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Responses of spleen cells from mice with X-linked B-cell defect to polyclonal B-cell activators, purified protein derivative of tuberculin, and dextran sulfate
CELLULAR
IMMUYOLOGY
61, 375-385 (1981)
Responses of Spleen Cells from Mice with X-linked B-Cell Defect to Polyclonal B-Cell Activators, Purified Protein Derivative of Tuberculin, and Dextran Sulfate H~DEO NARIUCHI Laboratory
AND TERUTAKA
KAKIUCHI
of Biological Products. The Institute of Medical Science, The University 4-6-l) Shiroganedai, Minatoku. Tokyo, 108 Japan Received December I I. 1980; accepted January
of Tokyo,
23, 1981
Polyclonal responsesto LPS, PPD, and DxS of spleen cells from mice expressing a X-linked B-cell defect were examined. Spleen cells from young (CBA/N X BALB/c)F, male mice responded slightly lower to LPS, significantly lower to PPD than the ceils from age-matched F, female mice, and showed no response to DxS stimulation. This hypo- or unresponsiveness of F, male cells to PPD or DxS could not be explained by a shift in the dose-responseor time kinetics of the responding cells, and also could not be due to the defect in the function of T cells or macrophages. Suppressor T cells to polyclonal response to PPD or DxS could not be shown in Fr male spleen cells. The responseof F, male cells to PPD was dramatically improved with age but not to DxS. These results suggest that B cells responsive to DxS may belong to a distinct subpopulation from the cells responsive to LPS or PPD.
INTRODUCTION The CBA/N strain of mouse carries a X-linked recessivedefect and the defective phenotype has been shown to be expressed in B cells (1, 2). The B cells in adult CBA/N mice have been considered to have defect in their maturation, since they have a high ratio of IgM to IgD on their surface (3) and express only low densities of B-cell differentiation markers, such as Ia, Mls, Lyb3, and Lyb5 (1,4-6). These defective B cells have the ability to respond to some but not to other thymusindependent (TI) antigens. The formers are classified now as TI-1 (trinitrophenyl [TNP]-lipopolysaccharide [LPS], TNP-Brucella abortus) (7, 8) and the latters TI-2 (TNP-Ficoll, SSS-III) (9, 10). TI-2 antigens have been suggestedto stimulate a more mature population of B cells than TI-1 antigens do (11). Polyclonal B-cell activators (PBAs) have recently been widely used as a tool for studying the mechanism of B-cell activation. Spleen cells from CBA/N mice have been reported to show low responses to PBAs (LPS, lipoprotein, Nocardia mitogen) (7, 12) or to fail to respond some other B-cell mitogens (anti-p, 2-mercaptoethanol) (13, 14). These defective functions of B cells in CBA/N mice in response to antigens and PBAs mentioned above have recently been reported to be due to a delay in the maturation of B cells and the functions were shown to increase with age (15, 16). The different PBAs have been suggested to stimulate B cells in different stages 375 OOOS-8749/81/100375-ll$O2.00/0 Copyright 0 1981 by Academac Press. Inc. All rights of reprcduct~on in any form reserved
376
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AND
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of differentiation (17, 18). LPS has been shown to stimulate broad populations of B cells in an intermediate stage of differentiation. Lipoprotein and Nocardia mitogen seem to stimulate similar B-cell population to the cells responsive to LPS (12). Dextran sulfate (DxS) has been suggested to activate primitive B cells, whereas purified protein derivative of tuberculin (PPD) activate only mature B cells (17-19). In the experiments reported here, we have studied the responsivenessof spleen cells from (CBA/N X BALB/c)F, male mice to three different kinds of PBAs, LPS, PPD and DxS. The results in this communication show that B cells from young F, male mice respond poorly to PPD and fail to respond to DxS. Spleen cells from 12-month-old F, male mice could not respond to DxS either, although PPD responseof spleen cells from F, male mice was improved with age. The hypoor unresponsiveness could not be due to the defect in T cells or macrophages, or to the presence of suppressor T cells. MATERIALS
AND METHODS
Mice. C57BL/6J male mice, 9 weeksof age, were supplied by the animal breeding unit of our institute. CBA/N mice were kindly given by Dr. Nakano, Jichi Medical School, Tochigi, Japan, and used at 8 weeks of age. (CBA/N X BALB/c)F, mice were bred in our laboratory and used at 8-l 2 weeks of age, unless otherwise stated. Male and female F, mice were also used at 9 and 12 months of age. Mitogens. LPS was prepared from Salmonella minnesota R 595 according to the methods of Westphal by Dr. R. Wheat, Duke University, Durham, North Carolina. It was routinely used at 10 pg/culture, unless otherwise mentioned. PPD was obtained from Mitsui Seiyaku, Tokyo. Unless otherwise stated, it was used at 10 pg/culture. DxS (MW: 500,000) was purchased from Pharmacia, Uppsala, Sweden, and used at 5 hg/culture, unless otherwise specified. Anti-Thy 1.2. The anti-Thy 1.2 ascites was prepared in AKR mice as described previously (20). Treatment of spleen cells with anti-Thy 1.2 and complement. Spleen cell suspension (1 X 10’ cells/ml) was incubated for 45 min at 37°C with equal volumes of anti-Thy 1.2 and rabbit complement previously absorbed with mouse spleen cells, washed three times, and used for cultures. Ascites without antibody activity was prepared in a similar manner to anti-Thy 1.2. Depletion of adherent cells. Adherent cells were depleted from spleen cell populations by passing through a Sephadex G- 10 column as described previously (21). Phagocytic cells were detected by supravital staining with neutral red according to the methods of Arnaiz-Villena et al. (22). Preparation of peritoneal adherent cells. The peritoneal exudate cells were washed out from unstimulated mice by 10 ml of RPM1 1640 each mouse. Peritoneal washings were pooled and the cells were spun down and resuspended in the medium containing 10% fetal calf serum (FCS) (Associated Biomedic Systems, Buffalo, N.Y.) at a concentration of 2 X 10’ cells/ml. Three milliliters of the suspension was poured into a plastic dish (Falcon No. 3002) and incubated at 37°C for 2 hr. After nonadherent cells were removed by vigorous washing, adherent cells were recovered by the use of rubber policeman, washed, and used for cultures. Cell cultures. Spleen cells were cultured as described previously (23). In brief,
377
B-CELL SUBSETS RESPONSIVE TO DIFFERENT PBAs
I X lo6 cells were cultured in 0.2 ml of medium RPM1 1640 containing 20% FCS, 5 x lo-’ M 2-mercaptoethanol, and kanamycin (100 pg/ml) in a flat-bottom Microtest II plate (Falcon No. 3042) in a humidified atmosphere of 5% CO,. in air at 37°C for 3 days, unless otherwise mentioned. Plaque-forming cell (PFC) assay. Direct PFC response to TNP was assayed by using a modification of the hemolytic plaque assay of Jerne and Nordin (24). TNPconjugated sheep red blood cells were prepared according to the methods of Rittenberg and Pratt (25). RESULTS Responses of Spleen Cells from CBA/N Mice to LPS, PPD, and DxS
In the first experiments polyclonal responsesof spleen cells from CBA/N mice were compared with those of C57BL/6J spleen cells to different kinds of PBAs in terms of anti-TNP PFC response. Spleen cells from CBA/N or C57BL/6J mice were cultured with LPS ( 10 pg/culture) PPD ( 10 pglculture), or DxS (5 pg/ culture) for 3 days and assayed for anti-TNP PFC. The quantities of PBAs were determined by preliminary experiments to be the optimal stimulatory doses for C57BL/6J spleen cells to develop anti-TNP PFC. As shown in Table 1, spleen cells from C57BL/6J mice responded well to both LPS and PPD, and they also responded significantly to DxS, whereas the cells from CBA/N mice showed low response to PPD and they did not respond to DxS at all. In order to confirm these observations, spleen cells from male and female (CBA/N X BALB/c)F, mice were cultured with LPS, PPD, or DxS for 3 days. As shown in Table 2 spleen cells from F, male mice responded quite poorly to PPD and not to DxS. On the other hand, the cells from female responded well both to PPD and DxS. Thus spleen cells from mice with X-linked B-cell defect were shown to be hypoor unresponsive to our regular doses of PPD and DxS. It is possible, however, that F, male spleen cells may have different dose-response profiles from Fi female cells to these PBAs. Dose-Responses of Spleen Cells from Male and Female F, Mice to LPS, PPD. and DxS
For studying the dose-response profiles of male and female F, mouse spleen cells, the cells from these mice were cultured with different doses of LPS (0.01-100 TABLE 1 Difference between C57BL/6J and CBA/N Spleen Cells in Polyclonal Responses to LPS, PPD. and DxS Anti-TNP PFC 1- SD”/culture to Strain
No PBA
LPSb
PPD*
DxSb
C57BLJ6J CBA/N
85 f 18 42 + 16
1067 f 52 2100 f 261
1017 -t 163 325 f 50
293 k 25 472 8
’ Results are expressed as mean f SD of triplicate assays. b Doses of PBAs; LPS; IO pg, PPD; 10 pg. DxS; 5 fig/culture. Spleen cells from C57BL/6J or CBA/N mice were cultured for 3 days and assayed for anti-TNP PFC.
