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In vitro effect of cadmium on primary antibody response to T-cell independent antigen (DNP-Ficoll)
Toxicology
Letters,
21
24 (1985) 21-24
Elsevier
TOXLett.
1334
IN VITRO EFFECT OF CADMIUM ON PRIMARY ANTIBODY TO T-CELL INDEPENDENT ANTIGEN (DNP-FICOLL) (Cadmium;
antibody
HIDEKAZU
response;
RESPONSE
DNP-Ficoll)
FUJIMAKI
The National Institute for Environmental Studies, Yatabe-machi, Tsukuba, Ibaraki 305 (Japan) (Received
July 5th,
(Accepted
August
1984) 27th,
1984)
SUMMARY The effect of various independent
antigen
significantly viabilities that
concentrations
(DNP-Ficoll)
enhanced
of cadmium
by 8 pM cadmium,
were decreased
in vitro exposure
on the in vitro primary
was investigated.
by cadmium
to cadmium
Anti
but suppressed
exposure
produces
DNP-Ficoll
response
response
by 20 and 40 pM cadmium.
with dose-dependent
different
antibody
antibody
effects
relationships.
on antibody
to T-cell
in mice was However,
cell
The results suggest
responses.
INTRODUCTION
Many reports have shown that cadmium modulated antibody response [l-3], decreased responses to the mitogens PHA and PWM [4], and conversely potentiated blastogenesis by mitogens Con A and LPS [5]. However, the mechanism of the effect of cadmium exposure on the immune response remains unknown. Fujimaki et al. [6] have recently shown that in vitro antibody response to SRBC was enhanced by 4 and 8 PM cadmium, but suppressed by 20 and 40 ,um cadmium. The enhancement of antibody response was mainly caused by the activation of B cells. To explore the mechanism of the enhancement of immune response by cadmium exposure, the effect of cadmium exposure on in vitro primary antibody response to DNP-Ficoll was studied. DNP-Ficoll as well as PVP is a potent T-independent antigen [7]. Abbreviations:
ConA,
charide;
plaque-forming
pokeweed
PFC,
mitogen;
03784274/85/S
concanavalin
SRBC,
03.30
A; DNP,
cells;
PHA,
sheep red blood
0 Elsevier
Science
dinitrophenyl; phytohemagglutinin; cells.
Publishers
B.V.
FCS, fetal calf serum; PVP,
LPS, lipopolysac-
polyvinylpyrrolidone;
PWM,
22
MATERIALS
AND METHODS
Male BALB/c mice, 2 to 3 months old, were obtained from Charles River Japan Inc. The mice were killed under ether anesthesia. Spleens from 6 to 7 mice were pooled and the cells were prepared by methods previously described [6]. After various concentrations of cadmium chloride and DNP-AECM-Ficoll(O.05 pg/well), purchased from Biosearch, as antigen were added to spleen cells (a total of 2 x 107/well), culture plates (Linbro Division Flow Lab., Inc.) were incubated for 5 days at 37°C in a 5% COz-humidified incubator. The culture medium employed was RPMI-1640 containing 10% heat-inactivated FCS, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 pg/ml). A direct PFC assay was used to enumerate antibody response as previously described [8]. Cell viability was determined by trypan blue dye exclusion test and was expressed as the percentage of viable cells divided by total counted cells. Statistical evaluations were examined by Student’s r-test. RESULTS
AND DISCUSSION
The results are summarized in Table. I. Primary PFC response to DNP-Ficoll as T-independent antigen was enhanced by 8 PM cadmium, but was significantly suppressed by 20 and 40 PM cadmium. Cell viabilities were decreased by cadmium exposure with dose-dependent relationships. The higher cadmium doses (20 and 40 PM) would by cytotoxic to spleen cells. These results were coincident with the previous observation of the effect of cadmium on in vitro antibody response to SRBC as T-dependent antigen [6]. Therefore, it appears from these data that in vitro exposure to low doses of cadmium enhances the antibody response, but exposure to high doses of cadmium suppresses it. As
TABLE EFFECT
1 OF CADMIUM
EXPOSURE
ON
IN VITRO
PRIMARY
PFC
RESPONSE
TO
DNP-
FICOLLa Cadmium WI)
dose
Number of PFC per wellb
% of
Cell viability’
control
% of control
270 f
27
1
380 i
25*
100 141
45 i
8
35 I
4
78
20
193 f
12*
71
22 i
1**
49
40
43 i
12**
16
11 i
1**
24
and DNP-Ficoll
for 5 days at 37°C.
