Induction of Nitric Oxide Production by Bovine ... - ScienceDirect

J. Dairy Sci. 84:1430–1437  American Dairy Science Association, 2001.

Induction of Nitric Oxide Production by Bovine Mammary Epithelial Cells and Blood Leukocytes1 V. Boulanger,* L. Bouchard,*,2 X. Zhao† and P. Lacasse* *Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, P.O. Box 90, 2000 Route 108 East, Lennoxville, Quebec, J1M 1Z3, Canada †Department of Animal Science, McGill University, 21 111 Lakeshore Rd, Ste-Anne de Bellevue, Quebec, H9X 3V9, Canada

ABSTRACT A recent study from our laboratory has shown that significant amounts of nitric oxide are released by somatic cells recovered during endotoxin-induced mastitis. The present study was undertaken to investigate which cell type(s) among milk somatic cell population can produce nitric oxide under inflammatory conditions. Nitric oxide release from mammary epithelial cell lines and from bovine neutrophils and monocytes extracted from blood was measured in response to cytokines and Escherichia coli lipopolysaccharides. An epithelial cell line isolated from bovine mammary gland, FbE cells, was found to release nitric oxide after exposure to interleukin-1β. This nitric oxide production was completely abolished by addition of L-N6-(1-iminoethyl) lysine, a potent inducible nitric oxide synthase inhibitor. Bovine monocytes produced nitric oxide in response to recombinant bovine interferon-γ alone or in combination with E. coli lipopolysaccharides. In these cells, nitric oxide release was reduced by the addition of inducible nitric oxide synthase inhibitors L-N6-(1iminoethyl) lysine and aminoguanidine. Lipopolysaccharides and recombinant bovine interferon-γ increased nitric oxide synthase mRNA in neutrophils, but nitric oxide release could not be detected under any of the experimental conditions used. These results show that bovine epithelial cells and mononuclear phagocytes produce nitric oxide under inflammatory conditions and suggest that these cell populations are responsible for nitric oxide release observed during mastitis. (Key words: nitric oxide, somatic cells, mastitis, inducible nitric oxide synthase)

Received October 30, 2000. Accepted February 4, 2001. Corresponding author: P. Lacasse; e-mail: [email protected]. 1 Dairy and Swine Research and development Centre contribution No. 688. 2 Present address: De´partement de Microbiologie et d’Infectiologie, Faculte´ de Me´decine Universite´ de Sherbrooke, Sherbrooke Quebec, J1H 5N4, Canada.

Abbreviation key: DMEM = Dulbecco’s modified Eagle’s medium, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, HBSS = Hanks balanced salt solution, iNOS = inducible nitric oxide synthase, L-NIL = L-N6(1-iminoethyl) lysine, LPS = lipopolysaccharide, NO = nitric oxide, NOx = nitrite plus nitrate, PMA = phorbol 12-myristate 13-acetate, rBoIFN-γ = recombinant bovine interferon-γ, rBoIL-1β = recombinant bovine interleukin-1β, rBoIL-2 = recombinant bovine interleukin-2, RT = reverse transcription, SOD = superoxide dismutase, TNF-α = tumor necrosis factor α. INTRODUCTION Mastitis is a devastating disease affecting the dairy cow and represents a significant economic loss to dairy producers. Mastitis is an inflammatory response of the mammary tissue to physiological and(or) metabolic changes, traumas or, more frequently, to injuries caused by microorganisms. During inflammatory response, the animal immune system is mobilized to protect the host and eliminate the pathogen. Macrophages, which are the first to encounter and phagocytose the pathogen, release chemotactical substances, including cytokines, which massively recruit neutrophils to the infected gland (Burvenich et al., 1994; Craven, 1983; Shuster et al., 1997). Mechanisms that normally protect the host from infection and eliminate bacteria can also cause extensive tissue injury in some situations. The inflammatory reaction accompanying mastitis causes mammary secretory cell damage, resulting in reduced milk production (Oliver and Calvinho, 1995; RajalaShultz et al., 1999). Nitric oxide (NO) is a potent biological effector regulating blood vessel dilatation, serving as a neuronal messenger and playing a complex role in inflammatory response (Dawson and Dawson, 1995). A family of three enzymes termed NO synthases produces NO from Larginine. Two isoforms are calcium/calmodulin-dependent and are constitutively expressed. The inducible isoform of NO synthase (iNOS) is expressed in a wide

