Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2001;70:367-373.)
© 2001 by Society for Leukocyte Biology

Activation of human neutrophils in vitro and dieldrin-induced neutrophilic inflammation in vivo

Martin Pelletier*, Charles J. Roberge{dagger}, Marc Gauthier*, Karen Vandal{ddagger}, Philippe A. Tessier{ddagger} and Denis Girard*

* INRS-Institut, Armand-Frappier/Santé Humaine, Université du Québec, Pointe-Claire;
{dagger} Association du Cancer de l’Est du Québec, Rimouski; and
{ddagger} Centre de Recherche en Infectiologie, Université Laval, Sainte-Foy, Québec, Canada

Correspondence: Denis Girard, Ph.D., INRS-Institut Armand-Frappier/Santé Humaine, 245 boul Hymus, Pointe-Claire, Québec H9R 1G6, Canada. E mail: Denis.Girard@INRS-Sante.Uquebec.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many chemicals of environmental concern are known to alter the immune system and are considered toxic molecules because they affect immune cell functions. Inflammation related to environmental chemical exposure, however, is poorly documented, except that from air pollutants. In this study, we found that the organochlorine insecticide dieldrin could not alter the ability of human neutrophils to phagocytose opsonized sheep red blood cells at nonnecrotic concentrations (0.1, 1, 10, and 50 µM). However, dieldrin was found to increase human neutrophil superoxide production, RNA synthesis, and proinflammatory cytokine interleukin-8 production. The normal apoptotic rate of neutrophils evaluated by both cytology and flow cytometry (CD-16 staining) was not altered by dieldrin treatments, and this was correlated with its inability to inhibit spreading of neutrophils onto glass. Using the murine air pouch model, we found that dieldrin induces a neutrophilic inflammation. Taken together, these results demonstrated that dieldrin is a proinflammatory contaminant. To our knowledge, this is the first report establishing that dieldrin is a contaminant exhibiting proinflammatory properties. In addition, it is the first time that the murine air pouch model has been successfully used to confirm that a chemical of environmental concern can induce an inflammatory response in vivo.

Key Words: pollutants • organochloride • O2- production • IL-8 • murine air pouch


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The toxicity of chemicals of environmental concern to the immune system has been primarily evaluated in animals and, to a lesser extent, in humans. In particular, the effects of various pollutants on B, T, natural killer, and monocyte-macrophage cells have been the focus of several reports, but neutrophils have been largely neglected. Recent data indicate that inflammation occurs in response to such chemicals [1 2 3 4 5 6 ]. Particularly, different dusts and other air pollutants are known to induce leukocyte infiltration into rodent airways [3 4 5 6 7 ].

Neutrophils are greatly involved in inflammation. They are the most abundant leukocytes of the blood and the first type of cell to arrive at an inflammatory site. These cells are known to exert phagocytosis, produce free radicals, synthesize various products such as cytokines and chemokines, and release proteolytic enzymes via degranulation [8 9 10 11 ]. Neutrophils also spontaneously undergo apoptosis in both in vitro and in vivo conditions [12 , 13 ]. This important mechanism controls the cell turnover rate of neutrophils. Although the responses of neutrophils to different agonists and pathogens are generally beneficial for host defense, they can be deleterious to the organism if these cells are inappropriately activated. In this sense, production of free radicals and proteolytic enzymes used as defenses against bacteria and microorganisms can be highly toxic to surrounding cells and tissues [14 , 15 ]. Consequently, it is important to identify neutrophil agonists.

Chlorinated compounds are recognized as environmental pollutants because they are associated with acute toxicity. Nevertheless, little is known about their effects on host defense mechanisms [16 ]. Dieldrin is a chlorinated chemical that belongs to the category of environmental pollutants designated as persistent organic pollutants (POPs) because these compounds accumulate in the food chain, persist in the environment, and reach higher trophic levels (in animals higher in the food chain). Many countries have therefore either restricted or banned the use of POPs [17 ]. Nevertheless, humans are still at risk of exposure to POPs. These chemicals enter the human body through the respiratory system or contaminated food or directly through the epidermis.

