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Different responses of CR(−) and CR(+) B cells to LPS stimulation
CELLULAR
IMMUNOLOGY
71, 80-88 (1982)
Different Responses of CR(-) and CR(+) B Cells to LPS Stimulation TERUTAKA Laboratory
KAKIUCHI
AND HIDEO NARIUCHI’
of Biological Products, Institute of Medical University of Tokyo, Tokyo 108, Japan
Received February
Science,
I, 1982; accepted May I I, 1982
Proliferative response of B cells with or without CR [CR(+) or CR(-) B cells] was compared in their polyclonal response when they were stimulated with lipopolysaccharide (LPS). CR(+) B cells responded better in proliferation and more poorly in polyclonal antibody formation than did CR( -) B cells. The dissociation between proliferation and antibody formation in LPS responsewas not due to the shift of the time kinetics nor the exhaustion of the culture medium. T cells and macrophages did not take part in the dissociation, since macrophage depletion from nu/nu mouse spleen cells could not modify the dissociation. The polyclonal antibody response of CR(-) B cells was more resistant to irradiation than that of CR(+) B cells. These results suggest that among LPS-responsive B cells there are CR(-) B-cell subset(s) more mature than CR(+) B cells.
INTRODUCTION There are several contradictory results on the differences between B cells with or without CR in the responsivenessto various antigens (l-4) and to LPS (2, 57). In regard to the B-cell maturation, CR(-) B cells have been reported to be less mature than CR(+) B cells (8, 9). In our previous report (lo), CR(-) B cells have been shown to be different from CR( +) B cells in the antibody-forming cells (AFC) responsesto purified protein derivative of tuberculin (PPD), dextran sulfate (DxS), and LPS, that is, CR(-) B cells responded well to LPS and poorly to PPD or DxS, and CR(+) B cells responded well both to PPD and DxS but seemed to respond poorly to LPS. In these experiments, it was suggested to be dissociation between antibody formation and proliferation in the LPS response of CR(-) and CR(+) B cells. In the present studies, therefore, CR(-) and CR(+) B cells were compared to each other in terms of [ 3H]thymidine ([ 3H]TdR) incorporation and antibody responseto LPS and the dissociation was confirmed. The sensitivities of LPS responses of CR(-) and CR(+) B cells to irradiation in terms of antibody formation were also studied. The maturational stages of CR(-) and CR(+) B cells responsive to LPS were discussed. ’ To whom all correspondence should be addressed: Laboratory of Biological Products, Institute of Medical Science, University of Tokyo, 4-6-1, Shiroganedai, Minatoku, Tokyo 108, Japan. 80 0008-8749/82/l lOOSO-09$02.00/0 Copyright 0 1982 by Academic Press, Inc. All fights of reproduction in any form rwe~cd.
LPS RESPONSE
OF CR(-)
MATERIALS
AND
CR(+)
B CELLS
81
AND METHODS
Mice. C57BL/6j female mice and mice with a nu/nu gene on a C57BL/6j background were supplied by the animal breeding unit of our institute, and used at 8 to 16 weeks of age. DDD mice were also supplied by the unit. Mitogens. LPS from Escherichia coli 055:B5 prepared by phenol extraction was purchased from Difco Laboratories, Detroit, Michigan. LPS was used at 5 pg/ culture. The quantity of LPS was determined by preliminary experiments to be the optimal stimulatory dose for C57BL/6j spleen cells. Erythrocyte-antibody-complement complexes (EAC). EAC were prepared as described previously ( 11). Briefly, sheep erythrocytes (E) were sensitized with a subagglutinating concentration of IgM anti-E antibody, and the resultant cells (EA) were treated with CS-deficient DDD mouse serum for 30 min at 37°C. Irradiation of cells. Cells were irradiated with varying doses in suspension using Gamma cell 40, Atomic Energy of Canada Ltd., Ottawa, Canada. Cultures. One million cells were cultured in 0.2 ml of medium RPM1 1640 containing 20% fetal calf serum (Associated Biomedic Systems, Buffalo, N.Y.), 5 X lo-’ M 2-mercaptoethanol, and kanamycin ( 100 pg/culture) in flatbottom Microtest II plates (Falcon 3042) in a humidified atmosphere of 5% CO1 in air at 37°C. After 3 days incubation the cells were assayed for anti-TNP PFC using TNP-sheep erythrocytes, unless otherwise stated. For the assessmentof proliferative response, 5 X lo5 cells were cultured under the same conditions to the antibody response except that the medium contained 2% fetal calf serum without 2-mercaptoethanol. The response was assessedby [3H]TdR incorporation (0.25 &i/ culture; specific activity, 2.0 Ci/mmol). Detection and depletion of an enrichment with lymphocytes bearing CR (CRL). CRL were detected by rosette-formation with EAC, and the depletion of and enrichment with EAC-rosette-forming cells (EAC-RFC) were carried out as described previously (10) with slight modifications. Briefly, spleen cells were mixed with an equal volume of 10% E and centrifuged over Ficoll-Hypaque; cells in the interface fraction (5 X lo7 cells/ml) were mixed with an equal volume of 10% EAC, incubated for 20 min at 37°C to form rosettes, and then layered on top of an equal volume of Ficoll-Hypaque and centrifuged at 2000g for 20 min at 20°C. The cells from the medium Ficoll-Hypaque interface (EAC interface) and those from the pellet fraction (EAC pellet) were used as CRL-depleted and -enriched cells, respectively, after they were treated with NH&Z1 to lyse erythrocytes. Residual CRL in the CRL-depleted fraction were less than 2.2% in all experiments, when cells from the fraction were rerosetted with EAC. Usually, CRL were 45 to 50% of total spleen cells. For the control experiments EA were used instead of EAC. Cells from the interface (EA-interface) and the pellet (EA-pellet) fractions were used for experiments. Depletion of adherent cells. Adherent cells were depleted from spleen cell population by the passage through a Sephadex G-10 column as described previously ( 12). RESULTS Comparison of antibody and proliferation responses of CRL-depleted and -enriched fractions of spleen cells to LPS stimulation. In our previous experiments
82
KAKIUCHI
AND
NARIUCHI
