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Volume 17, Number 3,
Issue of February 1, 1997
pp. 975-982
Copyright ©1997 Society for Neuroscience
Tumor Necrosis Factor Enhances the Capsaicin Sensitivity of Rat
Sensory Neurons
Grant D. Nicol1,
John
C. Lopshire2, and
Carl M. Pafford2
1 Department of Pharmacology and Toxicology and
2 Medical Neurobiology Program, School of Medicine, Indiana
University, Indianapolis, Indiana 46202-5120
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The capacity of the proinflammatory cytokines, tumor necrosis
factor 
(TNF) and interleukin 1
(IL-1), to modulate the sensitivity of isolated sensory neurons grown in culture to the excitatory chemical agent capsaicin was examined. Alterations in
capsaicin sensitivity were assessed by quantifying the number of
neurons labeled with cobalt after exposure to capsaicin and by
recording the whole-cell response from a single neuron to the focal
application of capsaicin. A 24 hr pretreatment of the neuronal cultures
with TNF (10 or 50 ng/ml), but not IL-1 (10 or 50 ng/ml), produced a concentration-dependent increase in the number of
cobalt-labeled neurons after exposure to 100 nM capsaicin.
The peak increase in the number of labeled neurons was attained after a
4 hr treatment with 10 ng/ml TNF. Similarly, pretreatment with
TNF (10 ng/ml for 4, 12, and 24 hr) produced a greater than twofold
increase in the average peak amplitude of the inward current evoked by 100 nM capsaicin. Both the TNF-induced increase in
labeling and current amplitude were blocked by treating the neuronal
cultures with indomethacin before the addition of TNF. Enhancement
of the capsaicin-evoked current also was blocked by the specific cyclo-oxygenase-2 inhibitor SC-236. These results indicate that TNF
can enhance the sensitivity of sensory neurons to the excitation produced by capsaicin and that this enhancement likely is mediated by
the neuronal production of prostaglandins. Isolated sensory neurons
grown in culture may prove to be a useful model system in which to
explore how prolonged exposure to mediators associated with chronic
inflammation alter the regulatory pathways that modulate the
excitability of the nervous system.
Key words:
tumor necrosis factor ;
interleukin 1;
capsaicin;
sensitization;
prostaglandins;
cyclo-oxygenase-2;
membrane
excitability
INTRODUCTION
Tumor necrosis factor and interleukin 1
serve as potent intermediaries between tissue injury and the resulting
physiological indices of inflammation, such as neutrophil activation,
plasma extravasation, and vasodilatation (Dinarello, 1987
, 1991; Le and Vil
ek, 1987; Kimball, 1991). In rheumatoid arthritis, a well characterized pathology exemplifying the effects of chronic
inflammation, the levels of tumor necrosis factor (TNF) and
interleukin 1 (IL-1) are elevated in synovial fluid (Dayer and
Demczuk, 1984; Arend and Dayer, 1990; Feldman et al., 1990). In animal
models of synovitis, injection of TNF or IL-1 into the joints
produces many of the symptoms observed with rheumatoid arthritis (Arend and Dayer, 1990). The responses associated with inflammation include a
heightened sensitivity to painful stimuli, a condition known as
hyperalgesia (Treede et al., 1992). Indeed, in animal models of
peripheral hyperalgesia, the injection of IL-1 or TNF lowers the
response threshold to noxious stimulation (Ferreira et al., 1988;
Schweizer et al., 1988; Follenfant et al., 1989; Cunha et al.,
1992).
Although the cellular mechanisms whereby TNF and IL-1 enhance the
sensitivity to noxious stimuli are unknown, this sensitization may
involve prostaglandins. Both TNF and IL-1 enhance the release of
arachidonic acid and the synthesis of eicosanoids, especially prostaglandin E2 (PGE2; Dayer et al., 1985),
via the induction of phospholipase A2 and cyclo-oxygenase
activities, respectively (Chang et al., 1986; Burch et al., 1988; Raz
et al., 1988; Burch and Tiffany, 1989). The cytokine-induced release of
PGE2 may sensitize cells and thus potentiate the response
to other inflammatory agents, such as bradykinin (Higgs et al., 1984;
Salmon and Higgs, 1987; Smith, 1992). Indeed, previous injection of
PGE2 into a rat's hind paw decreases the withdrawal time
in response to noxious stimulation (Ferreira et al., 1978). Similarly,
in single unit recordings from sensory nerves, pretreatment with
PGE2 increases the firing activity evoked by either
bradykinin (Handwerker, 1976; Mense, 1981) or mechanical stimulation
(Pateromichelakis and Rood, 1982; Heppelmann et al., 1985).
Furthermore, in isolated sensory neurons grown in culture, the number
of action potentials elicited by either elevated potassium
concentration or focally applied bradykinin is increased after
pretreatment with PGE2 (Baccaglini and Hogan, 1983; Nicol
and Cui, 1994). Thus, the sensitizing actions of PGE2 are
directly on the sensory neuron.
