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TOXICOLOGICAL SCIENCES 86(1), 75–83 (2005)
doi:10.1093/toxsci/kfi177
Advance Access publication April 20, 2005
Dose-Dependent Modulation of the In Vitro Cytokine Production of
Human Immune Competent Cells by Lead Salts
Nasr Y. A. Hemdan,*,† Frank Emmrich,* Khadiga Adham,‡ Gunnar Wichmann,† Irina Lehmann,† Azza El-Massry,‡
Hossam Ghoneim,‡ Jörg Lehmann,§ and Ulrich Sack*,1
*Institute of Clinical Immunology and Transfusion Medicine—IKIT, University of Leipzig, D-04103 Leipzig, Germany; †Department of Environmental
Immunology, UFZ-Centre for Environmental Research Leipzig-Halle, D-04318 Leipzig, Germany; ‡Department of Zoology, Faculty of Science,
University of Alexandria, Moharram Bey, Alexandria, Egypt; §Labor Diagnostik GmbH, D-04103 Leipzig, Germany
Received January 31, 2005; accepted April 14, 2005
Lead pollution constitutes a major health problem that has been
intensively debated. To reveal its effects on the immune response,
the influence of lead on the in vitro cytokine production of human
peripheral mononuclear blood cells was investigated. Isolated cells
were exposed to lead acetate or lead chloride for 24 h in the
presence of either heat-killed Salmonella enteritidis (hk-SE) or
monoclonal antibodies (anti-CD3, anti-CD28, anti-CD40) as cell
activators. Our results showed that while higher lead doses are
toxic, lower ones evoke immunomodulatory effects. All tested lead
doses significantly reduced cell vitality and/or proliferation and
affected secretion of proinflammatory, T helper cell type (TH)1
and TH2 cytokines. Expression of interferon (IFN)-g, interleukin
(IL)-1b, and tumor necrosis factor (TNF)-a was reduced at lower
lead doses in both models of cell stimulation. Although hk-SE
failed to induce detectable IL-4 levels, monoclonal antibody–
induced IL-4, IL-6, and IL-10 secretion increased in the presence
of lower lead doses. Also, levels of hk-SE-induced IL-10 and IL-6
secretion were increased at lower lead doses. Thus, exposure to
lower doses leads to suppression of the TH1 cytokine IFN-g and
the proinflammatory cytokines TNF-a and IL-1b. The elevated
production of IL-4 and/or IL-10 can induce and maintain a TH2
immune response and might contribute to increased susceptibility
to pathologic agents as well as the incidence of allergic hypersensitivity and/or TH2-dominated autoimmune diseases.
Key Words: allergy; autoimmune diseases; cytokine; immune
response; lead salts; T helper cells.
INTRODUCTION
Heavy metal exposure has long been known to be toxic to
humans and animals, causing diseases and occasionally death
The authors certify that all research involving human subjects was done
under full compliance with all government policies and the Helsinki
Declaration.
1
To whom correspondence should be addressed at Institute of Clinical
Immunology and Transfusion Medicine— IKIT, University of Leipzig, MaxBürger-Forschungszentrum, Johannisallee 30, D-04103 Leipzig, Germany.
Fax: þ49 341 97 25828. E-mail: [email protected].
(Yucesoy et al., 1997b). Many studies have addressed the toxic
effects of heavy metals on specific organism(s) (Fischer and
Skreb, 2001); other studies have documented the direct
immunotoxicity caused by short-term exposure to heavy metals
(Marth et al., 2001); still others observed genotoxic (Karakaya
et al., 2005), apoptotic effects (de la Fuente et al., 2002), or
even chronic effects resulting from long-term exposure (Koller
and Roan, 1977). There is growing concern about the subtle
health effects of lead, cadmium, and mercury. Lead has been
used in the past as an additive in gasoline and eventually
became widely dispersed throughout the environment. However, since the replacement of leaded gasoline with unleaded
gasoline in many countries in the mid-1980s, lead exposure
from that source has virtually disappeared (RAMP, 1996;
Ransom et al., 1987). This has led to a welcome decrease in the
median blood lead concentration (Rogan and Ware, 2003).
