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Vol. 288, Issue 2, 502-508, February 1999
Experimental and Biological Program, Department of Psychology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
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Abstract |
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 Top
 Abstract
 Introduction
Materials and methods
Results
Discussion
References
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In the present study, we tested the hypothesis that lipopolysaccharide (LPS)-induced expression of nitric oxide synthase (iNOS) by splenocytes is modulated through the activation of endogenous opioids in the central nervous system. The initial studies determined the parameters of LPS-induced expression of iNOS by splenocytes. Rats were injected with LPS at doses of 0, 1, 10, 100, and 1000 µg/kg, and measures of both iNOS mRNA and protein showed a dose-dependent increase in expression. In a time course study, rats received 100 µg/kg LPS and were killed at 0, 2, 4, 8, and 16 h postinjection. Both iNOS mRNA and protein expression was detectable at the 2-h time point, with peak expression occurring at 8 h. To evaluate the involvement of endogenous opioids, the opioid receptor antagonist naltrexone was administered at 0, 0.1, 1, or 10 mg/kg s.c. in combination with LPS (100 µg/kg), with a second injection of naltrexone at the same dose 4 h after the injection of LPS. Naltrexone induced a pronounced dose-dependent reduction in iNOS mRNA and protein expression by splenocytes. The modulation of iNOS expression occurs via central opioid receptors as intracerebroventricular administration but not peripheral administration of N-methylnaltrexone, the quaternary form of naltrexone that does not readily cross the blood-brain barrier, reduced the expression of iNOS. For all of the manipulations, nitrite/nitrate levels in the plasma showed effects similar to those for iNOS mRNA and protein. Collectively, these findings indicate that central opioid receptors are involved in the in vivo regulation of splenic nitric oxide production.
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Introduction |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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In
recent years, the involvement of nitric oxide in immune processes has
become apparent. For example, the role of nitric oxide in infectious
disease is demonstrated by studies showing that it is involved in the
cytostatic activity of macrophages on parasitic growth (Vincendeau et
al., 1992
). The development of autoimmune disease also has been linked
to alterations in the expression of nitric oxide, as spontaneous
autoimmune disease in MRL-lpr/lpr mice has been shown to be dependent
on enhanced expression of nitric oxide (Weinberg et al., 1994). In
addition to the impact of nitric oxide directly on infectious organisms and immunoreactive tissues, nitric oxide has important regulatory functions in the immune system. Numerous studies have shown that nitric
oxide limits the proliferative activity of lymphocytes (Albina and
Henry, 1991; Pascual et al., 1993).
Despite extensive knowledge of the biochemistry of nitric oxide and the
undeniable importance of nitric oxide for defense against disease,
little is known about the diversity of in vivo factors involved in the
regulation of nitric oxide production by cells of the immune system.
Much attention has been focused on the role of cytokines in the
regulation of nitric oxide production using in vitro culture systems.
For example, it has been established that interferon-
and tumor
necrosis factor increase the production of nitric oxide by macrophages
(Albina and Henry, 1991; Geller et al., 1993) and that interleukin-4
and interleukin-10 inhibit nitric oxide production by macrophages
(Al-Ramadi et al., 1992; Gazzinelli et al., 1992; Oswald et al., 1992).
However, there is evidence that explanations based on cytokine
regulation alone do not adequately account for the in vivo expression
of inducible nitric oxide. For example, Liu et al. (1993, 1997) found
that lipopolysaccharide (LPS) treatment in vivo induces a very rapid expression of mRNA for inducible nitric oxide synthase (iNOS), the
enzyme responsible for nitric oxide formation. This contrasts sharply
with in vitro studies showing that LPS and cytokines are often
relatively slow and weak stimulators of iNOS mRNA expression in a
number of culture systems (Geller et al., 1993). Thus, the in vivo
milieu provides an abundance of unexplored factors that serve as potent
regulators of nitric oxide production in the immune system.
