was estimated by measuring thymidine incorporation, and the differentiation of these cells into immunoglobulin-forming cells was determined by the enumeration of PFC to DNP. The results, as shown in Fig. 3, show that anti-p antibody produced little inhibition of thymidine incorporation at any concentration tested, whereas the PFC response was markedly suppressed even at low concentrations. Similar experiments were performed with antibody to yla and with normal rabbit serum. A wide concentration range of anti-y,, antiserum and normal rabbit serum had no effect on either thymidine incorporation or PFC number, in response to LPS stimulation. Therefore, anti-p antibody caused the dissociation between proliferation and antibody formation in the response of spleen cells from young mice to LPS stimulation. DISCUSSION Although an age-related decline in immune function has been demonstrated in mice, the mechanisms remain obscure. Lymphoid cell populations from old mice were defective in their response to antigenic or T cell-mitogenic stimuli in vitro
800
DAYS
AFTER
CULTURE
FIG. 1. In these experiments spleen cells from I-week- or 23-month-old mice were cultured with or without various concentrations of LPS for up to 5 days. The number of PFCs to DNP per culture was determined at daily intervals. Stimulated with 4 fig LPSlculture, %week-old mouse spleen cells l ), 23-month-old (O--0). Stimulated with 0.4 pg LPWculture, g-week-old (x x), (0 23-month-old ( x --- x). No LPS stimulation, &week-old (A A), 23-month-old (A--A). Each point represents the average of triplicate experiments.
LPS RESPONSE
IN OLD MOUSE SPLEEN
CELLS
299
HYDROXYUREA M0-4#)
FIG. 2. In these experiments the effects of hydroxyurea on thymidine incorporation (a) and the DNP PFC response (b) to LPS stimulation was studied. The cultures for [3H]TdR incorporation and for PFC determination were initiated using different cell densities and various concentrations of hydroxyurea, from 0.1 x lO-4 to 5 x 10e4 M per culture. The cell culture densities were 1 x lo6 cells/culture (0 l ), 5 x 10Vculture (0 0), and2.5 x 10Vculture (X x). Cultures were maintained for 3 days, with [3H]TdR present for the last 17 hr. PFCs were determined on Day 3. Each point represents the average of triplicate experiments.
a
10-5 10-Q 10-3 10-2 CR NUU4tL RABBIT SECWl
FIG. 3. In these experiments the effects of antiimmunoglobulin antisera and normal rabbit serum were determined on the LPS-induced [3H]thymidine incorporation (a) and the DNP PFC response (b) of spleen cells from I-week-old mice. The thymidine incorporation studies were measured on 3-day cultures, with [3H]TdR present for the last 17 hr. The DNP PFC determinations were on 3-day cultures. The serum was added to the cultures at the initiation. Anti-IgM (0 l ), anti-IgG (0 0), and normal rabbit serum (X x). Each point represents the average of triplicate experiments.
