The cytoplasmic domain of FcgammaRIIA (CD32) participates in phagolysosome formation.

Signaling motifs located within the cytoplasmic domain of certain receptors contribute to lysosome fusion. Most studies have described lysosome fusion with respect to endocytic receptors. Phagolysosome fusion has not been extensively studied. To test the hypothesis that the tail of FcgammaRIIA participates in phagolysosomal fusion, a "reverse" genetic complementation system was used. It was previously shown that complement receptor type 3 (CR3) can rescue the phagocytic activity of a mutant FcgammaRIIA lacking its cytoplasmic domain (tail-minus form). This system has allowed us to study Fcgamma receptor-dependent phagocytosis and phagolysosome fusion in the presence and absence of the cytoplasmic domain of FcgammaRIIA. Fluorescent dextran was used to label lysosomes. After target internalization, wild-type FcgammaRIIA-mediated phagolysosome formation was observed as indicated by colocalization of fluorescent dextran and the phagosome. In addition, when studying mutants of FcgammaRIIA containing a full-length cytoplasmic tail with the 2 ITAM tyrosines mutated to phenylalanine, (1) phagocytosis was abolished, (2) CR3 restored phagocytosis, and (3) lysosomal fusion was similar to that observed with the wild-type receptor. In contrast, in the presence of CR3 and the tail-minus form of FcgammaRIIA, internalized particles did not colocalize with dextran. Electron microscopy revealed that the lysosomal enzyme acid phosphatase colocalized with immunoglobulin G-coated targets internalized by wild-type FcgammaRIIA but not by tail-minus FcgammaRIIA and CR3. Thus, the tail of FcgammaRIIA contributes to phagolysosome fusion by a mechanism that does not require a functional ITAM sequence.

Identification of colchicine in placental blood from patients using herbal medicines.

While characterizing natural antiinflammatory substances in human placental blood, we discovered a factor that affected human neutrophils and their adherence. Rigorous chemical and stereochemical analyses revealed this factor to be the well-known alkaloid, colchicine. When samples from individual patients were analyzed, significant levels (49-763 microg/L) of colchicine could be found in placental blood of patients using nonprescription herbal dietary supplements during pregnancy. We confirmed the presence of colchicine in commercially available ginkgo biloba. Due to its potential harmful effects, it would appear that such supplements should be avoided by women who are pregnant or are trying to conceive.

Neutrophil oscillations: temporal and spatiotemporal aspects of cell behavior.

Neutrophil activation is an essential event in inflammatory responses. How cells coordinate, integrate, manage, and distribute information on physiologically-relevant timescales are not well understood. Although neutrophil oscillators have been known for many years, their biological roles have not been identified. We suggest that intracellular oscillators (such as NAD(P)H, pH, calcium, and so on) account for functional oscillations (e.g., superoxide and NO production, cytolytic marker release, pericellular proteolysis, and actin assembly). In addition to these well-known temporal oscillations, we have recently discovered self-organized traveling chemical waves in neutrophils; these waves respond to extracellular signals and have distinct origins that coincide with a cell's uropod, lamellipodium, or adherence site. The fundamental physico-chemical features of cell chemistry will have an increasing role in our understanding of leukocyte function.

Tumor cell invasion of model 3-dimensional matrices: demonstration of migratory pathways, collagen disruption, and intercellular cooperation.

We report a novel 3-dimensional model for visualizing tumor cell migration across a nylon mesh-supported gelatin matrix. To visualize migration across these model barriers, cell proteolytic activity of the pericellular matrix was detected using Bodipy-BSA (fluorescent upon proteolysis) and DQ collagen (fluorescent upon collagenase activity). For 3-dimensional image reconstruction, multiple optical images at sequential z axis positions were deconvoluted by computer analysis. Specificity was indicated using well-known inhibitors. Using these fluorescent proteolysis markers and imaging methods, we have directly demonstrated proteolytic and collagenolytic activity during tumor cell invasion. Moreover, it is possible to visualize migratory pathways followed by tumor cells during matrix invasion. Using cells of differing invasive potentials (uPAR-negative T-47D wild-type and uPAR-positive T-47D A2--1 cells), we show that the presence of the T-47D-A2--1 cells facilitates the entry of T-47D wild-type cells into the matrix. In some cases, wild-type cells follow T-47D A2--1 cells into the matrix whereas other T-47D-wild-type cells appear to enter without the direct intervention of T-47D A2--1 cells. Thus, we have developed a new 3-dimensional model of tumor cell invasion, demonstrated protein and collagen disruption, mapped the pathways followed by tumor cells during migration through an extracellular matrix, and illustrated cross-talk among tumor cell populations during invasion.