In some of our work, we have made the unique observation that the
administration of morphine alters the production of nitric oxide by
splenic macrophages (Fecho et al., 1994). In subsequent investigations,
we showed that microinjection of the mu-opioid receptor
agonist
[D-Ala2,N-MePhe4,Gly-ol5]enkephalin
into the central nervous system produced an elevation of nitric oxide
production by splenic macrophages, whereas the microinjection of
kappa- and delta-selective opioid agonists did not significantly alter nitric oxide production (Schneider and Lysle,
in press). The effect of
[D-Ala2,N-MePhe4,Gly-ol5]enkephalin
was blocked by the opioid receptor antagonist naltrexone, indicating
that the effect was opioid receptor mediated (Schneider and Lysle, in
press). These observations indicate that opioids modulate the
production of nitric oxide by macrophages and that the effect is
mediated by mu-opioid receptors in the central nervous system. These observations suggest a further hypothesis that the in
vivo induction of iNOS expression involves endogenous opioid activity
within the central nervous system.
The present study tests the hypothesis that iNOS expression involves
endogenous opioid activity within the central nervous system by
evaluating the effect of administration of the opioid receptor
antagonist naltrexone on LPS-induced expression of iNOS by splenocytes.
To evaluate the contribution of opioid receptors located in the central
nervous system, the effect of intracerebroventricular (i.c.v.) and
peripheral administration of N-methylnaltrexone on LPS-induced expression of iNOS by splenocytes was determined. N-methylnaltrexone is the quaternary form of naltrexone
that does not readily cross the blood-brain barrier (Brown and
Goldberg, 1985). The prediction, based largely on our prior work, was
that blockage of opioid receptors, particularly those in the central nervous system, would inhibit the expression of nitric oxide synthase by splenocytes. Because the literature provided only limited
information about the appropriate dose of LPS and the time course of
LPS-induced expression of iNOS, the initial assessments determined the
dose-effect relationship and time course of LPS-induced iNOS
expression. To measure nitric oxide production in vivo, RT-PCR (reverse
transcription-polymerase chain reaction) and Western blotting
techniques was used to measure iNOS mRNA and protein expression in
spleen tissue, and Griess reagent was used to measure nitrite/nitrate
in plasma.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Animals. Male Lewis rats 65 days old and 225 to 250 g in weight were purchased from Charles Rivers Laboratories (Raleigh, NC). On arrival, animals were individually caged in a colony room where a reversed light-dark (12-h) cycle was maintained through artificial illumination. Food and water were provided ad libitum during both a 2-week acclimation period and the experimental procedure.
Drug Administration. To determine the doses and time course of LPS-induced expression of nitric oxide, rats received an s.c. injection saline or LPS serotype 055:B5 (Difco Laboratories, Detroit, MI) at doses of 1, 10, 100, or 1000 µg/kg (n = 4). The animals were killed 4 h after the injection. In a second study, rats received an s.c. injection of 100 µg/kg LPS, and the animals were killed immediately or 2, 4, 8, or 16 h after the injection (n = 4). For both studies, the spleen was obtained for the extraction of RNA and protein, and a sample of peripheral blood from the abdominal aorta was used for the determination of nitrite/nitrate levels.
To evaluate the effect of naltrexone, rats received an s.c. injection of saline or naltrexone at doses of 0.1, 1, or 10 mg/kg (n = 4) in combination with an s.c. injection LPS (100 µg/kg), with a second injection of naltrexone at the same dose 4 h after the injection of LPS. Eight hours after the injection of LPS, the rats were killed, and the spleen and a sample of peripheral blood were obtained for analysis. To determine the involvement of central opioid receptors, N-methylnaltrexone was administer i.c.v. in combination with an s.c. injection of LPS (100 µg/kg). Rats were stereotaxically implanted with a single 22-gauge cannula (Plastics One) into the left lateral ventricle [anteroposterior +0.9, mediolateral +1.5, dorsoventral
3.2] under anesthesia induced with a 1 ml/kg injection
of a 1:1 (v/v) mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml). Coordinates are expressed as millimeters from bregma (Paxinos and
Watson, 1986). After a 1-week recovery period, rats were given an
injection of saline or N-methylnaltrexone (0.01, 0.1, 1, or 10 µg) in a 5 µl volume via a 28-gauge (Plastics One) injector that
protruded 1 mm beyond the tip of the cannula (n = 4).
The injector was connected by a length of tubing to a Hamilton syringe. N-Methylnaltrexone was administered i.c.v. at the time of an
s.c. injection LPS (100 µg/kg) with a second i.c.v. injection of
N-methylnaltrexone 4 h later. Eight hours after the
injection of LPS, rats were killed, and the spleen and a sample of
blood were obtained for plasma. The selection of doses of
N-methylnaltrexone were based on studies showing that an
i.c.v. dose of 1.0 µg of N-methylnaltrexone is sufficient
to block the immunomodulatory effect of morphine administration (Fecho
et al., 1996a).
