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Clinical and Diagnostic Laboratory Immunology, March 2001, p. 446-448, Vol. 8, No. 2
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.2.446-448.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cytokine Expression in the Mouse Brain in Response
to Immune Activation by Corynebacterium parvum
W. S.
Sheng,*
S.
Hu,
J. M.
Ding,
C. C.
Chao, and
P. K.
Peterson
Institute for Brain and Immune Disorders,
Minneapolis Medical Research Foundation, and the University of
Minnesota Medical School, Minneapolis, Minnesota 55404
Received 19 June 2000/Returned for modification 28 August
2000/Accepted 22 November 2000
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ABSTRACT |
Cytokine expression in the brain has been suggested to mediate
various sickness behaviors. Here we report that intraperitoneal injection of a Corynebacterium parvum antigen in C57BL/6
mice was followed by prolonged upregulation of cytokines in the
cerebral cortex and subcortical structures in a time course that
coincided with reduced spontaneous running activity.
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TEXT |
The response of the immune system to
microbial invasion is often accompanied by a series of sickness
behaviors including reduced physical activity, daytime sleepiness,
diminished food intake, and a decline in social activity (4,
8). The exact mechanism(s) underlying these sickness behaviors
is unknown. However, upregulation of cytokine expression in the central
nervous system (CNS) has been postulated to mediate a number of these
behavioral responses to infection (3, 5, 10, 12).
The sensation of fatigue or exhaustion is a prominent symptom in a
number of infectious and autoimmune diseases and has been a disabling
manifestation of certain idiopathic disorders, such as chronic fatigue
syndrome (6). We have developed a mouse model to study
immunologically mediated fatigue using heat-killed Corynebacterium parvum as the immune stimulant (1,
11). Our previous studies have demonstrated that intraperitoneal
(i.p.) inoculation of C. parvum greatly reduced spontaneous
running activity for as long as 2 to 3 weeks and that interleukin-1
(IL-1) and tumor necrosis factor alpha (TNF-
) mRNA expression
was observed in the brain at 10 days postinoculation (11).
We have also shown that peripheral administration of neutralizing
antibodies against IL-1 or TNF- failed to restore the animals'
running activity (11), suggesting that cytokines in the
CNS may play an important mediating role in immunologically mediated
fatigue. In this study, we sought to investigate the kinetics of
C. parvum-induced expression of cytokines in selected brain
regions that may be involved in immunologically mediated fatigue.
Time course of cytokine mRNA expression in the mouse
brain.
Previously, we had shown, by using reverse
transcription-PCR (RT-PCR) analysis, that i.p. inoculation of a
commercially purchased C. parvum antigen in C57BL/6 mice was
associated with expression of TNF-, IL-1, and transforming growth
factor (TGF-) mRNA in the brain at 10 days postinoculation
(11). We now used a quantitative RNase protection assay
(RPA) to evaluate the expression of mRNA of these cytokines and of
IL-1 and TNF receptors in the mouse brain at different time points
after a single i.p. injection of C. parvum antigen (2 mg/mouse). Fifty micrograms of extracted total RNA (2) was
used in the multiprobe RPA according to the manufacturer's protocol
(PharMingen, San Diego, Calif.). Results were quantified for each
cytokine (IL-1, TNF-, TGF-), cytokine receptor (IL-1 receptor,
TNF receptor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH,
used as an internal control) in control (saline injection) and C. parvum-challenged mice. The average ratio of the mRNA of each
cytokine or cytokine receptor to GAPDH mRNA in the control mice was
used as the baseline.
The C. parvum antigen was prepared in our laboratory
according to the protocol from Ribi Immuno-Chem Research (Hamilton,
Mont.) and was shown at a dose of 2 mg/mouse inoculated i.p. to induce a >50% reduction in daily spontaneous running activity over a 2-week
period using an exercise wheel to measure running distance (1) (Fig. 1). The level of
IL-1 mRNA expression, relative to that for the control,
increased about 2-fold at 24 h after C. parvum antigen
injection, peaked at 4.5-fold 15 days after C. parvum
inoculation, and then gradually returned to control levels at 22 days
postinjection (Fig. 2). TNF- mRNA
expression was first detected at 6 days postinjection, peaked at a
level eightfold higher than control values 15 days postinjection, and returned to near the control level by day 22. A similar profile was
found for TGF- mRNA, although at a lesser magnitude. In contrast to IL-1, TNF-, and TGF-, no change in IL-6 mRNA was
detected (data not shown). Expression levels of IL-1 receptor mRNA
and TNF receptor mRNA also showed two- to threefold increases after C. parvum injection and remained elevated throughout the
sampling periods, except that IL-1 receptor mRNA declined to
near-control levels at day 22. The time course of expression of
IL-1, TNF-, TGF-, and IL-1 receptor mRNA levels appeared
to coincide with the reduction of spontaneous running behavior shown in
Fig. 1.