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array of cell types after stimulation with inflammatory mediators and bacterial products (Bochsler et al., 1996; Nath and Powledge, 1997). Involvement of cytokines such as interleukin-1β, tumor necrosis factor α (TNFα), and interferon-γ during mastitis has been documented (Shuster et al., 1993; Sordillo and Peel, 1992). Interleukin-1 and TNF-α cause a rapid translocation of NF-κB a nuclear factor, implicated in the control of several inflammatory genes like iNOS (Gilad et al., 1998). Overproduction of NO was observed in several inflammatory diseases (Dawson and Dawson, 1995; Hobbs et al., 1999). Toxic effects of NO occur through the formation of peroxynitrite, a powerful oxidant that causes diverse chemical reactions in biological systems, including protein and DNA nitrosylation as well as lipid peroxidation (Maeda and Akaike, 1998; Murphy, 1999). It is presently not known whether peroxynitrite is a damage-causing agent during bovine mastitis. Recently, our laboratory clearly showed that significant amounts of NO are released in milk during clinical (Lacasse et al., 1997) and endotoxin-induced mastitis (Bouchard et al., 1999). We have also shown that somatic cells recovered from lipopolysaccharide (LPS)infused quarters can release NO into the culture medium. The cell type(s) responsible for this NO production is(are) still unknown. Milk somatic cells consist of several cell types, including macrophages, neutrophils, lymphocytes, and a smaller percentage of epithelial cells. During mastitis, neutrophils become the major cell type found in mammary secretions and can constitute more than 90% of the total somatic cells (Kehrli and Shuster, 1994; Paape et al., 1981). In the study reported here, two mammary epithelial cell lines (FbE and MAC-T) and blood leukocytes (neutrophils and monocytes) were used to verify involvement of these cell types in NO production observed during mastitis. Bovine monocytes from peripheral blood can be induced by cytokines and phagocytic stimulus to produce NO (Goff et al., 1996; Zhao et al., 1996). However, there is no report of NO release by bovine neutrophils and mammary epithelial cells. The objectives of this study were to determine whether mammary epithelial cell lines or blood leukocytes would release NO upon stimulation with LPS and cytokines involved in mastitis. MATERIALS AND METHODS Mammary Epithelial Cell Culture Two different bovine cell lines were used to study NO production by mammary epithelial cells: MAC-T and FbE. The epithelial cell line MAC-T is widely used (Huynh et al., 1991), and FbE cells are spontaneously immortal cells positive for vimentin and ZO-I (Woodward,

1996). Cells were grown on tissue culture plastic in complete Dulbecco’s modified Eagle medium (DMEM; Sigma Chemical Co., St. Louis, MO) supplemented with 10% (vol/vol) fetal bovine serum, 1 µg/ml of insulin, and 1% (vol/vol) antibiotic/antimycotic. Reagents for In Vitro Activation All reagents, including recombinant bovine interferon-γ (rBoIFN-γ, generously supplied by Novartis, Basel, Switzerland), recombinant bovine interleukin1β (rBoIL-1β), interleukin-2 (rBoIL-2, American Cyanamid Co., Princeton, NJ), and LPS (Escherichia coli 055:B5, Sigma) were dissolved in PBS. Aminoguanidine (Sigma) and L-N6-(1-iminoethyl) lysine (L-NIL, Calbiochem, San Diego, CA) were used as competitive inhibitor of L-arginine to demonstrate the specificity of the NO synthesis pathway. Isolation of Leukocytes from Peripheral Blood Leukocytes were isolated from whole blood according to a method described by Carlson and Kaneko (1973) with some modifications. Briefly, blood samples were collected from the caudal vein of healthy lactating Holstein cows into EDTA-coated Vacutainer tubes. Whole blood was layered on Ficoll-Paque Plus (Amersham Pharmacia, Montreal, Canada) and centrifuged 40 min at 500 × g. For monocytes isolation, interface (containing monocytes and lymphocytes) between plasma and Ficoll-Paque Plus was washed in Hanks balanced salt solution (HBSS) and centrifuged, and cells were resuspended in DMEM-F12. This cell suspension was layered on tissue culture plates previously treated with fetal bovine serum for 1 h at 37°C. Monocytes were allowed to adhere for 1 h at 37°C in a 5% CO2 atmosphere. Nonadherent cells were removed, and adherent cells (monocytes) were rinsed with warm DMEM-F12 and removed from plastic by incubating in DMEM-F12 containing 0.01 M EDTA for 20 min at 37°C followed with vigorous shaking. Monocytes were washed twice in HBSS and suspended (1 × 106 cells/ml) in DMEMF12 for NO production assays. Neutrophils were isolated by hypotonic lysis of erythrocytes in the red pellet with Tris-buffered 0.15 M ammonium chloride. Cells were washed twice in HBSS with centrifugation at 350 × g for 10 min and suspended (1 × 106 cells/ml) in DMEM-F12 for NO production assays. This procedure typically produced cell fractions with >99% viability as determined by trypan blue dye exclusion. NOx Determination Nitric oxide production was evaluated by measuring its more stable metabolites, nitrite and nitrate (NOx), Journal of Dairy Science Vol. 84, No. 6, 2001

