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in IL-6- and IL-2-Mediated Cell Activation1
*
Tokyo University of Pharmacy and Life Science, Tokyo, Japan; and
Department of Biological Sciences, Wayne State University, Detroit, MI 48202
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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20 s. IFN-
tripled the NAD(P)H oscillatory amplitude for these
cells. Although IL-6 had no effect, incubation of cells with IFN-
and IL-6 increased both oscillatory amplitude and frequency. Parallel
changes were noted after treatment with IFN- and IL-2. However,
IL-1ß and TNF-
did not display frequency doubling with or without
IFN- exposure. To determine whether frequency doubling required
complete IFN- 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- plus IL-6 and IFN- plus IL-2 enhanced NO release in
comparison to IFN- 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have proposed that amplitude and frequency modulation of intracellular metabolite (e.g., NADPH) concentrations may encode signaling information (11, 12, 13). We have speculated, for certain energy-demanding leukocyte functions (e.g., migration and adherence), that cell metabolite flux is the chemical signal for a cell function to appear while the associated kinases and phosphatases are the conduit through which the signal passes (11). For example, NADPH oxidase phosphorylation may "tune" the oxidase’s Kd to intercept the "signal" metabolic oscillations, which are then decoded into a series of superoxide bursts (12). Superoxide oscillations may, in turn, explain the multiple, periodic cytolytic events observed during cell-mediated cytotoxicity (13). This interpretation is in agreement with the finding that NAD(P)H oscillations are in-phase with oxidative oscillations (12) and that superoxide production follows NAD(P)H amplitude and frequency changes (12, 14). Thus, neutrophil metabolic oscillations may be linked to cellular functions such as kinase/phosphatase pathways, oxidant production, pericellular proteolysis, cytolysis, and microfilament extension (11, 12, 13). Furthermore, neutrophils optimally detect time-varying chemical fields displaying this same period (15). Cells are also capable of detecting external electric fields when frequency and phase matched with intracellular metabolic oscillators (16). If these oscillators are a central element in cell function, it should be possible to identify patients with abnormal metabolic oscillations. Indeed, neutrophils from pediatric-onset pyoderma gangrenosum patients display aberrations in intracellular oscillators and motility or shape that can be ameliorated by pharmacologic or physical perturbations that restore sinusoidal oscillations (17, 18). Hence, leukocyte metabolic clocks may contribute to cell signaling and drive certain functions.
This study explores the mechanism of cytokine synergy in cell activation. Although cytokine cooperation is well-known (19, 20), the mechanisms responsible for processing multiple signals is not. We show that prior enhancement of oscillatory metabolic amplitudes participates in IL-6- and IL-2-mediated metabolic frequency doubling (i.e., activation) effect on cells. Furthermore, metabolic oscillatory changes parallel downstream physiologic changes in NO production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine and human cytokines were obtained from R&D Systems (Minneapolis, MN). FMLP, PxB,3 LPS (Escherichia coli serotype 026:B6), and cycloheximide were obtained from Sigma (St. Louis, MO). Diaminofluorescein-2 diacetate (DAF-2 DA) was obtained from Daiichi Kagaku Yakuhin (Tokyo, Japan).
Cell culture
RAW264.7 macrophages were grown in RPMI 1640 containing 10% FCS and 1% PSA (penicillin G/streptomycin/amphotericin B) (Life Technologies, Grand Island, NY). For spectrophotometric assays, cells were grown in 24-well plates. For microscopy experiments, cells were grown for 24 h attached to glass coverslips.
Leukocyte isolation
Peripheral blood monocytes and neutrophils were obtained using two Ficoll-Hypaque solutions of different buoyant densities (Histopaque 1077 and 1119; Sigma) and centrifugation. Cells were washed twice by centrifugation and then resuspended in HBSS (Life Technologies). Trypan blue staining indicated that 95–99% of the cells were viable.
Spectrophotometric assay for NO
Macrophages (106/ml) were placed on
culture plates and treated with recombinant murine IFN- at 10 U/ml
and with 1000 U/ml IL-2 or 25 ng/ml of recombinant murine IL-6,
IL-1ß, or TNF- for 24 h. Cell-free culture supernatants were
collected for NO measurement. NO release was determined by assaying
supernatants for nitrite content. Briefly, 40 µl of cell-free
supernatant was reacted for 10 min at room temperature with an equal
volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene
diaminedihydrochloride, 2.5% phosphoric acid) as described
(21). Optical densities were measured at 540 nm.
