# Suppressive Effect of Hydroquinone, a Benzene Metabolite, on In Vitro Inflammatory Responses Mediated by Macrophages, Monocytes, and Lymphocytes

Authors: Jae Youl Cho
Journal: Mediators of Inflammation
 (2008)

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## Abstract

We investigated the inhibitory effects of hydroquinone on cytokine release, phagocytosis, NO production, ROS generation, cell-cell/cell fibronectin adhesion, and lymphocyte proliferation. We found that hydroquinone suppressed the production of proinflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6], secretion of toxic molecules [nitric oxide (NO) and reactive oxygen species (ROS)], phagocytic uptake of FITC-labeled dextran, upregulation of costimulatory molecules, U937 cell-cell adhesion induced by CD18 and CD29, and the proliferation of lymphocytes from the bone marrow and spleen. Considering that (1) environmental chemical stressors reduce the immune response of chronic cigarette smokers and children against bacterial and viral infections and that (2) workers in petroleum factories are at higher risk for cancer, our data suggest that hydroquinone might pathologically inhibit inflammatory responses mediated by monocytes, macrophages, and lymphocytes.


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## Body

## 1. Introduction

Monocytes, macrophages, and lymphocytes are the major types
of cells that mediate innate and adaptive immunity. Upon stimulation by bacterial,
fungal, or viral infections, macrophages at the infection site release many
different proinflammatory cytokines [e.g., tumor necrosis factor (TNF)-α and interleukin
(IL)-1] and cytotoxic and inflammatory molecules [e.g., nitric oxide (NO) and
oxygen species intermediates (ROS)] via the activation of the pattern recognition receptor (PRR). Monocytes
continuously migrate to the inflamed site via increased adhesion events
mediated by interactions with adhesion molecules such as selectins and
integrins. The migrating monocytes eventually differentiate into macrophages for
activation and further mediation of inflammatory responses. Macrophages that
have been fully activated by bacterial or fungal cell products such as LPS,
peptidoglycan (PEG), or β-glucan upregulate
surface expression of costimulatory molecules such as CD80, CD86, and major
histocompatibility molecule class II in order to enhance T cell proliferation. Activated
T cells also differentiate into Th1 or Th2 cells to modulate B cell function
for antibody production.Recent
decades have seen increased prevalence of several immunopathological diseases including
cancers, allergic diseases such as eczema and atopic dermatitis, and
respiratory tract infections in chronic cigarette smokers and children [1]. It is thought that
multiple environmental and genetic factors, in addition to smoking, are the main
reasons for these phenomena. Numerous chemically toxic molecules are particularly
known to have pathological effects. Benzene is known to be a human carcinogen [2]. Even though it is
chemically hazardous, occupational and habitual exposure to benzene still
occurs in the petrochemical and petroleum refining industry as well as through
some foods such as coffee [3].Hydroquinone
(benzene-1,4-diol) (Figure1) is a major component of cigarette smoke and coffee,
and a metabolite of benzene via a cytochrome p450-mediated pathway. It is also
a heavily used industrial chemical, a petroleum by-product, and an ubiquitous
environmental pollutant. Evidence suggesting that cigarette smoking is allergenic
and that benzene metabolites are strong haptens [4] stimulated our interest in
hydroquinone as an immunotoxicological agent in allergic diseases. Previous
reports have suggested that hydroquinone enhances Th2 response-mediated
allergic diseases via blockade of interferon (IFN)-γ production
in Th1 cells, enhanced interleukin-4 production in CD4+ T cells, increased
immunoglobulin E levels in antigen-primed mice, and blockade of IL-12
production via suppression of nuclear factor (NF)-κB binding
activity [5]. In addition, hydroquinone-mediated
inhibition of the production of several cytokines and toxic molecules such as
IL-1β, IL-2, and
NO [6–8] suggests that benzene
metabolites might interrupt global immune responses in addition to inducing
allergic disease. To date, the pathological importance and mechanisms of
benzene metabolites in the modulation of immune responses remain unclear. In the
present study, we, therefore, investigated the effect of hydroquinone, a
representative reactive benzene metabolite, on the modulation of inflammatory
processes mediated by monocytes, macrophages, and lymphocytes.Figure 1
Chemical structure of hydroquinone.


## 2. Materials and Methods

### 2.1. Materials

L-cysteine, phorbol 12-myristate
13-acetate (PMA), curcumin, 1,4-dithiothreitol (DTT),α-tocopherol, dihydrorhodamine 123 (DHR123), peptidoglycan (PGN), zymosan,
lipopolysaccharide (LPS), α-tocopherol,
sodium nitroprusside (SNP), concanavalin (Con) A, fluorescein isothiocyanate
(FITC)-label dextran, and
hydroquinone were obtained from Sigma Chemical Co. (St. Louis, Mo, USA). BAY
11-7082 (BAY) was from Calbiochem (La Jolla, Calif, USA). RAW264.7 and U937 cells were
purchased from ATCC (Rockville,
Md, USA).
All other chemicals were of reagent grade. Aggregation-inducing antibody against
CD29 (MEM 101A, purified IgG1) was used as reported previously [9–11]. FITC-labeled antibodies to CD80,
CD86, CD29, and CD18 were obtained from Immunotech (Marseilles, France).


### 2.2. Animals

C57BL/6 male mice (6–8 weeks old, 17–21 g) were
obtained from DAEHAN BIOLINK (Chungbuk,
Korea). The mice
were maintained in plastic cages under conventional conditions. Water and
pelleted diets (Samyang, Daejeon,
Korea) were
supplied ad libitum. Studies
were performed in accordance with guidelines established by the Kangwon
University Institutional Animal Care and Use Committee.