Cutting edge: evidence for a signaling partnership between urokinase receptors (CD87) and L-selectin (CD62L) in human polymorphonuclear neutrophils.

Leukocyte urokinase plasminogen activator receptors (uPARs) cluster at adhesion interfaces and at migratory fronts where they participate in adhesion, chemotaxis, and proteolysis. uPAR aggregation triggers activation signaling even though this glycolipid-anchored protein must associate with membrane-spanning proteins to access the cell interior. This study demonstrates a novel partnership between uPAR and L-selectin in human polymorphonuclear neutrophils. Fluorescence resonance energy transfer demonstrated a direct physical association between uPAR and L-selectin. To examine the role of L-selectin in uPAR-mediated signaling, uPAR was cross-linked and intracellular Ca(2+) concentrations were measured by spectrofluorometry. A mAb reactive against the carbohydrate binding domain (CBD) of L-selectin substantially inhibited uPAR-mediated Ca(2+) mobilization, whereas mAbs against the beta(2) integrin complement receptor 3 (CR3), another uPAR-binding adhesion protein, had no effect. Similarly, fucoidan, a sulfated polysaccharide that binds to L-selectin CBD, inhibited the Ca(2+) signal. We conclude that uPAR associates with the CBD region of L-selectin to form a functional signaling complex.

Pulsed DC electric fields couple to natural NAD(P)H oscillations in HT-1080 fibrosarcoma cells.

Previously, we have demonstrated that NAD(P)H levels in neutrophils and macrophages are oscillatory. We have also found that weak ultra low frequency AC or pulsed DC electric fields can resonate with, and increase the amplitude of, NAD(P)H oscillations in these cells. For these cells, increased NAD(P)H amplitudes directly signal changes in behavior in the absence of cytokines or chemotactic factors. Here, we have studied the effect of pulsed DC electric fields on HT-1080 fibrosarcoma cells. As in neutrophils and macrophages, NAD(P)H levels oscillate. We find that weak (approximately 10(-5) V/m), but properly phased DC (pulsed) electric fields, resonate with NAD(P)H oscillations in polarized and migratory, but not spherical, HT-1080 cells. In this instance, electric field resonance signals an increase in HT-1080 pericellular proteolytic activity. Electric field resonance also triggers an immediate increase in the production of reactive oxygen metabolites. Under resonance conditions, we find evidence of DNA damage in HT-1080 cells in as little as 5 minutes. Thus the ability of external electric fields to effect cell function and physiology by acting on NAD(P)H oscillations is not restricted to cells of the hematopoietic lineage, but may be a universal property of many, if not all polarized and migratory eukaryotic cells.

Dissipative metabolic patterns respond during neutrophil transmembrane signaling.

Self-organization is a common theme in biology. One mechanism of self-organization is the creation of chemical patterns by the diffusion of chemical reactants and their nonlinear interactions. We have recently observed sustained unidirectional traveling chemical redox [NAD(P)H - NAD(P)(+)] waves within living polarized neutrophils. The present study shows that an intracellular metabolic wave responds to formyl peptide receptor agonists, but not antagonists, by splitting into two waves traveling in opposite directions along a cell's long axis. Similar effects were noted with other neutrophil-activating substances. Moreover, when cells were exposed to an N-formyl-methionyl-leucyl-phenylalanine (FMLP) gradient whose source was perpendicular to the cell's long axis, cell metabolism was locally perturbed with reorientation of the pattern in a direction perpendicular to the initial cellular axis. Thus, extracellular activating signals and the signals' spatial cues are translated into distinct intracellular dissipative structures.

Mercury inhibition of neutrophil activity: evidence of aberrant cellular signalling and incoherent cellular metabolism.

