Elevated glucose concentrations promote receptor-independent activation of adherent human neutrophils: an experimental and computational approach.

Neutrophil activation plays integral roles in host tissue damage and resistance to infectious diseases. As glucose uptake and NADPH availability are required for reactive oxygen metabolite production by neutrophils, we tested the hypothesis that pathological glucose levels (>or=12 mM) are sufficient to activate metabolism and reactive oxygen metabolite production in normal adherent neutrophils. We demonstrate that elevated glucose concentrations increase the neutrophil's metabolic oscillation frequency and hexose monophosphate shunt activity. In parallel, substantially increased rates of NO and superoxide formation were observed. However, these changes were not observed for sorbitol, a nonmetabolizable carbohydrate. Glucose transport appears to be important in this process as phloretin interferes with the glucose-specific receptor-independent activation of neutrophils. However, LY83583, an activator of glucose flux, promoted these changes at 1 mM glucose. The data suggest that at pathophysiologic concentrations, glucose uptake by mass action is sufficient to activate neutrophils, thus circumventing the normal receptor transduction mechanism. To enable us to mechanistically understand these dynamic metabolic changes, mathematical simulations were performed. A model for glycolysis in neutrophils was created. The results indicated that the frequency change in NAD(P)H oscillations can result from the activation of the hexose monophosphate shunt, which competes with glycolysis for glucose-6-phosphate. Experimental confirmation of these simulations was performed by measuring the effect of glucose concentrations on flavoprotein autofluorescence, an indicator of the rate of mitochondrial electron transport. Moreover, after prolonged exposure to elevated glucose levels, neutrophils return to a "nonactivated" phenotype and are refractile to immunologic stimulation. Our findings suggest that pathologic glucose levels promote the transient activation of neutrophils followed by the suppression of cell activity, which may contribute to nonspecific tissue damage and increased susceptibility to infections, respectively.

Myeloperoxidase accumulates at the neutrophil surface and enhances cell metabolism and oxidant release during pregnancy.

Pregnancy is a unique immunological state. Pregnancy neutrophils differ from those of non-pregnant women as they cannot be fully activated for oxidant production, but yet have higher levels of unstimulated oxidant production. Although reduced activation is due to decreased hexose monophosphate shunt activity, the mechanism enhancing basal oxidant levels is unknown. We hypothesize that myeloperoxidase (MPO) trafficking affects the basal oxidant release by maternal neutrophils. Immunofluorescence microscopy has demonstrated MPO at the surface of pregnancy neutrophils, whereas non-pregnancy cells do not exhibit surface MPO. Adherent pregnancy neutrophils were characterized by high-amplitude metabolic oscillations, which were blocked by MPO inactivation. Conversely, metabolic oscillatory amplitudes of control neutrophils were heightened by incubation with PMA or exogenous MPO. Importantly, MPO decoration of cell surfaces and high-amplitude metabolic oscillations were observed for neutrophils from pregnant but not from non-pregnant mice. However, cells from pregnant MPO knockout mice did not exhibit MPO expression or high-amplitude metabolic oscillations. Unstimulated neutrophils from pregnant women were found to release reactive oxygen metabolites (ROM) and reactive nitrogen intermediates (RNI), but cells from non-pregnant women did not. MPO inhibition returned ROM and RNI formation to non-pregnant levels. Hence, MPO trafficking influences metabolic activity and oxidant production in pregnancy.

IFN-gamma 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-gamma. We hypothesize that synergistic cellular responses to IFN-gamma 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 approximately 20 s. IFN-gamma 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-gamma. 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-gamma priming for oxidant production. MPO inhibitors blocked cell responses to IFN-gamma and CpG DNA. In the absence of IFN-gamma 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-gamma 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-gamma plus CpG enhanced NO and reactive oxygen metabolite release compared with IFN-gamma treatment alone. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy.

Trophoblast contact deactivates human neutrophils.

