Effects of 2,2',4,4'-Tetrachlorobiphenyl on Granulocytic HL-60 Cell Function and Expression of Cyclooxygenase-2
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《毒物学科学杂志》
Department of Pharmacology and Toxicology, Center for Integrative Toxicology and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824
ABSTRACT
Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that affect a number of cellular systems, including neutrophils. It has been demonstrated that noncoplanar PCBs (i.e., ortho- substituted PCBs) alter function of primary rat neutrophils. The objectives of these experiments were to determine if responses in a human, neutrophil-like cell line exposed to PCBs were similar to those reported for rat neutrophils and to explore further PCB-mediated alterations in neutrophil function. The human promyelocytic leukemia cell line (HL-60) was differentiated with DMSO to a neutrophil-like phenotype. Treatment of differentiated HL-60 cells with 2,2',4,4'-tetrachlorobiphenyl, a noncoplanar, ortho-substituted PCB congener, caused an increase in f-Met-Leu-Phe-induced degranulation, as measured by release of myeloperoxidase (MPO). Treatment with the coplanar, non-ortho-substituted congener 3,3',4,4'-tetrachlorobiphenyl had no effect on MPO release. 2,2',4,4'-Tetrachlorobiphenyl caused a time- and dose-dependent release of [3H]-arachidonic acid (3H-AA). A significant increase in 3H-AA release was observed after 60 min of exposure, and concentrations of 10 μM or larger increased 3H-AA release. In contrast, 3,3',4,4'-tetrachlorobiphenyl had no effect on 3H-AA release. The effect of PCBs on mRNA levels for cyclooxygenase-2 (COX-2) was examined using semiquantitative RT-PCR. COX-2 mRNA was significantly elevated in response to 2,2',4,4'-tetrachlorobiphenyl in a concentration-dependent manner. COX-2 expression was maximal by 30 min of exposure to 2,2',4,4'-tetrachlorobiphenyl. COX-2 protein and activity were also increased after exposure to 2,2',4,4'-tetrachlorobiphenyl; COX-1 protein and activity were unaffected. 3,3',4,4'-Tetrachlorobiphenyl did not increase COX-2 mRNA levels. These results demonstrate that a noncoplanar PCB alters the functional status of granulocytic HL-60 cells, causing enhanced degranulation and upregulation of COX-2, whereas a coplanar PCB lacks this activity. These data suggest that noncoplanar PCBs alter HL-60 cell function and COX-2 expression via an Ah-receptor-independent mechanism.
Key Words: PCBs; neutrophils; cyclooxygenase-2; noncoplanar; arachidonic acid.
INTRODUCTION
Polychlorinated biphenyls (PCBs) are ubiquitous, man-made, environmental contaminants that have been shown to affect a number of cellular systems. PCBs were used widely for over 40 years due to their heat resistant properties, low conductivity, and chemical inertness. These properties made PCBs a very useful component of safety fluids used to insulate and cool heavy electrical equipment such as transformers and capacitors (Hutzinger et al., 1974). The lipophilic nature of PCBs and their resistance to chemical transformation allow them to bioaccumulate in the food chain (Kannan et al., 1998). This has led to exposures of humans and wildlife to PCBs. PCBs have been detected in human blood, milk, adipose tissue, and placental tissue (Giesy and Kannan, 1998; Laden et al., 1999).
PCBs exhibit a wide range of biological effects in animals. They are probable human carcinogens (Cogliano, 1998) and cause a number of noncancer effects, including dermal lesions and ocular effects in humans, and hepatotoxicity and endocrine effects in rats (Morse et al., 1996; Takamatsu et al., 1985; Twaroski et al., 2001). In addition, PCBs have effects on the reproductive system (Faqi et al., 1998), nervous system (Jacobson and Jacobson, 1997), cardiovascular system (Warshaw et al., 1979), and both the specific and nonspecific branches of the immune system (Vos and VanLoveren, 1998).
The specific effects PCBs produce are related to their structure. PCBs can be divided into two broad classes based on the presence or absence of chlorine atoms in the ortho- position. Congeners that lack chlorines in the ortho- positions are coplanar and bind with high affinity to the aryl hydrocarbon (Ah) receptor to induce changes in cellular function. Congeners containing chlorines substituted at the ortho- positions cannot attain a coplanar configuration and are, in general, poor ligands for the Ah receptor. The mechanisms by which these noncoplanar PCBs cause functional changes in cells are not well understood (Bandiera et al., 1982).
Among the effects caused by ortho-substituted, noncoplanar PCB congeners is the alteration in function of polymorphonuclear neutrophils (Brown and Ganey, 1995; Kristoffersen et al., 2002; Olivero and Ganey, 2001; Olivero-Verbel and Ganey, 1998; Voie et al., 2000). The primary role of neutrophils is to attack and destroy invading microorganisms. They are normally quiescent and exhibit their biological activity when activated. Alteration in neutrophil function can have serious consequences for the organism. Failure of neutrophils to activate results in compromise of the innate immune system, leading to increased risk of infection. At the other end of the spectrum, inappropriate activation of neutrophils can lead to inflammatory disease states and injury to host tissue. Noncoplanar PCBs cause many changes in function of primary rat neutrophils including stimulation of degranulation, production of reactive oxygen species such as superoxide anion, release of arachidonic acid (AA), and altered response to subsequent stimulation with other agents (Brown and Ganey, 1995; Ganey et al., 1993; Olivero and Ganey, 2000; Tithof et al., 1998). Given the role of neutrophils in host defense and the requirement for activation, understanding the mechanisms by which neutrophils become activated in response to exposure to PCBs is important.