Nitrite content was quantitated by comparison with a standard curve
generated using sodium nitrite (0–100 µM).
Electrode configuration
Electric fields were applied using two parallel platinum electrodes or Ag/AgCl electrodes (MF-2063; Bioanalytical Systems, West Lafayette, IN) as described for optical microscopy (18, 22, 23). A power supply (Grass Medical Instruments, Quincy, MA) was used to apply a pulsed square wave DC electric fields at 2 x 10-3 V/m. Electric field intensities were determined by measuring the current (23) using an electrometer (model 6517A; Keithley Instuments, Cleveland, OH). Pulse application was performed manually to coincide with the trough in NAD(P)H autofluorescence intensity for each cell under study.
Microscopic assay for NO production
Single-cell NO production experiments were performed using 2% gelatin matrices, similar to that previously described (12). Fluid-phase gelatin was mixed with 15 µM DAF-2 DA at 45°C then allowed to cool to 37°C, where it is a semisolid. DAF-2 DA has been previously shown to become fluorescent upon exposure to NO, but not other reactive species such as superoxide and hydrogen peroxide (24).
Microscopy
Cells were examined using an axiovert fluorescence microscope (Carl Zeiss, New York, NY) with mercury illumination and a quartz epifluorescence condenser interfaced to a Perceptics Biovision system (Knoxville, TN) (11). Quantitative experiments were performed using a 40x objective to provide depth of field and a 37°C stage. NAD(P)H autofluorescence was detected using 365DF20 and 405DF35 filters and a 405 long-pass dichroic mirror (11). Fluorescence levels were quantitated using a Hamamatsu (Bridgewater, NJ) photomultiplier tube held in a Products for Research (Danvers, MA) housing coupled to a microscope (11). Cells were illuminated individually. Background photon count rates were taken from a neighboring area that contained no cells. A Mac 9600 computer and MacLab system were used to record and analyze kinetic changes in fluorescence levels.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IFN- enhances oscillatory NAD(P)H amplitudes
Cell illumination at 
360 nm leads to autofluorescence emission
(>400 nm) that is largely accounted for by pyridine nucleotides (NADH
+ NADPH) (25, 26). This physical attribute of NAD(P)H has
been variously exploited to monitor metabolic oscillations (e.g.,
3, 9, 10, 11, 12, 14, 16, 17, 18). We have employed this strategy as a
nonperturbative method to study metabolic signaling in response to
cytokine stimulation.
Previous studies have suggested that IFN-, IL-6, IL-2, IL-1ß, and
TNF- can prime or activate cell functions, often in synergy with
other ligands (e.g., 19, 20, 27, 28, 29, 30, 31, 32). Enhanced leukocyte
function is presumably mediated by chemical events downstream from
receptor ligation, which includes metabolic responses. To test the
effects of cytokines on metabolic oscillations, RAW264.7 macrophages
were treated with various cytokines (Table I
). In contrast to neutrophil-activating
substances such as FMLP, immune complexes, and yeast (11, 14, 33), these murine cytokines did not influence NAD(P)H
oscillation periods (Table I). Although IL-6, IL-2, IL-1ß, and
TNF- did not affect NAD(P)H oscillations, cell exposure to IFN-
did significantly increase the amplitude of metabolic oscillations
(p < 0.001; line 8 vs line 3 in Table I).
IFN- dose-response studies were conducted from 0–25 U/ml. A dose of
12.5 U/ml was found to be sufficient to induce a maximal change in
NAD(P)H amplitude. However, as previously observed for other
IFN--mediated changes in cell physiology (19, 32, 34),
this required an incubation time of 4 h at 37°C. Thus, IFN-
affects the amplitude of metabolic oscillations in RAW264.7 cells.
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-mediated NAD(P)H amplitude modulation.
Cells were first exposed to IFN- for 3.5 h in an incubator then
transferred to a microscope stage at 37°C. As shown in Fig. 1A and Table I
, IFN-
dramatically increased the amplitude of NAD(P)H oscillations. These
amplitude changes progressed quickly to a steady-state level. However,
when cycloheximide (0.5 µg/ml) was added with IFN-, no changes in
amplitude were observed (Table I). Thus, protein synthesis appears to
be required for NAD(P)H amplitude modulation by IFN-, which is also
consistent with the 4 h incubation time required for these
changes.