In a subsequent study, N-methylnaltrexone (10 mg/kg) or
saline was administered s.c. at the time of an s.c. injection LPS (100 µg/kg) with a second injection of the same dose of
N-methylnaltrexone 4 h later (n = 5).
Eight hours after the injection of LPS, rats were killed, and the
spleen and a sample of blood were obtained for plasma.
RT-PCR.
Total RNA was extracted from a section of each
spleen using Tri-Reagent (Molecular Research Center, Inc., Cincinnati,
OH), a modification of the original method described by Chomczynski and
Sacchi (1987). The RNA was quantified spectrophotometrically (GeneQuant
II; Pharmacia-Biotech, Piscataway, NJ). For the RT-PCR, RT was
performed using Oligo(dT)18 primer and Moloney
murine leukemia virus-reverse transcriptase after the protocol of the
1st Strand cDNA Synthesis Kit from Clontech (Palo Alto, CA). The
resulting cDNA was amplified for 20 cycles using a PCR primer set for
iNOS, 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3' and
5'-GGGTGTCAGAGTCTTGTGCCTTTGG-3' that was synthesized by the Nucleic
Acids Core Facility (Lineberger Cancer Center, University of North
Carolina, at Chapel Hill). Taq DNA polymerase
(Boehringer-Mannheim, Indianapolis, IN) was used in the reaction. The
cycle temperatures were 94°C, 60°C, and 72°C for the denaturing,
annealing, and extending temperatures, respectively. The cycle times
were 30 s, 30 s, and 1.5 min for the denaturing, annealing,
and extending times, respectively. Moreover, given the time required to
start the relatively large number of samples, a hot-start method using
Ampliwax PCR gems (Perkin-Elmer, Branchburg, NJ) was used. Positive and
negative controls also were included. The PCR products were run on a
2.0% agarose gel in 0.5× Tris-Borate-EDTA buffer, stained with
ethidium bromide (0.5 µg/ml), and visualized under ultraviolet
transillumination. Product levels were quantified by densitometry using
UltraLum (KS 3000) gel documentation system and Sigmagel software
(Jandel Scientific). The data are expressed as a ratio of 
-actin
expression, which served as a housekeeping control. A PCR primer set
from Clontech was used to amplify -actin mRNA. The sequence of the -actin primers was 5'-TTGTAACCAACTGGGACGATATGG-3' and
5'-GATCTTGATCTTCATGGTGCTAGG-3'. The DNA molecular weight marker (DNA
marker VI; Boehringer-Mannheim) was used to estimate the size of the products.
Western Blot.
For the iNOS protein determinations, protein
was extracted from a section of spleen tissue using freeze/thaw lysis
in Tris buffer containing antiproteinases. Total protein was determined using the BioRad Protein Assay (BioRad, Hercules, CA). Then, 20 µg of
protein was loaded on a sodium dodecyl sulfate-polyacrylamide gel and
electrophoretically separated and blotted to nitrocellulose membrane
(Schleicher & Schuell, Keene, NH). The membrane was blocked with 10%
dry milk and reacted with a polyclonal rabbit anti-iNOS antibody
(Transduction Laboratories, Cincinnati, OH). The membrane then was
incubated with donkey anti-rabbit antibody conjugated to horseradish
peroxidase (Amersham, Cleveland, OH). Membranes were exposed to
Hyperfilm ECL, and peroxidase activity was detected using the enhanced
chemiluminescence analysis system (Amersham). A positive control
containing protein extracted from the RAW 264.7 (ATCC TIB71; American
Type Culture Collection, Rockville, MD) macrophage cell line that had
been stimulated for 12 h with interferon- (10 ng/ml; Gibco Life
Technologies, Gaithersburg, MD) and LPS (1.0 µg/ml serotype 055:B5;
Difco Laboratories, Detroit, MI), along with Kaleidoscope prestained
standards (BioRad), was used to identify the 130-kDA iNOS protein.
Product levels were quantified by scanning the film and performing
densitometry analysis (Sigmagel; Jandel Scientific).
Nitrite Production Assay.