Dieldrin has traditionally been used in agriculture as an insecticide and has been detected in air, water, soil, fish, birds, and humans (particularly in human breast milk) [18 , 19 ]. Food, especially dairy products and meats, is the primary source of human exposure to dieldrin [20 ], but it can also originate from the metabolism of aldrin, another POP targeted by the United Nations Environment Program. Dieldrin and aldrin are toxic to humans [21 ]. Symptoms of aldrin and dieldrin intoxication include headache, dizziness, nausea, and convulsions [21 ], but there is limited evidence that dieldrin affects the immune system. Dieldrin is known to activate rat neutrophils in vitro [22 ], causing a marginal concentration-dependent increase in superoxide (O2-) production by neutrophils. In addition, this chemical has been found to act as a priming agent in a concentration-dependent fashion to the bacterial tripeptide N-formyl-methionine-leucine-phenylalanine agonist and phorbol 12-myristate 13-acetate (PMA). This effect is dependent on extracellular calcium [22 ]. Furthermore, it was recently found that dieldrin can induce the liberation of arachidonic acid and that more than one isoform of phospholipase A2 is activated by this chemical in rat neutrophils [23 ]. To date, there has been no information available concerning the interaction between dieldrin and human neutrophils and no clear evidence that this chemical possesses proinflammatory properties, although inflammatory disorders are increasingly associated with and/or related to contaminant exposure, especially air pollutants that induce an acute inflammatory response in human airways [23a].

The activation of human neutrophils by chemicals of environmental concern is gaining increasing attention [24 25 26 27 28 29 30 31 32 ]. We have recently initiated studies focusing on the interactions between environmental contaminants and neutrophils to determine whether neutrophils respond differently to various pollutants and to compare their responses with those to other stimuli such as cytokines and lectins [33 ]. We reported that Na2SO3 can activate human neutrophils to produce O2- and enhance phagocytosis without altering the apoptotic rate [26 ]. The same pollutant has been found to inhibit de novo protein synthesis in human neutrophils but is cytotoxic for immature promyelocytic human leukocyte 60 cells [27 ]. In addition, we recently observed that toxaphene, another POP, is a potent neutrophil agonist, because it induces O2- via protein kinase Cs and tyrosine kinases, enhances phagocytosis, and induces apoptosis [28 ] by a mechanism that is as yet unknown. Because dieldrin induces some neutrophil responses in animals, these cells are of great importance in inflammation, and because dieldrin is among the most unwanted pollutants identified by the United Nations Environment Program [17 ], we investigated how it can alter human neutrophil cell responses in vitro and evaluated its potential to induce a proinflammatory response in vivo.

Herein, we describe results indicating that dieldrin acts as a human neutrophil agonist because it was found to induce O2- production, RNA synthesis, and production of the chemokine interleukin (IL)-8 without modulating phagocytosis, apoptosis, or cell spreading. In addition, our use of the murine air pouch model allowed us to conclude that dieldrin can induce neutrophilic inflammation in vivo. This is the first study identifying dieldrin as a human neutrophil agonist. In addition, this is the first time the murine air pouch model has been used in an immunotoxicological study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and agonists
Dieldrin [freshly prepared in a final concentration of <0.1% (v/v) ethyl alcohol (EtOH)] was purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (specific activity of 9x106 U/mg) was a gift from the Genetics Institute (Boston, MA). It was used as a positive control in some experiments at a concentration of 65 ng/mL as previously published [26 , 27 , 34 ]. Lipopolysaccharide (LPS) and PMA were purchased from Sigma-Aldrich Canada. IL-4 was from R&D Systems Inc. (Minneapolis, MN).