Trophoblasts are fetal epithelial cells that form an interface between mother and offspring. To evaluate their anti-inflammatory capacity, we tested the hypothesis that trophoblasts deactivate neutrophils using single-cell assays. Several biophysical (Ca2+ and NAD(P)H oscillation frequency) and physiological (oxidant production) markers of activated neutrophils revert to a nonactivated phenotype as activated cells make contact with trophoblasts. Indistinguishable results were obtained using syncytiotrophoblasts and in experiments using trophoblasts and neutrophils from the same mother to recapitulate the semiallogeneic system. These changes suggest reduced hexose monophosphate shunt (HMS) activity. We discovered that two metabolic regulatory points, glucose transport and HMS enzyme trafficking, are affected by trophoblasts. This restriction in HMS activity deactivates neutrophils, thereby limiting oxidative DNA damage within trophoblasts.

TLR4 mediates human retinal pigment epithelial endotoxin binding and cytokine expression.

PURPOSE: It was previously demonstrated that toll-like receptor 4 (TLR4) is involved in species-specific human retinal pigment epithelial (HRPE) photoreceptor outer segment recognition and oxidant production. This study was performed to demonstrate the classical role of TLR4 in HRPE endotoxin (lipopolysaccharide; LPS) binding leading to HRPE proinflammatory cytokine secretion. METHODS: Cultured HRPE cells were examined for TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. HRPE cells labeled with fluorescent monoclonal antibodies (mAbs) to TLR4 and its associated adhesion molecule, CD14, were analyzed by real-time microscopy and resonance energy transfer (RET), determining associations of fluorescent LPS, TLR4, and CD14. LPS-induced HRPE secretion of interleukin (IL)-8 was measured with and without specific blocking mAb to TLR4 and CD14. HRPE TLR4 expression was measured after LPS exposure in the presence and absence of blocking antibodies to TLR4 and CD14. RESULTS: All three HRPE cell lines demonstrated constitutive TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. Examination of HRPE cells labeled with fluorescent mAb to TLR4, CD14, and LPS demonstrated RET among the three molecules, indicating that LPS-CD14 binding preceded LPS-TLR4 binding and the close association of CD14 and TLR4. LPS-induced IL-8 was significantly reduced (P < 0.05) in the presence of blocking mAb to TLR4 or CD14. Upregulation of HRPE TLR4 gene and protein expression occurred in response to LPS exposure and was inhibited by mAb to TLR4 or CD14. CONCLUSIONS: HRPE TLR4 is a multifunctional molecule that participates in photoreceptor outer segment membrane recognition, oxidant production, LPS recognition, and cytokine production. These roles indicate potential involvement in retinal degenerative and inflammatory processes.

Ion channel clustering enhances weak electric field detection by neutrophils: apparent roles of SKF96365-sensitive cation channels and myeloperoxidase trafficking in cellular responses.

We have tested Galvanovskis and Sandblom's prediction that ion channel clustering enhances weak electric field detection by cells as well as how the elicited signals couple to metabolic alterations. Electric field application was timed to coincide with certain known intracellular chemical oscillators (phase-matched conditions). Polarized, but not spherical, neutrophils labeled with anti-K(v)1.3, FL-DHP, and anti-TRP1, but not anti-T-type Ca(2+) channels, displayed clusters at the lamellipodium. Resonance energy transfer experiments showed that these channel pairs were in close proximity. Dose-field sensitivity studies of channel blockers suggested that K(+) and Ca(2+) channels participate in field detection, as judged by enhanced oscillatory NAD(P)H amplitudes. Further studies suggested that K(+) channel blockers act by reducing the neutrophil's membrane potential. Mibefradil and SKF93635, which block T-type Ca(2+) channels and SOCs, respectively, affected field detection at appropriate doses. Microfluorometry and high-speed imaging of indo-1-labeled neutrophils was used to examine Ca(2+) signaling. Electric fields enhanced Ca(2+) spike amplitude and triggered formation of a second traveling Ca(2+) wave. Mibefradil blocked Ca(2+) spikes and waves. Although 10 microM SKF96365 mimicked mibefradil, 7 microM SKF96365 specifically inhibited electric field-induced Ca(2+) signals, suggesting that one SKF96365-senstive site is influenced by electric fields. Although cells remained morphologically polarized, ion channel clusters at the lamellipodium and electric field sensitivity were inhibited by methyl-beta-cyclodextrin. As a result of phase-matched electric field application in the presence of ion channel clusters, myeloperoxidase (MPO) was found to traffic to the cell surface. As MPO participates in high amplitude metabolic oscillations, this suggests a link between the signaling apparatus and metabolic changes. Furthermore, electric field effects could be blocked by MPO inhibition or removal while certain electric field effects were mimicked by the addition of MPO to untreated cells. Therefore, channel clustering plays an important role in electric field detection and downstream responses of morphologically polarized neutrophils. In addition to providing new mechanistic insights concerning electric field interactions with cells, our work suggests novel methods to remotely manipulate physiological pathways.