To provide an additional
assessment of nitric oxide production, the level of nitrate/nitrite in
plasma was determined using the Griess reagent assay (Green et al.,
1982). Nitrate and nitrite are formed nonenzymatically when nitric
oxide is exposed to oxygen; thus, plasma levels of these products
indicate the level of nitric oxide production. Total nitrite/nitrate
levels were determined by the conversion of nitrate to nitrite using
nitrate reductase in the presence of reduced nicotinamide adenine
dinucleotide phosphate and flavinadenine dinucleotide and then an
assessment using Griess reagent (Tracey et al., 1995). More
specifically, 6 µl of plasma was mixed with 44 µl of
dH2O, 20 µl of 0.31 M phosphate buffer (pH
7.5), and 10 µl of 0.86 mM reduced nicotinamide adenine dinucleotide phosphate (Boehringer-Mannheim), 10 µl of 0.11 mM flavinadenine dinucleotide (Sigma), and 10 µl of 1.0 U/ml nitrate reductase (Boehringer-Mannheim) in individual wells of a 96-well plate. Samples
were incubated for 1.5 h at room temperature. Then, 200 µl of
Griess reagent consisting of a 1:1 (v/v) solution of 1% sulfanilamide
in 5.0% phosphoric acid and 0.1%
N-(1-napthyl)ethyl-enedamine dihydrochloride in distilled
water was added to the samples. Absorbance was measured, after 10 min
of incubation at room temperature, at 550 nm using a Biotech plate
reader (model EL312), and the micromolar (µM) concentration of
nitrite was determined for each sample based on a standard curve.
Recovery of nitrate was greater than 95% in this assay.
Statistical Treatment of Data. Analysis of variance was performed on all data sets. Standard polynomial contrasts were performed on those analyses that showed a significant main effect of dose of naltrexone or N-methylnaltrexone. The level of significance for all statistical tests was set at a probability of .05.
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Results |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Figure 1 shows the dose-dependent
effects of LPS on the expression of iNOS mRNA as determined by RT-PCR.
The electrophoresis of the RT-PCR products is shown via an image of the
ethidium bromide-stained agarose gel visualized under ultraviolet
transillumination. The top image shows the results using the iNOS
primers, and the bottom image shows the results for -actin. The
first lane of each gel is the DNA molecular weight marker (DNA marker
VI; Boehringer-Mannheim). The amplified products using the iNOS primer
set are shown in the subsequent lanes of the top image starting with a
positive iNOS and negative control, followed by the samples from the
rats treated with increasing doses of LPS. There is a distinct product of the expected size (496 bp). The bottom image shows a distinct product of the expected size (764 bp) for -actin. The densitometric analysis of the bands showed no significant effect of LPS on the expression of -actin; however, the analysis of the ratio of
iNOS/-actin expression showed a significant effect of LPS on iNOS
expression: F(4,15) = 55.65 (p < .0001). Moreover,
there was a significant linear component to the main effect of LPS
[F(1,15) = 180.53, p < .0001] indicating a linear
relationship between the dose of LPS and iNOS expression. The ratio
data are shown in graphic form at the bottom of Fig. 1. The cloned iNOS
PCR product was found to have 99% identity with the corresponding
region of the reported sequence for rat iNOS (Genbank Accession No.
D14051; Nunokawa et al., 1993).
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Figure 2 shows the image and densitometric analysis of the Western blot for iNOS protein. The positive control showed the expected 130-kDa band for iNOS protein. The densitometric analysis of the bands showed a significant effect of LPS on the expression of iNOS [F(4,15) = 16.96, p < .0001]. Moreover, there was a significant linear component to the effect of LPS [F(1,15) = 56.34, p < .0001] indicating a linear relationship between the dose of LPS and iNOS expression.
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Figure 3 shows the effect of LPS on nitrite/nitrate levels in the plasma. The analysis showed a significant effect of LPS on the level of nitrite/nitrate in the plasma [F(4,15) = 16.54, p < .0001]. There was a significant linear component to the effect of LPS [F(1,15) = 33.98, p < .0001] indicating a linear relationship between the dose of LPS and nitrite/nitrate iNOS expression. There also was a significant quadratic component [F(1,15) = 24.78, p < .001] suggesting that the intermediate doses of LPS produced a slight decrease in the level of nitrite/nitrate, with the higher doses producing a pronounced increase in the levels.