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in cell-free supernatants. For NOx assays, cells were subcultured in 24-well culture plate in DMEM-F12 without serum as it contains nitrates that interfere with the assay. Cells were exposed to cytokines and(or) LPS for various periods of time (indicated in the figure legend) and conditioned media were collected and centrifuged at 400 × g for 5 min. Using a spectrometric method based on the Greiss reaction (Green et al., 1982), we evaluated NOx concentration. First, nitrate was reduced to nitrite with nitrate reductase (nitrate oxidoreductase from Aspergillus species: Boehringer Mannheim Canada: EC 1.6.6.2) as described (Gillam et al., 1993). Briefly, 75-µl samples were incubated with 25 µl of nitrate reductase buffer (0.4 U/ml of nitrate reductase, 420 µM NADPH, 160 mM potassium phosphate buffer, pH 7.5) at room temperature for 1 h in a microplate. An equal volume (100 µl) of Greiss reagent (1% sulfanilamide, 0.1% n-(1-naphtyl)-ethylenediamine, 2% phosphoric acid) was added, the reaction was allowed to proceed 10 min, and total nitrite was evaluated by reading optical density of samples at 540 nm. The final NOx concentration was determined by comparison with a NaNO2 standard curve (0 to 100 µM in DMEM-F12) freshly prepared before each experiment. Basal level of NOx in DMEM-F12 alone was measured and subtracted from sample concentration.

ation cycles for iNOS and 24 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. The denaturation cycles were performed at 94°C for 1 min (duration of the first cycle was 2 min), primer annealing at 60°C for 1 min and extension at 72°C for 1 min. An aliquot of 10 µl of PCR product was electrophoresed on 1% agarose gel containing 1 µg of ethidium bromide per milliliter. Bands were visualized and photographed by UV transillumination. The level of mRNA expression for iNOS was determined by densitometric image analysis (BioRad GS-670 imaging densitometer and Molecular Analyst 1.5 software, Hercules, CA), where the constitutively expressed GAPDH gene expression was also used. The mRNA expression levels for iNOS are presented as relative units after normalization to the observed GAPDH level. Statistical Analysis All data are expressed as mean ± SEM. Data were analyzed with the GLM procedure of SAS (SAS, 1985). Means were separated by least significant differences protected by an overall F value (P < 0.05). The number of replicates for each treatment and number of cow used in each experiment is included in the figure. RESULTS

Detection of Inducible Nitric Oxide Synthase Gene Expression by Reverse-Transcription (RT)-PCR Cellular RNA was isolated from bovine monocytes and neutrophils using TRIzol reagent (Gibco BRL, Grand Island, NY). Preparations of RNA were treated with RNase-free DNase (Gibco BRL). First strand DNA synthesis was performed using 1.0 to 1.5 µg of total RNA with 0.5 µg oligo(dT)12–18 as a primer and Superscript II RNase H− reverse transcriptase (Gibco BRL). The RT reaction was done in a 50-µl volume using 1× RT buffer (50 mM Tris-HCl, pH 8.3, 3 mM MgCl2, 75 mM KCl, and 10 mM DTT), 0.5 mM dNTP, 30 U of RNAguard, and 200 U Superscript II. The reaction was incubated for 50 min at 42°C, followed by inactivation of the reverse transcriptase at 70°C for 5 min. First-strand DNA was used as PCR template with sense (5′-GGAAGCAGTAACAAAGGAGATAG-3′) and antisense (5′-CATAGCGGATGAGCTGGGCG-3′) primers that were specific for bovine iNOS gene (designed from GenBank using PC1 Gene software). For PCR, 1.5 µl of the RT reaction was used in a volume of 50 µl (1× PCR Pharmacia buffer, 30 pmol of each primer, 1.25 U of Taq polymerase (QIAGEN, Ontario, Canada), and 0.4 mM dNTP). The preparation was overlaid with mineral oil and amplified in Perkin-Elmer DNA thermal cycler after determination of optimal condition for linearity [around 31 denaturJournal of Dairy Science Vol. 84, No. 6, 2001

