ToxSci Advance Access originally published online on March 12, 2007
Toxicological Sciences 2007 97(2):364-374; doi:10.1093/toxsci/kfm048
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3,4-Dichloropropionaniline Suppresses Normal Macrophage Function
,1
* Department of Microbiology, Immunology and Cell Biology
Department of Biochemistry and Molecular Pharmacology
Center for Immunopathology and Microbial Pathogenesis, West Virginia University, PO Box 9177, Morgantown, West Virginia 26506-9177
1 To whom correspondence should be addressed. Fax: (304) 293-7823. E-mail: jbarnett{at}hsc.wvu.edu.
Received December 14, 2006; accepted March 5, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Macrophages are a critical part of the innate immune response and natural surveillance mechanisms. As such, proper macrophage function is crucial for engulfing bacterial pathogens through phagocytosis and destroying them by generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The production of a number of cytokines by macrophages, such as tumor necrosis factor-
(TNF-), interleukin (IL)-1ß, and IL-6, plays an important role in the initiation of the acquired immune response creating an inflammatory environment favorable for fighting a bacterial infection. 3,4-Dichloropropionaniline (DCPA) suppresses several inflammatory parameters, including TNF- production through a mechanism where nuclear factor-
B (NF-B)–DNA binding is inhibited but not entirely abrogated. The goal of the present study was to evaluate the effects of DCPA on the inflammatory mediators of macrophages, including ROS and RNS in both murine peritoneal exudate cells and the human monocytic cell line, THP-1. The ability to perform phagocytosis and directly kill Listeria monocytogenes was also assessed. The results indicate that DCPA decreases the ability of both types of macrophages to phagocytize beads and generate both types of reactive species, which was correlated with a decrement in listericidal activity. These results demonstrate that DCPA has profound effects on macrophage function and provide insight into the potential mechanisms of immunosuppression by DCPA. Key Words: bactericidal; phagocytosis; Listeria monocytogenes; dichloropropionaniline; macrophage; THP-1 cell line.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3,4-Dichloropropionaniline (DCPA) or propanil is a selective postemergent herbicide used on approximately 70% of the U.S. rice crops, which ranks it as the 17th most commonly used conventional pesticide used in the U.S. Agricultural Market Sector (EPA, 2004). With this level of usage, the potential for either occupational or environmental exposure is high and therefore, it is important to understand the possible consequences of contact with this compound on the immune system. Macrophages are critical for normal maintenance of homeostasis as well as immune regulation and tumor surveillance. Whereas phagocytosis of apoptotic cells is a normal homeostatic function of macrophages, phagocytosis and clearance of bacteria and viruses is a crucial function of innate immunity. Through cytokine and chemokine production, direct cellular contacts, and antigen presentation, macrophages orchestrate the initiation of adaptive immune responses (Adams and Hamilton, 1984
; Celada and Nathan, 1994; Ganz, 1993). Clearly, alterations in any of the normal functions of these cells can lead to serious, even fatal consequences.
Defects in normal macrophage function contribute to chronic granulomatous disease (CGD) and result in altered immunity to bacterial and fungal pathogens. Patients with CGD develop granulomas consisting of fused monocytes and macrophages, which have engulfed bacteria but are unable to destroy them as a result of a defect in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Activation of NADPH oxidase is normally induced by microbial products such as bacterial lipopolysaccharide (LPS) or by cytokines such as interferon-
, interleukin (IL)-1ß, or IL-8 (Bonizzi et al., 2000). Superoxide produced by NADPH oxidase diffuses into the bacterium and kills it by inactivating essential enzymes (Miller and Britigan, 1997; Roos et al., 1984). Superoxide can be converted to hydrogen peroxide (H2O2), which can be a more reactive oxidant. H2O2 oxidizes cellular membranes and enzymes and causes DNA damage by interacting with Fe2+ to form the toxic ferryl radical which inhibits membrane transport processes (Forman and Torres, 2002; Imlay et al., 1988; Weiss and Peppin, 1986). Hydroxyl radicals (·OH) are also very potent oxidants implicated in the oxidation of bacterial proteins, DNA, and lipids (Ohya et al. 1998a). All these reactive oxygen species (ROS) are critical for macrophage bactericidal mechanisms against the facultative intracellular pathogen, Listeria monocytogenes (Alvarez-Dominguez et al., 2000; Ohya et al., 1998a).