Real-time control of neutrophil metabolism by very weak ultra-low frequency pulsed magnetic fields.

In adherent and motile neutrophils NAD(P)H concentration, flavoprotein redox potential, and production of reactive oxygen species and nitric oxide, are all periodic and exhibit defined phase relationships to an underlying metabolic oscillation of approximately 20 s. Utilizing fluorescence microscopy, we have shown in real-time, on the single cell level, that the system is sensitive to externally applied periodically pulsed weak magnetic fields matched in frequency to the metabolic oscillation. Depending upon the phase relationship of the magnetic pulses to the metabolic oscillation, the magnetic pulses serve to either increase the amplitude of the NAD(P)H and flavoprotein oscillations, and the rate of production of reactive oxygen species and nitric oxide or, alternatively, collapse the metabolic oscillations and curtail production of reactive oxygen species and nitric oxide. Significantly, we demonstrate that the cells do not directly respond to the magnetic fields, but instead are sensitive to the electric fields which the pulsed magnetic fields induce. These weak electric fields likely tap into an endogenous signaling pathway involving calcium channels in the plasma membrane. We estimate that the threshold which induced electric fields must attain to influence cell metabolism is of the order of 10(-4) V/m.

Oxidant release is dramatically increased by elevated glucose concentrations in neutrophils from pregnant women.

OBJECTIVE: To evaluate the mechanism of oxidative stress at glucose levels accompanying diabetic pregnancy. Specifically, we hypothesize that elevated glucose overwhelms hexose monophosphate shunt (HMS) down-regulation observed during pregnancy. METHODS: Peripheral blood cells from normal healthy pregnant women were exposed to heightened glucose levels to provide an in vitro model of the effects of diabetic pregnancy. Changes in NAD(P)H, reactive oxygen species (ROS) and nitric oxide (NO) production were evaluated in single cells. RESULTS: Altered metabolic dynamics, as judged by NAD(P)H autofluorescence of neutrophils from both pregnant and non-pregnant women, were observed during incubation with 14 mM glucose, a pathophysiologic level. In parallel, increased production of ROS and NO was observed. The ROS and NO levels attained in cells from pregnant women were greater than those observed in cells from non-pregnant women. Inhibitors of the HMS and NAD(P)H oxidase blocked these effects. These metabolic and oxidant changes required approximately one minute, suggesting that transient glucose spikes during pregnancy could trigger this response. CONCLUSIONS: Elevated glucose levels enhance HMS activity and oxidant production in cells from pregnant women. This mechanism may be generally applicable in understanding the role of diabetes in materno-fetal health.

Human, but not bovine, photoreceptor outer segments prime human retinal pigment epithelial cells for metabolic activation and massive oxidant release in response to lipopolysaccharide and interferon-gamma.

Reactive oxygen metabolites (ROMs) may contribute to several eye diseases, such as age-related macular degeneration, although the underlying mechanisms are unclear. The present study shows that human photoreceptor outer segments (POS) prime human retinal pigment epithelial (RPE) cells for massive ROM release in response to lipopolysaccharide (LPS) and interferon-gamma. However, no ROM priming of human RPE cells is observed for bovine POS. ROM production appears to be linked with underlying metabolic oscillations involving the hexose monophosphate shunt.