Indeed, the last decade was particularly fruitful in providing
new information on the manifold toxic influences of lead
(Lidsky and Schneider, 2003). However, reviewing these
studies has revealed some inconsistency. For example, some
publications reported that immunomodulatory effects could be
found neither in vitro nor in vivo, even when very high doses
were applied (Borella et al., 1990; Yucesoy et al., 1997a). Most
investigators, however, have concluded that an effect exists.
In vitro investigations employing human peripheral blood
mononuclear cells (PBMC) or T cell clones (McCabe and
Lawrence, 1991), as well as in vivo studies using wild-type
(Gupta et al., 2002; Heo et al., 1996; Kim and Lawrence, 2000;
McCabe et al., 1999; Miller et al., 1998) or transgenic animals
(Heo et al., 1998), have documented direct, although sometimes contradictory, effects of lead exposure on cytokine
profiles or TH cell differentiation.
The differentiation of naı̈ve cell responses into TH1 or TH2
branches determines (1) resistance or susceptibility of hosts to
infection, (2) development of allergic reactions, and (3) the
degree of tissue damage that occurs in many autoimmune
diseases (Abbas et al., 1996; Singh et al., 1999). The balance
between allergy-promoting TH2 cells and infection-fighting
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76
HEMDAN ET AL.
TH1 cells is considered to be a critical component of the
immune system. Hitherto, many reports have addressed the
effects of lead on the TH1–TH2 cell balance, but again some
showed contradictory results. The seminal work in this area is
that of McCabe and Lawrence (1991) who found, through the
employment of antigen-specific T cell clones, that exposure to
lead resulted in a TH2 activation preference. Corroborating
these findings, Yucesoy et al. (1997b) found that TH1 cytokines
were reduced in workers occupationally exposed to lead, although this skewing occurred only in the presence of antigens.
In contrast, preferential production of TH1 cytokines as a result
of lead exposure was reported by Krocova et al. (2000).
The current increase in disease susceptibility in polluted
areas implicates the environment and demonstrates the necessity for further studies to reveal the effects this pollutant may
have and the mechanisms involved. Cytokine profiles can be
differentially affected by heavy metals at concentrations that do
not affect other arms of the immune system (Shen et al., 2001).
Therefore, examining cytokine changes may offer a more
sensitive analysis of risk to human health than direct observation of other immune parameters. This study attempts to
evaluate the in vitro immunomodulatory activities of lead,
and especially its effects on TH1–TH2 homeostasis as observed
from changes in cytokine profiles. Two forms of lead salts and
two stimulation protocols were applied.
SERVA, Heidelberg, Germany). After incubation overnight, the MTT assay
results were read using an enzyme-linked immunosorbent assay (ELISA)Reader (Spectra Image; Tecan, Crailsheim, Germany) with a 570-nm filter and
a 450-nm reference filter.
Detection of cytokine release by ELISA. The cytokines IL-1b, IL-4, IL-6,
IL-10, IFN-c, and TNF-a in culture supernatants were determined using ELISA
kits (OptEIAKits; BD Biosciences, Heidelberg, Germany) according to the
manufacturer’s instructions. 3,3#,5,5#-Tetramethylbenzidine (TMB; Pharmingen, BD Biosciences, Heidelberg, Germany) was used as a substrate. The
optical density was measured by ELISA-Reader with a 450-nm filter and a
620-nm reference filter.