Production of reactive nitrogen species (RNS) is also important for control of microbial proliferation. Nitric oxide (NO) is produced by the inducible nitric oxide synthase (iNOS or NOS2) in the presence of O2, which catalyzes enzymatic oxidation of L-arginine to L-citrulline, using NADPH-derived electrons (Marletta, 1994). Increased iNOS expression and NO production are observed at sites of infection in animal models, such as toxoplasmosis and leishmaniasis, or in human infections such as tuberculosis (Nicholson et al., 1996). NO readily reacts with O2 resulting in production of transient and unstable N2O3 and, ultimately, unreactive NO2– ion product. Activated macrophages will also produce superoxide, which reacts with NO to form the reactive and toxic peroxynitrite (ONOO–) which in turn decomposes into additional nitrates and nitrites (Hibbs et al., 1988; Miwa et al., 1987; Stuehr and Marletta, 1987). Expression of the iNOS gene in macrophages is regulated mainly at the transcriptional level, particularly by nuclear factor-B (NF-B), which binds to multiple sites on the iNOS gene promoter (Xie et al., 1993). iNOS is similar to NADPH oxidase, in that it is stimulated by proinflammatory cytokines such as interferon- (IFN-), tumor necrosis factor- (TNF-), and IL-1ß, as well as by LPS (Fang, 1997).
Major listericidal mechanisms include the production of ROS and RNS by activated macrophages (Alvarez-Dominguez et al., 2000; Muller et al., 1999; Ohya et al., 1998b; Ouadrhiri et al., 1999). Although ROS and RNS have been identified as major anti-bacterial defense mechanisms, the details of how these agents work in activated macrophages are not completely understood. Both (IFN-) activation of macrophages and production of TNF- are necessary for clearance of L. monocytogenes (Buchmeier and Schreiber, 1985; Kiderlen et al., 1984; Nakane et al., 1988, 1989). Therefore, reductions in macrophage cytokine production, reactive oxygen, and/or nitrogen species generation result in increased susceptibility to L. monocytogenes infection.
The most common routes of exposure to DCPA are thought to be inhalational and dermal during manufacturing and application and to a lesser extent oral through drinking water and food. Upon entry into the individual, propanil is rapidly metabolized in the liver by the enzyme acylamidase into the major metabolite 3,4-dichloroaniline (3,4-DCA). Both DCPA and 3,4-DCA are often detected in the urine of the general population living in the agricultural areas as well as occupationally exposed people (Turci et al., 2006; Wittke et al., 2001). In nonoccupationally exposed people, the urine levels of 3,4-DCA were reported to reach a maximum level of 6.19 µg/l (Turci et al., 2006). Thus, it is apparent that the general population in areas that use DCPA carry a measurable body burden of 3,4-DCA.
The experiments reported herein were conducted in vitro, and the relationship of the in vitro concentrations used with expected body burdens found in humans is a relevant question. However, given that blood levels of a pesticide, which would be more relevant for study, may exceed the urine levels (McMullin et al., 2003), and that data on in vivo organ concentrations are unavailable, extrapolating from measured human urine levels to in vitro exposure concentrations is imprecise.
DCPA reduces several macrophage functions, including TNF- and IL-6 production, IP3-mediated calcium release, and nuclear NF-B translocation (Frost et al., 2001; Xie et al., 1997a,b). The present report was designed to determine the direct effects of DCPA on different macrophage functions. The results demonstrate that DCPA reduced phagocytic ability, ROS and RNS generation, and listericidal activity of both human and mouse macrophages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell line culture and maintenance.
The human monocytic cell line THP-1 was obtained from the American Type Culture Collection (Rockville, MD). The cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 media (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan UT), 10mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 100 U/ml penicillin, 100 µg/ml streptomycin, 2mM L-glutamine, and 5 x 10–5 M 2-mercaptoethanol (all obtained from Sigma, St Louis, MO) (complete media) and incubated at 37°C in an atmosphere of 5% CO2.
Animals.
C57Bl/6 female and male mice were purchased from Charles River Farms (Wilmington, DE). Animals were allowed to acclimate for 7 days before experimental procedures were initiated. Food and water were provided ad libitum. Experimental procedures were conducted between the ages of 7 and 12 weeks. All animal experiments were reviewed and approved by the West Virginia University Animal Care and Use Committee.
Primary macrophage harvest and preparation.
Resident and inflammatory primary macrophages (peritoneal exudate cells [PEC]) were utilized. Inflammatory macrophages were elicited by injecting 1.5 ml of aged 4% sterile thioglycollate broth ip 4 days before macrophage harvest. To harvest the PEC, 10 ml sterile Dulbecco's phosphate-buffered saline (PBS) (DPBS, BioWhittaker) containing 10 U/ml heparin or 100µM EDTA (Sigma) was injected into the mouse's exposed peritoneum and syringe aspirated. Resulting cell suspensions were washed twice in complete media, plated at the appropriate density in tissue culture dishes, and allowed to adhere for 3 h at 37°C in 5% CO2. Nonadherent cells were removed by the addition of two bursts of warm PBS and vacuum aspiration. Macrophage enrichment was monitored by nonspecific esterase staining and was 
95%. Viability was monitored by trypan blue exclusion and was routinely 95%.