NO Production by Mammary Epithelial Cells The NOx release by two bovine mammary epithelial cell lines, FbE and MAC-T, was studied. FbE cells consistently produced significant amounts of NOx in response to rBoIL-1β in a dose-dependent manner (P < 0.01; Figure 1). This rBoIL-1β-induced NOx release was completely abolished (P < 0.01) when the cells were exposed to L-NIL, a potent iNOS inhibitor. However, rBoIL-2 (200 ng/ml) and LPS (40 µg/ml) failed to induce NOx production by FbE cells (data not shown). In experiments with MAC-T cells, no detectable amounts of NOx were measured when incubated with LPS or cytokines (rBoIFN-γ, rBoIL-1β, rBoIL-2; data not shown). NO Production by Bovine Monocytes and Neutrophils As shown is Figure 2, moderate amounts of NOx were released when monocytes were stimulated with rBoIFN-γ alone (P < 0.01). No significant amount of NOx (P > 0.25) was detected in the conditioned media from unstimulated monocytes or cells stimulated with LPS alone. The addition of LPS to rBoIFN-γ-induced monocytes had variable effects on NOx release. Indeed, in some animals, LPS increased rBoIFN-γ-induced NO production, whereas in other, LPS had the opposite

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Surprisingly, the addition of neutrophils to rBoIFN-γinduced monocytes inhibited NOx accumulation in media (P < 0.05). When both cell types were exposed to rBoIFN-γ and LPS, more NOx was released compared with monocytes alone (P < 0.05). iNOS mRNA Levels Expression of iNOS, the enzyme responsible for NO production, was investigated in both neutrophils and monocytes (Figure 5). In response to rBoIFN-γ with or without LPS, iNOS mRNA level increased in bovine monocytes. Surprisingly, combination of rBoIFN-γ and LPS increased similar levels of iNOS mRNA in both cell types (Figures 5 and 6). In unstimulated monocytes, iNOS mRNA was detected, whereas it was undetectable in nonactivated neutrophils. DISCUSSION Figure 1. Nitrite plus nitrate (NOx) release by bovine mammary epithelial cell line FbE. Confluent cells were incubated with graded concentrations of recombinant bovine interleukin (rBoIL)-1β (0, 20, and 200 ng/ml) or 20 ng rBoIL-1β + 100 µM L-N6-(iminoethyl) lysine (L-NIL) per milliliter for 24 h at 37°C and 5% CO2. Supernatants were assayed for the accumulation of NOx. Individual experiments were performed in quadruplicate. aP < 0.01 compared with control. bP < 0.01 compared with treatment with 200 ng rBoIL-1β per milliliter.

effect. To demonstrate L-arginine-dependent NO production, aminoguanidine and L-NIL, two analogues of L-arginine, were added to the media simultaneously with rBoIFN-γ. Aminoguanidine and L-NIL suppressed NOx release by rBoIFN-γ-induced monocytes in a dosedependent manner (Figure 3) and reduced NOx accumulation below the detection limit (1 µM) at a concentration of 5 mM (Figure 3A) and of 250 µM (Figure 3B), respectively. The effect of bovine neutrophils on NOx production by monocytes in a coculture system (1 × 106 neutrophils: 1 × 106 monocytes) was investigated. Cells were exposed to rBoIFN-γ, LPS, or the combination rBoIFN-γ and LPS. None of the stimulatory conditions used in this study induced NO production by bovine neutrophils (Figure 4), even after 96 h in culture. The addition of the enzyme superoxide dismutase (SOD; Calbiochem; EC 1.15.1.1) to the media did not result in measurable accumulation of NOx (data not shown). In other experiments, NOx accumulation was not detected when bovine neutrophils were exposed to phorbol 12-myristate 13-acetate (PMA), formyl-methyl-leucine-phenylalanine, rBoIL-1β, or rBoIL-2 (data not shown). Moreover, incubation of neutrophils with different concentration of serum (1, 2.5, 5, and 10%) and LPS resulted in a nondetectable increase in NOx level (data not shown).

