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Stimulation of Human and Mouse Lymphocytes by Ribosomal Proteins
Immunobiol., vol. 156, pp. 464-469 (1979) Dept. Haematology, Klinikum Steglitz, Free University, Berlin, Germany
Stimulation of Human and Mouse Lymphocytes by Ribosomal Proteins G. SIEBER and H. RDHL Received March 27, 1979 . Accepted September 26, 1979
Abstract Ribosomal 30S and 50S subunits as well as total proteins from the large subunit of E. coli found to be potent mitogens mitogens for both human adult and cord ribosomes were found cord blood lymphocytes. On the other hand, the RNA fraction of the 50S subunit had only a weak stimulatory capacity suggesting suggesting that that the protein moiety contains the stimulatory component. Experiments capacity with lymphocyte subpopulations obtained by nylon wool filtration or E-rosette separation target cells are T lymphocytes. First First experiments with have shown that, in human, the main target mouse spleen cells indicate that, in mice, T and B lymphocytes can be stimulated by ribosomal proteins.
Introduction The mechanism of polyclonal lymphocyte activation which leads to proliferation and division of resting lymphocytes is as yet unknown (for review see 3). Most of our knowledge concerning the mechanism of activation has been obtained from studies using plant lectins. There are at least several hundred plant extracts of protein or glycoprotein nature, but only a few of these interact with lymphocytes and induce a mitogenic response. Thus, the structural requirements for lymphocyte activation are still unclear. We have examined the capacity of ribosomal subunits and especially of ribosomal proteins isolated from E. coli bacteria to stimulate human and mouse lymphocytes. The results indicate that these substances are potent mitogens for human peripheral lymphocytes and for mouse spleen cells.
Materials and Methods Preparation of ribosomal subunits Ribosomes were isolated from bacterium E. coli K 12, strain strain A 19, and separated into their 50S subunits were either either precipitated with polyethylenglysubunits as described described (7). 30S and 50S col-6000 or or pelleted pelleted by ultracentrifugation. The preparation of the total proteins (TP 50) and (23S and 5S) 5S) from the large subunits followed the procedure described described by the phenolized RNA (23S
Abbreviations: 30S and 50S - small and large subunit of the E. coli ribosome; TP 50 - total concanavalin A; LPS - lipopolysaccharide; PHAproteins prepared from 50S; Con A - concanavalin phytohemaglutinin; JH-TdR - 3H-thymidine.
Mitogenic Activity of Ribosomal Proteins
465
DOHME and and NIERHAUS (2). Acetic acid precipitated RNA (235 and 55) from the 50S subunit DOHME by SIEBER and and NIERHAUS (8). All preparations were were sterilized by by was prepared as described by millipore filtration.
Human lymphocyte cultures Heparinized venous blood was obtained from healthy adults as well as from umbilical cords. Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation. Lymphocyte were obtained either by by nylon wool filtration or E-rosette separation as subpopulations were described (5). The resultant T cell suspensions contained predominantly T cells forming red blood cells (E-RFC) and and less than 2% 2% Ig-positive B lymphocytes and rosettes with sheep red 1% E-RFC and a monocytes. B cell enriched lymphocyte suspensions contained less than 1% monocytes, (up to 40%). In some experiments, the percentage of monocyvariable number of monocytes (up the cells in petri dishes for for 90 tes in these cell suspensions was reduced by prior incubation of the minutes at 37°C. The resultant suspensions contained less than 10% monocytes.