Cell stimulation and exposure to DCPA.
PEC or THP-1 cells were simultaneously stimulated with LPS from Escherichia coli Serotype 055:B5 (Sigma) and treated with DCPA (99% purity, ChemServices, West Chester, PA) diluted in absolute ethanol (Mallinckrodt, Paris, KY) at concentrations indicated in the "Results" section. Control cultures received an equivalent amount of absolute ethanol only and are referred to as vehicle or 0µM DCPA. The final concentration of ethanol for all cultures was 0.10%. LPS was dissolved in RPMI 1640 at 1 mg/ml and added to cultures at 1 µl/ml of culture media. The possible cytotoxic effects of DCPA alone during the culture period were determined and no significant difference in viability was measured at any DCPA concentration used for this study.
TNF-–specific ELISA.
THP-1 cells were plated at 1.5 x 106 cells/3 ml in six-well plates then differentiated by addition of 100 ng/ml of phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h. Differentiation of the cells led to their adherence to tissue culture dishes. Monolayers were washed vigorously with PBS and cultured in complete media for 72 h. Differentiated cell were stimulated with 1 µg/ml LPS (Sigma) in the presence of DCPA or vehicle and incubated for 6, 12, and 24 h at 37°C in 5% CO2. Supernatants were collected and assayed by human TNF ELISA set with detection limit of 7.8 pg/ml (cat. 555212, BD Biosciences PharMingen, San Diego, CA) according to manufacturer's instructions. The plates were then read at an optical density of 450 nm on a µQuant plate reader (BioTek Instruments, Inc., Winooski, VT).
Bacteria.
L. monocytogenes, strain EGD, were grown overnight in 10 ml of brain-heart infusion (BHI) broth (Difco Laboratories, Detroit, MI) with aeration at 37°C. One milliliter of this culture was inoculated into 50 ml of sterile BHI broth and incubated at 37°C with aeration until culture reached an OD600 of approximately 0.4 (2–4 h). An OD600 absorbance value of 1.000 is equivalent to 1 x 109 bacteria/ml. The culture was diluted to the appropriate number of viable L. monocytogenes.
Phagocytosis.
THP-1 cells were differentiated by addition of 100 ng/ml PMA (Sigma) as described above. On the day of the phagocytosis assay, differentiated THP-1 cells or PEC (previously prepared and allowed to adhere overnight) were trypsinized, washed in PBS, seeded in 5-ml snap-cap tubes at 1 x 106 cells/ml in PBS + 1% FBS. Phagocytosis assays were conducted as described with minor modifications (Antonini et al., 2000). Briefly, carboxylate-modified, 2.0 µm yellow-green FluoroSpheres (Molecular Probes, Eugene, OR) were added at a concentration of approximately 30 beads per cell. LPS, DCPA, and vehicle treatments were added as indicated. Cells were incubated on a rocker in constant motion at 37°C in 5% CO2 for 2 h. Cells were pelleted by centrifugation at 500 x g, washed twice to remove any free beads, and allowed to adhere to sterile coverslips placed in six-well plates for 2 h. Adherent cells were washed twice with warm PBS, fixed with 2% paraformaldehyde for 30 min, stained with the fluorochrome nile red (0.1 µg/ml; Molecular Probes) for 5 min, and washed with PBS. Coverslips were then mounted to glass slides and stored in the dark (a maximum of 48 h before analysis). Confocal microscopy using a Zeiss LSM 510 microscope equipped with an argon laser (Zeiss, Thornwood, NY) was performed to evaluate the cells which were scored as having 0, 1–2, 3–4, and 5 beads per cell. A weighted phagocytic index was calculated by multiplying the number of cells with 0 beads by 0; the number of cells with 1–2 beads per cell by 1; the number of cells with 3–4 beads per cell by 2; and the number of cells with five or more beads per cell by 4, summing these products, and dividing by 200 (the total number of cells counted).
Determination of Listericidal activity.
