Response to Con A and LPS in senescent mice
Eur. J. Immunol. 1977. 7: 301-304
301
10 stark, O., Kien, V.,Frenzl, B. and BrdiEka, R., in Dausset, J.,
5. References 1 Charlton, R.K. and Zmijewski, C.M., Science 1970.170: 636. 2 van Rood, J.J., van Leeuwen, A. andvan Santen, M.C.T., Nature 1970.226: 366. 3 Schultz, J.S. and Shreffler, D.C., Transplantation 1972.13: 186. 4 Aster, R.H., Miskovich, B.H. and Rodey, G.E., Transplantation 1973.16: 205.
Hamburger, J. and Mathe', G. (Eds.), Advance in Transplantation, Munksgaard, Copenhagen 1968, p. 331. 11 stark, O., Kienovi, D., Kren, V. and Frenzl, B., Folia biol. Prague 1970.16: 1. 12 Chut&, J., Folia biol. Prague 1973. 19: 231. 13 Sanderson, A.R., Immunology 1965. 9: 287. 14 Wigzell, H., Transplantation 1965.3: 423.
5 Oh, S.K., Pellegino, M.A., Ferrone, S., Sevier, E.D. and Reisfeld, R.A., Eur. J. Immunol. 1975.5: 161.
15 Kamrin, B.B.,Proc. Soc. Exp. Biol. Med. 1959.100: 58.
6 Callahan, G.N., Ferrone, S., Allison, J.P. and Reisfeld, R.A., Transplantation 1975.20: 431.
17 Mannick. J.A. and Schmid, K., Transplantation 1967. 5 : 1231.
7 Calne, R.Y., Davis, D.R., Hadjiyannakis, E.,Sells, R.A., White, D.,
Herbertson, B.M., Millard, P.R., Joysey. V.C., Davies, D.A.L., Binns, R.M. and Festenstein, H., Nature 1970.227 903.
16 Mowbray, J.F., Transplantation 1963. I : 15. 18 Nelken, D. and Glaser, M., Transplantation 1972.14: 268. 19 Hanna, N., Kalderon, R. and Nelken, D., Immunology 1975.29: 433. 20 Pokorna, Z. and Vojtigkov& M., Folia biol. Prague 1966. 12: 88.
8 Hakk, M.. Chutd, J., HoI& V. and Shdebk, M.,Nature 1976. 262: 295.
21 Law, L.W., Apella, E., Strober, S., Wright, P.W. and Fischetti, T., Proc. Nut. Acad. Sci. US 1972.69: 1858.
9 HGek, M., Chutni, J., SGde&k, M. and HOG%,V., Folia biol. &ague 1976.22: 411.
22 Law, L.W., Apella, E., Cohen, J. and Bern, J.M., Nature-New Biol. 1973.246: 174.
Carmella Abaham', Y. Talo and Harriet Gerrhon' Technion, Israel Institute of Technology, School of Medicine' and Department of Pediatrios, Rothschikl Hospital0, Haifa
Reduced in vibo response to concanavalin A and lip0 polysaccharide in senescent mice: a function of reduced number of responding cells" The proliferative capacity of spleen cells from C57BL/6J female mice of various ages (3-28 months) to the polyclonal mitogens concanavalin A (ConA) and lipopolysaccharide (LPS) was examined. It was found that both the T and B cell population of the spleen demonstrate a n age-related decrease i n their capacity t o respond in vitro. Peak responses t o both mitogens occurs at about 1 year of age. This age-related reduction in response is expressed in t h e degree of incorporation of [3H]thymidine i n t o DNA, the total number of cells generated in vitro, the number of labeled cells per culture a n d t h e number of b h s t cells per culture. The day of peak response i n vitro does not change with age. Studies of the cell cycle of cells responding to Con A and t o LPS from 1 2 and 28-monthold mice demonstrate that the generation time of individual proliferating cells does not alter with age. Nor does i t differ for the B cells responding t o LPS or t h e T cells responding t o Con A. These studies also demonstrate that the proliferating cells from senescent mice are equally capable of repeated cell divisions as are the cells from the I-year-old adult mouse. It is concluded that the defect in senescent mice which leads t o a reduced i n vitro response t o t h e polyclonal mitogens LPS and Con A is a reduction in the number of responding cells and not a n alteration in t h e capacity of those cells which d o respond t o divide.
1. Introduction Aging mammals are characterized by several immunologically related disorders such as an increased incidence of neoplasia and autoimmune diseases a n d a n augmented susceptibility t o infectious diseases [l]. Studies of the immune reactivity of senescent animals, and in particular studies of mice and hu-
[I
16261
*
This work was supported by Grants from the Israel National Academy of Sciences and the Israel Cancer Society. Correspondence: Harriet Gershon, Technion, Israel Institute of Technology, The Aba Khoushy School of Medicine, Haifa, Israel Abbreviations: Comk Concanavalin A LPS: Lipopolysaccharide [3H)dThd: Tritiated thymidine cpm: Counts/min
mans, have demonstrated a n age-related decrease in the capacity to produce humoral [2, 31 as well as cell-mediated immune responses [3-51. In vitro studies reveal that populations of b o t h T and B lymphocytes from senescent mice are deficient in their capacity t o respond t o polyclonal mitogens, as indicated b y t h e incorporation of tritiated thymidine [3H]dThd into DNA [6-81. In order to better understand t h e nature of the defect in immune reactivity of senescent animals, we have performed a series of experiments designed t o determine whether t h e age-related decrease in response t o polyclonal mitogens is d u e t o a decreased number of ~ymphocytes capable of undergoing blast transformation a n d cell division, or t o a n altered capacity of individual cells t o divide, thus leading t o longer generation times and reduced response.