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Figure 4 shows the time-dependent effect
of LPS on the expression of nitric oxide. Figure 4A shows the ratio of
iNOS/-actin expression at the different time points. Figure 4B shows
the results of densitometric analysis of the Western blot for iNOS
protein. Figure 4C shows the time-dependent effect of LPS on
nitrite/nitrate levels in the plasma. The analysis showed a significant
effect of time on each measure [Fs(4,15) < 3.46, p < .05]. The results indicate that the peak expression of iNOS mRNA and
protein and plasma nitrite/nitrate levels occurs at about 8 h
after the injection of LPS.
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Figure 5 shows the effect of naltrexone
on LPS-induced expression of nitric oxide. Figure 5A shows the effect
of LPS on the expression of iNOS mRNA. The analysis of the ratio of
iNOS/-actin showed a significant effect of naltrexone [F(3,16) = 22.37, p < .0001]. There was a significant linear
component to the main effect of naltrexone [F(1,16) = 65.08, p < .0001] indicating that increasing doses of
naltrexone induced a decrease in iNOS mRNA expression. Contrasts showed
that the dose of 0.1 mg/kg naltexone was not significantly different
from the saline treatment, but the 1.0 mg/kg dose of naltrexone was
significantly different from the saline treatment [F (1,16) = 21.07, p < .001]. Moreover, the naltrexone dose of 10 mg/kg
was significantly different from the 1.0 mg/kg doses [F(1,16) = 7.73, p < .03]. The densitometric analysis of the bands
showed no significant effect of naltrexone on the expression of
-actin, indicating that the change in the ratio was the result of a
change in iNOS expression.
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Figure 5B shows the results of the densitometric analysis of the bands from the Western blot for iNOS protein. The analysis showed that naltrexone induced a significant decrease in the expression of iNOS protein [F(3,16) = 22.27, p < .0001]. There was a significant linear component to the effect of naltrexone [F(1,16) = 58.97, p < .0001] indicating that the expression of iNOS protein was linearly related to the dose of naltrexone. The linear effect was confirmed by contrasts that showed the dose of 0.1 mg/kg naltexone was not significantly different from the saline treatment, but the 1.0 mg/kg dose of naltrexone was significantly different from the saline treatment [F(1,16) = 7.24, p < .02]. Moreover, the naltrexone dose of 10 mg/kg was significantly different from the 1.0 mg/kg doses [F(1,16) = 24.01, p < .001].
Figure 5C shows the effect of naltrexone on serum nitrite/nitrate levels in the plasma. The analysis showed that naltrexone induced a significant reduction in the level of nitrite/nitrate in the plasma [F(3,16) = 26.00, p < .0001]. There was a significant linear component to the effect of naltrexone [F(1,16) = 66.53, p < .0001] indicating that the plasma nitrite/nitrate was linearly related to the dose of naltrexone. Contrasts showed that the dose of 0.1 mg/kg naltexone was not significantly different from the saline treatment, but the 1.0 mg/kg dose of naltrexone was significantly different from the saline treatment [F(1,16) = 7.784, p < .01]. The naltrexone dose of 10 mg/kg was significantly different from the 1.0 mg/kg doses [F(1,16) = 24.30, p < .001].
Figure 6 shows the effect of i.c.v.
administration of N-methylnaltrexone on LPS-induced
expression of nitric oxide. Figure 6A shows that the effects of LPS on
the expression of iNOS mRNA. The analysis of the ratio of
iNOS/-actin showed that N-methylnaltrexone induced a
decrease in the ratio [F(4,15) = 8.00, p < .0001].
Moreover, there is a significant linear component to the main effect of N-methylnaltrexone [F(1,15) = 18.02, p < .001] indicating that increasing doses of
N-methylnaltrexone induced a decrease in iNOS mRNA
expression. Although there was a significant linear trend, the
dose-effect function was more complicated, as contrasts showed that the
dose of 0.01 µg of N-methylnaltexone was significantly different from the saline treatment [F(1,15) = 5.47, p < .05], but the 0.1-µg dose of N-methylnaltrexone was
not significantly different from the saline treatment. Moreover, the
N-methylnaltrexone dose of 1.0 µg was significantly
different from the saline treatment [F(1,15) = 5.06, p < .05], and the 10-µg dose showed an even greater decrease in the
expression compared with the 1.0-µg dose [F(1,15) = 6.50, p < .03]. The densitometric analysis of the bands
showed no significant effect of N-methylnaltrexone on the
expression of -actin indicating that the change in the ratio was the
result of a change in iNOS expression.
