Having previously associated metabolic oscillations with cell locomotion, we hypothesized that patients with abnormalities in neutrophil trafficking may display aberrant intracellular oscillations. A pyoderma gangrenosum patient exhibiting aberrant leukocyte trafficking in vivo and skin ulceration without infection was identified. This patient's neutrophils constitutively overexpressed and clustered the leukocyte integrins CR3 and CR4 and failed to display appropriate integrin-to-GPI receptor interactions. Increased levels of tyrosine phosphorylation were observed. NAD(P)H oscillations, which are sinusoidal in normals, were chaotic with multiple frequency components in this patient's neutrophils. Normal cell shape and sinusoidal NAD(P)H oscillations were restored by providing a pulsed electric field to drive metabolic oscillations and by temperature reduction. N-acetyl-D-glucosamine disrupted CR3 clusters and sinusoidal NAD(P)H oscillations returned. Anecdotal reports suggest that local hypothermia is clinically useful for this patient. These data define the first metabolic oscillation-associated disease and suggest that pyoderma gangrenosum can be classified as a dynamical disease at the cellular level.

Migrating neutrophils utilize beta2 integrins for substrate attachment and urokinase receptors (uPAR) to focus pericellular proteolysis. Our studies show that CR3 associates with uPAR on resting cells, whereas uPAR associates with CR4 at lamellipodia of migrating cells. Using resonance energy transfer (RET) microscopy, we show that the molecular proximity between CR4 and uPAR oscillates on migrating cells, thus suggesting that CR4 molecules periodically bind/release uPAR. Cell contact with fibrinogen, endothelial cells, chemotactic factors and indomethacin, and treatment with sub-optimal doses of signal transduction inhibitors, affect the oscillations' period, amplitude, and/or waveform. The oscillations were indistinguishable in period and 180 degrees out-of-phase with cytosolic NAD(P)H autofluorescence oscillations. Thus, CR4 and CR3 identify a neutrophil's axis of migration and CR4 may restrain uPAR at lamellipodia. Oscillations in signal transduction and energy metabolism may coordinate cell adherence, local proteolysis, oxidant release, actin assembly, and cell extension.

Theoretical studies have predicted spatiotemporal organization of cell metabolism. Using a rapidly gated CCD camera, we demonstrate for the first time sustained traveling waves of NAD(P)H autofluorescence and protons in individual morphologically polarized living cells. Chemical concentration fronts moved in the direction of cell orientation, thus correlating dissipative structures with cell shape.

To better understand the mechanism of leukocyte migration in complex environments, model extracellular matrices were prepared using gelatin, Hanks' solution, Bodipy-BSA (fluorescent upon proteolysis), and dihydrotetramethylrosamine or hydroethidine (fluorescent upon oxidation). Using quantitative microfluorometry, neutrophil-mediated extracellular pulses of reactive oxygen metabolites (ROMs) and pericellular proteolysis were periodically observed showing that these functions occur as quantal bursts. However, chronic granulomatous disease neutrophils, which do not produce ROMs, did not display ROM deposition. Matrices show an alternating pattern of green (proteolytic) and red (oxidative) fluorescence, indicating these functions are out of phase. Electric fields phase-matched with metabolic oscillations, which increase the amplitude of intracellular NAD(P)H oscillations, increase ROM deposition and pericellular proteolysis; this further supports the link between intracellular chemical oscillators and extracellular functions. This phase relationship may allow ROMs to inactivate protease inhibitors, followed by protease activation.

Using high sensitivity fluorescence imaging with shutter speeds ∼600,000 times faster than those of video frames, we have characterized Ca2+ waves within cells in exquisite detail to reveal Ca2+ signaling routes. Polarized neutrophils exhibited a counterclockwise rotating ryanodine-sensitive juxtamembrane Ca2+ wave during temporal calcium spikes. During stimulation with fMLP, a chemotactic factor, two Ca2+ waves traveling in opposite directions around the perimeter of the cell emanated from sites of stimulation (the clockwise wave is verapamil sensitive). Phagocytosed targets exhibit counterclockwise Ca2+ waves traveling about their periphery originating from the plasma membrane. This study: 1) outlines the technology to observe Ca2+ signaling circuitry within small living cells; 2) shows that extracellular spatial information in the form of a chemotactic factor gradient is transduced into intracellular chemical patterns, which provides fresh insights in signaling; 3) suggests that a line of communication exits between the cell surface and phagosomes; and 4) suggests that spatiotemporal Ca2+ patterns contribute to drug actions.

Pregnancy is associated with changes in host susceptibility to infections and inflammatory disease. We hypothesize that metabolic enzyme trafficking affects maternal neutrophil activation. Specifically, immunofluorescence microscopy has shown that glucose-6-phosphate dehydrogenase (G-6-PDase), the rate-controlling step of the hexose monophosphate shunt (HMS), is located near the cell periphery in control neutrophils but is found near the microtubule-organizing centers in cells from pregnant women. Cytochemical studies confirmed that the distribution of the G-6-PDase antigen is coincident with functional G-6-PDase activity. Metabolic oscillations within activated pregnancy neutrophils are higher in amplitude, though lower in frequency, than activated control neutrophils, suggesting limited HMS activity. Analysis of radioisotope-labeled carbon flux from glucose to CO2 indicates that the HMS is intact in leukocytes from pregnant women, but its level is not enhanced by cell stimulation. Using extracellular fluorescent markers, activated pregnancy neutrophils were found to release reactive oxygen metabolites (ROMs) at a lower rate than activated control neutrophils. However, basal levels of ROM production in polarized pregnancy neutrophils were greater than in control neutrophils. Microtubule-disrupting agents reversed the observed changes in G-6-PDase trafficking, metabolic oscillations, and ROM production by maternal neutrophils. G-6-PDase trafficking appears to be one mechanism regulating ROM production by maternal neutrophils

Although much progress has been made in elucidating the biochemical properties of lipid rafts, there has been less success in identifying these structures within living cell membranes, which has led to some concern regarding their existence. One difficulty in analyzing lipid rafts using optical microscopy is their small size. We now test the existence of lipid rafts in polarized neutrophils, which redistribute lipid raft markers into comparatively large lamellipodia. Optical microspectrophotometry of Laurdan-labeled neutrophils revealed a large blue shift at lamellipodia relative to cell bodies. This blue shift disappeared after exposure to methyl-β-cyclodextrin (mβCD), which disrupts lipid rafts. The Ca2+ channel transient receptor potential-like channel-1, a lipid raft marker, traffics to lamellipodia, but redistributes uniformly about cells after exposure to mβCD. This is accompanied by disruption of Ca2+ waves normally initiated at lamellipodia. Thus, mβCD-sensitive lipid-ordered domains are present at and participate in signaling from the lamellipodia of living neutrophils.

ABSTRACT2؉ channel blockers nicardipine, SKF96365, diltiazem, and verapamil had no effect at appropriate doses. These results indicate that certain LVA and NVG channels regulate HT1080 cell motility. In addition to providing novel information regarding cancer cell motility, we suggest that it may be possible to design drugs that inhibit a key Ca 2؉ wave, thereby enhancing the efficacy of emerging therapeutic protocols.