Therefore, to determine whether the enhanced sensitivity produced by
the proinflammatory cytokines TNF or IL-1 results from a direct
action on the sensory neurons or via a secondary mediator, we examined
the excitation produced by capsaicin in embryonic rat sensory neurons
grown in culture as a measure of neuronal sensitivity. Capsaicin was
used because it selectively stimulates most, but not all, C-fibers and
some A
fibers, the neurons associated with nociceptive signaling and
neurogenic inflammation (Holzer, 1991). Capsaicin activates a
nonselective cationic channel that gives rise to an inward current
(Heyman and Rang, 1985; Marsh et al., 1987; Bevan and Forbes, 1988;
Wood et al., 1988). Activation of this channel by capsaicin in the
presence of extracellular cobalt results in the labeling of sensory
neurons; therefore, putative nociceptive neurons can be distinguished
from other neurons in a given population (Wood et al., 1988; Hingtgen
and Nicol, 1994). In this report we demonstrate that pretreatment with
TNF, but not IL-1, increased the number of neurons labeled with
cobalt after exposure to capsaicin. Furthermore, the peak amplitude of the capsaicin-evoked current produced by submaximal concentrations was
enhanced after treatment with TNF. These findings suggest that
TNF can directly enhance the sensitivity of sensory neurons to
excitatory chemical agents.
MATERIALS AND METHODS
Isolation and culture of embryonic rat sensory neurons.
The procedures for isolation and culture of rat sensory neurons
have been described previously (Vasko et al., 1994). Briefly, the
dorsal root ganglia (DRG) from E15-E17 fetal rats were dissected free and placed in a dish containing sterile calcium-free, magnesium-free HBSS at 4°C. The DRGs were incubated in HBSS containing 0.025% trypsin for 25 min at 37°C. The digestion was terminated with the
addition of 0.25% trypsin inhibitor; then the cells were washed and
centrifuged. Ganglia were washed once with HBSS and then resuspended in
DMEM (Life Technologies, Grand Island, NY) supplemented with 2 mM glutamine, 50 µg/ml penicillin and streptomycin, 10%
heat-inactivated fetal bovine serum, 50 µM
5-fluoro-2
-deoxyuridine, 150 µM uridine, and 250 ng/ml
7S-nerve growth factor (Harlan Bioproducts for Science, Indianapolis,
IN). Individual cells were obtained by mechanical agitation with a
fire-polished pipette until a cloudy suspension was observed. For the
cobalt-labeling studies, ~300,000 cells were plated into each well
(35 mm diameter) of a six well Falcon culture dish coated with
poly-D-lysine (100 µg/ml in sterile water). For the
electrophysiological recordings, ~300,000 cells were plated in a
collagen-coated culture dish containing small plastic coverslips. Cells
were grown at 37°C in a 5% CO2 atmosphere, and the
medium was changed every 2 d. These procedures have been approved
by the Animal Care and Use Committee of Indiana University School of
Medicine.
Cobalt labeling of sensory neurons. These experiments were
performed via methods that have been described previously (Hingtgen and
Nicol, 1994). Briefly, after 5 d in culture, cells were washed with normal Ringer's solution of the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH at 7.4 with NaOH. Then
cells were exposed to Ringer's solution containing 100 nM
capsaicin and 10 mM CoCl2 for 8 min. The
capsaicin-cobalt Ringer's solution was removed, the cells were washed
with Ringer's solution, and then the cells were exposed to Ringer's
solution containing 1% ammonium sulfide for 5 min. After the cobalt
precipitation, cells were washed with PBS (100 mM
NaH2PO4 and 155 mM NaCl, pH 7.4)
and fixed with 4% paraformaldehyde in PBS for 20 min. The cobalt
staining was enhanced by incubating the cells in a developer solution
(15 mM hydroquinone, 38 mM citric acid, and 280 mM sucrose) for 15 min at 60°C. Then the cells were
exposed to developer containing 0.1% (w/v) AgNO3 for ~30
min at 60°C. The intensity of the staining was inspected visually,
and enhancement was stopped by washing the cells with fresh developer.
Neuronal staining was determined by counting the number of positive
(dark-brown staining) and negative (clear or yellowish) cells in five
random fields per well (~80-100 cells per field) from dishes of
neuronal cultures established on at least three different days.
To examine the effects of TNF or IL-1 on the capsaicin
sensitivity of these sensory neurons, we pretreated cell cultures with
various concentrations of these agents for various times before
exposure to capsaicin (day 4 in culture). Untreated cultures obtained
from the same neuronal harvest served as the control cells for both the
labeling and electrophysiological studies. In those experiments
examining the effects of cyclo-oxygenase inhibition on the actions of
TNF, neuronal cultures were treated with either indomethacin or
SC-236 (a specific cyclo-oxygenase-2 inhibitor) for 1 hr before the
addition of TNF. To explore the effects of carba prostacyclin
(CPGI2, a nonhydrolyzable analog of prostacyclin or
prostaglandin I2) alone or in combination with TNF on
the capsaicin-cobalt loading, cells were exposed to 10 nM
CPGI2 or 10 nM CPGI2 and 10 ng/ml
TNF for 24 hr before exposure to capsaicin and cobalt.