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Figure 6B shows the results of the densitometric analysis of the bands from the Western blot for iNOS protein. The analysis showed a significant effect of N-methylnaltrexone on the expression of iNOS protein [F(3,15) = 5.74, p < .01]. Moreover, there was a significant linear component to the effect of N-methylnaltrexone [F(1,15) = 13.94, p < .001] indicating that the reduction in the expression of iNOS protein was linearly related to the dose of N-methylnaltrexone. Contrasts showed that the dose of 0.01 µg of N-methylnaltrexone was significantly different from the saline treatment [F(1,15) = 6.76, p < .03], but the 0.1-µg dose was not significantly different from the saline treatment. The N-methylnaltrexone dose of 1.0 µg was significantly different from the saline treatment [F(1,15) = 6.15, p < .03], and the 10-µg dose showed an even greater decrease in the expression compared with the 1.0-µg dose [F(1,15) = 8.78, p < .02].
Figure 6C shows the effect of N-methylnaltrexone on serum nitrite/nitrate levels in the plasma. The analysis showed that N-methylnaltrexone produced a significant reduction in the level of nitrite/nitrate in the plasma [F(4,15) = 4.56, p < .05]. There was a significant linear component to the effect of N-methylnaltrexone [F(1,15) = 13.33, p < .01] indicating that the reduction in the level of plasma nitrite/nitrate was linearly related to the dose of N-methylnaltrexone. Contrast showed that each dose of N-methylnaltrexone was significantly different from the saline treatment [Fs(1,15) > 5.01, ps < 0.05].
In contrast to the findings with i.c.v. administration of N-methylnaltrexone, the peripheral administration of N-methylnaltrexone (10 mg/kg) did not significantly alter the production of nitric oxide, as measured by iNOS mRNA expression using RT-PCR, Western blot for iNOS protein, and nitrite/nitrate production [Fs < 1], suggesting that peripheral opioid receptors are not involved in the regulation of nitric oxide production by splenocytes.
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Discussion |
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|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
The present study tested the hypothesis that LPS-induced expression of iNOS by splenocytes is modulated through the activation of endogenous opioids in the central nervous system. The initial studies showed that the injection of LPS produced dose- and time-dependent effects on the expression of both iNOS mRNA and protein in the spleen, as well as the accumulation of nitrite/nitrate in plasma. Interestingly, the opioid antagonist naltrexone was found to induce a pronounced dose-dependent reduction in iNOS mRNA and protein expression and the level of nitrite/nitrate in the plasma. Furthermore, the data indicate that the modulation of iNOS expression occurs via central opioid receptors; N-methylnaltrexone administered i.c.v. showed effects similar to those found with peripheral administration of naltrexone. Moreover, the peripheral administration of N-methylnaltrexone did not significantly alter the expression of iNOS mRNA, iNOS protein, or the level of plasma nitrate/nitrite. Taken together, these findings demonstrate that central opioid receptors are involved in the in vivo regulation of splenic nitric oxide production.
These findings raise several important issues, such as how peripheral
injection of LPS stimulates central opioid receptors and how the
activation of central opioid receptors induces alterations in the
production of iNOS by splenocytes. Although the pathways by which LPS
signals the brain have not been conclusively identified, there is
growing evidence for an afferent pathway via the peripheral nervous
system. For example, subdiaphragmatic vagotomy has been shown to
decrease LPS- or interleukin-1-induced fever (Watkins et al., 1994;
Sehic and Blatteis, 1996) and reduced food intake (Bret-Dibat et
al., 1995). Furthermore, subdiaphragmatic vagotomy has been shown to
decrease LPS- or cytokine-induced elevation of ACTH release, plasma
corticosteroid levels, and hypothalamic norepinephrine depletion
(Fleshner et al., 1995; Gaykema et al., 1995) and c-fos
expression in the brain (Wan et al., 1994; Gaykema et al., 1995). Thus,
it is possible that LPS induces activation of opioid receptors in the
brain via the vagus nerve.