there seemed to be dissociation between antibody formation and proliferation in LPS responses of CR(+) and CR(-) B cells. In the first experiments, therefore, spleen cells were fractionated into CRL-depleted and -enriched populations, and then they were cultured with LPS (5 pg/culture) for the assessmentsof t3H]TdR incorportion and PFC responses. When cells from each fraction were rerosetted with EAC, 50.7% of unfractionated spleen cells formed rosettes, and 49.8% of the E-interface, 23.6% of the E-pellet, 0.3% of the EAC-interface, and 36.0% of the EAC-pellet fractions were CRL. At the same time sIg positive cells from each fraction were 53.2, 54.6, 51.1, 16.2, and 63.7%, respectively. The results of PFC and [ 3H]TdR incorportion responsesare shown in Table 1. The PFC responsesof cells from CRL-enriched fraction was significantly lower (P < 0.01) than that of cells from the CRL-depleted fraction. On the contrary, [3H]TdR incorporation of cells from the CRL-enriched fraction was better than that of cells from the CRLdepleted fraction. It can be clearly concluded that CRL-depleted cells respond better to LPS in antibody formation and more poorly in proliferation than do CRLenriched cells. These results seem to reflect the different responsivenessof CR(+) B cells and CR(-) B cells to LPS. There are, however, several possibilities to be excluded: (i) CR(+) B cells may differ from CR(-) B cells in the time kinetics of their response to LPS, as shown by Hammerling et al. (5), (ii) the poor PFC responseof CR(+) B cells to LPS may be due to the exhaustion of culture medium caused by the vigorous proliferation of CR(+) B cells, (iii) T cells or macrophages may take part in reducing proliferative response of CR( -) B cells or PFC response TABLE LPS Response of CRL-Depleted
Cell fraction
LPS
Unfractionated
+
E interface
+
E pellet
+
EA interface
+
EA pellet
+
EAC interface
+
EAC pellet
+
1 and -Enriched
Anti-TNP PFC/culture + SD*
Spleen Cells” cpm/culture zk SDb
31 f 8 960 f 61
286 + 31 15,222 f 1786
37 + 10 913 + 35
13,229 f 3801
663 f 43 5193 f 206
404 f 91 4,341 + 416
55 f 10 1273 f 101
166 f 20 9,139 + 229
53 + 8 413 + 25
129 f 95 4,740 f 1434
40 f 5 2233 f 155 123 + 26 423 f 84
994 *
99 f 4,443 f 61 f
188
11 1065 5
12,940 f 765
’ Spleen cells were rosetted with EAC and separated into CRL-depleted (EAC interface) and CRLenriched (EAC pellet) fractions. EA were used for control. Cells from each fraction were cultured for 3 days with LPS (5 ag/culture) and assayed for anti-TNP PFC and [3H]TdR incorporation. b Mean + standard deviation of triplicate assays.
LPS RESPONSE
OF CR(-)
AND
CR(+)
B CELLS
83
of CR(+) B cells, since T cells are enriched in the CRL-depleted fraction, and T cells or macrophages have been reported to modulate LPS response (13-I 5). In the following experiments, these possibilities were examined. Time kinetics of the LPS response. The difference between CR(+) B cells and CR( -) B cells in LPS responsecould possibly be caused by the shift of time kinetics of the responding cells. The time course of LPS response of cells from the CRLdepleted and -enriched fractions was studied both in terms of anti-TNP PFC and [3H]TdR incorporation from Day 1 to Day 4 of culture. The results are shown in Fig. 1. In the PFC response, cells from the CRL-depleted fraction reached their peak on Day 3 and declined thereafter as well as the cells from E-interface fraction and unfractionated spleen cells, and the number of anti-TNP PFC at the peak response of the CRL-depleted fraction was 2.5 to 3 times more than that of the E-interface fraction or unfractionated spleen cells. Cells from the CRL-enriched fraction reached their peak response on Day 2, but the number of PFC was much fewer not only than that from the CRL-depleted fraction but also than that from the E-interface fraction or unfractionated spleen cells throughout the culture period. In the proliferative response, all cell fractions reached their peak responseson Day 2 and declined thereafter. The peak response of cells from the CRL-enriched fraction was as high as that of cells from the E-interface fraction or unfractionated spleen cells, and significantly higher (P < 0.01) than that of cells from the CRLdepleted fraction. These results indicate that the difference between CR(+) B cells and CR(-) ones in the LPS response cannot be explained by the shift of time kinetics. There is a possibility, however, that vigorous proliferation of cells from the CRL-enriched fraction might exhaust their culture medium and prevent them from differentiation into AFC. To examine the possibility, cells from each fraction were cultured with LPS at lower cell densities, 5 X 105, 2 X 105, and 1 X lo5 cells/culture, and time courses of their responses were studied by the assessmentsof anti-TNP PFC and [3H]TdR incorporation. At any cell density the cells from the CRL-depleted fraction responded better (P < 0.01) than the cells from any other fraction in terms of PFC, whereas in [3H]TdR incorporation the cells from the CRL-enriched fraction responded significantly better (P < 0.01) than those from the CRL-depleted fraction (data not shown). These findings indicate that it cannot be possible that extensive proliferation of cells from the CRL-enriched fraction exhaust the culture medium to inhibit their differentiation into AFC. Therefore, the difference between CRL-depleted and CRL-enriched fractions in LPS response was indicated not to be due to the culture conditions. LPS response of CRL-depleted and -enriched cells in the absence of T cells and macrophages. Although it is well known that LPS can induce B-cell proliferation
and differentiation in the absence of T cells and macrophages, T cells have been reported to enhance LPS responseof B cells ( 13). When spleen cells were separated into CRL-depleted and -enriched fractions, T cells might be enriched in the CRLdepleted fraction, and macrophages might be depleted from the fraction because they also bear CR. Therefore, the difference between CRL-depleted and -enriched cells in LPS response might be due to the different ratio of T cells or macrophages in these fractions. In the next experiments LPS responses of CRL-depleted and -enriched fractions were examined in the absence of both T cells and macrophages.