saline
a Spleen cells were cultured ’ The results represent All data
were analyzed
with cadmium
the mean
100
of- SE of six wells.
by Student’s
f-test:
*P
**P
‘Cell viability was determined by the trypan blue dye exclusion of viable cells divided by total counted cells.
test and was expressed
as the percentage
23
previously suggested by Fujimaki et al. [6], the enhancement of primary PFC response by 8 FM cadmium may be due to the effect on B cells. The results of in vitro effect of the low doses of cadmium on the antibody response correspond to those of Lawrence [ll] who found that in vitro lead exposure (10-4-10-6 M) enhanced the primary PFC response and B-cell mitogenic response. Concerning the mechanism of the effect of cadmium exposure on lymphocytes, Gaworski and Sharma [4] have suggested that cadmium may interfere with the activity of membrane-bound ATPase, or alter the synthesis of cellular DNA and thereby influence production of antibodies. Cadmium has been shown to have an affinity for microtubules which are thought to be essential to many cell membraneassociated phenomena, and immunoglobulin production and release are very sensitive to changes in both intracellular and extracellular calcium concentrations, which also can be altered by low levels of cadmium [9]. Moreover, Nelson et al. [lo] demonstrated that cadmium interferes with the interaction between the cation and specific sites on the surface of cells. These studies suggest that cadmium acts directly on the membrane of lymphocytes. As DNP-Ficoll is a T-independent antigen, it is possible to postulate that cadmium significantly influences the Ievels of antibody production by B celis through the interaction with the cell membranes. ACKNOWLEDGEMENTS
The author thanks Dr. K. Kubota for his encouragement for his reading of the manuscript.
and Dr. M. Murakami
REFERENCES
1 L.D.
Kolier,
Immunosuppression
produced
by lead, cadmium
and mercury,
Am.
J. Vet. Res., 34
(1973) 1457-1458. 2 L.D. Keller,
J.H.
Exon and J.G. Roan,
Antibody
suppression
by cadmium,
Arch.
Environ.
Health,
30 (1975) 598-601. 3 L.D.
Keller,
posure 4 C.L.
J.H.
Exon and J.C.
to lead or cadmium, Gaworski
phocytes,
and
Toxicol.
5 L.D. Keller,
R.P. Appi.
6 H. Fujimaki,
Sharma,
Humoral
Environ.
response,
P.R.B. antigen
(1975) 1585-1589. 8 H. Fujimaki and Arch.
of heavy
response
in mice after
single dose ex-
151 (1976) 339-342. metals
on [3H]thymidine
uptake
in lym-
46 (1978) 305-313.
Res.,
Mitogen
stimulation
of lymphocytes
in CBA mice exposed
19 (1979) 177-188.
and K. Kubota, Toxicoi.
antibody
Exp. Biol. Med.,
The effect
Pharmacol.,
M. Murakami
of the antibody
response,
Sot.
J.R. Roan and N.I. Kerkvliet,
to lead and cadmium,
7 R. Sharon, independent
Roan,
Proc.
Appl.
In vitro evaluation
Pharmacol.,
of cadmium-induced
augmentation
62 (1982) 288-293.
MeMaster, A.M. Kask, J.D. Owens and W.E. Paul, DNP-Lys-Ficoll: a Twhich elicits both IgM and anti-DNP antibody-secreting cells, J. Immunol., 114 F. Shimizu,
Environ.
Effect
Health,
of acute
exposure
36 (1981) 114-l 19.
to nitrogen
dioxide
on primary
antibody
24
9 B.E.
Bozelka,
mediated 10 D.J.
P.M.
immunity
Nelson,
Immun.,
and
L.W. Res.,
L. Kiremidjian-Schumacher
macrophage-mediated 11 D.A. Lawrence,
Burkholder
in mice, Environ. cytotoxicity
toward
In vivo and in vitro effects
31 (1981) 136-143.
Chang,
Cadmium,
a metallic
inhibitor
of antibody-
17 (1978) 390-402.
and G. Stotzky, tumor
Effects
cells, Environ.
of lead on humoral
of cadmium,
lead, and zinc on
Res., 28 (1982) 154-163. and cell-mediated
immunity,
Infect.