Electrophysiology. The procedures for whole-cell patch-clamp
recordings of rat sensory neurons have been described in detail previously (Nicol and Cui, 1994). Briefly, a coverslip with the DRG
neurons (5 d in culture) was placed in a recording chamber, where the
neurons were superfused with normal Ringer's solution. Using the
whole-cell patch-clamp technique (Hamill et al., 1981), we recorded
membrane currents from a holding potential of 
60 mV with a List EPC-7
(List Electronic, Darmstadt, Germany) patch-clamp amplifier. Recording
pipettes were pulled from borosilicate disposable pipettes and had
resistances of 3-5 M
when filled with the following solution (in
mM): 140 KCl, 5 MgCl2, 4 ATP, 0.3 GTP, 2.5 CaCl2, 5 EGTA (calculated free Ca2+
concentration of 100 nM), and 10 HEPES, at pH 7.2 with KOH.
The cell capacitance was compensated by the nulling circuitry of the recording amplifier.
Capsaicin was applied focally to the neuron by placing a pipette (5-10
µm in diameter) filled with Ringer's solution containing 100 nM capsaicin and 1 mM trypan blue within 10-30
µm of the cell body. Then positive pressure was applied to the
pipette to eject the solution for which trypan blue served to visualize
this process. The focal application of trypan blue alone had no effect
on these sensory neurons (Nicol and Cui, 1994). Capsaicin was diluted
to a concentration of 100 nM, because this value is
slightly less than the EC50 obtained for the
concentration-response relation for capsaicin in embryonic sensory
neurons (J. C. Lopshire and G. D. Nicol, unpublished observations).
While continuously superfusing the bath with Ringer's solution, we
obtained two responses, separated by ~2 min, to the focal application
of 100 nM capsaicin. After obtaining the test responses, we
changed the superfusate to a Ringer's solution containing 1 µM capsaicin to determine the maximal response. Responses
from only a single neuron were obtained from each coverslip to avoid
any possible desensitization that might occur after the application of
1 µM capsaicin. All experiments were done at room
temperature (~23°C).
Only the results obtained from neurons that satisfied the following
criteria are presented in this report. First, after establishing the
whole-cell configuration, neurons had to maintain zero-current potentials more hyperpolarized than 45 mV for at least 4-5 min before setting the holding potential to 60 mV. Second, the amplitudes of the two responses obtained to the focal application of 100 nM capsaicin had to be within ± 10% of their average
value.
Analysis. Statistical differences among the numbers of
neurons labeled with cobalt for the various experimental treatments were determined by a 
2 analysis. In this set of studies,
one or two wells of cells from each neuronal harvest served as a
control group for each experimental treatment. To determine
significance for each experimental treatment relative to their
respective controls, we obtained a 2 value for each
contingency table. The amplitudes of the inward currents elicited by
two applications of 100 nM capsaicin were averaged to
obtain the mean response to capsaicin. To account for the varying
sensitivity of different sensory neurons to capsaicin, we normalized
the mean response to the maximal response obtained with the bath
application of 1 µM capsaicin. Statistical differences between the control recordings and those obtained under various treatment conditions were determined by one-way ANOVA. When a significant difference was obtained, a Student-Newman-Keuls or Dunnett's post hoc test was performed. Values of
p < 0.05 were judged to be statistically
significant.
Chemicals. Carba prostacyclin was obtained from Cayman
Chemical (Ann Arbor, MI); tumor necrosis factor- and
interleukin-1 (recombinant murine) were obtained from R & D Systems
(Minneapolis, MN). The cyclo-oxygenase-2 inhibitor SC-236 was a
generous gift of Dr. Peter Isakson (G. D. Searle, St. Louis, MO). All
other chemicals were obtained from Sigma (St. Louis, MO). Carba
prostacyclin, capsaicin, and SC-236 were dissolved in
1-methyl-2-pyrrolidinone (HPLC grade, Aldrich, Milwaukee, WI) to obtain
concentrated stock solutions. Then these stock solutions were diluted
with Ringer's solution to yield the appropriate concentration.
RESULTS
Tumor necrosis factor enhances the number of
capsaicin-sensitive neurons
Previously, we demonstrated that capsaicin selectively labeled
with cobalt the small-diameter sensory neurons isolated from the dorsal
root ganglia of embryonic rats (Hingtgen and Nicol, 1994). In the
present study, the capacity of the proinflammatory cytokines, tumor
necrosis factor (TNF) and interleukin-1 (IL-1), to
modulate the sensitivity of isolated sensory neurons to the excitatory
agent capsaicin was examined by quantifying the number of neurons
labeled with cobalt after treatment with capsaicin. Under control
conditions, the average percentage of cells labeled by 100 nM capsaicin was 15.5 ± 1.2% (range 10-26%;
n = 14 neuronal harvests), indicating that, from
preparation to preparation, 100 nM capsaicin labeled a
relatively stable number of cells from the total neuronal population.
As illustrated in Figure 1, pretreatment with TNF
enhanced the number of capsaicin-sensitive neurons (Fig. 1A), whereas IL-1 was without effect (Fig.