One fate of AA released in response to PCB exposure is that it may serve as a precursor for eicosanoid synthesis, which is catalyzed by the cyclooxygenase enzymes. One isoform of cyclooxygenase, cyclooxygenase-2 (COX-2) is present in negligible amounts in quiescent neutrophils, but can be induced by a variety of inflammatory stimuli (Smith et al., 2000; Vane et al., 1998). It was of interest to determine whether COX-2 expression is affected by PCBs. In this work we used the human, promyelocytic leukemia cell line, HL-60, to examine the effect of in vitro exposure to PCBs on COX-2 mRNA levels. HL-60 cells have the capacity to differentiate to a number of different, functionally and morphologically distinct forms (Collins, 1987) including a neutrophil-like phenotype, and differentiated HL-60 cells have been a useful model with which to study neutrophil responses (Arroyo et al., 2002; Mullick et al., 2004).
MATERIALS AND METHODS
Chemicals.
2,2',4,4'-Tetrachlorobiphenyl (2244-TCB; >99% pure) and 3,3',4,4'-tetrachlorobiphenyl (3344-TCB; >99% pure) were purchased from ChemService (West Chester, PA). N-formyl-methionyl-leucyl-phenylalanine (fMLP) was purchased from Sigma Chemical Co. (St. Louis, MO). [3H-5,6,8,9,11,12,14,15]-Arachidonic acid (3H-AA; 180–240 Ci/mmol) was purchased from DuPont (Boston, MA). All other chemicals were of the highest grade commercially available.
HL-60 cells.
HL-60 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA). Cells between passage 20 and 45 were grown in Iscove's Modified Dulbecco's Medium supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 0.1% gentamicin and 0.9% antibiotic-antimycotic (Invitrogen Corporation, Carlsbad, CA). Cultures were maintained in a humidified incubator at 37°C in a controlled atmosphere of 5% CO2. Cells were induced to differentiate along the granulocyte pathway by culturing in the presence of 1.25% dimethyl sulfoxide (DMSO) for 5 days. Cells were then maintained an additional 2 days in the absence of DMSO. After this procedure, approximately 80% of the cells had nuclear appearance characteristic of neutrophils. In addition, these cells reduced nitro blue tetrazolium, a functional marker of differentiation to a granulocytic phenotype (Hua et al., 2000).
Exposure to polychlorinated biphenyls.
Stock solutions of the PCBs were prepared by dissolution in dimethylformamide (DMF). The differentiated HL-60 cells were suspended in HBSS in borosilicate glass test tubes, 12 x 75 mm (VWR, Chicago, IL), and 1 μl of the stock solution was added per ml of cells to achieve the desired concentration. Control cells
Cellular degranulation.
Degranulation was measured by the release of the enzyme, myeloperoxidase (MPO). Differentiated HL-60 cells (2 x 106) were suspended in HBSS and exposed to PCBs at 37°C for 30, 60, 90, or 120 min. fMLP (10 nM) was added for the final 30 min of treatment to stimulate degranulation. Immediately after treatment, cells were centrifuged for 10 min at 4000 x g. The cell-free supernatant fluids were collected, and MPO activity was measured according to the method of Henson et al. (1978). Release of MPO in the medium was expressed as the percentage of total MPO activity that was present in an equivalent number of cells lysed with 10 μl of Triton X-100 and ultrasonication.
Superoxide anion production.
Superoxide anion generation by HL-60 cells was measured as the reduction of cytochrome c in the presence or absence of superoxide dismutase (SOD; Babior et al., 1976). Differentiated HL-60 cells were suspended in Ca2+- and Mg2+-containing HBSS in the presence of cytochrome c (10 mg/ml) and of PCB or vehicle. Experiments were performed in 96-well plates, and for every sample two wells were incubated: one to which SOD (840 U/ml) was added and one to which an equal volume of vehicle was added. The amount of superoxide anion produced was estimated from the amount of cytochrome c reduced as determined from the difference in absorbance between the two wells, using an extinction coefficient of 18.5 cm–1 mM–1.
Labeling of HL-60 cells with 3H-arachidonic acid.
Differentiated HL-60 cells (10 x 106/ml) were suspended in HBSS containing 0.1% bovine serum albumin (BSA) and incubated for 120 min at 37°C with 0.5 μCi/ml 3H-AA. At the end of the labeling period, cells were washed twice and resuspended in HBSS containing 0.1% BSA. At the end of the incubation period, radioactivity in an aliquot of cells was determined in a scintillation counter to determine cellular uptake of radiolabel. Uptake was routinely 70% of added 3H-AA.
Determination of arachidonic acid release from HL-60 cells.
Cumulative release of 3H-AA was measured in HL-60 cells (2 x 106) exposed for 30, 60, 90, or 120 min at 37°C to PCBs. At the end of the incubations, samples were placed on ice and centrifuged, and radioactivity in the cell-free supernatant fluids was determined by liquid scintillation counting. Release is expressed as a percentage of the total radioactivity in the labeled cells.
Determination of COX-2 mRNA.