Mouse lymphocyte cultures C57Bl!6J mice, DBA 2/J, C57Bl/6J/nu-nu mice and and the the heterozygous litter mates of the the C57BlI6J Ry, Denmark. - Single spleen cell suspensions latter (nu/-) were obtained from Bomholtgard, Ry, were prepared and in some experiments passed over a nylon wool column in of these mice were order to obtain T cell enriched lymphocyte suspensions.
culture technique Lymphocyte culture Cells were were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated and antibiotics. 1 X 106 cells/ml were cultured in pooled human AB serum, L-glutamine and round-bottom microplates at a volume of 0.2 ml/well. The The cultures were incubated at 37°C in and received 1 ~C/well of 3H-Tdr (spec. act. 2.0 Ci/mM; an atmosphere of 5% CO2 in air and Radiochemical Centre, Amersham, England) for the final 18 hours of incubation. The cultures for the were harvested by by using a multiple harvesting device (Skatron, Norway), and and the the radioactivity were 6 counts per minute was counted in a liquid scintillation counter. Results are expressed as I'-, (cpm in stimulated cultures minus cpm cpm in unstimulated controls of quadruplicate samples). varied between 500 and and 2000 cpm. cpm. The thymidine-uptake in unstimulated control cultures varied
Results
Stimulation of human lymphocytes In unfractionated lymphocyte suspensions from healthy adults, the ribosomal subunits as well as the ribosomal proteins were found to stimulate DNA synthesis and blast transformation. The magnitude of the responses was comparable to that achieved with phytomitogens; optimal stimulation was obtained after 5-7 days of incubation. Dose response ug/ml, curves revealed that the total proteins showed a sharp optimum at 5 !lg/ml, while the 30S and 50S subunits did stimulate at higher concentrations (100-200 !lg/ml). ug/ml). The RNA preparations, on the other hand, had a weak stimulatory capacity, and, in a number of experiments, a marginal response could be observed. The overall results are shown in Table 1. Lymphocytes from all adults as well as cord blood lymphocytes could be stimulated, thus indicating that these substances act as mitogens and not as antigens. - To define the target cells, T lymphocyte enriched cell suspensions obtained by nylon wool filtration or E-rosette separation were incubated with 30S, 50S
466 . G. SIEBER SIEBER and H . R ROHL OHL
responsee of human peripheral and cord blood lymphocyt lymphocytes ribosomal Table 1. Proliferating respons es to ribo somal subu nits , ribosomal proteins and Con A subunits, 50 S
30 S (100 !J.g/ ug/ ml)
TP 50 (5 !J.g/ml) ug/ml)
r-RNA (100 !J.g/ml) ug/ ml)
Con A (20 !J.g/ml) ug/ml)
Adults Ad ult s (n = 17)
43.470 43.470"* (9.58Q-(9.58076.420)
33.2500 33.25 (10.000(10.00Q-49.910)
40.415 (11.28Q-(11.28093.760)
6.500 (2.92Q-(2.92010.090)
63.660 (36.52Q-(36.520115.280)
Cord blood (n = 4)
34.275 34.2 75 (26.34348.860)
25.684 (11.00343.871) 43 .871)
30.983 (13.75667.034)
n. d. d.
81.323 (16.626156.740)
~ cpm cpm;; the range is given by the numbers in parenthesis. ':. Results are expressed as the mean ,1. ,; days, all other cultures after 7 days of Con A-stimulated cultures were harvested after 5 days, donee.. incubation. n. d. = not don
and TP 50. As shown by a typical experiment in Table 2, all preparations induced a good response in T cell enriched cell suspensions, whereas isolated B cells did not respond to any of the substances. That the high proportion of monocytes present in the B cell enriched cell suspensions did not suppress the responses could be excluded by experiments in which the monocytes were removed by prior incubation of the cells in petri dishes. Also, these further purified B cell suspensions could not be stimulated significantly by the ribosomal subunits or proteins (data not shown).
Stimulation of mouse spleen cells Experiments with spleen cells from various strains of mice revealed that 30S, lymphocytes (Table 3). 305, 50S and TP 50 are mitogenic for mouse lymphocytes Optimal stimulation was achieved after 3 days of incubation with the same doses found to be optimal for human lymphocytes; the RNA preparation had no or a weak stimulatory effect (data not shown). As to the target cells of these substances, our findings findings suggest that in mice, both T and B lymphocytes can be stimulated. Thus, spleen cells from ath athymic ymic nude mice 30S, 50S, and TP 50 though the responses were weak could be stimulated by 305, response lymphocytee subpopulations obtained by by Table 2. Lymphoproliferative respon se of human lymphocyt nylon ny lon wool filtration and E-rosette E-rosette separation. The results of twO two typical typ ical experiments are shown 50 S
30 S (100 !J.g/ ug/rnl ml))
TP 50 TP (5 !J.g/ ug/rnl ml))
PHA (1 (1 !J.g/ml)
Unnseparated separated U Nylon N ylon wool filt.