300
NARIUCHI
AND
ADLER
(l-3). T, B, and adherent cells are known to be required for a normal antibody response. Among these three cell populations, adherent cells from old mice have been shown to support in vitro antibody responses as effectively as those from young mice (11). Therefore, the cause of an age-related decline in antibody formation could result from a deficiency of T and/or B cell function. Since the proliferative response of spleen cells to the T cell mitogens and allogeneic cells declines with age, it is attractive to assume that a defect in T cells is responsible for the decrease in immune function. However, since our understanding of T-B cell interactions is limited, it is difficult to place the total responsibility of a poor antibody response on a malfunctioning T cell population. Age-related changes in B cells have been reported by several authors. Antibody responses to a T-independent antigen in NZB mice have been reported to decrease with age (12). The number of functional precursors of antibody-forming cells in the spleen was shown to be diminished with age (6). However, the number of B cells did not seem to change appreciably with age when B cells were detected by the presence of surface immunoglobulins or complement receptors (4), and the proliferative response of spleen cells from old mice to LPS did not decrease with age (5). These inconsistent findings led us to investigate the LPS response of spleen cells from old mice. LPS is known to stimulate B cells from young mice to proliferate (13) and differentiate into immunoglobulin-forming cells (14). These two different functional results showed that the antibody-forming capacity of most of the spleen cells from the old mice was reduced, although the LPS-induced proliferative capacity was retained. The reduced PFC response could not be explained in terms of alterations in the kinetics of the responding cells, but there are several other possible explanations for the reduced response: (i) Culture conditions could be unsatisfactory for the cells from the old mice; (ii) spleen cells from old mice have different types of B cell subpopulations; (iii) spleen cells from old mice contain suppressors of antibody-forming cells or elaborate inhibitory substances; and (iv) spleen cells from old mice have defects in metabolism, cell surface receptors, or different signals from the receptors. The first possibility is unlikely to be the case because there was do difference between cells from young or old mice in the proliferative response to LPS and the cells from some of the old mice showed PFC response almost equal to that seen with the cells from young mice. The second possibility is difficult to evaluate because of the lack of information on subpopulations of B cells. Preliminary results have shown that the number of cells sensitive to the cytotoxic effects of our anti-B cell antiserum decreased in spleens from old mice (15, 16). A density distribution analysis using a BSA discontinuous gradient showed that the frequency of less dense cells increased at the expense of the more dense cell population in the spleen cells from old mice (5). These experimental results could provide indirect evidence for the possibility of a B cell population shift. However, spleen lymphocyte population studies using membrane immunofluorescense for detection of surface immunoglobulins and C3 rosette formation showed no difference in the numbers of B cells in the spleens from young or old mice (4). The third possibility might also involve changes in spleen cell subpopulations. Suppressor cells do appear to be a significant factor in some spleen cell assays in older mice (17). Also, inhibitory substances can be elaborated to a greater extent in spleen cell cultures from older mice (18). In both cases, however, the induction of suppressor or the stimulation of the release of an inhibitor usually
LPS RESPONSE
IN OLD MOUSE SPLEEN
CELLS
301
relies on a T cell activator rather than LPS. If suppressor cells are present, or inhibitors are released, they seem to affect only the PFC response, since the thymidine incorporation is not reduced in the spleen cell cultures from the old mice. In terms of evaluating the fourth possibility, the effects of hydroxyurea and an anti-p chain antiserum on B cell proliferation and differentiation were studied. Hydroxyurea inhibits the biosynthesis of DNA in mammalian cells (19). When spleen cells from young mice were stimulated with LPS in the presence of a 0.5 x lop4 M concentration of hydroxyurea, the dissociation between the PFC and the proliferative response could be shown. The hydroxyurea reduced the PFC response, but the thymidine incorporation was, if anything, usually augmented. These results suggest the presence of a metabolic pathway which is essential for antibody formation but which cannot be evaluated simply by a thymidine incorporation study. Using treatment with