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The effects of stannous chloride on the humoral immune response of mice
Toxicology Letters, 21 (1984) 279-285
279
Elsevier TOXLett 1210
THE EFFECTS OF STANNOUS CHLORIDE IMMUNE RESPONSE OF MICE (Primary immune response; secondary hemolytic reaction; hemagglutination)
ON THE HUMORAL
immune response;
plaque-forming
cells;
OSAMU HAYASHI, MOMOKO CHIBA and MASAKAZU KIKUCHI Department
of Hygiene,
Juntendo
University School of Medicine,
I-I Hongo 2-chome,
Bunkyo-ku,
Tokyo 113 (Japan)
(Received October 1lth, 1983) (Revision received January 3rd, 1984) (Accepted January 3Oth, 1984)
SUMMARY The effects of stannous chloride (SnClz) on primary and secondary immune responses to sheep red blood cells (SRBCs) were examined in mice. 1.~. administration of 167 pmol/kg/day of SnC12 for 3 consecutive days suppressed the number of splenic indirect plaque-forming cells (PFCs) in the primary response and the hemagglutination (HA) and hemolytic reaction (HR) titers of non-2-mercaptoethanol(2-ME)-treated sera in both the primary and secondary immune response. However, tin affected neither the indirect PFC response nor the antibody titers of the 2-ME-treated sera after the secondary immunization, which was performed on day 6 following the primary immunization. These results suggest that the subacute administration of tin suppresses part of the immune response in which IgM antibody production plays an important role, and that the production of IgG in the secondary immune response is little affected, while the IgG production in the primary response is suppressed or delayed.
INTRODUCTION
In addition to the toxicity to the heme biosynthetic pathway and the CNS, lead (Pb) is toxic to the immune systems of man and animals. The immunotoxicity of Pb has been investigated and reviewed recently by Koller et al. [ 12,131. The toxicity of inorganic tin (SnClz), which belongs to the same chemical group (IVa) in the periodic chart as Pb, has, however, not been widely studied because the toxicity of tin is regarded as low. Tin (Sn) is widely used in the lining of cans for food preserva-
Abbreviations: ALAD, b-aminolevulinic acid dehydratase; CNS, central nervous system; EAC, erythrocyte-antibody-complement; HA, hemagglutination; HR, hemolytic reaction; 2-ME, 2-mercaptoethanol; PBS, phosphate-buffered saline; PFCs, plaque-forming cells; PHA, phytohemagglutinin; SRBCs, sheep red blood cells. 0378-4274/84/$
03.00 0 Elsevier Science Publishers B.V.
280
tion, stabilizing agents for plastics, etc. Thus, there is a possibility of exposure to Sn and its compounds in our daily life [9]. Chiba et al. [2,4] reported the effects of several metals on ALAD activity both in vitro and in vivo and found that both Sn, especially bivalent tin, and Pb significantly inhibited ALAD activity in rabbit blood. Furthermore, Seinen [19] has reviewed the immunotoxicity of alkyltin compounds. In contrast to organotin compounds, little work has been done on the toxic effects of Sn on immune responses [6]. The purpose of the present study was to examine the toxic effects of SnClz on the primary and secondary immune responses of mice to SRBCs. MATERIALS AND METHODS
Animals 240 male ddY mice (Nippon Bio-Supp. Center Co. Ltd., Japan), aged 6 weeks, were kept in a climatic chamber maintained at 25 + 1°C and 60& 5% relative humidity. Food pellets (CE-2, Nippon Clea Co. Ltd., Japan) and distilled water were available ad lib. After 1 week in the climatic chamber, the mice were divided into two groups. 120 mice (the Sn group) were given 167 pmol/kg/day of SnCl2 - 2HzO dissolved in deionized water (pH 2.6) i.p. for 3 consecutive days. The Sn solution was prepared just before injection. The remaining animals (the control group) were given a highly diluted (2-3 drops of HCl/lOO ml distilled water) hydrochloric acid solution (pH 2.0) i.p. for 3 consecutive days. These solutions were administered at a dose of 0.1 ml/10 g body weight. Immunization