56.500':'" 56.500 58.200
32.100 45.100
51.300 45 .200 45.200
86.925 64.718
Unseparated U nseparated «B» (Eros. sep.) «B" (E-ros. «T "» (Eros. sep .) (E-ros. sep.)
25.600 0 45.200
28.300 0 44.100
16.500 0 17.800
100.300 0 99.000
" Results are expressed as ,1. ~ cpm. PHA-stimulated cultures were harvested after 3 days, all otherr cultures after after 7 days of incubation. othe incub ation.
Mitogenic Activity Mitogen ic Acti vity of Ribosomal Ribosom al Proteins . 467 were Table 3. Lymphoproliferative response of mouse spleen cells to mitogens. Cultures were harvested after three days of incubation incubation;; the results results are expr expressed har vested after essed the mean .::). ~ cpm and represent repre sent optimal stimulation of dose response curves
PHA Con A LPS TP 50 TP 50 S 30 S
C 57 Bl
DBA
Nu /Nu/-
Nu/nu
66.663 124.072 41.220 41.045 53 .636 53.636 38.583
49.393 118.121 29.770 36.322 27.896 15.665
55.328 66.597 18.045 27.593 22.092 n. d.
152 0 43.810 7.007 8.089 5.083
Bl spleen cells to ribosomal subunits and ribosomal proteins. Table 4. Response of C 57 BI twoo experiments (mean .::). were results of tw Shown are the results ~ cpm) in which a part of the cells were The cultures were harvested after after three days of incubation passed over a nylon passed ny lon wool column. The
50 S 30 S TP TP 50
Exp. I Unseparated Un separated Nylon c.
Exp, Exp. II Un separated Unseparated
Nylon c.
19.456 29.895 34.632
18.275 27.763 24.450
18.633 26.900 16.294
15.191 38.797 14.996
C57BI, DBA or in the heterozygous in comparison to those observed in C57BI, litter mates of the nude mice. On the other hand, nylon column purified splenic T cells from C57BI mice, containing less than 3% of Ig-positive B cells, could be stimulated to the same extent as unseparated cell suspensions (Table 4). Discussion
Our results indicate that 30S and 50S ribosomal subunits and the total proteins from the 50S subunit of bacterium E. coli are mitogenic for human and mouse lymphocytes. Since lymphocytes from all adults as well as cord blood lymphocytes could be stimulated, it can be concluded that these substances act as mitogens and probably not as antigens. The stimulatory capacity of the preparations used was most likely not due to contamination by other substances. Thus, polyethylenglycol, which was used as a precipitating agent, had no stimulatory effect on human and mouse lymphocytes (data not shown). It is also quite unlikely that the by a contaminating LPS since, firstly, LPS do not stimulation is caused by stimulate human peripheral lymphocytes significantly and - secondly -, LPS would not activate isolated T cells in mice (Table 4). Contamination of RNA with proteins was checked in the two dimensional protein gel were found (8). Proteins were electrophoresis and no traces of proteins were A28o:A26o:A230 o:A26o:A230 was better than 10:1:1 thus accepted as "pure" if the ratio A28 were practically free of RNA. indicating that the proteins were
SIEBER and H. RDHL RUHL 468 . G. SIEBER
Our results have further shown that the ribosomal subunits and proteins stimulate human peripheral T cells, while B cells did not respond. The latter lymphocyte subpopulation seems to be totally unresponsive as measured by thymidine uptake, since isolated B cells could not be stimulated even in the presence of syngeneic mitomycintreated T cells (data not shown). The activation of T cells seems to be relatively independent of the presence of monocytes, since in most experiments, monocyte depleted nylon wool filtered cells could be stimulated to the same extent as unseparated cell suspensions. Further experiments have nevertheless shown that after a second passage over a