302
C. Abraham, Y. Tal and
Eur. J. Immunol. 1977. 7: 301-304
H.Gershon
2. Materials and mathods 2.1. Animals
All experiments were performed with t h e lymphocytes of C57BL/6J female mice of various ages. These mice were bred and raised under specific pathogen-free conditions a t the Weizmann Institute of Science. The mean life-span of this mouse colony is 24 months.
2.2. Culture conditions and mitogens used Viable spleen cells (1 06)were cultured i n 1 ml tissue culture medium with or without the addition of mitogen for u p t o 120 h at 37 "C in a water-saturated atmosphere of 5 % COz in air. Tissue culture medium consisted of Eagle's minimum essential medium with Earle's salts, glutamine and nonessential amino acids (F-15 Gibco, Grand Island, N.Y.,) supplemented with 1 5 % heat-inactivated fetal calf serum (Gibco) plus 20 pg/ml asparagine (Fluka AG,Buchs, Switzerland), 2-mercaptoethanol (Fluka), at a final concentration of l o 4 M, 1Os units/l penicillin G (Rafa Laboratories Ltd., Jerusalem) and 0.1 g/l dihydro-streptomycin (Teva L.td., Jerusalem). Cultures were performed in either Nunclon 1 4 1 0 or Corning 25200 disposable plastic tissue culture tubes. Results obtained in both types of tubes were identical and data were pooled.
To individual cultures either 1 0 pg E. coli 026:36 lipopolysaccharide-W (Difco, Detroit, Mich.), activated according t o Anderson et al. 191 or I0 p g ConA (Miles-Yeda) were added.
with 95 % ethanol and drying for 30 min at 1 0 0 "C, t h e filters were placed in vials containing 1 0 m l scintillation fluid (5 g PPO, 500 mg POPOP in 1 1toluene) and cpm monitored o n a Packard Tricarb scintillation counter. 2.5. Determination of the numbers of cells and blasts per
culture At various times after the initiation of cultures, individual tubes were removed from the incubator and their cells counted on a Z F Model Coulter counter with an orifice of 100 p. The aperture current set a t 64 a n d the amplification at 0.707. Discriminations which allowed the enumeration of total cells (threshold setting 8) and blast cells (threshold setting 24) were used for counting each culture.
2.6. Determination of the number and percent-labeled cells Cultures labeled with 1 pCi [3H]dThd were washed free of unincorporated radioactive material by washing 3 times with 4 m l Hanks' balanced salt solution. The final cell pellet was resuspended in horse serum. Cytological preparations were made i n a Cytospin centrifuge (Shandon), fixed in methanol and air-dried. Autoradiography was performed with Ilford Nuclear Research Emulsion G5 diluted 1 : 3 with distilled water. Slides were stored in the dark for 4 days and then developed, fixed and stained with May-Griinwald-Giemsa. Cells were considered labeled if t h e density of the grains above the cell was at least double that of the background. 2.7. Determination of cell cycle time
2.3. Preparation of spleen cells for culture Mice were sacrificed by cervical dislocation and spleens were removed asceptically. Cell suspensions were obtained b y pressing t h e spleens through a stainless steel strainer i n t o cold tissue culture medium. These suspensions were allowed t o stand in the cold in conical centrifuge tubes for 3 t o 5 min in order t o allow cell clumps t o settle. The supernatant fluid containing single cell suspension was transferred t o a separate test tube, cells were counted in both Turk's solution and trypan blue and appropriate dilutions made in either tissue culture medium or medium plus Con A or LPS t o give 1O6 viable spleen cells/ml. One ml aliquots of cell suspension was distributed t o tissue culture tubes and incubated as described above. All cultures were performed and assayed in triplicate. 2.4. Determination of degree of [ 3H]dThd incorporation
At various times after initiation of t h e cultures, 1 pCi of [3H]dThd diluted t o a specific activity of 1.68 Ci/mmole (New England Nuclear or Nuclear Research Center, Israel) with cold dThd was added t o individual cultures and incorporation allowed t o proceed for 16 h under standard culture conditions. Cultures were terminated by centrifugation at 6 0 0 x g for I0 min at 4 "C. The supernatant fluid was discarded and t h e cell pellet resuspended in ice-cold saline containing 20 pg/ml dThd. T h e cells were filtered o n t o GF/B fiber glass filters on a multiple membrane filter (Yeda Scientific Instrument, Rehovot, Israel) washed well with t h e saline-dThd solution and acid precipitated with cold 5 % trichloroacetic acid containing 2 0 pg/ml dThd. After washing
The cell cycle of cells stimulated with either Con A or LPS was determined b y t h e method of Wilson et al. [ l o ] as follows. 0.1 pCi [3H]dThd was added t o the cultures for a pulse period of 30 min, 48 h after t h e initiation of cultures stimulated with Con A or 7 2 h after the initiation of LPS-stimulated cultures. The pulse was terminated b y washing t h e cultures 3 times with ice-cold Hanks' balanced salt solution and then reconstituting the cultures t o their original 1 ml volume with tissue culture medium plus t h e appropriate mitogen. Cultures were returned t o t h e incubator and at various time intervals after t h e radioactive pulse, individual cultures were exposed t o 100 pg colchicine (Merck, Darmstadt, FRG) for 2 h. Cultures were then terminated by centrifugation in the cold a t 600 x g for 10 min and resuspension in horse serum. Cytological preparations were prepared in the Cytospin centrifuge and autoradiography performed. The percent-labeled mitoses was determined by scoring 100-400 mitoses/culture. Mitoses were deemed labeled if the density of grains over them was at least twice that of the background. In addition, the mitotic index, expressed as percent total mitoses, was calculated by counting t h e total number of mitoses (labeled and unlabeled)/lOOO cells.