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primes macrophages for IL-6- and IL-2-mediated metabolic
frequency doubling
Our previous studies (11, 12, 13, 14, 33) suggest that
metabolic frequency doubling is a hallmark of leukocyte activation. For
example, FMLP, immune complexes, and ß-glucans increase metabolic
oscillation frequency, whereas certain antiinflammatory agents prevent
these changes. Although frequency changes were not observed when cells
were incubated with murine IL-6, IL-2, IL-1ß, or TNF- alone, we
hypothesized that signaling events associated with dual cytokine
exposure could synergize to yield outputs that neither cytokine alone
possessed. This is consistent with numerous studies illustrating
cytokine synergy (e.g., 19, 20). Because previous studies
established that IFN- primes leukocytes (e.g., 31), we
first exposed cells to IFN- (4 h at 15 U/ml) followed by the
addition of a second cytokine. The addition of IL-6 to IFN--treated
macrophages led to frequency doubling within 3 min (Fig. 1A,
Table I). Similarly, exposure of IFN--primed cells to IL-2 (50
ng/ml) led to metabolic frequency doubling within 3 min (Fig. 2A, Table I). Specificity is
suggested by the fact that IL-1ß and TNF- had no effect on
IFN--primed macrophages (Table I). Thus, IFN- and IL-6 or IFN-
and IL-2 synergize to increase the amplitude and frequency of NAD(P)H
oscillations in macrophages.
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and LPS can synergistically
enhance NO production by macrophages (e.g., 20, 32).
Therefore, we examined the effect of LPS on NAD(P)H oscillations. LPS
(100 ng/ml) doubled the frequency of metabolic oscillations without
effecting the oscillation’s amplitude (Table I). This behavior is
similar to that of FMLP and immune complexes (11, 12, 14, 33). Although treatment with IL-6 or IL-2 alone did not effect
metabolic oscillations, we were concerned that subthreshold LPS
contamination in the cytokine preparations may have synergistically
interacted with the IFN--treated macrophages to double the metabolic
oscillation frequency. To deflect this concern, we included PxB (2
µg/ml) in these assays. PxB blocks the frequency doubling effect of
LPS on cell metabolism (Table I). Moreover, PxB inclusion in the
presence of IFN- and IL-6 or IFN- and IL-2 had no effect on
metabolic amplitude or frequency (data not shown). Thus, LPS
contamination cannot explain the frequency doubling effects of IL-6 and
IL-2 on NAD(P)H oscillations.
IFN- primes human leukocytes for IL-6- and IL-2-mediated
metabolic frequency doubling
The studies described above show that IFN- increases the
amplitude of NAD(P)H oscillations in RAW264.7 macrophages and that
IFN- can synergize with certain cytokines to double metabolic
oscillation frequency. Although alterations in metabolic amplitude and
frequency have been noted in human neutrophils (11, 12, 14, 16, 17, 18), it remains possible that these synergistic cytokine
effects on metabolic signaling are limited to this transformed cell
line. To test this possibility, we exposed human neutrophils and
monocytes to human IFN-, IL-6, and/or IL-2. Human cytokines were
employed in this system because murine IL-6 has no effect on human
leukocytes (30). Fig. 3
shows a series of experiments illustrating the effects of cytokine
exposure on metabolic oscillations of neutrophils and monocytes.
Polarized neutrophils and monocytes show NAD(P)H autofluorescence
oscillations of 20 s that are not inflluenced by the addition of
IL-6 (25 ng/ml) or IL-2 (50–100 ng/ml). However, exposure to IFN-
(30 µg/ml) for >4 h led to increased metabolic oscillation
amplitudes. Subsequent treatment of IFN--primed cells with IL-6 or
IL-2 led to high-amplitude, high-frequency metabolic oscillations.
Thus, cytokine synergy is associated with metabolic cross-talk in
multiple cell types.