THP-1 or PEC (5 x 105 cells/ml) were activated with 100 U/ml rhIFN- (BD PharMingen) or rmIFN- (BD PharMingen), respectively, for 24 h in RPMI 1640 media containing 10% FBS (no antibiotics). This activation did not cause adhesion of THP-1 cells, which continued to grow in loose suspension as described (Ouadrhiri et al., 1999). THP-1 cells were washed with PBS, resuspended in 1 ml PBS containing 1% FBS, and infected with 2.5 x 106 L. monocytogenes, strain EGD/ml. Activated PEC were trypsinized, washed in PBS, resuspended in PBS containing 1% FBS, and also infected with 2.5 x 106 L. monocytogenes, strain EGD/ml. LPS stimulation and vehicle and DCPA treatments were initiated at the same time as initial infection. Cells were transferred to 5-ml snap-cap tubes and incubated at 37°C in 5% CO2 on a rocker for 1 h to allow for infection. Cells were then centrifuged at 500 x g and washed twice with PBS. The final pellet was resuspended in PBS containing 25 µg/ml gentamicin. Initial infection levels (1 h) were obtained by lysing cells in 500 µl of ice-cold, sterile, distilled water and plating 50 µl of the lysate. Remaining treatment groups were returned to incubation conditions in the gentamicin solution. Cell lysates were prepared at 3 and 5 h after infection. Lysates were plated on Trypticase soy agar plates (Difco Laboratories, Detroit, MI) using an Autoplate 4000 (Spiral Biotech, Bethesda, MD) in triplicate. Plates were incubated at 37°C for 24 h, and colonies were counted using a CASBA 4 plate scanner and CIA-BEN colony imaging and analysis software (Spiral Biotech). Resulting L. monocytogenes colony-forming units (CFU) per 1 ml were calculated and compared across treatments.
Respiratory burst.
Luminol, 5-amino-2-3-dihydro-1,4 phthalazinedione (Sigma), was used to amplify chemiluminescence (CL) signals generated by activation of the NADPH oxidase (Allen and Loose, 1976; Dahlgren and Karlsson, 1999). The CL signal generated by luminol occurs as the compound accepts an electron from free radical species as they return to ground state. To measure respiratory burst, we differentiated THP-1 cells or harvested PEC as previously described. All cells were plated at 5 x 105 cells/ml in 35-mm tissue culture dishes. Cells were washed with PBS before elicitation of the respiratory burst. In a total reaction volume of 2 ml, respiratory burst activity was elicited by addition of 100µM PMA simultaneously with PBS, 5µM luminol, indicated concentrations of LPS, and either ethanol or propanil. Dishes were placed in a prewarmed luminometer (Berthold, Co., Wilbad, Germany), and CL readings were taken every minute for 20 min. Data shown represent CL curves generated from these readings over time to peak values. Control experiments to determine if DCPA alone quenched the CL signal showed no interference of the assay by the DCPA.
Intracellular ROS measurement.
THP-1 cells were plated at 2.25 x 105 cells/ml in Lab-Tek chambered cover glass system (four chamber; Nalge Nunc International Corp., Naperville, IL) in phenol red-free complete RPMI and differentiated by addition of PMA as described above. Cells were washed with PBS three times and incubated for 40 min at 37°C in 5% CO2 in the dark with 5µM 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA, Invitrogen, Carlsbad, CA). CM-H2DCFDA is a nonfluorescent dye which enters the cells by passive diffusion; it becomes fluorescent only when it is oxidized by ROS, particularly by H2O2, (·OH), or their downstream free radical products. After incubation with the dye, cells were washed three times with PBS, and Hanks' Balanced Salt Solution (BioWhittaker) was added to the wells. Cells were treated with different doses of DCPA or vehicle control and simultaneously stimulated with 1 µg/ml LPS and 100µM PMA. Cultures were examined with a Zeiss Axiovert 100M microscope equipped with a laser scanning confocal attachment (model LSM 510; Zeiss) to locate the cells and analyze their images overtime at 512 x 512 pixel resolution. CM-H2DCFDA was excited with the 488-nm line of an argon/krypton mixed-gas laser; emission was collected with a 505 nm long-pass filter. Images were collected every 2 min for up to 26 min, 14 images in total were taken for the course of each experiment, and cells were treated and stimulated after the first scan. Pixel time was 1.76 µs with the scan time of 983.04 msec for each image taken. Plan-Neofluar x40/0.75 objective was used to obtain the images. Fluorescence emission from 25 to 30 cells per experiment was analyzed by LPS 510 software, and at least three experiments were performed per treatment group.
NO release from mouse peritoneal macrophages.
Resident PEC were harvested as previously described and cultured in 5 ml of complete RPMI in 60-mm plates. The cells were stimulated with 1 µg/ml LPS and simultaneously treated with 0, 5, 25, or 100µM DCPA and incubated for 12, 24, and 48 h for NO induction. Each treatment was plated in duplicate and analyzed in triplicate. The supernatant of each culture was transferred to a plastic tube, centrifuged at 2000 rpm, and the supernatant was stored at – 80°C assayed for NO. Greiss reagent was used to measure nitrite (NO2–) as a stable form of NO. Briefly, 100 µl test sample was mixed with 150 µl of Greiss reagent (0.1% naphthylethylenediamine chloride) (Sigma) in 60% acetic acid and 1% sulfanilamide (Sigma) in 30% acetic acid for 10 min, and color development was assessed at 540 nm with a µQuant plate reader (BioTek Instruments). A standard curve was generated with a serial dilution of sodium nitrite dissolved in culture medium.