Neutrophil isolation
Neutrophils were isolated from venous blood of healthy volunteers by dextran sedimentation and centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech Inc., Baie d’Urfé, Québec) as previously described [26 27 28 ]. Blood donations were obtained from informed and consenting individuals according to institutionally approved procedures. Cell viability was monitored by trypan blue exclusion, and the purity (>98%) was verified by cytology from cytocentrifuged preparations colored by Diff-Quik staining (Baxter, Miami, FL). We concluded that dieldrin (0–50 µM) and its diluent EtOH are not toxic to neutrophils under the various conditions used in this study because cell viability was always >98%. In preliminary experiments, we found that the optimal concentration of dieldrin was 50 µM with respect to the O2- production assay (data not shown).

Phagocytosis of sheep erythrocytes
Sheep red blood cells (SRBCs) (Quelab, Montréal, Canada) were opsonized with a 1/200 dilution of a rabbit immunoglobulin G anti-SRBC antibody (Sigma-Aldrich Canada) for 45 min at 37°C as previously described [26 27 28 29 ]. Neutrophils (4x106 in RPMI1640 alone) that had been pretreated for 30 min with buffer, diluent, IL-4 (10 ng/mL), or increasing concentrations of dieldrin (0–50 µM) were incubated with 20 x 106 opsonized SRBCs for 45 min as described above. The samples were centrifuged at 200 g for 10 min at 4°C. Supernatants were discarded, and osmotic shock was performed on the pellets by treating them with 500 µL of H2O for 15 s and then immediately adding 10 mL of Hanks’ balanced saline solution (HBSS). The samples were washed, and the final pellets were resuspended in 1 mL of HBSS. Duplicate cytocentrifuged samples were prepared in aliquots of ~200 µL and processed as for apoptosis (described below). Phagocytosis was measured by counting the number of neutrophils ingesting at least one opsonized SRBC. IL-4 was used as a positive control because it has been previously reported to enhance neutrophil phagocytosis [35 ].

O2- production
O2- production was monitored in relation to the reduction of cytochrome c as previously reported [26 27 28 ]. Briefly, neutrophil suspensions (106 cells/mL in HBSS supplemented with 1.6 mM CaCl2) were incubated in the presence or absence of 10 µg/mL of O2- dismutase (Sigma-Aldrich Canada) with 130 µM cytochrome c (Sigma-Aldrich Canada) for 15 or 30 min at 37°C in the presence of buffer, diluent, PMA (10-7 M), or increasing concentrations of dieldrin. The absorbance of cytochrome c was monitored at 550 nm, and the number of O2- anions produced was calculated by the difference between corresponding wells with or without O2- dismutase using an extinction coefficient of 21.1.

RNA synthesis
RNA synthesis was evaluated by measuring the incorporation of [5-3H]uridine (Amersham Corp., Oakville, Canada) into total RNA essentially as previously described [34 ]. Briefly, 100 µL of a 5 x 106-cells/mL suspension were incubated in 96-well microtiter plates in the presence of 1 µCi of [3H]uridine along with buffer, diluent, GM-CSF, or increasing concentrations of dieldrin for 4 h at 37°C in an atmosphere with 5% CO2. After incubation, the cells were collected onto borosilicate glass fiber paper with a multiple-cell culture harvester (Skatron Instruments Inc., Sterling, VA). Sections of the filter corresponding to each microwell were then punched out and placed into scintillation counting vials with 4 mL of ScintiSafe Econo 1 (Fisher Scientific Canada, St. Laurent, Québec). The results were expressed as stimulation indices (cpm from tested cells/cpm from cells treated with diluent alone).

Assessment of neutrophil apoptosis by cytology and by CD16 expression
Freshly isolated human neutrophils (100 µL of a 10x106/mL suspension in RPMI 1640 supplemented with 10% autologous serum) were incubated for 24 h in the presence or absence of increasing concentrations of dieldrin, and apoptosis was evaluated by cytology. Preparations of neutrophils were cytocentrifuged using a Cyto-tek® centrifuge (Miles Scientific, Elkhart, IN) as previously described [26 27 28 , 34 ] and were stained with a Diff-Quik staining kit according to the manufacturer’s instructions. The cells were examined by light microscopy at 400x final magnification. Apoptotic neutrophils were defined as cells containing one or more characteristically dark-stained pyknotic nuclei. An ocular with a 10- by 10-square grid was used to count at least five different fields (>100 cells) for assessment of apoptotic cells. The results were expressed as the percentage of neutrophils in apoptosis.

