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Proliferative response of normal rabbit and mouse bone marrow cells in vitro
CELLULAR
5, 48C-486 (1972)
IMMUNOLOGY
Proliferative
Response Bone
of Normal
Marrow
Cells
Rabbit
and Mouse
in Vitro 1
J. J. MOND' AND G. J. THORBECKE~ Department
of Pathology,
New
New York University School York, New York 10016
Received
March
of Medicine,
22, 1972
Bone marrow cells from both normal rabbits and mice are stimulated by exposure to antigens in vitro. Responsiveness is not removed by prior injection of the antigen into the animals. The specificity of this phenomenon is doubtful in view of a lack of additive effects between antigens, unrelatedness to immunogenicity of the antigen, and occurrence in cells from immunologically immature rabbits.
It has been concluded from a number of observations that, in the rabbit, unlike the mouse, the bone marrow is a major source of T-cells. Among the data leading to this conclusion are: (a) the fact that bone marrow cells proliferate in response to various antigens in vitro (1) ; (b) the ability of transferred bone marrow to induce antibody-forming cells of recipient origin to sheep erythrocytes in X-irradiated rabbits (2) ; (c) removal of both activities from the bone marrow by preexposure to antigen in Z&JO (2) or by passage through antigen-coated columns (3). There are several problems in accepting this conclusion. It is known that bone marrow cells in vitro tend to respond with increased proliferation to a variety of nonimmunogenic as well as immunogenic substances, and that this proliferation is probably due to hematopoietic colony-forming precursor cells (4, 5). Responsiveness of rabbit thymocytes in vitro in the mixed lymphocyte reaction (6) and to phytohemagglutinin (7) is similar to that found in mice. Thymectomy followed by X-irradiation in adult rabbits and mice leads to comparable changes in the histology of their peripheral lymphoid tissue (8). In addition, it seems difficult to, understand why a previous injection should deplete cells from the bone marrow rather than cause the presence of circulating memory cells as is the case in other rodents (9, lo), particularly after immunization of the animals with antigen in complete Freund’s adjuvant. The present studies were undertaken in an effort to determine whether proliferation of normal bone marrow cells to a variety of antigens in vitro can be achieved 1 Supported by U.S.P.H.S. Grant AI-3078 s Recipient of NIH Training Grant 5 TO 5GM01668 from the National Institute of General Medical Science. 3 Health Research Council Career Scientist of the City of New York. 480 Co yright Alfrights
Q 1972 by Academic Press of reprodurtinn in anv ford
Inc. reserved.
BONE
MARROW
PROLIFERATIVE
RESPONSE
4X1
in species other than the rabbit. The specificity of the response was also investigated. The experiments to be described demonstrate that normal mouse bone marrow cells proliferate in response to several antigens. Depletion of responsiveness of bone marrow cells is not regularly obtained by prior antigen injection in either rabbits or mice. MATERIALS AND
METHODS
Aniwmls and immunization. New Zealand rabbits ranging in age from 1 week to 1 year were used. Allotypes on y-globulin were determined by double diffusion in agar employing reference antisera kindly provided by Dr. A. Kelus (Base1 Institute for Immunology, Basel, SwitzerIand). Rabbits were immunized with human gamma globulin (HGG, generously provided by Dr. G. A. Jamieson, American National Red Cross, Bethesda, MD) either intravenously with 25.0 mg HGG or subcutaneously with 0.5 mg HGG in complete Freund’s adjuvant. Balb/c mice were obtained from Cumberland Farms, Clinton, TE. In some experiments LAF, mice obtained from Jackson Laboratories (Bar Harbor, ME) was used. Immunization was by intravenous injection of HGG. Preparation
of Bone iWarrozv Cell Cultures