378
NARIUCHI
AND TABLE
Difference
KAKIUCHI 2
between Male and Female (CBA/N X BALB/c)F, Spleen Cells in Responses to Polyclonal Activators, LPS, PPD. and DxS Anti-TNP
PFC/culture”
Expt I
Expt 2
Activator
Male
Female
Male
Female
No LPS PPD DxS
IO 760 90 I3
53 I003 683 417
I3 740 43 I3
67 823 513 483
“Spleen cells from male or female F, mice were cultured with LPS (IO &culture), PPD (10 rg/ culture), or DxS (5 pg/culture) for 3 days and assayed for anti-TNP PFC, and the results are expressed as the mean of triplicate assays of the cells pooled from three culture wells.
pg/culture), PPD (0.5-50 ~g/culture), or DxS (0.1-100 pug/culture) for 3 days and assayed for anti-TNP PFC. The results are shown in Fig. 1. In the response to LPS stimulation, F, male spleen cells responded well and reached their peak response, 1445 + 144 PFC/culture, at 10 pg LPS/culture, although their responseswere apparently lower than those of F, female cells in all LPS doses. In PPD response, there was great difference between male and female F, spleen cells. Male cells responded poorly, when they were cultured with 10 pg
; 3
1200-
5
a t k
PPD BOO-
400 4
c
&.&-*.Y’
J
1200SW-
400 -
M’TOGEN
DOSE(pg/c”lt”rs)
FIG. I. Dose-response profile of LPS, PPD. or DxS response. Spleen cells from (CBA/N X BALB/ c)F, male (0) or female (0) mice were cultured with various doses of LPS, PPD, or DxS for 3 days and assayed for anti-TNP PFC. Each point represents the mean PFC, and vertical bar, standard deviation of triplicate assays.
B-CELL
SUBSETS
RESPONSIVE
TO
DIFFERENT
PBAs
379
PPD, 117 f 25 PFC/culture, or with 50 ~g PPD, 223 + 31 PFC/culture. On the other hand, female cells responded significantly even at a low dose as 0.5 pg of PPD, 313 f 70 PFC/culture, and the response was increased as dose of PPD almost to the comparable level to the LPS response, 1050 + 125 PFC/culture at 50 pg PPD/culture. The most conspicuous difference between male and female spleen cells was observed in the response to DxS. Female cells showed a characteristic dose-response profile. They responded well in a narrow range of the doses. They reached their peak response, 1230 f 200 PFC/culture, at 5 pg DxS/culture and the response was reduced dramatically with 10 pg or more DxS. In contrast to the high responsiveness of female cells, male cells did not respond to DxS at all in a wide range of doses from 0.1 to 100 pg per culture. These results indicate that hypoor unresponsiveness of F, male spleen cells to LPS, PPD, or DxS could not be explained by a shift of the dose-response curve. Kinetics of Anti-TNP PFC Responses to LPS, PPD, and DxS of Spleen Cells from Male or Female F, Mice
The hypo- or unresponsiveness of F, male cells could possibly be explained by a shift in time kinetics of the responding cells. To study the possibility, time courses of the responses of male and female cells to LPS (10 pg/culture), PPD (10 pg/ culture), and DxS (5 pg/culture) were examined from Day 1 to Day 4 after initiation of cultures. As shown in Fig. 2, the PFC responsesof F, male cells to LPS and PPD were significantly lower than those of female cells with the peak response occurring at Day 3, and the difference between male and female cells was much bigger in PPD response than in the LPS response throughout the culture period. In the DxS response, no significant response could be observed in F, male cell cultures at any day from Day 1 to Day 4, whereas female cells responded significantly from Day 2 to Day 4.
1600 _
E 5 G 2 P
1200.
800.
400.
Days
afler
culture
FIG. 2. Kinetics of anti-TNP PFC response to LPS, PPD, or DxS. Spleen cells from male or female (CBA/N X BALB/c)F, mice were cultured with LPS (IO&culture) (6, A; 9, A), PPD (10 &culture) d, 0; 9, W) or DxS (5 pglculture) (8, 0; 9, 0). or without PBA (X). Each point represents the mean PFC, and vertical bar, standard deviation of triplicate assays.