nylon wool column, the responses were markedly diminished; they could be restored by the addition of a small number of syngeneic monocytes (manuscript in preparation). These diminished responses were especially seen when TP 50 was used. This may indicate that mitogens with a low molecular weight (6,000-32,000 for for TP 50) are more sensitive to removal of monocytes than substances with high molecular weights (1000,000-2000,000 for all subunits). Interestingly, another small molecular weight T cell mitogen - zinc chloride - was found to be extremely sensitive to removal of monocytes (4). The results further indicate that, in mice, probably T lymphocytes are the main target cells. The evidence for this was derived from experiments in which the spleen cells from normal C57BI mice were passed over a nylon wool column. Though in the resultant cell suspensions the percentage of Igpositive B cells was substantially reduced (less than 3 % Ig-positive cells), the responses were as high as in unseparated cell suspensions. - On the other hand, further experiments have shown that spleen cells from athymic nude mice could also be activated by the ribosomal subunits and ribosomal proteins. This was a consistent finding in all experiments; that the responses were weak (stimulation indices between 5 and 12), might indicate that few optimal B cell activation by these substances requires the presence of a few contaminant T cells. (23S and The 505 ribosomal subunits of E. coli contain ribosomal RNA (235 55) and a bulk of 34 proteins (for review see 1). Obviously, the protein moiety is the stimulatory component, since RNA obtained by two different preparation procedures did not stimulate significantly. This is consistent with the finding that a large number of proteins have been located on the surface of the particle and are accessible for antibodies (1). Weare now investigating whether groups of proteins or single proteins can act as mitogens. In this case, single ribosomal proteins would be a useful can tool to elucidate the structural prerequisites of a protein for mitogenicity, since most of them are well characterized with respect to their physicochemical parameters (1). Acknowledgment GUDRUN BaCHERT for her excellent technical The authors would like to thank Mrs. GUDRUN assistance. assistance.
Mitogenic Activity of Ribosomal Proteins . 469
References 1.
2. 3. 4. 5.
6. 7. 8.
BRIMACOMBE, R., K. H. NIERHAUS, R. A. GARRET, and H. G. WITTMANN. 1976. The ribosome of Escherichia coli. Prog. Nucl. Acid Res. Mol. BioI. 18: 1. DOHME, F., and K. H. NIERHAUS. 1976. Total reconstitution and assembly of 50S subunits DOHME,F., J. Mol. Mol. BioI. 107: 585. from E. coli ribosomes in vitro. J. J. J., J., and D. L. ROSENSTREICH. 1976. Signale regulating in vitro activation of OPPENHEIM, J. lymphocytes. Prog. Allergy 20: 65. RUHL, H., and H. H. KIRCHNER. 1978. Monocyte-dependent stimulation of human T cells RUHL, by zinc. Clin. Clin. expo expo Immunol. 132: 484. by RUHL, H., H. H. SCHOLLE, G. BOCHERT, and W. VOGT. 1978. Activation of lymphocyte RUHL, leucemia. Zeitschr. Immunitatsforsubpopulations in patients with chronic lymphocytic leucemia. schung 154: 75. SAKANE, T., and 1. GREEN. 1978. Protein A from Staphylococcus Aureus - a mitogen for J. Immunol. 120: 302. human T lymphocytes and B lymphocytes but not L lymphocytes. J. H. NIERHAUS. 1973. Protein involved in the binding of dihydroSCHREINER, G., and K. H. Mol. BioI. 81: 71. streptomycin to ribosomes of Escherichia coli. J. Mol. H. NIERHAUS. 1978. Kinetic and thermodynamic parameters of the SIEBER, G., and K. H. assembly in vitro of the large subunit from Escherichia coli ribosomes. Biochemistry 17: 3505.