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Although the above studies correlate IFN--mediated amplitude
modulation with IL-6- and IL-2-mediated frequency doubling, it is
unclear whether these changes require amplitude modulation or an
independent parameter whose expression simply parallels that of NAD(P)H
amplitude modulation. To address this issue, we sought to heighten
NAD(P)H amplitudes by independent means. This was accomplished by
exposing macrophages to a frequency and phase-matched electric field
that heightens NAD(P)H oscillatory amplitudes in the absence of
receptor ligation (12, 14, 16). Pulsed DC electric fields
(2 x 10-3 V/m with 20 ms duration) were applied
to cells at troughs of NAD(P)H autofluorescence intensity (12, 14, 16). Data are shown in Fig. 1B and Table I. As
Fig. 1B shows, the NAD(P)H oscillatory amplitude markedly
increases in the presence of an appropriate phase-matched electric
field; maximal peak height is reached within <2 min. About 2–3 min
after IL-6 addition, the metabolic frequency doubles (
10
s). When the electric field is terminated, the high-amplitude,
high-frequency oscillations rapidly revert to the low-amplitude,
low-frequency state. Importantly, these parallel amplitude/frequency
modulation events are reversible. As shown later in this same trace,
when the electric field is again applied to this same cell, the
frequency doubling reappears immediately. Similar findings were
observed for all cells. Moreover, exposure of cycloheximide-pretreated
cells to electric fields elicited the same changes in NAD(P)H amplitude
and IL-6-dependent frequency alterations (Table I). Parallel
experiments using IL-2 and electric field application yielded
indistinguishable results (Fig. 2B, Table I). Electric field
and IFN--mediated NAD(P)H amplitude modulation differ in both their
induction period and protein synthesis dependence. We suggest that
metabolic amplitude modulation, in the absence of IFN- receptor
ligation or altered gene expression, is sufficient to synergize with
IL-6 or IL-2 to elicit metabolic frequency doubling.
IFN- and IL-6 or IL-2 synergize to increase macrophage NO
production
To test the physiological role of amplitude and frequency changes,
we measured NO production by RAW264.7 macrophages in response to
various cytokine treatments. Experiments were conducted in the presence
and absence of PxB to control for the potential effect of LPS
contamination. LPS (100 ng/ml) induced NO production by macrophages,
but was blocked by PxB (2 µg/ml) (Table II). The murine cytokines IL-6, IL-1ß,
and IL-2, when incubated with macrophages, had no significant effect on
NO production as judged by nitrite formation (Table II). However,
IFN- (10 U/ml) increased NO release to 11.3 ± 0.3 µM in the
presence of PxB; this confirms the fact that IFN- is responsible for
increased NO production. Cell exposure to IL-1ß or TNF- after
treatment with IFN- gave results similar to that of IFN- alone
when PxB was included during cell treatment (Table II). In contrast,
IL-6 and IL-2 were found to synergize with IFN- to potentiate NO
release (Table II), which could not be explained by LPS contamination.
Thus, certain cytokine combinations can synergize to enhance NO
release.
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The studies described above correlate: 1) IFN--mediated
increases in NAD(P)H amplitude with heightened NO production and 2)
IL-6- and IL-2-mediated metabolic frequency doubling with enhanced NO
production. To more closely link metabolic oscillations with NO
production, we assessed NO production by single cells. Cells were
observed on microscope slides at 37°C surrounded by a gelatin matrix
containing DAF-2 DA, which reports local NO availability. The gel,
which resembles an extracellular matrix, limits the extent of probe
diffusion during experimentation. Microfluorometric observations of
IFN- (15 U/ml, 4 h)- and IL-6 (25 ng/ml)-treated cells revealed
a stepwise increase in NO-mediated accumulation of pericellular
fluorescence (Fig. 4A,
trace c). However, treatment of cells with buffer alone
caused little or no NO production (Fig. 4A, trace
a; Fig. 4B, trace a). Exposure to IFN-
alone (Fig. 4A, trace b; Fig. 4B,
trace b) led to significant NO production, although at a
lower frequency than that of IFN-- and IL-6-treated cells.
Similarly, NO release doubling was also observed for cells treated with
IFN- (15 U/ml, 4 h) and IL-2 (50 ng/ml) (Fig. 4B,
trace c). Thus, the combination of IFN- with either IL-6
or IL-2 leads to heightened NO release. Moreover, the frequency of NO
release matches that previously reported for neutrophil NAD(P)H and
superoxide production oscillations (12).
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C, trace a),
IFN- (15 U/ml, 4 h) did have a small effect on local NO release
(Fig. 4C, trace b). As noted above for RAW264.7
cells, exposure to IFN- (15 U/ml, 4 h) and IL-6 (25 ng/ml) led
to increased NO release (Fig. 4C, trace c).