and immunological procedures
One day after the last administration, all mice were injected i.p. with 0.2 ml of a 15% (v/v) suspension of SRBCs in saline per 10 g body weight as a primary immunization. The secondary immunization was performed on day 6 following the primary immunization. After each immunization, 10 mice from the Sn and 10 from the control group were killed and bled from the femoral artery to collect serum samples on appropriate days, i.e., days 3, 4, 5, 7, 9, 10, 11, and 13, to measure the primary and secondary immune responses. The spleen from each mouse was immediately removed, and the number of direct PFCs, which was regarded as the response of IgM antibody-producing cells, and that of indirect PFCs, which was regarded as the responses of both IgG and IgM anitbody-producing cells, were measured by a modification of Jerne’s plaque assay described by Cunningham and Szenberg [5] and the method of Sterzl et al. [20], respectively. To destroy IgM as described by Grubb and Swahn [9], 0.1 ml of the serum sample was treated with 2-ME in PBS as a 2-ME-treated serum sample, and the remaining 0.1 ml of the serum was treated with PBS only as a non-2-ME-treated sample. HA assay and HR
281
of the sera were performed with microtiter plates by the method of Jacobs and Lunde [ll] and Fujita et al. [7], respectively. Statistical analyses Statistical analyses of these results were performed
by Student’s t-test.
RESULTS
Changes of body and spleen weights after administration
of tin
The body weight of the Sn group was significantly reduced for the first 4 days after the cessation of the Sn injection when compared with the control group. After that, the body weight gain of the Sn group was almost equal to that of the control group, and no significant difference was observed between them on day 5 (not shown). The total mortality after injection of the Sn solution was 6.7%. The spleen weight of the Sn group increased up to 1.6 times that of the control group (Fig. 1). A significant increase of the spleen weight/g body weight and that of the number of spleen cells per spleen was also observed in the Sn group starting on the fifth day after the final administration of Sn. The effects of Sn on the primary and the secondary immune responses As shown in Fig. 2, administration of 167 pmol/kg/day of Sn for 3 consecutive days tended to depress the number of direct PFCs in both the primary and seconSpleen weight (mg) 300
-
200
-
100
x!qy
i
5 6AdB
101112
14
20
SRBC
SRBC
Days after the last dose of tin Fig.
1. Spleen weight
Sn group;
0,
control
of the experimental group;
mice. Each value represents
*P < 0.05;**P -z0.01.
the mean
f
SE of 10 mice.
0,
Indirect PFC/spleen
282
(B)
(x104) 80.0 1
Direct PFCfspleen
I
(A)
,o.o_~ ~ __
(X104) 20.0.
o,:+
.
345 1st SRBC
-* ’ 2nd7
9 10 11
13
19
19 1st SRBC
SRBC
Days after primary
2nd SRBC Oays after primary
immunization
inwnization
Fig. 2. Effects of Sn on the number of PFCs in the primary and the secondary immune responses. (A) Number of direct PFCs; (B) number of indirect PFCs. Sn was injected for three consecutive days followed by primary immunization, Each value represents the mean f SE of 10 mice. Symbols as in Fig. 1. HA titer of non-Z-ME-treated
(2”)
HA titer of 2-ME-treated
(A)
sera
* * kB-:-;-.l
(B)
(2"1
6.OL
4.0
sera
-
**
_ 2.0
*t
~ 19 1st 2nd SRBC SRBC HR titer of non-Z-ME-treated
1st SRBC
sera
(C)
’ “’ 9
0
2nd SRBC
HR titer of Z-ME-treated
1011
-
19
13
(D)
sera
'b'."d
4.0
.
,(.... .4----........_.....(.(.. i’: ,,;
2.0
.