various anti-immunoglobulin antisera, the mouse splenic B cell response to LPS can be inhibited (20, 21). In our experiments an anti-p antiserum was able to inhibit the differentiation of splenic B cells into antibody-forming cells without affecting their thymidine incorporation. These findings are in accordance with those of Andersson el al. (20) and Kearney et al., who demonstrated using a different technique, that antibodies to p chain suppressed the synthesis of cytoplasmic immunoglobulins (22). Their results suggested that the suppression of the PFC response which we see in our experiments is not due to the disturbance of immunoglobulin secretion but to a lack of synthesis. It has been shown that LPS receptor on B cells is separated from the surface immunoglobulin molecules (23), and in our work, the proliferative response of spleen cells to LPS was not inhibited by anti-p antibodies. Therefore, anti-p chain antibodies seem to have inhibitory effects on the B cell response at an intracellular level rather than an interference of membrane interaction. By using either a low concentration of hydroxyurea or anti-p chain antibodies, it is possible to force the spleen cells from young mice to behave in the same manner as the cells from the old mice in the absence of these agents. Since both reagents are considered to affect the metabolism of B cells and do not allow a differentiation of B cells into antibody-forming cells, this could indicate that B cells from old mice have a defect(s) in their metabolic pathway which is essential for antibody formation but is not necessary for proliferation. Thus, changes in B cell function in old mice could be a qualitative difference rather than a quantitative decrease or loss of a cell type. The mechanism of a metabolic difference between B cells from young and old mice is still not known, but will form the basis of future research in this area. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Hori, Y., Perkins, E. H., and HalsaIl, M. K., Proc. Sot. Exp. Biol. Med. 144, 48, 1973. Adler, W. H., Takiguchi, T., and Smith, R. T., J. Immunol. 107, 1357, 1971. Walters, C. S., and Claman, H. N., J. Immunol. 115, 1438, 1975. Stutman, O., J. Immunol. 109, 602, 1972. Makinodan, T., and Adler, W. H., Fed. Proc. 34, 153, 1975. Kishimoto, S., and Yamamura, Y., Clin. Exp. Immunol. 8, 9.57, 1971. Andersson, J., Sjoberg, O., and Moller, G., Eur. J. Immunol. 2, 349, 1972. Jeme N. K., Nordin, A. A., and Henry, C., In “Cell-Bound Antibodies” (B. Amos and H. Koprowski, Eds.), p. 109. Winter Institute Press, 1963. 9. Plotz, P. H., Talal, N., and Asofsky, R. J., .I. Immunol. 100, 744, 1968. 10. Inman, .I. K., Merchant, B., Claflin, L., and Tacey, S. E., Immunochemistry 10, 165, 1973.
302 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
NARIUCHI
AND ADLER
Heidrick, M. L., and Makinodan, T., J. Immunol. 111, 1502, 1973. Blankwater, M. J., Levert, L. A., and Hijmans, W., Immunology 28, 847, 1975. Peavy, D. L., Adler, W. H., Shands, J. W., and Smith, R. T., Ceil. Immunol. 11, 86, 1974. Andersson, J., Sjoberg, O., and Moller, G., Eur. J. Immunol. 2, 349, 1972. Kakiuchi, T., Nariuchi, H., and Tamura, N., J. Immunol. 116, 1224, 1976. Adler, W. H., Jones, K. H., and Nariuchi, H., In “Recent Advances in Clinical Immunology” (R. A. Thompson, Ed.), p. 77. Churchill, Livingstone, Edinburgh, London, and New York, 1977. Hirano, T., and Nordin, A. A., J. Immunol. 117, 1093, 1976. Heine, J. W., and Adler, W. H., J. Zmmunol. 118, 1366, 1977. Krahoff, I. H., Brown, N. C., and Reichard, P., Cancer Res. 28, 1559, 1968. Andersson, J., Bullock, W. W., and Melchers, F., Eur. J. Immunol. 4, 715, 1974. Schrader, J. W., J. Immunol. 115, 323, 1975. Kearney, J. F., Cooper, M. D., and Lawton, A. R., J. Immunol. 116, 1664, 1976. Sidman, C. L., and Unanue, E. R., J. Exp. Med. 144, 882, 1976.
IMMUNOLOGY
45,
295-302 (1979)
Dissociation between Old Mouse Spleen
Proliferation and Antibody Formation Cells in Response to LPS Stimulation
H. NARIUCHI Laboratory of Biological Japan, and Gerontology
Products, Research
Institute Center, Received
by
AND W. H. ADLER of Medical Science, University of Tokyo, 108 Tokyo, Baltimore City Hospitals, Baltimore, Maryland 21224 October
3, 1978
Both lipopolysaccharide (LPS)-induced proliferation and antibody formation by C57B l/6 spleen cells from old mice were studied by measuring thymidine incorporation and plaque-forming cells (PFCs) to the Z$dinitrophenyl group (DNP). There was no significant difference in the proliferative response of spleen cells from young or old mice. Anti-DNP antibody formation by spleen cells from the old mice was greatly reduced. The reduced PFC response could not explained by a shift in kinetics of the responding cells. A similar dissociation could be obtained with LPS-stimulated spleen cells from young mice by using an anti-p serum or a low concentration of hydroxyurea in the culture medium.
