Andrei L. Kindzelskii

Toll-like receptor 4 (TLR4) of retinal pigment epithelial cells participates in transmembrane signaling in response to photoreceptor outer segments.

Retinal pigment epithelial (RPE) cells mediate the recognition and clearance of effete photoreceptor outer segments (POS), a process central to the maintenance of normal vision. Given the emerging importance of Toll-like receptors (TLRs) in transmembrane signaling in response to invading pathogens as well as endogenous substances, we hypothesized that TLRs are associated with RPE cell management of POS. TLR4 clusters on human RPE cells in response to human, but not bovine, POS. However, TLR4 clustering could be inhibited by saturating concentrations of an inhibitory anti-TLR4 mAb. Furthermore, human POS binding to human RPE cells elicited transmembrane metabolic and calcium signals within RPE cells, which could be blocked by saturating doses of an inhibitory anti-TLR4 mAb. However, the heterologous combination of bovine POS and human RPE did not trigger these signals. The pattern recognition receptor CD36 collected at the POS–RPE cell interface for both homologous and heterologous samples, but human TLR4 only collected at the human POS–human RPE cell interface. Kinetic experiments of human POS binding to human RPE cells revealed that CD36 arrives at the POS–RPE interface followed by TLR4 accumulation within 2 min. Metabolic and calcium signals immediately follow. Similarly, the production of reactive oxygen metabolites (ROMs) was observed for the homologous human system, but not the heterologous bovine POS–human RPE cell system. As (a) the bovine POS/human RPE combination did not elicit TLR4 accumulation, RPE signaling, or ROM release, (b) TLR4 arrives at the POS–RPE cell interface just before signaling, (c) TLR4 blockade with an inhibitory anti-TLR4 mAb inhibited TLR4 clustering, signaling, and ROM release in the human POS–human RPE system, and (d) TLR4 demonstrates similar clustering and signaling responses to POS in confluent RPE monolayers, we suggest that TLR4 of RPE cells participates in transmembrane signaling events that contribute to the management of human POS.

A Model of the Oscillatory Metabolism of Activated Neutrophils

We present a two-compartment model to explain the oscillatory behavior observed experimentally in activated neutrophils. Our model is based mainly on the peroxidase-oxidase reaction catalyzed by myeloperoxidase with melatonin as a cofactor and NADPH oxidase, a major protein in the phagosome membrane of the leukocyte. The model predicts that after activation of a neutrophil, an increase in the activity of the hexose monophosphate shunt and the delivery of myeloperoxidase into the phagosome results in oscillations in oxygen and NAD(P)H concentration. The period of oscillation changes from >200 s to 10-30 s. The model is consistent with previously reported oscillations in cell metabolism and oxidant production. Key features and predictions of the model were confirmed experimentally. The requirement of the hexose monophosphate pathway for 10 s oscillations was verified using 6-aminonicotinamide and dexamethasone, which are inhibitors of glucose-6-phosphate dehydrogenase. The role of the NADPH oxidase in promoting oscillations was confirmed by dose-response studies of the effect of diphenylene iodonium, an inhibitor of the NADPH oxidase. Moreover, the model predicted an increase in the amplitude of NADPH oscillations in the presence of melatonin, which was confirmed experimentally. Successful computer modeling of complex chemical dynamics within cells and their chemical perturbation will enhance our ability to identify new antiinflammatory compounds.

Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells Their Apparent Roles in Cell Motility and Invasion

Intracellular Ca2+ signals have been associated with cell polarization and locomotion. As cell motility underlies metastasis, we have sought to better characterize the Ca2+ signaling events in HT1080 fibrosarcoma cells. We have tested the hypothesis that low voltage-activated (LVA) and nonvoltage-gated (NVG) channels of HT1080 cells participate in dynamic Ca2+-signaling events leading to cell migration and invasion. Immunofluorescence microscopy has shown that HT1080 cells express LVA T-type Ca2+ channels uniformly about the cell periphery, whereas the transient receptor potential-1 (a NVG cation channel) protein appears as punctate spots about a cell’s periphery. HT1080 cells exhibit periodic intracellular Ca2+ spikes. High-speed imaging revealed that the Ca2+ spikes were composed of a single Ca2+ wave traveling unidirectionally about the periphery of the cytoplasm in a clockwise fashion (as viewed from basal to apical surfaces). The T-type Ca2+ channel blocker mibefradil inhibited Ca2+ spikes and waves on cells and, in parallel, inhibited cell motility and invasion in a dose-dependent manner. Similar changes were noted with the NVG cation channel blockers Gd3+ and carboxyamido-triazole. The combination of LVA and NVG blockers further reduced Matrigel invasiveness. However, the Ca2+ 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 Ca2+ wave, thereby enhancing the efficacy of emerging therapeutic protocols.

Apparent role of traveling metabolic waves in oxidant release by living neutrophils

Cell metabolism self-organizes into two types of dissipative structures: chemical oscillations and traveling metabolic waves. In the present study we test the hypothesis that traveling NAD(P)H waves within neutrophils are associated spatially and temporally with the release of reactive oxygen metabolites (ROMs). Using high-speed optical microscopy and taking advantage of the autofluorescence of NAD(P)H, we have observed the propagation of NAD(P)H waves within cells. When NAD(P)H waves reach the lamellipodium of morphologically polarized neutrophils, a diffusing plume of superoxide is released as evidenced by the conversion of hydroethidine in the extracellular environment to ethidium bromide. Parallel results were obtained by using high-speed emission microspectrophotometry. These experiments indicate that the spatial and temporal properties of NAD(P)H waves are transformed into ROM pulses in the extracellular environment. Propagating NAD(P)H waves allow neutrophils to specifically deliver substrate to the lamellipodium at high concentrations, thus facilitating the local and periodic release of ROMs in the direction of cell movement and/or a target.