Intracellular cytokine staining. Intracellular cytokines were evaluated by
flow cytometry as follows: PBMC (2 3 106/ml) were seeded in TPP cell-culture
tubes (Techno Plastic Products AG, Trasadingen, Switzerland). After 24 h of
exposure to lead at 37°C/5% CO2, cells received 1.0 ml fresh medium
containing 1.0 lM ionomycin (Calbiochem, Bad Soden, Germany), 2.5 lM
phorbol-12-myristate 13-acetate (PMA) and 2.5 lM monensin (Sigma Aldrich,
Deisenhofen, Germany). After another incubation period of 4 h, cells were
fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, and
stained with fluorescein isothiocyanate (FITC)-labeled mAbs against the CD3
(UCHT1), and PE-labeled mAbs against the cytokines IFN-c (45.15) or IL-4
(4D9) (Beckman-Coulter, Krefeld, Germany). Mouse IgG1 PE-labeled isotype
control was used. Fluorescence-labeled cells were analyzed by flow cytometer
(FACSCalibur, Becton Dickinson, Germany).
Statistical analysis. All experiments were carried out in triplicate. Data
analysis was performed with the STATISTICA 5.1 software (Statsoft,
Hamburg, Germany). Differences between control samples and lead-treated
cells were tested with the Wilcoxon test for paired samples with an entry
criterion of 0.05.
MATERIALS AND METHODS
Cell culture. Peripheral blood mononuclear cells were separated from
buffy coats of healthy donors using Ficoll-density gradient (Amersham
Biosciences, Uppsala, Sweden). Buffy coats were obtained from the Blood
Bank of the University Clinic of Leipzig, Germany. Isolated cells were
suspended in HybridoMed DIF 1000 (Biochrom, Berlin, Germany) and
cultured at a density of 1 3 106 PBMC/ml in 48-well flat-bottom microtiter
plates (Greiner Bio-one GmbH, Nürtingen, Germany). Cells were stimulated
either with monoclonal antibodies (mAbs) against CD3 (OKT3, mouse IgG1;
Ortho Biotech, Bridgewater, NJ), CD28 (clone CD28.2, mouse IgG1;
Immunotech, Marseille, France) and CD40 (clone B-B20, Trinova Biochem,
Gieben, Germany), each at a final concentration of 100 ng/ml, or with heatkilled Salmonella enteritidis (hk-SE) at a final concentration of 1.25 3 105
colony-forming units (CFU)/ml.
Application of lead salts. Stock solutions of lead acetate (Sigma-Aldrich,
Deisenhofen, Germany) and lead chloride (Merck-Schuchardt, Hohenbrunn,
Germany) were prepared in deionized water. Immediately before application,
14 serial doses were made using the culture medium: 5.0 mg–1.5 ng and
0.5 mg–0.15 ng/ml for both salts, respectively. As control samples, cells
received either media alone (nonstimulated control 1) or mAbs or hk-SE
(stimulated control 2).
MTT cell vitality/proliferation test. The method employed is that described by Mosmann (1983) and modified by Wichmann et al. (2002). After
24 h of exposure to lead, 750 ll of cell-free culture supernatant was harvested.
The remaining cells were treated with 25 ll/well MTT solution (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 5 g/l phosphate buffered saline (PBS); Sigma, Deisenhofen, Germany) and were incubated in the
dark at 37°C. After 4 h, the metabolic conversion of MTT into purple formazan
crystals by active cells was terminated by adding 300 ll/well stop solution
(10% w/v sodium dodecylsulfate (SDS) in 50% v/v N,N#-dimethylformamide,
RESULTS
Cell Vitality
Twenty-four–hour exposure to lead doses above 50.0 lg/ml
exhibited strong cytotoxic effects (Fig. 1), but lower doses
ranging from 15 pg/ml to 50 lg/ml significantly inhibited cell
vitality and/or proliferation in a dose-dependent manner. These
effects were independent of the kind of cell activators used or the
metal salts tested. Similar results were obtained in both cases.
Cytokine Release
Changes in in vitro cytokine release by mAb or hk-SE–
stimulated human PBMC after short-term exposure to one or
both metal forms are represented as percentages of the
accompanying controls (Figs. 2 and 3). The normal cytokine
ranges of these controls, where cells are stimulated either by
mAb or hk-SE, but not by lead, are represented in Table 1. A
dose-dependent stimulation or inhibition of cytokine production resulted from exposure to lead.