INTRODUCTION Because antibody formation in mice decreases with age, there has been a great deal of interest in the cellular basis of this age-related decline of the immune response. The proliferative responses of T cells to the mitogens, phytohemagglutinin (PHA) and concanavalin A (Con A), and to allogeneic cells in mixed lymphocyte cultures (MLC) have been generally shown to decrease with age (l-3). The significance, however, of these impaired T cell functions in the age-related reduction of antibody formation is still unclear. Age-related changes in B cell function are less well documented. The representation of the B cell population carrying immunoglobulin receptors does not seem to change appreciably with age in various lymphoid organs (4). The proliferative response to LPS, a B cell mitogen, of mouse spleen cells cultures does not decrease with age (5). The presence of a young-type response to LPS of cells from old mice, however, does not correlate with other B cell functions, such as the age-related decrease in the number of functional precursors of antibody-forming cells in spleen (6, 7). In this work we have studied both proliferation and antibody formation in response to LPS stimulation. We found that there is a dissociation between the proliferative response and the antibody-forming ability of spleen cells from old mice, and the possible mechanisms of the dissociation were investigated in an attempt to elucidate the B cell deficiency in the old mice. MATERIALS
AND METHODS
Mice. C57B1/6 male mice purchased from Cumberland View Farms, Clinton, Tenn., were used at 22-24 months of age. They were brought into the colony of the 295 OOOB-8749/79/080295-08$02.00/0 Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
296
NARIUCHI
AND
ADLER
Gerontology Research Center at 48-56 weeks of age and housed under standard conditions. Mice with gross pathology were excluded from these studies. The young mice used were 8 to lZweek-old C57B l/6 obtained either from the same source as the old mice or from Charles River, Mass. Cell preparation. Single cell suspensions were prepared by teasing spleens with forceps in Eagle’s minimal essential medium and passing the suspension through a 25gauge needle. LPS stimulation. For thymidine incorporation studies, 5 x lo5 spleen cells were cultured in 0.2 ml medium with 4pg LPS in Microtest II plates (Falcon No. 3040) in a humidified atmosphere of 5% CO, in air at 37°C. Five-tenths microcurie [3H]thymidine ([3H]TdR) (1.9 Ci/mmol) was added to each culture on Day 2, and the cells were harvested 17 hr later using a cell harvester (Brandel, M24 V, Rockville, Md.). The cellular material precipitated by 5% trichloroacetic acid on glass-fiber filters was dried, then solubilized with NCS in vials, and the radioactivity was determined by scintillation counting. LPS was prepared from Salmonella Minnesota R595 according to the methods of Westphal by Dr. R. Wheat, Duke University, Durham, N.C. For the antibody formation studies, 1.0 x lo6 spleen cells were cultured in 0.2 ml medium as detailed above. After variable periods of culture, from 1 to 5 days, the cells from four replicate wells were pooled, washed, and assayed for DNP PFC. Culture medium. For the thymidine incorporation studies, RPMI-1640 supplemented with 2% human serum which had been heated at 56°C for 30 