3. Results 3.1. Decreased response t o Con A and LPS with age
The in vifro response of C57BL/6J spleen cells t o LPS and Con A shows an age-related decline whether measured by the maximum cell density reached in culture or the maximal
Eur. J. Immunol. 1977. 7: 301-304
Response to ConA and LPS in senescent mice
amount of [3H]dThd incorporation i n t o acid insoluble material (Tables 1 and 2). This drop in response is also reflected in the number of cells incorporating [3H]dThd and the number of blast cells/culture. Maximal responses t o both the B cell mitogen LPS and the T cell mitogen Con A are observed between 6 and 1 2 months of age. The response t o Con A is maintained at near optimal levels through 19-20 months and drops sharply thereafter, whereas the response t o LPS at 19-20 months already demonstrates the beginnings of the age-related decrease in response which becomes more acute with increasing age. Table 1. The in vitro response of spleen cells from C57BL/6J female mice of various ages to E. coli LPS on day of peak responsea)
Age
in months 3 6 11-12
19-20 23-24 26-28
NO. of cells x 104 CO.~) ~xp. 20 3.2 3.0 2.9 3.8 2.2
12.9 10.7 8.0 7.3 6.8 4.0
Incorporation of [3H]dThd (cpm x CO. ~xp. 1.3 1.7 1.9 1.1 1.2 0.4
9.1 12.6 10.3 1 I .o 5.2 2.5
No. of labeled cells x 10-5
6.8 5.0 7.8 4.9 1.9 1.7
Blabt
cells (c;)
50 63 62 56 52 44
303
pable of responding t o the mitogens is reduced in senescent mice, i.e. fewer cells are responding. (b) The cells in the older mice which respond are altered in their capacity t o undergo blast transformation. This alteration may lead t o a longer generation time per cell and, therefore, a lower response per culture. ( c ) Those cells from senescent mice which undergo blast transformation are defective in their capacity t o undergo repeated cell divisions, thus leading t o an abortive response. In order t o differentiate between these various possible causes of the decreased in vitro response of B and T lymphocytes from senescent mice t o mitogens, a series of experiments t o determine the length of the cell cycle of the responding cells and their capacity t o undergo recycling was initiated. As can be seen in Figs. 1 and 2, the length of the cell cycle of Con A and LPS-stimulated lymphocytes is the same whether the lymphocyte donors are 12 months old and at the peak of their capacity t o respond, or senescent at 28 months of age. The individual stages of the cell cycle are also identical for the cells from old and young donors as attested by the virtual overlapping of the curves. These experiments also permit the evaluation of the capacity of the dividing lymphocytes t o undergo repeated cell divisions. In the case of the Con Astimulated cultures we were able t o define two consecutive cycles of cell division of equal length in the ct.llx from old
a ) The peak response for all age groups was observed on day 4-5.
The results are expressed as the arithmetic mean of 3-4 individual experiments per age group. As a rule, individual mice were used for each experiment. However, certain experiments were performed with pools of from 2-4 spleens from mice of a single age. Each age group represents the resultsof from 4-10 mice. b) Co. = control; Exp. = experimental. Table 2. The in v i m response of spleen cells from C57BL/6J female mice of various ages to Con A on day of peak responsea)
m-
3
8070-
E m-
1 so3 40-
-8
30-
g 20-
months
No. of cells x 10-5 C0.b) Exp.
3 6 11 -12 19-20 23-24 26-28 34
2.6 4.0 3.5 3.5 4.4 2.5 3.6
Age in
13.0 16.4 14.0 14.0 7.9 4.8 6
Incorporation of ['HIdThd (cpm x 10-4) co. Exp. 0.9 1.o 1.4 1.o
18.0 20.4 21.9 22.1
0.6
1.1
0.3
9.0 5.2
0.9
No. of labeled cells
Blast cells
x 10-5
(%)
5.2
68 65 65 67 47 45 41
11 13.2 9.9 4.0 5.1
4
a ) The peak response for all age groups was observed on day 3-4.
The results are expressed as the arithmetic mean of 3-4 individual experiments per age group representing results from 3-8 mice except for age groups 3 and 34 months. At 3 months 25 mice were used, and at 34 months we report but a single experiment representing the pooled spleens of two mice. b) Co. = control; Exp. = experimental.
Figure 1 . The cell cycle of splenic lymphocytes stimulated with Con A
from 12-month-old ( 0 - - 0 )and 28-monthald (0-0) C57BL/6J mice. The results are the arithmetic mean of two individual experiments.
I00 -
mn
8
80-
0
i
p
70-
60-
; -
50-
l
I
I
4
6
8
I
I
10
I2
l
I
I
I
I
16
18
20
22
24
3.2. Cell cycle studies In order t o further characterize the nature of the age-related decline in reactivity of both B and T lymphocytes t o polyclonal mitogens, it is of importance t o determine whether the defect in response is detectable at the level of the single cell as well as a t the level of the response of t h e entire cell population. There are several possible explanations for the decline in response. (a) The relative proportion of cells ca-
10 2
111626.21
14
Hours post pulse
Figure 2 . The cell cycle of splenic lymphocytes stimulated with LPS
from 12-monthold (0- 0)and 28-month-old (t--.C57BL/6J ) mice. The results are the arithmetic mean of two individual experiments.
