Stimulation of neutrophil actin polymerization and degranulation by opsonized and unopsonized Candida albicans hyphae and zymosan

Abstract

We previously showed that unopsonized Candida albicans hyphae stimulated a delayed rise in the putative neutrophil second messengers Ca2+ and inositol 1,4,5-trisphosphate and subsequent O2- release, as compared with opsonized hyphae or zymosan. Therefore, cytoskeletal and degranulation temporal responses to these stimuli were examined. Unopsonized zymosan elicited no neutrophil responses under the experimental condition used. Neutrophil actin polymerization (quantitated by fluorescent measurements of NBD phallacidin) was rapid after stimulation by opsonized hyphae or zymosan (peaking at 1 and 2 min, respectively). This corresponded to observed changes in microscopic actin polymerization, measured with rhodamine phalloidin, which progressed from initially diffuse to collarlike to cylinderlike staining patterns surrounding the hyphae. Compared with opsonized hyphae, unopsonized hyphae resulted in a delayed appearance of the last two visible patterns (P less than 0.05) and in quantitative actin polymerization despite similarly rapid initial contact and spreading over the hyphae by neutrophils. Unlike other neutrophil responses, degranulation did not follow the delayed patterns of responses to stimulation with unopsonized hyphae. In the absence of the release of the cytoplasmic marker lactate dehydrogenase, the release of beta-glucuronidase, an azurophil granule marker, gradually and progressively rose in response to all of the stimuli but unopsonized zymosan. The low but significant levels observed were within a range consistent with published results for degranulation responses to particulate stimuli without cytochalasin B. A quantitative immunoassay of lactoferrin, a specific granule marker, detected no release into supernatants, and immunofluorescent staining indicated concomitant depletion of lactoferrin from neutrophil granules and binding to hyphal and neutrophil surfaces after stimulation by unopsonized hyphae. Thus, the delayed actin polymerization response to unopsonized hyphae occurred subsequent to neutrophil attachment and spreading and resembled the temporal sequence of other neutrophil responses linked to the respiratory burst. In contrast, the degranulation responses to all stimuli appeared to begin and progress gradually after observed attachment and spreading of the neutrophil over hyphal surfaces without a clear temporal relationship to rises in cytoplasmic Ca2+ or F-actin. In addition, the avid binding of released lactoferrin to cell surfaces eliminates its value as a quantitative marker of enzyme release but raises the possibility that it might participate in fungicidal activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Independence of neutrophil respiratory burst oxidant generation from the early cytosolic calcium response after stimulation with unopsonized Candida albicans hyphae

“…On the basis of the model of neutrophil activation derived from stimulation with the soluble chemotactic factor fMLP, substantial evidence suggests that a rise in cytosolic free calcium is necessary but not sufficient for initiation of the respiratory burst (1,18,25). Likewise, results of our own work with particulate stimuli had been at least consistent with this postulated interdependence of the early calcium transient and other postactivation events, since opsonized zymosan, as well as opsonized or unopsonized C. albicans hyphae, evoked temporally related increases in cytosolic free calcium, 1,4,5-inositol trisphosphate, actin polymerization, and superoxide generation (28,39). However, we now report an active respiratory burst response by neutrophils activated with unopsonized C. albicans hyphae despite total ablation of any cytosolic calcium rise detectable with indo-1.…”

“…that elicited from BAPTA-loaded neutrophils by opsonized zymosan (P < 0.01) or hyphae (P < 0.001). As in our previous studies (28,34,39), unopsonized zymosan, which neither attached to nor was ingested by neutrophils under the experimental conditions used, evoked no responses.…”

“…These considerations seem particularly significant since our previous data suggest that opsonized and unopsonized hyphae may activate divergent signal transduction pathways in neutrophils, pathways that may differ from those initiated * Corresponding author. by other agonists as well (28,32,33,35,39). A hypothetical model of neutrophil activation based on a large body of evidence obtained with the soluble chemotactic peptide fMet-Leu-Phe (fMLP) (6,7,23,45,46,51,52,60) as well as with particulate opsonized zymosan (36,41,42) has been proposed.…”

Innate and adaptive immunity in female genital tract: cellular responses and interactions

“…Fallopian neutrophils also contained lower amounts of LF and myeloperoxidase, two important granule‐associated molecules. Their decreased content in Fallopian neutrophils suggests that the cells have undergone some amount of degranulation (353). The somewhat increased levels of CD45 are also consistent with this idea, as others have noted that freshly isolated blood neutrophils have relatively low surface levels of CD45 and that there is a pool of CD45 in cytoplasmic granules (354).…”

Stimulation of cell motility and expression of late markers of differentiation in human oral keratinocytes byCandida albicans

“…Studies with human larynx epithelioid carcinoma (HEp‐2) cells that were treated with C. albicans or C. albicans metabolites revealed changes in the actin cytoskeleton, reduced membrane ruffling and decreased cell motility (Tsarfaty et al ., 2000; Sandovsky‐Losica et al ., 2002). C. albicans has also been shown to cause a reorganization of the cytoskeleton in neutrophils and macrophages (Kolotila and Diamond, 1988; Schindler and Segal, 2008; Wozniok et al ., 2008) and to elicit the formation of an actin mantle around hyphae (Park et al ., 2005). However, the role(s) of keratinocyte differentiation in candidiasis have not been considered.…”