As shown in Figure 2, after exposure to lead acetate, the
release of the proinflammatory cytokines TNF-a by mAbsstimulated PBMC was significantly reduced at lead doses
above 1.5 ng/ml, IL-1b above 5.0 ng/ml, and IL-6 from 150.0
ng/ml to 15.0 mg/ml. The exception was the stimulation of IL-6
release at doses lower than 150.0 ng/ml. Furthermore, IFN-c
release was significantly reduced at all tested doses of lead
MODULATION OF CYTOKINE PRODUCTION BY LEAD SALTS
120
% of controls
100
80
60
40
mAbs cell vitality
0.
00
0. 15
0
0. 05
01
0. 5
0
0. 5
15
0.
5
1.
5
5.
15 0
5 .0
150.0
5 0.0
15 00.
0
50 00.
00 5
.0
20
(a)
Pb (µg/ml)
77
After exposure to lead chloride (Fig. 3), the release of TNF-a
and IL-1b at all doses above 150.0 pg/ml and the release of IL6 at doses above 1.5 ng/ml was significantly inhibited. Exceptions were the significant stimulation of TNF-a and IL-6
release at higher doses, in the range 0.5/ml to 150 lg/ml (Fig.
3a and 3c) and IL-6 at lower doses of 150 and 50 pg/ml. IFN-c
production by hk-SE–stimulated cells was reduced at all
doses; this inhibition was significant at doses from 150 pg/ml
to 150 ng/ml. Again, although hk-SE failed to produce detectable levels of IL-4 after exposure to lead chloride, IL-10 release
was significantly stimulated at lower doses and inhibited at
higher doses (Fig. 3e). Estimating the ratios %IFN-c/%IL-10
revealed the polarization of the immune response toward IL-10
production; at low doses of 150.0 pg/ml and up to 15.0 ng/ml,
a significant decrease in the ratios resulted (Fig. 3f).
120
Intracellular Cytokines
% of controls
100
80
60
40
hk-SE cell vitality
0.
00
0. 01
0 5
0. 00
00 5
0. 15
0
0. 05
01
0. 5
0
0. 5
15
0.
5
1.
5
5.
15 0
50.0
.
150
500
0
20
(b)
Pb (µg/ml)
FIG. 1. The effect of lead (Pb) on the vitality and/or proliferation of human
peripheral blood mononuclear cells (PBMC). The medians and the 10th, 25th,
75th, and 90th percentiles of 12 sample donors of monoclonal antibody
(mAbs)-stimulated cells exposed to Pb acetate (a), and results of 10 sample
donors of heat-killed Salmonella enteritidis (hk-SE)–stimulated cells exposed
to Pb chloride (b) are shown as percentages of the controls. The asterisks (*)
indicate the significance as tested by the Wilcoxon test ( p ¼ 0.05).
from 1.5 ng to 5.0 mg/ml. In contrast, secretion of IL-10 and
IL-4 was significantly increased at all doses below 150.0 and
15.0 lg/ml, respectively. The production of all cytokines is
drastically inhibited at lead doses above 150.0 lg/ml (Figs. 2
and 3). Estimating the ratio of %IFN-c/%IL-4 provided
a picture of the TH1–TH2 balance. Because a balance between
the two cell types is hypothesized to exist, a sample was
obtained at all tested doses below 150.0 lg/ml (Fig. 2g);
a value lower than unity for a given sample indicated a probable
deviation toward the TH2 type. Similar results were obtained
when comparing the ratios of %IFNc and %IL-10 (Fig. 2h),
which could also indicate a TH2 polarized immune response at
all lead doses tested. With minor differences up to one dilution
step, we obtained the same results by applying lead chloride
(data not shown).
Cytokine profiles of hk-SE stimulated PBMC after 24 h of
exposure to lead acetate and lead chloride were also identical.