380
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AND
KAKlUCHl
Thus, hypo- or unresponsiveness of F, male cells to LPS, PPD, and DxS could not be explained by a shift of kinetics of the responding cells. Effect of Treatment with Anti-Thy sponses to PPD and DxS
1.2 and Complement on the Polyclonal Re-
Human B cells have recently been reported to require T-cell help for polyclonal response to PPD (26) and mitogenic response of murine B cells to DxS has been reported to be controlled by suppressor T cells (27). In the next experiments, therefore, the possibility of the participation of T cells in the hypo- or unresponsiveness of B cells to PPD or DxS was studied. Spleen cells from male or female F, mice were treated with anti-Thy 1.2 and complement to deplete T cells, cultured with PPD or DxS for 3 days and assayed for anti-TNP PFC. Experiments for PPD response and DxS response were carried out at separate occasions, and the results are shown in Table 3. The efficiency of T-cell depletion was monitored by mitogenic responsesof the cells before and after the treatment with anti-Thy 1.2. The PHA response of these cells was confirmed to be abrogated by the treatment, while they responded well to LPS. The anti-Thy 1.2 treatment could neither enhance the responsesof F, male cells nor suppressed those of F, female cells both to PPD and DxS stimulations. These results suggest that T cells do not play a critical role both in hyporesponsiveness of F, male cells to PPD and in their unresponsiveness to DxS. Effect of Adherent Cells on Polyclonal Responses to PPD and DxS Low responsivenessof F, male cells to PBAs could possibly be due to the defect of the function of accessory cells of these mice. Adherent cell dependency of polyclonal responses of F, spleen cells to PPD and DxS was studied in our culture TABLE Effect of the Treatment
3
with Anti-Thy 1.2 and Complement on Polyclonal of F, Spleen Cells to PPD and DxS Anti-TNP
PFC/culture” DxS
PPD Expt 2
Expt I Treatment
Anti-Thy
1.2 + Cb
Male
Female
(80)
(Z)
880 (100)
20 (17)
953 (73)
(ii)
(Z)
730 (83)
(E)
Male
Female
Male
110 (5)
860 (95)
107 (32)
413 (78)
(ii)
45 (5)
655 (65)
23 (3)
430
NMAtC (E)
605 (140)
(60) (128)
Female 760
657 (::)
Expt 2
Expt 1
Female
Male
Responses
913
(80) 827
(80)
” Spleen cells from male or female F, mice were cultured with PPD (10 erg/culture) or DxS (5 pg/ culture), or without PBA for 3 days and assayed for anti-TNP PFC. The results are expressed as the mean of triplicate assays of cells pooled from three culture wells. The number in parentheses represents the mean PFC of cultures which have not received any PBA. ’ Rabbit complement previously absorbed with mouse spleen cells.
B-CELL
SUBSETS
RESPONSIVE
TO DIFFERENT
PBAs
381
No of M + added(x10-31
FIG. 3. Macrophage dependency of PPD and DxS response. Spleen cells from (CBA/N X BALB/ c)F, female mice were passed through a Sephadex G-10 column to deplete adherent cells and cultured for 3 days with PPD (m) (IO bg/culture) or DxS (0) (5 pg/culture) in the presence of various numbers of PAC or in the absence of PAC. and they were assayed for anti-TNP PFC on Day 3. Untreated F, female spleen cells were also cultured for 3 days with PPD (8) or DxS (0).
system. Spleen cells from F, female were passedthrough a Sephadex G-10 column to deplete adherent cells, treated with NH,+21 to lyse red blood cells, washed, and cultured with PPD (10 pg/culture) or DxS (5 pg/culture) for 3 days. The cells were also reconstituted with various numbers of peritoneal adherent cells (PAC), 5-100 X lo3 PAC/culture, from F, female mice and cultured with PPD or DxS. The response of spleen cells to PPD was sometimes decreased slightly by the depletion of adherent cells (from 1097 + 110 to 770 + 66 PFC/culture), as shown in Fig. 3. The reduced PPD response, however, was not consistent finding and it could not be restored by the addition of adherent cells. On the other hand, DxS response was reduced by 80% by passing spleen cells through a Sephadex G- 10 column. The reduced response was restored by the addition of 10 x lo3 PAC and the response was suppressed again by 50-100 X lo3 PAC. In these experiments, depletion of adherent cells from spleen cell population was monitored by the antibody formation response to DNP-Ficoll, which is known to be macrophage dependent, and the response of F, female cells was diminished to 14% of that of untreated spleen cells by the passage through the column. The reduced anti-DNP response was fully restored by the addition of 30 X lo3 PAC. The above results indicate that polyclonal response to DxS is absolutely dependent upon the presence of adherent cells and that PPD response is adherent cell independent one or the dependency is quite low. In the next experiments, the effect of PAC from male or female F, mice on PPD and DxS responsesof female or male spleen cells was studied. Adherent cells from male or female mice were added to female or male spleen cells passed through a Sephadex G-10 column and these cells were cultured with PPD ( IO pg/culture) or DxS (5 pg/culture) for 3 days and assayed for anti-TNP PFC. The results of
382
NARIUCHI
AND KAKIUCHI TABLE 4
Effect of Adherent Cells from Male or Female (CBA/N X BALB/c)F, Mice on the Responsesof Female or Male Spleen Cells to PPD and DxS Anti-TNP PFC/culture” PPD Treatment of cells
Origin of adherent cellsb
G-IO passage G- 10 passage G- 10 passage
Female Male
DxS
Expt 1
Expt 2
Expt I
Expt 2
Male
Female
Male
Female
Male
Female
Male
Female
52 9
433 273 387 313
77
737
30
550
I 10
593 20 360 507
50
23 33 17
460 60 317 433
13 37
671 600
20
3 20 3
a Spleen cells from male or female F, mice were cultured with PPD (10 ag/culture) or DxS (10 rg/ culture) after they were passed through a Sephadex G-10 column with or without addition of PAC for 3 days and assayed for anti-TNP PFC. The results are expressed as the mean of triplicate assays of the cells pooled from three culture wells. *The numbers of PAC added for culture of PPD response and DxS response were 5 X IO3 and 1 X I 04, respectively.
these experiments are shown in Table 4. PAC from either male or female mice could not restore the low responses of male spleen cells to PPD and DxS, while male PAC did restore the reduced response as well as female PAC to DxS of female spleen cells depleted of adherent cells. Background PFC for untreated and adherent cell-depleted spleen cells from male F, mice were 7 and 3 per culture, respectively, and from female mice, 20 and 7, respectively, in Exp. 1. In Exp. 2, the background PFC of untreated and adherent cell-depleted spleen cells were 13 and 3 per culture, respectively, for male mice and 77 and 40, respectively, for female mice. Essentially no difference could be observed between male and female PAC in the accessory cell function on polyclonal response to DxS, when various numbers of PAC, 5-50 X 10’ PAC/culture, were added to male or female spleen cells depleted of adherent cells (data not shown). Thus adherent cells were indicated not to play a critical role in either hyporesponsivenessof F, male spleen cells to PPD or the unresponsiveness to DxS. Effect of Age on the Responses of F, Male Spleen Cells to LPS, PPD. and DxS
The defective responsesof B cells from CBA/N mice to PBAs have been reported to be due to a delay in the maturation of the cells and their responses could be improved with age (15, 16). In the next experiments, therefore, the effect of age on LPS, PPD, and DxS responsesof F, male spleen cells were studied. Spleen cells were obtained from 2-, 9-, or 12-month-old F, male mice and cultured with LPS (10 g/culture), PPD (10 g/culture), or DxS (5 g/culture) for 3 days. The antiTNP PFC responsesof these cells were compared with those of age-matched female spleen cells. The results are shown in Fig. 4. The effects of age on the responses of F, male cells to these three PBAs were quite different to each other. Spleen cells from 2-month-old male mice responded rather well to LPS and the response increased slightly with age. However, a great increase was obtained in the response
B-CELL SUBSETS RESPONSIVE TO DIFFERENT
2M OLD
9M OLD
PBAs
383
12M OLD
FIG. 4. Effect of age on the responsesof (CBA/N X BALB/c)F, male spleen cells to PBAs. Two-, 9-, and 12-month-old F, male (m) or female (I@ spleen cells were cultured with LPS (10 pg/culture), PPD (IO rglcuulture), DxS (5 ~g/culture), or without PBA for 3 days and assayed for anti-TNP PFC. Five mice were used for each group and the results are presented as the mean values, and vertical bars represent standard deviations.
