- Email: [email protected]
W F1 mice
Im,um~,h~el Letter,. ~ 119841 97 100 Else~ let I inlet 480
LACK
OF SYNERGY
BETWEEN
T AND
2-MERCAPTOETHANOL
B CELLS
IN OLD
IN THE
NZB/W
RESPONSE
TO
F! MICE
Norikazu N A G A T A and Yoshihiro H A M A S H I M A Department ¢,1"Pathology. Fa~uhl or" Iledtcine. Kv~m~ L ntver~nt. .Sa~ vo-ku, l~loto 606. Japan {Received 28 Februar3. 1984~ (Modified ~ersion receixed 27 March 1984) (Accepted 28 March 19841
I. Summary The responses of NZB × NZW (NZB. W) F I mice to 2-mercaptoethanol (2-ME) were examined from the x iewpoint of T - B cell interaction. Young ( I-mthold) NZB. W F I mice responded to 2-ME in an almost similar pattern to that of BALB. c mice, although a slightly higher rate of D N A synthesis was obserxed in B cell-enriched cultures (75% B cells) containing 2-ME than in those of B A L B ; c mice. Old (9-ruth-old) N Z B / W F I mice showed an absent synergistic effect of Y and B cells in the response to 2-ME. These results indicate an abnormality of T - B cell interaction particularly in old N Z B / W Fj mice.
2. Introduction The B cells of autoimmune mice are reported to be in a state of polyclonal activation, and this abnormality is considered to be, at least, a partial cause of autoimmune disease [I,2]. In addition, many abnornml T cell functions ha~e also been reviewed [3,4]. Recently, B cell differentiation factor produced by M R L, M p-lpr; lpr mouse T cells was reported, suggesting a relationship between polyclonal B cell activation and abnormal T cell functions [5]. Mitogenic effects of 2-mercaptoethanol (2-ME) on spleen cells have been shown to be dependent on the cellular interaction o f T and B cells. Moreover, T Key *,ords" NZB W Fi mice teraction 0165 2478
84
2-mercaptoethanol
]--B cell in-
$3.00 ,D Elsevier Soence Pubhsher,, B.V.
cells have helper effects on polyclonal activation of B cells in the response to 2-ME [6]. The use of 2-ME is considered to provide a good model of T - B cell interaction. We have studied the cellular interaction between T and B ceils of NZB,' W F I mice, a strain of autoimmune mice, using 2-ME, and we discuss the significance of evaluating T-B cell interaction of autoimmune mice.
3. Materials and Methods
3. I. Mice and cell cuhure Female NZB × NZW (NZB,.'W) F I and B A L B c mice ~ere obtained from the breeding colonies of our animal facilities. Single-cell suspensions were obtained from spleens using a loosely fitting glass homogenizer. The spleen cells were treated with Tris-buffered ammonium chloride (pH 7.2) to eliminate erythrocyte contamination and washed twice. The culture medium used was R P M I 1640 (Grand Island Biological Co., Grand Island, NY) supplemented with 50 U,' ml penicillin, 50 #g., ml streptom)cin, 20 mM Hepes and 5c~ heat-inactivated fetal calf serum (Grand Island Biological Co.). 3.2. Preparation of T- or B-enriched populations Spleen cell populations enriched for T cells were prepared by passage o~er a nylon wool column by the method of Julius et al. [7]. B cell-enriched populations were prepared by treating spleen cells with monoclonal anti-Th.~ 1.2 antibody (Olac 1976 Ltd., Blackthorn, Bicester, U.K.) (clone F7D5) and rabbit 97
complement. Briefly, spleen cells I I ~ 10" cells ml) were incubated with anti-Thy 1.2 antibod} (1:250) at 4 ° C for I h. After washing twice, the cells ~ere treated ~ith rabbit complement (1:15) at 37 ~C for 30 rain. T cell-enriched populations were composed of 85 90c~- T cells, and B cell-enriched populations contained 0-IC~ T cells, determined from a c3totoxicity test using anti-Th,~ 1.2 antibod~ and complement. 3.3. ,lIea3ttrement o f D,V.4 srtTtheMs Separated spleen cells ~ere cultured in microculture plates (Nunc Products. Roskilde, Denmark) in x arious ratios of T and B cells at a densit.~ of 5 / 105 cells well in 0.2 ml culture medium ~ith or without 2-ME (Nakarai Chemicals, Ltd., Japan). Each test was performed in duplicate. Twelve hours before the termination of 72 h culture, each culture was labeled with 1.0 #Ci of [3H]thymidine and the cells were harvested by a multiple cell harvester (Flow Laboratories, Inc., McLean, VA). Incorporation of radioactivity was measured in a Beckman liquid scintillation counter.