Differentiated HL-60 cells (5 x 106) were suspended in HBSS and treated at 37°C for 30, 60, 90, or 120 min with 3344-TCB or 2244-TCB at the concentrations indicated in the figure legends. At the end of the incubation period, total cellular RNA was isolated using Tri-Reagent (Sigma Chemical Co., St. Louis, MO). Concentration of isolated RNA was determined spectrophotometrically by measuring absorbance at 260 nm. cDNA was synthesized by reverse transcription at 42°C for 45 min in a 20-μl reaction mixture containing 1 μg total RNA and 100 units MMLV reverse transcriptase (Promega Corp., Madison, WI). After heating at 99°C for 5 min for denaturing, followed by cooling at 5°C, the cDNA was used for amplification. For PCR reactions, 5 μl of denatured cDNA was amplified in a 25-μl final volume with 2.5 units of Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA), 1μM of each primer and Taq polymerase buffer containing 25 mM MgCl2 with 2 mM of each dNTP (dATP, dCTP, dGTP, and dTTP; Promega Corp.). PCR was performed in a thermal cycler (GeneAmp 9700, Applied Biosystems, Foster City, CA), using a program of 35 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by a 10-min extension at 72°C. The amplified products were subjected to electrophoresis on a 1.5% agarose gel and detected and photographed under UV light. Densitometry on detected bands was performed using Bio-Rad Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA).
The following primers were used for COX-2: 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3' (forward primer), 5'-AGATCATCTCTGCCTGAGTATCTT-3' (reverse primer). The predicted size of the fragment was 301 bp.
For -actin, the primers were 5'-GACGAGGCCCAGAGCAAGAGAG-3' (forward primer), 5'-ACGTACATGGCTGGGGTGTTG-3' (reverse primer). The predicted size of the fragment was 284 bp (Vergne et al., 1998).
COX-2 Western blotting.
Differentiated HL-60 cells (50 x 106) were suspended in HBSS and treated at 37°C for 30, 60, 90, or 120 min with 2244-TCB at the concentrations indicated in the figure legends. Immediately after treatment, cells were centrifuged for 10 min at 4000 x g. Supernatant was discarded, and the cell pellet was lysed with 100 μl of 2% SDS. Proteins (30 μl) were separated on 10% Bis-Tris polyacrylamide gels (NuPAGE, Invitrogen Corporation, Carlsbad, CA) and electrophoretically transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). After blocking, the membranes were incubated with a primary anti-human COX-2 monoclonal antibody (1:1000 dilution; Cayman Chemical, Ann Arbor, MI) and then a secondary antibody (peroxidase-conjugated goat anti-mouse IgG; 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). The blots were developed using an ECL detection kit (Amersham Biosciences, Little Chalfont, UK). The blots were stripped and successively reprobed with an anti-human -actin monoclonal antibody (Sigma Chemical Co. St. Louis, MO) then with a corresponding secondary antibody. The levels of COX-2 bands were measured densitometrically as described above and corrected using levels of B-actin as an internal standard.
Cyclooxygenase activity assay.
Differentiated HL-60 cells (15 x 106) were suspended in HBSS and treated at 37°C for 30, 60, 90, or 120 min with 2244-TCB at the concentrations indicated in the figure legends. Immediately after treatment, cells were centrifuged for 10 min at 4000 x g. Supernatant was discarded, and the cell pellet was resuspended in 200 μl of assay buffer then lysed via sonication. COX activity was then measured using a COX activity assay (Cayman Chemical, Ann Arbor, MI) according to manufacturer's instructions. Activities of COX-1 and COX-2 were differentiated using the isoform-specific inhibitors DuP-697 and SC-560 according to manufacturer's instructions.
Statistical analysis.
Data are expressed as mean ± SEM. Results were analyzed by one-way analysis of variance. Group means for the superoxide anion production results were compared using Dunnett's post hoc test; all other data were compared using Tukey's post hoc test. Appropriate transformations were performed on all data that did not follow a normal distribution (e.g., percent data). For all studies, the criterion for statistical significance was p < 0.05.
RESULTS
Degranulation of HL-60 Cells
In the absence of PCB exposure, differentiated HL-60 cells released 27% of total MPO in response to fMLP, a peptide that activates neutrophils. Exposure to the noncoplanar PCB congener 2244-TCB resulted in an increase in degranulation that was significant by 90 min (Fig. 1). 2244-TCB concentrations greater than 3 μM were required to effect increases in degranulation (Fig. 2). 3344-TCB did not affect fMLP-induced degranulation at any time or dose examined (Figs. 1 and 2).
Superoxide Anion Production
In the absence of PCB exposure, differentiated HL-60 cells produced <5nmol of superoxide per 106 cells (Fig. 3). Exposure to 2244-TCB resulted in a significant increase in superoxide anion production. The coplanar PCB congener 3344-TCB did not affect superoxide anion production (Fig. 3).
3H-AA Release
In the absence of PCB exposure, HL-60 cells released <10% of total 3H-AA (Fig. 4) after 2 h. Exposure of differentiated HL-60 cells to 2244-TCB resulted in an increase in the release of 3H-AA. Statistically significant increases in 3H-AA release were observed in cells exposed to 2244-TCB for longer than 60 min (Fig. 4). Concentrations of 2244-TCB greater than 3 μM produced a significant increase in 3H-AA release from HL-60 cells (Fig. 5). Exposure to 3344-TCB did not cause statistically significant changes in 3H-AA release at any time or concentration examined (Figs. 4 and 5).
Effects of PCBs on COX-2 mRNA Expression
COX-2 mRNA levels did not change over 2 h in vehicle-treated HL-60 cells. A significant increase in the level of COX-2 mRNA was observed after 30 or 60 min exposure to 2244-TCB (Fig. 6A). In cells treated longer then 60 min, expression of COX-2 mRNA returned to baseline. This effect was observed with 10 or 30 μM 2244-TCB (Fig. 7A). 3344-TCB had no statistically significant effect on COX-2 mRNA levels at any time or concentration examined.
Effects of PCBs on COX-2 Protein Expression
COX-2 protein levels did not change over 2 h in vehicle-treated HL-60 cells (data not shown). COX-2 protein was increased after 30, 60, or 90 min exposure to 2244-TCB. After 120 min exposure to 2244-TCB, protein levels were decreased compared to previous time points, but still elevated compared to untreated cells (Fig. 6B). This effect was only observed with 30 μM 2244-TCB (Fig. 7B).