of F, male cells to PPD with age. The response increased nearly IO-fold from a barely significant level in 2-month-old mice to the comparable level to 12-monthold female response. On the other hand, F, male spleen cells did not show a significant response to DxS, even when the cells from 12-month-old mice were used. These results could indicate that the cells responsive to LPS could appear first, followed by the cells responsive to PPD, and DxS responsive cells could appear last or they could not appear in X-linked B-cell-defective mice. DISCUSSION Since the discovery of B-cell defect in CBA/N mice, many attempts have been made to characterize the defect for studying the differentiation process of murine B cells. According to the responsivenessof these mice, TI antigens were subdivided into two groups, TI-1 and TI-2 (7-lo), and TI-1 antigens have been suggested to stimulate immature B cells resembling the B cells found in CBA/N mice (8). On the other hand, B cells in different stages of differentiation have been suggested to be stimulated by different kinds of B-cell mitogens. In the present study, we have shown that spleen cells from young (CBA/N X BALB/c)F, mice respond quite poorly to PPD and they do not respond to DxS. The hypo- or unresponsiveness could not be explained by a shift in dose-response or in time kinetics of the responding cells. Murine splenic T cells have been reported to be able to enhance the polyclonal antibody response of B cells to LPS (28) and the T cells from mice with X-linked B-cell defect did not enhance the response (29). Murine spleen cells, however, required no T-cell help for PPD or DxS responsein our experiments, DxS response of F, male spleen cells was not enhanced after they were treated with anti-Thy 1.2 and complement, indicating that their unresponsiveness to DxS could not be due to the presence of suppressor T cells, although mitogenic response of murine B cells to DxS has been reported to be controlled by suppressor T cells (27) and the response of F1 female cells was apt to be increased by the treatment with anti-Thy
384
NARIUCHIAND
KAKIUCHI
1.2 and complement. These results indicate that defect of young F, male spleen cells in PPD and DxS responsescould not be due to the T-cell function. The role of macrophages has recently been emphasized in many immunological phenomena. The polyclonal antibody response of murine spleen cells to PPD did not require the presence of macrophage in our culture system, and excess amount of PAC seemed to suppress the response. The DxS response required apparently the presence of macrophages. Macrophages from male and female young F, mice were equally effective in the restoration of reduced DxS response of Sephadex Glo-passed F, female cells, and F, female macrophages did not restore the response of Fr male cells. These results showed that macrophages have nothing to do with the unresponsiveness of F, male spleen cells to DxS. These results on the effects of T cells and macrophages on PPD and DxS responsesindicate that B cells from young F, male themselves are hypo- or unresponsive to these PBAs. The defect in the functions of B cells from CBA/N mice has been reported to be due to a slow maturation of the cells (15, 16) or to a deviation in B-cell differentiation at a certain stage of their development (30). The effect of age, therefore, on the PPD and DxS responses was studied. The PPD response of F, male cells increased dramatically with age and the cells from 12-month-old F, male mice showed the comparable response to those from age-matched F, female mice. F, male spleen cells, however, remained unresponsive to DxS, even when they were from 12-month-old mice. These results indicate that B cells responsive to DxS appear later than the cells responsive to PPD in F, male mice, or they could not develop in these mice. The apparent difference in the rate of maturation or the defect of differentiation may be a reflection of the different subpopulations of B cells that are responsive to PPD and DxS, or may suggest that the environment of F, male mice can affect the rate at which the maturation of B cells responsive to these PBAs occur. B cells responsive to DxS have been reported to appear earlier in ontogeny than the cells responsive to PPD (17) and they have also been shown to be less sensitive to removal of Ia-positive cells than the cells responsive to LPS (31), indicating that the cells responsive to DxS are less mature than the cells responsive to PPD or LPS. Thus the development of some immature B-cell population(s) responsive to DxS is considered to be more affected than the mature B cells responsive to PPD or LPS. In response to T-dependent and T-independent phosphorylcholin antigens, cells from CBA/N mice do not make IgM or IgG antibody (32) but do make IgE antibody (33). B cells producing IgE antibody were shown to be derived from B cells bearing surface IgM (34) and the treatment of new-born mice with anti-p antibody blocked the development of IgE response (35) indicating that IgE-producing cells would appear after IgM-producing cells. Taken together these and our findings, it is conceivable that the B-cell defect due to X-linked gene may not be lie solely in the delay of normal B-cell maturation during ontogeny but that different subpopulations of B cells are affected differently by the mutant gene product(s). It could be possible that there are several separated differentiation pathways for functionally specialized different B-cell subpopulations. In X-linked B-cell-defective mice, some of these pathways could remain undeveloped and some others could develop slower than in normal mice. These two apparently different processesof B-cell maturation could possibly be explained by the defect in a naturally occurring amplification process of B-cell clone. The idea might partly be supported by the lack of B-cell colony formation in vitro (35) and
B-CELL SUBSETS RESPONSIVE
TO DIFFERENT
PBAs
385
low background division in culture (15) and also by our findings that background PFC of F, male spleen cells were consistently lower than those of female even at 12-month-old mice (Fig. 4). The discovery of the site and action of the defect caused by X-linked gene would possibly throw light on an understanding of the processes involved in normal B-cell differentiation. ACKNOWLEDGMENTS The technical assistance of Miss Keiko Sakoda is gratefully acknowledged. This work was supported in part by grants from the Ministry of Education and the Ministry of Public Health and Welfare of Japan.
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Il. Mosier, D. E., Zitron, I. M., Mond, J. J., Ahmed, A., Scher, I., and Paul, W. E., Immunol. Rev. 37, 89, 1977. 12. Huber, B., and Melcher, F., Eur. J. Immunol. 9, 827, 1979. 13. Sieckman, D. G., Scher, I., Asofsky, R., Mosier, D. E., and Paul, W. E., J. Exp. Med. 148, 1628,
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26. 27.
28. 29.
30. 31. 32. 33.
34. 35.
