Effects of nitric oxide on bovine polymorphonuclear functions

Abstract

The effects of nitric oxide (NO) on the functionality of polymorphonuclear neutrophils (PMNs) in bovine milk or blood were investigated. In 2 experiments, mastitis was induced by infusing both hind quarters with saline containing Escherichia coli endotoxins. In addition, the left hind quarter was infused with aminoguanidine, an inhibitor of the inducible form of NO synthase (iNOS). At various times after infusion, somatic cells were isolated from milk samples, and superoxide (O2-) production induced by phorbol myristate acetate was evaluated. In both experiments, the addition of aminoguanidine had no inhibitory effect on the number of milk somatic cells or on their O2- production. The effect of NO and iNOS inhibitors on the functionality of bovine PMNs isolated from blood was investigated in vitro. The neutrophils did not produce NO. A neutrophil:monocyte co-culture system was used to study the effect of NO derived from monocytes on O2- production by bovine neutrophils. Neither NO derived from activated monocytes nor the iNOS inhibitors aminoguanidine and L-N6-(1-iminoethyl)lysine had an effect on the ability of bovine neutrophils to release O2-. Moreover, aminoguanidine did not affect the ability of bovine neutrophils to phagocytose bacteria. These results suggest that inhibition of NO release during inflammation does not interfere with the migration of immune cells to the site of infection or the ability of these cells to destroy pathogens. Thus, NO does not appear to play a major role in the control of the functions of bovine neutrophils.

Figure 1

Effect of intramammary infusion of Escherichia coli 055:B5 lipo-polysaccharide (LPS) and aminoguanidine on superoxide (O₂⁻) production by cells stimulated (black bars) with phorbol myristate acetate (PMA) or not stimulated with PMA (white bars) and harvested from milk samples. In experiment 1, milk samples were collected from 3 cows 4 h after infusion of 15 μg of LPS either alone or with 1500 mg of aminoguanidine. In experiment 2, milk samples were collected from 4 cows 6 h after infusion of 100 μg of LPS either alone or with 3000 mg of aminoguanidine. Superoxide assay was performed in triplicate.

Figure 2

Somatic cell count (SCC) in milk samples from mammary gland hind quarters infused with LPS either alone (white bars) or with aminoguanidine (black bars) in 2 experiments, with 3 cows and 4 cows, respectively. Time 0 is the time of infusion.

Figure 3

Superoxide production by bovine neutrophils in inflammatory conditions. Cells were isolated from bovine blood and incubated with Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 alone or in combination with LPS (2 μg/mL), recombinant bovine interferon-gamma (rBoINF-γ, 100 U/mL), or both LPS and rBoINF-γ for 48 h. The cells were washed, and O₂⁻ production by PMA-stimulated cells (black bars) and unstimulated cells (grey bars) was measured. Data represent the mean for 3 cows; each treatment was performed in triplicate.

Figure 4

Superoxide production by bovine monocytes in inflammatory conditions. Cells were isolated from bovine blood and incubated as described for Figure 3. The cells were washed, and O₂⁻ production by PMA-stimulated cells (black bars) and unstimulated cells (grey bars) was measured. Data represent the mean for 3 cows; each treatment was performed in triplicate.

Figure 5

Nitric oxide production by bovine neutrophils and monocytes co-incubated in inflammatory conditions. Cells were isolated from bovine blood and incubated as described for Figure 3. The cell culture medium was harvested and the concentration of nitrate + nitrite (NO_x) measured. Data represent the mean for 3 cows; each treatment was performed in triplicate.

Figure 6

Superoxide production by bovine neutrophils and monocytes co-incubated in inflammatory conditions. Cells were isolated from bovine blood and incubated as described for Figure 3. The cells were washed, and O₂⁻ production by PMA-stimulated cells (black bars) and unstimulated cells (grey bars) was measured. Data represent the mean for 3 cows; each treatment was performed in triplicate.

Figure 7

Effect of inhibitors of the inducible isoform of nitric oxide synthase (iNOS) on O₂⁻ production by bovine neutrophils. Cells were isolated from bovine blood and incubated in DMEM/F12 for 16 h. Two hours before O₂⁻ assay, the cells were washed and incubated with medium alone (control), 2 mM aminoguanidine, or 100 μM l-N⁶-(1-iminoethyl)lysine (l-NIL). The cells were washed, and O₂⁻ production by PMA-stimulated cells (black bars) and unstimulated cells (white bars) was measured. Data represent the mean for 3 cows; each treatment was performed in triplicate.