Chemotactic Process

Abstract: Chemotaxis is one of the cardinal functions of leukocytes, which enables them to be recruited efficiently to the right place at the right time. Analyzing chemotactic activities is important not only for the study on leukocyte migration but also for many other applications including development of new drugs interfering with the chemotactic process. However, there are many technical limitations in the conventional in vitro chemotaxis assays. Here we applied a new optical assay to investigate chemotactic activities induced in differentiated HL-60 cells. Methods: HL-60 cells were stimulated with 0.8% dimethylformamide (DMF) for 4 days. The cells were analyzed for morphology, flow cytometry as well as chemotactic activities by a time-lapse videomicroscopic assay using a chemotactic microchamber bearing a fibronectin-coated cover slip and an etched silicon chip. Results: Videomicroscopic observation of the real cellular motions in a stable concentration gradient of chemokines demonstrated that HL-60 cells showed chemotaxis to inflammatory chemokines (CCL3, CCL5 and CXCL8) and also a homeostatic chemokine (CXCL12) after DFM-induced differentiation to granulocytic cells. The cells moved randomly at a speed of 6.99±1.24μm/min (n=100) in the absence of chemokine. Chemokine stimulation induced directional migration of differentiated HL-60 cells, while they still wandered very much and significantly increased the moving speeds. Conclusion: The locomotive patterns of DMF-stimulated HL-60 cells can be analyzed in detail throughout the course of chemotaxis by the use of a time-lapse videomicroscopic assay. DMF-stimulated HL-60 cells may provide a convenient in vitro model for chemotactic studies of neutrophils.

Abstract: SUMMARY: Free-swimming zoospores of several isolates of the oomycetous water mould Achlya demonstrate mutual attraction (adelphotaxis) which may result in auto-aggregation. In the apparent absence of exogenous chemotactic signals macroscopic globular aggregates or microscopic plaques of cysts are formed. Auto-aggregation was favoured by high concentrations of zoospores in suspension; the growth rate of spontaneous aggregates was estimated at 4--10 x 103spores min−1; growth of an aggregate was limited to about 7 min. By means of chemotactic assays, attractant activity for zoospores was detected in the vicinity of aggregates, and in zoospore or cyst supernatants. The attractant activity extracted from spore aggregates was heat-stable. In the absence of exogenous nutrients, the macroscopic aggregates of an homothallic isolate produced sporangia and oogonia. Aggregation was readily initiated as a result of zoospore chemotaxis towards capillary tubes containing exogenous attractant in an agar medium. Aggregating zoospores frequently assumed a slender swarm configuration moving directly towards the aggregate at the capillary-tube tip. Analysis of video recordings showed that many zoospores joining the swarm were attracted directly by the swarm rather than by the exogenous attractant. Such aggregation was a time-limited process, the duration of which was extended by the presence of amino acids in the zoospore suspension. Under natural conditions zoospore aggregation may be a dual-component chemotactic process triggered by exogenous attractants and amplified by chemical signals from the aggregating spores. The possible consequences of adelphotaxis and auto-aggregate formation, namely enhanced inoculum potential, syngamy and spore production, are discussed.