In vitro expression of tumor necrosis factor-alpha, interleukin 1beta, and interleukin 8 mRNA by bovine macrophages following exposure to Porphyromonas levii.

The objective was to evaluate the pro-inflammatory response of bovine macrophages towards Porphyromonas levii, an etiologic agent of acute interdigital phlegmon, by evaluating the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and interleukin 8 (IL-8). Bovine macrophages detect the presence of bacteria, such as P. levii, and respond by upregulating transcription of the genes for pro-inflammatory cytokines TNF-alpha and IL-1beta in addition to the neutrophil chemoattractant IL-8. Monocytes were isolated from blood obtained from Holstein steers. These cells were cultured and differentiated into macrophages over 7 d, followed by exposure to P. levii, Escherichia coli lipopolysaccharide (LPS), or tissue culture medium for 1, 1.5, 2, 4, 6, 8,12, or 24 h. Total RNA was extracted, and reverse transcriptase polymerase chain reaction was conducted to examine the presence of TNF-alpha, IL-1beta, or IL-8 mRNA. Products were visualized on agarose gels to determine the presence or absence of cytokine mRNA amplified DNA. Bovine macrophages, when exposed to P. levii or E. coli LPS, produced mRNA for TNF-alpha, IL-1beta, and IL-8. Expression of all 3 cytokines was higher in the P. levii and LPS-exposed macrophages at all time points examined, compared with tissue culture medium-treated cells. Expression of these cytokines by macrophages is likely directly involved in orchestration of the immune response, and particularly in neutrophil recruitment to affected tissues in acute interdigital phlegmon.

Chemotaxis, phagocytosis, and oxidative metabolism in bovine macrophages exposed to a novel interdigital phlegmon (foot rot) lesion isolate, Porphyromonas levii.

OBJECTIVE: To examine the host response toward Porphyromonas levii, by evaluating chemotaxis, phagocytosis, and oxidative burst of bovine macrophages in vitro. SAMPLE POPULATION: Cultured bovine macrophages obtained from monocytes harvested from blood samples of 15 Holstein steers. Porphyromonas levii was isolated from the foot rot lesion of an acutely affected feedlot steer. PROCEDURE: Monocytes were cultured for macrophage differentiation over 7 days. Porphyromonas levii was cultured in strict anaerobic conditions for experimentation. Chemotaxis was evaluated by quantifying macrophage migration toward P. levii in Boyden chambers. Phagocytosis was assessed by quantification of macrophages engulfing P. levii following incubation with or without anti-P. levii serum or purified IgG. Oxidative burst was measured by use of the nitroblue tetrazolium reduction assay. RESULTS: Chemotaxis toward P. levii was not significantly different from control values at any of the tested bacterial concentrations. Phagocytosis of P. levii was approximately 10% at a 10:1 bacterium to macrophage ratio and did not change significantly over time. When higher proportions of P. levii were tested for phagocytosis, the 1,000:1 bacterium to macrophage ratio had a significant increase, compared with the 10:1 test group. Opsonization of P. levii with high-titeranti-P. levii serum or anti-P. levii IgG produced a significant increase in macrophage phagocytosis. Oxidative production significantly increased compared with control in the 1,000:1 test group only. CONCLUSIONS AND CLINICAL RELEVANCE: Porphyromonas levii may evade host detection by decreased chemotaxis, phagocytosis, and oxidative burst by macrophages. Acquired immunity may be beneficial for clearance of P. levii in foot rot lesions in cattle.