dIou.sc cell czdlturcs. Femoral and tibia1 bones were removed and the bone marrow cells were flushed out with Hanks’ balanced salt solution (HBSS). Bone marrow plugs were dispersed by passage through a 25-gauge needle. The cells were filtered through sterile gauze, washed twice in HBSS, and suspended to a concentration of lo6 cells/ml in RPM1 1640 + 10% FCS (Associated Biomedics Systems, Buffalo, NY). Cells were distributed in l.O-ml aliquots into 12 X 75-mm culture tubes (Falcon Plastics, Los Angeles, CA), and cultured after addition of antigen, at 37°C in 5% CO,-95% air atmosphere. On the fourth day of incubation 1 PCi of tritiated thymidine (Schwarz BioResearch Inc., Orangeburg, NY sp act 0.36 Ci/mmole) was added to each tube and incubation was continued for an additional l&24 hr. Rabbit ccl1 cz&ures. Bone marrow was taken from the femur and teased into a cell suspension. The cells were filtered through sterile gauze, washed twice in HBSS! and suspended in medium at a concentration of lo6 cells/ml. The medium used for rabbit cells was Dulbecco’s modified Eagle’s medium with 10% NRS (bl serum). Culture additives. Anti-b5 was prepared as previously described. Antigens used included killed Brucella abortus organisms (BA) (kindly donated by Dr. C. E. Watson from the United States Dept. of Agriculture), dinitrophenylated hemocyanin (DNP-KLH), DNP-bovine gamma globulin (DNP-BGG), DNP-human serum albumin (DNP-HSA). All culture additives were added in 0.05-ml aliquots to 1.0 ml of cell suspension. illraswrment
of $H Thyvtzidine Incorporation
.1t the end of the culture period, the cells were centrifuged, washed twice in phosphate-buffered saline, pH 7.2, and resuspended in 1.0 ml of 0.5 M perchloric
482
MOND
AND
THORBECKE
acid. The tubes were then left at 4°C overnight. The precipitates were washed twice with 0.25 M perchloric acid and once with methanol. After the final washing the precipitates were left to dry overnight at room temperature, The next day 0.5 ml hyamine was added to each tube and the tubes were incubated for 2 hr at room temperature. Aliquots of 0.2 ml were pipetted into glass vials containing 10 ml of toluene-liquifluor scintillation fluid (New England Nuclear, Boston, MA). The amount of radioactivity was determined in a Tri-Carb liquid scintillation counter (Packard Instrument Co., Downers Grove, IL), RESULTS
Proliferative
Response of Bone Marrow
Cells to Antigens
The results in Table 1 clearly show that both rabbit and mouse marrow cells from uninjected animals respond with enhanced proliferation to the presence of antigens during the culture period. Of the antigens tried, HGG (1.0 mg) was most regularly stimulatory to bone marrow cells of both species. DNP conjugates of KLH, BGG, and HSA (not in tables) were all stimulatory in doses of 0.1-1.0 mg per tube. DNP-HSA was found to stimulate better at 1.0 mg than at 0.1 mg, DNP-KLH was sometimes toxic at the l.O-mg level. Other antigens, which stimulated, but were only examined in an occasional experiment, included sheep erythrocyte stromata (6) and BA. An additional experiment was performed with mouse bone marrow cells preincubated at 37°C for 2% hr with 250 pg of bromodesoxyuridine (BUDR) per ml. Such pretreated cells still gave a response to HGG suggesting that the responding cells were not proliferating at a rapid rate prior to the initiation of culture ( 11) . The results in Table 2 show that the modifications of HGG generally used to render it either “immunogenic” (aggregated) or “tolerogenic” (spun) (12, 13) did not influence the degree of the response obtained. This suggests that the proliferative response of normal bone marrow cells is independent of the immunogenicity of the stimulant. TABLE THE PKOLIFERATIVE
1
RESPONSES OF NORMAL MOUSE OR RABBIT TO VARIOUS ANTIGENS in Vitro 5
Normal rabbit bone marrow Antigen
MARROW
CELLS
mouse bone marrow
added Expt
No antigen DNP-KLH
Normal
BONE
(0.1 mg)
HGG (1.0 mg) DNP-BGG (1.0 mg)
180 685
1
Expt 2 1855 4330
515
0 Results are expressed in counts per minute culture. h DNP-BGG (0.2 mg) was used.
Expt 3
Expt 4
Expt 5
Expt 6
190
560 1230
480 3425
955 2170
1090
2705
1995 2045
8350 b
of 3H-thymidine
incorporated
during
Day 4 in
BONE
MARROW
PROLIFERATIVE
TABLE
483
RESl’Oh-SE
2
Com.4~1~0~ OF THE in Vitro PKOLIFERATIVE RESPONSES OF NONAL RABBIT HGG (AHGG) OH Mous~s BONE MARKOR. CELLS a TO AGGREGATED AND ULTRACENTRIFUGED HGG (SPCX HGG)
Antigen
added
Expt
None AHGG b (1.0 mg) Spun HGG c (1.0 mg)
Expt 8 (cpm)
7 (cpm)
1790 3800 10535
765 1400 1960
‘* Expt 7 utilized rabbit, Expt 8 utilized mouse bone marrow cells. 6 HGG was prepared at a concentration of 10.0 mg/ml in saline and allowed to incubate for 1.5 min at 63°C. r HGG was spun in a preparative ultracentrifuge at 25,000 rpm for 2 hr and only the top !,.i fractic,ll 1vi15 used.