:f,j ;:
1st SRBC immunization
.............(/“” a **
r-
0
Days after primary
P..... .......~
?nd SRBC
9 10 11
Days after primary
13
19
inmwniration
Fig. 3. Effects of Sn on the HA and HR titers in the primary and the secondary immune responses. (A) HA titer of non-2.ME-treated sera; (B) HA titer of Z-ME-treated sera; (C) HR titer of non-2-ME-tr~ted sera; (D) HR titer of 2-ME-treated sera. Sn was injected for three consecutive days followed by primary immunization. Each value represents the mean zt SE of 10 mice. Symbols as in Fig. 1.
283
dary immune responses (Fig. 2A) and significantly suppressed that of indirect PFCs on days 4 and 5 after the primary immunization (Fig. 2B). The administration of Sn also suppressed the HA and HR titers of the non-2-ME-treated sera significantly on days 4 (P < 0.01 for HA and HR) and 5 (P < 0.05 for HA) after the primary immunization (Fig. 3, A and C). The number of indirect PFCs in the secondary imSn also suppressed the HA and HR titers of the non-2-ME-treated sera significantly on days 4 (P < 0.01 for HA and HR) and 5 (P c 0.05 for HA) after the primary immunization (Fig. 3, A and C). The number of indirect PFCs in the secondary immune response, however, was not affected (Fig. 2B). The antibody titers of the 2-ME-treated sera were not affected in either the primary or secondary immune response, as shown in Fig. 3, B and D, except for the suppression of the HR titer on day 13 (Fig. 3D), although those of the non-2-ME-treated ones were suppressed on days 10 (P < 0.05 for HA), 11 (P < 0.05 for HR), and 13 (P < 0.05 for HA and P c 0.01 for HR) in the secondary response (Fig. 3, A and C). DISCUSSION
Administration of 167 pmol/kg/day of Sn for 3 consecutive days suppressed the circulating antibody titers in the primary immune response. Reduction of the number of direct PFCs (Fig. 2A) and/or decrease of antibody titer of the non-2-MEtreated sera (Fig. 3, A and C) was observed on days 10 and 13 in the secondary immune response. These phenomena might be due to the reduction of IgM antibody production. On the other hand, the number of indirect PFCs was suppressed in the primary immune response (Fig. 2B). Neither the number of indirect PFCs (Fig. 2B) nor the HA antibody titers of the 2-ME-treated sera (Fig. 3B) in the secondary immune response, however, were much affected. These results suggest that subacute administration of Sn suppresses part of the immune response in which IgM antibody production plays an important role and that the production of IgG in the secondary immune response is little affected, while that in the primary response is suppressed or delayed. Immunological memory for IgM is presumably suppressed by Sn administration, but that for IgG is so slightly affected that the IgG antibody response of the Sn group following secondary immunization was not markedly different from that of the control. This is in contrast to Pb, which has been shown by other workers to reduce IgG antibody in the secondary immune response of mice or rats [15,18]. Pb at a large dosage (1300 ppm) in the drinking water for 10 weeks impaired the memory response after the inoculation of mice with SRBCs [16], and exposure to high Pb concentrations (2000 ppm) for 30 days significantly decreased the responses of splenic lymphocytes to the mitogen, PHA, in mice [8]. Dimitrov et al. [6] reported that a single i.p. injection of 200 fig of SnClz tended to suppress or barely affect the number of IgM-PFCs and slightly increase that of IgG-PFCs and rosette formation after primary immunization in mice. This is different from the findings in the present study but a lower level of SnC12, as opposed