min and with penicillin (100 U/ml) and streptomycin (100 &m) was used. For the antibody formation studies, spleen cells were cultured in RPMI-1640 with 20% fetal calf serum (Gibco), 5 x 10e5 M 2-mercaptoethanol, and penicillin and streptomycin. PFC assay. Cells producing antibody to DNP were assayed by a plaque technique according to the methods of Jerne and Nordin (8) as modified by Plotz et al. (9) using sheep red blood cells conjugated with DNP-alanyl-glycyl-glycyl BOC hydrazide ( 10). Reagents. Medium RPMI-1640 was purchased from Grand Island Biologicals. The fetal calf serum was obtained from Associated Biomedic System, Inc., N.Y., or Grand Island Biologicals, N.Y. Anti-p serum and anti-y,, serum were purchased from Litton Bionetics, and the specificities were confirmed by Ouchterlony analysis. Hydroxyurea was purchased from Sigma Chemicals, St. Louis, MO. RESULTS Thymidine incorporation and antibody production by spleen cells from yoing and old mice in response to LPS stimulation. Spleen cells from mice 8 weeks and 22 to
24 months of age were cultured with 4 pg LPS for 3 days. Both the proliferative response and the antibody formation response were assayed using the cells from individual mice by measuring thymidine incorporation and the number of PFCs to DNP. The results shown inTable 1 are from two representative experiments carried out separately on different occasions. In each experiment spleen cells from three individual young mice were cultured and the results were combined. The data in Table 1 demonstrate no significant differences in the proliferative responses of spleen cells from either the young or the old mice. The responses of the spleen cells from some of the old mice actually exceeded those from the young mice. However,
LPS RESPONSE
297
IN OLD MOUSE SPLEEN CELLS TABLE
1
Dissociation between LPS-Induced Thymidine Incorporation and PFC Response to DNP in Aged Mice”,” Thymidine incorporation Mouse No. 1 2 3 4 5 6 7 8 9 10’
Background 2882 5917 4518 2500 7103 6511 8300 3724 2269 2387
2 448 2 778 f 902 T 69 4 228 2 2561 + 1540 -t 357 % 74 + 515
(cpm) Stimulated
104020 70865 78857 157145 113053 82232 95144 65069 149065 76252
k -t + t 2 -c 5 + t r
34927 8234 14126 3734 4499 2435 4842 4726 7764 5655
PFCiculture 301 120 124 232 70 1302 1252 248 225 1068
2 + k 2 + it k + k 2
48 11 36 77 34 31 23 2 10 107
o Twenty-two- to twenty-four-month-old C57B1/6. b One x lo6 spleen cells were cultured with 4 pg of LPS for 3 days. Results for old mice are expressed as average values of triplicate assays and standard deviations. Results for young mice are the averages of six individual mice. c Eight-week-old C57B1/6.
the anti-DNP antibody-forming cell number was greatly reduced in most of the cell cultures from the old mouse spleens, with usually only about one-fifth to one-tenth the number of PFCs seen in cultures of the young cells. The background numbers of PFCs in the unstimulated cultures were from 70 to 150 for cells from both young and old mouse spleens. However, since it was possible that the spleen cells from the old mice have an LPS response with different kinetics, the next series of experiments was designed to determine if this could explain the differences in the PFC response which had been obtained. Kinetics
of LPS-induced anti-DNPproduction
by cellsfrom young and old mice.