Efect Cells
of Antigen
The experiments cells from rabbits at varying times was not noticeably taken 1 day after this antigen (Expts Attempts Responses
to
Preinjectiofz
in Yivo
on the in Vitro
Response
of Bone Marvow
presented in Table 3 were performed with bone marrow and mice, which had received either HGG or DNP-KLH before death. The responsiveness of the bone marrow cells affected by such injections. Even rabbit bone marrow cells an intravenous injection of 25.0 mg HGG still responded to 9 and IO).
Establish
Inzm.mological
Specificity
the
of
Bone
Marrozrl
Cell
The results in Fig. 1 show that the simultaneous addition of two antigens (HGG 1.0 mg and DNP-KLH 0.1 mg) , each of which gave stimulation on its own, did not result in an additive effect on the proliferative response. It should be noted that in other studies in this laboratory an additive effect of phytohemagglutinin and anti-b5 has readily been shown with rabbit lymph node cells (14). TABLE
3
1.4~~ OF S~CIFIC IKHIRITION AFTEK PREINJECTION a OF ANTIGENS INTO RABBITS .\lmr ON THE PROLIFEKATIVE RESPOKSIVENESS OF THEIR BONE h'fARROw CELLS in
Radioactk-ity :Intigen
Incorporated
011 Vitro
‘(
(cpm)
added Expt 9
None HGG (1.0 mg) 1)X1’-KLH (0.1 mg)
338 1880 500
Expt
10
1175 17220
Expt
11
Expt
12
Espt
885 2500
3500 13770
905 2335
2620
5220
1655
13
o The following were the preinjection schedules for the experiments described above. Expts. 9, 10, 25.0 mg HGG intravenously on Da)- -1, Expt . 11, 10.0 mg DNP-KLH intravenously on Day -1, Espt. 12, 5.0 mg HGG subcutaneously on Day -3, Expt. 13, 0.5 mg spun HGG intrave IiOUSl~ “11 Day -10. h Experiments 9, 10, 11, 12 were performed with rabbit bone marrow cells. Experiment 13 was done with mouse bone marrow cells.
484
MOND
AND
THORBECKE
.20 “b 16 ;
12
68 0 NO Antigen
FIG. 1. Response of rabbit
HGG o.lmQ
bone marrow
HGG l.hQ
DNP-KLH DNP-KLH o.hQ O.hQ + HGG+ l.Omg O.lmg
cells to combination
of
HGG and DNP-KLH.
Although the presence of similar amounts of anti-b5 in the cultures of rabbit spleen cells was previously shown to completely suppress a secondary plaqueforming cell response to sheep erythrocytes in vitro (6), its presence throughout the incubation period with the bone marrow cells did not block the proliferative response to HGG (Table 4). Most forms of immunological competence show a relatively rapid maturation in neonatal animals. Rabbit lymphoid tissue is poorly developed at birth and does not reach optimal levels of responsiveness until 4 weeks later. It, therefore, was of interest to study the response of bone marrow cells to antigens before the animals reached immunological maturity. In all of four experiments, performed with bone marrow cells from rabbits l-4 weeks of age, a variable degree of stimulation was obtained by addition of 1 mg HGG. The radioactivity in stimulated cells was 1.4-5.1 times that in control cells, with an average degree of stimulation of 2.5X. The cells from l-week-old rabbits responded at least as well as those from the older rabbits. The response of these same bone marrow cells to anti-b5 was also tested and found to be low or absent, as was also described previously (7). The early appearance of this responsiveness to HGG again suggests the possibility that this is a nonspecific rather than an immunological phenomenon. A possible lack of dependency on immunological maturation was also suggested by the fact that bone marrow from 3 to 4-week-old mice responded as well as did cells from young adult mice. TABLE INABILITY Antigen
OF AivrI-b5’” TO BLOCK THE in Vitro PROLIFERATIVE RESPONSE OF HOMOZYGOUS b5 RABBIT BONE MARROW CELLS TO ANTIGEN
added
Expt 9
None HGG
(1.0 mg)
Anti-b5 Anti-b5
+ 1.0 mg HGG
0 Anti-b5
4
was used at a dilution
of l/20.