304
Eur. J. Immunol. 1977.7: 301-304
C. Abraham, Y. Tal and H. Cershon
and young adult donors. The LPS-stimulated cultures underwent three identical consecutive cell divisions within a 24hour period in cultures of spleen cells from senescent (28 month) and adult (12 month) mice. I t therefore appears that those cells which divide in culture under the influence of the mitogens Con A and LPS demonstrate the same cell cycle time and equal capacity t o undergo repeated cell divisions, regardless of whether the cells are obtained from adult (1 2 month) o r senescent ( 2 8 month) mice. These results support the conclusion that there are fewer cells with the capacity t o divide in the cultures of senescent animals. This conclusion is further substantiated by calculation of the total proportion of cells in mitoses in the various cultures. After a 2-hour exposure t o colchicine, the mitotic index of Con A-stimulated spleen cells was 12.5 % for cells from 12-month-old mice and 6.2 % for cells from 28-monthold mice. The mitotic index in LPSstimulated cultures was 7.7 % for cells from 12-month-old mice and 4.6 % for cells from 28-month-old animals.
4. Discussion
The in vitro responses of C57BL/6J spleen cells t o the T cell mitogen Con A and the B cell mitogen LPS decrease with age. The responses t o these t w o mitogens remain fairly stable from 6 months of age t o a year and a half and then drop sharply. The B cell response to LPS decreases in parallel t o the T cell response t o Con A. This finding differs somewhat from the findings of Gerbase-Delima e t al. [7] who claim a more gradual and less severe age-related decrease in t h e B cell response than in the T cell response t o polyclonal mitogens. It should also be noted that the incorporation of [jH]dThd in control cultures of senescent mice is also low, both at 7 2 and 96 h after the initiation of cultures. This may reflect a reduced response t o antigenic o r mitogenic components of the tissue culture medium especially t o the fetal calf serum. I t has been our experience that different batches of fetal calf serum induce different levels of background response. The senescent animals respond with lower background cpm than young adults, regardless of the batch of serum used*.
capacity of T cells nor of B cells from adult and senescent mice was detected. I t thus appears that the age-related decline in the in vitro response of murine lymphoid cells t o polyclonal mitogens cannot be attributed t o an altered cell generation time nor t o a reduced capacity for repeated cell division. The decreased response elicited by lymphoid cell populations from senescent mice is thus most probably due t o a reduced number of responding cells, which is also reflected in a lower mitotic index in cultures from senescent mice, while those cells which d o respond, d o so with equal potential t o that of the cells of the young animal. The conclusion that those cells in the senescent animal which are capable of responding, d o so equally well as the cells of younger animals at the prime of their response, is also supported in other experimental systems. We have similar results in murine mixed lymphocyte cultures**. Goodman and Makinodan [ 1 11 and Menon e t al. [ 121 have reached this conclusion for the cytotoxic capacity of T cells. One can infer the same for antibody-producing cells from the report of Price and Makinodan [ 131 as well as from our own studies on the specificity of IgM production in C57BL mice of various ages***. The reports of Harrison on the proliferative capacity of erythropoietic and lymphopoietic stem cells from mice of various ages also lead t o the same conclusion [ 14, 151. Received December 25,1976;in revised form March 3,1977.
5. References
The experiments in which the cell cycle was determined also provided information about the capacity of the lymphoid cells from adult a n d senescent mice t o undergo repeated rounds of cell division. N o differences between the recycling
1 Walford, R.L., The Immunologic Theory of Aging, Munksgaard, Copenhagen 1969. 2 Nordin, A.A. and Makinodan, T., Fed. Proc. 1974.33: 2033. 3 Mackay, I.R., Gerontologia-Basel 1972.18:285. 4 Joris, F.and Gerard, J.P., Int. Arch. Allergy Appl. Immunol.1975. 48:584. 5 Konen, T.G., Smith, G.S. and Walford, R.L.,J. Immunol. 1973. 110: 1216. 6 Hori, J., Perkins, E.H. and Halsall, M.K., Proc. SOC.Exp. Biol. Med. 1973.144:48. 7 Gerbase-Delima,M., Wilkenson, J.,Smith, G.S. and Walford, R.L., J. Gerontol. 1974.29:261. 8 Smith, A.M., J. Immunol.1976.116:469. 9 Anderson, J., Melchers, F., Galanos, C. and Luderitz, 0.. J. Exp. Med. 1973.137:943. 10 Wilson, D.B., Blyth, J.L. and Nowell, P.C., J. Exp. Med. 1973. 128: 1157. 11 Goodman, S.A. and Makinodan, T., Clin. Exp. lmmunol.1975. 1 9 : 533. 12 Menon, M., Jaroslow, B.N. and Koesterer, R., J. Gerontol. 1974. 29: 499. 13 Rice, G.B. and Makinodan, T., J. Immunol.1972. 108:403. 14 Harrison, D.E., J. Gerontol. 1975.30: 279. 15 Harrison, D.E. and Doubleday, J.U.,J. Immunol.1975.114:1314.
* Abraham, C . and Gershon, H., unpublished data.
** Merhav, S.and Gershon, H.,in prcparation. *** Zharhary, D. and Gershon, H., in preparation.
Experiments comparing the cell cycle of T cells responding t o Con A and B cells responding t o LPS from mice 1 2 and 2 8 months of age, demonstrate that both T cells and B cells from mice of both age groups divide with the same generation time of 9 h. The experiments performed here also permitted an estimate of the length of the various stages of the cell cycle which also appear t o be identical in both age groups for both T and B cells, with Gz lasting approximately 1.5 h ; S being of about 5-6 h duration and G I and M requiring the remaining time.