Intracellular cytokine staining was carried out to investigate
the differentiation preference of T cells by lead. Of the total T
lymphocytes, the proportions of IFN-c–producing and IL-4–
producing cells were estimated and expressed as percentages of
the controls (Fig. 4). With few exceptions, a dose-dependent
decrease in IFN-c–producing CD3þ T cells was found after
lead exposure. In contrast, the number of IL-4–producing T
cells increased after exposure to non-toxic doses of lead (from
150.0 pg/ml to 15.0 ng/ml); it then decreased with the increase
in lead toxicity (at doses higher than 15.0 lg/ml). Estimating
the %IFN-c/%IL-4 ratio could clearly represent the preferential
proliferation of IL-4–producing T cells in comparison to IFNc–producing cells (Fig. 4c). A decrease in the ratio resulted at
all tested doses, except at 15.0 ng/ml and 150.0 lg/ml, where
about 90% of the tested samples showed a polarized TH2
response, reflected by the increase in the percentage of IL-4–
producing T cells.
DISCUSSION
Heavy metals may influence cells by penetrating the interior
of the cell through calcium channels and/or by reacting with
surface structures of the cell, such as the cell receptors or
associated ligands (Kerper and Hinkle, 1997; Marth et al.,
2001). The hypothesis tested here is whether lead promotes
TH2-dependent response over TH1 response, which may
contribute to the increased incidence of atopy and allergic
diseases in polluted areas.
Lead Reduces Cell Vitality and/or Proliferation
One of the mechanisms by which lead and other heavy
metals can affect the immune system, is through effects on cell
vitality and proliferation. Our results demonstrate an inhibitory
effect of lead on the vitality and/or proliferation of human
PBMC. However, previous investigations showed conflicting
78
HEMDAN ET AL.
140
500
400
100
% of controls
% of controls
120
80
60
40
200
100
20
mAbs TNF-α
(a)
300
mAbs IL-10
(e)
140
250
200
100
% of controls
80
60
40
150
100
50
20
mAbs IL-1β
(b)
200
1.4
180
%IFNγ / %IL-4 ratio
% of controls
140
120
100
80
60
40
20
0.6
0.4
(g)
%IFNγ / %IL-10 ratio
% of controls
0.8
1.2
120
100
80
60
40
%IFNγ / %IL-10
1.0
0.8
0.6
0.4
0.2
mAbs IFN-γ
0.
00
0. 15
0
0. 05
01
0. 5
0
0. 5
15
0.
5
1.
5
5.
15 0
5 .0
150.0
5 0.0
1500.
0
5000.
00 5
.0
(d)
1.0
0.2
mAbs IL-6
140
20
% IFNγ / %IL-4
1.2
160
(c)
mAbs IL-4
(f)
Pb (µg/ml)
(h)
0.
00
0. 15
0
0. 05
01
0. 5
0
0. 5
15
0.
5
1.
5
5.
15 0
5 .0
150.0
5 0.0
1500.0
5000.
00 5
.0
% of controls
120
Pb (µg/ml)
FIG. 2. The effect of lead (Pb) acetate on the mAbs-stimulated release of TNF-a (a), IL-1b (b), IL-6 (c), IFN-c (d), IL-10 (e), and IL-4 (f) by human PBMC.
Box-plots show the medians and the 10th, 25th, 75th, and 90th percentile ranges of 12 samples, each with three replicates. TH1:TH2 ratios were evaluated from
%IFN-c/%IL-4 (g) and %IFN-c/%IL-10 (h). Symbols above each plot show whether, at each dose, cytokine release was significantly stimulated (#) or suppressed
(*) compared with the controls ( p < 0.05 in Wilcoxon test).
MODULATION OF CYTOKINE PRODUCTION BY LEAD SALTS
500
SE TNF-α
509
200
1046
180
400
160
350
140
% of controls
% of controls
450
300
250
200
150
120
100
80
60
100
(a)
40
50
140
(d)
% of controls
% of controls
80
60
40
150
100
50
(b)
hk -S E IL-10
(e)
200
15.0
180
12.0
%IFNγ / %IL-10 ratio
160
140
120
100
80
60
40
9.0
6.0
3.0
2.0
1.5
1.0
00
0. 01
0 5
0. 00
00 5
0. 15
0
0. 05
01
0. 5
0
0. 5
15
0.