Expt
14
338
190
1880
1090
775
350
3200
7760
BONE
M.4RROW
PROLIFERATIVE
RESPONSE
485
DISCUSSION
The present findings show that normal mouse bone marrow gives a similar proliferative response to antigen as does normal rabbit bone marrow. Several of the properties of this response were comparable in the two species. There was thus no indication that a fundamental difference existed concerning the role of the bone marrow in the immune response of the mouse and the rabbit, such as was suggested previously by Richter and Abdou ( 15 ) . In fact, the present findings provide no evidence that the tendency of bone marrow cells to show enhanced mitotic activity in the presence of HGG or Dh’Pprotein conjugates has any immunological basis, since each individual observation speaks against specificity of the phenomenon. 1. The major argument previously used by Richter and co-workers (2) in favor of specificity was that the presence of antigen in the animal removed from the bone marrow the ability to respond to that antigen. Similarly, the passage of bone marrow cells through antigen-containing columns also specifically removed this property (3). Under the conditions of the present experiments, the in viva attempts at removal of responsiveness by previous injection of the antigens did not prove effective in either the rabbmitor the mouse. 2. Additive effects of two simultaneously added antigens were not observed. This is contrary to what has been observed for the proliferative responses of thymus-derived peripheral blood lymphocytes to two different major histocompatibihty antigens. which give additive effects in the mixed lymphocyte reaction i16j. Similarly, additive effects have been observed in the response to antigens of lymphocytes taken from delayed hypersensitive individuals ( 17). It should also be noted that additive effects in the proliferative response are obtained \vhen B and T lymphocytes are responding simultaneously to the combination of PHA and anti-Ig ( 1-t. 1s). 3. Aggregated and nonaggregated forms of HGG were equally effective in causing the proliferative response in bone marrow in vitro, whereas these motlifi( 12. cations of HGG are known to greatly influence its immunogenicity in ni7v 13). 3. Bone marrow from neonatal animals was equally responsive as that of adults. This is unlike immunologically specific functions expressed in peripheral lymphoid organs, since these require some degree of maturation of the immune system prior to optimal performance ( 19 ) . 5. Finally, the presence of anti-b5 throughout the incubation period did not interfere with the proliferative response of b5 homozygous rabbit bone marrow cells. This same antiserum completely blocks the ability of immune spleen cells to give a secondary plaque-forming cell response to sheep erythrocytes in vitro (6). However, it does not interfere with mixed lymphocyte reactions of thymus cells (6). Therefore, the inability of this antiserum to suppress the bone marrow resl>onse to HGG does not rule out specificity of the proliferative response due to thymus-derived cells. In combination, these observations tend to rule out specificity of the hone marrow proliferative response at both the T- and the B-lymphocyte level. The nonspecific nature of this response was recently also suggested 1)~ other investi-
486
MOND
AND
THORBECKE
gators (20) who obtained stimulation of bone marrow cells with nonimmunogenic haptens. It seems likely that the known tendency of colony-forming cells in bone marrow to proliferate in response to a number of varied stimuli (4, 5) is responsible for the nonspecific type of stimulation of DNA-synthesis obtained in the present experiments. REFERENCES 1. 2. .3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Singhal, S. K., and Richter, M., J. Exp. Med. 138, 1099, 1968. Abdou, N. I., and Richter, M., J. Ext. Med. 129, 757, 1%9. Richter, M., J. Zmmulzol. 105, 25, 1970. McNeill, T. A., Immunology 18, 39, 1970. Metcalf, D., Immmwlogy 20, 727, 1970. Mond, J. J., Luzzati, A. L., and Thorbecke, G. J., J. Immmwl. 108, 567, 1972. Kaplan, R E., and Thorbecke, G. J. Cell. Immzmol. 1, 632, 1970. and Electron Microscopy of the Immune Response.” Veldman, J. E., “Histophysiology N. V. Dijkstra Nicmeyer, Groningen. Doctoral dissertation 1970. Miller, H. C., and Cudkowicz, G., J. Exp. Med. 133, 973, 1971. Tubergen, D., and Feldman, J. D., J. E-Q. Med. 134, 1144, 1971. Puck, T. T., and Kao, F. Proc. Nat. Acad. Sci. 58, 1227, 1967. Golub, E. S., Mishell, R. I., Weigle, W. O., and Dutton, R. W., J. Zmmunol. 100, 133, 1968. Dresser, D W., Immunology 5, 378, 1962 Mond, J. J., Kaplan, R. E., and Thorbecke, G. J., Manuscript in preparation. Richter, M., and Abdou, N. I., J. Exp. Med. 129, 1261, 1969. Wilson, D. B., and Nowell, P. C., J. Exp. Med. 131, 391, 1970. Moller, G., and Kashiwagi, N., Immunology 22, 441, 1972. Daguillard, F., and Richter, M., J. Exp. Med. 130, 1187, 1%9. Sterzl, J., and Silverstein, A. M. In Advances in Immunology (J. H. Humphrey and F. J. Dixon, Jr., Eds.), Vol. 6, p. 337. Academic Press, New York, 1967. Eisen, S. A., Lyle, L. R., and Parker, C. W., Fed. Proc. 30, 649 (abstr), 1971.