### 2.3. Preparation of Peritoneal Macrophages

Peritoneal exudates were obtained from C57BL/6
male mice (7-8 weeks old,
17–21 g) by lavage four days after intraperitoneal injection of 1 mL sterile 4%
thioglycollate broth (Difco Laboratories, Detroit,
Mich, USA).
Cells were washed and resuspended in RPMI 1640 containing 2% FBS and plated in
100 mm tissues culture dishes for 4 hours at37°C in a 5% CO2 humidified atmosphere.


### 2.4. Cell Culture

RAW 264.7 and
U937 cells obtained from American Type Culture Collection (Rockville,
Md, USA)
were cultured with RPMI medium supplemented with 10% heat-inactivated fetal bovine
serum (Gibco, Grand Island,
NY, USA),
glutamine, and antibiotics (penicillin and streptomycin) at37°C with 5% CO2.


### 2.5. Cytokine Production

The inhibitory effect of hydroquinone on TNF-α production
was determined as previously described [12]. Hydroquinone was
solubilized with vehicle (100% ethanol) and diluted with RPMI1640. RAW264.7
cells (2×106 cells/mL) were incubated with LPS (2.5 μg/mL) in
the presence or absence of hydroquinone for 6 hours. Supernatants were assayed
for the levels of TNF-α, IL-1β, and IL-6 using a mouse TNF-α ELISA kit
(Amersham, Little Chalfont, Buckinghamshire,
UK).


### 2.6. No Assay

RAW 264.7
cells were preincubated with or without tested compounds (hydroquinone and
L-NAME) for 30 minutes and continuously activated with LPS (2.5μg/mL) for 24 hours. Nitrite determination
was carried out using Griess reagent [13]. The absorbance of the product dye
was measured at 540 nm using a flow-through spectrophotometer.


### 2.7. MTT Assay

Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay, as described previously [12].


### 2.8. Extraction of Total RNA and Semiquantitative RT-PCRAmplification

The total RNA from LPS-treated RAW264.7 cells was prepared by
adding TRIzol reagent (Gibco BRL), according to the manufacturer’s protocol.
Semiquantitative RT reactions were conducted using MuLV reverse transcriptase
as reported previously [14]. Primers (Table 1)
(Bioneer, Daejeon, Korea) were used as previously reported [15].Table 1
Primers used for RT-PCR
analysis (F: forward,
R: reverse).
GenePrimer sequencesTNF-αF5′-TTGACCTCAGCGCTGAGTTG-3′R5′-CCTGTAGCCCACGTCGTAGC-3′IL-1βF5′-CAGGATGAGGACATGAGCACC-3′R5′-CTCTGCAGACTCAAACTCCAC-3′IL-6F5′-GTACTCCAGAAGACCAGAGG-3′R5′-TGCTGGTGACAACCACGGCC-3′iNOSF5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′R5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3′GAPDHF5′-CACTCACGGCAAATTCAACGGCAC-3′R5′-GACTCCACGACATACTCAGCAC-3′


### 2.9. ROS Determination

The level of intracellular ROS was
determined by the change in fluorescence resulting from oxidation of the
fluorescent probe DHR123 [16]. Briefly, 5×105 cells/well were incubated
with hydroquinone for 30 minutes and then with SNP (125 μM) for an additional 6 hours. After a final
incubation with 2.5 μM
DHR123 for 1 hour, the intracellular ROS level was determined using flow
cytometry.


### 2.10. Phagocytic Uptake

To measure the phagocytic activity
of RAW264.7 cells, a  previously reported  method was used with slight
modifications [17]. RAW 264.7
cells (1×106 cells/mL) were preincubated with or without hydroquinone
for 30 minutes, and further incubated for 6 hours. Finally, the cells were
further incubated with FITC-dextran (1 mg/mL) for 30 minutes at 37°C. The
incubation was stopped by addition of 2 mL of ice-cold PBS, and the
cells were washed four times with cold PBS. After fixing the cells with 3.7%
formaldehyde, phagocytic uptake was analyzed using
a FACScan device (Becton Dickinson, San Jose, Calif,
USA).


### 2.11. Cell-Cell or Cell-Extracellular Matrix Protein (Fibronectin) Adhesion Assay

U937 cell
adhesion assay was performed as previously reported [9, 18]. Briefly, U937
cells maintained in complete RPMI1640 medium (supplemented with 100 U/mL of penicillin and 100 μg/mL of streptomycin, and 10% FBS) were preincubated with hydroquinone for 1 hour at 37°C
and further incubated with activating (agonistic) antibodies (1 μg/mL)
in a 96-well plate. After a 3-hour incubation, cell-cell clusters were determined
by homotypic cell-cell adhesion assay using a hemocytometer [18] and analyzed with
an inverted light microscope equipped with a COHU high-performance CCD
(Diavert) video camera. For the cell-fibronectin adhesion assay, hydroquinone-treated
U937 cells (5×105 cells/well) were seeded on a fibronectin
(50 μg/mL)-coated
plate and incubated for 3 hours [19]. After removal of unbound cells with PBS, attached cells were
treated with 0.1% crystal violet for 15 minutes. The OD value at 540 nm was
measured by a Spectramax 250 microplate reader.


### 2.12. Flow Cytometric Analysis

Surface
levels of CD80, CD86, CD29, and CD18 on U937 or RAW264.7 cells were determined
by flow cytometric analysis as reported previously [18]. Stained cells were analyzed on
a FACScan device (Becton
Dickinson, San Jose, Calif., USA).


### 2.13. Preparation of Lymphocytes from Bone Marrow and Spleen and their Proliferation Assay

Splenocytes or bone marrow-derived cells (5×105 cells/well) were prepared from the spleens or bone marrow of mice
killed by cervical dislocation under sterile conditions, as described
previously [12]. Briefly, the splenocytes or
bone marrow-derived cells were released by teasing them into RPMI-1640 medium supplemented with 20 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]
(HEPES) buffer. The splenocytes or bone marrow-derived cells (5×106 cells/mL) were cultured in 96-well plates in the presence
and absence of
T or B lymphocyte mitogens (concanavalin A (Con A) and LPS) with hydroquinone in a total volume of 200 μL/well under
the same conditions for 48 hours. Proliferation was determined by MTT assay.