4. Results
T- and B-enriched populations obtained from 2mth-old B A L B c mice s~ere cultured at xarious ratios in the presence or absence of 6 × I0 s M 2-ME. We used a mixture of spleen cells from 3 mice of each strain. As shown in Fig.. I, there ssas a s.~nergi~tic effect dependent on the ratios of T and B cells in the response to 2-ME. The maximum incorporauon of [3H]thymidine occurred in the cultures containing 75t~ T cells and 25c~-. B cells. This result is compatible ~ith the report of Goodman and Weigle [6]. Similar experiments ssere carried out using l-ruthold
T
T
7o 7" o
E
/
\,
/
7.,
lO
iN
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] +,'
\\r
j +
//'
5
,%-
\
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i
/
/
T
'1" • ,
i
i
i
i
i
i
|
T. en~ 10,-,
-~
6,"
.~B
,-'
B, ":
2 ~-
~,~
-~:.
,).~
T .:e,~
IOO
75.
fit],
25
O
B cen+
O
25
50
75
IO,2,
Fig. I S),nerg,', bep.,,een 1- a n d B cells Ln the response to 2 - M E of 2-ruth-old B A L B c mice T a n d B cell-enriched spleen cell populauons ,.~ere cultured in xar3ing, proportions for "7'2 h in the pre,ence I,D) or a b s e n c e IOl of 5 ," 10 "~ M 2 - M E . E x p e r i m e n t s ",,,ere d o n e m duplicate. Each '.alue represents the m e a n c o u n t
98
/
,5
0
m m +_ S D
\
/ / /
,-
1
r ]
1
/
+,
.g
,
,/'
/
%
.
h
'['
Fig,. 2 S} nerg`!, bet~,,een 1- and B cells m the response to 2 - M E ol I-ruth-old N Z B W F~ mice. T and B cell-enriched ~pleen cell populauon,, ',,.ere cultured m ,.ar.~ mg proportions for 72 h m the presence c.-',p or absence
IO o K E "2
g s
/
5
/:
"i"
=~ =2 E
1
0
i
I
L
i
-
,r, ~ii s 11,-11-i
7~
~,'i
-,~
i- l
'
B,:ens
25
5')
T5
I,D0
,'_"
Fig 3 Lack ol ,.~ nerg.~ belv, een r a n d B cells m the response (o 2-1ME o l 9 - m t h - o l d N Z B %~, F t m~ce T a n d B cell.-ennched ~,pleen cell populalton,, ~,~ere c u h u r e d m ',ar~,mg proporlion~ Ior ,"2 h m the presence ( C , ) o r a b ~ e n c e ( O l oi 5 . 10 s M 2 - M E . E ~ . p e n m e n l , ,.,.ere d o n e m d u p h c a t e Each ~alue represents the meancoun(
mm_+SD.