Using different concentrations of LPS for initial stimulation, and assaying for DNP PFC number from Day 1 to Day 5 after initiation of the culture, spleen cells from both young and old mice reached their peak responses at Day 3 and declined thereafter. Results of a representative experiment are shown in Fig. 1. The PFC responses of the old mouse spleen cells were much lower throughout the culture period, with the peak value occurring at Day 3. There were evident differences in the responses of the cells from the young and old animals to different concentrations of LPS. When a low concentration of LPS was used, the cells from the old animals would not develop any PFCs, while the cells from the young mice responded well. Effects of hydroxyurea on cellular thymidine incorporation and antibody formation. Spleen cells from young C57B l/6 mice were cultured with 4 pg LPS for 3
days at various densities in the presence of various concentrations of hydroxyurea. Results are shown in Fig. 2. Concentrations of hydroxyurea lower than 0.5 x 1O-4 M had no inhibitory effect on thymidine incorporation, while the PFC response to DNP was suppressed. When lop4 M or higher concentrations of hydroxyurea were added to the culture medium, both the thymidine incorporation and the PFC responses were suppressed. Therefore, the hydroxyurea had a greater effect on the
298
NARIUCHI
AND ADLER
antibody formation, and in the presence of low concentrations of hydroxyurea the cells from the young mice behave in the same fashion as do the cells from old animals, in that they will incorporate thymidine in response to LPS stimulation but will not form anti-DNP PFCs. Effects of anti-immunoglobulin antiserum on the LPS response of spleen cells. Spleen cells from young C57B1/6 mice were cultured with 4 pg LPS in the presence of anti-p antibody. The proliferative response again was estimated by measuring thymidine incorporation, and the differentiation of these cells into immunoglobulin-forming cells was determined by the enumeration of PFC to DNP. The results, as shown in Fig. 3, show that anti-p antibody produced little inhibition of thymidine incorporation at any concentration tested, whereas the PFC response was markedly suppressed even at low concentrations. Similar experiments were performed with antibody to yla and with normal rabbit serum. A wide concentration range of anti-y,, antiserum and normal rabbit serum had no effect on either thymidine incorporation or PFC number, in response to LPS stimulation. Therefore, anti-p antibody caused the dissociation between proliferation and antibody formation in the response of spleen cells from young mice to LPS stimulation. DISCUSSION Although an age-related decline in immune function has been demonstrated in mice, the mechanisms remain obscure. Lymphoid cell populations from old mice were defective in their response to antigenic or T cell-mitogenic stimuli in vitro
800
DAYS
AFTER
CULTURE
FIG. 1. In these experiments spleen cells from I-week- or 23-month-old mice were cultured with or without various concentrations of LPS for up to 5 days. The number of PFCs to DNP per culture was determined at daily intervals. Stimulated with 4 fig LPSlculture, %week-old mouse spleen cells l ), 23-month-old (O--0). Stimulated with 0.4 pg LPWculture, g-week-old (x x), (0 23-month-old ( x --- x). No LPS stimulation, &week-old (A A), 23-month-old (A--A). Each point represents the average of triplicate experiments.
LPS RESPONSE
IN OLD MOUSE SPLEEN
CELLS
299
HYDROXYUREA M0-4#)
FIG. 2. In these experiments the effects of hydroxyurea on thymidine incorporation (a) and the DNP PFC response (b) to LPS stimulation was studied. The cultures for [3H]TdR incorporation and for PFC determination were initiated using different cell densities and various concentrations of hydroxyurea, from 0.1 x lO-4 to 5 x 10e4 M per culture. The cell culture densities were 1 x lo6 cells/culture (0 l ), 5 x 10Vculture (0 0), and2.5 x 10Vculture (X x). Cultures were maintained for 3 days, with [3H]TdR present for the last 17 hr. PFCs were determined on Day 3. Each point represents the average of triplicate experiments.
a
10-5 10-Q 10-3 10-2 CR NUU4tL RABBIT SECWl
FIG. 3. In these experiments the effects of antiimmunoglobulin antisera and normal rabbit serum were determined on the LPS-induced [3H]thymidine incorporation (a) and the DNP PFC response (b) of spleen cells from I-week-old mice. The thymidine incorporation studies were measured on 3-day cultures, with [3H]TdR present for the last 17 hr. The DNP PFC determinations were on 3-day cultures. The serum was added to the cultures at the initiation. Anti-IgM (0 l ), anti-IgG (0 0), and normal rabbit serum (X x). Each point represents the average of triplicate experiments.