### 2.14. Statistic Analysis

Student’st-test and one-way
ANOVA were used to determine the statistical significance between experimental
and control groups. P values
of 0.05 or less were considered to be statistically significant.


## 2.1. Materials

L-cysteine, phorbol 12-myristate
13-acetate (PMA), curcumin, 1,4-dithiothreitol (DTT),α-tocopherol, dihydrorhodamine 123 (DHR123), peptidoglycan (PGN), zymosan,
lipopolysaccharide (LPS), α-tocopherol,
sodium nitroprusside (SNP), concanavalin (Con) A, fluorescein isothiocyanate
(FITC)-label dextran, and
hydroquinone were obtained from Sigma Chemical Co. (St. Louis, Mo, USA). BAY
11-7082 (BAY) was from Calbiochem (La Jolla, Calif, USA). RAW264.7 and U937 cells were
purchased from ATCC (Rockville,
Md, USA).
All other chemicals were of reagent grade. Aggregation-inducing antibody against
CD29 (MEM 101A, purified IgG1) was used as reported previously [9–11]. FITC-labeled antibodies to CD80,
CD86, CD29, and CD18 were obtained from Immunotech (Marseilles, France).


## 2.2. Animals

C57BL/6 male mice (6–8 weeks old, 17–21 g) were
obtained from DAEHAN BIOLINK (Chungbuk,
Korea). The mice
were maintained in plastic cages under conventional conditions. Water and
pelleted diets (Samyang, Daejeon,
Korea) were
supplied ad libitum. Studies
were performed in accordance with guidelines established by the Kangwon
University Institutional Animal Care and Use Committee.


## 2.3. Preparation of Peritoneal Macrophages

Peritoneal exudates were obtained from C57BL/6
male mice (7-8 weeks old,
17–21 g) by lavage four days after intraperitoneal injection of 1 mL sterile 4%
thioglycollate broth (Difco Laboratories, Detroit,
Mich, USA).
Cells were washed and resuspended in RPMI 1640 containing 2% FBS and plated in
100 mm tissues culture dishes for 4 hours at37°C in a 5% CO2 humidified atmosphere.


## 2.4. Cell Culture

RAW 264.7 and
U937 cells obtained from American Type Culture Collection (Rockville,
Md, USA)
were cultured with RPMI medium supplemented with 10% heat-inactivated fetal bovine
serum (Gibco, Grand Island,
NY, USA),
glutamine, and antibiotics (penicillin and streptomycin) at37°C with 5% CO2.


## 2.5. Cytokine Production

The inhibitory effect of hydroquinone on TNF-α production
was determined as previously described [12]. Hydroquinone was
solubilized with vehicle (100% ethanol) and diluted with RPMI1640. RAW264.7
cells (2×106 cells/mL) were incubated with LPS (2.5 μg/mL) in
the presence or absence of hydroquinone for 6 hours. Supernatants were assayed
for the levels of TNF-α, IL-1β, and IL-6 using a mouse TNF-α ELISA kit
(Amersham, Little Chalfont, Buckinghamshire,
UK).


## 2.6. No Assay

RAW 264.7
cells were preincubated with or without tested compounds (hydroquinone and
L-NAME) for 30 minutes and continuously activated with LPS (2.5μg/mL) for 24 hours. Nitrite determination
was carried out using Griess reagent [13]. The absorbance of the product dye
was measured at 540 nm using a flow-through spectrophotometer.


## 2.7. MTT Assay

Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay, as described previously [12].


## 2.8. Extraction of Total RNA and Semiquantitative RT-PCRAmplification

The total RNA from LPS-treated RAW264.7 cells was prepared by
adding TRIzol reagent (Gibco BRL), according to the manufacturer’s protocol.
Semiquantitative RT reactions were conducted using MuLV reverse transcriptase
as reported previously [14]. Primers (Table 1)
(Bioneer, Daejeon, Korea) were used as previously reported [15].Table 1
Primers used for RT-PCR
analysis (F: forward,
R: reverse).
GenePrimer sequencesTNF-αF5′-TTGACCTCAGCGCTGAGTTG-3′R5′-CCTGTAGCCCACGTCGTAGC-3′IL-1βF5′-CAGGATGAGGACATGAGCACC-3′R5′-CTCTGCAGACTCAAACTCCAC-3′IL-6F5′-GTACTCCAGAAGACCAGAGG-3′R5′-TGCTGGTGACAACCACGGCC-3′iNOSF5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′R5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3′GAPDHF5′-CACTCACGGCAAATTCAACGGCAC-3′R5′-GACTCCACGACATACTCAGCAC-3′


## 2.9. ROS Determination

The level of intracellular ROS was
determined by the change in fluorescence resulting from oxidation of the
fluorescent probe DHR123 [16]. Briefly, 5×105 cells/well were incubated
with hydroquinone for 30 minutes and then with SNP (125 μM) for an additional 6 hours. After a final
incubation with 2.5 μM
DHR123 for 1 hour, the intracellular ROS level was determined using flow
cytometry.


## 2.10. Phagocytic Uptake

To measure the phagocytic activity
of RAW264.7 cells, a  previously reported  method was used with slight
modifications [17]. RAW 264.7
cells (1×106 cells/mL) were preincubated with or without hydroquinone
for 30 minutes, and further incubated for 6 hours. Finally, the cells were
further incubated with FITC-dextran (1 mg/mL) for 30 minutes at 37°C. The
incubation was stopped by addition of 2 mL of ice-cold PBS, and the
cells were washed four times with cold PBS. After fixing the cells with 3.7%
formaldehyde, phagocytic uptake was analyzed using
a FACScan device (Becton Dickinson, San Jose, Calif,
USA).