to a higher one, probably activates the immune system in mice. Heils [lo] reported that radioactive tin was observed to some extent in the spleen 48 h after a single i.v. injection of ‘r3SnC12 in rats. Chiba et al. [3] reported that a relatively high deposition of Sn was found in the spleen of rabbits, with the maximum concentration being observed 5 days after injection of 5 pmol Sri/kg/day for 6 consecutive days. Although there might be a difference in the effects of Sn on immune response between mice and other animals, accumulation of Sn in the spleen may suppress or delay antibody production. A transient depression of HR titers of the sera of the Sn group was observed on day 13 in the secondary response (Fig. 3, C and D), regardless of 2-ME treatment of the sera. This phenomenon cannot be satisfactorily explained at present but there is some evidence that metals such as Pb, Cd and Hg suppress antibody titers to some antigens, viruses, SRBC or y-globulin [12,17,21]. Koller [12,14] suggested that the direct effect of Pb or Cd on B cells could account in part for the decrease of EAC rosettes formed by splenic B-lymphocytes from mice exposed to the metal in contrast to those from controls. Our unpublished data indicated that 0.5 mM SnC12 decreased the HR titer to SRBCs in vitro to 90%, and 2.5 mM SnC12 to 10%. From these facts, it is supposed that Sn might directly suppress the antibody titer of hemolysis or inhibit the action of complement on erythrocyte-antibody complex. But there is no evidence for a transient increase of Sn concentration in the blood on about the 13th day after its administration. Another notable phenomenon, hypertrophy of the spleen, was observed in the mice administered Sn (Fig. 1). It had been reported that pathological change, a bluish-grey appearance, was seen in the spleen of rabbits exposed to tin compounds, stannous tartrate or stannous citrate, and that this might be due to the deposition of the injected Sn in the reticuloendothelial cells [l]. REFERENCES 1 J.M. Barnes and H.B. Stoner, The toxicology of tin compounds, Pharmacol. Rev., 11 (1959) 211. 2 M. Chiba and M. Kikuchi, Effect of tin compounds on activity of S-aminolevulinate dehydratase in blood, Biochem. Biophys. Res. Commun., 82 (1978) 1057. 3 M. Chiba, K. Ogihara, Y. Inaba, T. Nishima and M. Kikuchi, The organ distribution of tin and the effect of tin on concentrations of several essential elements in rabbit, Toxicology (1983) in press. 4 M. Chiba, K. Ogihara and M. Kikuchi, In vitro effects of tin, lead, and mercury on the activity of 5aminolevulinate hydrolyase in erythrocytes (in Japanese), Sangyo-Igaku, 21 (1979) 182. 5 A.J. Cunningham and S. Szenberg, Further improvements on the plaque technique for detecting single antibody-forming cells, Immunology, 14 (1968) 599. 6 N.V. Dimitrov, C. Meyer, F. Nahhas, C. Miller and B.A. Averil, Effect of tin on immune response of mice, Clin. Immunol. Immunopathol., 20 (1981) 39. 7 K. Fujita, K. Yamashita and M. Kikuchi, The influence of thermal environment on the immune response of mice, 1. Cellular and humoral effects of low ambient temperature, Jap. J. Hyg., 29 (1974) 491. 8 C.L. Gaworski and R.P. Sharma, The effects of heavy metals on [‘Hlthymidine uptake in lymphocytes, Toxicol. Appl. Pharmacol., 46 (1978) 305.