5
1.
5
5.
15 0
5 .0
15 0.0
500.5
0.
0
0.
00
0. 01
0 5
0. 00
00 5
0. 15
0
0. 05
01
0. 5
0
0. 5
15
0.
5
1.
5
5
15.0
50.0
.
150
500
0
Pb (µg/ml)
%IFN-γ / %IL-10
0.5
hk-SE IL-6
0.
(c)
hk-SE IFN-γ
200
100
20
% of controls
20
250
hk-SE IL-1β
120
20
79
(f)
Pb (µg/ml)
FIG. 3. The effect of lead (Pb) chloride on the hk-SE–stimulated release of TNF-a (a), IL-1b (b), IL-6 (c), IFN-c (d), and IL-10 (e) by human PBMC. Medians
and the 10th, 25th, 75th, and 90th percentile ranges of 12 samples are given, each with three replicates. TH1:TH2 ratios were calculated from %IFN-c/%IL-10 (f).
Symbols above each plot show whether, at each dose, cytokine release was significantly stimulated (#) or suppressed (*) compared with the controls ( p < 0.05 in
Wilcoxon test).
results. It was found in some studies that lead did not affect cell
vitality or growth (Borella et al., 1990; Smith and Lawrence,
1988), whereas inhibitory or stimulatory effects of lead on
immune competent cells were described in other reports. For
example, a significant inhibitory effect of lead on rat splenocytes derived from rats exposed to 10 or 1000 lg/ml lead was
demonstrated by Exon et al. (1985). Similarly, lead was found
to enhance B cell proliferation (Lawrence, 1981) and T cell
proliferation (Warner and Lawrence, 1986) in mice. Tellingly,
lead augmented the proliferation of rat spleen cells at doses of
0.5 to 200 lg/ml, but it inhibited proliferation at doses above
200 lg/ml (Razani-Boroujerdi et al., 1999). These data provide
a reason for the contradictory results: conflicting findings may
be attributed to differences in lead doses or in the experimental
models used. Our results confirm that exposure to lower as well
as higher lead doses, namely, from 150 pg/ml up to 5 mg/ml,
significantly inhibits cell vitality and/or proliferation. We
propose that the capability of lead to impair cell vitality may
80
HEMDAN ET AL.
Cytokine
IL-1b
IL-4
IL-6
IL-10
IFN-c
TNF-a
mAb-activated cells
median (10–90%) (pg/ml)
hk-SE-activated cells
median (10–90%) (pg/ml)
1194 (768–9555)
90 (23–198)
2049 (1355–4487)
230 (157–621)
2473 (670–7764)
10446 (4307–12030)
8341 (2912–25888)
—
3125 (1244–6611)
276 (145–531)
301 (105–2121)
5224 (2369–12562)
120
100
% of controls
TABLE 1
The Ranges of the Different Cytokines Produced by mAb- and hkSE–Activated Cells after a 24-h Incubation Period that were Used
as Controls in Figures 1–4 to Demonstrate the Effect of Lead on
Human PBMC
80
60
40
IFN-γ
5 samples
Mean value
20
(a)
300
The median, the 10th and the 90th percentile values of each cytokine are
represented as measured by ELISA.
100
IL-4
5 samples
Mean value
50
(b)
%IFN-γ / %IL-4
1.6
5 samples
Mean value
1.4
1.2
1.0
0.8
0.6
0.4
.0
15
00
.0
0.
0
15
5
1.
15
(c)
1
0. 5
00
15
0.
01
5
0.