Exposure to environmental heavy metals has been reported to affect the immune system. Here, we tested the hypothesis that Hg(+2), acting through membrane proteins, disrupts metabolic dynamics and downstream cell functions in human neutrophils. We found that HgCl(2) inhibited: (1) polarization and (2) immunoglobulin (Ig)G-mediated phagocytosis of sheep erythrocytes in a dose-dependent manner from 2.5 to 10 microM. Because these activities have been linked with pro-inflammatory signalling, we also studied the effects of HgCl(2) on intracellular signalling by measuring protein tyrosine phosphorylation. HgCl(2) at doses = 1 microM increased tyrosine phosphorylation. We also studied the effect of HgCl(2) on neutrophil metabolism by measuring NAD(P)H autofluorescence as an indicator of intracellular NAD(P)H concentration. After HgCl(2) treatment, we found that normal sinusoidal NAD(P)H oscillations became incoherent. We recently reported that the NAD(P)H oscillation frequency is affected by cell migration and activation, which can in turn be regulated by integrin-mediated signalling. Therefore, we examined the effects of HgCl(2) on cell surface distribution of membrane proteins. After exposure to environmentally relevant concentrations of HgCl(2) we found that CR3, but not other membrane proteins (e.g. uPAR, Fc gamma RIIA and the formyl peptide receptor), became clustered on cell surfaces. We suggest that HgCl2 disrupts integrin signalling/functional pathways in neutrophils.

Overview of the physical state of proteins within cells.

Proteins, the end product of gene expression, play a pivotal role in cellular structure and function. To understand how proteins work it is necessary to understand their physical state within the cell. This unit reviews the classification of proteins, how that is related to the hydropathicity of the protein, other factors that affect the heterogeneity of proteins, protein assemblies, methods for altering the solubility of proteins, and limitations of in vitro manipulations of proteins.

Interferon-gamma and sinusoidal electric fields signal by modulating NAD(P)H oscillations in polarized neutrophils.

Metabolic activity in eukaryotic cells is known to naturally oscillate. We have recently observed a 20-s period NAD(P)H oscillation in neutrophils and other polarized cells. Here we show that when polarized human neutrophils are exposed to interferon-gamma or to ultra-low-frequency electric fields with periods double that of the NAD(P)H oscillation, the amplitude of the NAD(P)H oscillations increases. Furthermore, increases in NAD(P)H amplitude, whether mediated by interferon-gamma or by an oscillating electric field, signals increased production of reactive oxygen metabolites. Hence, amplitude modulation of NAD(P)H oscillations suggests a novel signaling mechanism in polarized cells.

Lipopolysaccharide induces physical proximity between CD14 and toll-like receptor 4 (TLR4) prior to nuclear translocation of NF-kappa B.

CD14, a GPI-linked protein, plays a pivotal role in LPS-mediated signaling by potentiating leukocyte adherence, activation, and cytokine production. Recent studies have identified the Toll-like receptor 4 (TLR4) as a membrane cofactor in LPS-mediated transmembrane signaling in cytokine induction, although the mechanism responsible for this cooperation is unknown. Using fluorescence resonance energy transfer (RET) techniques, we demonstrate that LPS triggers a physical association between CD14 and TLR4. Because LPS stimulation upregulates CD14 and TLR4 expression, it was necessary to control for the possibility that these newly expressed molecules were associated with one another independent of LPS stimulation. Although the calcium ionophore A23187 increased the expression of CD14 and TLR4, they did not exhibit energy transfer. However, following A23187 treatment, LPS promoted physical proximity between CD14 and TLR4. Therefore, we suggest that a close interaction between CD14 and TLR4 participates in LPS signaling, leading to nuclear translocation of NF-kappaB.

Imaging sustained dissipative patterns in the metabolism of individual living cells.

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.

Mercuric chloride damages cellular DNA by a non-apoptotic mechanism.

Mercury is a xenobiotic metal that is well known to adversely affect the immune system, however, little is known as to the molecular mechanism. Recently, it has been suggested that mercury may induce immune dysfunction by triggering apoptosis in immune cells. Here, we studied the effects of Hg(2+) (HgCl(2)) on U-937 cells, a human cell line with monocytic characteristics. We found that these cells continued to proliferate when exposed to low doses of mercury between 1 and 5 microM. Using the single cell gel electrophoresis (SCGE) or 'comet' assay, we found that mercury damaged DNA at these levels. Between 1 and 50 microM Hg(2+), comet formation was concentration-dependent with the greatest number of comets formed at 5 microM mercury. However, the appearance of mercury-induced comets was qualitatively different from those of control cells treated with anti-fas antibody, suggesting that although mercury might damage DNA, apoptosis was not involved. This was confirmed by the finding that cells treated with 5 microM mercury were negative for annexin-V binding, an independent assay for apoptosis. These data support the notion that DNA damage in surviving cells is a more sensitive indicator of environmental insult than is apoptosis, and suggests that low-concentrations of ionic mercury may be mutagenic.

Ebola virus secretory glycoprotein (sGP) diminishes Fc gamma RIIIB-to-CR3 proximity on neutrophils.