300
NARIUCHI
AND
ADLER
(l-3). T, B, and adherent cells are known to be required for a normal antibody response. Among these three cell populations, adherent cells from old mice have been shown to support in vitro antibody responses as effectively as those from young mice (11). Therefore, the cause of an age-related decline in antibody formation could result from a deficiency of T and/or B cell function. Since the proliferative response of spleen cells to the T cell mitogens and allogeneic cells declines with age, it is attractive to assume that a defect in T cells is responsible for the decrease in immune function. However, since our understanding of T-B cell interactions is limited, it is difficult to place the total responsibility of a poor antibody response on a malfunctioning T cell population. Age-related changes in B cells have been reported by several authors. Antibody responses to a T-independent antigen in NZB mice have been reported to decrease with age (12). The number of functional precursors of antibody-forming cells in the spleen was shown to be diminished with age (6). However, the number of B cells did not seem to change appreciably with age when B cells were detected by the presence of surface immunoglobulins or complement receptors (4), and the proliferative response of spleen cells from old mice to LPS did not decrease with age (5). These inconsistent findings led us to investigate the LPS response of spleen cells from old mice. LPS is known to stimulate B cells from young mice to proliferate (13) and differentiate into immunoglobulin-forming cells (14). These two different functional results showed that the antibody-forming capacity of most of the spleen cells from the old mice was reduced, although the LPS-induced proliferative capacity was retained. The reduced PFC response could not be explained in terms of alterations in the kinetics of the responding cells, but there are several other possible explanations for the reduced response: (i) Culture conditions could be unsatisfactory for the cells from the old mice; (ii) spleen cells from old mice have different types of B cell subpopulations; (iii) spleen cells from old mice contain suppressors of antibody-forming cells or elaborate inhibitory substances; and (iv) spleen cells from old mice have defects in metabolism, cell surface receptors, or different signals from the receptors. The first possibility is unlikely to be the case because there was do difference between cells from young or old mice in the proliferative response to LPS and the cells from some of the old mice showed PFC response almost equal to that seen with the cells from young mice. The second possibility is difficult to evaluate because of the lack of information on subpopulations of B cells. Preliminary results have shown that the number of cells sensitive to the cytotoxic effects of our anti-B cell antiserum decreased in spleens from old mice (15, 16). A density distribution analysis using a BSA discontinuous gradient showed that the frequency of less dense cells increased at the expense of the more dense cell population in the spleen cells from old mice (5). These experimental results could provide indirect evidence for the possibility of a B cell population shift. However, spleen lymphocyte population studies using membrane immunofluorescense for detection of surface immunoglobulins and C3 rosette formation showed no difference in the numbers of B cells in the spleens from young or old mice (4). The third possibility might also involve changes in spleen cell subpopulations. Suppressor cells do appear to be a significant factor in some spleen cell assays in older mice (17). Also, inhibitory substances can be elaborated to a greater extent in spleen cell cultures from older mice (18). In both cases, however, the induction of suppressor or the stimulation of the release of an inhibitor usually
LPS RESPONSE
IN OLD MOUSE SPLEEN
CELLS
301