15
0.2
00
0
The mechanisms by which lead affects the TH1–TH2 balance
may include some or all of the following: (1) direct inhibition
of TH1 cells by lead; (2) stimulation of TH2 cell activities,
which induce cytokine release, which in turn inhibits TH1 cell
activity; and (3) an indirect inhibition of TH1 cells mediated by
impairment of antigen-presenting cells (APC). In light of previous investigations (e.g., McCabe et al., 1999; Shen et al.,
2001), and as demonstrated here, it seems that the most rational
approach to revealing the cellular interactions involved would
be to measure cytokine profile changes. We found that shortterm exposure of PBMC to low lead doses had immunomodulatory effects that might reflect skewness of the immune
response toward the TH2 type.
In mAbs-activated cells, a significant increase in IL-4, IL-10,
and IL-6 release occurred after exposure to lead acetate or lead
chloride. Production of the TH1 cytokine IFN-c and of the
proinflammatory cytokines TNF-a and IL-1b was significantly
inhibited. Slightly different results were obtained from hk-SEstimulated cells. These included stimulation of TNF-a and IL-6
release at higher lead doses, the undetectable production of IL-4,
and the relative increase in IFN-c production, which is evident
by comparing the IFN-c/IL-10 ratios between the two models
of cell stimulation. Therefore, we suggest that different lead
immunomodulatory pathways become active according to the
microenvironment of cell stimulation.
Evaluating the intracellular cytokines could also reveal the
proliferation preference of TH2 cells at lower lead doses.
Therefore, suppression of the IFN-c release, together with the
stimulation of IL-4 and/or IL-10 release indicated by ELISA,
may be due to a reduction in the number of IFN-c–producing
cells and preferential proliferation of IL-4–producing and/or
150
0.
Lead Exerts a Differential Effect on TH1-TH2 Balance
200
%IFNγ / %IL-4 ratio
be the result of (1) the direct interaction between the lead ion and
mAbs or hk-SE, and/or (2) interference with cell metabolism.
% of controls
250
Pb (µg/ml)
FIG. 4. The effect of lead (Pb) acetate on the mAbs-induced production of
IFN-c (a) and IL-4 (b) by human PBMC as evaluated by intracellular cytokine
staining. Medians and the 10th, 25th, 75th, and 90th percentiles of five samples
are represented as percentages of intracellular cytokine-PE–labeled cells of the
total isothiocyanate (FITC)-labeled CD3þ T cells. TH1:TH2 ratios (c) were
calculated from %IFN-c/%IL-4.
IL-10–producing cells (TH2 cells), or by changing cytokine
production potency of both TH cell subsets.
Some previous investigations reported no influence of lead
on the immune response (Blakley et al., 1982; Yucesoy et al.,
1997a). Our results, however, are in agreement with other data
MODULATION OF CYTOKINE PRODUCTION BY LEAD SALTS
documenting an inclination toward TH2 immune response
following exposure to lead. In direct opposition to these data,
Guo et al. (1996) and Krocova et al. (2000) found a preferential
production of TH1 cytokines; TNF-a was produced by macrophages as well as by T cells. Our assertion that high lead doses
stimulate TNF-a production by hk-SE–stimulated cells confirms the studies of Guo et al. (1996) and indicates a relative
increase in the activity of hk-SE stimulated macrophages at
high lead doses. The dissimilarity in reactivity of IFN-c and
TNF-a may result from the differing methods or crossregulatory processes from other segments of the immune
system that are compromised in our models. But, the finding
that IL-10 inhibits cytokine production by TH1 cells (Mosmann
and Moore, 1991) and the reciprocal regulation of TH1 and TH2
cells (Dubey et al., 1991; Manetti et al., 1993) rather confirms
our results concerning the inhibition of IFN-c and TNF-a levels
and the preferential proliferation of TH2 in association with
lower nontoxic doses of lead.
The Role of IL-4, -6, and -10 in Lead-Induced
Immunomodulation
The preferential stimulation of TH2 immune response by
lead is suggested here by the increase in IL-4, IL-6, and IL-10
production observed after exposure to lead. Indeed, many
investigations have addressed IL-4 as the major determinant for
differentiation of antigen-stimulated naive T cells into TH2
cells (Le Gros et al., 1990). The importance of IL-10 in the
TH1–TH2 immune balance, however, has long been debated.