RUHL, Hamatologische Abteilung, Klinikum Steglitz, Hindenburgdamm 30, Dr. HARTMUT RUHL, Berlin 45, Federal Republic of Germany 1000 Berlin
Stimulation of Human and Mouse Lymphocytes by Ribosomal Proteins G. SIEBER and H. RDHL Received March 27, 1979 . Accepted September 26, 1979
Abstract Ribosomal 30S and 50S subunits as well as total proteins from the large subunit of E. coli found to be potent mitogens mitogens for both human adult and cord ribosomes were found cord blood lymphocytes. On the other hand, the RNA fraction of the 50S subunit had only a weak stimulatory capacity suggesting suggesting that that the protein moiety contains the stimulatory component. Experiments capacity with lymphocyte subpopulations obtained by nylon wool filtration or E-rosette separation target cells are T lymphocytes. First First experiments with have shown that, in human, the main target mouse spleen cells indicate that, in mice, T and B lymphocytes can be stimulated by ribosomal proteins.
Introduction The mechanism of polyclonal lymphocyte activation which leads to proliferation and division of resting lymphocytes is as yet unknown (for review see 3). Most of our knowledge concerning the mechanism of activation has been obtained from studies using plant lectins. There are at least several hundred plant extracts of protein or glycoprotein nature, but only a few of these interact with lymphocytes and induce a mitogenic response. Thus, the structural requirements for lymphocyte activation are still unclear. We have examined the capacity of ribosomal subunits and especially of ribosomal proteins isolated from E. coli bacteria to stimulate human and mouse lymphocytes. The results indicate that these substances are potent mitogens for human peripheral lymphocytes and for mouse spleen cells.
Materials and Methods Preparation of ribosomal subunits Ribosomes were isolated from bacterium E. coli K 12, strain strain A 19, and separated into their 50S subunits were either either precipitated with polyethylenglysubunits as described described (7). 30S and 50S col-6000 or or pelleted pelleted by ultracentrifugation. The preparation of the total proteins (TP 50) and (23S and 5S) 5S) from the large subunits followed the procedure described described by the phenolized RNA (23S
Abbreviations: 30S and 50S - small and large subunit of the E. coli ribosome; TP 50 - total concanavalin A; LPS - lipopolysaccharide; PHAproteins prepared from 50S; Con A - concanavalin phytohemaglutinin; JH-TdR - 3H-thymidine.
Mitogenic Activity of Ribosomal Proteins
465
DOHME and and NIERHAUS (2). Acetic acid precipitated RNA (235 and 55) from the 50S subunit DOHME by SIEBER and and NIERHAUS (8). All preparations were were sterilized by by was prepared as described by millipore filtration.
Human lymphocyte cultures Heparinized venous blood was obtained from healthy adults as well as from umbilical cords. Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation. Lymphocyte were obtained either by by nylon wool filtration or E-rosette separation as subpopulations were described (5). The resultant T cell suspensions contained predominantly T cells forming red blood cells (E-RFC) and and less than 2% 2% Ig-positive B lymphocytes and rosettes with sheep red 1% E-RFC and a monocytes. B cell enriched lymphocyte suspensions contained less than 1% monocytes, (up to 40%). In some experiments, the percentage of monocyvariable number of monocytes (up the cells in petri dishes for for 90 tes in these cell suspensions was reduced by prior incubation of the minutes at 37°C. The resultant suspensions contained less than 10% monocytes.
Mouse lymphocyte cultures C57Bl!6J mice, DBA 2/J, C57Bl/6J/nu-nu mice and and the the heterozygous litter mates of the the C57BlI6J Ry, Denmark. - Single spleen cell suspensions latter (nu/-) were obtained from Bomholtgard, Ry, were prepared and in some experiments passed over a nylon wool column in of these mice were order to obtain T cell enriched lymphocyte suspensions.
