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pubmed:abstractText |
Canine synovial membrane explants were exposed to high- or low-passage Borrelia burgdorferi for 3, 6, 12, and 24 h. Spirochetes received no treatment, were UV light irradiated for 16 h, or were sonicated prior to addition to synovial explant cultures. In explant tissues, mRNA levels for the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-1beta, and IL-8 were surveyed semiquantitatively by reverse transcription-PCR. Culture supernatants were examined for numbers of total and motile (i.e., viable) spirochetes, TNF-like and IL-1-like activities, polymorphonuclear neutrophil (PMN) chemotaxis-inducing activities, and IL-8. During exposure to synovial explant tissues, the total number of spirochetes in the supernatants decreased gradually by approximately 30%, and the viability also declined. mRNAs for TNF-alpha, IL-1alpha, IL-1beta, and IL-8 were up-regulated in synovial explant tissues within 3 h after infection with untreated or UV light-irradiated B. burgdorferi, and mRNA levels corresponded to the results obtained with bioassays. During 24 h of coincubation, cultures challenged with untreated or UV light-irradiated spirochetes produced similar levels of TNF-like and IL-1-like activities. In contrast, explant tissues exposed to untreated B. burgdorferi generated significantly higher levels of chemotactic factors after 24 h of incubation than did explant tissues exposed to UV light-treated spirochetes. In identical samples, a specific signal for IL-8 was identified by Western blot analysis. High- and low-passage borreliae did not differ in their abilities to induce proinflammatory cytokines. No difference in cytokine induction between untreated and sonicated high-passage spirochetes was observed, suggesting that fractions of the organism can trigger the production and release of inflammatory mediators. The titration of spirochetes revealed a dose-independent cytokine response, where 10(3) to 10(7) B. burgdorferi organisms induced similar TNF-like activities but only 10(7) spirochetes induced measurable IL-1-like activities. The release of chemotactic factors was dose dependent and was initiated when tissues were infected with at least 10(5) organisms. We conclude that intact B. burgdorferi or fractions of the bacterium can induce the local up-regulation of TNF-alpha, IL-1alpha, and IL-1beta in the synovium but that the interaction of viable spirochetes with synovial cells leads to the release of IL-8, which probably is a prime initiator of PMN migration during acute Lyme arthritis.
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