KAKIUCHI
AND NARIUCHI
5
4
2 3 s x
= 2 p. ” 1
1
,
1
2 Days of
3 culture
41
1
2 Days of
3
4d
culture
FIG. 1. Time kinetics of anti-TNP PFC (A) and [‘H]TdR incorporation response (B). Spleen cells unfractionated (X) and cells from the E-interface (*), EAC-interface (0), and EAC-pellet (0) fraction were cultured with LPS, and assayed for anti-TNP PFC and [‘H]TdR incorporation on Days 1, 2, 3, and 4 of culture. When cells were cultured without LPS, anti-TNP PFC and [‘H]TdR incorporation were less than 127 PFC/culture and 2240 cpm/culture, respectively. Each point represents a mean value of triplicate assays and vertical bars represent standard deviation.
Spleen cells from nu/nu mice on a C57BL/6 background were depleted of macrophages by the passage through a Sephadex G-10 column, and the cells were separated into CRL-depleted and -enriched fractions. The efficiency of the macrophage depletion was monitored by the polyclonal PFC response to DxS, since the response was absolutely dependent on the presence of macrophages in our culture system as reported by Persson et al. (16). Cells passed through the column did not respond to DxS. The cells from each fraction were cultured with LPS for 3 days and assayedfor anti-TNP PFC and [ 3H]TdR incorporation. The results are shown in Table 2. Macrophage depletion from unseparated spleen cells did not affect the responses both in the number of PFC and the [3H]TdR incorporation (P > 0.05). Cells from the CRL-depleted fraction responded signficantly better (P < 0.01) in anti-TNP PFC and significantly more poorly in [3H]TdR incorporation (P < 0.01) than cells from the E-interface fraction. On the other hand, cells from the CRL-enriched fraction responded more poorly in PFC, and equally well in [3H]TdR incorporation to those from the E-interface fraction. These findings were confirmed by using spleen cells from normal mice treated with anti-Thy 1.2 and complement (data not shown).
LPS RESPONSE
OF CR(-)
AND
CR(+)
85
B CELLS
These results indicate that T cells and macrophages do not take part in the different LPS responses between cells from the CRL-depleted and -enriched fractions, and that CR(-) B cells respond to LPS better in antibody formation and more poorly in proliferation than CR(+) B cells. E$ect of irradiation on LPS response of CRL-depleted and -enriched cells. As described above, CR(-) B cells seem to differentiate into AFC rather than proliferate in LPS response, and CR(+) B cells respond to LPS inversely. Murine B cells have been reported to differentiate into AFC without proliferation in LPS response (17). In the next experiments, therefore, CR(+) and CR(-) B cells were irradiated and then cultured with LPS to see if CR(-) B cells can differentiate into AFC without proliferation. Spleen cells were separated into CRL-depleted and -enriched fractions, irradiated at various doses, and then they were cultured with LPS. After 3 days they were assayed for anti-TNP PFC. The results are shown in Fig. 2. In any fraction the number of PFC was increased slightly at 200 R irradiation, and then decreased with increasing doses of irradiation, and could not be decreased any more at 1000 R. With 1000 R or more irradiation cells from the CRL-depleted fraction could show better PFC responsethan those from the E-interface fraction (P < 0.01) and cells from the CRL-enriched fraction could not respond. These results indicate that the PFC response of CR(-) B cells to LPS is more resistant to irradiation than that of CR(+) B cells. DISCUSSION In the experiments reported here, CR(+) B cells seemed to proliferate better than CR(-) ones, but more antibody-forming cells could be induced from CR(-) B cells than from CR(+) ones in the response to LPS stimulation. Since SRBC were used both for the preparations of EAC and indicator cells for PFC assay, specific SRBC PFC could possibly modify the enumeration of polyclonal anti-TNP TABLE
2
LPS Response of CRL-Enriched or -Depleted Spleen Cells from nu/nu Mice after the Depletion of Adherent Cells”
LPS
Anti-TNP PFC/culture f SD
cpm/culture + SD
Unfractionated
+
60 -c 5 920 + 159
592 + 371 12,254 + 5299
Unfractionated G- 10 passed
+
25 + 9 920 f 72
353 + 106 29,528 2 4589
G- 10 passed E interface
+
23 f 8 933 f 81
52+ 18 9,505 + 482
G- 10 passed EAC interface
+
27 f 3 2333 k 122
G- 10 passed EAC pellet
+
63 f 19 160 f 35
Cell fraction
75 + 22 358 f 107 49 f 74 7,747 + 274
’ Spleen cells from nu/nu mice passed through a Sephadex G-10 column were fractionated as described in the footnote to Table 1.
and cultured
86
KAKIUCHI
AND NARIUCHI l
; E - Interface
o ; EAC-Interface l
0 4
; EAC-Pellet
0
l -•
\\
./‘\‘\“\ .
l
0
0
l -• b
I
‘* _ __ _ _ _ _. _ _ 1*.
t
.
.
I
0
2
4
6
10
Radiation
Dose ( R )
15 x 100
FIG. 2. LPS response of cells from the E-interface fraction (*), the EAC interface (O), and EAC pellet (0) after irradiation. Cells from each fraction were irradiated with 0 to 1500 R, cultured with (-) or without (- - -) LPS for 3 days, and assayed for anti-TNP PFC.