Immunoblotting.
PEC were harvested as described above and plated at 6.5 x 106 cells/5 ml in 60-mm dishes (Becton Dickinson Labware, Franklin Lakes, NJ) in complete RPMI and allowed to adhere for 3 h at 37°C in 5% CO2. Nonadherent cells were removed by addition of three bursts of warm DPBS and aspirated by pipette; 5 ml of complete RPMI was added to the wells, and cells were incubated for 1 h before treatments were administrated. The cells were stimulated with 1 µg/ml LPS and simultaneously treated with 5, 25, or 100µM DCPA or ethanol and incubated for 6, 12, and 24 h for iNOS protein detection. Whole-cell extracts were prepared using lysis buffer (Cell Signaling Technology, Beverly, MA) according to manufacturer instructions, and protein levels were quantitated using bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Whole-cell lysates (15 µg) were separated by 8% polyacrylamide gels using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Pall Corporation, Pensacola, FL) overnight at 0.1 A. The membranes were then blocked by incubation for 45 min with 5% nonfat dry Carnation milk (Nestle, Glendale, CA) in Tris-buffered saline (TBS: 20mM Tris-HCl, pH 7.6, 137mM NaCl) containing 0.1% Tween 20 (TBS-Tween) and then rinsed three times with TBS-Tween. The protein blots were cut across to incubate the upper half overnight with rabbit polyclonal IgG anti-NOS2 (Santa Cruz Biotechnology, Santa Cruz, CA; sc-650; 1:2000 dilution) and the lower half with goat polyclonal IgG anti-actin (Santa Cruz Biotechnology; sc-1616; 1:1000 dilution) in TBS-Tween plus 5% bovine serum albumin. After incubation with the appropriate secondary antibodies (Sigma, A0545 and A5420), blots were developed using Phototope-HRP detection kit for Western blots (Cell Signaling) and visualized by exposing to x-ray film (Kodak BioMax Light Film, Eastman Kodak Company, Rochester, NY) for 15 s to 3 min. Densitometric analysis was completed with Optimas software (Media Cybernetics, Silver, MD).
Statistical analysis.
Data were analyzed using JMP (SAS, Inc., Belmont, CA) and SigmaStat (SPSS, Inc., Chicago, IL) statistical analysis software. Significance of interactions between treatment groups was assessed using an analysis of variance. For all analyses, the minimum criterion of significance was set at p < 0.05. Each experiment was repeated at least twice with similar results. Data shown represent means of at least three experiments or representative experiments, as indicated. Error bars represent standard deviation unless otherwise noted to represent standard error, where appropriate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DCPA Suppresses TNF-
Production by THP-1 CellsThe production of TNF-
, a key inflammatory mediator produced by activated macrophages, is reduced when murine macrophages are exposed to DCPA (Xie et al., 1997a,b). To provide a closer link to possible effects on humans, a human cell line was used throughout this study. To determine if DCPA caused a similar reduction in TNF- in human cells, we measured TNF- production from the human monocytic cell line, THP-1 (Fig. 1). DCPA reduced TNF- production from THP-1 cells at all time points tested. In a representative experiment, DCPA caused a reduction in TNF- production from 2465 ± 107, 2279 ± 196, and 1998 ± 18.68 pg/ml in the vehicle control cells to 1637 ± 125, 1321 ± 142, and 1202 ± 168 pg/ml at 6, 12, and 24 h, respectively, in the 100µM DCPA–treated cells. The difference in TNF- levels between the 0 and both 50 and 100µM DCPA was statistically significant at all time points (p < 0.001). A statistically significant reduction in TNF- production was also observed in the 25µM DCPA–treated cells at 12 h (p < 0.001). Differences in TNF- production between the 0 and 5 or 25µM concentrations were not significantly reduced at 6 and 24 h.
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In vitro Exposure to DCPA Reduces Phagocytic Capabilities
A major function of macrophages in vivo is phagocytosis of bacteria and cellular debri. To determine if DCPA affected the ability of PMA-differentiated THP-1 cells and PEC to phagocytize fluorescent 2.0 µm beads, a weighted phagocytic index was measured. The weighted phagocytic index allows a quantification of the amount of phagocytosis (number of beads per cell) in addition to the number of macrophages that are capable of phagocytosis. DCPA caused significant reductions (p < 0.05) in the ability of THP-1 and PEC cells to phagocytize beads. The weighted phagocytic index for THP-1 cells was reduced from 0.730 ± 0.303 in the vehicle control to 0.320 ± 0.07 and 0.267 ± 0.068 in the 50 and 100µM DCPA group, respectively (Fig. 2A). Significant differences (p < 0.05) were also evident with the DCPA-treated PEC, from an average of 1.084 ± 0.108 in the vehicle control group to 0.724 ± 0.071 and 0.534 ± 0.072 in the 50 and 100 µM treatments, respectively (Fig. 2B).