Although IL-10 has been reported to be secreted by both CD4þ
subsets (Yssel et al., 1992), the finding that IL-10 inhibits
cytokine production by TH1 cells (Fiorentino et al., 1989;
Mosmann and Moore, 1991) would support its role in inducing
TH2 immune responses. Thus, it is reasonable here to suggest
a skewed TH2 immune response after lead exposure, even in the
absence of detectable levels of IL-4 in the hk-SE–stimulation
model. Therefore, not all cytokines are indicative of impairment in TH cell balance and, in some instances, cytokine levels
could be misleading. Mihara et al. (1991) found that IL-6
inhibited delayed-type hypersensitivity reactions that are
known to be elicited by TH1 responses. Therefore, the elevated
IL-6 levels may be a further indication of a TH2-polarized
response, which suggests a heightened stress based on the
absence of a priming level of IFN-c.
In asthma, TH1 type reactions are deficient and TH2 reactions
predominate. TH2 reactions are driven by IL-4, IL-5, IL-10, and
IL-13 and result in an increase in IgE production. Indeed,
significantly elevated levels of IgE have been found in leadexposed workers, although no significant relationship between
blood lead levels and serum IL-4 or IFN-c levels was observed
(Heo et al., 2004). The range of lower doses of lead used in our
study is comparable to the blood lead levels found in workers
occupationally exposed to lead—range: 0.07 to >0.8 lg/ml
(Basaran and Undeger, 2000; Heo et al., 2004; Palus et al.,
81
2003; Sata et al., 1998; Stollery et al., 1991; Truckenbrodt
et al., 1984). Thus, the lower TH1:TH2 ratio we obtained
confirms some previous reports of immunomodulation by lead
and raises the possibility for the development of allergic and/or
autoimmune diseases as suggested by Heo et al. (1996).
Lead Reduces the Proinflammatory Response
Exposure to lead resulted in a significant inhibition in the
production of the proinflammatory cytokines TNF-a and IL1b, which are mediators of the inflammatory response and
which are released from different cell types (Schuerwegh et al.,
2003). The importance of IL-1b, a primary mediator of
immune response to injury and infection (Brough et al.,
2003), and TNF-a in immune response stems largely from
their ability to promote TH1 versus TH2 pathways by antigen
presenting cells (APC; Cervi et al., 2004). These cytokines
increase the efficiency with which an APC can bind and
activate TH cells. It seems then, that lead exerts an inhibitory
effect on APC, leading to the inhibition of TNF-a and IL-1b
release and consequently to clonal proliferation.
Overall, we conclude that lead skews the immune response
toward the TH2 pathway, leading to a weakened ability of the
body to defend against various infections and an enhanced risk
of allergic diseases. The use of two forms of the metal confirms
the hypothesis that it is the action of the metal ion that is
operative, regardless of the applied form. This was not the case
when applying two models of immune stimulation; both
revealed identical effects, but they demonstrated that lead
may act through different pathways including different cytokine mediators. Identification of the intermediate molecules, as
well as the mechanisms that establish such susceptibility or the
initiation of the TH1–TH2 imbalance, may be an important goal
for future experiments.
ACKNOWLEDGMENTS
We thank Katrin Bauer (IKIT, University of Leipzig, Germany) and Sylke
Drubig (UFZ, Department of Environmental Immunology, Leipzig, Germany)
for technical assistance and Dr. Sonya Faber (Coordination Centre for Clinical
Trials, Leipzig, Germany) and Ms. Audrey Braun (University of Leipzig,
Germany) for reviewing the manuscript. Ulrich Sack, as well as this work, has
been funded by a grant from the Interdisziplinäres Zentrum für Klinische
Forschung Leipzig (IZKF) of the Medical Faculty, University of Leipzig,
Germany (project Z10). Nasr Y. A. Hemdan was funded by DAAD (Deutscher
Akademischer Austausch Dienst, The German Academic Exchange Service).
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