PFC response. The possibility, however, seems unlikely on the following basis; (i) the difference between CR(-) and CR(+) B cells in PFC response was confirmed using TNP-HRBC as indicator cells as described previously (lo), (ii) B cells responsive to SRBC have been reported to bear CR on their surface (2, 17), (iii) prior to the separation into CR(-) and CR( +) populations spleen cells were mixed with SRBC and centrifuged on the density gradient. SRBC specific B cells would be depleted by the procedure. The efficiency of CRL depletion could be a matter, because the percentage of EAC-RFC in the EAC-pellet fraction was lower than expected in rerosetting with EAC. A possible explanation for the low percentage of EAC-RFC would be that the debris of red blood cells lysed with NH4CL might still be on the CR of some B cells in the EAC-pellet fraction and inhibit rerosetting with EAC. The idea was
LPS RESPONSE
OF CR(-)
AND
CR(+)
B CELLS
87
supported by the fact that the cells surrounded by the ghost red cells were observed under a phase contrast microscope. It can be concluded, therefore, that there was much more CRL in the EAC-pellet fraction than the number of EAC-RFC in rerosetting with EAC. Some investigators have reported different results from ours. Hammerling et al. have reported that CR(-) B cells can respond to LPS as well as CR( +) B cells but with a delay of 24 hr both in proliferation and Ig synthesis (5). In their experiments only 25% of total spleen cells formed rosettes with EAC, whereas 45 to 50% were CRL in our system. The different efficiency in CRL depletion would explain the different results. Brandon et al. have claimed that CR( +) B cells respond to LPS better than CR(-) B cells both in proliferation and Ig synthesis (6). In their experiments, however, Ig synthesis of B cells was measured by the incorporation of [3H]leucine into immunoglobulin with a 4-hr pulse on Day 6 of culture. Thus, their assay system was quite different from ours. Lewis et al. have described lower proliferative response of CR(+) B cells to LPS than of CR(-) B cells (2). In their report the results were presented by stimulation index and they did not mention background thymidine incorporation. The high background of CR(+) Bcell fraction, [3H]TdR incorporation, or PFC assay made results difficult to interpret in our previous results (10). In our present studies, it was shown that there are two separate B-cell subsets responsive to LPS stimulation. B cells in one subset have CR and they proliferate vigorously but differentiate poorly into PFC on LPS stimulation, and the cells in another subset do not have CR and they differentiate efficiently into AFC but proliferate poorly in the LPS response. These results suggest that CR(-) B cells are more mature than CR(+) B cells at least in the B-cell population responsive to LPS stimulation. On the other hand CR(+) B cells are generally believed to be more mature than CR( -) B cells because CR( +) B cells appear late in ontogeny (8, 9). This contradiction could be solved by the assumption that there are some mature B-cell subsets which have no CR, that is, B cells mature in order, CR( -) - CR( +) --t CR( -). The possibility could be supported by the radioresistancy of PFC response of CR(-) B cells to LPS shown in this report, which is consistent with the findings by Quintans and Lefkovitz (17). In addition, CR has been shown to be on precursor B cells but not on AFC (18, 19). Mason also has shown that CR(-) B cells can generate secondary IgG PFC response (3). These results would support the concept that CR(+) B cells lose CR during their differentiation into AFC. In this context, it seemslikely that LPS stimulation to CR(+) B cells causes their proliferation rather than differentiation and the stimulation to the most mature CR(-) B cells causes differentiation with little proliferation. The accumulation of the findings on the difference between CR(+) and CR(-) B cells in their functional properties would throw light on the functional significance of CR. REFERENCES 1. 2. 3. 4. 5.
Parish, C. R., and Chilcott, A. B., Cell. Immunol. 20, 290, 1975. Lewis, G. K., Ranken, R., Nitecki, D. E., and Goodman, J. W., J. Exp. Med. 144, 382, 1976. Mason, D. W., .I. Exp. Med. 143, 1111, 1976. Simon, M. M., and Hgmmerling, U., J. Itnmunol. 119, 1700. Hgmmerling, U., Chua, R., and Hoffman, M. K., J. Immunol. 120, 750, 1978.
88 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19.
KAKIUCHI
AND NARIUCHI
Brandon, D. L., Edwards, A. J., and Parkhouse, R. M. E., Immunology 36, 865, 1979. Nariuchgi, H., and Kakiuchi, T., Cell. Zmmunol. 54, 264, 1980. Hammerling, U., Chin, A. F., and Abbot, J., Proc. Nat. Acad. Sci. USA 73, 2008, 1976. Yang, W. C., Miller, S. C., and Osmond, D. G., J. Exp. Med. 148, 1251, 1978. Kakiuchi, T., and Nariuchi, H., J. Zmmunol. 127, 954, 1981. Kakiuchi, T., Nariuchi, H., and Matuhasi, T., Immunobiology 156, 342, 1979. Ly, J. A., and M&hell, R. I., J. Zmmunol. Methods 5, 239, 1974. Goodman, M. G., and Weigle, W. O., J. Zmmunol. 122, 2548, 1979. Yoshinaga, M., Yoshinaga, A., and Waksman, B. H., J. Exp. Med. 136, 956, 1972. Baird, L. G., and Kaplan, A. M., Cell. Zmmunol. 28, 22, 1977. Persson, U. C. L., Hammerstrom, L. L. G., and Smith, C. I. C., J. Zmmunol. 119, 1138, 1977. Quintans, J., and Lefkovitz, I., J. Immunol. 113, 1373, 1974. Parish, C. R., and Hayward, J. A., Proc. R. Sot. London Ser. B 187, 65, 1974. McConnel, I., and Hurd, C. M., Immunology 30, 825, 1976.