285 9 R. Grubb and B. Swahn, Destruction of some agglutinins but not of others by two sulfhydryl compounds, Acta Pathol. Microbial. Stand., 43 (1958) 305. 10 R.A. Hiles, Absorption, distribution and excretion of inorganic tin in rats, Toxicol. Appl. Pharmacol., 27 (1974) 366. 11 L. Jacobs and M.N. Lunde, A hemagglutination test for toxoplasmosis, J. Parasitol., 43 (1957) 308. 12 L.D. Koller, Immunotoxicology of heavy metals, Int. J. Immunopharmacol., 2 (1980) 269. 13 L.D. Koller, In vitro assessment of humoral immunity following exposure to heavy metals, Environ. Health Perspect., 43 (1982) 37. 14 L.D. Koller and J.A. Brauner, Decreased B-lymphocyte response after exposure to lead and cadmium, Toxicol. Appl. Pharmacol., 42 (1977) 621. 15 L.D. Koller and S. Kovacic, Decreased antibody formation in mice exposed to lead, Nature, 250 (1974) 148. 16 L.D. Koller, J.A. Brauner and J.H. Exon, Immune response in aged mice exposed to lead, J. Toxicol. Env. Health, 3 (1977) 535. 17 L.D. Koller and J.G. Vos, Immunologic effects of metals, Immunol. Consid. Toxicol., 1 (1981) 67. 18 L.I. Luster, R.E. Faith and C.A. Kimmel, Depression of humoral immunity in rats following chronic developmental lead exposure, J. Environ. Pathol. Toxicol., 1 (1978) 397. 19 W. Seinen, Immunotoxicity of alkyltin compounds, Immunol. Consid. Toxicol., 1 (1981) 103. 20 J. Sterzl, I. Riha, D.W. Dressor and H.H. Wortis, A localized haemolysis in gel method for the detection of cells producing 7s antibody, Nature, 208 (1965) 858. 21 H.W. Williams, W.T. Caraway and W.A. de Young, Inactivation of antibodies. A causative factor of brain pathology in acute lead intoxication, Arch. Neurol. Psychiat., 72 (1954) 579.
279
Elsevier TOXLett 1210
THE EFFECTS OF STANNOUS CHLORIDE IMMUNE RESPONSE OF MICE (Primary immune response; secondary hemolytic reaction; hemagglutination)
ON THE HUMORAL
immune response;
plaque-forming
cells;
OSAMU HAYASHI, MOMOKO CHIBA and MASAKAZU KIKUCHI Department
of Hygiene,
Juntendo
University School of Medicine,
I-I Hongo 2-chome,
Bunkyo-ku,
Tokyo 113 (Japan)
(Received October 1lth, 1983) (Revision received January 3rd, 1984) (Accepted January 3Oth, 1984)
SUMMARY The effects of stannous chloride (SnClz) on primary and secondary immune responses to sheep red blood cells (SRBCs) were examined in mice. 1.~. administration of 167 pmol/kg/day of SnC12 for 3 consecutive days suppressed the number of splenic indirect plaque-forming cells (PFCs) in the primary response and the hemagglutination (HA) and hemolytic reaction (HR) titers of non-2-mercaptoethanol(2-ME)-treated sera in both the primary and secondary immune response. However, tin affected neither the indirect PFC response nor the antibody titers of the 2-ME-treated sera after the secondary immunization, which was performed on day 6 following the primary immunization. These results suggest that the subacute administration of tin suppresses part of the immune response in which IgM antibody production plays an important role, and that the production of IgG in the secondary immune response is little affected, while the IgG production in the primary response is suppressed or delayed.
INTRODUCTION
In addition to the toxicity to the heme biosynthetic pathway and the CNS, lead (Pb) is toxic to the immune systems of man and animals. The immunotoxicity of Pb has been investigated and reviewed recently by Koller et al. [ 12,131. The toxicity of inorganic tin (SnClz), which belongs to the same chemical group (IVa) in the periodic chart as Pb, has, however, not been widely studied because the toxicity of tin is regarded as low. Tin (Sn) is widely used in the lining of cans for food preserva-
Abbreviations: ALAD, b-aminolevulinic acid dehydratase; CNS, central nervous system; EAC, erythrocyte-antibody-complement; HA, hemagglutination; HR, hemolytic reaction; 2-ME, 2-mercaptoethanol; PBS, phosphate-buffered saline; PFCs, plaque-forming cells; PHA, phytohemagglutinin; SRBCs, sheep red blood cells. 0378-4274/84/$
03.00 0 Elsevier Science Publishers B.V.