## 2.11. Cell-Cell or Cell-Extracellular Matrix Protein (Fibronectin) Adhesion Assay

U937 cell
adhesion assay was performed as previously reported [9, 18]. Briefly, U937
cells maintained in complete RPMI1640 medium (supplemented with 100 U/mL of penicillin and 100 μg/mL of streptomycin, and 10% FBS) were preincubated with hydroquinone for 1 hour at 37°C
and further incubated with activating (agonistic) antibodies (1 μg/mL)
in a 96-well plate. After a 3-hour incubation, cell-cell clusters were determined
by homotypic cell-cell adhesion assay using a hemocytometer [18] and analyzed with
an inverted light microscope equipped with a COHU high-performance CCD
(Diavert) video camera. For the cell-fibronectin adhesion assay, hydroquinone-treated
U937 cells (5×105 cells/well) were seeded on a fibronectin
(50 μg/mL)-coated
plate and incubated for 3 hours [19]. After removal of unbound cells with PBS, attached cells were
treated with 0.1% crystal violet for 15 minutes. The OD value at 540 nm was
measured by a Spectramax 250 microplate reader.


## 2.12. Flow Cytometric Analysis

Surface
levels of CD80, CD86, CD29, and CD18 on U937 or RAW264.7 cells were determined
by flow cytometric analysis as reported previously [18]. Stained cells were analyzed on
a FACScan device (Becton
Dickinson, San Jose, Calif., USA).


## 2.13. Preparation of Lymphocytes from Bone Marrow and Spleen and their Proliferation Assay

Splenocytes or bone marrow-derived cells (5×105 cells/well) were prepared from the spleens or bone marrow of mice
killed by cervical dislocation under sterile conditions, as described
previously [12]. Briefly, the splenocytes or
bone marrow-derived cells were released by teasing them into RPMI-1640 medium supplemented with 20 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]
(HEPES) buffer. The splenocytes or bone marrow-derived cells (5×106 cells/mL) were cultured in 96-well plates in the presence
and absence of
T or B lymphocyte mitogens (concanavalin A (Con A) and LPS) with hydroquinone in a total volume of 200 μL/well under
the same conditions for 48 hours. Proliferation was determined by MTT assay.


## 2.14. Statistic Analysis

Student’st-test and one-way
ANOVA were used to determine the statistical significance between experimental
and control groups. P values
of 0.05 or less were considered to be statistically significant.


## 3. Results

### 3.1. Effect of Hydroquinone on Macrophage Activation

Previously, we reported that hydroquinone strongly diminishes
LPS-induced TNF-α production
with an IC50 value of 14.8 μM.
In this study, we  examined its inhibitory effect on other inflammatory
cytokines including IL-1β and IL-6.  Figure 2(a) shows that hydroquinone suppressed
production of cytokines without altering cell viability (data not shown). The
inhibition of these cytokines also appeared at the transcriptional level (Figure 2(b)), as we had previously shown for TNF-α production.Effect of hydroquinone on the production of cytokines and
their mRNA expression in LPS-activated RAW264.7 cells.
(a) RAW264.7
cells (2×106 cells/mL) were incubated with indicated concentrations
of HQ (hydroquinone) in the presence or absence of LPS
(2 μg/mL)
for 12 hours. Secreted levels of TNF-α, IL-1β,
and IL-6 in culture supernatant were determined by ELISA. (b) The mRNA levels of
proinflammatory cytokines from the RAW264.7 cells were determined by
semiquantitative RT-PCR. The
results show one representative experiment of three. p*<.05 and p**<.01  represent significant
difference as compared to control.
(a)(b)Since we had also previously reported
that hydroquinone could modulate NO production, we next characterized
hydroquinone inhibition of NO production under various stimulation conditions.
Figure3(a) shows that hydroquinone inhibited zymosan-induced NO production, whereas
LPS-induced NO production was only weakly blocked, suggesting that dectin-1 and
TLR2-mediated NO production pathways might be more sensitive targets of
hydroquinone. Furthermore, hydroquinone-mediated inhibition of peritoneal
macrophage-mediated NO production was also observed (Figure 3(b)). Thus,
hydroquinone (as well as the PI3K inhibitors LY294002 and wortmannin) concentration-dependently
suppressed NO production in LPS-treated macrophages, whereas the antioxidant α-tocopherol did not affect NO production, even under
effective antioxidative conditions. Moreover, hydroquinone also diminished LPS-induced
iNOS expression (Figure 3(c)), as reported previously [8].Effects
of hydroquinone on the production of NO in LPS-treated RAW 264.7 cells or
peritoneal macrophages. (a) and (b) RAW264.7 cells or peritoneal macrophages (1×106) were pretreated with various
concentrations of HQ
(hydroquinone) in the presence or absence of LPS (2 μg/mL), PGN
(10 μg/mL),
or
zymosan (400 μg/mL) for
24 hours. The level of NO was determined by Griess reagent as described in
Section 2. (c) The mRNA levels of proinflammatory
cytokines from RAW264.7 cells were determined by semiquantitative RT-PCR. The results show one representative
experiment of three. p*<.05 and p**<.01 represent
significant difference as compared to control.
(a)(b)(c)ROS generation is an important event
in inflammatory responses. We, therefore, examined the ROS scavenging activity of
hydroquinone. As reported previously, hydroquinone effectively scavenged
radical activity as determined by DPPH assay (Figure4(a)). Furthermore, this
compound also neutralized radical generation in SNP-treated RAW264.7 cells, as was
the case for α-tocopherol (Figure 4(b)).Effects
of hydroquinone on ROS generation in SNP-induced RAW 264.7 cells. (a) Antioxidant
effect of hydroquinone was examined by DPPH assay as described in Section2. (b)
RAW264.7 cells (1×106) were pretreated with various
concentrations of HQ
(hydroquinone) in the presence or absence of SNP (100 μM) for 30
minutes. The level of generated ROS was determined by flowcytometric analysis
as described in Section 2 . p*<.05 and p**<.01 represent
significant difference as compared to control.
(a)(b)Since phagocytosis is representative
of inflammatory responses, the effects of hydroquinone on phagocytic uptake of
FITC-dextran were investigated. Figure5 shows that hydroquinone concentration-dependently
suppressed dextran uptake, suggesting that it could block the initial response
of macrophage activation. Figure 6 shows that hydroquinone and BAY 11-7082 suppressed
the upregulation of CD80 and CD86 by LPS-induced dextran uptake. However, hydroquinone
did not block normal expression of CD80 and CD86 and rather enhanced it in both
RAW264.7 (Figure 6(a)) and U937 cells (Figure 6(b)).Effect of hydroquinone on the phagocytic uptake of FITC-labeled dextran. RAW264.7 cells (1×106)
were incubated with in the indicated
concentrations of HQ (hydroquinone) in the presence or absence of 1 mg/mL of FITC-labeled dextran for 30 minutes. The uptake of dextran was detected by flow cytometric analysis as
described in Section 2. p**<.01 represents significant difference as compared to control.
(a)(b)Effect of hydroquinone on surface expression of costimulatory molecules. (a) and (b) RAW264.7 or U937 cells (1×106)
were incubated with indicated
concentrations of HQ (hydroquinone) in the presence or absence of LPS (2 μg/mL) for 24 hours. The surface expression of CD80 and CD86 was determined
by flow cytometric analysis as described in Section 2. p**<.01 represents significant
difference as compared to control.
(a)(b)Figure7 shows that 25 μM hydroquinone failed to suppress morphological changes of
RAW264.7 cells induced by PMA-mediated actin cytoskeleton rearrangement. These
data suggest that the pharmacological effects of hydroquinone might occur in a
signal-dependent manner.Figure 7
Effect of hydroquinone on PMA-induced morphological changes. RAW264.7 cells (1×105)
were incubated with indicated
concentrations of HQ (hydroquinone) in the presence or absence of PMA (10 ng/mL) for 72 hours. The images
of the cells in culture
were obtained using an inverted phase contrast microscope attached to a video
camera.