~ a s o b s e n e d ( Fig. 3). Although the m a x i m u m incorporation occurred at the ratio of 75Q T cells and 25% B cells in the presence of 2-ME, the level was much lower than that of BALB c and ~oung NZB W F~ mice, suggesting an absence of synergism between T and B cells,
5. Discussion Several recent reports as described belo~ suggest an important perspective in the analysis of autoimmune phenomena. Some factors produced b~ M R L Mp-lpr/lpr mouse T cells induce the differentiation of B cells [5]. A b n o r m a l T - B cell cooperation in a chronic graft-versus-host reaction induces a systemic lupus erythematosus-like disease [8]. AntissDNA antibody production induced by bacterial lipopolysaccharide can be enhanced by some T cell factors in normal mice [9]. All these findings indicate that it is ~ery important to assess the cellular collaboration bet~een T and B cells in autoimmune mice. Mitogenic effects of 2-ME are dependent on unstimulated-lymphocytes and T - B cell interaction, but not on macrophages [6,10]. By using a simple model
of T - B cell interaction, our present study has re~ealed that old NZB W F] mice shov, an abnormal pattern of synergy between T and B cells in the response to 2-ME. This abnormalit~ can be attributed to an abnormal T - B cell interaction and or the existence of an excess of activated lymphocytes [I.2]. However, the latter possibility is unlikely because old NZB W Fj mice did not show higher levels of [)H]th~midine uptake than those of young NZB W F i mice in the absence of 2-ME ( Figs. 2 and 3). The autologous (s.vngeneic) mixed I,vmphoc.~ te reaction ( A M L R ) has been reported to be absent or greath reduced in autoimmune strains ~ith age [I 1,12]. Although our experimental system resembles that of A M L R , there are some differences. Smith and Pasternak [I I] reported the poor A M L R of NZB mice even at a young age. However, in our present stud.~ young NZB W F i mice sho~ed a similar synergistic phenomenon in the response to 2M E. There are other critical points distinguishing them. The addition of polyethylene glycol (PEG) to cultures is essential to detect A M L R of mouse b m phoc~tes, and the stimulating cells appear to be macrophages and or la-bearing cells [12]. Hinderer, ~e did not add PEG to cultures, and it has been reported that macrophages are not related to the synergistic effect in the response to 2-ME [61. Therefore, the results of this study are not attributable to the loss of A M L R of old autoimmune mice. This study has sho~ n an abnormality of T - B cell interaction in old NZB W F i mice in the response to 2-ME, and it opens up the possibilit) of e~aluating the cellular interaction of T and B cells in autoimmune mice.
Acknowledgements This work was supported by grants from the Ministr.s of Health and Welfare and from the Ministry of Education, Science and Culture, Japan.
References [ l ] Cohen. P L. andZfff. M. l l g ~ ) J [2]
Immunol 119.
1534 153 "7 Izu[, S , M c C o n a h e ~ . P. J. a n d D i \ o n . F. J. I I'-):s).l Im-
munol 121.2213 2219.
99
[3] Steinberg.. A. D . Huston, D. P.. Taurog. J D., Co,.,.der3. J S, and Ra'..echi:. E S. ( 19811 m" Immunological Re',.ie'a-, IG Moiler. Ed ) tol. 55, pp. 121 154. Munksgaard. Copenhagen. [4] Theofilopoulo,.A. N and Di'~on, F . J . I l g g l i m Immunological Re'-.ie'.,,.~ IG. Moiler. Ed ) ",.ol. 55. pp 179 216. M unksgaard. Copenhagen. [5] Prud'homme. G J , Park, C. L.. Yleser. T M . koller. R.. Db:on. F. J. and Theol]h:,poulo-,. A N. 119,'t31J. E',.p Med 15",. ,"30 ",4.". [6] Goodman. M G. and V,,elgle, ~.%.O. 119~9t J [mmunc, I 122. 1433 1439.