CD16 expression is known to be down-regulated in apoptotic neutrophils [36 , 37 ], so it has been used to confirm the inability of dieldrin to modulate the neutrophil apoptotic rate [36 , 37 ]. After 24 h of incubation in the presence or absence of dieldrin, the cells were suspended at concentrations of 1.5 x 106/mL, washed, and preincubated for 30 min at 4°C (light protected) with 20% autologous serum to prevent nonspecific binding via Fc receptors. The cells were then washed and incubated with 2 µL of fluorescein isothiocyanate-mouse anti-human CD16 monoclonal antibody (mAb) (PharMingen Canada, Mississauga, Ontario) for 30 min at 4°C (light protected) before fluorescein-activated cell sorter analysis. Flow-cytometric analysis (10,000 events) was performed using a FACScan (Becton Dickinson, San Jose, CA).

Neutrophil spreading onto glass
The inability of neutrophils to spread onto glass was previously found to correlate with induction of apoptosis [38 , 39 ]. We evaluated this response by incubating cells (5x106/mL in RPMI 1640 supplemented with 1% autologous serum) in 24-well plates at 37°C in an atmosphere with 5% CO2 in the presence of buffer, diluent, LPS (1 µg/mL), or increasing concentrations of dieldrin for 12 h, the length of time previously found to be optimal for this assay [38 , 39 ]. Then 10 µL of the cell suspension were loaded onto a hemacytometer and incubated for 5 min at 37°C. Immediately after incubation, the cells were examined under a light microscope and recorded as spread (irregularly shaped) or nonspread (round shaped).

Production of IL-8
IL-8 production was measured in freshly isolated neutrophils (10x106/mL in RMPI 1640 supplemented with 5% fetal calf serum) treated with buffer, 65 ng/mL of GM-CSF, or increasing concentrations of dieldrin for 24 h. The concentration of IL-8 released in the external milieu was determined using a commercially available enzyme-linked immunosorbent assay kit (sensitivity <10 pg/mL) (R&D Systems) according to the manufacturer’s instructions. All assays were performed at least in duplicate.

In vivo neutrophilic inflammation
We decided to use the murine air pouch model [40 41 42 ] for evaluating potential proinflammatory effects of dieldrin. C57Bl/6 mice (6–8 weeks old) were obtained from Charles River, Canada (St.-Constant). The mice (five/group) were anesthetized with isofurane, and the dorsal nuchal region was shaved and swabbed with isopropyl alcohol. Sterilized air (3 mL filtered through a 0.2-µm Millipore filter) was injected subcutaneously in the back using a 26-gauge needle to make an air pouch on day 0 and day 3. At day 6, 1 mL of dieldrin (50 µM) or its diluent [0.1% EtOH in phosphate-buffered saline (PBS)] or 1 µg/mL of LPS (positive control) was injected into the air pouches of mice 6 or 12 h before the mice were killed by CO2 asphyxiation. The air pouches were washed once with 1 mL and then twice with 2 mL of PBS containing 5 mM EDTA, and the exudates were centrifuged at 100 g for 10 min at room temperature. The cells were resuspended in 1 mL of PBS, stained with acetic blue, and counted. The cells (2x105) were centrifuged onto microscope slides and stained with Diff-Quik to allow quantification of granulocytic and monocytic populations. To further characterize the leukocyte subpopulations, the cells were suspended in PBS supplemented with 0.1% bovine serum albumin (BSA) and stained as previously described [40 ] for 30 min at 4°C with purified rat anti-mouse Ly-6G (Gr-1) mAb directed against murine neutrophils (PharMingen Canada) or rat anti-mouse F4/80 antigen antibody directed against murine monocyte/macrophages (PharMingen Canada). The cells were washed three times with PBS supplemented with 1% BSA before being incubated with goat anti-rat-fluorescein isothiocyanate antibodies diluted in the same buffer for 30 min at 4°C. The cells were washed three times, and analysis was performed with an EPICS 753 flow cytometer (Coulter, Miami Lakes, FL).