Eur. J. Immunol. 1977. 7: 301-304
301
10 stark, O., Kien, V.,Frenzl, B. and BrdiEka, R., in Dausset, J.,
5. References 1 Charlton, R.K. and Zmijewski, C.M., Science 1970.170: 636. 2 van Rood, J.J., van Leeuwen, A. andvan Santen, M.C.T., Nature 1970.226: 366. 3 Schultz, J.S. and Shreffler, D.C., Transplantation 1972.13: 186. 4 Aster, R.H., Miskovich, B.H. and Rodey, G.E., Transplantation 1973.16: 205.
Hamburger, J. and Mathe', G. (Eds.), Advance in Transplantation, Munksgaard, Copenhagen 1968, p. 331. 11 stark, O., Kienovi, D., Kren, V. and Frenzl, B., Folia biol. Prague 1970.16: 1. 12 Chut&, J., Folia biol. Prague 1973. 19: 231. 13 Sanderson, A.R., Immunology 1965. 9: 287. 14 Wigzell, H., Transplantation 1965.3: 423.
5 Oh, S.K., Pellegino, M.A., Ferrone, S., Sevier, E.D. and Reisfeld, R.A., Eur. J. Immunol. 1975.5: 161.
15 Kamrin, B.B.,Proc. Soc. Exp. Biol. Med. 1959.100: 58.
6 Callahan, G.N., Ferrone, S., Allison, J.P. and Reisfeld, R.A., Transplantation 1975.20: 431.
17 Mannick. J.A. and Schmid, K., Transplantation 1967. 5 : 1231.
7 Calne, R.Y., Davis, D.R., Hadjiyannakis, E.,Sells, R.A., White, D.,
Herbertson, B.M., Millard, P.R., Joysey. V.C., Davies, D.A.L., Binns, R.M. and Festenstein, H., Nature 1970.227 903.
16 Mowbray, J.F., Transplantation 1963. I : 15. 18 Nelken, D. and Glaser, M., Transplantation 1972.14: 268. 19 Hanna, N., Kalderon, R. and Nelken, D., Immunology 1975.29: 433. 20 Pokorna, Z. and Vojtigkov& M., Folia biol. Prague 1966. 12: 88.
8 Hakk, M.. Chutd, J., HoI& V. and Shdebk, M.,Nature 1976. 262: 295.
21 Law, L.W., Apella, E., Strober, S., Wright, P.W. and Fischetti, T., Proc. Nut. Acad. Sci. US 1972.69: 1858.
9 HGek, M., Chutni, J., SGde&k, M. and HOG%,V., Folia biol. &ague 1976.22: 411.
22 Law, L.W., Apella, E., Cohen, J. and Bern, J.M., Nature-New Biol. 1973.246: 174.
Carmella Abaham', Y. Talo and Harriet Gerrhon' Technion, Israel Institute of Technology, School of Medicine' and Department of Pediatrios, Rothschikl Hospital0, Haifa
Reduced in vibo response to concanavalin A and lip0 polysaccharide in senescent mice: a function of reduced number of responding cells" The proliferative capacity of spleen cells from C57BL/6J female mice of various ages (3-28 months) to the polyclonal mitogens concanavalin A (ConA) and lipopolysaccharide (LPS) was examined. It was found that both the T and B cell population of the spleen demonstrate a n age-related decrease i n their capacity t o respond in vitro. Peak responses t o both mitogens occurs at about 1 year of age. This age-related reduction in response is expressed in t h e degree of incorporation of [3H]thymidine i n t o DNA, the total number of cells generated in vitro, the number of labeled cells per culture a n d t h e number of b h s t cells per culture. The day of peak response i n vitro does not change with age. Studies of the cell cycle of cells responding to Con A and t o LPS from 1 2 and 28-monthold mice demonstrate that the generation time of individual proliferating cells does not alter with age. Nor does i t differ for the B cells responding t o LPS or t h e T cells responding t o Con A. These studies also demonstrate that the proliferating cells from senescent mice are equally capable of repeated cell divisions as are the cells from the I-year-old adult mouse. It is concluded that the defect in senescent mice which leads t o a reduced i n vitro response t o t h e polyclonal mitogens LPS and Con A is a reduction in the number of responding cells and not a n alteration in t h e capacity of those cells which d o respond t o divide.
1. Introduction Aging mammals are characterized by several immunologically related disorders such as an increased incidence of neoplasia and autoimmune diseases a n d a n augmented susceptibility t o infectious diseases [l]. Studies of the immune reactivity of senescent animals, and in particular studies of mice and hu-
[I
16261
*
This work was supported by Grants from the Israel National Academy of Sciences and the Israel Cancer Society. Correspondence: Harriet Gershon, Technion, Israel Institute of Technology, The Aba Khoushy School of Medicine, Haifa, Israel Abbreviations: Comk Concanavalin A LPS: Lipopolysaccharide [3H)dThd: Tritiated thymidine cpm: Counts/min
mans, have demonstrated a n age-related decrease in the capacity to produce humoral [2, 31 as well as cell-mediated immune responses [3-51. In vitro studies reveal that populations of b o t h T and B lymphocytes from senescent mice are deficient in their capacity t o respond t o polyclonal mitogens, as indicated b y t h e incorporation of tritiated thymidine [3H]dThd into DNA [6-81. In order to better understand t h e nature of the defect in immune reactivity of senescent animals, we have performed a series of experiments designed t o determine whether t h e age-related decrease in response t o polyclonal mitogens is d u e t o a decreased number of ~ymphocytes capable of undergoing blast transformation a n d cell division, or t o a n altered capacity of individual cells t o divide, thus leading t o longer generation times and reduced response.