Previous studies have shown that Ebola virus' secretory glycoprotein (sGP) binds to Fc gamma RIIIB (CD16b) and inhibits L-selectin shedding. In this study, we test the hypothesis that sGP interferes with the physical linkage between CR3 and Fc gamma RIIIB. 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 Fc gamma RIIIB 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 Fc gamma RIIIB. In contrast, cells exposed to sGP showed a significant reduction in the CR3-Fc gamma RIIIB RET signal using these methods. Interestingly, colocalization and cocapping of CR3 and Fc gamma RIIIB were not affected, suggesting that the proximity of these two receptors is reduced without triggering dissociation. Thus, sGP alters the physical linkage between Fc gamma RIIIB and CR3.

Extremely low frequency pulsed DC electric fields promote neutrophil extension, metabolic resonance and DNA damage when phase-matched with metabolic oscillators.

Application of extremely low frequency pulsed DC electric fields that are frequency- and phase-matched with endogenous metabolic oscillations leads to greatly exaggerated neutrophil extension and metabolic resonance wherein oscillatory NAD(P)H amplitudes are increased. In the presence of a resonant field, migrating cell length grows from 10 to approximately 40 microm, as does the overall length of microfilament assemblies. In contrast, cells stop locomotion and become spherical when exposed to phase-mismatched fields. Although cellular effects were not found to be dependent on electrode type and buffer, they were sensitive to temporal constraints (phase and pulse length) and cell surface charge. We suggest an electromechanical coupling hypothesis wherein applied electric fields and cytoskeletal polymerization forces act together to overcome the surface/cortical tension of neutrophils, thus promoting net cytoskeletal assembly and heightened metabolic amplitudes. Metabolic resonance enhances reactive oxygen metabolic production by neutrophils. Furthermore, cellular DNA damage was observed after prolonged metabolic resonance using both single cell gel electrophoresis ('comet' assay) and 3'-OH DNA labeling using terminal deoxynucleotidyl transferase. These results provide insights into transmembrane signal processing and cell interactions with weak electric fields.

Immune complex size and complement regulate cytokine production by peripheral blood mononuclear cells.

We have previously shown that immune complexes isolated from children with juvenile rheumatoid arthritis are heterogeneous in their size, composition, and proinflammatory capacities. The experiments described here were undertaken to clarify further the roles of size and composition in determining the proinflammatory effects of immune complexes. We incubated peripheral blood mononuclear cells (PBMCs) with different soluble immune complex preparations: opsonized complexes, which were formed in the presence of serum, unopsonized complexes, which were formed in the absence of serum, and immune precipitates solubilized by complement after their formation. ELISA assays showed that immune complexes formed in the presence of complement were less efficient than unopsonized complexes in inducing IL-1beta and IL-8 secretion from leukocytes. Solubilized immune precipitates showed intermediate capacity to stimulate the release of both cytokines. Complexes formed in heat-inactivated serum were as efficient as unopsonized complexes in eliciting cytokine secretion from the cells. The capacity of complement to regulate cytokine secretion from leukocytes was related, at least in part, to immune complex size. Sucrose density gradients showed unopsonized complexes and solubilized immune precipitates were larger than opsonized immune complexes. In contrast, fluid-phase binding of C4 to immune complexes, which did not appreciably change immune complex size, substantially increased IL-1beta secretion from PBMC.

Amplitude and frequency modulation of metabolic signals in leukocytes: synergistic role of IFN-gamma in IL-6- and IL-2-mediated cell activation.

Many stimuli cause intracellular concentration oscillations of second messengers or metabolites, which, in turn, may encode information in their amplitudes and frequencies. We now test the hypothesis that synergistic cellular responses to dual cytokine exposure correlate with cross-talk between metabolic signaling pathways of leukocytes. Polarized RAW264.7 macrophages and human neutrophils and monocytes exhibited NAD(P)H autofluorescence oscillation periods of congruent with20 s. IFN-gamma tripled the NAD(P)H oscillatory amplitude for these cells. Although IL-6 had no effect, incubation of cells with IFN-gamma and IL-6 increased both oscillatory amplitude and frequency. Parallel changes were noted after treatment with IFN-gamma and IL-2. However, IL-1beta and TNF-alpha did not display frequency doubling with or without IFN-gamma exposure. To determine whether frequency doubling required complete IFN-gamma signaling or simply metabolic amplitude modulation, an electric field was applied to cells at NAD(P)H troughs, which has been shown to enhance NAD(P)H amplitudes. Electric field application led to frequency doubling in the presence of IL-6 or IL-2 alone, suggesting that amplitude modulation is crucial to synergism. Because NADPH participates in electron trafficking to NO, we tested NO production during cytokine exposure. Although IL-6 and IL-2 alone had no effect, IFN-gamma plus IL-6 and IFN-gamma plus IL-2 enhanced NO release in comparison to IFN-gamma treatment alone. When NO production was examined for single cells, it incrementally increased with the same phase and period as NAD(P)H. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy and entrain NO production.