Statistical analysis
Statistical analysis was performed with SigmaStat for Windows version 2.0 using a one-way analysis of variance. Statistical significance was established at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Neutrophil phagocytic function is not altered by dieldrin
Phagocytosis is an important function of neutrophils. We decided to investigate whether dieldrin could alter the ability of human neutrophils to phagocytose opsonized SRBCs. As illustrated in Figure 1 , this chemical did not significantly modulate phagocytosis. As expected, IL-4 was found to increase phagocytosis, because the percentage of cells ingesting at least one SRBC was 39.3 ± 4.3%, when compared with 23 ± 4.8% for control neutrophils.



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  		Figure 1. Dieldrin does not alter the neutrophil phagocytosis of opsonized SRBCs. Phagocytosis was evaluated by determining ingestion of opsonized SRBCs by neutrophils pretreated for 30 min with buffer (Ctrl), diluent (Dil), 10 ng/mL of IL-4, or increasing concentrations of dieldrin as described in Materials and Methods. Results are expressed as the percentage of phagocytosis (cells ingesting at least one opsonized SRBC/number of cells countedx100) and are means plus or minus SE (n=3 different blood donors).

 
Dieldrin induces ;O2- production
To better investigate how dieldrin could influence neutrophil cell physiology, we next studied its effect on neutrophil respiratory burst activity by measuring O2- production. As illustrated in Figure 2 , dieldrin can induce such a response at 10 and 50 µM but not at 0.1 and 1 µM. In fact, after 30 min of stimulation with 10 or 50 µM dieldrin, we observed that this chemical can induce O2- production as strongly as the classical neutrophil agonist PMA (Fig. 2B) .



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  		Figure 2. Activation of O2- production by dieldrin. Freshly isolated human neutrophils were treated with buffer (Ctrl), the diluent (Dil), or concentrations of 0.1, 1, 10, or 50 µM dieldrin for 15 (A) or 30 (B) min, and the O2- production was measured as described in Materials and Methods. Results are means plus or minus SE (n=4 different blood donors).

 
Dieldrin is an activator of RNA synthesis
Modulation of RNA synthesis, as assessed by measurement of [3H]uridine incorporation, can be used as a marker of neutrophil activation [34 ]. The results shown in Figure 3 illustrate that dieldrin can activate RNA synthesis in a concentration-dependent manner. The indices of stimulation (mean plus or minus SE; n=4) increased from 1.3 ± 0.2 to 3.5 ± 0.2 when the cells were treated with dieldrin at 0.1 or 50 µM, respectively. As expected [34 ], the potent agonist GM-CSF was found to significantly increase neutrophil RNA synthesis (index: 8.2±0.5).



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  		Figure 3. Induction of RNA synthesis by diedrin. Neutrophils were stimulated with agonists for 4 h in the presence of 5-[3H]uridine, and RNA synthesis was evaluated as described in Materials and Methods. Results are means plus or minus SE (n=4 different blood donors). Ctrl, buffer; Dil, diluent; GM, GM-CSF.

 
Neutrophil apoptotic rate and spreading onto glass are not modulated by dieldrin
Because we found that dieldrin could induce the O2- production and that free radicals can induce neutrophils to undergo apoptosis [43 ], we next investigated whether dieldrin could alter neutrophil apoptotic rates. As illustrated in Figure 4 , after 24 h of culture, ~40–70% of the neutrophils were apoptotic, as measured by cytology and by loss of CD16 expression (inset). Dieldrin could not significantly modulate the neutrophil apoptotic rate. As expected, GM-CSF was found to delay the normal neutrophil apoptotic rate. In addition, because it was previously found that apoptotic neutrophils lose their ability to spread onto glass [38 , 39 ], we investigated whether dieldrin could decrease this response. The results shown in Figure 5 are in agreement with the fact that dieldrin cannot alter the neutrophil apoptotic rate. The number of spread cells remained stable after treatment with dieldrin and was similar to the number of spread cells treated with the diluent. This reinforces the observation that this chemical is not a modulator of neutrophil apoptosis and that it is not cytotoxic to these cells at the concentrations used.