IMMUNOLOGY
71, 80-88 (1982)
Different Responses of CR(-) and CR(+) B Cells to LPS Stimulation TERUTAKA Laboratory
KAKIUCHI
AND HIDEO NARIUCHI’
of Biological Products, Institute of Medical University of Tokyo, Tokyo 108, Japan
Received February
Science,
I, 1982; accepted May I I, 1982
Proliferative response of B cells with or without CR [CR(+) or CR(-) B cells] was compared in their polyclonal response when they were stimulated with lipopolysaccharide (LPS). CR(+) B cells responded better in proliferation and more poorly in polyclonal antibody formation than did CR( -) B cells. The dissociation between proliferation and antibody formation in LPS responsewas not due to the shift of the time kinetics nor the exhaustion of the culture medium. T cells and macrophages did not take part in the dissociation, since macrophage depletion from nu/nu mouse spleen cells could not modify the dissociation. The polyclonal antibody response of CR(-) B cells was more resistant to irradiation than that of CR(+) B cells. These results suggest that among LPS-responsive B cells there are CR(-) B-cell subset(s) more mature than CR(+) B cells.
INTRODUCTION There are several contradictory results on the differences between B cells with or without CR in the responsivenessto various antigens (l-4) and to LPS (2, 57). In regard to the B-cell maturation, CR(-) B cells have been reported to be less mature than CR(+) B cells (8, 9). In our previous report (lo), CR(-) B cells have been shown to be different from CR( +) B cells in the antibody-forming cells (AFC) responsesto purified protein derivative of tuberculin (PPD), dextran sulfate (DxS), and LPS, that is, CR(-) B cells responded well to LPS and poorly to PPD or DxS, and CR(+) B cells responded well both to PPD and DxS but seemed to respond poorly to LPS. In these experiments, it was suggested to be dissociation between antibody formation and proliferation in the LPS response of CR(-) and CR(+) B cells. In the present studies, therefore, CR(-) and CR(+) B cells were compared to each other in terms of [ 3H]thymidine ([ 3H]TdR) incorporation and antibody responseto LPS and the dissociation was confirmed. The sensitivities of LPS responses of CR(-) and CR(+) B cells to irradiation in terms of antibody formation were also studied. The maturational stages of CR(-) and CR(+) B cells responsive to LPS were discussed. ’ To whom all correspondence should be addressed: Laboratory of Biological Products, Institute of Medical Science, University of Tokyo, 4-6-1, Shiroganedai, Minatoku, Tokyo 108, Japan. 80 0008-8749/82/l lOOSO-09$02.00/0 Copyright 0 1982 by Academic Press, Inc. All fights of reproduction in any form rwe~cd.
LPS RESPONSE
OF CR(-)
MATERIALS
AND
CR(+)
B CELLS
81
AND METHODS
Mice. C57BL/6j female mice and mice with a nu/nu gene on a C57BL/6j background were supplied by the animal breeding unit of our institute, and used at 8 to 16 weeks of age. DDD mice were also supplied by the unit. Mitogens. LPS from Escherichia coli 055:B5 prepared by phenol extraction was purchased from Difco Laboratories, Detroit, Michigan. LPS was used at 5 pg/ culture. The quantity of LPS was determined by preliminary experiments to be the optimal stimulatory dose for C57BL/6j spleen cells. Erythrocyte-antibody-complement complexes (EAC). EAC were prepared as described previously ( 11). Briefly, sheep erythrocytes (E) were sensitized with a subagglutinating concentration of IgM anti-E antibody, and the resultant cells (EA) were treated with CS-deficient DDD mouse serum for 30 min at 37°C. Irradiation of cells. Cells were irradiated with varying doses in suspension using Gamma cell 40, Atomic Energy of Canada Ltd., Ottawa, Canada. Cultures. One million cells were cultured in 0.2 ml of medium RPM1 1640 containing 20% fetal calf serum (Associated Biomedic Systems, Buffalo, N.Y.), 5 X lo-’ M 2-mercaptoethanol, and kanamycin ( 100 pg/culture) in flatbottom Microtest II plates (Falcon 3042) in a humidified atmosphere of 5% CO1 in air at 37°C. After 3 days incubation the cells were assayed for anti-TNP PFC using TNP-sheep erythrocytes, unless otherwise stated. For the assessmentof proliferative response, 5 X lo5 cells were cultured under the same conditions to the antibody response except that the medium contained 2% fetal calf serum without 2-mercaptoethanol. The response was assessedby [3H]TdR incorporation (0.25 &i/ culture; specific activity, 2.0 Ci/mmol). Detection and depletion of an enrichment with lymphocytes bearing CR (CRL). CRL were detected by rosette-formation with EAC, and the depletion of and enrichment with EAC-rosette-forming cells (EAC-RFC) were carried out as described previously (10) with slight modifications. Briefly, spleen cells were mixed with an equal volume of 10% E and centrifuged over Ficoll-Hypaque; cells in the interface fraction (5 X lo7 cells/ml) were mixed with an equal volume of 10% EAC, incubated for 20 min at 37°C to form rosettes, and then layered on top of an equal volume of Ficoll-Hypaque and centrifuged at 2000g for 20 min at 20°C. The cells from the medium Ficoll-Hypaque interface (EAC interface) and those from the pellet fraction (EAC pellet) were used as CRL-depleted and -enriched cells, respectively, after they were treated with NH&Z1 to lyse erythrocytes. Residual CRL in the CRL-depleted fraction were less than 2.2% in all experiments, when cells from the fraction were rerosetted with EAC. Usually, CRL were 45 to 50% of total spleen cells. For the control experiments EA were used instead of EAC. Cells from the interface (EA-interface) and the pellet (EA-pellet) fractions were used for experiments. Depletion of adherent cells. Adherent cells were depleted from spleen cell population by the passage through a Sephadex G-10 column as described previously ( 12). RESULTS Comparison of antibody and proliferation responses of CRL-depleted and -enriched fractions of spleen cells to LPS stimulation. In our previous experiments
82
KAKIUCHI
AND
NARIUCHI