Letters,
21
24 (1985) 21-24
Elsevier
TOXLett.
1334
IN VITRO EFFECT OF CADMIUM ON PRIMARY ANTIBODY TO T-CELL INDEPENDENT ANTIGEN (DNP-FICOLL) (Cadmium;
antibody
HIDEKAZU
response;
RESPONSE
DNP-Ficoll)
FUJIMAKI
The National Institute for Environmental Studies, Yatabe-machi, Tsukuba, Ibaraki 305 (Japan) (Received
July 5th,
(Accepted
August
1984) 27th,
1984)
SUMMARY The effect of various independent
antigen
significantly viabilities that
concentrations
(DNP-Ficoll)
enhanced
of cadmium
by 8 pM cadmium,
were decreased
in vitro exposure
on the in vitro primary
was investigated.
by cadmium
to cadmium
Anti
but suppressed
exposure
produces
DNP-Ficoll
response
response
by 20 and 40 pM cadmium.
with dose-dependent
different
antibody
antibody
effects
relationships.
on antibody
to T-cell
in mice was However,
cell
The results suggest
responses.
INTRODUCTION
Many reports have shown that cadmium modulated antibody response [l-3], decreased responses to the mitogens PHA and PWM [4], and conversely potentiated blastogenesis by mitogens Con A and LPS [5]. However, the mechanism of the effect of cadmium exposure on the immune response remains unknown. Fujimaki et al. [6] have recently shown that in vitro antibody response to SRBC was enhanced by 4 and 8 PM cadmium, but suppressed by 20 and 40 ,um cadmium. The enhancement of antibody response was mainly caused by the activation of B cells. To explore the mechanism of the enhancement of immune response by cadmium exposure, the effect of cadmium exposure on in vitro primary antibody response to DNP-Ficoll was studied. DNP-Ficoll as well as PVP is a potent T-independent antigen [7]. Abbreviations:
ConA,
charide;
plaque-forming
pokeweed
PFC,
mitogen;
03784274/85/S
concanavalin
SRBC,
03.30
A; DNP,
cells;
PHA,
sheep red blood
0 Elsevier
Science
dinitrophenyl; phytohemagglutinin; cells.
Publishers
B.V.
FCS, fetal calf serum; PVP,
LPS, lipopolysac-
polyvinylpyrrolidone;
PWM,
22
MATERIALS
AND METHODS
Male BALB/c mice, 2 to 3 months old, were obtained from Charles River Japan Inc. The mice were killed under ether anesthesia. Spleens from 6 to 7 mice were pooled and the cells were prepared by methods previously described [6]. After various concentrations of cadmium chloride and DNP-AECM-Ficoll(O.05 pg/well), purchased from Biosearch, as antigen were added to spleen cells (a total of 2 x 107/well), culture plates (Linbro Division Flow Lab., Inc.) were incubated for 5 days at 37°C in a 5% COz-humidified incubator. The culture medium employed was RPMI-1640 containing 10% heat-inactivated FCS, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 pg/ml). A direct PFC assay was used to enumerate antibody response as previously described [8]. Cell viability was determined by trypan blue dye exclusion test and was expressed as the percentage of viable cells divided by total counted cells. Statistical evaluations were examined by Student’s r-test. RESULTS
AND DISCUSSION
The results are summarized in Table. I. Primary PFC response to DNP-Ficoll as T-independent antigen was enhanced by 8 PM cadmium, but was significantly suppressed by 20 and 40 PM cadmium. Cell viabilities were decreased by cadmium exposure with dose-dependent relationships. The higher cadmium doses (20 and 40 PM) would by cytotoxic to spleen cells. These results were coincident with the previous observation of the effect of cadmium on in vitro antibody response to SRBC as T-dependent antigen [6]. Therefore, it appears from these data that in vitro exposure to low doses of cadmium enhances the antibody response, but exposure to high doses of cadmium suppresses it. As
TABLE EFFECT
1 OF CADMIUM
EXPOSURE
ON
IN VITRO
PRIMARY
PFC
RESPONSE
TO
DNP-
FICOLLa Cadmium WI)
dose
Number of PFC per wellb
% of
Cell viability’
control
% of control
270 f
27
1
380 i
25*
100 141
45 i
8
35 I
4
78
20
193 f
12*
71
22 i
1**
49
40
43 i
12**
16
11 i
1**
24
and DNP-Ficoll
for 5 days at 37°C.