1978. Goodman, M. G., Fidler, J. M., and Weigle, W. O., J. Immunol. 121, 1905, 1978. Whitlock, C. A., and Watson, J. D., J. Exp. Med. 150, 1483, 1979. Fidler, J. M., Morgan, E. L., and Weigle, W. O., J. Immunol. 124, 13, 1980. Gronowitz, E. A., Coutinho, A., and MBller, G., Stand. J. Immunol. 3, 413, 1974. Gronowitz, E., and Coutinho, A., &and. J. Immunol. 5, 55, 1976. Andersson, J., Lernhardt, and Melcher, F., J. Exp. Med. 150, 1339, 1979. Kakiuchi, T., Nariuchi, H., and Tamura, N., J. Immunol. 116, 1224, 1976. Ly, J. A., and Mishell, R. I., J. fmmunol. Methods 5, 239, 1974. Arneiz-Villena, A., GyiingySssy, M. I. C., and Playfair, J. H. L., Chin Exp. Immunol. 18, 177, 1974. Nariuchi, H., and Adler, W. H., Cell. Immunol. 45, 295, 1979. Jerne, N. K., and Nordin, A. A., Science 140, 405, 1963. Rittenberg, N. B., and Pratt, K. L., Proc. Sot. Exp. Biol. Med. 132, 575, 1969. Nishikawa, S., Hirata, T., and Izumi, T., J. Immunol. 123, 1106, 1979. Diamantslein, T., Willinger, E., and Reiman, J. Exp. Med. 150, 1571, 1979. Goodman, M. G., and Weigle, W. 0.. J. Immunol. 122, 2548, 1979. Goodman, M. G., and Weigle, W. O., J. Immunol. 123, 2484, 1979. Lindsten, T., and Andersson, B., J. Exp. Med. 150, 1285, 1979. Bick, P. H., and Shreffler, D. C., J. Immunol. 123, 2298, 1979. Quintans,J., and Kaplan, R. B., Cell. Immunol. 38, 294, 1978. Kishimoto, T., Shigemoto, S., Watanabe, T., and Yamamura, Y., J. Immunol. 123, 1039, 1979. Ishizaka, K., Ishizaka, T., Okudaira, H., and Bazin, H., J. Immunol. 120, 655, 1978. Manning, D. D., Manning, J. K., Reed, N. D., J. Exp. Med. 144, 288, 1976.
IMMUYOLOGY
61, 375-385 (1981)
Responses of Spleen Cells from Mice with X-linked B-Cell Defect to Polyclonal B-Cell Activators, Purified Protein Derivative of Tuberculin, and Dextran Sulfate H~DEO NARIUCHI Laboratory
AND TERUTAKA
KAKIUCHI
of Biological Products. The Institute of Medical Science, The University 4-6-l) Shiroganedai, Minatoku. Tokyo, 108 Japan Received December I I. 1980; accepted January
of Tokyo,
23, 1981
Polyclonal responsesto LPS, PPD, and DxS of spleen cells from mice expressing a X-linked B-cell defect were examined. Spleen cells from young (CBA/N X BALB/c)F, male mice responded slightly lower to LPS, significantly lower to PPD than the ceils from age-matched F, female mice, and showed no response to DxS stimulation. This hypo- or unresponsiveness of F, male cells to PPD or DxS could not be explained by a shift in the dose-responseor time kinetics of the responding cells, and also could not be due to the defect in the function of T cells or macrophages. Suppressor T cells to polyclonal response to PPD or DxS could not be shown in Fr male spleen cells. The responseof F, male cells to PPD was dramatically improved with age but not to DxS. These results suggest that B cells responsive to DxS may belong to a distinct subpopulation from the cells responsive to LPS or PPD.
INTRODUCTION The CBA/N strain of mouse carries a X-linked recessivedefect and the defective phenotype has been shown to be expressed in B cells (1, 2). The B cells in adult CBA/N mice have been considered to have defect in their maturation, since they have a high ratio of IgM to IgD on their surface (3) and express only low densities of B-cell differentiation markers, such as Ia, Mls, Lyb3, and Lyb5 (1,4-6). These defective B cells have the ability to respond to some but not to other thymusindependent (TI) antigens. The formers are classified now as TI-1 (trinitrophenyl [TNP]-lipopolysaccharide [LPS], TNP-Brucella abortus) (7, 8) and the latters TI-2 (TNP-Ficoll, SSS-III) (9, 10). TI-2 antigens have been suggestedto stimulate a more mature population of B cells than TI-1 antigens do (11). Polyclonal B-cell activators (PBAs) have recently been widely used as a tool for studying the mechanism of B-cell activation. Spleen cells from CBA/N mice have been reported to show low responses to PBAs (LPS, lipoprotein, Nocardia mitogen) (7, 12) or to fail to respond some other B-cell mitogens (anti-p, 2-mercaptoethanol) (13, 14). These defective functions of B cells in CBA/N mice in response to antigens and PBAs mentioned above have recently been reported to be due to a delay in the maturation of B cells and the functions were shown to increase with age (15, 16). The different PBAs have been suggested to stimulate B cells in different stages 375 OOOS-8749/81/100375-ll$O2.00/0 Copyright 0 1981 by Academac Press. Inc. All rights of reprcduct~on in any form reserved
376
NARIUCHI
AND
KAKIUCHI
of differentiation (17, 18). LPS has been shown to stimulate broad populations of B cells in an intermediate stage of differentiation. Lipoprotein and Nocardia mitogen seem to stimulate similar B-cell population to the cells responsive to LPS (12). Dextran sulfate (DxS) has been suggested to activate primitive B cells, whereas purified protein derivative of tuberculin (PPD) activate only mature B cells (17-19). In the experiments reported here, we have studied the responsivenessof spleen cells from (CBA/N X BALB/c)F, male mice to three different kinds of PBAs, LPS, PPD and DxS. The results in this communication show that B cells from young F, male mice respond poorly to PPD and fail to respond to DxS. Spleen cells from 12-month-old F, male mice could not respond to DxS either, although PPD responseof spleen cells from F, male mice was improved with age. The hypoor unresponsiveness could not be due to the defect in T cells or macrophages, or to the presence of suppressor T cells. MATERIALS
AND METHODS
Mice. C57BL/6J male mice, 9 weeksof age, were supplied by the animal breeding unit of our institute. CBA/N mice were kindly given by Dr. Nakano, Jichi Medical School, Tochigi, Japan, and used at 8 weeks of age. (CBA/N X BALB/c)F, mice were bred in our laboratory and used at 8-l 2 weeks of age, unless otherwise stated. Male and female F, mice were also used at 9 and 12 months of age. Mitogens. LPS was prepared from Salmonella minnesota R 595 according to the methods of Westphal by Dr. R. Wheat, Duke University, Durham, North Carolina. It was routinely used at 10 pg/culture, unless otherwise mentioned. PPD was obtained from Mitsui Seiyaku, Tokyo. Unless otherwise stated, it was used at 10 pg/culture. DxS (MW: 500,000) was purchased from Pharmacia, Uppsala, Sweden, and used at 5 hg/culture, unless otherwise specified. Anti-Thy 1.2. The anti-Thy 1.2 ascites was prepared in AKR mice as described previously (20). Treatment of spleen cells with anti-Thy 1.2 and complement. Spleen cell suspension (1 X 10’ cells/ml) was incubated for 45 min at 37°C with equal volumes of anti-Thy 1.2 and rabbit complement previously absorbed with mouse spleen cells, washed three times, and used for cultures. Ascites without antibody activity was prepared in a similar manner to anti-Thy 1.2. Depletion of adherent cells. Adherent cells were depleted from spleen cell populations by passing through a Sephadex G- 10 column as described previously (21). Phagocytic cells were detected by supravital staining with neutral red according to the methods of Arnaiz-Villena et al. (22). Preparation of peritoneal adherent cells. The peritoneal exudate cells were washed out from unstimulated mice by 10 ml of RPM1 1640 each mouse. Peritoneal washings were pooled and the cells were spun down and resuspended in the medium containing 10% fetal calf serum (FCS) (Associated Biomedic Systems, Buffalo, N.Y.) at a concentration of 2 X 10’ cells/ml. Three milliliters of the suspension was poured into a plastic dish (Falcon No. 3002) and incubated at 37°C for 2 hr. After nonadherent cells were removed by vigorous washing, adherent cells were recovered by the use of rubber policeman, washed, and used for cultures. Cell cultures. Spleen cells were cultured as described previously (23). In brief,