### 3.2. Effect of Hydroquinone on the Cell Adhesion of Monocytes

Since we
previously reported the effects of hydroquinone on CD29-mediated cell-cell
adhesion, in this study we evaluated fibronectin-cell adhesion and
CD18-mediated cell-cell aggregation. Figures8(a) and
8(b) show that CD18-mediated
cell-cell adhesion  induced by PMA and CD29-mediated cell-cell adhesion
was strongly suppressed by hydroquinone treatment. In contrast, hydroquinone
did not block cell-fibronectin adhesion events (Figure 8(c)), indicating that
hydroquinone might specifically modulate cell-cell adhesion events and not
cell-matrix adhesion. Because these adhesion models are known to depend on CD29
and CD18, the effects of hydroquinone on surface expression of these adhesion
molecules were additionally
examined. Figure 8(d) shows that hydroquinone treatment significantly diminished
surface levels of CD29 and CD18; such downregulation might comprise an
inhibitory mechanism for monocyte adhesion.Effect of hydroquinone
on cell-cell or cell-fibronectin adhesion events and surface levels of
integrins (CD18 and CD29). (a) and (b) U937 cells (1×106)
were incubated with indicated
concentrations of HQ (hydroquinone) in the
presence or absence of a proaggregation (activating) antibody against CD29
(MEM101A (1 μg/mL))  for
2 hours. The extent of cell-cell aggregation was determined by quantitative
cell-cell adhesion assay as described in Materials and Methods. (b) Images of cells in culture were obtained using an inverted phase contrast
microscope attached to a video camera. (c) U937 cells pretreated with
hydroquinone were seeded on fibronectin
(50 μg/mL)-coated
plates and further incubated for 3 hours. Attached cells were determined by
crystal violet assay, as
described in Section 2. (d) U937 cells were incubated with hydroquinone (12.5 μM) for 3 hours.
The surface expression
of adhesion molecules (CD18 and CD29) was determined by flow cytometric
analysis as described in Section 2. p**<.01 represents significant difference as compared to control.
(a)(b)(c)(d)


### 3.3. Effect of Hydroquinone on the Proliferation of Lymphocytes from Bone Marrow or Spleen

Lymphocyte
proliferation is important for prolonged immune responses. Therefore, we determined
whether hydroquinone could modulate lymphocyte proliferation induced by the T
cell mitogen Con A or the B cell mitogen LPS. Figure9(a) shows that hydroquinone concentration-dependently
suppressed the normal viability of splenocytes and bone marrow-derived cells.
Figure 9(b) shows, however, that T cell and B cell responses to hydroquinone varied
according to tissue origin. LPS-induced B cell proliferation from bone
marrow-derived cells was not blocked by 25 μM hydroquinone, whereas Con
A-induced T cell proliferation was greatly suppressed. In contrast, Con A- and
LPS-induced proliferation of T and B cells from splenocytes showed similar levels
of inhibition, suggesting that sensitivity of lymphocytes to hydroquinone might
be tissue dependent.Effect of hydroquinone on the viablility or proliferation
of splenic and bone marrow-derived lymphocytes induced by Con A and LPS. (a) Splenic
lymphocytes (5×106 cells/mL) were incubated with hydroquinone
under normal culture conditions. (b) Bone
marrow-derived lymphocytes (5×106 cells/mL) were incubated
with hydroquinone in the presence or absence of Con A (1 μg/mL) and LPS (10 μg/mL). Cell viability or proliferation was
determined by MTT assay. p*<.05 and p**<.01 represent
significant difference as compared to normal or control.
(a)(b)