culture technique Lymphocyte culture Cells were were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated and antibiotics. 1 X 106 cells/ml were cultured in pooled human AB serum, L-glutamine and round-bottom microplates at a volume of 0.2 ml/well. The The cultures were incubated at 37°C in and received 1 ~C/well of 3H-Tdr (spec. act. 2.0 Ci/mM; an atmosphere of 5% CO2 in air and Radiochemical Centre, Amersham, England) for the final 18 hours of incubation. The cultures for the were harvested by by using a multiple harvesting device (Skatron, Norway), and and the the radioactivity were 6 counts per minute was counted in a liquid scintillation counter. Results are expressed as I'-, (cpm in stimulated cultures minus cpm cpm in unstimulated controls of quadruplicate samples). varied between 500 and and 2000 cpm. cpm. The thymidine-uptake in unstimulated control cultures varied
Results
Stimulation of human lymphocytes In unfractionated lymphocyte suspensions from healthy adults, the ribosomal subunits as well as the ribosomal proteins were found to stimulate DNA synthesis and blast transformation. The magnitude of the responses was comparable to that achieved with phytomitogens; optimal stimulation was obtained after 5-7 days of incubation. Dose response ug/ml, curves revealed that the total proteins showed a sharp optimum at 5 !lg/ml, while the 30S and 50S subunits did stimulate at higher concentrations (100-200 !lg/ml). ug/ml). The RNA preparations, on the other hand, had a weak stimulatory capacity, and, in a number of experiments, a marginal response could be observed. The overall results are shown in Table 1. Lymphocytes from all adults as well as cord blood lymphocytes could be stimulated, thus indicating that these substances act as mitogens and not as antigens. - To define the target cells, T lymphocyte enriched cell suspensions obtained by nylon wool filtration or E-rosette separation were incubated with 30S, 50S
466 . G. SIEBER SIEBER and H . R ROHL OHL
responsee of human peripheral and cord blood lymphocyt lymphocytes ribosomal Table 1. Proliferating respons es to ribo somal subu nits , ribosomal proteins and Con A subunits, 50 S
30 S (100 !J.g/ ug/ ml)
TP 50 (5 !J.g/ml) ug/ml)
r-RNA (100 !J.g/ml) ug/ ml)
Con A (20 !J.g/ml) ug/ml)
Adults Ad ult s (n = 17)
43.470 43.470"* (9.58Q-(9.58076.420)
33.2500 33.25 (10.000(10.00Q-49.910)
40.415 (11.28Q-(11.28093.760)
6.500 (2.92Q-(2.92010.090)
63.660 (36.52Q-(36.520115.280)
Cord blood (n = 4)
34.275 34.2 75 (26.34348.860)
25.684 (11.00343.871) 43 .871)
30.983 (13.75667.034)
n. d. d.
81.323 (16.626156.740)
~ cpm cpm;; the range is given by the numbers in parenthesis. ':. Results are expressed as the mean ,1. ,; days, all other cultures after 7 days of Con A-stimulated cultures were harvested after 5 days, donee.. incubation. n. d. = not don
and TP 50. As shown by a typical experiment in Table 2, all preparations induced a good response in T cell enriched cell suspensions, whereas isolated B cells did not respond to any of the substances. That the high proportion of monocytes present in the B cell enriched cell suspensions did not suppress the responses could be excluded by experiments in which the monocytes were removed by prior incubation of the cells in petri dishes. Also, these further purified B cell suspensions could not be stimulated significantly by the ribosomal subunits or proteins (data not shown).
Stimulation of mouse spleen cells Experiments with spleen cells from various strains of mice revealed that 30S, lymphocytes (Table 3). 305, 50S and TP 50 are mitogenic for mouse lymphocytes Optimal stimulation was achieved after 3 days of incubation with the same doses found to be optimal for human lymphocytes; the RNA preparation had no or a weak stimulatory effect (data not shown). As to the target cells of these substances, our findings findings suggest that in mice, both T and B lymphocytes can be stimulated. Thus, spleen cells from ath athymic ymic nude mice 30S, 50S, and TP 50 though the responses were weak could be stimulated by 305, response lymphocytee subpopulations obtained by by Table 2. Lymphoproliferative respon se of human lymphocyt nylon ny lon wool filtration and E-rosette E-rosette separation. The results of twO two typical typ ical experiments are shown 50 S
30 S (100 !J.g/ ug/rnl ml))
TP 50 TP (5 !J.g/ ug/rnl ml))
PHA (1 (1 !J.g/ml)
Unnseparated separated U Nylon N ylon wool filt.
