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Infection with Borrelia burgdorferi is suspected to be a cause of lameness and arthritis in cattle. Interleukin-1 (IL-1) activity has been detected in joint fluids from human patients affected by various arthritides, including Lyme arthritis. In addition, human monocytes and murine macrophages have been reported to release IL-1 activity when incubated with B. burgdorferi in vitro. To address a possible mechanism by which B. burgdorferi might cause a bovine arthritic syndrome, we determined whether bovine peripheral blood monocytes released IL-1 activity when coincubated with B. burgdorferi in vitro. High-passage and low-passage isolates of B. burgdorferi stimulated release of IL-1 activity from bovine monocytes. The amount of IL-1 activity released was dependent on the number of borreliae added to the monocyte cultures. In addition, live and heat-killed B. burgdorferi cells stimulated release of similar amounts of IL-1. We also obtained no evidence that soluble components released from in vitro-cultured B. burgdorferi stimulated IL-1 release from bovine monocytes. A recombinant IL-1 receptor antagonist blocked the proliferative activity of monocyte-conditioned medium in a thymocyte costimulation assay, thus demonstrating that the costimulatory activity detected was due to IL-1.