Affiliation:
1. Department of Medicine, University of California, Los Angeles School of Medicine 90024.
Abstract
Although purified defensins are effective microbicides in vitro, their operation within intact phagocytes has not been established. To address this question, we inserted cDNA encoding human defensin HNP-1 into a pBabe/neo retroviral vector and transduced it into RAW 264.7 cells, a murine macrophage line that lacks endogenous defensins. We isolated five independent clones of HNP-1-transduced cells, all of which secreted prodefensin and contained small amounts of fully processed HNP-1. The two clones that produced the largest amounts of defensin (clones 5 and 14), together with wild-type RAW cells and pBabe/neo-transduced RAW cells (control), were used for the present study. All cells were grown in Dulbecco's modified Eagle's medium-F12 medium that contained 10% heat-inactivated fetal bovine serum and gentamicin. The medium used for the transduced cells contained aminoglycoside G418 in lieu of gentamicin. Both wild-type and transduced cells were placed in antibiotic-free medium 96 h prior to challenge with a yeast-phase strain of Histoplasma capsulatum. Phagocytosis of yeast cells was allowed to proceed for 90 min and was followed by washing and further incubation for 18.5 h. Whereas the phagocytic index did not differ significantly among the four cell populations under study, the mean level of intracellular growth of H. capsulatum in the defensin-transduced RAW cells was significantly lower than those observed for any other cell types (P < 0.05). These findings constitute the first instance of xenogeneic expression of an antimicrobial peptide by phagocytes and suggest that macrophages can be armed with defensins to enhance their ability to restrict certain intracellular pathogens.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Immunology,Microbiology,Parasitology
Cited by
35 articles.
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