### 3.4. Effect of Thiol-Containing Compounds on Hydroquinone-Mediated Inhibition

Finally, the  importance of thiolation by hydroquinone was
explored using NO production and bone marrow cell proliferation. Interestingly,
two thiol-containing compounds, L-cysteine, and DTT abolished the inhibitory
effects of hydroquinone on NO production and T cell proliferation from bone
marrow-derived cells (Figure10). These results suggest that hydroquinone
inhibition might require thiolation  of intracellular target proteins.Figure 10
Effect
of thiol-containing compounds on hydroquione-mediated inhibition of NO
production  and lymphocyte proliferation. (a) RAW264.7 cells (1×106)
were pretreated with L-cysteine (800 μM)
or DTT (100 μM) and HQ
(hydroquinone, 25 μM) in the presence
or absence of LPS
(2.5 μg/mL) for
24 hours. The level of NO was determined by Griess reagent as described in Section
2. (b) Bone marrow-derived cells were
incubated with L-cysteine (800 μM) or DTT (100 μM) and
hydroquinone (25 μM) in the presence or absence of Con A (1 μg/mL) for 24 hours. p**<.01 and p##<.01 represent
significant difference as compared to control or HQ-treated group.Figure 11
Schematic diagram of hydroquinone-mediated inhibition.


## 3.1. Effect of Hydroquinone on Macrophage Activation

Previously, we reported that hydroquinone strongly diminishes
LPS-induced TNF-α production
with an IC50 value of 14.8 μM.
In this study, we  examined its inhibitory effect on other inflammatory
cytokines including IL-1β and IL-6.  Figure 2(a) shows that hydroquinone suppressed
production of cytokines without altering cell viability (data not shown). The
inhibition of these cytokines also appeared at the transcriptional level (Figure 2(b)), as we had previously shown for TNF-α production.Effect of hydroquinone on the production of cytokines and
their mRNA expression in LPS-activated RAW264.7 cells.
(a) RAW264.7
cells (2×106 cells/mL) were incubated with indicated concentrations
of HQ (hydroquinone) in the presence or absence of LPS
(2 μg/mL)
for 12 hours. Secreted levels of TNF-α, IL-1β,
and IL-6 in culture supernatant were determined by ELISA. (b) The mRNA levels of
proinflammatory cytokines from the RAW264.7 cells were determined by
semiquantitative RT-PCR. The
results show one representative experiment of three. p*<.05 and p**<.01  represent significant
difference as compared to control.
(a)(b)Since we had also previously reported
that hydroquinone could modulate NO production, we next characterized
hydroquinone inhibition of NO production under various stimulation conditions.
Figure3(a) shows that hydroquinone inhibited zymosan-induced NO production, whereas
LPS-induced NO production was only weakly blocked, suggesting that dectin-1 and
TLR2-mediated NO production pathways might be more sensitive targets of
hydroquinone. Furthermore, hydroquinone-mediated inhibition of peritoneal
macrophage-mediated NO production was also observed (Figure 3(b)). Thus,
hydroquinone (as well as the PI3K inhibitors LY294002 and wortmannin) concentration-dependently
suppressed NO production in LPS-treated macrophages, whereas the antioxidant α-tocopherol did not affect NO production, even under
effective antioxidative conditions. Moreover, hydroquinone also diminished LPS-induced
iNOS expression (Figure 3(c)), as reported previously [8].Effects
of hydroquinone on the production of NO in LPS-treated RAW 264.7 cells or
peritoneal macrophages. (a) and (b) RAW264.7 cells or peritoneal macrophages (1×106) were pretreated with various
concentrations of HQ
(hydroquinone) in the presence or absence of LPS (2 μg/mL), PGN
(10 μg/mL),
or
zymosan (400 μg/mL) for
24 hours. The level of NO was determined by Griess reagent as described in
Section 2. (c) The mRNA levels of proinflammatory
cytokines from RAW264.7 cells were determined by semiquantitative RT-PCR. The results show one representative
experiment of three. p*<.05 and p**<.01 represent
significant difference as compared to control.
(a)(b)(c)ROS generation is an important event
in inflammatory responses. We, therefore, examined the ROS scavenging activity of
hydroquinone. As reported previously, hydroquinone effectively scavenged
radical activity as determined by DPPH assay (Figure4(a)). Furthermore, this
compound also neutralized radical generation in SNP-treated RAW264.7 cells, as was
the case for α-tocopherol (Figure 4(b)).Effects
of hydroquinone on ROS generation in SNP-induced RAW 264.7 cells. (a) Antioxidant
effect of hydroquinone was examined by DPPH assay as described in Section2. (b)
RAW264.7 cells (1×106) were pretreated with various
concentrations of HQ
(hydroquinone) in the presence or absence of SNP (100 μM) for 30
minutes. The level of generated ROS was determined by flowcytometric analysis
as described in Section 2 . p*<.05 and p**<.01 represent
significant difference as compared to control.
(a)(b)Since phagocytosis is representative
of inflammatory responses, the effects of hydroquinone on phagocytic uptake of
FITC-dextran were investigated. Figure5 shows that hydroquinone concentration-dependently
suppressed dextran uptake, suggesting that it could block the initial response
of macrophage activation. Figure 6 shows that hydroquinone and BAY 11-7082 suppressed
the upregulation of CD80 and CD86 by LPS-induced dextran uptake. However, hydroquinone
did not block normal expression of CD80 and CD86 and rather enhanced it in both
RAW264.7 (Figure 6(a)) and U937 cells (Figure 6(b)).Effect of hydroquinone on the phagocytic uptake of FITC-labeled dextran. RAW264.7 cells (1×106)
were incubated with in the indicated
concentrations of HQ (hydroquinone) in the presence or absence of 1 mg/mL of FITC-labeled dextran for 30 minutes. The uptake of dextran was detected by flow cytometric analysis as
described in Section 2. p**<.01 represents significant difference as compared to control.
(a)(b)Effect of hydroquinone on surface expression of costimulatory molecules. (a) and (b) RAW264.7 or U937 cells (1×106)
were incubated with indicated
concentrations of HQ (hydroquinone) in the presence or absence of LPS (2 μg/mL) for 24 hours. The surface expression of CD80 and CD86 was determined
by flow cytometric analysis as described in Section 2. p**<.01 represents significant
difference as compared to control.
(a)(b)Figure7 shows that 25 μM hydroquinone failed to suppress morphological changes of
RAW264.7 cells induced by PMA-mediated actin cytoskeleton rearrangement. These
data suggest that the pharmacological effects of hydroquinone might occur in a
signal-dependent manner.Figure 7
Effect of hydroquinone on PMA-induced morphological changes. RAW264.7 cells (1×105)
were incubated with indicated
concentrations of HQ (hydroquinone) in the presence or absence of PMA (10 ng/mL) for 72 hours. The images
of the cells in culture
were obtained using an inverted phase contrast microscope attached to a video
camera.