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In vitro Exposure to DCPA Reduces Anti-Bacterial Capabilities
The effect of DCPA on the ability of THP-1 and PEC macrophages to kill L. monocytogenes was measured as an indicator of the biological consequences of DCPA exposure. IFN-
plus LPS-stimulated macrophages that were treated with vehicle had significant bacterial reductions by 5 h (p < 0.05) (Figs. 3A and 3B). Treatment of both THP-1 cells and PEC cells with 100µM DCPA abrogated or reduced their listericidal activity (Figs. 3A and 3B). Fewer bacteria were initially phagocytosed by THP-1 cells and PEC treated with 50 or 100µM DCPA than by cells treated with vehicle (p < 0.05) (Figs. 3A and 3B; 1 h). THP-1 cells treated with 100µM DCPA actually showed that the bacteria continued to multiply in these cells at least until the 3-h time point, while vehicle-treated cells showed a consistent decline in the number of intracellular bacteria. PEC cells treated with 50µM DCPA showed a steady decline in the number of intracellular bacteria. Those treated with 100µM phagocytosed a much smaller number of bacteria (1-h value), and there appeared to be a slight increase in their CFU over time. These results demonstrate that DCPA causes inhibition of listericidal activity of macrophages and further support the effects of DCPA on phagocytosis described above.
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DCPA Exposure Inhibits the Generation of a Respiratory Burst
Functional aspects of macrophage biology include destruction of phagocytosed organisms via generation of reactive oxygen and nitrogen species. Exposure to 100µM DCPA virtually shuts down the respiratory burst activity of both cell types, whereas 50µM DCPA had an intermediate effect on the respiratory burst (Figs. 4A and 4B). The response of THP-1 cells treated with 25µM DCPA was similar to vehicle control cells (data not shown). The respiratory burst of PEC was more sensitive to the effects of DCPA, resulting in significantly different dose-dependent decreases in integrated relative light units upon exposure to 50 and 100µM DCPA (Fig. 4B).
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Intracellular ROS Production is Inhibited by DCPA
The respiratory burst, due to ROS production in phagocytic cells by H2O2 and hydroxyl radicals, is an important function of macrophages. Figure 5A shows representative confocal image fields of PEC cells at 0, 10, and 20 min after LPS stimulation with or without DCPA. Amount of fluorescence produced by the cells during oxidation of CM-H2DCFDA dye directly corresponded to the amount of ROS generated. Figure 5B shows the increase in measured fluorescent intensity with respect to time of PEC cells treated with the vehicle. PEC cells exposed for 20 min to vehicle and LPS demonstrated a twofold increase in fluorescent intensity from 53.73 ± 2.76 to 103.47 ± 15.63, indicating an induction of ROS production (Fig. 5B). PEC treated with 25 or 100µM DCPA showed a significant decrease in ROS generation over the 26 m assayed (p < 0.05). Further, 5µM DCPA–treated cells also demonstrated a decrease in ROS generation for 26 min (data not shown). Altogether, these data confirm the luminol CL data and provide more specific characterization of the respiratory burst in the presence of DCPA. THP-1 cells show a decrease in ROS generation (fluorescent intensity) at all concentrations of DCPA; however, none of these decreases were significantly different (data not shown). The lack of significance for the 100µM–treated THP-1 cells may be due to a high variance in the data as there is a constant decrease in fluorescence intensity during the initial 14 min of the experiment; however, the lack of an apparent concentration-dependent decrease at 5 and 25µM would imply that THP-1 cells are fairly resistant to DCPA with regard to ROS generation.