1C). After a 24 hr pretreatment with TNF, the number of
capsaicin-sensitive neurons was increased in a concentration-dependent
manner. TNF at 1 ng/ml did not enhance the number of positively
stained neurons (36 of 292 neurons, 12.3% positively labeled), whereas
the number was increased significantly by twofold for 10 ng/ml (883 of
2556 neurons, 34.5% labeled) and 50 ng/ml TNF (448 of 970 neurons,
46.2% labeled). In another series of experiments, the time course of
the TNF-induced sensitization was determined. As illustrated in
Figure 1B, it appeared that the maximal increase in
the number of labeled neurons (27.3%, 163 of 597 neurons) was attained
after a 4 hr treatment with 10 ng/ml TNF. Similar values for the
number of labeled neurons were observed after 12 hr (25.7%, 177 of 689 neurons) and 24 hr exposures (26.7%, 172 of 645 neurons). In contrast
to the sensitization produced by TNF, only 17.7% (91 of 514 neurons) and 12.6% (101 of 799 neurons) of the neurons were labeled
with cobalt after a 24 hr exposure to 10 and 50 ng/ml IL-1,
respectively (Fig. 1C). In control experiments, sensory
neurons exposed to either 100 nM capsaicin in the absence
of cobalt or 10 mM CoCl2 in the absence of
capsaicin produced no labeling (data not shown).
Fig. 1.
TNF increases the number of neurons labeled
with cobalt after exposure to 100 nM capsaicin. The
percentage of labeled neurons for each control condition is represented
by the open bars. A, The percentage of
neurons positively labeled with cobalt after a 24 hr pretreatment with
TNF. The hatched bars represent the percentages
obtained for the following concentrations of TNF: 1 ng/ml
(coarse), 10 ng/ml (medium), or 50 ng/ml
(crossed). B, Time course for the
sensitization produced by treatment with 10 ng/ml TNF for the
indicated times in hours. These experiments were performed in a set of
neurons that differed from those shown in A.
C, The percentage of neurons that were positively
labeled after a 24 hr pretreatment with 10 and 50 ng/ml of IL 1,
shown as the coarse- and fine-hatched
bars, respectively. The asterisks indicate a
statistical difference at p < 0.05.
[View Larger Version of this Image (24K GIF file)]
In many instances, the physiological actions of cytokines are
believed to be intertwined with prostaglandins. This interdependency may result from either a cytokine-mediated activation of the
cyclo-oxygenase pathway, i.e., the production of prostaglandins (Dayer
et al., 1985; Burch et al., 1988; Burch and Tiffany, 1989), or
prostaglandins may act synergistically to enhance the actions of the
cytokine (Burch and Tiffany, 1989). To investigate the possibility that prostaglandins mediated the TNF-induced increase in labeled neurons, we pretreated cell cultures with 30 µM indomethacin, a
nonselective blocker of cyclo-oxygenase activity, for 1 hr before the
addition of TNF to the cultures. As shown in Figure
2A, indomethacin blocked the
sensitization produced by TNF. The number of labeled neurons was
reduced significantly from a value of 28.6% after treatment with
TNF to 17% after indomethacin and TNF (189 of 1114 neurons). Exposure to 30 µM indomethacin alone had no significant
effect on the number of labeled neurons (125 of 937 neurons, 13.3%
positive).
Fig. 2.
Indomethacin blocks the TNF-induced increase in
labeled neurons. These data represent the percentage of neurons labeled
with cobalt after exposure to 100 nM capsaicin. The
open bars show the percentage of neurons obtained for
the various control conditions. A, Suppression of the
TNF-mediated enhancement of labeling by indomethacin. The
fine-hatched bar, labeled TNF, represents
the percentage after a 24 pretreatment with 10 ng/ml TNF; the
coarse-hatched bar, labeled TNF INDO,
demonstrates the effects of 30 µM indomethacin in
combination with 10 ng/ml TNF; and the fine-hatched
bar, labeled INDO, represents the percentage
obtained after a 24 hr treatment with 30 µM indomethacin
alone. B, The enhancement of labeling by
CPGI2, TNF, and a combination of CPGI2 and TNF.
The fine-hatched bar, labeled
CPGI2, represents the percentage
after a 24 hr pretreatment with 10 nM CPGI2;
the coarse-hatched bar, labeled TNF,
shows the percentage after a 24 treatment with 10 ng/ml TNF; and the
fine-hatched bar, labeled TNF
CPGI2, illustrates the percentage
obtained for the combined treatment with 10 nM CPGI2 and 10 ng/ml TNF. The asterisks
indicate significant differences between the control and experimental
treatments (p < 0.05).