relies on a T cell activator rather than LPS. If suppressor cells are present, or inhibitors are released, they seem to affect only the PFC response, since the thymidine incorporation is not reduced in the spleen cell cultures from the old mice. In terms of evaluating the fourth possibility, the effects of hydroxyurea and an anti-p chain antiserum on B cell proliferation and differentiation were studied. Hydroxyurea inhibits the biosynthesis of DNA in mammalian cells (19). When spleen cells from young mice were stimulated with LPS in the presence of a 0.5 x lop4 M concentration of hydroxyurea, the dissociation between the PFC and the proliferative response could be shown. The hydroxyurea reduced the PFC response, but the thymidine incorporation was, if anything, usually augmented. These results suggest the presence of a metabolic pathway which is essential for antibody formation but which cannot be evaluated simply by a thymidine incorporation study. Using treatment with various anti-immunoglobulin antisera, the mouse splenic B cell response to LPS can be inhibited (20, 21). In our experiments an anti-p antiserum was able to inhibit the differentiation of splenic B cells into antibody-forming cells without affecting their thymidine incorporation. These findings are in accordance with those of Andersson el al. (20) and Kearney et al., who demonstrated using a different technique, that antibodies to p chain suppressed the synthesis of cytoplasmic immunoglobulins (22). Their results suggested that the suppression of the PFC response which we see in our experiments is not due to the disturbance of immunoglobulin secretion but to a lack of synthesis. It has been shown that LPS receptor on B cells is separated from the surface immunoglobulin molecules (23), and in our work, the proliferative response of spleen cells to LPS was not inhibited by anti-p antibodies. Therefore, anti-p chain antibodies seem to have inhibitory effects on the B cell response at an intracellular level rather than an interference of membrane interaction. By using either a low concentration of hydroxyurea or anti-p chain antibodies, it is possible to force the spleen cells from young mice to behave in the same manner as the cells from the old mice in the absence of these agents. Since both reagents are considered to affect the metabolism of B cells and do not allow a differentiation of B cells into antibody-forming cells, this could indicate that B cells from old mice have a defect(s) in their metabolic pathway which is essential for antibody formation but is not necessary for proliferation. Thus, changes in B cell function in old mice could be a qualitative difference rather than a quantitative decrease or loss of a cell type. The mechanism of a metabolic difference between B cells from young and old mice is still not known, but will form the basis of future research in this area. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Hori, Y., Perkins, E. H., and HalsaIl, M. K., Proc. Sot. Exp. Biol. Med. 144, 48, 1973. Adler, W. H., Takiguchi, T., and Smith, R. T., J. Immunol. 107, 1357, 1971. Walters, C. S., and Claman, H. N., J. Immunol. 115, 1438, 1975. Stutman, O., J. Immunol. 109, 602, 1972. Makinodan, T., and Adler, W. H., Fed. Proc. 34, 153, 1975. Kishimoto, S., and Yamamura, Y., Clin. Exp. Immunol. 8, 9.57, 1971. Andersson, J., Sjoberg, O., and Moller, G., Eur. J. Immunol. 2, 349, 1972. Jeme N. K., Nordin, A. A., and Henry, C., In “Cell-Bound Antibodies” (B. Amos and H. Koprowski, Eds.), p. 109. Winter Institute Press, 1963. 9. Plotz, P. H., Talal, N., and Asofsky, R. J., .I. Immunol. 100, 744, 1968. 10. Inman, .I. K., Merchant, B., Claflin, L., and Tacey, S. E., Immunochemistry 10, 165, 1973.
302 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
NARIUCHI
AND ADLER
Heidrick, M. L., and Makinodan, T., J. Immunol. 111, 1502, 1973. Blankwater, M. J., Levert, L. A., and Hijmans, W., Immunology 28, 847, 1975. Peavy, D. L., Adler, W. H., Shands, J. W., and Smith, R. T., Ceil. Immunol. 11, 86, 1974. Andersson, J., Sjoberg, O., and Moller, G., Eur. J. Immunol. 2, 349, 1972. Kakiuchi, T., Nariuchi, H., and Tamura, N., J. Immunol. 116, 1224, 1976. Adler, W. H., Jones, K. H., and Nariuchi, H., In “Recent Advances in Clinical Immunology” (R. A. Thompson, Ed.), p. 77. Churchill, Livingstone, Edinburgh, London, and New York, 1977. Hirano, T., and Nordin, A. A., J. Immunol. 117, 1093, 1976. Heine, J. W., and Adler, W. H., J. Zmmunol. 118, 1366, 1977. Krahoff, I. H., Brown, N. C., and Reichard, P., Cancer Res. 28, 1559, 1968. Andersson, J., Bullock, W. W., and Melchers, F., Eur. J. Immunol. 4, 715, 1974. Schrader, J. W., J. Immunol. 115, 323, 1975. Kearney, J. F., Cooper, M. D., and Lawton, A. R., J. Immunol. 116, 1664, 1976. Sidman, C. L., and Unanue, E. R., J. Exp. Med. 144, 882, 1976.