5, 48C-486 (1972)
IMMUNOLOGY
Proliferative
Response Bone
of Normal
Marrow
Cells
Rabbit
and Mouse
in Vitro 1
J. J. MOND' AND G. J. THORBECKE~ Department
of Pathology,
New
New York University School York, New York 10016
Received
March
of Medicine,
22, 1972
Bone marrow cells from both normal rabbits and mice are stimulated by exposure to antigens in vitro. Responsiveness is not removed by prior injection of the antigen into the animals. The specificity of this phenomenon is doubtful in view of a lack of additive effects between antigens, unrelatedness to immunogenicity of the antigen, and occurrence in cells from immunologically immature rabbits.
It has been concluded from a number of observations that, in the rabbit, unlike the mouse, the bone marrow is a major source of T-cells. Among the data leading to this conclusion are: (a) the fact that bone marrow cells proliferate in response to various antigens in vitro (1) ; (b) the ability of transferred bone marrow to induce antibody-forming cells of recipient origin to sheep erythrocytes in X-irradiated rabbits (2) ; (c) removal of both activities from the bone marrow by preexposure to antigen in Z&JO (2) or by passage through antigen-coated columns (3). There are several problems in accepting this conclusion. It is known that bone marrow cells in vitro tend to respond with increased proliferation to a variety of nonimmunogenic as well as immunogenic substances, and that this proliferation is probably due to hematopoietic colony-forming precursor cells (4, 5). Responsiveness of rabbit thymocytes in vitro in the mixed lymphocyte reaction (6) and to phytohemagglutinin (7) is similar to that found in mice. Thymectomy followed by X-irradiation in adult rabbits and mice leads to comparable changes in the histology of their peripheral lymphoid tissue (8). In addition, it seems difficult to, understand why a previous injection should deplete cells from the bone marrow rather than cause the presence of circulating memory cells as is the case in other rodents (9, lo), particularly after immunization of the animals with antigen in complete Freund’s adjuvant. The present studies were undertaken in an effort to determine whether proliferation of normal bone marrow cells to a variety of antigens in vitro can be achieved 1 Supported by U.S.P.H.S. Grant AI-3078 s Recipient of NIH Training Grant 5 TO 5GM01668 from the National Institute of General Medical Science. 3 Health Research Council Career Scientist of the City of New York. 480 Co yright Alfrights
Q 1972 by Academic Press of reprodurtinn in anv ford
Inc. reserved.
BONE
MARROW
PROLIFERATIVE
RESPONSE
4X1
in species other than the rabbit. The specificity of the response was also investigated. The experiments to be described demonstrate that normal mouse bone marrow cells proliferate in response to several antigens. Depletion of responsiveness of bone marrow cells is not regularly obtained by prior antigen injection in either rabbits or mice. MATERIALS AND
METHODS
Aniwmls and immunization. New Zealand rabbits ranging in age from 1 week to 1 year were used. Allotypes on y-globulin were determined by double diffusion in agar employing reference antisera kindly provided by Dr. A. Kelus (Base1 Institute for Immunology, Basel, SwitzerIand). Rabbits were immunized with human gamma globulin (HGG, generously provided by Dr. G. A. Jamieson, American National Red Cross, Bethesda, MD) either intravenously with 25.0 mg HGG or subcutaneously with 0.5 mg HGG in complete Freund’s adjuvant. Balb/c mice were obtained from Cumberland Farms, Clinton, TE. In some experiments LAF, mice obtained from Jackson Laboratories (Bar Harbor, ME) was used. Immunization was by intravenous injection of HGG. Preparation
of Bone iWarrozv Cell Cultures