saline
a Spleen cells were cultured ’ The results represent All data
were analyzed
with cadmium
the mean
100
of- SE of six wells.
by Student’s
f-test:
*P
**P
‘Cell viability was determined by the trypan blue dye exclusion of viable cells divided by total counted cells.
test and was expressed
as the percentage
23
previously suggested by Fujimaki et al. [6], the enhancement of primary PFC response by 8 FM cadmium may be due to the effect on B cells. The results of in vitro effect of the low doses of cadmium on the antibody response correspond to those of Lawrence [ll] who found that in vitro lead exposure (10-4-10-6 M) enhanced the primary PFC response and B-cell mitogenic response. Concerning the mechanism of the effect of cadmium exposure on lymphocytes, Gaworski and Sharma [4] have suggested that cadmium may interfere with the activity of membrane-bound ATPase, or alter the synthesis of cellular DNA and thereby influence production of antibodies. Cadmium has been shown to have an affinity for microtubules which are thought to be essential to many cell membraneassociated phenomena, and immunoglobulin production and release are very sensitive to changes in both intracellular and extracellular calcium concentrations, which also can be altered by low levels of cadmium [9]. Moreover, Nelson et al. [lo] demonstrated that cadmium interferes with the interaction between the cation and specific sites on the surface of cells. These studies suggest that cadmium acts directly on the membrane of lymphocytes. As DNP-Ficoll is a T-independent antigen, it is possible to postulate that cadmium significantly influences the Ievels of antibody production by B celis through the interaction with the cell membranes. ACKNOWLEDGEMENTS
The author thanks Dr. K. Kubota for his encouragement for his reading of the manuscript.
and Dr. M. Murakami
REFERENCES
1 L.D.
Kolier,
Immunosuppression
produced
by lead, cadmium
and mercury,
Am.
J. Vet. Res., 34
(1973) 1457-1458. 2 L.D. Keller,
J.H.
Exon and J.G. Roan,
Antibody
suppression
by cadmium,
Arch.
Environ.
Health,
30 (1975) 598-601. 3 L.D.
Keller,
posure 4 C.L.
J.H.
Exon and J.C.
to lead or cadmium, Gaworski
phocytes,
and
Toxicol.
5 L.D. Keller,
R.P. Appi.
6 H. Fujimaki,
Sharma,
Humoral
Environ.
response,
P.R.B. antigen
(1975) 1585-1589. 8 H. Fujimaki and Arch.
of heavy
response
in mice after
single dose ex-
151 (1976) 339-342. metals
on [3H]thymidine
uptake
in lym-
46 (1978) 305-313.
Res.,
Mitogen
stimulation
of lymphocytes
in CBA mice exposed
19 (1979) 177-188.
and K. Kubota, Toxicoi.
antibody
Exp. Biol. Med.,
The effect
Pharmacol.,
M. Murakami
of the antibody
response,
Sot.
J.R. Roan and N.I. Kerkvliet,
to lead and cadmium,
7 R. Sharon, independent
Roan,
Proc.
Appl.
In vitro evaluation
Pharmacol.,
of cadmium-induced
augmentation
62 (1982) 288-293.
MeMaster, A.M. Kask, J.D. Owens and W.E. Paul, DNP-Lys-Ficoll: a Twhich elicits both IgM and anti-DNP antibody-secreting cells, J. Immunol., 114 F. Shimizu,
Environ.
Effect
Health,
of acute
exposure
36 (1981) 114-l 19.
to nitrogen
dioxide
on primary
antibody
24
9 B.E.
Bozelka,
mediated 10 D.J.
P.M.
immunity
Nelson,
Immun.,
and
L.W. Res.,
L. Kiremidjian-Schumacher
macrophage-mediated 11 D.A. Lawrence,
Burkholder
in mice, Environ. cytotoxicity
toward
In vivo and in vitro effects
31 (1981) 136-143.
Chang,
Cadmium,
a metallic
inhibitor
of antibody-
17 (1978) 390-402.
and G. Stotzky, tumor
Effects
cells, Environ.
of lead on humoral
of cadmium,
lead, and zinc on
Res., 28 (1982) 154-163. and cell-mediated
immunity,
Infect.