280
tion, stabilizing agents for plastics, etc. Thus, there is a possibility of exposure to Sn and its compounds in our daily life [9]. Chiba et al. [2,4] reported the effects of several metals on ALAD activity both in vitro and in vivo and found that both Sn, especially bivalent tin, and Pb significantly inhibited ALAD activity in rabbit blood. Furthermore, Seinen [19] has reviewed the immunotoxicity of alkyltin compounds. In contrast to organotin compounds, little work has been done on the toxic effects of Sn on immune responses [6]. The purpose of the present study was to examine the toxic effects of SnClz on the primary and secondary immune responses of mice to SRBCs. MATERIALS AND METHODS
Animals 240 male ddY mice (Nippon Bio-Supp. Center Co. Ltd., Japan), aged 6 weeks, were kept in a climatic chamber maintained at 25 + 1°C and 60& 5% relative humidity. Food pellets (CE-2, Nippon Clea Co. Ltd., Japan) and distilled water were available ad lib. After 1 week in the climatic chamber, the mice were divided into two groups. 120 mice (the Sn group) were given 167 pmol/kg/day of SnCl2 - 2HzO dissolved in deionized water (pH 2.6) i.p. for 3 consecutive days. The Sn solution was prepared just before injection. The remaining animals (the control group) were given a highly diluted (2-3 drops of HCl/lOO ml distilled water) hydrochloric acid solution (pH 2.0) i.p. for 3 consecutive days. These solutions were administered at a dose of 0.1 ml/10 g body weight. Immunization
and immunological procedures
One day after the last administration, all mice were injected i.p. with 0.2 ml of a 15% (v/v) suspension of SRBCs in saline per 10 g body weight as a primary immunization. The secondary immunization was performed on day 6 following the primary immunization. After each immunization, 10 mice from the Sn and 10 from the control group were killed and bled from the femoral artery to collect serum samples on appropriate days, i.e., days 3, 4, 5, 7, 9, 10, 11, and 13, to measure the primary and secondary immune responses. The spleen from each mouse was immediately removed, and the number of direct PFCs, which was regarded as the response of IgM antibody-producing cells, and that of indirect PFCs, which was regarded as the responses of both IgG and IgM anitbody-producing cells, were measured by a modification of Jerne’s plaque assay described by Cunningham and Szenberg [5] and the method of Sterzl et al. [20], respectively. To destroy IgM as described by Grubb and Swahn [9], 0.1 ml of the serum sample was treated with 2-ME in PBS as a 2-ME-treated serum sample, and the remaining 0.1 ml of the serum was treated with PBS only as a non-2-ME-treated sample. HA assay and HR
281
of the sera were performed with microtiter plates by the method of Jacobs and Lunde [ll] and Fujita et al. [7], respectively. Statistical analyses Statistical analyses of these results were performed
by Student’s t-test.
RESULTS
Changes of body and spleen weights after administration
of tin
The body weight of the Sn group was significantly reduced for the first 4 days after the cessation of the Sn injection when compared with the control group. After that, the body weight gain of the Sn group was almost equal to that of the control group, and no significant difference was observed between them on day 5 (not shown). The total mortality after injection of the Sn solution was 6.7%. The spleen weight of the Sn group increased up to 1.6 times that of the control group (Fig. 1). A significant increase of the spleen weight/g body weight and that of the number of spleen cells per spleen was also observed in the Sn group starting on the fifth day after the final administration of Sn. The effects of Sn on the primary and the secondary immune responses As shown in Fig. 2, administration of 167 pmol/kg/day of Sn for 3 consecutive days tended to depress the number of direct PFCs in both the primary and seconSpleen weight (mg) 300
-
200
-
100
x!qy
i
5 6AdB
101112
14
20
SRBC
SRBC
Days after the last dose of tin Fig.
1. Spleen weight
Sn group;
0,
control
of the experimental group;
mice. Each value represents
*P < 0.05;**P -z0.01.
the mean
f
SE of 10 mice.
0,
Indirect PFC/spleen
282
(B)
(x104) 80.0 1
Direct PFCfspleen
I
(A)
,o.o_~ ~ __
(X104) 20.0.
o,:+
.