[View Larger Version of this Image (27K GIF file)]
Another series of experiments examined the possibility that the
TNF-induced sensitization might be enhanced further by cotreatment with a prostaglandin. Neuronal cultures were treated with 10 nM CPGI2 alone or in combination with 10 ng/ml
TNF for 24 hr. This concentration was chosen because a previous
study demonstrated that 10 nM CPGI2 produced a
two- to threefold enhancement in the release of substance P or
calcitonin gene-related peptide (CGRP) when sensory neurons grown in
culture were stimulated by capsaicin (Hingtgen and Vasko, 1994). As
illustrated in Figure 2B, CPGI2 alone
significantly increased the number of labeled neurons to 38.1% (463 of
1216 neurons). The combination of CPGI2 and TNF had no
additional effect on the increase in neuronal number (510 of 1251 neurons, 40.8% positive), as compared with TNF alone (41.8%
positive). It does not seem likely that values between 30 and 40% were
maximal, because previous work in adult, neonatal, or embryonic sensory
neurons demonstrated that, with a higher concentration of capsaicin (1 µM), >50% of the neurons can be labeled with cobalt
(Winter, 1987; Wood et al., 1988; Hingtgen and Nicol, 1994).
Tumor necrosis factor sensitizes the neuronal response
to capsaicin
The results presented above demonstrate that TNF increased the
number of neurons that were labeled by cobalt after exposure to
capsaicin. To further investigate the possibility that the increased
number of positively labeled neurons resulted from a TNF-mediated
increase in the sensitivity of these neurons to capsaicin, we obtained
the response to focally applied capsaicin from a single neuron via the
whole-cell patch-clamp recording technique (Hamill et al., 1981). A
representative response to capsaicin obtained under control conditions
is illustrated in Figure 3 (top
panel). At a holding potential of 60 mV, the focal application of 100 nM capsaicin (2 sec duration) elicited a
peak inward current of 850 pA that slowly recovered to baseline. At the
peak of the response there was an increase in the membrane current
noise, suggesting that this inward current resulted from the opening of
capsaicin-gated ion channels. Then the cell was superfused for another
2 min with Ringer's solution to ensure complete recovery. At this
point, the superfusate was changed to Ringer's containing 1 µM capsaicin, for which a maximal inward current of 2300 pA was elicited. In recordings from eight neurons under control
conditions, the inward current elicited by 100 nM capsaicin
had an average value of 610 ± 163 pA (range 65-1238 pA).
Exposure of these neurons to 1 µM capsaicin produced an
average maximal response of 1701 ± 311 pA (range 700-3000 pA).
In comparison with the control recordings, the neuronal response to
capsaicin was enhanced greatly after a 24 hr treatment with 10 ng/ml
TNF (Fig. 3, bottom panel). In this representative
neuron, a 2 sec pulse of 100 nM capsaicin applied focally
elicited an inward current of 1725 pA; the bath application of 1 µM capsaicin produced a maximal response of 2450 pA.
After a 24 hr treatment with TNF, the average response to 100 nM capsaicin was 1446 ± 339 pA (range 400-4000 pA,
n = 10), whereas the average maximal response was 1898 ± 387 pA (range 600-4800 pA). This large increase in the inward current evoked by the focal application of capsaicin after TNF treatment did not result from an overall enhancement of the capsaicin response, because the average maximum response to 1 µM capsaicin obtained under control conditions was not
significantly different from that after TNF treatment.
Fig. 3.
TNF enhances the amplitude of the capsaicin
response. The top panel illustrates a representative
response to the focal application of 100 nM capsaicin
obtained under control conditions. The bottom panel
shows the response from a different neuron to the focal application of
capsaicin after a 24 hr treatment with 10 ng/ml TNF. The
bars labeled CAP indicate the timing and
duration of the applications of capsaicin. Both neurons were held at
60 mV; inward currents are shown as downward.
[View Larger Version of this Image (8K GIF file)]
The notion that TNF enhanced the capsaicin sensitivity of sensory
neurons is supported further by examining the response amplitudes to
the focal application of 100 nM capsaicin as a function of
the response amplitudes to the bath application of 1 µM
capsaicin (i.e., the maximum response). As shown in Figure
4, the amplitudes of the responses obtained under
control conditions are fit by a linear regression line having a slope
of 0.47 and a Pearson's correlation coefficient of 0.90. In contrast,
after treatment with TNF the response amplitudes are larger and are
now fit by a linear regression line having a slope of 0.77 and a
correlation coefficient of 0.88. Therefore, these results indicate that
treatment with TNF heightened the sensitivity of sensory neurons to
this excitatory agent without altering the magnitude of the maximal response.
Fig. 4.
TNF enhances the response to capsaicin without
altering the maximal response. The amplitude of the response to the
focal application of 100 nM capsaicin is plotted as a
function of the maximal response obtained in that same neuron for the
bath application of 1 µM capsaicin. The filled
circles represent those responses obtained under control
conditions (n = 8), whereas the filled triangles illustrate the responses after a 24 hr treatment with 10 ng/ml TNF (n = 10). The lines
through the points are the linear regressions in which
the fitting parameters are listed in the text. The broken
lines represent the 95% confidence limits for each regression
line.
[View Larger Version of this Image (26K GIF file)]
The time course for sensitization of the capsaicin-evoked current by
TNF was very similar to that observed for the cobalt-labeling studies. As shown in Figure 5, the capsaicin response
was enhanced by nearly twofold after a 4 hr treatment with 10 ng/ml
TNF. Likewise, after 12 and 24 hr treatments with 10 ng/ml TNF,
the relative amplitudes of the capsaicin-elicited current were doubled.