The involvement of central opioid receptors in the modulation of
splenic nitric oxide also requires a neural/endocrine pathway by which
central opioid activity induces alterations of immune status. The
sympathetic nervous system and the hypothalamic-pituitary-adrenal axis
are two possible pathways that may mediate central opioid-induced immune alterations. Activation of central opioid receptors has been
shown to increase the activity of both the sympathetic nervous system
and the hypothalamic-pituitary-adrenal axis (George and Way, 1959;
Appel et al., 1986). Moreover, lymphoid organs are innervated by
sympathetic nerve fibers (Felten et al., 1985), and immunocytes express
functional alpha and beta adrenergic receptors with which the sympathetic neurotransmitters epinephrine and
norepinephrine interact (Hadden et al., 1970; McPherson and Summers,
1982; Abrass et al., 1985). The evidence from our laboratory indicates
that beta adrenergic receptor antagonists block
opioid-induced decreases in splenic lymphocyte proliferation,
suggesting that some of the central effects of opioids are mediated by
the sympathetic nervous system (Fecho et al., 1993). In addition, both
chlorisondamine, a ganglionic blocker, and adrenalectomy antagonize the
suppressive effects of morphine on the proliferative response of
splenic lymphocytes (Fecho et al., 1996b). There also are a number of
in vitro studies showing that exposure to catecholamines induce changes
in macrophage activity. For example, it has been shown that epinephrine
and norepinephrine stimulate murine macrophages to suppress the growth of Mycobacterium avium (Miles et al., 1996). Other studies
have shown that norepinephrine and epinephrine increase tumor
necrosis factor and hydrogen peroxide production in rat
macrophages (Hu et al., 1991; Costa Rosa et al., 1992). These results
suggest that sympathoadrenal activity may be involved in the
alterations of nitric oxide production after the administration of LPS.
This suggestion is consistent with findings showing that the systemic injection of LPS at doses between 10 and 100 µg to rats induces an
increase in splenic nerve activity up to about 175% of the control
level (MacNeil et al., 1996).
Nitric oxide plays an important role in disease processes. For example,
the role of nitric oxide in infectious disease is demonstrated by
studies showing that it is involved in the cytostatic activity of
macrophages on parasitic growth (Vincendeau et al., 1992). Moreover,
mice lacking inducible nitric oxide synthase have markedly reduced
resistance to parasitic and bacterial infection (MacMicking et
al., 1995; Wei et al., 1995). In addition to the direct impact of
nitric oxide on infectious organisms, nitric oxide has important
regulatory functions in the immune system. There is evidence strongly
supporting a role for nitric oxide in the suppression of antibody
formation to tetanus toxoid and sheep red blood cells after
immunization with Salmonella typhimurium (Al-Ramadi et al.,
1992; Eisenstein et al., 1994). In addition, studies have shown that
nitric oxide limits the proliferative activity of lymphocytes (Albina
and Henry, 1991; Pascual et al., 1993). The present work suggests that
opioid antagonists can be used to regulate nitric oxide production in
vivo. This suggestion is consistent with other studies showing that
ventriculocisternal administration of naloxone protects against severe
hypotension during endotoxin shock (Janssen and Lutherer, 1980).
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Footnotes |
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Accepted for publication August 20, 1998.
Received for publication March 17, 1998.
1 This research was supported by National Institute on Drug Abuse Grants DA10167 and DA07481. D.T.L. is the recipient of a Research Scientist Development Award DA00334 from the National Institute on Drug Abuse.
Send reprint requests to: Donald T. Lysle, Ph.D, Department of Psychology, Davie Hall, CB#3270, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3270. E-mail: dlysle{at}isis.unc.edu
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Abbreviations |
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iNOS, inducible nitric oxide synthase; i.c.v., intracerebroventricular; RT, reverse transcription; PCR, polymerase chain reaction; LPS, lipopolysaccharide.
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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induced corticosterone elevation and hypothalamic NE depletion is vagally mediated.
Brain Res Bull
37:
605-610[Medline].-activated macrophages.
J Immunol
148:
1792-1796[Abstract].
-adrenergic receptors simulates the anti-mycobacterial activity of murine peritoneal macrophages.
J Neuroimmunol
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19-24[Medline]. to inhibit macrophage cytotoxic activity.
J Immunol
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3578-3582[Abstract].This article has been cited by other articles:
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