Binding interactions of murine natural killer cells with the fungal target Cryptococcus neoformans

Abstract

Murine natural killer (NK) cells have been shown to inhibit the growth of the yeastlike organism Cryptococcus neoformans both in vivo and in vitro. An essential first step in NK cell-mediated damage of cryptococcal cells is the binding of the NK cell to the cryptococcal cell. The studies presented here focused on the binding event. Electron photomicrographs and three-dimensional reconstructions of NK cell-C. neoformans conjugates show that NK cells bind to cryptococci through many microvilli. This is in contrast to the broad membrane-membrane interactions which form the binding site of NK cell-YAC-1 tumor cell conjugates. NK cell binding to cryptococci is much slower than NK cell binding to YAC-1 targets. Maximal conjugate formation with cryptococcal targets is reached after 2 h, whereas maximal conjugate formation with YAC-1 targets is obtained after 20 min. Once maximum NK cell-C, neoformans conjugate formation is obtained, another 4 h is required before damage to the cryptococcal cells can be detected with the CFU assay. These data indicate that the binding and action of NK cells on C. neoformans cells requires considerably more time than is necessary for similar events to occur in the NK cell-tumor cell model. NK cell membrane integrity is necessary for NK cells to bind to tumor targets, since some disruption of membrane integrity with 0.1 M dimethyl sulfoxide reduces conjugate formation and tumor cell lysis. In contrast, 0.1 M dimethyl sulfoxide did not diminish NK cell binding to cryptococcal targets; however, it significantly reduced cryptococcal growth inhibition. Although we have observed several differences in NK cell binding to the cryptococcal target compared with NK cell binding to tumor cell targets, there are some similarities in binding interactions of NK cells with the two different targets. Disulfide bonding appears to play a role in the binding of NK cells to both targets, since 5 mM 2-mercaptoethanol, a reagent that reduces disulfide bonds, prevented NK cells from binding to the tumor targets as well as the cryptococcal targets. Actin filaments, components of the cytoskeletal network, must be intact for NK cells to bind to YAC-1 cells or cryptococci. Taken together, our data confirm that binding of NK cells to the cryptococcal target is prerequisite to the stages that result in damage to the cryptococcal cell and that there are similarities and differences in NK cell-binding interactions with structurally different target cells.