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DCPA Inhibits Nitrite Production
RNS are important anti-microbial factors and their production over time can be monitored by measuring nitrite accumulation in culture supernatants of activated macrophages. The production of nitrite by PEC was significantly decreased in 100 µm DCPA–treated cells compared to vehicle-treated control cells by 40% at 12 h (p < 0.05) (Fig. 6). Cells treated with 25µM and 100µM DCPA for 24 h resulted in 15 and 22% decreases, respectively (p < 0.01) in nitrate production (Fig. 6). Treatment with 5µM DCPA did not decrease nitrate production at any time point (data not shown). Nitrite production by THP-1 cells was not measured because human macrophages do not produce NO when stimulated in vitro (Stuehr and Marletta, 1987
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DCPA Inhibits iNOS Protein Expression
In activated macrophages, NO is produced by iNOS, an enzyme converting L-arginine to L-citrulline. To determine whether the reduced NO production by PEC exposed to DCPA was due to the inhibition of iNOS protein expression, cellular lysates were analyzed by Western blotting. In these experiments, PEC were treated with 5, 25, or 100µM DCPA or vehicle and simultaneously stimulated with LPS for 6, 12, and 24 h. iNOS protein expression was undetected in unstimulated PEC; however, gradual induction of expression was observed over a period of time as a result of LPS stimulation (Figs. 7A and 7B). A 6 h, treatment resulted in detectable levels of iNOS demonstrating 10 and 14% inhibition at 25 and 100µM DCPA, respectively. A 12 h, treatment resulted in a dose-dependent decrease in iNOS protein levels demonstrating the largest decrease of 29% in 100µM DCPA–treated cells (p < 0.05) and 9% in 25µM DCPA–treated cells compared to vehicle-treated controls. The longest exposure to DCPA, 24 h, demonstrated a 12% decrease at 100µM DCPA (p < 0.05). The lowest does of DCPA, 5µM, did not decrease expression in comparison to control-treated cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Appropriate macrophage function is critical for normal immune functions and homeostasis as macrophages participate in various aspects of innate and adaptive immunity as well as tumor surveillance. The present report demonstrates that DCPA decreased TNF-
production from the human THP-1 cell line, similar to results shown previously in murine cells (Frost et al., 2001; Xie et al., 1997a). DCPA also caused reductions in phagocytic ability, ROS and RNS production, and listericidal activity in both the THP-1 cell line and primary murine PEC. The nuclear factor primarily responsible for activating TNF- transcription is NF-B. Previous work has demonstrated that DCPA causes reductions in nuclear levels of NF-B in THP-1 cells (Frost et al., unpublished data). Since DCPA inhibits NF-B activation, it likely plays a role in the reduction of TNF-, and investigations are underway to further elucidate the mechanism of suppression of TNF- production by DCPA.
The THP-1 cell line is a human monocytic cell line that has been extensively characterized (Tsuchiya et al., 1980, 1982). These cells have been shown to mobilize NF-B into the nucleus upon stimulation with LPS and produce cytokines typical of activated human macrophages (Gatanaga et al., 1991; Steer et al., 2000). Further differentiation of the cells with PMA leads to a mature, differentiated macrophage phenotype (Tsuchiya et al., 1982). We wanted to demonstrate the effects of propanil in both murine and human models. Primary PEC were used for murine studies because we had previously established the effects of propanil on cytokine production and other immune parameters with these cells (Frost et al., 2001; Xie et al., 1997a,b). THP-1 cells were chosen for a human model due to the existing body of literature regarding their monocytic phenotype and characterizing their cytokine response, respiratory burst activity, and phagocytic abilities (Condino-Neto et al., 1998; Delbosc et al., 2002; Gatanaga et al., 1991; Steer et al., 2000; Tsuchiya et al., 1980). The use of THP-1 cells also did not impose the restrictions of utilizing primary human monocytes.
DCPA has also been shown to ablate the IP3-mediated calcium release in macrophages despite the generation of IP3 equivalent to control cells (Xie et al., 1997b). An extensive body of literature has established a critical role for calcium (Ca2+) in various aspects of the generation of a respiratory burst in macrophages and neutrophils (Dahlgren et al., 1992; Dusi et al., 1993; Foyouzi-Youssefi et al., 1997; Geiszt et al., 1999; Movitz et al., 1997). Calcium has specifically been implicated to play a role in the translocation of the p47phox cytosolic component of the NADPH oxidase to the membrane-bound cytochrome b portion (Zhou et al., 1997). Recently, Granfeldt et al. (2002) further evaluated the differential activation of plasma membrane- and granule-localized NADPH oxidase, showing that intracellular NADPH oxidase activity could be activated by an increase in intracellular Ca2+ via the capacitative Ca2+ entry, whereas plasma membrane-localized NADPH oxidase activity required the capacitative Ca2+ influx in addition to other signaling events. The results demonstrating reductions in respiratory burst activity have been generated using luminol, which can cross the plasma membrane (Dahlgren and Karlsson, 1999), as the chemiluminescent detector; therefore, the measurements may reflect a decrease in activity of either intracellular or plasma membrane-localized NADPH oxidase activity, or both. This decrease in NADPH oxidase activity is potentially a result of the altered calcium dynamics induced by DCPA.