302
C. Abraham, Y. Tal and
Eur. J. Immunol. 1977. 7: 301-304
H.Gershon
2. Materials and mathods 2.1. Animals
All experiments were performed with t h e lymphocytes of C57BL/6J female mice of various ages. These mice were bred and raised under specific pathogen-free conditions a t the Weizmann Institute of Science. The mean life-span of this mouse colony is 24 months.
2.2. Culture conditions and mitogens used Viable spleen cells (1 06)were cultured i n 1 ml tissue culture medium with or without the addition of mitogen for u p t o 120 h at 37 "C in a water-saturated atmosphere of 5 % COz in air. Tissue culture medium consisted of Eagle's minimum essential medium with Earle's salts, glutamine and nonessential amino acids (F-15 Gibco, Grand Island, N.Y.,) supplemented with 1 5 % heat-inactivated fetal calf serum (Gibco) plus 20 pg/ml asparagine (Fluka AG,Buchs, Switzerland), 2-mercaptoethanol (Fluka), at a final concentration of l o 4 M, 1Os units/l penicillin G (Rafa Laboratories Ltd., Jerusalem) and 0.1 g/l dihydro-streptomycin (Teva L.td., Jerusalem). Cultures were performed in either Nunclon 1 4 1 0 or Corning 25200 disposable plastic tissue culture tubes. Results obtained in both types of tubes were identical and data were pooled.
To individual cultures either 1 0 pg E. coli 026:36 lipopolysaccharide-W (Difco, Detroit, Mich.), activated according t o Anderson et al. 191 or I0 p g ConA (Miles-Yeda) were added.
with 95 % ethanol and drying for 30 min at 1 0 0 "C, t h e filters were placed in vials containing 1 0 m l scintillation fluid (5 g PPO, 500 mg POPOP in 1 1toluene) and cpm monitored o n a Packard Tricarb scintillation counter. 2.5. Determination of the numbers of cells and blasts per
culture At various times after the initiation of cultures, individual tubes were removed from the incubator and their cells counted on a Z F Model Coulter counter with an orifice of 100 p. The aperture current set a t 64 a n d the amplification at 0.707. Discriminations which allowed the enumeration of total cells (threshold setting 8) and blast cells (threshold setting 24) were used for counting each culture.
2.6. Determination of the number and percent-labeled cells Cultures labeled with 1 pCi [3H]dThd were washed free of unincorporated radioactive material by washing 3 times with 4 m l Hanks' balanced salt solution. The final cell pellet was resuspended in horse serum. Cytological preparations were made i n a Cytospin centrifuge (Shandon), fixed in methanol and air-dried. Autoradiography was performed with Ilford Nuclear Research Emulsion G5 diluted 1 : 3 with distilled water. Slides were stored in the dark for 4 days and then developed, fixed and stained with May-Griinwald-Giemsa. Cells were considered labeled if t h e density of the grains above the cell was at least double that of the background. 2.7. Determination of cell cycle time
2.3. Preparation of spleen cells for culture Mice were sacrificed by cervical dislocation and spleens were removed asceptically. Cell suspensions were obtained b y pressing t h e spleens through a stainless steel strainer i n t o cold tissue culture medium. These suspensions were allowed t o stand in the cold in conical centrifuge tubes for 3 t o 5 min in order t o allow cell clumps t o settle. The supernatant fluid containing single cell suspension was transferred t o a separate test tube, cells were counted in both Turk's solution and trypan blue and appropriate dilutions made in either tissue culture medium or medium plus Con A or LPS t o give 1O6 viable spleen cells/ml. One ml aliquots of cell suspension was distributed t o tissue culture tubes and incubated as described above. All cultures were performed and assayed in triplicate. 2.4. Determination of degree of [ 3H]dThd incorporation
At various times after initiation of t h e cultures, 1 pCi of [3H]dThd diluted t o a specific activity of 1.68 Ci/mmole (New England Nuclear or Nuclear Research Center, Israel) with cold dThd was added t o individual cultures and incorporation allowed t o proceed for 16 h under standard culture conditions. Cultures were terminated by centrifugation at 6 0 0 x g for I0 min at 4 "C. The supernatant fluid was discarded and t h e cell pellet resuspended in ice-cold saline containing 20 pg/ml dThd. T h e cells were filtered o n t o GF/B fiber glass filters on a multiple membrane filter (Yeda Scientific Instrument, Rehovot, Israel) washed well with t h e saline-dThd solution and acid precipitated with cold 5 % trichloroacetic acid containing 2 0 pg/ml dThd. After washing
The cell cycle of cells stimulated with either Con A or LPS was determined b y t h e method of Wilson et al. [ l o ] as follows. 0.1 pCi [3H]dThd was added t o the cultures for a pulse period of 30 min, 48 h after t h e initiation of cultures stimulated with Con A or 7 2 h after the initiation of LPS-stimulated cultures. The pulse was terminated b y washing t h e cultures 3 times with ice-cold Hanks' balanced salt solution and then reconstituting the cultures t o their original 1 ml volume with tissue culture medium plus t h e appropriate mitogen. Cultures were returned t o t h e incubator and at various time intervals after t h e radioactive pulse, individual cultures were exposed t o 100 pg colchicine (Merck, Darmstadt, FRG) for 2 h. Cultures were then terminated by centrifugation in the cold a t 600 x g for 10 min and resuspension in horse serum. Cytological preparations were prepared in the Cytospin centrifuge and autoradiography performed. The percent-labeled mitoses was determined by scoring 100-400 mitoses/culture. Mitoses were deemed labeled if the density of grains over them was at least twice that of the background. In addition, the mitotic index, expressed as percent total mitoses, was calculated by counting t h e total number of mitoses (labeled and unlabeled)/lOOO cells.