377
B-CELL SUBSETS RESPONSIVE TO DIFFERENT PBAs
I X lo6 cells were cultured in 0.2 ml of medium RPM1 1640 containing 20% FCS, 5 x lo-’ M 2-mercaptoethanol, and kanamycin (100 pg/ml) in a flat-bottom Microtest II plate (Falcon No. 3042) in a humidified atmosphere of 5% CO,. in air at 37°C for 3 days, unless otherwise mentioned. Plaque-forming cell (PFC) assay. Direct PFC response to TNP was assayed by using a modification of the hemolytic plaque assay of Jerne and Nordin (24). TNPconjugated sheep red blood cells were prepared according to the methods of Rittenberg and Pratt (25). RESULTS Responses of Spleen Cells from CBA/N Mice to LPS, PPD, and DxS
In the first experiments polyclonal responsesof spleen cells from CBA/N mice were compared with those of C57BL/6J spleen cells to different kinds of PBAs in terms of anti-TNP PFC response. Spleen cells from CBA/N or C57BL/6J mice were cultured with LPS ( 10 pg/culture) PPD ( 10 pglculture), or DxS (5 pg/ culture) for 3 days and assayed for anti-TNP PFC. The quantities of PBAs were determined by preliminary experiments to be the optimal stimulatory doses for C57BL/6J spleen cells to develop anti-TNP PFC. As shown in Table 1, spleen cells from C57BL/6J mice responded well to both LPS and PPD, and they also responded significantly to DxS, whereas the cells from CBA/N mice showed low response to PPD and they did not respond to DxS at all. In order to confirm these observations, spleen cells from male and female (CBA/N X BALB/c)F, mice were cultured with LPS, PPD, or DxS for 3 days. As shown in Table 2 spleen cells from F, male mice responded quite poorly to PPD and not to DxS. On the other hand, the cells from female responded well both to PPD and DxS. Thus spleen cells from mice with X-linked B-cell defect were shown to be hypoor unresponsive to our regular doses of PPD and DxS. It is possible, however, that F, male spleen cells may have different dose-response profiles from Fi female cells to these PBAs. Dose-Responses of Spleen Cells from Male and Female F, Mice to LPS, PPD. and DxS
For studying the dose-response profiles of male and female F, mouse spleen cells, the cells from these mice were cultured with different doses of LPS (0.01-100 TABLE 1 Difference between C57BL/6J and CBA/N Spleen Cells in Polyclonal Responses to LPS, PPD. and DxS Anti-TNP PFC 1- SD”/culture to Strain
No PBA
LPSb
PPD*
DxSb
C57BLJ6J CBA/N
85 f 18 42 + 16
1067 f 52 2100 f 261
1017 -t 163 325 f 50
293 k 25 472 8
’ Results are expressed as mean f SD of triplicate assays. b Doses of PBAs; LPS; IO pg, PPD; 10 pg. DxS; 5 fig/culture. Spleen cells from C57BL/6J or CBA/N mice were cultured for 3 days and assayed for anti-TNP PFC.
378
NARIUCHI
AND TABLE
Difference
KAKIUCHI 2
between Male and Female (CBA/N X BALB/c)F, Spleen Cells in Responses to Polyclonal Activators, LPS, PPD. and DxS Anti-TNP
PFC/culture”
Expt I
Expt 2
Activator
Male
Female
Male
Female
No LPS PPD DxS
IO 760 90 I3
53 I003 683 417
I3 740 43 I3
67 823 513 483
“Spleen cells from male or female F, mice were cultured with LPS (IO &culture), PPD (10 rg/ culture), or DxS (5 pg/culture) for 3 days and assayed for anti-TNP PFC, and the results are expressed as the mean of triplicate assays of the cells pooled from three culture wells.
pg/culture), PPD (0.5-50 ~g/culture), or DxS (0.1-100 pug/culture) for 3 days and assayed for anti-TNP PFC. The results are shown in Fig. 1. In the response to LPS stimulation, F, male spleen cells responded well and reached their peak response, 1445 + 144 PFC/culture, at 10 pg LPS/culture, although their responseswere apparently lower than those of F, female cells in all LPS doses. In PPD response, there was great difference between male and female F, spleen cells. Male cells responded poorly, when they were cultured with 10 pg
; 3
1200-
5
a t k
PPD BOO-
400 4
c
&.&-*.Y’
J
1200SW-
400 -
M’TOGEN
DOSE(pg/c”lt”rs)
FIG. I. Dose-response profile of LPS, PPD. or DxS response. Spleen cells from (CBA/N X BALB/ c)F, male (0) or female (0) mice were cultured with various doses of LPS, PPD, or DxS for 3 days and assayed for anti-TNP PFC. Each point represents the mean PFC, and vertical bar, standard deviation of triplicate assays.
B-CELL
SUBSETS
RESPONSIVE
TO
DIFFERENT
PBAs
379
PPD, 117 f 25 PFC/culture, or with 50 ~g PPD, 223 + 31 PFC/culture. On the other hand, female cells responded significantly even at a low dose as 0.5 pg of PPD, 313 f 70 PFC/culture, and the response was increased as dose of PPD almost to the comparable level to the LPS response, 1050 + 125 PFC/culture at 50 pg PPD/culture. The most conspicuous difference between male and female spleen cells was observed in the response to DxS. Female cells showed a characteristic dose-response profile. They responded well in a narrow range of the doses. They reached their peak response, 1230 f 200 PFC/culture, at 5 pg DxS/culture and the response was reduced dramatically with 10 pg or more DxS. In contrast to the high responsiveness of female cells, male cells did not respond to DxS at all in a wide range of doses from 0.1 to 100 pg per culture. These results indicate that hypoor unresponsiveness of F, male spleen cells to LPS, PPD, or DxS could not be explained by a shift of the dose-response curve. Kinetics of Anti-TNP PFC Responses to LPS, PPD, and DxS of Spleen Cells from Male or Female F, Mice
The hypo- or unresponsiveness of F, male cells could possibly be explained by a shift in time kinetics of the responding cells. To study the possibility, time courses of the responses of male and female cells to LPS (10 pg/culture), PPD (10 pg/ culture), and DxS (5 pg/culture) were examined from Day 1 to Day 4 after initiation of cultures. As shown in Fig. 2, the PFC responsesof F, male cells to LPS and PPD were significantly lower than those of female cells with the peak response occurring at Day 3, and the difference between male and female cells was much bigger in PPD response than in the LPS response throughout the culture period. In the DxS response, no significant response could be observed in F, male cell cultures at any day from Day 1 to Day 4, whereas female cells responded significantly from Day 2 to Day 4.
1600 _
E 5 G 2 P
1200.
800.
400.