## 3.2. Effect of Hydroquinone on the Cell Adhesion of Monocytes

Since we
previously reported the effects of hydroquinone on CD29-mediated cell-cell
adhesion, in this study we evaluated fibronectin-cell adhesion and
CD18-mediated cell-cell aggregation. Figures8(a) and
8(b) show that CD18-mediated
cell-cell adhesion  induced by PMA and CD29-mediated cell-cell adhesion
was strongly suppressed by hydroquinone treatment. In contrast, hydroquinone
did not block cell-fibronectin adhesion events (Figure 8(c)), indicating that
hydroquinone might specifically modulate cell-cell adhesion events and not
cell-matrix adhesion. Because these adhesion models are known to depend on CD29
and CD18, the effects of hydroquinone on surface expression of these adhesion
molecules were additionally
examined. Figure 8(d) shows that hydroquinone treatment significantly diminished
surface levels of CD29 and CD18; such downregulation might comprise an
inhibitory mechanism for monocyte adhesion.Effect of hydroquinone
on cell-cell or cell-fibronectin adhesion events and surface levels of
integrins (CD18 and CD29). (a) and (b) U937 cells (1×106)
were incubated with indicated
concentrations of HQ (hydroquinone) in the
presence or absence of a proaggregation (activating) antibody against CD29
(MEM101A (1 μg/mL))  for
2 hours. The extent of cell-cell aggregation was determined by quantitative
cell-cell adhesion assay as described in Materials and Methods. (b) Images of cells in culture were obtained using an inverted phase contrast
microscope attached to a video camera. (c) U937 cells pretreated with
hydroquinone were seeded on fibronectin
(50 μg/mL)-coated
plates and further incubated for 3 hours. Attached cells were determined by
crystal violet assay, as
described in Section 2. (d) U937 cells were incubated with hydroquinone (12.5 μM) for 3 hours.
The surface expression
of adhesion molecules (CD18 and CD29) was determined by flow cytometric
analysis as described in Section 2. p**<.01 represents significant difference as compared to control.
(a)(b)(c)(d)


## 3.3. Effect of Hydroquinone on the Proliferation of Lymphocytes from Bone Marrow or Spleen

Lymphocyte
proliferation is important for prolonged immune responses. Therefore, we determined
whether hydroquinone could modulate lymphocyte proliferation induced by the T
cell mitogen Con A or the B cell mitogen LPS. Figure9(a) shows that hydroquinone concentration-dependently
suppressed the normal viability of splenocytes and bone marrow-derived cells.
Figure 9(b) shows, however, that T cell and B cell responses to hydroquinone varied
according to tissue origin. LPS-induced B cell proliferation from bone
marrow-derived cells was not blocked by 25 μM hydroquinone, whereas Con
A-induced T cell proliferation was greatly suppressed. In contrast, Con A- and
LPS-induced proliferation of T and B cells from splenocytes showed similar levels
of inhibition, suggesting that sensitivity of lymphocytes to hydroquinone might
be tissue dependent.Effect of hydroquinone on the viablility or proliferation
of splenic and bone marrow-derived lymphocytes induced by Con A and LPS. (a) Splenic
lymphocytes (5×106 cells/mL) were incubated with hydroquinone
under normal culture conditions. (b) Bone
marrow-derived lymphocytes (5×106 cells/mL) were incubated
with hydroquinone in the presence or absence of Con A (1 μg/mL) and LPS (10 μg/mL). Cell viability or proliferation was
determined by MTT assay. p*<.05 and p**<.01 represent
significant difference as compared to normal or control.
(a)(b)


## 3.4. Effect of Thiol-Containing Compounds on Hydroquinone-Mediated Inhibition

Finally, the  importance of thiolation by hydroquinone was
explored using NO production and bone marrow cell proliferation. Interestingly,
two thiol-containing compounds, L-cysteine, and DTT abolished the inhibitory
effects of hydroquinone on NO production and T cell proliferation from bone
marrow-derived cells (Figure10). These results suggest that hydroquinone
inhibition might require thiolation  of intracellular target proteins.Figure 10
Effect
of thiol-containing compounds on hydroquione-mediated inhibition of NO
production  and lymphocyte proliferation. (a) RAW264.7 cells (1×106)
were pretreated with L-cysteine (800 μM)
or DTT (100 μM) and HQ
(hydroquinone, 25 μM) in the presence
or absence of LPS
(2.5 μg/mL) for
24 hours. The level of NO was determined by Griess reagent as described in Section
2. (b) Bone marrow-derived cells were
incubated with L-cysteine (800 μM) or DTT (100 μM) and
hydroquinone (25 μM) in the presence or absence of Con A (1 μg/mL) for 24 hours. p**<.01 and p##<.01 represent
significant difference as compared to control or HQ-treated group.Figure 11
Schematic diagram of hydroquinone-mediated inhibition.