Effects of PCBs on COX-2 Activity
The activity of the peroxidase component of COX was used as a measure of total COX activity. Activity of COX-2 did not change over 2 h in vehicle-treated HL-60 cells (data not shown). An increase in the levels of COX-2 activity was observed after 60, 90, or 120 min exposure to 2244-TCB (Fig. 8A). This effect was only observed with 30 μM 2244-TCB (Fig. 8B). Activity of COX-1 did not change at any concentration of 2244-TCB examined (data not shown).
DISCUSSION
In the present study, the function of a human cell line differentiated to a neutrophil-like phenotype was affected by exposure to the noncoplanar PCB congener 2244-TCB, but not by the coplanar congener 3344-TCB. These results have not been previously described in the human neutrophillic HL-60 cell line. 2244-TCB caused a time- and dose-dependent increase in cellular degranulation and induced superoxide anion production (Figs. 1–3). These results are similar to previous observations of the effects of PCBs on primary rat neutrophils, in which several mono- or di-ortho-substituted PCBs stimulated superoxide anion production and/or increased superoxide anion generation in response to phorbol myristate acetate (Brown et al., 1998; Ganey et al., 1993). None of a variety of coplanar PCBs, including 3344-TCB, affected the function of rat neutrophils (Brown et al., 1998; Brown and Ganey, 1995). In primary human neutrophils noncoplanar PCB congeners, including 2244-TCB, increased oxidative burst (Voie et al., 1998, 2000) confirming that differentiated HL-60 cells respond similarly to primary human neutrophils. 2244-TCB and 2,3,4,5-TCB increase degranulation in rat neutrophils (Brown and Ganey, 1995), but changes in degranulation caused by PCBs have not been reported previously in human cells. Thus, with respect to oxidative burst and degranulation, responses in differentiated HL-60 cells, a human model of the neutrophil, are similar to those observed in rat neutrophils as well as in primary human neutrophils.
Several potential mediators of PCB-induced superoxide anion production have been described, including increased intracellular Ca2+ with subsequent activation of Ca2+-dependent proteins such as calmodulin (Olivero and Ganey, 2001) and release of AA due to activation of phospholipases (Tithof et al., 1998). In the present study, the noncoplanar PCB congener 2244-TCB, but not the coplanar congener 3344-TCB, increased the release of 3H-AA in a time- and dose-dependent manner (Figs. 4 and 5). Similar time- and dose-dependent increases in 3H-AA release have been reported in primary rat neutrophils, but not in human cells, treated with Aroclor 1242 as well as with individual, noncoplanar PCB congeners (Olivero et al., 2002; Olivero and Ganey, 2001; Tithof et al., 1998). Release of 3H-AA, similar to that observed in PCB-treated HL-60 cells, has also been observed in rat uterine strips (Bae et al., 1999) and in cerebellar granule cells treated with Aroclor mixtures (Kodavanti and Derr-Yellin, 2002; Kodavanti and Tilson, 2000).
One major fate of released AA is its conversion into eicosanoids, such as prostaglandins and thromboxanes, by cyclooxygenases or cytochrome P450 monooxygenases. In neutrophils, the eicosanoids produced by COX-2 play an important modulatory role in inflammation. COX-2 is an immediate early gene that is present in negligible amounts in quiescent neutrophils. Upon activation of the cell, COX-2 is rapidly upregulated. In the present study 2244-TCB rapidly and transiently increased levels of COX-2 mRNA and protein in a time- and dose-dependent manner (Figs. 6 and 7). Additionally time- and dose-dependent increases in COX activity were observed (Fig. 8), and these were greatly inhibited by the COX-2-specific inhibitor DuP-697. Previously observed effects of PCBs on COX-2 regulation have involved activation of the Ah receptor (Kietz and Fischer, 2003; Kwon et al., 2002). Because 2244-TCB, a poor ligand for the Ah receptor, caused changes in COX-2, whereas 3344-TCB, a more potent Ah receptor ligand, did not, it is unlikely that Ah receptor activation is involved in the increases in COX-2 observed in the present study. In mast cells, another immune cell critical in the initiation of inflammatory responses, COX-2 mRNA was markedly induced after exposure to the noncoplanar PCB congener 2,2',4,4',5,5'-hexachlorobiphenyl (Kwon et al., 2002). A similar induction of COX-2 mRNA was observed in rabbit blastocysts that were exposed to a mixture of seven different noncoplanar PCB congeners (Kietz and Fischer, 2003). Interestingly, COX-2 mRNA was also elevated in rabbit blastocysts by treatment with a mixture of three coplanar PCB congeners. This was not the case in differentiated HL-60 cells exposed to 3344-TCB, indicating differences in COX-2 regulation between HL-60 cells and rabbit blastocysts.
In summary, exposure to a noncoplanar PCB in vitro alters the function of granulocytic HL-60 cells in several ways. Exposing differentiated HL-60 cells to the noncoplanar PCB congener 2244-TCB increased degranulation and superoxide anion production, stimulated the release of 3H-AA, and induced COX-2 mRNA as well as protein and activity. Exposure to 3344-TCB had no effect on any of these endpoints in HL-60 cells. These effects mirror those seen with other noncoplanar PCB congeners in a variety of cell systems. Neutrophils play a vital role in the immune response as well as in inflammation, and any alterations in neutrophil function can have dire implications, either leading to increased susceptibility to infection, or contributing to destruction of healthy host cells.