dIou.sc cell czdlturcs. Femoral and tibia1 bones were removed and the bone marrow cells were flushed out with Hanks’ balanced salt solution (HBSS). Bone marrow plugs were dispersed by passage through a 25-gauge needle. The cells were filtered through sterile gauze, washed twice in HBSS, and suspended to a concentration of lo6 cells/ml in RPM1 1640 + 10% FCS (Associated Biomedics Systems, Buffalo, NY). Cells were distributed in l.O-ml aliquots into 12 X 75-mm culture tubes (Falcon Plastics, Los Angeles, CA), and cultured after addition of antigen, at 37°C in 5% CO,-95% air atmosphere. On the fourth day of incubation 1 PCi of tritiated thymidine (Schwarz BioResearch Inc., Orangeburg, NY sp act 0.36 Ci/mmole) was added to each tube and incubation was continued for an additional l&24 hr. Rabbit ccl1 cz&ures. Bone marrow was taken from the femur and teased into a cell suspension. The cells were filtered through sterile gauze, washed twice in HBSS! and suspended in medium at a concentration of lo6 cells/ml. The medium used for rabbit cells was Dulbecco’s modified Eagle’s medium with 10% NRS (bl serum). Culture additives. Anti-b5 was prepared as previously described. Antigens used included killed Brucella abortus organisms (BA) (kindly donated by Dr. C. E. Watson from the United States Dept. of Agriculture), dinitrophenylated hemocyanin (DNP-KLH), DNP-bovine gamma globulin (DNP-BGG), DNP-human serum albumin (DNP-HSA). All culture additives were added in 0.05-ml aliquots to 1.0 ml of cell suspension. illraswrment
of $H Thyvtzidine Incorporation
.1t the end of the culture period, the cells were centrifuged, washed twice in phosphate-buffered saline, pH 7.2, and resuspended in 1.0 ml of 0.5 M perchloric
482
MOND
AND
THORBECKE
acid. The tubes were then left at 4°C overnight. The precipitates were washed twice with 0.25 M perchloric acid and once with methanol. After the final washing the precipitates were left to dry overnight at room temperature, The next day 0.5 ml hyamine was added to each tube and the tubes were incubated for 2 hr at room temperature. Aliquots of 0.2 ml were pipetted into glass vials containing 10 ml of toluene-liquifluor scintillation fluid (New England Nuclear, Boston, MA). The amount of radioactivity was determined in a Tri-Carb liquid scintillation counter (Packard Instrument Co., Downers Grove, IL), RESULTS
Proliferative
Response of Bone Marrow
Cells to Antigens
The results in Table 1 clearly show that both rabbit and mouse marrow cells from uninjected animals respond with enhanced proliferation to the presence of antigens during the culture period. Of the antigens tried, HGG (1.0 mg) was most regularly stimulatory to bone marrow cells of both species. DNP conjugates of KLH, BGG, and HSA (not in tables) were all stimulatory in doses of 0.1-1.0 mg per tube. DNP-HSA was found to stimulate better at 1.0 mg than at 0.1 mg, DNP-KLH was sometimes toxic at the l.O-mg level. Other antigens, which stimulated, but were only examined in an occasional experiment, included sheep erythrocyte stromata (6) and BA. An additional experiment was performed with mouse bone marrow cells preincubated at 37°C for 2% hr with 250 pg of bromodesoxyuridine (BUDR) per ml. Such pretreated cells still gave a response to HGG suggesting that the responding cells were not proliferating at a rapid rate prior to the initiation of culture ( 11) . The results in Table 2 show that the modifications of HGG generally used to render it either “immunogenic” (aggregated) or “tolerogenic” (spun) (12, 13) did not influence the degree of the response obtained. This suggests that the proliferative response of normal bone marrow cells is independent of the immunogenicity of the stimulant. TABLE THE PKOLIFERATIVE
1
RESPONSES OF NORMAL MOUSE OR RABBIT TO VARIOUS ANTIGENS in Vitro 5
Normal rabbit bone marrow Antigen
MARROW
CELLS
mouse bone marrow
added Expt
No antigen DNP-KLH
Normal
BONE
(0.1 mg)
HGG (1.0 mg) DNP-BGG (1.0 mg)
180 685
1
Expt 2 1855 4330
515
0 Results are expressed in counts per minute culture. h DNP-BGG (0.2 mg) was used.
Expt 3
Expt 4
Expt 5
Expt 6
190
560 1230
480 3425
955 2170
1090
2705
1995 2045
8350 b
of 3H-thymidine
incorporated
during
Day 4 in
BONE
MARROW
PROLIFERATIVE
TABLE
483
RESl’Oh-SE
2
Com.4~1~0~ OF THE in Vitro PKOLIFERATIVE RESPONSES OF NONAL RABBIT HGG (AHGG) OH Mous~s BONE MARKOR. CELLS a TO AGGREGATED AND ULTRACENTRIFUGED HGG (SPCX HGG)
Antigen
added
Expt
None AHGG b (1.0 mg) Spun HGG c (1.0 mg)
Expt 8 (cpm)
7 (cpm)
1790 3800 10535
765 1400 1960
‘* Expt 7 utilized rabbit, Expt 8 utilized mouse bone marrow cells. 6 HGG was prepared at a concentration of 10.0 mg/ml in saline and allowed to incubate for 1.5 min at 63°C. r HGG was spun in a preparative ultracentrifuge at 25,000 rpm for 2 hr and only the top !,.i fractic,ll 1vi15 used.
