Immunopathology of mastitis: insights into disease recognition and resolution.

Mastitis is an inflammation of the mammary gland commonly caused by bacterial infection. The inflammatory process is a normal and necessary immunological response to invading pathogens. The purpose of host inflammatory responses is to eliminate the source of tissue injury, restore immune homeostasis, and return tissues to normal function. The inflammatory cascade results not only in the escalation of local antimicrobial factors, but also in the increased movement of leukocytes and plasma components from the blood that may cause damage to host tissues. A precarious balance between pro-inflammatory and pro-resolving mechanisms is needed to ensure optimal bacterial clearance and the prompt return to immune homeostasis. Therefore, inflammatory responses must be tightly regulated to avoid bystander damage to the milk synthesizing tissues of the mammary gland. The defense mechanisms of the mammary gland function optimally when invading bacteria are recognized promptly, the initial inflammatory response is adequate to rapidly eliminate the infection, and the mammary gland is returned to normal function quickly without any noticeable clinical symptoms. Suboptimal or dysfunctional mammary gland defenses, however, may contribute to the development of severe acute inflammation or chronic mastitis that adversely affects the quantity and quality of milk. This review will summarize critical mammary gland defense mechanisms that are necessary for immune surveillance and the rapid elimination of mastitis-causing organisms. Situations in which diminished efficiency of innate or adaptive mammary gland immune responses may contribute to disease pathogenesis will also be discussed. A better understanding of the complex interactions between mammary gland defenses and mastitis-causing pathogens should prove useful for the future control of intramammary infections.

Effect of infection with bovine leukosis virus on lymphocyte proliferation and apoptosis in dairy cattle.

OBJECTIVE: To determine effects of infection with bovine leukosis virus (BLV) on lymphocyte proliferation and apoptosis in dairy cattle. ANIMALS: 27 adult Holstein cows. PROCEDURES: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from lactating Holstein cows seronegative for BLV (n = 9 cows), seropositive for BLV and aleukemic (aleukemic; 9), and seropositive for BLV and persistently lymphocytotic (PL; 9). Isolated PBMCs were assayed for mitogen-induced proliferation and were analyzed by means of flow cytometry. The PBMCs from a subset of each group were assayed for apoptosis, caspase-9 activity, and expression of selected genes related to apoptosis. RESULTS: PL cows had significantly higher total lymphocyte counts and significantly lower proportions of T-lymphocyte populations than did BLV-negative and aleukemic cows. Both groups of BLV-infected cows had significantly higher proportions of B cells and major histocompatibility complex II-expressing cells than did BLV-negative cows. Proliferation with concanavalin A was significantly lower for PL cows, compared with proliferation for BLV-negative cows. Pokeweed mitogen-induced proliferation was significantly higher for aleukemic and PL cows than for BLV-negative cows. Gene expression of apoptosis-inhibitory proteins BCL2 and BCL2L1 was significantly higher for aleukemic cows and expression of BCL2 was significantly higher for PL cows than for BLV-negative cows. CONCLUSIONS AND CLINICAL RELEVANCE: Cattle infected with BLV had marked changes in PBMC populations accompanied by alterations in proliferation and apoptosis mechanisms. Because the relative distribution and function of lymphocyte populations are critical for immune competence, additional studies are needed to investigate the ability of BLV-infected cattle to respond to infectious challenge.

Ethyl pyruvate decreases proinflammatory gene expression in lipopolysaccharide-stimulated equine monocytes.

Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation protocol and enriched to 76% purity by adhesion to tissue culture dishes. Isolated monocytes were incubated with 0, 1, 5, 10 and 50 mM ethyl pyruvate. Cell viability, production of caspase 3/7, and caspase-3 gene expression were determined. In a separate experiment, monocytes were stimulated with LPS (0.1 ng/ml for 1h) followed by incubation with 0, 1, 5, or 10 mM ethyl pyruvate for 1 h. Proinflammatory gene expression was determined by real-time PCR. Ethyl pyruvate at 50 mM adversely affected monocyte viability. Ethyl pyruvate at 10mM or less had no significant effect on monocyte viability, and did not increase activity of caspase 3/7 nor caspase-3 gene expression. Incubation with LPS alone induced a significant upregulation in proinflammatory gene expression. Subsequent treatment of monocytes with ethyl pyruvate significantly reduced IL-8 expression in LPS stimulated monocytes at 5 mM, and IL-8, TNF-α and COX-2 at 10 mM. No beneficial effect on expression of IL-1ß or IL-6 was detected. Overall, 10 mM ethyl pyruvate did not adversely affect monocyte viability and suppressed LPS-induced proinflammatory gene expression. Ethyl pyruvate may be a beneficial anti-inflammatory therapy in equine endotoxemia.

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

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.