In this experiment, FbE cells, a bovine mammary epithelial cell line, produced NOx when stimulated with rBoIL-1β. This rBoIL-1β-induced NOx release was completely abolished when cells were exposed to L-NIL, an iNOS inhibitor, demonstrating a L-arginine-dependent pathway. Other proinflammatory agents such as rBoIL-

Figure 2. Effect of lipopolysaccharide (LPS) and recombinant bovine interferon (rBoIFN)-γ on nitrite plus nitrate (NOx) release by bovine monocytes. Cells (1 × 106/ml) were isolated from bovine blood and incubated in the presence of medium alone (Dulbecco’s modified Eagle’s medium/F12), and (per ml) 2 µg of LPS, 100 U of rBoIFN-γ, or 2 µg of LPS + 100 U rBoIFN-γ for 48 h at 37°C and 5% CO2. Data represent the mean for eight cows, and each treatment was done in triplicate. aP < 0.01 compare with control. Journal of Dairy Science Vol. 84, No. 6, 2001

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upon stimulation with IFN-γ in the presence of TNFα, NO production could be induced in bovine retinal pigmented epithelial cells (Goureau et al., 1992) and in COMMA-D, a murine mammary epithelial cell line (Low et al., 1997). However, in both studies, IL-1β alone failed to induce NO production. The results demonstrated that rBoIFN-γ induce a moderate NOx production in bovine blood monocytes. However, LPS alone failed to induce NOx release, and in combination with rBoIFN-γ, LPS had variable effects on NO release. Jungi et al. (1996) have observed that increase of iNOS mRNA in bovine monocytes is dependent on the differentiation stage of the cell. Therefore, the difference in LPS effect between animals might be the result of variation in the differentiation stages of the isolated cells. Other studies reported that rBoIFNγ alone is a weak stimulator of NO synthesis by bovine monocytes (Goff et al., 1996; Jungi et al., 1996; Zhao et al., 1996), but the addition of LPS or TNF-α to rBoIFN-γ induced higher NO release. In the current experiment, reduction of NOx accumulation by L-NIL and aminoguanidine has confirmed that NOx production in bovine monocytes is L-arginine dependent. Neutrophils represent 90% of the total SCC during mastitis; we then investigated their ability to release NO. Bovine neutrophils did not generate detectable lev-

Figure 3. Dose-dependent effect of aminoguanidine and L-N6(iminoethyl) lysine (L-NIL) on recombinant bovine interferon (rBoIFN)-γ-induced nitrite plus nitrate (NOx) release by bovine monocytes. 1 × 106 cells/ml were isolated from bovine blood and incubated for 48 h in Dulbecco’s modified Eagle’s medium F12 supplemented with rBoIFN-γ (100 U/ml) in the absence or presence of A) aminoguanidine or B) L-NIL. Data represent the mean for two cows, and each treatment was done in duplicate.

2, LPS, and rBoIFN-γ failed to induce NO production in FbE cells. However, the other bovine epithelial cell line, MAC-T, did not release NO under any conditions used. This cell line is immortalized with simian virus 40 large T antigen, whereas FbE are spontaneously immortal cells. Large T-transformed cells may not be able to release NOx because cytokines cannot initiate cell activation. According to published results, large T antigen has an inhibitory effect on the IL-1β-induced expression of cytokines in synovial fibroblasts from rheumatoid arthritis tissue transformed by the SV40 large T antigen (Asamitsu et al., 1999). Other epithelial cell types can produce NO. Studies demonstrated that Journal of Dairy Science Vol. 84, No. 6, 2001

Figure 4. Effect of lipopolysaccharide (LPS) and recombinant bovine interferon (rBoIFN)-γ on nitrite plus nitrate (NOx) produced by (white) neutrophils (1 × 106/ml), (black) monocytes (1 × 106/ml), or (gray) monocytes (1 × 106/ml) and neutrophils (1 × 106/ml) coculture. Cells were isolated from bovine blood and incubated for 48 h with Media alone (Dulbecco’s modified Eagle’s medium/F12), and (per ml) 2 µg of LPS, 100 U of rBoIFN-γ, or 2 µg of LPS + 100 U of rBoIFN-γ for 48 h at 37°C and 5% CO2. Data represent the mean for four cows, and each treatment was done in triplicate. aP < 0.01 compare with control. bP < 0.05 compare with monocytes alone with the same treatment.