ACKNOWLEDGMENTS
We thank Mary Kinser for technical assistance. This work was supported by Superfund grant ESO4911 from the NIH. Steven Bezdecny was supported by training grant T32 ES007255 from the NIEHS.
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ABSTRACT
Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that affect a number of cellular systems, including neutrophils. It has been demonstrated that noncoplanar PCBs (i.e., ortho- substituted PCBs) alter function of primary rat neutrophils. The objectives of these experiments were to determine if responses in a human, neutrophil-like cell line exposed to PCBs were similar to those reported for rat neutrophils and to explore further PCB-mediated alterations in neutrophil function. The human promyelocytic leukemia cell line (HL-60) was differentiated with DMSO to a neutrophil-like phenotype. Treatment of differentiated HL-60 cells with 2,2',4,4'-tetrachlorobiphenyl, a noncoplanar, ortho-substituted PCB congener, caused an increase in f-Met-Leu-Phe-induced degranulation, as measured by release of myeloperoxidase (MPO). Treatment with the coplanar, non-ortho-substituted congener 3,3',4,4'-tetrachlorobiphenyl had no effect on MPO release. 2,2',4,4'-Tetrachlorobiphenyl caused a time- and dose-dependent release of [3H]-arachidonic acid (3H-AA). A significant increase in 3H-AA release was observed after 60 min of exposure, and concentrations of 10 μM or larger increased 3H-AA release. In contrast, 3,3',4,4'-tetrachlorobiphenyl had no effect on 3H-AA release. The effect of PCBs on mRNA levels for cyclooxygenase-2 (COX-2) was examined using semiquantitative RT-PCR. COX-2 mRNA was significantly elevated in response to 2,2',4,4'-tetrachlorobiphenyl in a concentration-dependent manner. COX-2 expression was maximal by 30 min of exposure to 2,2',4,4'-tetrachlorobiphenyl. COX-2 protein and activity were also increased after exposure to 2,2',4,4'-tetrachlorobiphenyl; COX-1 protein and activity were unaffected. 3,3',4,4'-Tetrachlorobiphenyl did not increase COX-2 mRNA levels. These results demonstrate that a noncoplanar PCB alters the functional status of granulocytic HL-60 cells, causing enhanced degranulation and upregulation of COX-2, whereas a coplanar PCB lacks this activity. These data suggest that noncoplanar PCBs alter HL-60 cell function and COX-2 expression via an Ah-receptor-independent mechanism.
Key Words: PCBs; neutrophils; cyclooxygenase-2; noncoplanar; arachidonic acid.
INTRODUCTION
Polychlorinated biphenyls (PCBs) are ubiquitous, man-made, environmental contaminants that have been shown to affect a number of cellular systems. PCBs were used widely for over 40 years due to their heat resistant properties, low conductivity, and chemical inertness. These properties made PCBs a very useful component of safety fluids used to insulate and cool heavy electrical equipment such as transformers and capacitors (Hutzinger et al., 1974). The lipophilic nature of PCBs and their resistance to chemical transformation allow them to bioaccumulate in the food chain (Kannan et al., 1998). This has led to exposures of humans and wildlife to PCBs. PCBs have been detected in human blood, milk, adipose tissue, and placental tissue (Giesy and Kannan, 1998; Laden et al., 1999).
PCBs exhibit a wide range of biological effects in animals. They are probable human carcinogens (Cogliano, 1998) and cause a number of noncancer effects, including dermal lesions and ocular effects in humans, and hepatotoxicity and endocrine effects in rats (Morse et al., 1996; Takamatsu et al., 1985; Twaroski et al., 2001). In addition, PCBs have effects on the reproductive system (Faqi et al., 1998), nervous system (Jacobson and Jacobson, 1997), cardiovascular system (Warshaw et al., 1979), and both the specific and nonspecific branches of the immune system (Vos and VanLoveren, 1998).
The specific effects PCBs produce are related to their structure. PCBs can be divided into two broad classes based on the presence or absence of chlorine atoms in the ortho- position. Congeners that lack chlorines in the ortho- positions are coplanar and bind with high affinity to the aryl hydrocarbon (Ah) receptor to induce changes in cellular function. Congeners containing chlorines substituted at the ortho- positions cannot attain a coplanar configuration and are, in general, poor ligands for the Ah receptor. The mechanisms by which these noncoplanar PCBs cause functional changes in cells are not well understood (Bandiera et al., 1982).
Among the effects caused by ortho-substituted, noncoplanar PCB congeners is the alteration in function of polymorphonuclear neutrophils (Brown and Ganey, 1995; Kristoffersen et al., 2002; Olivero and Ganey, 2001; Olivero-Verbel and Ganey, 1998; Voie et al., 2000). The primary role of neutrophils is to attack and destroy invading microorganisms. They are normally quiescent and exhibit their biological activity when activated. Alteration in neutrophil function can have serious consequences for the organism. Failure of neutrophils to activate results in compromise of the innate immune system, leading to increased risk of infection. At the other end of the spectrum, inappropriate activation of neutrophils can lead to inflammatory disease states and injury to host tissue. Noncoplanar PCBs cause many changes in function of primary rat neutrophils including stimulation of degranulation, production of reactive oxygen species such as superoxide anion, release of arachidonic acid (AA), and altered response to subsequent stimulation with other agents (Brown and Ganey, 1995; Ganey et al., 1993; Olivero and Ganey, 2000; Tithof et al., 1998). Given the role of neutrophils in host defense and the requirement for activation, understanding the mechanisms by which neutrophils become activated in response to exposure to PCBs is important.