Complement deposition on immune complexes reduces the frequencies of metabolic, proteolytic, and superoxide oscillations of migrating neutrophils.

Neutrophils exhibit intrinsic sinusoidal metabolite concentration oscillations of 3 min in resting cells and an additional approximately 10- or 20-s oscillation in migrating/adhering cells. To better understand immune complex (IC)-mediated leukocyte activation, we have studied neutrophil metabolic oscillations in the presence of ICs either with or without fixed complement. Using a microscope photometer we quantitated NAD(P)H autofluorescence oscillations. Cells exposed to ICs exhibited metabolic oscillation periods of approximately 12 s in the absence of complement and approximately 22 s in the presence of complement opsonization. To determine if the effects could be associated with C3 deposition, we used ICs opsonized with only C3 or only C1 and C4. Untreated ICs, heat-inactivated complement-treated ICs, and C1,C4-treated ICs trigger rapid metabolic oscillations, as do fMLP and yeast; in contrast, ICs treated with full complement or C3 alone did not affect NAD(P)H oscillations in comparison to controls. The induction of higher frequency (approximately 10 s) NAD(P)H oscillations by ICs could be blocked by addition of anti-FcgammaRII, but not FcgammaRIII mAb fragments, suggesting the participation of FcgammaRII in cellular metabolic responses to ICs. Parallel changes in the frequencies of oxidant release and pericellular proteolysis were found for all of these stimuli. Thus, immune complex composition affects both intracellular metabolic signals and extracellular functional oscillations. We suggest that complement attenuates the phlogistic potential of ICs by reducing the frequency of cytoplasmic NAD(P)H oscillations.

Ultrasensitive detection of hydrogen peroxide-mediated DNA damage after alkaline single cell gel electrophoresis using occultation microscopy and TUNEL labeling.

DNA damage at the level of individual cells can be detected using the single cell gel electrophoresis (SCGE) or 'comet' assay. In the present study, we report novel variations on the conventional comet assay that can be used to enhance the microscopic detection of DNA damage. Hydrogen peroxide-treated peripheral blood leukocytes were used as a DNA damage model system. Cells were embedded in agarose, treated, and electrophoresed according to the procedure of Singh et al. [N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988), p. 184-191]. However, sites of strand breaks were directly labeled with the TUNEL (TdT-mediated fluorescein-dUTP nick end labeling) method. This labeling protocol revealed clumps and/or a series of stripes in the comet tail perpendicular to the direction of electrophoresis; these sites may account for the substructure seen in conventional comet assays. In a second comet variation, we passed an opaque disk into a field-conjugated plane of the microscope near the lamp, thus occluding the nucleus' image. Nuclear occultation allows the intensified charge-coupled device (ICCD) camera gain to increase to a single photon detection level thus revealing low levels of DNA damage in the tail. These methods offer a substantial improvement in sensitivity.

Early membrane rupture events during neutrophil-mediated antibody-dependent tumor cell cytolysis.

Although cell-mediated cytolysis is a fundamental immune effector response, its mechanism remains poorly understood at the cellular level. In this report, we image for the first time transient ruptures, as inferred by cytoplasmic marker release, in tumor cell membranes during Ab-dependent cellular cytolysis. The cytosol of IgG-opsonized YAC tumor cells was labeled with tetra-methylrhodamine diacetate followed by the formation of tumor cell-neutrophil conjugates. We hypothesized that tumor cell cytolysis proceeds via a series of discrete membrane rupture/resealing events that contribute to marker release. To test this hypothesis, we occluded the fluorescence image of the labeled tumor cells by passing an opaque disk into a field-conjugated plane between the light source and the sample. Multiple small bursts of fluorescent label release from tumor cells could be detected using a photomultiplier tube. Similarly, multiple fluorescent plumes were observed at various sites around the perimeter of a target. These findings support a multihit model of target cytolysis and suggest that cytolytic release is not focused at specific sites. Cytolytic bursts were generally observed at 20-s intervals, which match the previously described reduced nicotinamide-adenine dinucleotide phosphate and superoxide release oscillation periods for neutrophils; we speculate that metabolic oscillations of the effector cell drive the membrane damage of the target.