345 1st SRBC
-* ’ 2nd7
9 10 11
13
19
19 1st SRBC
SRBC
Days after primary
2nd SRBC Oays after primary
immunization
inwnization
Fig. 2. Effects of Sn on the number of PFCs in the primary and the secondary immune responses. (A) Number of direct PFCs; (B) number of indirect PFCs. Sn was injected for three consecutive days followed by primary immunization, Each value represents the mean f SE of 10 mice. Symbols as in Fig. 1. HA titer of non-Z-ME-treated
(2”)
HA titer of 2-ME-treated
(A)
sera
* * kB-:-;-.l
(B)
(2"1
6.OL
4.0
sera
-
**
_ 2.0
*t
~ 19 1st 2nd SRBC SRBC HR titer of non-Z-ME-treated
1st SRBC
sera
(C)
’ “’ 9
0
2nd SRBC
HR titer of Z-ME-treated
1011
-
19
13
(D)
sera
'b'."d
4.0
.
,(.... .4----........_.....(.(.. i’: ,,;
2.0
.
:f,j ;:
1st SRBC immunization
.............(/“” a **
r-
0
Days after primary
P..... .......~
?nd SRBC
9 10 11
Days after primary
13
19
inmwniration
Fig. 3. Effects of Sn on the HA and HR titers in the primary and the secondary immune responses. (A) HA titer of non-2.ME-treated sera; (B) HA titer of Z-ME-treated sera; (C) HR titer of non-2-ME-tr~ted sera; (D) HR titer of 2-ME-treated sera. Sn was injected for three consecutive days followed by primary immunization. Each value represents the mean zt SE of 10 mice. Symbols as in Fig. 1.
283
dary immune responses (Fig. 2A) and significantly suppressed that of indirect PFCs on days 4 and 5 after the primary immunization (Fig. 2B). The administration of Sn also suppressed the HA and HR titers of the non-2-ME-treated sera significantly on days 4 (P < 0.01 for HA and HR) and 5 (P < 0.05 for HA) after the primary immunization (Fig. 3, A and C). The number of indirect PFCs in the secondary imSn also suppressed the HA and HR titers of the non-2-ME-treated sera significantly on days 4 (P < 0.01 for HA and HR) and 5 (P c 0.05 for HA) after the primary immunization (Fig. 3, A and C). The number of indirect PFCs in the secondary immune response, however, was not affected (Fig. 2B). The antibody titers of the 2-ME-treated sera were not affected in either the primary or secondary immune response, as shown in Fig. 3, B and D, except for the suppression of the HR titer on day 13 (Fig. 3D), although those of the non-2-ME-treated ones were suppressed on days 10 (P < 0.05 for HA), 11 (P < 0.05 for HR), and 13 (P < 0.05 for HA and P c 0.01 for HR) in the secondary response (Fig. 3, A and C). DISCUSSION
Administration of 167 pmol/kg/day of Sn for 3 consecutive days suppressed the circulating antibody titers in the primary immune response. Reduction of the number of direct PFCs (Fig. 2A) and/or decrease of antibody titer of the non-2-MEtreated sera (Fig. 3, A and C) was observed on days 10 and 13 in the secondary immune response. These phenomena might be due to the reduction of IgM antibody production. On the other hand, the number of indirect PFCs was suppressed in the primary immune response (Fig. 2B). Neither the number of indirect PFCs (Fig. 2B) nor the HA antibody titers of the 2-ME-treated sera (Fig. 3B) in the secondary immune response, however, were much affected. These results suggest that subacute administration of Sn suppresses part of the immune response in which IgM antibody production plays an important role and that the production of IgG in the secondary immune response is little affected, while that in the primary response is suppressed or delayed. Immunological memory for IgM is presumably suppressed by Sn administration, but that for IgG is so slightly affected that the IgG antibody response of the Sn group following secondary immunization was not markedly different from that of the control. This is in contrast to Pb, which has been shown by other workers to reduce IgG antibody in the secondary immune response of mice or rats [15,18]. Pb at a large dosage (1300 ppm) in the drinking water for 10 weeks impaired the memory response after the inoculation of mice with SRBCs [16], and exposure to high Pb concentrations (2000 ppm) for 30 days significantly decreased the responses of splenic lymphocytes to the mitogen, PHA, in mice [8]. Dimitrov et al. [6] reported that a single i.p. injection of 200 fig of SnClz tended to suppress or barely affect the number of IgM-PFCs and slightly increase that of IgG-PFCs and rosette formation after primary immunization in mice. This is different from the findings in the present study but a lower level of SnC12, as opposed