One fate of AA released in response to PCB exposure is that it may serve as a precursor for eicosanoid synthesis, which is catalyzed by the cyclooxygenase enzymes. One isoform of cyclooxygenase, cyclooxygenase-2 (COX-2) is present in negligible amounts in quiescent neutrophils, but can be induced by a variety of inflammatory stimuli (Smith et al., 2000; Vane et al., 1998). It was of interest to determine whether COX-2 expression is affected by PCBs. In this work we used the human, promyelocytic leukemia cell line, HL-60, to examine the effect of in vitro exposure to PCBs on COX-2 mRNA levels. HL-60 cells have the capacity to differentiate to a number of different, functionally and morphologically distinct forms (Collins, 1987) including a neutrophil-like phenotype, and differentiated HL-60 cells have been a useful model with which to study neutrophil responses (Arroyo et al., 2002; Mullick et al., 2004).
MATERIALS AND METHODS
Chemicals.
2,2',4,4'-Tetrachlorobiphenyl (2244-TCB; >99% pure) and 3,3',4,4'-tetrachlorobiphenyl (3344-TCB; >99% pure) were purchased from ChemService (West Chester, PA). N-formyl-methionyl-leucyl-phenylalanine (fMLP) was purchased from Sigma Chemical Co. (St. Louis, MO). [3H-5,6,8,9,11,12,14,15]-Arachidonic acid (3H-AA; 180–240 Ci/mmol) was purchased from DuPont (Boston, MA). All other chemicals were of the highest grade commercially available.
HL-60 cells.
HL-60 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA). Cells between passage 20 and 45 were grown in Iscove's Modified Dulbecco's Medium supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 0.1% gentamicin and 0.9% antibiotic-antimycotic (Invitrogen Corporation, Carlsbad, CA). Cultures were maintained in a humidified incubator at 37°C in a controlled atmosphere of 5% CO2. Cells were induced to differentiate along the granulocyte pathway by culturing in the presence of 1.25% dimethyl sulfoxide (DMSO) for 5 days. Cells were then maintained an additional 2 days in the absence of DMSO. After this procedure, approximately 80% of the cells had nuclear appearance characteristic of neutrophils. In addition, these cells reduced nitro blue tetrazolium, a functional marker of differentiation to a granulocytic phenotype (Hua et al., 2000).
Exposure to polychlorinated biphenyls.
Stock solutions of the PCBs were prepared by dissolution in dimethylformamide (DMF). The differentiated HL-60 cells were suspended in HBSS in borosilicate glass test tubes, 12 x 75 mm (VWR, Chicago, IL), and 1 μl of the stock solution was added per ml of cells to achieve the desired concentration. Control cells
Cellular degranulation.
Degranulation was measured by the release of the enzyme, myeloperoxidase (MPO). Differentiated HL-60 cells (2 x 106) were suspended in HBSS and exposed to PCBs at 37°C for 30, 60, 90, or 120 min. fMLP (10 nM) was added for the final 30 min of treatment to stimulate degranulation. Immediately after treatment, cells were centrifuged for 10 min at 4000 x g. The cell-free supernatant fluids were collected, and MPO activity was measured according to the method of Henson et al. (1978). Release of MPO in the medium was expressed as the percentage of total MPO activity that was present in an equivalent number of cells lysed with 10 μl of Triton X-100 and ultrasonication.
Superoxide anion production.
Superoxide anion generation by HL-60 cells was measured as the reduction of cytochrome c in the presence or absence of superoxide dismutase (SOD; Babior et al., 1976). Differentiated HL-60 cells were suspended in Ca2+- and Mg2+-containing HBSS in the presence of cytochrome c (10 mg/ml) and of PCB or vehicle. Experiments were performed in 96-well plates, and for every sample two wells were incubated: one to which SOD (840 U/ml) was added and one to which an equal volume of vehicle was added. The amount of superoxide anion produced was estimated from the amount of cytochrome c reduced as determined from the difference in absorbance between the two wells, using an extinction coefficient of 18.5 cm–1 mM–1.
Labeling of HL-60 cells with 3H-arachidonic acid.
Differentiated HL-60 cells (10 x 106/ml) were suspended in HBSS containing 0.1% bovine serum albumin (BSA) and incubated for 120 min at 37°C with 0.5 μCi/ml 3H-AA. At the end of the labeling period, cells were washed twice and resuspended in HBSS containing 0.1% BSA. At the end of the incubation period, radioactivity in an aliquot of cells was determined in a scintillation counter to determine cellular uptake of radiolabel. Uptake was routinely 70% of added 3H-AA.
Determination of arachidonic acid release from HL-60 cells.
Cumulative release of 3H-AA was measured in HL-60 cells (2 x 106) exposed for 30, 60, 90, or 120 min at 37°C to PCBs. At the end of the incubations, samples were placed on ice and centrifuged, and radioactivity in the cell-free supernatant fluids was determined by liquid scintillation counting. Release is expressed as a percentage of the total radioactivity in the labeled cells.
Determination of COX-2 mRNA.
Differentiated HL-60 cells (5 x 106) were suspended in HBSS and treated at 37°C for 30, 60, 90, or 120 min with 3344-TCB or 2244-TCB at the concentrations indicated in the figure legends. At the end of the incubation period, total cellular RNA was isolated using Tri-Reagent (Sigma Chemical Co., St. Louis, MO). Concentration of isolated RNA was determined spectrophotometrically by measuring absorbance at 260 nm. cDNA was synthesized by reverse transcription at 42°C for 45 min in a 20-μl reaction mixture containing 1 μg total RNA and 100 units MMLV reverse transcriptase (Promega Corp., Madison, WI). After heating at 99°C for 5 min for denaturing, followed by cooling at 5°C, the cDNA was used for amplification. For PCR reactions, 5 μl of denatured cDNA was amplified in a 25-μl final volume with 2.5 units of Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA), 1μM of each primer and Taq polymerase buffer containing 25 mM MgCl2 with 2 mM of each dNTP (dATP, dCTP, dGTP, and dTTP; Promega Corp.). PCR was performed in a thermal cycler (GeneAmp 9700, Applied Biosystems, Foster City, CA), using a program of 35 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by a 10-min extension at 72°C. The amplified products were subjected to electrophoresis on a 1.5% agarose gel and detected and photographed under UV light. Densitometry on detected bands was performed using Bio-Rad Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA).