Efect Cells
of Antigen
The experiments cells from rabbits at varying times was not noticeably taken 1 day after this antigen (Expts Attempts Responses
to
Preinjectiofz
in Yivo
on the in Vitro
Response
of Bone Marvow
presented in Table 3 were performed with bone marrow and mice, which had received either HGG or DNP-KLH before death. The responsiveness of the bone marrow cells affected by such injections. Even rabbit bone marrow cells an intravenous injection of 25.0 mg HGG still responded to 9 and IO).
Establish
Inzm.mological
Specificity
the
of
Bone
Marrozrl
Cell
The results in Fig. 1 show that the simultaneous addition of two antigens (HGG 1.0 mg and DNP-KLH 0.1 mg) , each of which gave stimulation on its own, did not result in an additive effect on the proliferative response. It should be noted that in other studies in this laboratory an additive effect of phytohemagglutinin and anti-b5 has readily been shown with rabbit lymph node cells (14). TABLE
3
1.4~~ OF S~CIFIC IKHIRITION AFTEK PREINJECTION a OF ANTIGENS INTO RABBITS .\lmr ON THE PROLIFEKATIVE RESPOKSIVENESS OF THEIR BONE h'fARROw CELLS in
Radioactk-ity :Intigen
Incorporated
011 Vitro
‘(
(cpm)
added Expt 9
None HGG (1.0 mg) 1)X1’-KLH (0.1 mg)
338 1880 500
Expt
10
1175 17220
Expt
11
Expt
12
Espt
885 2500
3500 13770
905 2335
2620
5220
1655
13
o The following were the preinjection schedules for the experiments described above. Expts. 9, 10, 25.0 mg HGG intravenously on Da)- -1, Expt . 11, 10.0 mg DNP-KLH intravenously on Day -1, Espt. 12, 5.0 mg HGG subcutaneously on Day -3, Expt. 13, 0.5 mg spun HGG intrave IiOUSl~ “11 Day -10. h Experiments 9, 10, 11, 12 were performed with rabbit bone marrow cells. Experiment 13 was done with mouse bone marrow cells.
484
MOND
AND
THORBECKE
.20 “b 16 ;
12
68 0 NO Antigen
FIG. 1. Response of rabbit
HGG o.lmQ
bone marrow
HGG l.hQ
DNP-KLH DNP-KLH o.hQ O.hQ + HGG+ l.Omg O.lmg
cells to combination
of
HGG and DNP-KLH.
Although the presence of similar amounts of anti-b5 in the cultures of rabbit spleen cells was previously shown to completely suppress a secondary plaqueforming cell response to sheep erythrocytes in vitro (6), its presence throughout the incubation period with the bone marrow cells did not block the proliferative response to HGG (Table 4). Most forms of immunological competence show a relatively rapid maturation in neonatal animals. Rabbit lymphoid tissue is poorly developed at birth and does not reach optimal levels of responsiveness until 4 weeks later. It, therefore, was of interest to study the response of bone marrow cells to antigens before the animals reached immunological maturity. In all of four experiments, performed with bone marrow cells from rabbits l-4 weeks of age, a variable degree of stimulation was obtained by addition of 1 mg HGG. The radioactivity in stimulated cells was 1.4-5.1 times that in control cells, with an average degree of stimulation of 2.5X. The cells from l-week-old rabbits responded at least as well as those from the older rabbits. The response of these same bone marrow cells to anti-b5 was also tested and found to be low or absent, as was also described previously (7). The early appearance of this responsiveness to HGG again suggests the possibility that this is a nonspecific rather than an immunological phenomenon. A possible lack of dependency on immunological maturation was also suggested by the fact that bone marrow from 3 to 4-week-old mice responded as well as did cells from young adult mice. TABLE INABILITY Antigen
OF AivrI-b5’” TO BLOCK THE in Vitro PROLIFERATIVE RESPONSE OF HOMOZYGOUS b5 RABBIT BONE MARROW CELLS TO ANTIGEN
added
Expt 9
None HGG
(1.0 mg)
Anti-b5 Anti-b5
+ 1.0 mg HGG
0 Anti-b5
4
was used at a dilution
of l/20.