However, an acute 10 min exposure to TNF had no significant effect
on the capsaicin response. In our previous studies, a 10 min exposure to PGE2 was sufficient to produce a maximal enhancement in
the number of action potentials evoked by bradykinin (Nicol and Cui, 1994; Cui and Nicol, 1995). Therefore, these results suggest that TNF does not have an immediate action on the sensitivity of these neurons to capsaicin, but, rather, the processes mediating this enhancement require a period of hours to become effective.
Fig. 5.
Time course for the TNF-induced sensitization
of the capsaicin response. The bars represent the
current elicited by the focal application of 100 nM
capsaicin that was normalized to the maximal response obtained to the
bath application of 1 µM capsaicin for different
experimental treatments. The open bar represents
untreated neurons under control conditions (n = 9).
The hatched bar, labeled 10 M, represents
the currents obtained after a 10 min exposure to 10 ng/ml TNF
(n = 5). The coarse-,
medium-, and fine-hatched bars denote
those currents recorded after 4 (n = 5), 12 (n = 5), and 24 (n = 5) hr
treatments, respectively, with 10 ng/ml TNF.
[View Larger Version of this Image (40K GIF file)]
The neuronal responses to capsaicin obtained under control conditions
and after TNF treatment from two different sets of neurons are
summarized in Figure 6A. After
normalization, the application of 100 nM capsaicin under
control conditions produced an average response that was 0.32 ± 0.05 (n = 8) of the maximal response. In those sensory
neurons treated with 10 ng/ml TNF for 24 hr, the average response to
100 nM capsaicin was increased significantly to 0.77 ± 0.07 (n = 10) of the maximal response. Thus, TNF
produced a greater than twofold enhancement in the sensitivity of
sensory neurons to the focal application of capsaicin.
Fig. 6.
TNF enhancement of the capsaicin response is
blocked by inhibition of cyclo-oxygenase. A, The
sensitizing effects of TNF on the normalized response to capsaicin
and its inhibition by indomethacin. The focal response to 100 nM capsaicin is expressed as the fraction of the maximal
response obtained with the bath application of 1 µM
capsaicin. The bars represent the following experimental
conditions: the control is shown as the open bar (n = 8); the fine-hatched bar,
labeled TNF, is after a 24 hr treatment with 10 ng/ml
TNF (n = 10); the coarse-hatched
bar is after treatment with 30 µM indomethacin
and 10 ng/ml TNF (n = 4); and the
fine-hatched bar, labeled INDO, is after
a 24 hr treatment with 30 µM indomethacin alone
(n = 4). B demonstrates in another
set of sensory neurons inhibition of the TNF-induced sensitization
by treatment with the selective COX-2 inhibitor SC-236. The open
bar represents the untreated control neurons
(n = 3). The fine-hatched bar
represents results obtained after a 24 hr treatment with 10 ng/ml
TNF (n = 5); the coarse-hatched
bar is after treatment with 300 nM SC-236 and 10 ng/ml TNF (n = 5). The asterisks
indicate statistical significance at p < 0.05.
[View Larger Version of this Image (26K GIF file)]
As with the cobalt-labeling studies, the possible contribution of
cyclo-oxygenase products to the TNF sensitization was examined by
pretreating the neuronal cultures with 30 µM indomethacin
before the addition of 10 ng/ml TNF. As shown in Figure
6A, indomethacin blocked the TNF-induced increase
in the response to 100 nM capsaicin such that, after
exposure to indomethacin and TNF, the average fractional response
was only 0.36 ± 0.07 (n = 4) of the maximum and
was similar to the control value of 0.32. A 24 hr treatment with 30 µM indomethacin alone had no significant effect on the average fractional response to 100 nM capsaicin (0.36 ± 0.06, n = 4). At this concentration, indomethacin
inhibits both the constitutively active cyclo-oxygenase-1 (COX-1) and
the inducible cyclo-oxygenase-2 [COX-2; Masferrer et al. (1994);
Seibert et al. (1994b); but see Meade et al. (1993)]. In a separate
series of experiments, a selective inhibitor of COX-2, SC-236 (Peter Isakson, personal communication), was used to distinguish which isoform
mediated the TNF-induced sensitization. This inhibitor of COX-2 has
an activity on human enzymes with an IC50 of 0.01 µM for COX-2 and 18 µM for COX-1 (T. D. Penning, J. J. Talley, S. R. Bertenshaw, J. S. Carter, P. W. Collins,
S. Docter, M. J. Graneto, L. F. Lee, J. W. Malecha, J. M. Miyashiro, R. S. Rogers, D. J. Rogier, S. S. Yu, G. D. Anderson, J. N. Cogburn, S. A. Gregory, C. M. Koboldt, W. E. Perkins, K. Seibert,
A. W. Veenhuizen, Y. Zhang, P. C. Isakson, unpublished data). In the
presence of 300 nM SC-236, the twofold increase in the
capsaicin sensitivity produced by TNF was blocked completely (Fig.