100
['] [~] [L;] [10] [11} [12]
.luhu~. M . g m l p , , o n . E and Hertzenberg. [ N c l 9 " 3 1 E u r I. Immunol 3.6-15 049 (-,leichmann. E . Ellen. E H . , a n a n d ' ~ e e n . J. P \~. ;an d e r f l g X 2 1 E u r J Immunol 12. 152 159 Nagata. N . Nakm. '~ .. Sam~. H and Hama',hmla. '~ I Ig>,2~ ImmunoicJ~, -~, 3,'-,~ 390 Goc, dman. M C,. Fidler. J M and ~3,el~21e. \~ () 119~xl J lmmunol. I"1. I,~gg 191)4 %rmth. J. B. and Paqernak. R D I I g ~ x ) J IrnmunLq. I"1. h,,,x9 I,',92 (Muncher. L. H..'C, lemberg. ~ D.. Hou,e.% B and Green. I [19,xInJ Immun,q 125 I.,32 I,',3,',
LACK
OF SYNERGY
BETWEEN
T AND
2-MERCAPTOETHANOL
B CELLS
IN OLD
IN THE
NZB/W
RESPONSE
TO
F! MICE
Norikazu N A G A T A and Yoshihiro H A M A S H I M A Department ¢,1"Pathology. Fa~uhl or" Iledtcine. Kv~m~ L ntver~nt. .Sa~ vo-ku, l~loto 606. Japan {Received 28 Februar3. 1984~ (Modified ~ersion receixed 27 March 1984) (Accepted 28 March 19841
I. Summary The responses of NZB × NZW (NZB. W) F I mice to 2-mercaptoethanol (2-ME) were examined from the x iewpoint of T - B cell interaction. Young ( I-mthold) NZB. W F I mice responded to 2-ME in an almost similar pattern to that of BALB. c mice, although a slightly higher rate of D N A synthesis was obserxed in B cell-enriched cultures (75% B cells) containing 2-ME than in those of B A L B ; c mice. Old (9-ruth-old) N Z B / W F I mice showed an absent synergistic effect of Y and B cells in the response to 2-ME. These results indicate an abnormality of T - B cell interaction particularly in old N Z B / W Fj mice.
2. Introduction The B cells of autoimmune mice are reported to be in a state of polyclonal activation, and this abnormality is considered to be, at least, a partial cause of autoimmune disease [I,2]. In addition, many abnornml T cell functions ha~e also been reviewed [3,4]. Recently, B cell differentiation factor produced by M R L, M p-lpr; lpr mouse T cells was reported, suggesting a relationship between polyclonal B cell activation and abnormal T cell functions [5]. Mitogenic effects of 2-mercaptoethanol (2-ME) on spleen cells have been shown to be dependent on the cellular interaction o f T and B cells. Moreover, T Key *,ords" NZB W Fi mice teraction 0165 2478
84
2-mercaptoethanol
]--B cell in-
$3.00 ,D Elsevier Soence Pubhsher,, B.V.
cells have helper effects on polyclonal activation of B cells in the response to 2-ME [6]. The use of 2-ME is considered to provide a good model of T - B cell interaction. We have studied the cellular interaction between T and B ceils of NZB,' W F I mice, a strain of autoimmune mice, using 2-ME, and we discuss the significance of evaluating T-B cell interaction of autoimmune mice.
3. Materials and Methods
3. I. Mice and cell cuhure Female NZB × NZW (NZB,.'W) F I and B A L B c mice ~ere obtained from the breeding colonies of our animal facilities. Single-cell suspensions were obtained from spleens using a loosely fitting glass homogenizer. The spleen cells were treated with Tris-buffered ammonium chloride (pH 7.2) to eliminate erythrocyte contamination and washed twice. The culture medium used was R P M I 1640 (Grand Island Biological Co., Grand Island, NY) supplemented with 50 U,' ml penicillin, 50 #g., ml streptom)cin, 20 mM Hepes and 5c~ heat-inactivated fetal calf serum (Grand Island Biological Co.). 3.2. Preparation of T- or B-enriched populations Spleen cell populations enriched for T cells were prepared by passage o~er a nylon wool column by the method of Julius et al. [7]. B cell-enriched populations were prepared by treating spleen cells with monoclonal anti-Th.~ 1.2 antibody (Olac 1976 Ltd., Blackthorn, Bicester, U.K.) (clone F7D5) and rabbit 97
complement. Briefly, spleen cells I I ~ 10" cells ml) were incubated with anti-Thy 1.2 antibod} (1:250) at 4 ° C for I h. After washing twice, the cells ~ere treated ~ith rabbit complement (1:15) at 37 ~C for 30 rain. T cell-enriched populations were composed of 85 90c~- T cells, and B cell-enriched populations contained 0-IC~ T cells, determined from a c3totoxicity test using anti-Th,~ 1.2 antibod~ and complement. 3.3. ,lIea3ttrement o f D,V.4 srtTtheMs Separated spleen cells ~ere cultured in microculture plates (Nunc Products. Roskilde, Denmark) in x arious ratios of T and B cells at a densit.~ of 5 / 105 cells well in 0.2 ml culture medium ~ith or without 2-ME (Nakarai Chemicals, Ltd., Japan). Each test was performed in duplicate. Twelve hours before the termination of 72 h culture, each culture was labeled with 1.0 #Ci of [3H]thymidine and the cells were harvested by a multiple cell harvester (Flow Laboratories, Inc., McLean, VA). Incorporation of radioactivity was measured in a Beckman liquid scintillation counter.