Days
afler
culture
FIG. 2. Kinetics of anti-TNP PFC response to LPS, PPD, or DxS. Spleen cells from male or female (CBA/N X BALB/c)F, mice were cultured with LPS (IO&culture) (6, A; 9, A), PPD (10 &culture) d, 0; 9, W) or DxS (5 pglculture) (8, 0; 9, 0). or without PBA (X). Each point represents the mean PFC, and vertical bar, standard deviation of triplicate assays.
380
NARIUCHI
AND
KAKlUCHl
Thus, hypo- or unresponsiveness of F, male cells to LPS, PPD, and DxS could not be explained by a shift of kinetics of the responding cells. Effect of Treatment with Anti-Thy sponses to PPD and DxS
1.2 and Complement on the Polyclonal Re-
Human B cells have recently been reported to require T-cell help for polyclonal response to PPD (26) and mitogenic response of murine B cells to DxS has been reported to be controlled by suppressor T cells (27). In the next experiments, therefore, the possibility of the participation of T cells in the hypo- or unresponsiveness of B cells to PPD or DxS was studied. Spleen cells from male or female F, mice were treated with anti-Thy 1.2 and complement to deplete T cells, cultured with PPD or DxS for 3 days and assayed for anti-TNP PFC. Experiments for PPD response and DxS response were carried out at separate occasions, and the results are shown in Table 3. The efficiency of T-cell depletion was monitored by mitogenic responsesof the cells before and after the treatment with anti-Thy 1.2. The PHA response of these cells was confirmed to be abrogated by the treatment, while they responded well to LPS. The anti-Thy 1.2 treatment could neither enhance the responsesof F, male cells nor suppressed those of F, female cells both to PPD and DxS stimulations. These results suggest that T cells do not play a critical role both in hyporesponsiveness of F, male cells to PPD and in their unresponsiveness to DxS. Effect of Adherent Cells on Polyclonal Responses to PPD and DxS Low responsivenessof F, male cells to PBAs could possibly be due to the defect of the function of accessory cells of these mice. Adherent cell dependency of polyclonal responses of F, spleen cells to PPD and DxS was studied in our culture TABLE Effect of the Treatment
3
with Anti-Thy 1.2 and Complement on Polyclonal of F, Spleen Cells to PPD and DxS Anti-TNP
PFC/culture” DxS
PPD Expt 2
Expt I Treatment
Anti-Thy
1.2 + Cb
Male
Female
(80)
(Z)
880 (100)
20 (17)
953 (73)
(ii)
(Z)
730 (83)
(E)
Male
Female
Male
110 (5)
860 (95)
107 (32)
413 (78)
(ii)
45 (5)
655 (65)
23 (3)
430
NMAtC (E)
605 (140)
(60) (128)
Female 760
657 (::)
Expt 2
Expt 1
Female
Male
Responses
913
(80) 827
(80)
” Spleen cells from male or female F, mice were cultured with PPD (10 erg/culture) or DxS (5 pg/ culture), or without PBA for 3 days and assayed for anti-TNP PFC. The results are expressed as the mean of triplicate assays of cells pooled from three culture wells. The number in parentheses represents the mean PFC of cultures which have not received any PBA. ’ Rabbit complement previously absorbed with mouse spleen cells.
B-CELL
SUBSETS
RESPONSIVE
TO DIFFERENT
PBAs
381
No of M + added(x10-31
FIG. 3. Macrophage dependency of PPD and DxS response. Spleen cells from (CBA/N X BALB/ c)F, female mice were passed through a Sephadex G-10 column to deplete adherent cells and cultured for 3 days with PPD (m) (IO bg/culture) or DxS (0) (5 pg/culture) in the presence of various numbers of PAC or in the absence of PAC. and they were assayed for anti-TNP PFC on Day 3. Untreated F, female spleen cells were also cultured for 3 days with PPD (8) or DxS (0).
system. Spleen cells from F, female were passedthrough a Sephadex G-10 column to deplete adherent cells, treated with NH,+21 to lyse red blood cells, washed, and cultured with PPD (10 pg/culture) or DxS (5 pg/culture) for 3 days. The cells were also reconstituted with various numbers of peritoneal adherent cells (PAC), 5-100 X lo3 PAC/culture, from F, female mice and cultured with PPD or DxS. The response of spleen cells to PPD was sometimes decreased slightly by the depletion of adherent cells (from 1097 + 110 to 770 + 66 PFC/culture), as shown in Fig. 3. The reduced PPD response, however, was not consistent finding and it could not be restored by the addition of adherent cells. On the other hand, DxS response was reduced by 80% by passing spleen cells through a Sephadex G- 10 column. The reduced response was restored by the addition of 10 x lo3 PAC and the response was suppressed again by 50-100 X lo3 PAC. In these experiments, depletion of adherent cells from spleen cell population was monitored by the antibody formation response to DNP-Ficoll, which is known to be macrophage dependent, and the response of F, female cells was diminished to 14% of that of untreated spleen cells by the passage through the column. The reduced anti-DNP response was fully restored by the addition of 30 X lo3 PAC. The above results indicate that polyclonal response to DxS is absolutely dependent upon the presence of adherent cells and that PPD response is adherent cell independent one or the dependency is quite low. In the next experiments, the effect of PAC from male or female F, mice on PPD and DxS responsesof female or male spleen cells was studied. Adherent cells from male or female mice were added to female or male spleen cells passed through a Sephadex G-10 column and these cells were cultured with PPD ( IO pg/culture) or DxS (5 pg/culture) for 3 days and assayed for anti-TNP PFC. The results of
382
NARIUCHI
AND KAKIUCHI TABLE 4
Effect of Adherent Cells from Male or Female (CBA/N X BALB/c)F, Mice on the Responsesof Female or Male Spleen Cells to PPD and DxS Anti-TNP PFC/culture” PPD Treatment of cells
Origin of adherent cellsb
G-IO passage G- 10 passage G- 10 passage
Female Male
DxS
Expt 1
Expt 2
Expt I
Expt 2
Male
Female
Male
Female
Male
Female
Male
Female
52 9
433 273 387 313
77
737
30
550
I 10
593 20 360 507
50
23 33 17
460 60 317 433
13 37
671 600
20
3 20 3
a Spleen cells from male or female F, mice were cultured with PPD (10 ag/culture) or DxS (10 rg/ culture) after they were passed through a Sephadex G-10 column with or without addition of PAC for 3 days and assayed for anti-TNP PFC. The results are expressed as the mean of triplicate assays of the cells pooled from three culture wells. *The numbers of PAC added for culture of PPD response and DxS response were 5 X IO3 and 1 X I 04, respectively.