The effects of DCPA on respiratory burst activity may also affect the listericidal capability of macrophages. Contributions of the various phagocyte oxidase (phox) proteins to the listericidal capacity of macrophages have been investigated using mice with targeted gene deletions (Dinauer et al., 1997; Endres et al., 1997; Shiloh et al., 1999). These studies demonstrated that macrophages harvested from gp91phox–/– mice were substantially defective in their ability to kill L. monocytogenes (Shiloh et al., 1999). Dinauer et al. (1997), also using gp91phox–/– mice, suggested that neutrophils and ROS controlled early in vivo Listeria infection. Macrophages and RNS production were suggested to be important in later stages of infection.
The CM-H2DCFDA fluorescent method demonstrated that within 2–3 min of LPS and PMA stimulation, H2O2 and hydroxyl radical were produced. DCPA treatment inhibited their production and that can potentially decrease the ability of macrophages to fight L. monocytogenes and other bacterial infections. Previous studies by Watson et al. (2000) demonstrated that exposure to DCPA, 2 or 7 days prior to L. monocytogenes infection did not affect bacterial colonization of the spleens and livers at 3 days postinfection when compared to control mice. However, it is not known if exposure to DCPA at the time of infection would have an immediate effect on the ability of macrophages to control early bacterial replication and thus increase susceptibility of the host. Myers et al. (2003) demonstrated that ROS were absolutely required for L. monocytogenes clearance by activated macrophages. In their model, gp91phox–/– mice defective in NADPH oxidase were unable to generate superoxide, resulting in bacterial escape from the phagolysosome and proliferation in the cytoplasm (Myers et al., 2003). A critical factor implicated in the escape of Listeria from the phagolysosome is lysteriolysin O (LLO), a sulfhydryl-activated pore-forming hemolysin secreted by L. monocytogenes. It has been reported that mutants lacking LLO remained in vacuoles without proliferating (Gaillard et al., 1987; Portnoy et al., 1988). Myers et al. (2003) suggest that ROS could inactivate LLO, thereby preventing the escape of Listeria into the cytosol. The present report studies the initial events in the course bacterial infection, which does not contradict with previously published report by Watson et al. (2000).
THP-1 cells generally were less sensitive than primary mouse cells. There are known changes in this cell line from normal blood monocytes that may explain that this difference includes the lack of functional p53, resulting in impaired signal transduction (Traore et al., 2005). THP-1 cells also failed to respond to chemokine stimulation to polymerize actin and demonstrate cell polarization (Vaddi and Newton, 1994). NADPH oxidase activity and superoxide production in THP-1 cells were also diminished compared to primary blood monocytes (Almeida et al., 2005).
Examination of NO2 accumulation in the media allowed for direct RNS detection upon LPS stimulation of primary mouse cells. A decrease in nitrite production can be explained by down regulation of iNOS enzyme, resulting in low levels of NO production. Studies on LPS-activated macrophages demonstrated that the iNOS gene has two consensus sequences for binding of NF-B. Upon LPS activation, the heterodimer p65/p50 has been reported to be responsible for NF-B binding to the iNOS promoter (Kim et al., 1997; Na et al., 1999; Spink et al., 1995; Xie et al., 1994). DCPA exposure caused a decrease in NF-B binding and its transcriptional activity (Frost et al., 2001) and could explain a decrease in NO production through inhibition of iNOS gene expression. A study by Ohya et al. (1998a) observed an induction of iNOS mRNA transcription and gene expression followed by accumulation of nitrite in culture media as a result of L. monocytogenes infection.
DCPA also decreases phagocytosis. Interestingly, phagocytosis in macrophages is regulated to some extent by activation of the signaling proteins syk and PI-3K (Aderem and Underhill, 1999). Activation of PI-3K leads to the downstream activation of PKC, Akt, and NF-B. The Internalin B protein of L. monocytogenes, which mediates internalization of the bacterium, also requires activation of PI-3K (Ireton et al., 1996). InlB ultimately leads to NF-B activation via PI-3K and Akt (Mansell et al., 2001). DCPA alters NF-B activation (Frost et al., 2001), and further studies have demonstrated that DCPA also affects Akt activity (Barnett et al., unpublished data). Studies examining the effects of DCPA on the upstream mediator PI-3K are ongoing. It is possible that the signaling alterations induced by DCPA exposure affect several pathways that culminate in the observed reductions in phagocytosis.
The data herein demonstrate the profound effects of DCPA on macrophage function. DCPA exposure results in decreased TNF- production, phagocytosis, ROS and RNS production, and listericidal activity. Additionally, the effect of DCPA on the respiratory burst suggests the potential use of DCPA as an inhibitor/antioxidant. Future laboratory experiments are aimed at furthering our understanding of respiratory burst regulation and redox signaling in cells using DCPA.
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NOTES |
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2 Present address: Department of Natural Sciences and Engineering Technology, Point Park University, Pittsburgh, PA 15222.
3 These authors contributed equally to this study.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the National Institute of Environmental Health Sciences (ES07512).
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REFERENCES |
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