56.500':'" 56.500 58.200
32.100 45.100
51.300 45 .200 45.200
86.925 64.718
Unseparated U nseparated «B» (Eros. sep.) «B" (E-ros. «T "» (Eros. sep .) (E-ros. sep.)
25.600 0 45.200
28.300 0 44.100
16.500 0 17.800
100.300 0 99.000
" Results are expressed as ,1. ~ cpm. PHA-stimulated cultures were harvested after 3 days, all otherr cultures after after 7 days of incubation. othe incub ation.
Mitogenic Activity Mitogen ic Acti vity of Ribosomal Ribosom al Proteins . 467 were Table 3. Lymphoproliferative response of mouse spleen cells to mitogens. Cultures were harvested after three days of incubation incubation;; the results results are expr expressed har vested after essed the mean .::). ~ cpm and represent repre sent optimal stimulation of dose response curves
PHA Con A LPS TP 50 TP 50 S 30 S
C 57 Bl
DBA
Nu /Nu/-
Nu/nu
66.663 124.072 41.220 41.045 53 .636 53.636 38.583
49.393 118.121 29.770 36.322 27.896 15.665
55.328 66.597 18.045 27.593 22.092 n. d.
152 0 43.810 7.007 8.089 5.083
Bl spleen cells to ribosomal subunits and ribosomal proteins. Table 4. Response of C 57 BI twoo experiments (mean .::). were results of tw Shown are the results ~ cpm) in which a part of the cells were The cultures were harvested after after three days of incubation passed over a nylon passed ny lon wool column. The
50 S 30 S TP TP 50
Exp. I Unseparated Un separated Nylon c.
Exp, Exp. II Un separated Unseparated
Nylon c.
19.456 29.895 34.632
18.275 27.763 24.450
18.633 26.900 16.294
15.191 38.797 14.996
C57BI, DBA or in the heterozygous in comparison to those observed in C57BI, litter mates of the nude mice. On the other hand, nylon column purified splenic T cells from C57BI mice, containing less than 3% of Ig-positive B cells, could be stimulated to the same extent as unseparated cell suspensions (Table 4). Discussion
Our results indicate that 30S and 50S ribosomal subunits and the total proteins from the 50S subunit of bacterium E. coli are mitogenic for human and mouse lymphocytes. Since lymphocytes from all adults as well as cord blood lymphocytes could be stimulated, it can be concluded that these substances act as mitogens and probably not as antigens. The stimulatory capacity of the preparations used was most likely not due to contamination by other substances. Thus, polyethylenglycol, which was used as a precipitating agent, had no stimulatory effect on human and mouse lymphocytes (data not shown). It is also quite unlikely that the by a contaminating LPS since, firstly, LPS do not stimulation is caused by stimulate human peripheral lymphocytes significantly and - secondly -, LPS would not activate isolated T cells in mice (Table 4). Contamination of RNA with proteins was checked in the two dimensional protein gel were found (8). Proteins were electrophoresis and no traces of proteins were A28o:A26o:A230 o:A26o:A230 was better than 10:1:1 thus accepted as "pure" if the ratio A28 were practically free of RNA. indicating that the proteins were
SIEBER and H. RDHL RUHL 468 . G. SIEBER
Our results have further shown that the ribosomal subunits and proteins stimulate human peripheral T cells, while B cells did not respond. The latter lymphocyte subpopulation seems to be totally unresponsive as measured by thymidine uptake, since isolated B cells could not be stimulated even in the presence of syngeneic mitomycintreated T cells (data not shown). The activation of T cells seems to be relatively independent of the presence of monocytes, since in most experiments, monocyte depleted nylon wool filtered cells could be stimulated to the same extent as unseparated cell suspensions. Further experiments have nevertheless shown that after a second passage over a nylon wool column, the responses were markedly diminished; they could