Expt
14
338
190
1880
1090
775
350
3200
7760
BONE
M.4RROW
PROLIFERATIVE
RESPONSE
485
DISCUSSION
The present findings show that normal mouse bone marrow gives a similar proliferative response to antigen as does normal rabbit bone marrow. Several of the properties of this response were comparable in the two species. There was thus no indication that a fundamental difference existed concerning the role of the bone marrow in the immune response of the mouse and the rabbit, such as was suggested previously by Richter and Abdou ( 15 ) . In fact, the present findings provide no evidence that the tendency of bone marrow cells to show enhanced mitotic activity in the presence of HGG or Dh’Pprotein conjugates has any immunological basis, since each individual observation speaks against specificity of the phenomenon. 1. The major argument previously used by Richter and co-workers (2) in favor of specificity was that the presence of antigen in the animal removed from the bone marrow the ability to respond to that antigen. Similarly, the passage of bone marrow cells through antigen-containing columns also specifically removed this property (3). Under the conditions of the present experiments, the in viva attempts at removal of responsiveness by previous injection of the antigens did not prove effective in either the rabbmitor the mouse. 2. Additive effects of two simultaneously added antigens were not observed. This is contrary to what has been observed for the proliferative responses of thymus-derived peripheral blood lymphocytes to two different major histocompatibihty antigens. which give additive effects in the mixed lymphocyte reaction i16j. Similarly, additive effects have been observed in the response to antigens of lymphocytes taken from delayed hypersensitive individuals ( 17). It should also be noted that additive effects in the proliferative response are obtained \vhen B and T lymphocytes are responding simultaneously to the combination of PHA and anti-Ig ( 1-t. 1s). 3. Aggregated and nonaggregated forms of HGG were equally effective in causing the proliferative response in bone marrow in vitro, whereas these motlifi( 12. cations of HGG are known to greatly influence its immunogenicity in ni7v 13). 3. Bone marrow from neonatal animals was equally responsive as that of adults. This is unlike immunologically specific functions expressed in peripheral lymphoid organs, since these require some degree of maturation of the immune system prior to optimal performance ( 19 ) . 5. Finally, the presence of anti-b5 throughout the incubation period did not interfere with the proliferative response of b5 homozygous rabbit bone marrow cells. This same antiserum completely blocks the ability of immune spleen cells to give a secondary plaque-forming cell response to sheep erythrocytes in vitro (6). However, it does not interfere with mixed lymphocyte reactions of thymus cells (6). Therefore, the inability of this antiserum to suppress the bone marrow resl>onse to HGG does not rule out specificity of the proliferative response due to thymus-derived cells. In combination, these observations tend to rule out specificity of the hone marrow proliferative response at both the T- and the B-lymphocyte level. The nonspecific nature of this response was recently also suggested 1)~ other investi-
486
MOND
AND
THORBECKE
gators (20) who obtained stimulation of bone marrow cells with nonimmunogenic haptens. It seems likely that the known tendency of colony-forming cells in bone marrow to proliferate in response to a number of varied stimuli (4, 5) is responsible for the nonspecific type of stimulation of DNA-synthesis obtained in the present experiments. REFERENCES 1. 2. .3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Singhal, S. K., and Richter, M., J. Exp. Med. 138, 1099, 1968. Abdou, N. I., and Richter, M., J. Ext. Med. 129, 757, 1%9. Richter, M., J. Zmmulzol. 105, 25, 1970. McNeill, T. A., Immunology 18, 39, 1970. Metcalf, D., Immmwlogy 20, 727, 1970. Mond, J. J., Luzzati, A. L., and Thorbecke, G. J., J. Immmwl. 108, 567, 1972. Kaplan, R E., and Thorbecke, G. J. Cell. Immzmol. 1, 632, 1970. and Electron Microscopy of the Immune Response.” Veldman, J. E., “Histophysiology N. V. Dijkstra Nicmeyer, Groningen. Doctoral dissertation 1970. Miller, H. C., and Cudkowicz, G., J. Exp. Med. 133, 973, 1971. Tubergen, D., and Feldman, J. D., J. E-Q. Med. 134, 1144, 1971. Puck, T. T., and Kao, F. Proc. Nat. Acad. Sci. 58, 1227, 1967. Golub, E. S., Mishell, R. I., Weigle, W. O., and Dutton, R. W., J. Zmmunol. 100, 133, 1968. Dresser, D W., Immunology 5, 378, 1962 Mond, J. J., Kaplan, R. E., and Thorbecke, G. J., Manuscript in preparation. Richter, M., and Abdou, N. I., J. Exp. Med. 129, 1261, 1969. Wilson, D. B., and Nowell, P. C., J. Exp. Med. 131, 391, 1970. Moller, G., and Kashiwagi, N., Immunology 22, 441, 1972. Daguillard, F., and Richter, M., J. Exp. Med. 130, 1187, 1%9. Sterzl, J., and Silverstein, A. M. In Advances in Immunology (J. H. Humphrey and F. J. Dixon, Jr., Eds.), Vol. 6, p. 337. Academic Press, New York, 1967. Eisen, S. A., Lyle, L. R., and Parker, C. W., Fed. Proc. 30, 649 (abstr), 1971.