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.

6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase form a supramolecular complex in human neutrophils that undergoes retrograde trafficking during pregnancy.

Neutrophils from pregnant women display reduced neutrophil-mediated effector functions, such as reactive oxygen metabolite (ROM) release. Because the NADPH oxidase and NO synthase produce ROMs and NO, the availability of their substrate NADPH is a potential regulatory factor. NADPH is produced by glucose-6-phosphate dehydrogenase (G-6-PDase) and 6-phosphogluconate dehydrogenase (6-PGDase), which are the first two steps of the hexose monophosphate shunt (HMS). Using immunofluorescence microscopy, we show that 6-PGDase, like G-6-PDase, undergoes retrograde transport to the microtubule-organizing centers in neutrophils from pregnant women. In contrast, 6-PGDase is found in an anterograde distribution in cells from nonpregnant women. However, lactate dehydrogenase distribution is unaffected by pregnancy. Cytochemical studies demonstrated that the distribution of 6-PGDase enzymatic activity is coincident with 6-PGDase Ag. The accumulation of 6-PGDase at the microtubule-organizing centers could be blocked by colchicine, suggesting that microtubules are important in this enzyme's intracellular distribution. In situ kinetic studies reveal that the rates of 6-gluconate turnover are indistinguishable in samples from nonpregnant and pregnant women, suggesting that the enzyme is functionally intact. Resonance energy transfer experiments showed that 6-PGDase and G-6-PDase are in close physical proximity within cells, suggesting the presence of supramolecular enzyme complexes. We suggest that the retrograde trafficking of HMS enzyme complexes during pregnancy influences the dynamics of NADPH production by separating HMS enzymes from glucose-6-phosphate generation at the plasma membrane and, in parallel, reducing ROM and NO production in comparison with fully activated neutrophils from nonpregnant women.

Cutting edge: optical microspectrophotometry supports the existence of gel phase lipid rafts at the lamellipodium of neutrophils: apparent role in calcium signaling.

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-beta-cyclodextrin (m beta CD), which disrupts lipid rafts. The Ca(2+) channel transient receptor potential-like channel-1, a lipid raft marker, traffics to lamellipodia, but redistributes uniformly about cells after exposure to m beta CD. This is accompanied by disruption of Ca(2+) waves normally initiated at lamellipodia. Thus, m beta CD-sensitive lipid-ordered domains are present at and participate in signaling from the lamellipodia of living neutrophils.

Identification of channels promoting calcium spikes and waves in HT1080 tumor cells: their apparent roles in cell motility and invasion.

Intracellular Ca(2+) signals have been associated with cell polarization and locomotion. As cell motility underlies metastasis, we have sought to better characterize the Ca(2+) 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 Ca(2+)-signaling events leading to cell migration and invasion. Immunofluorescence microscopy has shown that HT1080 cells express LVA T-type Ca(2+) 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 Ca(2+) spikes. High-speed imaging revealed that the Ca(2+) spikes were composed of a single Ca(2+) wave traveling unidirectionally about the periphery of the cytoplasm in a clockwise fashion (as viewed from basal to apical surfaces). The T-type Ca(2+) channel blocker mibefradil inhibited Ca(2+) 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 Gd(3+) and carboxyamido-triazole. The combination of LVA and NVG blockers further reduced Matrigel invasiveness. However, the Ca(2+) 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.

Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator.

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA-/-) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1). developed a novel methodology to detect pericellular proteolytic function, (2). demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3). demonstrated its usefulness in real-time studies of cell migration, and (4). showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis.

Signal processing times in neutrophil activation: dependence on ligand concentration and the relative phase of metabolic oscillations.