be restored by the addition of a small number of syngeneic monocytes (manuscript in preparation). These diminished responses were especially seen when TP 50 was used. This may indicate that mitogens with a low molecular weight (6,000-32,000 for for TP 50) are more sensitive to removal of monocytes than substances with high molecular weights (1000,000-2000,000 for all subunits). Interestingly, another small molecular weight T cell mitogen - zinc chloride - was found to be extremely sensitive to removal of monocytes (4). The results further indicate that, in mice, probably T lymphocytes are the main target cells. The evidence for this was derived from experiments in which the spleen cells from normal C57BI mice were passed over a nylon wool column. Though in the resultant cell suspensions the percentage of Igpositive B cells was substantially reduced (less than 3 % Ig-positive cells), the responses were as high as in unseparated cell suspensions. - On the other hand, further experiments have shown that spleen cells from athymic nude mice could also be activated by the ribosomal subunits and ribosomal proteins. This was a consistent finding in all experiments; that the responses were weak (stimulation indices between 5 and 12), might indicate that few optimal B cell activation by these substances requires the presence of a few contaminant T cells. (23S and The 505 ribosomal subunits of E. coli contain ribosomal RNA (235 55) and a bulk of 34 proteins (for review see 1). Obviously, the protein moiety is the stimulatory component, since RNA obtained by two different preparation procedures did not stimulate significantly. This is consistent with the finding that a large number of proteins have been located on the surface of the particle and are accessible for antibodies (1). Weare now investigating whether groups of proteins or single proteins can act as mitogens. In this case, single ribosomal proteins would be a useful can tool to elucidate the structural prerequisites of a protein for mitogenicity, since most of them are well characterized with respect to their physicochemical parameters (1). Acknowledgment GUDRUN BaCHERT for her excellent technical The authors would like to thank Mrs. GUDRUN assistance. assistance.
Mitogenic Activity of Ribosomal Proteins . 469
References 1.
2. 3. 4. 5.
6. 7. 8.
BRIMACOMBE, R., K. H. NIERHAUS, R. A. GARRET, and H. G. WITTMANN. 1976. The ribosome of Escherichia coli. Prog. Nucl. Acid Res. Mol. BioI. 18: 1. DOHME, F., and K. H. NIERHAUS. 1976. Total reconstitution and assembly of 50S subunits DOHME,F., J. Mol. Mol. BioI. 107: 585. from E. coli ribosomes in vitro. J. J. J., J., and D. L. ROSENSTREICH. 1976. Signale regulating in vitro activation of OPPENHEIM, J. lymphocytes. Prog. Allergy 20: 65. RUHL, H., and H. H. KIRCHNER. 1978. Monocyte-dependent stimulation of human T cells RUHL, by zinc. Clin. Clin. expo expo Immunol. 132: 484. by RUHL, H., H. H. SCHOLLE, G. BOCHERT, and W. VOGT. 1978. Activation of lymphocyte RUHL, leucemia. Zeitschr. Immunitatsforsubpopulations in patients with chronic lymphocytic leucemia. schung 154: 75. SAKANE, T., and 1. GREEN. 1978. Protein A from Staphylococcus Aureus - a mitogen for J. Immunol. 120: 302. human T lymphocytes and B lymphocytes but not L lymphocytes. J. H. NIERHAUS. 1973. Protein involved in the binding of dihydroSCHREINER, G., and K. H. Mol. BioI. 81: 71. streptomycin to ribosomes of Escherichia coli. J. Mol. H. NIERHAUS. 1978. Kinetic and thermodynamic parameters of the SIEBER, G., and K. H. assembly in vitro of the large subunit from Escherichia coli ribosomes. Biochemistry 17: 3505.
RUHL, Hamatologische Abteilung, Klinikum Steglitz, Hindenburgdamm 30, Dr. HARTMUT RUHL, Berlin 45, Federal Republic of Germany 1000 Berlin