In this study, we compared the apoptotic activities of clinical and environmental isolates of Vibrio vulnificus toward macrophages in vitro and in vivo. The clinical isolates induced apoptosis in macrophage-like cells in vitro and in macrophages in vivo. This suggests that macrophage apoptosis may be important for the clinical virulence of V. vulnificus.Many bacterial pathogens that infect mammals have developed specific traits to avoid the innate and specific immune defenses of the host (3,6,12,17). A characteristic common to several invasive enteric pathogens (e.g., Shigella, Salmonella, and Yersinia species) is the ability to induce macrophage apoptosis via a type III protein secretion system (8,9,16,21). Macrophage apoptosis in response to Shigella and Salmonella infections triggers severe inflammation via the action of proinflammatory cytokines (22). Yersiniae induce apoptosis in macrophages by suppressing the signaling pathway that leads to the production of proinflammatory cytokines (1, 10). Macrophage cell death may lead to either the induction or the inhibition of an inflammatory response. In studying the interaction between phagocytes and Vibrio vulnificus, most efforts have focused on the capsule (7,11,13,18). Encapsulated isolates of V. vulnificus are more resistant to phagocytosis by human polymorphonuclear leukocytes and murine peritoneal macrophages than are unencapsulated isolates (4,5,15). However, the cytotoxic effects on phagocytes have not been clearly demonstrated. In this study, we examined the apoptotic effects of nine isolates of V. vulnificus on macrophages.First, we examined each isolate of V. vulnificus for apoptotic activity toward a macrophage-like cell line, J774, by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis. Five clinical isolates of V. vulnificus, the K series of strains, were isolated from the blood of individual septicemic patients at Kurashiki Central Hospital in Japan between 1985 and 1999. Two environmental strains, E4 and E10, were isolated from seafood in Florida. Strain R41 was isolated from plankton in Okayama Prefecture in Japan. Strain G83 was isolated from seafood in the Republic of Korea. Bacteria in the logarithmic growth phase were obtained by cultivation with Luria-Bertani broth at 37°C. The desired bacterial concentration was checked by plating serial dilutions of the samples on agar and counting CFU after incubation. J774 cells were grown in Dulbecco's modified Eagle's medium (Gibco BRL Life Technologies, Rockville, Md.) supplemented with 2 mM glutamine, 2 mM sodium pyruvate, and 20% heattreated fetal calf serum. Cells were seeded in 24-well tissue culture plates at 10 5 cells/well. Each isolate of V. vulnificus was inoculated into the wells at a multiplicity of infection of approximately 1.0. After incubation at 37°C for 150 min, the Mebstain Apoptosis Kit (Immunotech, Marseilles, France) was used to label the free 3Ј-OH ends of DNA fragments with fluorescein as recommended by the manufacturer. The wellkno...
Vibrio vulnificus causes severe sepsis in humans. There are several reports about the relationship between host immunity and bacterial growth in V. vulnificus infection. However, the effect on leukocytes of V. vulnificus infection in vivo has not been elucidated. A murine model of V. vulnificus infection was used to investigate its effects on leukocytes in this study. Bacteria were recovered from the blood of mice 3 h after subcutaneous injection in the right lower flank. They were detected in 87 . 5 % (n ¼ 7/8) of mice at 6 h, but this value decreased to 12 . 5 % (n ¼ 1/8) at 12 h. In contrast, the number of lymphocytes in peripheral blood had already started to decrease at 3 h, and reached a minimum at 6-9 h post-inoculation. Typical DNA laddering, a hallmark of apoptosis, was also detected in thymocytes and splenocytes at 6 and 9 h, and showed a tendency to disappear by 12 h. Although the number of lymphocytes decreased in the model, the numbers of neutrophils did not. These results suggested that V. vulnificus has selective cytotoxicity for lymphocytes in peripheral blood in vivo, and the lymphocyte depletion was probably associated with apoptosis in vivo.
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