The following primers were used for COX-2: 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3' (forward primer), 5'-AGATCATCTCTGCCTGAGTATCTT-3' (reverse primer). The predicted size of the fragment was 301 bp.
For -actin, the primers were 5'-GACGAGGCCCAGAGCAAGAGAG-3' (forward primer), 5'-ACGTACATGGCTGGGGTGTTG-3' (reverse primer). The predicted size of the fragment was 284 bp (Vergne et al., 1998).
COX-2 Western blotting.
Differentiated HL-60 cells (50 x 106) were suspended in HBSS and treated at 37°C for 30, 60, 90, or 120 min with 2244-TCB at the concentrations indicated in the figure legends. Immediately after treatment, cells were centrifuged for 10 min at 4000 x g. Supernatant was discarded, and the cell pellet was lysed with 100 μl of 2% SDS. Proteins (30 μl) were separated on 10% Bis-Tris polyacrylamide gels (NuPAGE, Invitrogen Corporation, Carlsbad, CA) and electrophoretically transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). After blocking, the membranes were incubated with a primary anti-human COX-2 monoclonal antibody (1:1000 dilution; Cayman Chemical, Ann Arbor, MI) and then a secondary antibody (peroxidase-conjugated goat anti-mouse IgG; 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). The blots were developed using an ECL detection kit (Amersham Biosciences, Little Chalfont, UK). The blots were stripped and successively reprobed with an anti-human -actin monoclonal antibody (Sigma Chemical Co. St. Louis, MO) then with a corresponding secondary antibody. The levels of COX-2 bands were measured densitometrically as described above and corrected using levels of B-actin as an internal standard.
Cyclooxygenase activity assay.
Differentiated HL-60 cells (15 x 106) were suspended in HBSS and treated at 37°C for 30, 60, 90, or 120 min with 2244-TCB at the concentrations indicated in the figure legends. Immediately after treatment, cells were centrifuged for 10 min at 4000 x g. Supernatant was discarded, and the cell pellet was resuspended in 200 μl of assay buffer then lysed via sonication. COX activity was then measured using a COX activity assay (Cayman Chemical, Ann Arbor, MI) according to manufacturer's instructions. Activities of COX-1 and COX-2 were differentiated using the isoform-specific inhibitors DuP-697 and SC-560 according to manufacturer's instructions.
Statistical analysis.
Data are expressed as mean ± SEM. Results were analyzed by one-way analysis of variance. Group means for the superoxide anion production results were compared using Dunnett's post hoc test; all other data were compared using Tukey's post hoc test. Appropriate transformations were performed on all data that did not follow a normal distribution (e.g., percent data). For all studies, the criterion for statistical significance was p < 0.05.
RESULTS
Degranulation of HL-60 Cells
In the absence of PCB exposure, differentiated HL-60 cells released 27% of total MPO in response to fMLP, a peptide that activates neutrophils. Exposure to the noncoplanar PCB congener 2244-TCB resulted in an increase in degranulation that was significant by 90 min (Fig. 1). 2244-TCB concentrations greater than 3 μM were required to effect increases in degranulation (Fig. 2). 3344-TCB did not affect fMLP-induced degranulation at any time or dose examined (Figs. 1 and 2).
Superoxide Anion Production
In the absence of PCB exposure, differentiated HL-60 cells produced <5nmol of superoxide per 106 cells (Fig. 3). Exposure to 2244-TCB resulted in a significant increase in superoxide anion production. The coplanar PCB congener 3344-TCB did not affect superoxide anion production (Fig. 3).
3H-AA Release
In the absence of PCB exposure, HL-60 cells released <10% of total 3H-AA (Fig. 4) after 2 h. Exposure of differentiated HL-60 cells to 2244-TCB resulted in an increase in the release of 3H-AA. Statistically significant increases in 3H-AA release were observed in cells exposed to 2244-TCB for longer than 60 min (Fig. 4). Concentrations of 2244-TCB greater than 3 μM produced a significant increase in 3H-AA release from HL-60 cells (Fig. 5). Exposure to 3344-TCB did not cause statistically significant changes in 3H-AA release at any time or concentration examined (Figs. 4 and 5).
Effects of PCBs on COX-2 mRNA Expression
COX-2 mRNA levels did not change over 2 h in vehicle-treated HL-60 cells. A significant increase in the level of COX-2 mRNA was observed after 30 or 60 min exposure to 2244-TCB (Fig. 6A). In cells treated longer then 60 min, expression of COX-2 mRNA returned to baseline. This effect was observed with 10 or 30 μM 2244-TCB (Fig. 7A). 3344-TCB had no statistically significant effect on COX-2 mRNA levels at any time or concentration examined.
Effects of PCBs on COX-2 Protein Expression
COX-2 protein levels did not change over 2 h in vehicle-treated HL-60 cells (data not shown). COX-2 protein was increased after 30, 60, or 90 min exposure to 2244-TCB. After 120 min exposure to 2244-TCB, protein levels were decreased compared to previous time points, but still elevated compared to untreated cells (Fig. 6B). This effect was only observed with 30 μM 2244-TCB (Fig. 7B).