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  		Figure 4. Neutrophil apoptotic rate is not altered by dieldrin. Freshly isolated human neutrophils (10x106/mL) were incubated for 24 h in the presence of buffer (Ctrl), diluent (Dil), GM-CSF (GM), or increasing concentrations of dieldrin. Apoptosis was assessed by cytology (Diff-Quik staining) as described in Materials and Methods. Results are means plus or minus SE (n=4 different blood donors). The charts at left show one representative experiment out of three illustrating cell surface expression of CD16.

 


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  		Figure 5. Dieldrin does not alter neutrophil cell spreading. Cells were incubated for 12 h with buffer (Ctrl), diluent (Dil), LPS (1 µg/mL), or increasing concentrations of dieldrin, and the spreading of cells onto glass was assessed as described in Materials and Methods. Results are means plus or minus SE (n=4 different blood donors).

 
Production of IL-8 is increased by dieldrin
IL-8 is a potent chemotactic factor for neutrophils. Moreover, production of this proinflammatory cytokine is an important marker of neutrophil activation. We performed experiments to determine whether dieldrin could induce the secretion of IL-8 by human neutrophils. As illustrated in Figure 6 , dieldrin induced the secretion of IL-8 in a concentration-dependent manner, but this effect was statistically significant only at a concentration of 50 µM. All other treatments were not significant when compared with both the control and the diluent.



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  		Figure 6. IL-8 production is increased by dieldrin. Neutrophils were incubated for 24 h in the presence of buffer (Ctrl), diluent (Dil), or increasing concentrations of dieldrin. The supernatants were harvested, and IL-8 production was measured by ELISA as described in Materials and Methods. Results are means plus or minus SE (n=4 different blood donors).

 
Induction of neutrophil influx in vivo by dieldrin
The murine air pouch model was used to evaluate the potential inflammatory effect of dieldrin in vivo. Dieldrin was found to significantly increase the number of total leukocytes 6 h after injection into the air pouch. Leukocyte influx induced by dieldrin was, however, transitory, because the leukocyte accumulation observed in the air pouch returned to baseline levels within 12 h. As expected, leukocyte influx was observed after 6 h of LPS treatment, used here as a positive control (Fig. 7 ). Neutrophils constituted the main cell population that migrated to the pouch after 6 h in both dieldrin- and LPS-induced animals, because they represented ~76% and ~68%, respectively, of the total leukocytes in the pouch. Cells of the monocytic lineage were the other main population observed in the air pouches, and they represented <~24% and ~16% of the dieldrin- and LPS-induced cell populations, respectively.



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  		Figure 7. Dieldrin induces neutrophilic inflammation in vivo. The murine air pouch model was used for evaluating the potential proinflammatory effect of dieldrin by counting total leukocytes, neutrophils, and monocytes-macrophages as described in Materials and Methods. Mice were treated for 6 or 12 h with 50 µM dieldrin, the diluent (Dil), or 1 µg/mL of LPS. Results are means plus or minus SE (n=5 different blood donors). The inset shows a representative cytocentrifuged preparation of cells collected from one dieldrin-induced mouse after 6 h. The arrow indicates a cell of the monocytic lineage. Note the predominance of neutrophils.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, an increasing number of environmentally significant chemicals were found to possess some proinflammatory properties [8 9 10 ]. The present study was aimed at better understanding how the organochlorine insecticide dieldrin can alter the human neutrophil cell physiology and at verifying its proinflammatory potential. In this report, we provided evidence that dieldrin is a potent activator of O2- production, RNA synthesis, and IL-8 production in human neutrophils. In contrast, this compound did not modulate phagocytosis, apoptosis, or cell spreading, demonstrating that it was not cytotoxic at concentrations ranging from 0.1 to 50 µM. Proinflammatory properties of dieldrin were confirmed in vivo, because it was found to induce neutrophil accumulation in murine air pouches.