there seemed to be dissociation between antibody formation and proliferation in LPS responses of CR(+) and CR(-) B cells. In the first experiments, therefore, spleen cells were fractionated into CRL-depleted and -enriched populations, and then they were cultured with LPS (5 pg/culture) for the assessmentsof t3H]TdR incorportion and PFC responses. When cells from each fraction were rerosetted with EAC, 50.7% of unfractionated spleen cells formed rosettes, and 49.8% of the E-interface, 23.6% of the E-pellet, 0.3% of the EAC-interface, and 36.0% of the EAC-pellet fractions were CRL. At the same time sIg positive cells from each fraction were 53.2, 54.6, 51.1, 16.2, and 63.7%, respectively. The results of PFC and [ 3H]TdR incorportion responsesare shown in Table 1. The PFC responsesof cells from CRL-enriched fraction was significantly lower (P < 0.01) than that of cells from the CRL-depleted fraction. On the contrary, [3H]TdR incorporation of cells from the CRL-enriched fraction was better than that of cells from the CRLdepleted fraction. It can be clearly concluded that CRL-depleted cells respond better to LPS in antibody formation and more poorly in proliferation than do CRLenriched cells. These results seem to reflect the different responsivenessof CR(+) B cells and CR(-) B cells to LPS. There are, however, several possibilities to be excluded: (i) CR(+) B cells may differ from CR(-) B cells in the time kinetics of their response to LPS, as shown by Hammerling et al. (5), (ii) the poor PFC responseof CR(+) B cells to LPS may be due to the exhaustion of culture medium caused by the vigorous proliferation of CR(+) B cells, (iii) T cells or macrophages may take part in reducing proliferative response of CR( -) B cells or PFC response TABLE LPS Response of CRL-Depleted
Cell fraction
LPS
Unfractionated
+
E interface
+
E pellet
+
EA interface
+
EA pellet
+
EAC interface
+
EAC pellet
+
1 and -Enriched
Anti-TNP PFC/culture + SD*
Spleen Cells” cpm/culture zk SDb
31 f 8 960 f 61
286 + 31 15,222 f 1786
37 + 10 913 + 35
13,229 f 3801
663 f 43 5193 f 206
404 f 91 4,341 + 416
55 f 10 1273 f 101
166 f 20 9,139 + 229
53 + 8 413 + 25
129 f 95 4,740 f 1434
40 f 5 2233 f 155 123 + 26 423 f 84
994 *
99 f 4,443 f 61 f
188
11 1065 5
12,940 f 765
’ Spleen cells were rosetted with EAC and separated into CRL-depleted (EAC interface) and CRLenriched (EAC pellet) fractions. EA were used for control. Cells from each fraction were cultured for 3 days with LPS (5 ag/culture) and assayed for anti-TNP PFC and [3H]TdR incorporation. b Mean + standard deviation of triplicate assays.
LPS RESPONSE
OF CR(-)
AND
CR(+)
B CELLS
83
of CR(+) B cells, since T cells are enriched in the CRL-depleted fraction, and T cells or macrophages have been reported to modulate LPS response (13-I 5). In the following experiments, these possibilities were examined. Time kinetics of the LPS response. The difference between CR(+) B cells and CR( -) B cells in LPS responsecould possibly be caused by the shift of time kinetics of the responding cells. The time course of LPS response of cells from the CRLdepleted and -enriched fractions was studied both in terms of anti-TNP PFC and [3H]TdR incorporation from Day 1 to Day 4 of culture. The results are shown in Fig. 1. In the PFC response, cells from the CRL-depleted fraction reached their peak on Day 3 and declined thereafter as well as the cells from E-interface fraction and unfractionated spleen cells, and the number of anti-TNP PFC at the peak response of the CRL-depleted fraction was 2.5 to 3 times more than that of the E-interface fraction or unfractionated spleen cells. Cells from the CRL-enriched fraction reached their peak response on Day 2, but the number of PFC was much fewer not only than that from the CRL-depleted fraction but also than that from the E-interface fraction or unfractionated spleen cells throughout the culture period. In the proliferative response, all cell fractions reached their peak responseson Day 2 and declined thereafter. The peak response of cells from the CRL-enriched fraction was as high as that of cells from the E-interface fraction or unfractionated spleen cells, and significantly higher (P < 0.01) than that of cells from the CRLdepleted fraction. These results indicate that the difference between CR(+) B cells and CR(-) ones in the LPS response cannot be explained by the shift of time kinetics. There is a possibility, however, that vigorous proliferation of cells from the CRL-enriched fraction might exhaust their culture medium and prevent them from differentiation into AFC. To examine the possibility, cells from each fraction were cultured with LPS at lower cell densities, 5 X 105, 2 X 105, and 1 X lo5 cells/culture, and time courses of their responses were studied by the assessmentsof anti-TNP PFC and [3H]TdR incorporation. At any cell density the cells from the CRL-depleted fraction responded better (P < 0.01) than the cells from any other fraction in terms of PFC, whereas in [3H]TdR incorporation the cells from the CRL-enriched fraction responded significantly better (P < 0.01) than those from the CRL-depleted fraction (data not shown). These findings indicate that it cannot be possible that extensive proliferation of cells from the CRL-enriched fraction exhaust the culture medium to inhibit their differentiation into AFC. Therefore, the difference between CRL-depleted and CRL-enriched fractions in LPS response was indicated not to be due to the culture conditions. LPS response of CRL-depleted and -enriched cells in the absence of T cells and macrophages. Although it is well known that LPS can induce B-cell proliferation
and differentiation in the absence of T cells and macrophages, T cells have been reported to enhance LPS responseof B cells ( 13). When spleen cells were separated into CRL-depleted and -enriched fractions, T cells might be enriched in the CRLdepleted fraction, and macrophages might be depleted from the fraction because they also bear CR. Therefore, the difference between CRL-depleted and -enriched cells in LPS response might be due to the different ratio of T cells or macrophages in these fractions. In the next experiments LPS responses of CRL-depleted and -enriched fractions were examined in the absence of both T cells and macrophages.