6B). In another series of experiments, 10 µM SC-236 also produced a complete suppression of the
TNF-mediated enhancement (from 0.49 ± 0.03 to 0.21 ± 0.02), whereas SC-236 alone had no effect on the response to capsaicin (0.26 ± 0.03 vs 0.22 ± 0.02 for the control; data not
shown). Therefore, using both population studies involving
cobalt-labeling as well as electrophysiological studies examining the
response obtained from a single neuron, we show results that indicate
that pretreatment with TNF enhanced the sensitivity of sensory
neurons to the excitation elicited by capsaicin. Furthermore, the
delayed time course of sensitization likely results from a
TNF-mediated induction of COX-2 that ultimately leads to the
production of prostaglandins.
DISCUSSION
The results presented in this study demonstrate that TNF, but
not IL-1, can sensitize isolated sensory neurons to the excitation produced by capsaicin. With the use of two different assays of neuronal
sensitivity, TNF increased the number of neurons labeled by cobalt
after exposure to capsaicin as well as the amplitude of the inward
current evoked by the focal application of capsaicin to a single
neuron. Although the cellular pathways mediating this sensitization are
not well characterized, it seems unlikely that the increased
responsiveness results from an increase in the total number of
capsaicin receptors, because the maximal inward current elicited by 1 µM capsaicin was similar in the absence or presence of
TNF.
The TNF-induced sensitization likely is mediated by prostaglandins,
because enhancement of the capsaicin response was blocked by both
indomethacin, a nonselective inhibitor of cyclo-oxygenase, and SC-236,
a specific inhibitor of COX-2. These results suggest that TNF
somehow leads to the induction and activation of COX-2 to release
prostaglandins and, thus, enhances the response to capsaicin. Indeed,
sensory neurons grown in culture can synthesize, from labeled
arachidonic acid, many different prostanoids, most prominently
PGE2, with smaller levels of 6-keto PGF1
(a
breakdown product of PGI2), PGD2, and
PGF2 (Vasko et al., 1994). Also, these sensory neurons
were labeled heavily by a nonspecific antibody to cyclo-oxygenase
(Vasko et al., 1994). Thus, sensory neurons express cyclo-oxygenase
that is capable of generating prostaglandins. It is well documented
that prostaglandins, especially PGE2 and PGI2
(prostacyclin), play critical roles in the initiation of the heightened
sensitivity to stimulation in both in vivo and in
vitro models of neurogenic inflammation (Davies et al., 1984; Higgs et al., 1984). This notion is supported further by our
observations that the number of cobalt-labeled neurons was increased by
approximately twofold after a 24 hr pretreatment with the
proinflammatory prostaglandin CPGI2. Likewise, exposure to
CPGI2 on a much shorter time scale sensitizes isolated
sensory neurons. Previously, we reported that pretreatment of sensory
neurons with a higher concentration of CPGI2 (1 µM) for only 20 min produced a threefold increase the number of cobalt-labeled cells by capsaicin (Hingtgen and Nicol, 1994).
Currently, the intracellular transduction cascades linking the TNF
receptor and activation of cyclo-oxygenase are unknown. Recent evidence
indicates that exposure to inflammatory mediators stimulates
cyclo-oxygenase activity; however, the increased activity is limited to
the inducible isoform COX-2 (Fu et al., 1990; Kujubu et al., 1991;
O'Banion et al., 1991). Treatment with proinflammatory agents
(Crofford et al., 1994; Masferrer et al., 1994; Seibert et al.,
1994a,b; Feng et al., 1995) or tissue injury (Pritchard et al., 1994)
induces the synthesis of COX-2 (prostaglandin G/H synthase 2) but has
no effect on the constitutively active isoform COX-1 (prostaglandin G/H
synthase 1). These observations indicate that it is the induction of
COX-2, rather than increased activity of COX-1, which mediates the
release of prostaglandins during the inflammatory response. Indeed,
COX-2 is expressed in synovial tissues isolated from patients with
rheumatoid arthritis, and when explants of these synovial tissues are
grown in culture, IL-1 significantly increases the mRNA level of
COX-2, but not COX-1 (Crofford et al., 1994). The notion that TNF
promotes the induction of COX-2 in sensory neurons is supported by two
of our findings. First, our results demonstrate that selective
inhibition of COX-2 with the compound SC-236 prevents the
TNF-induced sensitization of the capsaicin response. Second, TNF
does not have an immediate action on the capsaicin sensitivity of these
neurons but, rather, requires ~4 hr to become effective. This time
course of cytokine action is supported by recent observations in which
a significant increase in the relative expression of COX-2 mRNA was
found 3 hr after producing carrageenan-induced inflammation in the paw of a rat (Seibert et al., 1994b). Also, in the isolated rat trachea, the capsaicin-evoked release of CGRP was potentiated after a 5 hr
exposure to either TNF or IL-1; however, there was no effect after 2 hr (Hua et al., 1996). When taken together, experimental observations indicate that proinflammatory mediators have no
significant effect on the levels or activity of COX-1; therefore, it
seems highly likely that the enhanced sensitivity to capsaicin results from the ability of TNF to induce the synthesis of COX-2.