The fact that dieldrin was found to induce RNA synthesis demonstrates that this chemical is a potent activator of human neutrophils. These data suggest that dieldrin induces neutrophil gene expression. We have observed similar results with sodium sulfite [26 , 27 ] and with cytokines such as IL-4 [44 ], IL-15 [34 ], tumor necrosis factor-{alpha} [45 ], N-formyl-methionine-leucine-phenylalanine [45 ], and GM-CSF [45 ]. Unlike these latter cytokines, dieldrin [this report] and sodium sulfite [27 ] did not alter the neutrophil apoptotic rate. However, sodium sulfite has been found to enhance phagocytosis of opsonized SRBCs by neutrophils [27 ], which was not the case for dieldrin. Taken together, these observations suggest that the dieldrin-induced neutrophil RNA synthesis is not initiated for modulating apoptosis or phagocytosis.

The ability of dieldrin to increase IL-8 production is of interest. This proinflammatory molecule is a powerful neutrophil chemoattractant. The fact that a chemical such as dieldrin can induce IL-8 production suggests that some environmental contaminants exert proinflammatory properties similar to those of other molecules such as cytokines, which was supported by our observation that dieldrin could induce in vivo neutrophilic inflammation. Although it is known that LPS can induce an inflammatory response that is still observable 12 h after initial challenge, dieldrin-induced leukocyte infiltration peaked after ~6 h of treatment and was no longer observable after 12 h. In concentration-dependent experiments (0.1–50 µM), we observed a potent in vivo inflammatory response only at the concentration of 50 µM dieldrin (data not shown). This is of interest because IL-8 production in dieldrin-induced neutrophils was significantly higher than in controls only at a concentration of 50 µM (Fig. 2) .

In contrast to another POP, toxaphene [28 ], but similar to the non-POP air pollutant sodium sulfite [26 ], dieldrin is not an inductor of neutrophil apoptosis. These observations indicate that neutrophils can respond differently to various chemicals. The mechanisms involved in these responses remain to be determined. In addition, our observations indicate that contaminants are not necessarily toxic to human neutrophils at relatively high concentrations because they do not automatically induce apoptosis or necrosis. It is tempting to speculate that neutrophils possess a mechanism of detoxification. In this sense, neutrophils are known to express the enzyme sulfite oxidase, which can be overexpressed or become more active upon stimulation with sodium sulfite. The mechanism by which neutrophils could detoxify dieldrin is totally unknown and remains purely speculative at this moment.

In this study, we demonstrated for the first time that an environmentally significant chemical that possesses some in vitro proinflammatory properties can induce in vivo inflammation. To our knowledge, this is the first time that the murine air pouch model has been used successfully to evaluate pollutant-induced in vivo inflammation. The air pouch model has been used in the past to evaluate the pro- or anti-inflammatory activities of pharmacological compounds. This model, in addition to our general proposed in vitro approach with the use of human neutrophils, should be used for immunotoxicological evaluation of pesticides and other related molecules because it could shed light on the effects of environmental contaminants on human health, particularly regarding inflammatory host responses.


   ACKNOWLEDGEMENTS
 
This study was partly supported by Fonds de la Recherche en Santé du Québec (FRSQ), Fonds pour la Formation de Chercheurs et l’Aide à la Recherche, FRSQ-Réseau de Recherche en Santé Environnementale, and Association du Cancer de l’Est du Québec. M. P. holds an MSc Fonds pour la Formation de Chercheurs et l’Aide à la Recherche, FRSQ Santé award. D. G. and P. A. T. are scholars from FRSQ and The Arthritis Society, respectively. We thank Mary Gregory for reading the manuscript.

Received September 1, 2000; revised April 25, 2001; accepted April 26, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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