Ebola Virus Secretory Glycoprotein (sGP) Diminishes FcγRIIIB-to-CR3 Proximity on Neutrophils

Previous studies have shown that Ebola virus’ secretory glycoprotein (sGP) binds to FcγRIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and FcγRIIIB. Neutrophils were stained with rhodamine-conjugated anti-CD16b mAb (which does not inhibit sGP binding) and fluorescein-conjugated anti-CR3 mAb reagents and then incubated in media with or without sGP. Physical proximity between fluorochrome-labeled CR3 and FcγRIIIB on individual cells was measured by resonance energy transfer (RET) imaging, quantitative RET microfluorometry, and single-cell imaging spectrophotometry. Cells incubated with control supernatants displayed a significant RET signal, indicative of physical proximity (<7 nm) between CR3 and FcγRIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-FcγRIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and FcγRIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between FcγRIIIB and CR3.

Intracellular Calcium Waves Accompany Neutrophil Polarization, Formylmethionylleucylphenylalanine Stimulation, and Phagocytosis: A High Speed Microscopy Study

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 alters glucose-6-phosphate dehydrogenase trafficking, cell metabolism, and oxidant release of maternal neutrophils

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 CO(2) 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.

IFN-γ Primes RAW264 Macrophages and Human Monocytes for Enhanced Oxidant Production in Response to CpG DNA via Metabolic Signaling: Roles of TLR9 and Myeloperoxidase Trafficking

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-γ. We hypothesize that synergistic cellular responses to IFN-γ and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of ∼20 s. IFN-γ increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-γ. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-γ priming for oxidant production. MPO inhibitors blocked cell responses to IFN-γ and CpG DNA. In the absence of IFN-γ exposure, the effects of CpG DNA could be duplicated by MPO addition to cell samples. Moreover, monocytes from MPO knockout mice were metabolically unresponsive to IFN-γ and CpG DNA. NAD(P)H frequency doubling responses due to CpG DNA were blocked by an inhibitor of the hexose monophosphate shunt. Because NAD(P)H participates in electron trafficking to NO and superoxide anions, we tested oxidant production. Although CpG DNA alone had no effect, IFN-γ plus CpG enhanced NO and reactive oxygen metabolite release compared with IFN-γ treatment alone. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy.

Signal sequence within FcγRIIA controls calcium wave propagation patterns: Apparent role in phagolysosome fusion

Calcium oscillations and traveling calcium waves have been observed in living cells, although amino acid sequences regulating wave directionality and downstream cell functions have not been reported. In this study we identify an amino acid sequence within the cytoplasmic domain of the leukocyte IgG receptor FcγRIIA that affects the amplitude of calcium spikes and the spatiotemporal dynamics of calcium waves in the vicinity of phagosomes. By using high-speed microscopy to map calcium-signaling routes within cells, we have discovered that bound IgG-coated targets trigger two calcium waves traveling in opposite directions about the perimeter of cells expressing FcγRIIA. After phagocytosis, one calcium wave propagates around the plasma membrane to the site of phagocytosis where it splits into two calcium signals: one traveling to and encircling the phagosome once, and the second continuing around the plasma membrane to the point of origin. However, in a genetically engineered form of FcγRIIA containing a mutation in the cytoplasmic L-T-L motif, the calcium signal travels around the plasma membrane, but is not properly routed to the phagosome. Furthermore, these calcium pattern-deficient mutants were unable to support phagolysosome fusion, although recruitment of phagolysosome-associated proteins lysosome-associated protein 1, Rab5, and Rab7 were normal. Our findings suggest that: (i) calcium signaling is a late step in phagolysosome fusion, (ii) a line of communication exists between the plasma membrane and phagosome, and (iii) the L-T-L motif is a signal sequence for calcium signal routing to the phagosome.

Cutting Edge: Fever-Associated Temperatures Enhance Neutrophil Responses to Lipopolysaccharide: A Potential Mechanism Involving Cell Metabolism

Although much progress has been made in elucidating the mechanisms underlying the physiological regulation of fever, there is little understanding of the biological utility of fever’s thermal component. Considering the evolutionary co-conservation of fever and innate immunity, we hypothesize that fever’s thermal component might in general augment innate immune function and, in particular, neutrophil activation. Accordingly, we have evaluated the effect of febrile temperatures on neutrophil function at the single-cell level. We find that reactive oxygen intermediates and NO release are greatly enhanced at febrile temperatures for unstimulated as well as LPS-stimulated adherent human neutrophils. Furthermore, our studies suggest that these changes in oxidant release are linked to upstream changes in NADPH dynamics. Inasmuch as reactive oxygen intermediates and NO production are important elements in innate immune responses to bacterial pathogens, we suggest that the febrile rise in core temperature is a broad-based systemic signaling mechanism to enhance innate immunity.