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Figure 5. Detection of inducible nitric oxide synthase (iNOS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA by reverse transcription PCR. mRNA was extracted from bovine blood neutrophils or monocytes. Cells were incubated for 6 h at 37°C with culture medium alone; lipopolysacharide (LPS) (2 µg/ml); recombinant bovine interferon (rBoIFN)-γ (100 U/ml); LPS (2 µg/ml) + rBoIFN-γ (100 U/ ml). In first lane, RNA was extracted from freshly isolated neutrophils or monocytes (no incubation). The number under each band represents the expression level relative to the housekeeping gene GAPDH. The optimal cycle number was 25 for amplification of GAPDH fragment and 43 cycles for amplification of iNOS fragment. The experiment was performed on four cows. Results from one typical cow are shown.

els of NOx after activation by different agents, including those providing a positive response in bovine monocytes, even after 96 h of exposition. Many teams have studied NO production by human and murine neutrophils but very few have worked with bovine neutrophils. Consistent with our results, Goff et al. (1996) did not observed NOx accumulation in medium of bovine neutrophils exposed to rBoIFN-γ, PMA, opsonized zymosan, or a combination of reagents. Despite that, LPS are potent stimulator of immune cells, including rodent neutrophils (Rodenas et al., 1996); they failed to induce NOx accumulation. LPS-initiated response is strongly enhanced by serum proteins called LPS-binding protein (Tapping and Tobias, 1997). Complexes of LPS-binding protein and LPS were shown to bind to cellular CD14 and enhance cell stimulation compared with that resulting from LPS alone (Schumann and Latz, 2000). Nevertheless, the addition of serum to LPS had no effect on NO release by bovine neutrophils. Expression of iNOS in both monocytes and neutrophils was investigated. Results showed that rBoINF-γ but not LPS increased iNOS mRNA levels in bovine monocytes. Despite variable effects on NOx accumulation, combination of LPS and rBoINF-γ consistently increased iNOS mRNA. Crespo et al. (2000) have reported that pretreatment of mouse bone marrow cul-

ture-derived macrophages with LPS dramatically reduces their ability to produce NO in response to subsequent stimulation with IFN-γ. In our case, LPS and rBoIFN-γ induced iNOS mRNA increase, but LPS might have variable effects on protein synthesis that could explain variable NOx accumulation. In freshly isolated monocytes, iNOS mRNA was expressed. After 6 h of incubation in media alone, no iNOS expression was observed. Expression of iNOS in freshly isolated monocytes could be attributed to stimulation with cytokines secreted by lymphocytes during the adherence step of monocyte isolation. The majority of the lymphocytes present in the adherence step are gammadelta T (γδT) cells capable of producing IFN-γ (Goff et al., 1996). Results indicated that contaminating γδT cells in monocyte cultures might influence monocyte iNOS mRNA levels. After 6 h in culture media without lymphocytes, iNOS mRNA in monocytes might have been down regulated, and no band would have been observed. Surprisingly, LPS and rBoIFN-γ increase iNOS mRNA in bovine blood neutrophils, whereas they did not produce a significant amount of NOx when incubated with LPS and rBoIFN-γ, even after 96 h in culture. To our knowledge, iNOS expression in bovine neutrophils has not been reported. Nitric oxide production Journal of Dairy Science Vol. 84, No. 6, 2001

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Figure 6. Level of inducible nitric oxide synthase (iNOS) relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. RNA was extracted from bovine blood neutrophils (black) or monocytes (white) of four different cows after exogenous stimulation for 6 h at 37°C with media alone (Dulbecco’s modified Eagle’s medium/ F12), and (per ml) 2 µg lipopolysaccharide (LPS), 100 U rBoINF-γ, or 2 µg LPS + 100 U rBoINF-γ. For the first bars, RNA was extracted from freshly isolated neutrophils or monocytes (no incubation). aP < 0.01 compare with control.