KAKIUCHI
AND NARIUCHI
5
4
2 3 s x
= 2 p. ” 1
1
,
1
2 Days of
3 culture
41
1
2 Days of
3
4d
culture
FIG. 1. Time kinetics of anti-TNP PFC (A) and [‘H]TdR incorporation response (B). Spleen cells unfractionated (X) and cells from the E-interface (*), EAC-interface (0), and EAC-pellet (0) fraction were cultured with LPS, and assayed for anti-TNP PFC and [‘H]TdR incorporation on Days 1, 2, 3, and 4 of culture. When cells were cultured without LPS, anti-TNP PFC and [‘H]TdR incorporation were less than 127 PFC/culture and 2240 cpm/culture, respectively. Each point represents a mean value of triplicate assays and vertical bars represent standard deviation.
Spleen cells from nu/nu mice on a C57BL/6 background were depleted of macrophages by the passage through a Sephadex G-10 column, and the cells were separated into CRL-depleted and -enriched fractions. The efficiency of the macrophage depletion was monitored by the polyclonal PFC response to DxS, since the response was absolutely dependent on the presence of macrophages in our culture system as reported by Persson et al. (16). Cells passed through the column did not respond to DxS. The cells from each fraction were cultured with LPS for 3 days and assayedfor anti-TNP PFC and [ 3H]TdR incorporation. The results are shown in Table 2. Macrophage depletion from unseparated spleen cells did not affect the responses both in the number of PFC and the [3H]TdR incorporation (P > 0.05). Cells from the CRL-depleted fraction responded signficantly better (P < 0.01) in anti-TNP PFC and significantly more poorly in [3H]TdR incorporation (P < 0.01) than cells from the E-interface fraction. On the other hand, cells from the CRL-enriched fraction responded more poorly in PFC, and equally well in [3H]TdR incorporation to those from the E-interface fraction. These findings were confirmed by using spleen cells from normal mice treated with anti-Thy 1.2 and complement (data not shown).
LPS RESPONSE
OF CR(-)
AND
CR(+)
85
B CELLS
These results indicate that T cells and macrophages do not take part in the different LPS responses between cells from the CRL-depleted and -enriched fractions, and that CR(-) B cells respond to LPS better in antibody formation and more poorly in proliferation than CR(+) B cells. E$ect of irradiation on LPS response of CRL-depleted and -enriched cells. As described above, CR(-) B cells seem to differentiate into AFC rather than proliferate in LPS response, and CR(+) B cells respond to LPS inversely. Murine B cells have been reported to differentiate into AFC without proliferation in LPS response (17). In the next experiments, therefore, CR(+) and CR(-) B cells were irradiated and then cultured with LPS to see if CR(-) B cells can differentiate into AFC without proliferation. Spleen cells were separated into CRL-depleted and -enriched fractions, irradiated at various doses, and then they were cultured with LPS. After 3 days they were assayed for anti-TNP PFC. The results are shown in Fig. 2. In any fraction the number of PFC was increased slightly at 200 R irradiation, and then decreased with increasing doses of irradiation, and could not be decreased any more at 1000 R. With 1000 R or more irradiation cells from the CRL-depleted fraction could show better PFC responsethan those from the E-interface fraction (P < 0.01) and cells from the CRL-enriched fraction could not respond. These results indicate that the PFC response of CR(-) B cells to LPS is more resistant to irradiation than that of CR(+) B cells. DISCUSSION In the experiments reported here, CR(+) B cells seemed to proliferate better than CR(-) ones, but more antibody-forming cells could be induced from CR(-) B cells than from CR(+) ones in the response to LPS stimulation. Since SRBC were used both for the preparations of EAC and indicator cells for PFC assay, specific SRBC PFC could possibly modify the enumeration of polyclonal anti-TNP TABLE
2
LPS Response of CRL-Enriched or -Depleted Spleen Cells from nu/nu Mice after the Depletion of Adherent Cells”
LPS
Anti-TNP PFC/culture f SD
cpm/culture + SD
Unfractionated
+
60 -c 5 920 + 159
592 + 371 12,254 + 5299
Unfractionated G- 10 passed
+
25 + 9 920 f 72
353 + 106 29,528 2 4589
G- 10 passed E interface
+
23 f 8 933 f 81
52+ 18 9,505 + 482
G- 10 passed EAC interface
+
27 f 3 2333 k 122
G- 10 passed EAC pellet
+
63 f 19 160 f 35
Cell fraction
75 + 22 358 f 107 49 f 74 7,747 + 274
’ Spleen cells from nu/nu mice passed through a Sephadex G-10 column were fractionated as described in the footnote to Table 1.
and cultured
86
KAKIUCHI
AND NARIUCHI l
; E - Interface
o ; EAC-Interface l
0 4
; EAC-Pellet
0
l -•
\\
./‘\‘\“\ .
l
0
0
l -• b
I
‘* _ __ _ _ _ _. _ _ 1*.
t
.
.
I
0
2
4
6
10
Radiation
Dose ( R )
15 x 100
FIG. 2. LPS response of cells from the E-interface fraction (*), the EAC interface (O), and EAC pellet (0) after irradiation. Cells from each fraction were irradiated with 0 to 1500 R, cultured with (-) or without (- - -) LPS for 3 days, and assayed for anti-TNP PFC.
