Pro-inflammatory and pro-apoptotic responses of TNF-α stimulated bovine mammary endothelial cells

Abstract

Coliform mastitis may be severe in periparturient cows due to enhanced expression of pro-inflammatory cytokines that contribute to disease pathogenesis. Tumor necrosis factor (TNF)-α is implicated with the severity of coliform mastitis by provoking inflammatory responses in affected tissues. The endothelium is an integral organ in regulating inflammatory responses and loss of endothelial integrity may be fatal. Studies in humans suggest that endothelial cell apoptosis may be a consequence of TNF-α exposure and contributes to the development of sepsis, however, its impact on bovine mammary endothelial cells (BMEC) is unknown. We sought to determine the inflammatory and apoptotic responses of primary BMEC exposed to TNF-α in vitro. Stimulation of endothelial monolayers with TNF-α resulted in significant increase of toll-like receptor 4, interleukin-6 and -8, and intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 gene expression in a time-dependent manner. Caspase-8 and caspase-3 mRNA expression, as well as caspase enzyme activity, also increased significantly following TNF-α stimulation. Cell viability assessed by ATP activity and BMEC apoptosis determined by flow cytometry revealed no significant changes across time with TNF-α stimulation. Results suggest that TNF-α stimulation, at the dose used in this study, can elicit a pro-inflammatory response in BMEC, but not induce apoptosis. The impact of TNF-α on mammary vascular function and the subsequent impact on the pathophysiology of severe coliform mastitis warrant further investigation.

Introduction

Mastitis continues to be a primary periparturient disease afflicting dairy cattle. Coliform intramammary infections are of particular interest due to their ability to cause peracute mastitis during the periparturient period and early lactation, which may lead to substantial losses in milk production, high rates of animal culling, sepsis, and death (De Schepper et al., 2008). Lipopolysaccharide (LPS) released from multiplying and dying gram-negative bacteria binds to the toll like receptor (TLR)-4 complex on numerous mammary gland cells (i.e., macrophages, epithelial, endothelial cells) and triggers an innate immune response (Bannerman and Goldblum, 2003, De Schepper et al., 2008). Activation of TLR4 results in cellular production and release of pro-inflammatory cytokines (i.e., tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8), and vascular adhesion molecules essential for recruitment of leukocytes to the infection site and destruction of pathogens. A delayed and inefficient innate immune response resulting in exponential bacterial growth, excessive endotoxin and cytokine release is associated with the severity of coliform mastitis during early lactation (Hill, 1981, Satta et al., 2008, Sordillo and Peel, 1992). Overproduction of pro-inflammatory mediators and release into the vasculature results in not only local tissue damage but also systemic disease.

Several lines of evidence suggest that TNF-α is one of the acute phase cytokines implicated in the severity of coliform mastitis. Enhanced production of TNF-α by monocytic cells occurred during the periparturient period following LPS stimulation (Sordillo et al., 1995). Significant increases in milk and serum concentrations of TNF-α occurred during severe experimental and naturally occurring coliform mastitis (Hoeben et al., 2000, Nakajima et al., 1997, Sordillo and Peel, 1992). Subcutaneous rbTNF-α injection into periparturient Holstein cows produced similar systemic effects as seen during coliform mastitis (Kushibiki et al., 2003). In addition, intramammary infusion of rbTNF-α resulted in decreased milk yield, increased neutrophil influx, increased plasma haptoglobin, increased intramammary systemic nitrite and nitrate and caused changes in various milk proteins (Watanabe et al., 2000). Collectively these studies support the contention that TNF-α is responsible for many of the local and systemic symptoms observed during severe periparturient coliform mastitis.

Resident mammary macrophages and epithelial cells are thought to be the primary cells involved in the initial recognition of intramammary coliform pathogens and are likely the initial sources of soluble TNF-α (Lahouassa et al., 2007). A wide variety of cells can respond to TNF-α including innate immune cells, mammary epithelial and endothelial cells. Indeed, the vascular endothelium is a major target for TNF-α and is implicated in the pathophysiology of sepsis (Madge and Pober, 2001). Excess intramammary production of TNF-α and other pro-inflammatory cytokines may damage the blood-milk barrier allowing cytokine access into the bloodstream (Blum et al., 2000). Whereas mammary leukocyte and epithelial cell populations were examined extensively with respect to cytokine-induced responses, the relevance of mammary endothelial cells during mastitis is poorly understood.

Activation of vascular endothelium by TNF-α occurs primarily through TNF-α receptors I and II, although limited data suggests TLR-4 activation may occur through cytokine binding. A primary endothelial response to TNF-α includes upregulation of additional pro-inflammatory cytokines as well as adhesion molecules recruiting leukocytes to the infection site (Madge and Pober, 2001, Van Kampen and Mallard, 2001b). Prolonged or excessive leukocyte-endothelial transmigration and cytokine release may result in vascular injury and endothelial dysfunction. Endothelial cell apoptosis is implicated in the pathogenesis of several human diseases, including acute respiratory distress syndrome, atherosclerosis, and hypertension (Bannerman and Goldblum, 2003). Tumor necrosis factor-α may result in endothelial programmed cell death by activation of multiple apoptotic pathways. The activation of TNFRI, for example, results in caspase-8 and caspase-3 activation with subsequent proteolysis and cell death (Madge and Pober, 2001). Bax is a proapoptotic protein and is opposed by the antiapoptotic protein, Bcl2 that can regulate caspase-9 activation. Upregulation of Bax and downregulation of Bcl2 was demonstrated in mammary gland biopsies from experimentally infected E. coli cows (Long et al., 2001).

The severity of coliform infections during the periparturient period results in financial burdens to the dairy industry. Tumor necrosis factor-α is associated with clinical severity, however, its exact role in coliform mastitis pathology is not completely known. Despite the significance of the endothelium to human inflammatory-based diseases, relatively little is known about the endothelium in regards to bovine mammary health. The purpose of this study was to determine if TNF-α could induce the expression of proinflammatory and/or apoptotic genes associated with mammary vascular dysfunction.