## 4. Discussion

In this study, we explored the effect of hydroquinone on
inflammatory responses mediated by monocytes, macrophages, and lymphocytes. Our
results suggest that hydroquinone can strongly suppress cellular responses
required for inflammation, including cytokine production (Figure2), NO
generation (Figure 3), ROS release (Figure 4), phagocytic uptake
(Figure 5), surface
expression of costimulatory molecules (Figure 6), U937 cell-cell adhesion
mediated by CD29 and CD18 (Figure 8), and the proliferation of lymphocytes
isolated from bone marrow and spleen (Figure 9).The molecular mechanism of hydroquinone-mediated downregulation of
cellular inflammatory responses is not yet fully understood. Recently, we and others
have reported that hydroquinone can reduce the DNA binding of NF-κB [5, 20]
and block the phosphorylation of Akt [8].
Although hydroquinone has been reported as a good NF-κB inhibitor, several lines of evidence support that it might block
upstream signaling of either NF-κB activation or innate and adaptive immune responses. In the present
study, the maximum anti-TNF-α
effect of hydroquinone was observed when treatment occurred before LPS administration;
this suggests that early signaling events occurring within 30 minutes might be
potential hydroquinone  targets [8].
Additionally, hydroquinone treatment still blocked NF-κB-independent cellular responses such as integrin-mediated cell adhesion [21]
and phagocytic uptake (Figure 5). Since the activation of PI3K/Akt, but not MAPKs,
is necessary for these inflammation-related cellular events as well as for
activation of NF-κB [22],
it is possible that hydroquinone might inhibit PI3K/Akt. This is supported by a
previous study in which hydroquinone diminished the phosphorylation of Akt, and
where the PI3K inhibitors LY294002 and wortmannin displayed broad spectrum
inhibition of inflammatory responses [8]
similar to that observed for hydroquinone in the present study.Recently, hydroquinone  has been reported to be a potent antioxidant with
radical  scavenging activities
[6, 23],
and to induce hemeoxygenase-1 expression. [8] similarly
to most antioxidants. However, we unexpectedly
found that hydroquinone itself was able to enhance radical generation in
RAW264.7 cells to similar extent as LPS [8],
suggesting a role as a strong pro-oxidant agent with chemical reactivity [24].
Interestingly, we found that hydroquinone reacts with sulfhydryl groups of
thiol compounds, such as L-cysteine, DTT, and N-acetyl-L-cysteine (Figure 10). This
chemical reactivity appears to depend on the position of hydroxyl groups in the
benzene backbone. Hydroxyl groups in the para position were confirmed to be the most reactive, since catechol with ortho groups and resorcinol with meta groups showed reduced inhibitory
effects [25].
Similar modes
of action were reported for sesquiterpene lactone compounds with reactive α-butyro-γ-lactone rings, where these involved the
direct binding of NF-κB to DNAvia direct thiolation of 165-cysteine residue of the p65 NF-κB subunit [12, 26]. The
effects of hydroquinone may thus occur as the result of direct binding to
L-cysteine residues critically involved in the activity of target proteins such
as  PI3K/Akt.We and other groups have proposed
that hydroquinone could have potential curative effects in inflammatory disease
[6, 8, 25]. Nonetheless, environmental toxicity
is an important consideration since hydroquinone is topically applied for
skin whitening. Because of its potential cancer risk, some countries (e.g., France) have banned
the use of this compound. The possible adverse effects are thought to arise
from its reactivity, as it is considered the most potent DNA-damaging
benzene  metabolite. Furthermore, the  reactivity of hydroquinone is
known to be  increased in
the presence of NO [27].Hydroquinone
is also a major component of cigarette smoke and may cause increased rates of
higher respiratory tract infection in chronic cigarette smokers [28, 29]. Our results suggest that these
immunopathological phenomena might be due to immunosuppressive effects of
hydroquinone. In the present study, hydroquinone suppressed most stages of
innate immunity mediated by monocytes and macrophages, and of adaptive immunity
by lymphocytes, as summarized in Figure 11. Our
results suggest that the initial
activation of macrophages by bacterial infection might be diminished by
hydroquinone exposure. Additionally, hydroquinone might inhibit the migration
of monocytes from the blood  to infected areas and the aggregation of these
cells at infection sites, since it blocked upregulation of integrins (CD18 and
CD29) and their functional activation as assessed by homotypic aggregation of
monocytic cells. Finally, hydroquinone reduced the normal viability of
lymphocytes from bone  marrow and spleen as well as their LPS- and Con A-induced
proliferation (Figure 9). Previous
reports have suggested that inhibition of IFN-γ production from activated T cells and IL-12 production from activated
macrophages was blocked by hydroquinone  treatment and supported an inhibitory
role in Th1-type lymphocytes [30–32].In conclusion, we report that hydroquinone
negatively regulates the functional activation of monocytes, macrophages, and
lymphocytes (summarized in Figure11) with respect to proinflammatory
cytokine production, secretion of cytotoxic molecules, phagocytic uptake,
costimulatory molecule expression, monocytic cell-cell adhesion, and lymphocyte
proliferation. These effects appear to be mediated by reactions with thiol
groups of L-cysteines in target proteins. Considering that (1) hydroquinone is a
major component of cigarette smoke and that (2) immunoprotection
of chronic cigarette smokers against lung infections is greatly reduced [29, 33],
our data suggest that hydroquinone and other benzene metabolites might play a
role in various immunotoxicological conditions.



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*Source: 298010-2009-01-14.xml*