Induction of Nitric Oxide Production Mediated by Tumor Necrosis Factor Alpha on Staphylococcal Enterotoxin C-Stimulated Bovine Mammary Gland Cells

Abstract

Mammary gland (MG) secretions (MGS) derived from secretory cows infected with coagulase-negative staphylococci (CoNS) showed somatic cell counts and lactoferrin similar to levels found in the MGS of secretory cows infected with Staphylococcus aureus. However, nitrite and nitrate (NOx) and staphylococcal enterotoxin C (SEC) were found in MGS infected with S. aureus at much higher levels than in cows infected with CoNS. These results suggested that NOx could be intimately correlated with the production of SEC in secretory cows infected with S. aureus. Therefore, we examined the production of NOx and the expression of proinflammatory cytokines and microsomal cytochrome P450 (CYP450) after injection of SEC into the MGS of secretory cows. We were able to detect NOx and the proinflammatory cytokine tumor necrosis factor alpha Staphylococcus aureus is the major causative bacteria of bovine mastitis. S. aureus infections result in changes of T-cell subpopulations and proinflammatory cytokine and chemokine production (27,35). Moreover, lymphocytes and leukocytes stimulated with staphylococcal enterotoxins produce proinflammatory cytokines (36) and nitric oxide (17), which show inflammatory effects. In ruminants, many strains of S. aureus isolated from mammary gland (MG) secretions (MGSs) produce staphylococcal enterotoxin C (SEC) (16,20,33). It was reported that SEC induced clinical signs of bovine mastitis such as an increase in the number of leukocytes in the MG (16). SEC binds to the specific V␤ chain of the T-cell receptor via the major histocompatibility complex class II molecules of antigen-presenting cells such as macrophages and activates bovine T lymphocytes (5,18,36). Then, the activated T lymphocytes and macrophages produce proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, gamma interferon, and tumor necrosis factor alpha (TNF-␣) (35,36). Therefore, it is suggested that the superantigenic activity of SEC induces bovine mastitis (5, 36). Thus, it is indicated that inflammatory cytokines such as IL-1, IL-6, and TNF-␣ are produced by activated leukocytes and lymphocytes in the early stages of staphylococcal mastitis.Down-regulation of intracellular cytochrome P450 (CYP450) expression is caused by IL-1, 25), which are produced in response to certain toxins such as staphylococcal enterotoxin B (31), aflatoxin B1 (15), and Escherichia coli lipopolysaccharide (LPS) (19). These cytokines also induce the expression of the inducible isoform of nitric oxide (NO) synthetase (iNOS), which results in the production of NO in certain cells including hepatocytes, macrophages, endothelial cells, and leukocytes during the cellular response. Several studies have suggested that NO production is mediated by the down-regulation of CYP450 expression in cytokine and LPS models of inflammation, based on the ability of exogenously administered NO to down-regulate CYP450 gene expression and attenuate the down-regulation by NO inhibitors (13,14). Then, the production of NO is regulated by the downregulation of CYP450 and cause...

Innate immunity of the bovine mammary gland

“…Concentration of NO is very low in colostrum or mature milk from healthy cows [12]. Production of NO is transiently increased in milk after E. coli LPS infusion, but NO is released in higher amounts and for longer periods in E. coli or S. aureus infected quarters [12,13,84]. Milk leucocytes were shown to release NO upon stimulation with bacterial products like LPS or staphylococcal enterotoxin [13,84].…”

“…Production of NO is transiently increased in milk after E. coli LPS infusion, but NO is released in higher amounts and for longer periods in E. coli or S. aureus infected quarters [12,13,84]. Milk leucocytes were shown to release NO upon stimulation with bacterial products like LPS or staphylococcal enterotoxin [13,84]. Macrophages are likely to contribute the bulk of milk leucocytes NO, because bovine macrophages are good producers of NO upon stimulation with LPS, on the contrary to neutrophils [16,74].…”

“…Bovine macrophages are also able to secrete NO when stimulated with components of Gram-positive bacteria, but they need co-stimulation with cytokines like IFN-γ [74]. The pro-inflammatory cytokine TNF-α is likely to synergize with staphylococcal enterotoxin C for induction of iNOS and NO production by bovine macrophages, possibly through activation of the NF-κB pathway [84]. A marked increase in iNOS mRNA of mammary secretory tissue was reported to occur as soon as 3 h after infusion of LPS through the teat canal [163].…”

Nitric oxide concentrations in mammary quarters during heifer mastitis

“…NOx is a free radical that mediates physiological and pathological changes in the body, is synthesized from L-arginine by inducible nitric oxide synthase (iNOS; Archer, 1993) and mediated from endothelial, epithelial, neuronal and phagocytic cells (Palmer et al, 1987;Onado and Inano, 1998). However, NOx becomes a cytotoxic mediator when it is released in excessive amounts during Escherichia coli (E. coli ) infections and in response to intramammary injection of lipopolysaccharide (LPS; Boulanger et al, 2001;Burvenich et al, 2003;Komine et al, 2004).…”

“…In addition to these radicals, the concentration of nitric oxide (NOx) increases in affected tissue. Total nitrite and nitrate is assumed to be valid as an estimate of NOx generation; nitrite oxide from nitrite and nitrate is referred to as NOx (Bouchard et al, 1999;Komine et al, 2004). NOx is a free radical that mediates physiological and pathological changes in the body, is synthesized from L-arginine by inducible nitric oxide synthase (iNOS; Archer, 1993) and mediated from endothelial, epithelial, neuronal and phagocytic cells (Palmer et al, 1987;Onado and Inano, 1998).…”