to a higher one, probably activates the immune system in mice. Heils [lo] reported that radioactive tin was observed to some extent in the spleen 48 h after a single i.v. injection of ‘r3SnC12 in rats. Chiba et al. [3] reported that a relatively high deposition of Sn was found in the spleen of rabbits, with the maximum concentration being observed 5 days after injection of 5 pmol Sri/kg/day for 6 consecutive days. Although there might be a difference in the effects of Sn on immune response between mice and other animals, accumulation of Sn in the spleen may suppress or delay antibody production. A transient depression of HR titers of the sera of the Sn group was observed on day 13 in the secondary response (Fig. 3, C and D), regardless of 2-ME treatment of the sera. This phenomenon cannot be satisfactorily explained at present but there is some evidence that metals such as Pb, Cd and Hg suppress antibody titers to some antigens, viruses, SRBC or y-globulin [12,17,21]. Koller [12,14] suggested that the direct effect of Pb or Cd on B cells could account in part for the decrease of EAC rosettes formed by splenic B-lymphocytes from mice exposed to the metal in contrast to those from controls. Our unpublished data indicated that 0.5 mM SnC12 decreased the HR titer to SRBCs in vitro to 90%, and 2.5 mM SnC12 to 10%. From these facts, it is supposed that Sn might directly suppress the antibody titer of hemolysis or inhibit the action of complement on erythrocyte-antibody complex. But there is no evidence for a transient increase of Sn concentration in the blood on about the 13th day after its administration. Another notable phenomenon, hypertrophy of the spleen, was observed in the mice administered Sn (Fig. 1). It had been reported that pathological change, a bluish-grey appearance, was seen in the spleen of rabbits exposed to tin compounds, stannous tartrate or stannous citrate, and that this might be due to the deposition of the injected Sn in the reticuloendothelial cells [l]. REFERENCES 1 J.M. Barnes and H.B. Stoner, The toxicology of tin compounds, Pharmacol. Rev., 11 (1959) 211. 2 M. Chiba and M. Kikuchi, Effect of tin compounds on activity of S-aminolevulinate dehydratase in blood, Biochem. Biophys. Res. Commun., 82 (1978) 1057. 3 M. Chiba, K. Ogihara, Y. Inaba, T. Nishima and M. Kikuchi, The organ distribution of tin and the effect of tin on concentrations of several essential elements in rabbit, Toxicology (1983) in press. 4 M. Chiba, K. Ogihara and M. Kikuchi, In vitro effects of tin, lead, and mercury on the activity of 5aminolevulinate hydrolyase in erythrocytes (in Japanese), Sangyo-Igaku, 21 (1979) 182. 5 A.J. Cunningham and S. Szenberg, Further improvements on the plaque technique for detecting single antibody-forming cells, Immunology, 14 (1968) 599. 6 N.V. Dimitrov, C. Meyer, F. Nahhas, C. Miller and B.A. Averil, Effect of tin on immune response of mice, Clin. Immunol. Immunopathol., 20 (1981) 39. 7 K. Fujita, K. Yamashita and M. Kikuchi, The influence of thermal environment on the immune response of mice, 1. Cellular and humoral effects of low ambient temperature, Jap. J. Hyg., 29 (1974) 491. 8 C.L. Gaworski and R.P. Sharma, The effects of heavy metals on [‘Hlthymidine uptake in lymphocytes, Toxicol. Appl. Pharmacol., 46 (1978) 305.
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