Intracellular NAD(P)H oscillations exhibited by polarized neutrophils display congruent with 20 s periods, which are halved to congruent with 10 s upon stimulation with chemotactic peptides such as FNLPNTL (N-formyl-nle-leu-phe-nle-tyr-lys). By monitoring this frequency change, we have measured accurately the time interval between stimulus and metabolic frequency changes. A microscope flow chamber was designed to allow rapid delivery of FNLPNTL to adherent cells. Using fluorescein as a marker, we found delivery to be complete and stable throughout the chamber within approximately 400 ms. Peptides were injected into the chamber at concentrations ranging from 10(-6) to 10(-9) M. Injections also varied with respect to the relative phase of a cell's NAD(P)H oscillations. The time interval between injection of 10(-6) M FNLPNTL and the acquisition of congruent with 10 s period metabolic oscillations was found to be 12.2+/-3.3 s when injections occurred at the NAD(P)H oscillation peak whereas the lag time was 22.5+/-4.8 s when coinciding with a trough. At 10(-8) M FNLPNTL, lag times were found to be 26.1+/-5.2 and 30.5+/-7.3 s for injections at NAD(P)H peaks and troughs, respectively. FNLPNTL at 10(-9) M had no effect on metabolic oscillations, consistent with previous studies. Our experiments show that the kinetics of transmembrane signal processing, in contrast to a simple transmembrane chemical reaction, can depend upon both ligand dose and its temporal relationship with intracellular metabolic oscillations.

Signal sequence within Fc gamma RIIA 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 Fc gamma RIIA 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 Fc gamma RIIA. 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 Fc gamma RIIA 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.

Human RPE cell lysis of extracellular matrix: functional urokinase plasminogen activator receptor (uPAR), collagenase and elastase.

Age-related macular degeneration (ARMD), proliferative vitreoretinopathy (PVR) and uveitis are characterized by RPE motility through the ECM of retinal lesions. The purpose of this study was to test the hypothesis that multiple proteolytic systems are functionally intact at the HRPE surface and peri-cellular region and that these activities are differentially modulated by IL-1beta. HRPE cells were evaluated: (1). as individual cells or cell extracts, (2). during migration across three-dimensional ECM-like layers and (3). in tissue sections. The urokirase plasminogen activator receptor (uPAR; CD87) was detected on HRPE cells as well as its functional activity. Although uPAR was associated with CD11b (CR3) on live resting cells, polarized migratory HRPE cells were found to dissociate uPAR from CR3; uPAR then translocated to anterior pole of the cell, where it enhanced PAI-1-inhibitable local proteolytic activity. The relative contribution of uPAR and collagenase in HRPE migration was evaluated using three-dimensional gelatin matrices. Interestingly, uPAR/uPA was found to play a key role in migration across these layers. IL-1 upregulated uPAR, collagenase, and elastase activities, suggesting that cytokines may affect the invasive program of HRPE cells in vivo. Immunohistochemistry for uPAR was performed in sections of human retina. Immunoreactive uPAR was present along the HRPE basolateral membrane in retinal sections and in sections of diseased retinal tissue at an enhanced level. Our results suggest that multiple proteolytic systems are present in association with HRPE and that the uPAR/uPA system may be particularly important.

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.

Intracellular calcium waves accompany neutrophil polarization, formylmethionylleucylphenylalanine stimulation, and phagocytosis: a high speed microscopy study.

Using high sensitivity fluorescence imaging with shutter speeds approximately 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.

Hexokinase translocation during neutrophil activation, chemotaxis, and phagocytosis: disruption by cytochalasin D, dexamethasone, and indomethacin.

Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophil's cytosol to its periphery in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP). Translocation was observed from 10(-6) to 10(-9)M fMLP. However, fMLP did not affect the intracellular distribution of lactate dehydrogenase. Hexokinase accumulated at the lamellipodium of cells exposured to an fMLP gradient whereas it localized to the phagosome after latex bead uptake. Thus, hexokinase is differentially translocated within cells depending upon the prevailing physiological conditions. Further studies noted that cytochalasin D, dexamethasone, and indomethacin blocked hexokinase translocation. Parallel regulation of reactive oxygen metabolite (ROM) production was shown. We speculate that hexokinase translocation participates in neutrophil activation.