4. Results
T- and B-enriched populations obtained from 2mth-old B A L B c mice s~ere cultured at xarious ratios in the presence or absence of 6 × I0 s M 2-ME. We used a mixture of spleen cells from 3 mice of each strain. As shown in Fig.. I, there ssas a s.~nergi~tic effect dependent on the ratios of T and B cells in the response to 2-ME. The maximum incorporauon of [3H]thymidine occurred in the cultures containing 75t~ T cells and 25c~-. B cells. This result is compatible ~ith the report of Goodman and Weigle [6]. Similar experiments ssere carried out using l-ruthold
T
T
7o 7" o
E
/
\,
/
7.,
lO
iN
/ 10
] +,'
\\r
j +
//'
5
,%-
\
'\I
i
/
/
T
'1" • ,
i
i
i
i
i
i
|
T. en~ 10,-,
-~
6,"
.~B
,-'
B, ":
2 ~-
~,~
-~:.
,).~
T .:e,~
IOO
75.
fit],
25
O
B cen+
O
25
50
75
IO,2,
Fig. I S),nerg,', bep.,,een 1- a n d B cells Ln the response to 2 - M E of 2-ruth-old B A L B c mice T a n d B cell-enriched spleen cell populauons ,.~ere cultured in xar3ing, proportions for "7'2 h in the pre,ence I,D) or a b s e n c e IOl of 5 ," 10 "~ M 2 - M E . E x p e r i m e n t s ",,,ere d o n e m duplicate. Each '.alue represents the m e a n c o u n t
98
/
,5
0
m m +_ S D
\
/ / /
,-
1
r ]
1
/
+,
.g
,
,/'
/
%
.
h
'['
Fig,. 2 S} nerg`!, bet~,,een 1- and B cells m the response to 2 - M E ol I-ruth-old N Z B W F~ mice. T and B cell-enriched ~pleen cell populauon,, ',,.ere cultured m ,.ar.~ mg proportions for 72 h m the presence c.-',p or absence
IO o K E "2
g s
/
5
/:
"i"
=~ =2 E
1
0
i
I
L
i
-
,r, ~ii s 11,-11-i
7~
~,'i
-,~
i- l
'
B,:ens
25
5')
T5
I,D0
,'_"
Fig 3 Lack ol ,.~ nerg.~ belv, een r a n d B cells m the response (o 2-1ME o l 9 - m t h - o l d N Z B %~, F t m~ce T a n d B cell.-ennched ~,pleen cell populalton,, ~,~ere c u h u r e d m ',ar~,mg proporlion~ Ior ,"2 h m the presence ( C , ) o r a b ~ e n c e ( O l oi 5 . 10 s M 2 - M E . E ~ . p e n m e n l , ,.,.ere d o n e m d u p h c a t e Each ~alue represents the meancoun(
mm_+SD.