3. Results 3.1. Decreased response t o Con A and LPS with age
The in vifro response of C57BL/6J spleen cells t o LPS and Con A shows an age-related decline whether measured by the maximum cell density reached in culture or the maximal
Eur. J. Immunol. 1977. 7: 301-304
Response to ConA and LPS in senescent mice
amount of [3H]dThd incorporation i n t o acid insoluble material (Tables 1 and 2). This drop in response is also reflected in the number of cells incorporating [3H]dThd and the number of blast cells/culture. Maximal responses t o both the B cell mitogen LPS and the T cell mitogen Con A are observed between 6 and 1 2 months of age. The response t o Con A is maintained at near optimal levels through 19-20 months and drops sharply thereafter, whereas the response t o LPS at 19-20 months already demonstrates the beginnings of the age-related decrease in response which becomes more acute with increasing age. Table 1. The in vitro response of spleen cells from C57BL/6J female mice of various ages to E. coli LPS on day of peak responsea)
Age
in months 3 6 11-12
19-20 23-24 26-28
NO. of cells x 104 CO.~) ~xp. 20 3.2 3.0 2.9 3.8 2.2
12.9 10.7 8.0 7.3 6.8 4.0
Incorporation of [3H]dThd (cpm x CO. ~xp. 1.3 1.7 1.9 1.1 1.2 0.4
9.1 12.6 10.3 1 I .o 5.2 2.5
No. of labeled cells x 10-5
6.8 5.0 7.8 4.9 1.9 1.7
Blabt
cells (c;)
50 63 62 56 52 44
303
pable of responding t o the mitogens is reduced in senescent mice, i.e. fewer cells are responding. (b) The cells in the older mice which respond are altered in their capacity t o undergo blast transformation. This alteration may lead t o a longer generation time per cell and, therefore, a lower response per culture. ( c ) Those cells from senescent mice which undergo blast transformation are defective in their capacity t o undergo repeated cell divisions, thus leading t o an abortive response. In order t o differentiate between these various possible causes of the decreased in vitro response of B and T lymphocytes from senescent mice t o mitogens, a series of experiments t o determine the length of the cell cycle of the responding cells and their capacity t o undergo recycling was initiated. As can be seen in Figs. 1 and 2, the length of the cell cycle of Con A and LPS-stimulated lymphocytes is the same whether the lymphocyte donors are 12 months old and at the peak of their capacity t o respond, or senescent at 28 months of age. The individual stages of the cell cycle are also identical for the cells from old and young donors as attested by the virtual overlapping of the curves. These experiments also permit the evaluation of the capacity of the dividing lymphocytes t o undergo repeated cell divisions. In the case of the Con Astimulated cultures we were able t o define two consecutive cycles of cell division of equal length in the ct.llx from old
a ) The peak response for all age groups was observed on day 4-5.
The results are expressed as the arithmetic mean of 3-4 individual experiments per age group. As a rule, individual mice were used for each experiment. However, certain experiments were performed with pools of from 2-4 spleens from mice of a single age. Each age group represents the resultsof from 4-10 mice. b) Co. = control; Exp. = experimental. Table 2. The in v i m response of spleen cells from C57BL/6J female mice of various ages to Con A on day of peak responsea)
m-
3
8070-
E m-
1 so3 40-
-8
30-
g 20-
months
No. of cells x 10-5 C0.b) Exp.
3 6 11 -12 19-20 23-24 26-28 34
2.6 4.0 3.5 3.5 4.4 2.5 3.6
Age in
13.0 16.4 14.0 14.0 7.9 4.8 6
Incorporation of ['HIdThd (cpm x 10-4) co. Exp. 0.9 1.o 1.4 1.o
18.0 20.4 21.9 22.1
0.6
1.1
0.3
9.0 5.2
0.9
No. of labeled cells
Blast cells
x 10-5
(%)
5.2
68 65 65 67 47 45 41
11 13.2 9.9 4.0 5.1
4
a ) The peak response for all age groups was observed on day 3-4.
The results are expressed as the arithmetic mean of 3-4 individual experiments per age group representing results from 3-8 mice except for age groups 3 and 34 months. At 3 months 25 mice were used, and at 34 months we report but a single experiment representing the pooled spleens of two mice. b) Co. = control; Exp. = experimental.
Figure 1 . The cell cycle of splenic lymphocytes stimulated with Con A
from 12-month-old ( 0 - - 0 )and 28-monthald (0-0) C57BL/6J mice. The results are the arithmetic mean of two individual experiments.
I00 -
mn
8
80-
0
i
p
70-
60-
; -
50-
l
I
I
4
6
8
I
I
10
I2
l
I
I
I
I
16
18
20
22
24
3.2. Cell cycle studies In order t o further characterize the nature of the age-related decline in reactivity of both B and T lymphocytes t o polyclonal mitogens, it is of importance t o determine whether the defect in response is detectable at the level of the single cell as well as a t the level of the response of t h e entire cell population. There are several possible explanations for the decline in response. (a) The relative proportion of cells ca-
10 2
111626.21
14
Hours post pulse
Figure 2 . The cell cycle of splenic lymphocytes stimulated with LPS
from 12-monthold (0- 0)and 28-month-old (t--.C57BL/6J ) mice. The results are the arithmetic mean of two individual experiments.