Effects of PCBs on COX-2 Activity
The activity of the peroxidase component of COX was used as a measure of total COX activity. Activity of COX-2 did not change over 2 h in vehicle-treated HL-60 cells (data not shown). An increase in the levels of COX-2 activity was observed after 60, 90, or 120 min exposure to 2244-TCB (Fig. 8A). This effect was only observed with 30 μM 2244-TCB (Fig. 8B). Activity of COX-1 did not change at any concentration of 2244-TCB examined (data not shown).
DISCUSSION
In the present study, the function of a human cell line differentiated to a neutrophil-like phenotype was affected by exposure to the noncoplanar PCB congener 2244-TCB, but not by the coplanar congener 3344-TCB. These results have not been previously described in the human neutrophillic HL-60 cell line. 2244-TCB caused a time- and dose-dependent increase in cellular degranulation and induced superoxide anion production (Figs. 1–3). These results are similar to previous observations of the effects of PCBs on primary rat neutrophils, in which several mono- or di-ortho-substituted PCBs stimulated superoxide anion production and/or increased superoxide anion generation in response to phorbol myristate acetate (Brown et al., 1998; Ganey et al., 1993). None of a variety of coplanar PCBs, including 3344-TCB, affected the function of rat neutrophils (Brown et al., 1998; Brown and Ganey, 1995). In primary human neutrophils noncoplanar PCB congeners, including 2244-TCB, increased oxidative burst (Voie et al., 1998, 2000) confirming that differentiated HL-60 cells respond similarly to primary human neutrophils. 2244-TCB and 2,3,4,5-TCB increase degranulation in rat neutrophils (Brown and Ganey, 1995), but changes in degranulation caused by PCBs have not been reported previously in human cells. Thus, with respect to oxidative burst and degranulation, responses in differentiated HL-60 cells, a human model of the neutrophil, are similar to those observed in rat neutrophils as well as in primary human neutrophils.
Several potential mediators of PCB-induced superoxide anion production have been described, including increased intracellular Ca2+ with subsequent activation of Ca2+-dependent proteins such as calmodulin (Olivero and Ganey, 2001) and release of AA due to activation of phospholipases (Tithof et al., 1998). In the present study, the noncoplanar PCB congener 2244-TCB, but not the coplanar congener 3344-TCB, increased the release of 3H-AA in a time- and dose-dependent manner (Figs. 4 and 5). Similar time- and dose-dependent increases in 3H-AA release have been reported in primary rat neutrophils, but not in human cells, treated with Aroclor 1242 as well as with individual, noncoplanar PCB congeners (Olivero et al., 2002; Olivero and Ganey, 2001; Tithof et al., 1998). Release of 3H-AA, similar to that observed in PCB-treated HL-60 cells, has also been observed in rat uterine strips (Bae et al., 1999) and in cerebellar granule cells treated with Aroclor mixtures (Kodavanti and Derr-Yellin, 2002; Kodavanti and Tilson, 2000).
One major fate of released AA is its conversion into eicosanoids, such as prostaglandins and thromboxanes, by cyclooxygenases or cytochrome P450 monooxygenases. In neutrophils, the eicosanoids produced by COX-2 play an important modulatory role in inflammation. COX-2 is an immediate early gene that is present in negligible amounts in quiescent neutrophils. Upon activation of the cell, COX-2 is rapidly upregulated. In the present study 2244-TCB rapidly and transiently increased levels of COX-2 mRNA and protein in a time- and dose-dependent manner (Figs. 6 and 7). Additionally time- and dose-dependent increases in COX activity were observed (Fig. 8), and these were greatly inhibited by the COX-2-specific inhibitor DuP-697. Previously observed effects of PCBs on COX-2 regulation have involved activation of the Ah receptor (Kietz and Fischer, 2003; Kwon et al., 2002). Because 2244-TCB, a poor ligand for the Ah receptor, caused changes in COX-2, whereas 3344-TCB, a more potent Ah receptor ligand, did not, it is unlikely that Ah receptor activation is involved in the increases in COX-2 observed in the present study. In mast cells, another immune cell critical in the initiation of inflammatory responses, COX-2 mRNA was markedly induced after exposure to the noncoplanar PCB congener 2,2',4,4',5,5'-hexachlorobiphenyl (Kwon et al., 2002). A similar induction of COX-2 mRNA was observed in rabbit blastocysts that were exposed to a mixture of seven different noncoplanar PCB congeners (Kietz and Fischer, 2003). Interestingly, COX-2 mRNA was also elevated in rabbit blastocysts by treatment with a mixture of three coplanar PCB congeners. This was not the case in differentiated HL-60 cells exposed to 3344-TCB, indicating differences in COX-2 regulation between HL-60 cells and rabbit blastocysts.
In summary, exposure to a noncoplanar PCB in vitro alters the function of granulocytic HL-60 cells in several ways. Exposing differentiated HL-60 cells to the noncoplanar PCB congener 2244-TCB increased degranulation and superoxide anion production, stimulated the release of 3H-AA, and induced COX-2 mRNA as well as protein and activity. Exposure to 3344-TCB had no effect on any of these endpoints in HL-60 cells. These effects mirror those seen with other noncoplanar PCB congeners in a variety of cell systems. Neutrophils play a vital role in the immune response as well as in inflammation, and any alterations in neutrophil function can have dire implications, either leading to increased susceptibility to infection, or contributing to destruction of healthy host cells.
ACKNOWLEDGMENTS
We thank Mary Kinser for technical assistance. This work was supported by Superfund grant ESO4911 from the NIH. Steven Bezdecny was supported by training grant T32 ES007255 from the NIEHS.
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