However, due to the fact that normal leukocytes produce smaller
quantities of NO than RAW264.7 cells, the traces have a lower intensity
and higher noise level. Relative phases of NAD(P)H oscillations and NO release revealed by single-cell measurements
To further link NAD(P)H oscillations with NO release, we
simultaneously followed NAD(P)H autofluorescence and NO release using
the techniques described above. These measurements were made by rapidly
switching the interference filters between the set-ups for NAD(P)H
autofluorescence and NO fluorescence emission detection, resulting in
the staggered appearance of the data (Fig. 5). Data are shown for IFN- (15 U/ml,
4 h)-treated (Fig. 5A) and IFN- (15 U/ml, 4 h)
plus IL-6 (25 ng/ml)-treated (Fig. 5B) RAW264.7 cells.
Parallel observations of NAD(P)H and NO production oscillations from
the same cells were recorded. Both NAD(P)H autofluorescence and NO
release were observed at the same frequency for cells under both
conditions. Moreover, the increases in DAF-2 DA fluorescence were
observed near NAD(P)H oscillation peaks for both IFN- (15 U/ml,
4 h) (Fig. 5A), IFN- plus IL-6 (Fig. 5B),
and IFN- plus IL-2 (data not shown) treatments. Thus, the phase of
NO production increments appear to parallel the points of maximal
NAD(P)H concentrations.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/IL-6 and IFN-/IL-2 synergisms leading
to enhanced NO production. Combined metabolic amplitude and frequency
modulation represents a novel ligand information processing mechanism
and may explain emergent physiological outcomes.
Recently, IFN-’s mechanism of gene activation, via
receptor-associated Janus kinase-mediated tyrosine phosphorylation of
STAT proteins and STAT protein translocation to the nucleus, has been
described (36, 37). However, the mechanisms linking the
IFN--responsive genes to phenotypic changes in cell behavior are
uncertain. This signaling pathway leads to leukocyte priming for
oxidant production and increased phagocytosis and tumoricidal activity
(31, 38, 39). Inasmuch as a protein synthesis-dependent
4-h incubation period with IFN- is required for metabolic amplitude
modulation, changes in gene expression are likely required for this
process. We predict that metabolic pathways are affected by
IFN--mediated changes in gene expression. Metabolic amplitude
modulation may account for certain phenotypic properties of
IFN--treated cells. We have previously shown that enhanced NAD(P)H
amplitudes parallel heightened neutrophil spreading, extension, and
superoxide production (12, 14, 16). Thus, when the NADPH
oxidase is "tuned" to the NADPH oscillatory region, the enhanced
NADPH levels lead to greater oxidant production. NADPH amplitude
modulation may be the physico-chemical mechanism responsible for the
priming effects of IFN- (e.g., 31, 39). Greater levels
of oxidant production may, in turn, explain the enhanced tumoricidal
activity of neutrophils and macrophages. Inasmuch as ATP oscillates at
the same frequency, but 180° out-of-phase with NAD(P)H
(26), a similar argument may be relevant to
IFN--enhanced ATP-driven functions such as phagocytosis. Thus,
metabolic amplitude modulation may provide the biochemical mechanism
responsible for certain priming and effector functions of IFN-.
IL-6- and IL-2-mediated frequency doubling was dependent upon IFN-
dose, IFN- incubation time, and protein synthesis. Although
IFN--mediated metabolic amplitude modulation accompanies IL-6- and
IL-2-mediated frequency doubling, these experiments alone cannot
establish amplitude modulation as a sufficient condition. We have found
that application of electric fields to cells at troughs of NAD(P)H
autofluorescence leads to enhanced NAD(P)H oscillatory amplitudes of
untreated cells (14, 16). Therefore, we employed this
strategy to alter NAD(P)H amplitudes in the absence of IFN-,
receptor ligation, or its inducible gene products. We have shown that
an applied electric field is able to substitute for IFN- treatment.
That is, heightened NAD(P)H amplitudes are sufficient to reconstitute
IL-6- and IL-2-mediated frequency doubling. These changes were observed
regardless of the order of IL-6 or IL-2 addition with respect to
electric field application and were reversible by switching the
electric field on and off (Figs. 1B and 2B). We
speculate that metabolic amplitude modulation is a key step in
IFN--mediated priming for IL-6- and IL-2-mediated metabolic
signaling.