~ a s o b s e n e d ( Fig. 3). Although the m a x i m u m incorporation occurred at the ratio of 75Q T cells and 25% B cells in the presence of 2-ME, the level was much lower than that of BALB c and ~oung NZB W F~ mice, suggesting an absence of synergism between T and B cells,
5. Discussion Several recent reports as described belo~ suggest an important perspective in the analysis of autoimmune phenomena. Some factors produced b~ M R L Mp-lpr/lpr mouse T cells induce the differentiation of B cells [5]. A b n o r m a l T - B cell cooperation in a chronic graft-versus-host reaction induces a systemic lupus erythematosus-like disease [8]. AntissDNA antibody production induced by bacterial lipopolysaccharide can be enhanced by some T cell factors in normal mice [9]. All these findings indicate that it is ~ery important to assess the cellular collaboration bet~een T and B cells in autoimmune mice. Mitogenic effects of 2-ME are dependent on unstimulated-lymphocytes and T - B cell interaction, but not on macrophages [6,10]. By using a simple model
of T - B cell interaction, our present study has re~ealed that old NZB W F] mice shov, an abnormal pattern of synergy between T and B cells in the response to 2-ME. This abnormalit~ can be attributed to an abnormal T - B cell interaction and or the existence of an excess of activated lymphocytes [I.2]. However, the latter possibility is unlikely because old NZB W Fj mice did not show higher levels of [)H]th~midine uptake than those of young NZB W F i mice in the absence of 2-ME ( Figs. 2 and 3). The autologous (s.vngeneic) mixed I,vmphoc.~ te reaction ( A M L R ) has been reported to be absent or greath reduced in autoimmune strains ~ith age [I 1,12]. Although our experimental system resembles that of A M L R , there are some differences. Smith and Pasternak [I I] reported the poor A M L R of NZB mice even at a young age. However, in our present stud.~ young NZB W F i mice sho~ed a similar synergistic phenomenon in the response to 2M E. There are other critical points distinguishing them. The addition of polyethylene glycol (PEG) to cultures is essential to detect A M L R of mouse b m phoc~tes, and the stimulating cells appear to be macrophages and or la-bearing cells [12]. Hinderer, ~e did not add PEG to cultures, and it has been reported that macrophages are not related to the synergistic effect in the response to 2-ME [61. Therefore, the results of this study are not attributable to the loss of A M L R of old autoimmune mice. This study has sho~ n an abnormality of T - B cell interaction in old NZB W F i mice in the response to 2-ME, and it opens up the possibilit) of e~aluating the cellular interaction of T and B cells in autoimmune mice.
Acknowledgements This work was supported by grants from the Ministr.s of Health and Welfare and from the Ministry of Education, Science and Culture, Japan.
References [ l ] Cohen. P L. andZfff. M. l l g ~ ) J [2]
Immunol 119.
1534 153 "7 Izu[, S , M c C o n a h e ~ . P. J. a n d D i \ o n . F. J. I I'-):s).l Im-
munol 121.2213 2219.
99
[3] Steinberg.. A. D . Huston, D. P.. Taurog. J D., Co,.,.der3. J S, and Ra'..echi:. E S. ( 19811 m" Immunological Re',.ie'a-, IG Moiler. Ed ) tol. 55, pp. 121 154. Munksgaard. Copenhagen. [4] Theofilopoulo,.A. N and Di'~on, F . J . I l g g l i m Immunological Re'-.ie'.,,.~ IG. Moiler. Ed ) ",.ol. 55. pp 179 216. M unksgaard. Copenhagen. [5] Prud'homme. G J , Park, C. L.. Yleser. T M . koller. R.. Db:on. F. J. and Theol]h:,poulo-,. A N. 119,'t31J. E',.p Med 15",. ,"30 ",4.". [6] Goodman. M G. and V,,elgle, ~.%.O. 119~9t J [mmunc, I 122. 1433 1439.
100
['] [~] [L;] [10] [11} [12]
.luhu~. M . g m l p , , o n . E and Hertzenberg. [ N c l 9 " 3 1 E u r I. Immunol 3.6-15 049 (-,leichmann. E . Ellen. E H . , a n a n d ' ~ e e n . J. P \~. ;an d e r f l g X 2 1 E u r J Immunol 12. 152 159 Nagata. N . Nakm. '~ .. Sam~. H and Hama',hmla. '~ I Ig>,2~ ImmunoicJ~, -~, 3,'-,~ 390 Goc, dman. M C,. Fidler. J M and ~3,el~21e. \~ () 119~xl J lmmunol. I"1. I,~gg 191)4 %rmth. J. B. and Paqernak. R D I I g ~ x ) J IrnmunLq. I"1. h,,,x9 I,',92 (Muncher. L. H..'C, lemberg. ~ D.. Hou,e.% B and Green. I [19,xInJ Immun,q 125 I.,32 I,',3,',