In many ways the physiological actions of TNF and IL-1 are
believed to be quite similar (Dinarello, 1987; Le and Vilek, 1987; Arend and Dayer, 1990). For example, studies using behavioral measures for the perception of noxious stimuli in intact animals report
that injection of either TNF or IL-1 produces a hyperalgesic response (Ferreira et al., 1988; Schweizer et al., 1988; Follenfant et
al., 1989; Cunha et al., 1992). In the intact animals, the cytokine-induced sensitization was blocked by pretreatment with indomethacin, also indicating a role for cyclo-oxygenase products. Similarly, in the isolated and perfused rat trachea,
lipopolysaccharide, IL-1, or TNF facilitated the release of CGRP
that was evoked by stimulation with capsaicin (Hua et al., 1996). These
results, then, suggest that the threshold had been lowered, wherein
TNF or IL-1 had a sensitizing effect on the sensory afferent
nerves, responding to the noxious or chemical stimulation. It is,
however, curious that TNF, but not IL-1, sensitizes the isolated
sensory neurons grown in culture to the excitation produced by
capsaicin. Compared with TNF, IL-1 had no significant effect on
the number of cobalt-labeled neurons, even at a relatively high
concentration (50 ng/ml). The lack of an IL-1 effect on the
excitability of sensory neurons is supported by a similar finding in
which, in recordings from cultured sympathetic neurons isolated from
rat superior cervical ganglia, the calcium current was potentiated by
long-term exposures (>4 hr) to TNF, but not to IL-1 (Soliven and
Albert, 1992). Therefore, our observations are consistent with the
notion that the sensitizing effects of IL-1 observed in the intact
animal or isolated tissue might result from an IL-1-induced production of a secondary mediator released from other cell types rather than from a direct action of IL-1 on the sensory neuron.
The intracellular transduction cascades that mediate the
prostaglandin-induced sensitization are poorly understood. Our previous observations suggest that these isolated sensory neurons grown in
culture are an excellent model system in which to investigate the
regulatory pathways activated by proinflammatory prostaglandins. Acute
treatment with PGE2 enhances the excitability of isolated sensory neurons grown in culture in a manner that is similar to that
observed in both in vivo and in vitro animal
models of pain and neurogenic inflammation. For example, a 10 min
pretreatment with 1 µM PGE2 produced a
threefold increase in the number of action potentials elicited by the
focal application of bradykinin (Nicol and Cui, 1994). Similarly, in
the intact animal, the frequency of action potentials recorded from
C-fibers in response to bradykinin was increased after the application
of PGE2 (Handwerker, 1976) or PGE1 (Chahl and
Iggo, 1977). Furthermore, acute treatment with either PGE2
or CPGI2 enhanced the bradykinin-evoked release of substance P and calcitonin gene-related peptide from isolated sensory
neurons grown in culture (Hingtgen and Vasko, 1994;Vasko et al., 1994).
These neuroactive peptides have been demonstrated to be important
mediators of neurogenic inflammation (Cuello, 1987; Foreman,
1987).
In a manner analogous to short-term applications, our current findings
describing the effects of sustained treatment (24 hr) with TNF and
CPGI2 suggest that isolated sensory neurons grown in
culture may prove to be a useful model system in which to explore how
prolonged exposure to mediators associated with chronic inflammation alters the pathways controlling cellular sensitivity and excitability. Investigation of the cellular mechanisms whereby proinflammatory cytokines, such as TNF, produce transcriptional changes that lead to
either the induction or downregulation of various enzyme systems, such
as COX-2, would enhance our understanding of the role played by sensory
neurons in the maintenance of neurogenic inflammation. Indeed, patients
afflicted with rheumatoid arthritis (a condition associated with
chronic inflammation of the joints) have elevated levels of
inflammatory cytokines and prostaglandins in their synovial fluids as
well as a tendency for chronic pain in those joints (Arend and Dayer,
1990; Feldman et al., 1990; Bhoola et al., 1992; Konttinen et al.,
1994).
In conclusion, the proinflammatory cytokine, TNF, directly enhances
the sensitivity of sensory neurons to the chemical excitatory agent
capsaicin via a cyclo-oxygenase-dependent pathway. It is possible that
this cytokine-mediated sensitization may be part of a more global
mechanism whereby regulatory pathways associated with the immune system
modulate the excitability of the nervous system.
FOOTNOTES
Received Oct. 31, 1996; accepted Nov. 18, 1996.
This work was supported by National Institutes of Health Grant
RO1-NS30527 (G.D.N.). J.C.L. was supported by the Indiana University Purdue University, Indianapolis (IUPUI) Research Investment Fund; C.M.P. was supported by the IUPUI Research Investment Fund and an
Indiana Medical Scholar Fellowship. We are grateful to Drs. Michael
Vasko and Angela Evans for discussions about signal transduction in
sensory neurons. We thank Dr. Peter Isakson for the generous gift of
SC-236.
Correspondence should be addressed to Dr. G. D. Nicol, Department of
Pharmacology and Toxicology, School of Medicine, Indiana University,
635 Barnhill Drive, Indianapolis, IN 46202-5120.
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