these experiments are shown in Table 4. PAC from either male or female mice could not restore the low responses of male spleen cells to PPD and DxS, while male PAC did restore the reduced response as well as female PAC to DxS of female spleen cells depleted of adherent cells. Background PFC for untreated and adherent cell-depleted spleen cells from male F, mice were 7 and 3 per culture, respectively, and from female mice, 20 and 7, respectively, in Exp. 1. In Exp. 2, the background PFC of untreated and adherent cell-depleted spleen cells were 13 and 3 per culture, respectively, for male mice and 77 and 40, respectively, for female mice. Essentially no difference could be observed between male and female PAC in the accessory cell function on polyclonal response to DxS, when various numbers of PAC, 5-50 X 10’ PAC/culture, were added to male or female spleen cells depleted of adherent cells (data not shown). Thus adherent cells were indicated not to play a critical role in either hyporesponsivenessof F, male spleen cells to PPD or the unresponsiveness to DxS. Effect of Age on the Responses of F, Male Spleen Cells to LPS, PPD. and DxS
The defective responsesof B cells from CBA/N mice to PBAs have been reported to be due to a delay in the maturation of the cells and their responses could be improved with age (15, 16). In the next experiments, therefore, the effect of age on LPS, PPD, and DxS responsesof F, male spleen cells were studied. Spleen cells were obtained from 2-, 9-, or 12-month-old F, male mice and cultured with LPS (10 g/culture), PPD (10 g/culture), or DxS (5 g/culture) for 3 days. The antiTNP PFC responsesof these cells were compared with those of age-matched female spleen cells. The results are shown in Fig. 4. The effects of age on the responses of F, male cells to these three PBAs were quite different to each other. Spleen cells from 2-month-old male mice responded rather well to LPS and the response increased slightly with age. However, a great increase was obtained in the response
B-CELL SUBSETS RESPONSIVE TO DIFFERENT
2M OLD
9M OLD
PBAs
383
12M OLD
FIG. 4. Effect of age on the responsesof (CBA/N X BALB/c)F, male spleen cells to PBAs. Two-, 9-, and 12-month-old F, male (m) or female (I@ spleen cells were cultured with LPS (10 pg/culture), PPD (IO rglcuulture), DxS (5 ~g/culture), or without PBA for 3 days and assayed for anti-TNP PFC. Five mice were used for each group and the results are presented as the mean values, and vertical bars represent standard deviations.
of F, male cells to PPD with age. The response increased nearly IO-fold from a barely significant level in 2-month-old mice to the comparable level to 12-monthold female response. On the other hand, F, male spleen cells did not show a significant response to DxS, even when the cells from 12-month-old mice were used. These results could indicate that the cells responsive to LPS could appear first, followed by the cells responsive to PPD, and DxS responsive cells could appear last or they could not appear in X-linked B-cell-defective mice. DISCUSSION Since the discovery of B-cell defect in CBA/N mice, many attempts have been made to characterize the defect for studying the differentiation process of murine B cells. According to the responsivenessof these mice, TI antigens were subdivided into two groups, TI-1 and TI-2 (7-lo), and TI-1 antigens have been suggested to stimulate immature B cells resembling the B cells found in CBA/N mice (8). On the other hand, B cells in different stages of differentiation have been suggested to be stimulated by different kinds of B-cell mitogens. In the present study, we have shown that spleen cells from young (CBA/N X BALB/c)F, mice respond quite poorly to PPD and they do not respond to DxS. The hypo- or unresponsiveness could not be explained by a shift in dose-response or in time kinetics of the responding cells. Murine splenic T cells have been reported to be able to enhance the polyclonal antibody response of B cells to LPS (28) and the T cells from mice with X-linked B-cell defect did not enhance the response (29). Murine spleen cells, however, required no T-cell help for PPD or DxS responsein our experiments, DxS response of F, male spleen cells was not enhanced after they were treated with anti-Thy 1.2 and complement, indicating that their unresponsiveness to DxS could not be due to the presence of suppressor T cells, although mitogenic response of murine B cells to DxS has been reported to be controlled by suppressor T cells (27) and the response of F1 female cells was apt to be increased by the treatment with anti-Thy
384
NARIUCHIAND
KAKIUCHI
1.2 and complement. These results indicate that defect of young F, male spleen cells in PPD and DxS responsescould not be due to the T-cell function. The role of macrophages has recently been emphasized in many immunological phenomena. The polyclonal antibody response of murine spleen cells to PPD did not require the presence of macrophage in our culture system, and excess amount of PAC seemed to suppress the response. The DxS response required apparently the presence of macrophages. Macrophages from male and female young F, mice were equally effective in the restoration of reduced DxS response of Sephadex Glo-passed F, female cells, and F, female macrophages did not restore the response of Fr male cells. These results showed that macrophages have nothing to do with the unresponsiveness of F, male spleen cells to DxS. These results on the effects of T cells and macrophages on PPD and DxS responsesindicate that B cells from young F, male themselves are hypo- or unresponsive to these PBAs. The defect in the functions of B cells from CBA/N mice has been reported to be due to a slow maturation of the cells (15, 16) or to a deviation in B-cell differentiation at a certain stage of their development (30). The effect of age, therefore, on the PPD and DxS responses was studied. The PPD response of F, male cells increased dramatically with age and the cells from 12-month-old F, male mice showed the comparable response to those from age-matched F, female mice. F, male spleen cells, however, remained unresponsive to DxS, even when they were from 12-month-old mice. These results indicate that B cells responsive to DxS appear later than the cells responsive to PPD in F, male mice, or they could not develop in these mice. The apparent difference in the rate of maturation or the defect of differentiation may be a reflection of the different subpopulations of B cells that are responsive to PPD and DxS, or may suggest that the environment of F, male mice can affect the rate at which the maturation of B cells responsive to these PBAs occur. B cells responsive to DxS have been reported to appear earlier in ontogeny than the cells responsive to PPD (17) and they have also been shown to be less sensitive to removal of Ia-positive cells than the cells responsive to LPS (31), indicating that the cells responsive to DxS are less mature than the cells responsive to PPD or LPS. Thus the development of some immature B-cell population(s) responsive to DxS is considered to be more affected than the mature B cells responsive to PPD or LPS. In response to T-dependent and T-independent phosphorylcholin antigens, cells from CBA/N mice do not make IgM or IgG antibody (32) but do make IgE antibody (33). B cells producing IgE antibody were shown to be derived from B cells bearing surface IgM (34) and the treatment of new-born mice with anti-p antibody blocked the development of IgE response (35) indicating that IgE-producing cells would appear after IgM-producing cells. Taken together these and our findings, it is conceivable that the B-cell defect due to X-linked gene may not be lie solely in the delay of normal B-cell maturation during ontogeny but that different subpopulations of B cells are affected differently by the mutant gene product(s). It could be possible that there are several separated differentiation pathways for functionally specialized different B-cell subpopulations. In X-linked B-cell-defective mice, some of these pathways could remain undeveloped and some others could develop slower than in normal mice. These two apparently different processesof B-cell maturation could possibly be explained by the defect in a naturally occurring amplification process of B-cell clone. The idea might partly be supported by the lack of B-cell colony formation in vitro (35) and
B-CELL SUBSETS RESPONSIVE
TO DIFFERENT
PBAs
385
low background division in culture (15) and also by our findings that background PFC of F, male spleen cells were consistently lower than those of female even at 12-month-old mice (Fig. 4). The discovery of the site and action of the defect caused by X-linked gene would possibly throw light on an understanding of the processes involved in normal B-cell differentiation. ACKNOWLEDGMENTS The technical assistance of Miss Keiko Sakoda is gratefully acknowledged. This work was supported in part by grants from the Ministry of Education and the Ministry of Public Health and Welfare of Japan.
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