The mechanism(s) used by the IL-6 and IL-2 receptors to detect prior
IFN- amplitude modulation is not known. However, a mechanism similar
to that we have proposed for integrin/NADPH oxidase activation may be
operating (11, 14). Briefly, the concentration of a
substrate (e.g., NADPH or ATP) draws near the
Kd of an important regulatory
component, such as a receptor-associated kinase, thus allowing receptor
function (such as metabolic signaling and NO production (Fig. 4)) to
appear.
A physical characteristic of IL-6- and IL-2-mediated cell activation is
metabolic frequency doubling. Similar responses have been observed for
activating stimuli such as FMLP, immune complexes, ß-glucans, and
yeast (11, 14, 33). One physiological response
accompanying NAD(P)H frequency doubling in response to dual cytokine
exposure is a heightened NO production (Table II). Synergistic
cytokine-mediated NO production enhancement was observed with IL-6 and
IL-2; cytokines that did not trigger frequency doubling had no effect.
Although this correlation is rigorous, it remains possible that all
cells experience metabolic frequency doubling while only a small
fraction of the cells experience heightened NO production. To deflect
this concern, we evaluated NO production at the single-cell level. We
found an incremental increase in NO production by individual cells that
increased in parallel with NAD(P)H frequency (Fig. 4).
As noted above, Cox et al. (20) have observed a
synergistic effect between IFN- and IL-2 that leads to a doubling of
reactive nitrogen intermediate production and a dramatic increase in
cell-mediated cytotoxicity. Although TNF- may play a role in
enhanced leukocyte function (20), TNF- alone and
TNF- plus IFN- did not effect metabolic signaling in leukocytes.
This suggests that TNF- acts downstream from metabolic
signaling.
We suggest that the increases in metabolic frequency and NO production
increments are mechanistically related. The formation of NO and
citrulline is catalyzed by NO synthase in the presence of NAD(P)H and
molecular oxygen (40, 41). Because our buffers contained
dissolved oxygen, we speculate that electron flux from NAD(P)H is rate
limiting; thus, increasing the substrate oscillation frequency
increases the NO production rate yielding the enhancement (IFN- vs
IFN- plus IL-6 or IFN- plus IL-2). Importantly, the approximate
phase alignment of NAD(P)H and NO production is consistent with an
oscillatory metabolic apparatus entraining NO release (11, 12, 14, 16).
A potential implication of these studies concerns the mechanism of
electric field-to-cell interactions. Our studies indicate that
application of a low-frequency pulsed DC electric field mimics the
NAD(P)H amplitude modulation effects obtained for IFN--primed cells.
Similarly, both exposure to an appropriate phase-matched electric field
and IFN- lead to enhanced assembly of microfilaments (16, 42). The synergism of electric fields and IL-6 may cause
reactive nitrogen and oxygen species production and DNA damage in vivo,
which may be relevant to biological effects of electromagnetic
fields.
Recently, there has been a great deal of interest in the potential role
of biological oscillators as signaling devices (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17).
This study extends the field to include cytokine-mediated signaling by
providing a potential mechanisms for IFN--mediated phenotypic
changes and IFN-/IL-6 and IFN-/IL-2 signaling synergy in
leukocytes. This approach may provide a means of simplifying the
bewildering array of cytokine interactions and dissecting their
mechanisms of action.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Howard R. Petty, Department of Biological Sciences, Wayne State University, Detroit, MI 48202. E-mail address:
3 Abbreviations used in this paper: PxB, polymyxin B; DAF-2 DA, diaminofluorescein-2 diacetate.
Received for publication February 18, 1999. Accepted for publication July 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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xß2; CD11c/CD18) oscillations on neutrophils in a mild form of pyoderma gangrenosum. J. Invest. Dermatol. 111:101.
-dependent production of reactive nitrogen intermediates mediates IFN- plus IL-2-induced murine macrophage tumoricidal activity. J. Immunol. 149:3290.[Abstract]
-treated macrophages. J. Immunol. 158:897.[Abstract]
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 A. Rosenspire, A. Kindzelskii, and H. Petty Pulsed DC electric fields couple to natural NAD(P)H oscillations in HT-1080 fibrosarcoma cells J. Cell Sci., January 4, 2001; 114(8): 1515 - 1520. [Abstract] [PDF]
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H. R. Petty and A. L. Kindzelskii Dissipative metabolic patterns respond during neutrophil transmembrane signaling PNAS, March 13, 2001; 98(6): 3145 - 3149. [Abstract] [Full Text] [PDF]
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