304
Eur. J. Immunol. 1977.7: 301-304
C. Abraham, Y. Tal and H. Cershon
and young adult donors. The LPS-stimulated cultures underwent three identical consecutive cell divisions within a 24hour period in cultures of spleen cells from senescent (28 month) and adult (12 month) mice. I t therefore appears that those cells which divide in culture under the influence of the mitogens Con A and LPS demonstrate the same cell cycle time and equal capacity t o undergo repeated cell divisions, regardless of whether the cells are obtained from adult (1 2 month) o r senescent ( 2 8 month) mice. These results support the conclusion that there are fewer cells with the capacity t o divide in the cultures of senescent animals. This conclusion is further substantiated by calculation of the total proportion of cells in mitoses in the various cultures. After a 2-hour exposure t o colchicine, the mitotic index of Con A-stimulated spleen cells was 12.5 % for cells from 12-month-old mice and 6.2 % for cells from 28-monthold mice. The mitotic index in LPSstimulated cultures was 7.7 % for cells from 12-month-old mice and 4.6 % for cells from 28-month-old animals.
4. Discussion
The in vitro responses of C57BL/6J spleen cells t o the T cell mitogen Con A and the B cell mitogen LPS decrease with age. The responses t o these t w o mitogens remain fairly stable from 6 months of age t o a year and a half and then drop sharply. The B cell response to LPS decreases in parallel t o the T cell response t o Con A. This finding differs somewhat from the findings of Gerbase-Delima e t al. [7] who claim a more gradual and less severe age-related decrease in t h e B cell response than in the T cell response t o polyclonal mitogens. It should also be noted that the incorporation of [jH]dThd in control cultures of senescent mice is also low, both at 7 2 and 96 h after the initiation of cultures. This may reflect a reduced response t o antigenic o r mitogenic components of the tissue culture medium especially t o the fetal calf serum. I t has been our experience that different batches of fetal calf serum induce different levels of background response. The senescent animals respond with lower background cpm than young adults, regardless of the batch of serum used*.
capacity of T cells nor of B cells from adult and senescent mice was detected. I t thus appears that the age-related decline in the in vitro response of murine lymphoid cells t o polyclonal mitogens cannot be attributed t o an altered cell generation time nor t o a reduced capacity for repeated cell division. The decreased response elicited by lymphoid cell populations from senescent mice is thus most probably due t o a reduced number of responding cells, which is also reflected in a lower mitotic index in cultures from senescent mice, while those cells which d o respond, d o so with equal potential t o that of the cells of the young animal. The conclusion that those cells in the senescent animal which are capable of responding, d o so equally well as the cells of younger animals at the prime of their response, is also supported in other experimental systems. We have similar results in murine mixed lymphocyte cultures**. Goodman and Makinodan [ 1 11 and Menon e t al. [ 121 have reached this conclusion for the cytotoxic capacity of T cells. One can infer the same for antibody-producing cells from the report of Price and Makinodan [ 131 as well as from our own studies on the specificity of IgM production in C57BL mice of various ages***. The reports of Harrison on the proliferative capacity of erythropoietic and lymphopoietic stem cells from mice of various ages also lead t o the same conclusion [ 14, 151. Received December 25,1976;in revised form March 3,1977.
5. References
The experiments in which the cell cycle was determined also provided information about the capacity of the lymphoid cells from adult a n d senescent mice t o undergo repeated rounds of cell division. N o differences between the recycling
1 Walford, R.L., The Immunologic Theory of Aging, Munksgaard, Copenhagen 1969. 2 Nordin, A.A. and Makinodan, T., Fed. Proc. 1974.33: 2033. 3 Mackay, I.R., Gerontologia-Basel 1972.18:285. 4 Joris, F.and Gerard, J.P., Int. Arch. Allergy Appl. Immunol.1975. 48:584. 5 Konen, T.G., Smith, G.S. and Walford, R.L.,J. Immunol. 1973. 110: 1216. 6 Hori, J., Perkins, E.H. and Halsall, M.K., Proc. SOC.Exp. Biol. Med. 1973.144:48. 7 Gerbase-Delima,M., Wilkenson, J.,Smith, G.S. and Walford, R.L., J. Gerontol. 1974.29:261. 8 Smith, A.M., J. Immunol.1976.116:469. 9 Anderson, J., Melchers, F., Galanos, C. and Luderitz, 0.. J. Exp. Med. 1973.137:943. 10 Wilson, D.B., Blyth, J.L. and Nowell, P.C., J. Exp. Med. 1973. 128: 1157. 11 Goodman, S.A. and Makinodan, T., Clin. Exp. lmmunol.1975. 1 9 : 533. 12 Menon, M., Jaroslow, B.N. and Koesterer, R., J. Gerontol. 1974. 29: 499. 13 Rice, G.B. and Makinodan, T., J. Immunol.1972. 108:403. 14 Harrison, D.E., J. Gerontol. 1975.30: 279. 15 Harrison, D.E. and Doubleday, J.U.,J. Immunol.1975.114:1314.
* Abraham, C . and Gershon, H., unpublished data.
** Merhav, S.and Gershon, H.,in prcparation. *** Zharhary, D. and Gershon, H., in preparation.
Experiments comparing the cell cycle of T cells responding t o Con A and B cells responding t o LPS from mice 1 2 and 2 8 months of age, demonstrate that both T cells and B cells from mice of both age groups divide with the same generation time of 9 h. The experiments performed here also permitted an estimate of the length of the various stages of the cell cycle which also appear t o be identical in both age groups for both T and B cells, with Gz lasting approximately 1.5 h ; S being of about 5-6 h duration and G I and M requiring the remaining time.