PFC response. The possibility, however, seems unlikely on the following basis; (i) the difference between CR(-) and CR(+) B cells in PFC response was confirmed using TNP-HRBC as indicator cells as described previously (lo), (ii) B cells responsive to SRBC have been reported to bear CR on their surface (2, 17), (iii) prior to the separation into CR(-) and CR( +) populations spleen cells were mixed with SRBC and centrifuged on the density gradient. SRBC specific B cells would be depleted by the procedure. The efficiency of CRL depletion could be a matter, because the percentage of EAC-RFC in the EAC-pellet fraction was lower than expected in rerosetting with EAC. A possible explanation for the low percentage of EAC-RFC would be that the debris of red blood cells lysed with NH4CL might still be on the CR of some B cells in the EAC-pellet fraction and inhibit rerosetting with EAC. The idea was
LPS RESPONSE
OF CR(-)
AND
CR(+)
B CELLS
87
supported by the fact that the cells surrounded by the ghost red cells were observed under a phase contrast microscope. It can be concluded, therefore, that there was much more CRL in the EAC-pellet fraction than the number of EAC-RFC in rerosetting with EAC. Some investigators have reported different results from ours. Hammerling et al. have reported that CR(-) B cells can respond to LPS as well as CR( +) B cells but with a delay of 24 hr both in proliferation and Ig synthesis (5). In their experiments only 25% of total spleen cells formed rosettes with EAC, whereas 45 to 50% were CRL in our system. The different efficiency in CRL depletion would explain the different results. Brandon et al. have claimed that CR( +) B cells respond to LPS better than CR(-) B cells both in proliferation and Ig synthesis (6). In their experiments, however, Ig synthesis of B cells was measured by the incorporation of [3H]leucine into immunoglobulin with a 4-hr pulse on Day 6 of culture. Thus, their assay system was quite different from ours. Lewis et al. have described lower proliferative response of CR(+) B cells to LPS than of CR(-) B cells (2). In their report the results were presented by stimulation index and they did not mention background thymidine incorporation. The high background of CR(+) Bcell fraction, [3H]TdR incorporation, or PFC assay made results difficult to interpret in our previous results (10). In our present studies, it was shown that there are two separate B-cell subsets responsive to LPS stimulation. B cells in one subset have CR and they proliferate vigorously but differentiate poorly into PFC on LPS stimulation, and the cells in another subset do not have CR and they differentiate efficiently into AFC but proliferate poorly in the LPS response. These results suggest that CR(-) B cells are more mature than CR(+) B cells at least in the B-cell population responsive to LPS stimulation. On the other hand CR(+) B cells are generally believed to be more mature than CR( -) B cells because CR( +) B cells appear late in ontogeny (8, 9). This contradiction could be solved by the assumption that there are some mature B-cell subsets which have no CR, that is, B cells mature in order, CR( -) - CR( +) --t CR( -). The possibility could be supported by the radioresistancy of PFC response of CR(-) B cells to LPS shown in this report, which is consistent with the findings by Quintans and Lefkovitz (17). In addition, CR has been shown to be on precursor B cells but not on AFC (18, 19). Mason also has shown that CR(-) B cells can generate secondary IgG PFC response (3). These results would support the concept that CR(+) B cells lose CR during their differentiation into AFC. In this context, it seemslikely that LPS stimulation to CR(+) B cells causes their proliferation rather than differentiation and the stimulation to the most mature CR(-) B cells causes differentiation with little proliferation. The accumulation of the findings on the difference between CR(+) and CR(-) B cells in their functional properties would throw light on the functional significance of CR. REFERENCES 1. 2. 3. 4. 5.
Parish, C. R., and Chilcott, A. B., Cell. Immunol. 20, 290, 1975. Lewis, G. K., Ranken, R., Nitecki, D. E., and Goodman, J. W., J. Exp. Med. 144, 382, 1976. Mason, D. W., .I. Exp. Med. 143, 1111, 1976. Simon, M. M., and Hgmmerling, U., J. Itnmunol. 119, 1700. Hgmmerling, U., Chua, R., and Hoffman, M. K., J. Immunol. 120, 750, 1978.
88 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19.
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AND NARIUCHI
Brandon, D. L., Edwards, A. J., and Parkhouse, R. M. E., Immunology 36, 865, 1979. Nariuchgi, H., and Kakiuchi, T., Cell. Zmmunol. 54, 264, 1980. Hammerling, U., Chin, A. F., and Abbot, J., Proc. Nat. Acad. Sci. USA 73, 2008, 1976. Yang, W. C., Miller, S. C., and Osmond, D. G., J. Exp. Med. 148, 1251, 1978. Kakiuchi, T., and Nariuchi, H., J. Zmmunol. 127, 954, 1981. Kakiuchi, T., Nariuchi, H., and Matuhasi, T., Immunobiology 156, 342, 1979. Ly, J. A., and M&hell, R. I., J. Zmmunol. Methods 5, 239, 1974. Goodman, M. G., and Weigle, W. O., J. Zmmunol. 122, 2548, 1979. Yoshinaga, M., Yoshinaga, A., and Waksman, B. H., J. Exp. Med. 136, 956, 1972. Baird, L. G., and Kaplan, A. M., Cell. Zmmunol. 28, 22, 1977. Persson, U. C. L., Hammerstrom, L. L. G., and Smith, C. I. C., J. Zmmunol. 119, 1138, 1977. Quintans, J., and Lefkovitz, I., J. Immunol. 113, 1373, 1974. Parish, C. R., and Hayward, J. A., Proc. R. Sot. London Ser. B 187, 65, 1974. McConnel, I., and Hurd, C. M., Immunology 30, 825, 1976.