was studied in bovine neutrophils but not iNOS expression (Goff et al., 1996). We have demonstrated here that LPS and rBoIFN-γ can increase iNOS mRNA in bovine neutrophils without stimulating NOx release. Mature neutrophils have a small Golgi apparatus and some mitochondria but very few ribosomes or rough endoplasmic reticulum (Tizard, 1996), thus they cannot synthesize large amounts of protein. Therefore, the translation of iNOS mRNA might have been impaired. Undetectable NOx accumulation might also be due to superoxide anion production. Some NO might be released by neutrophils but react with superoxide anions to form peroxynitrite (ONOO−). Superoxide anions are produced by PMA-activated bovine neutrophils under these conditions (Boulanger, 2000). To study NO release by rodent neutrophils, cells need to be incubated with SOD, a scavenger of superoxide anion (Rodenas et al., 1996). However, we repeated the experiment, adding SOD, and observed no difference in NOx concentration measured. A reaction of superoxide anion with NO might explain the reduction of NOx accumulation in rBoIFN-γ-stimulated coculture of neutrophils and monocytes compared with monocytes alone. However, when both cell types were exposed to rBoIFN-γ and LPS, an increase in NOx accumulation was observed. In this experiment, the Journal of Dairy Science Vol. 84, No. 6, 2001

highest levels of iNOS mRNA in neutrophils occured after rBoIFN-γ plus LPS stimulation. Pigatto et al. (1990) reported that LPS-stimulated monocytes and psoriatic human monocytes can stimulate neutrophil chemotaxis, phagocytosis, and superoxide anion production. Similarly, it is possible that in presence of monocytes, some neutrophil iNOS mRNA gets translated to proteins and leads to NO production. The potential contribution of lymphocytes, which are important components of milk somatic cell population, to NO production has not been investigated. However, Schuberth et al. (1998) found that NO production by cells isolated from bovine blood was correlated with the proportion of CD14+ monocytes but not with CD4+ and CD8+ lymphocytes. We investigated NO production by bovine monocytes, neutrophils, and mammary epithelial cells in order to identify which cell type(s) among milk somatic cells can release NO during endotoxin-induced mastitis. In conclusion, mammary epithelial cells and(or) mononuclear phagocytes contribute to NO production upon stimulation with LPS and cytokines involved in mastitis (IL-1 and IFN-γ). Nevertheless, the possibility that some NO release could be attributed to neutrophils or lymphocytes cannot be excluded. These results suggest that during mastitis, NO released by mammary epithelial cells and monocytes may damage mammary tissue. Keeping up with our long-term objective to reduce mastitis-associated tissue damage, the effect of NO and other free radicals on mammary epithelial cell damage are currently studied. ACKNOWLEDGMENTS NOVALAIT Inc. (Que´bec, Canada), and the Dairy Farmers of Canada supported this work. The FbE cells were kindly supplied by T. Woodward (McGill University, Montreal, Canada). We thank D. Petitclerc (Dairy and Swine Research Centre, Lennoxville, Canada) for his advice and Catherine Desrosiers and Lisette StJames for their excellent technical assistance. REFERENCES Asamitsu, K., S. Sakurada, K. Mashiba, K. Nakagawa, K. Torikai, K. Onozaki, and T. Okamoto. 1999. Alteration of the cellular response to interleukin-1 beta by SV40 large T antigen in rheumatoid synovial fibroblasts. Arch. Virol. 144:317–327. Bochsler, P. N., G. L. Mason, T.W.L. Olchowy, and Z. Yang. 1996. Bacterial lipopolysaccharide-stimulated nitric oxide generation is unrelated to concurrent cytotoxicity of bovine alveolar macrophages. Inflammation 20:177–189. Bouchard, L., S. Blais, C. Desrosiers, X. Zhao, and P. Lacasse. 1999. Nitric oxide production during endotoxin-induced mastitis in the cow. J. Dairy Sci. 82:2574–2581. Boulanger, V. 2000. Effect of proinflammatory agents on oxygen species production by bovine mammary epithelial and immune cells. M. Sc. Thesis. McGill Univ.

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Journal of Dairy Science Vol. 84, No. 6, 2001