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FIG. 1.
C. parvum (Cp) antigen-induced reduction of
spontaneous running activity. C. parvum antigen at 2 mg/mouse was injected i.p. (0.2 ml) in mice (day 0) after 15 days of
running adaptation. Control mice were injected with saline (0.2 ml).
Running activity was recorded for 30 days after injection. The mean of
preinjection running distances of individual mice was used as the
baseline. Ten mice were used per group.
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FIG. 2.
Cytokine mRNA expression in the brains of C. parvum antigen-challenged mice. Total RNA (50 µg) from control
(saline injection) or C. parvum antigen-challenged mouse
brains at various time points was used in the multitemplate RPA.
Expression of cytokines, their receptors, and GAPDH (as an internal
control) in individual mouse brain was quantified. The average ratio of
cytokine or cytokine receptor to GAPDH in the control mouse was used as
a baseline. There were four mice per group per time period. Asterisks,
expression below detectable threshold.
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Cytokine proteins in specific brain regions.
To determine
whether upregulation of cytokine mRNA following inoculation of
C. parvum antigen was associated with the production of
cytokines at the protein level, an enzyme-linked immunosorbent assay
(ELISA) method was used to measure cytokines in brain tissue (dissected
into cortex, subcortex, and cerebellum) at day 10 post-C. parvum antigen injection. A marked increase in IL-1 and TNF- protein production was found in the cerebral cortex and the subcortical brain region, while no significant change was seen in the cerebellum (Fig. 3). Consistent with the mRNA
findings, no increase in levels of IL-6, which was undetectable, was
found in any of the three brain regions (data not shown).
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FIG. 3.
Cytokine protein production in the cortex (Cx),
subcortex (SCx), and cerebellum (Cb) from C. parvum (Cp)
antigen-challenged mice. Protein extracts from three brain regions of
control (saline injection) or C. parvum antigen-challenged
mice at day 10 postinoculation were assayed for cytokine levels
(expressed as picograms per milligram of brain tissue protein) by
ELISA. *, P < 0.05; **, P < 0.001. Significance was calculated for differences from the
respective control (Student's t test; n = 5 per
group).
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Taken together, the results of these experiments indicate that the
immunologic response to an i.p. challenge with
C. parvum antigen is associated with a relatively rapid and sustained
upregulation
of IL-1
and TNF-
and/or their cognate receptors
within cortical
and subcortical regions of the brain. These findings
contrast
with those of studies of lipopolysaccharide as an immune
stimulant,
which have shown a rapid but transient upregulation of
TNF-
and
IL-1
in the mouse brain (
7,
9). The
observation that cytokine
upregulation in the mouse brain following
C. parvum challenge
was associated with a decrease in
running activity supports the
notion that cytokines within the CNS
serve as mediators of behavioral
responses that are seen in a variety
of infectious and immunologic
disorders. Further investigation is
needed, however, to determine
more specifically which brain structures
and cell types are involved
in cytokine expression and whether this
upregulation of brain
cytokines indeed plays a pivotal mediator role in
the decreased
physical activity observed as a neurobehavioral response
to the
sensation of
fatigue.
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ACKNOWLEDGMENTS |
This study was supported by U.S. Public Health grant AI35110.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Brain and Immune Disorders, Minneapolis Medical Research
Foundation, NeuroImmunology Lab, D-305, 914 South 8th St., D-3,
Minneapolis, MN 55404. Phone: (612) 337-7376. Fax: (612)
337-7372. E-mail: wsheng{at}biosci.cbs.umn.edu.
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Clinical and Diagnostic Laboratory Immunology, March 2001, p. 446-448, Vol. 8, No. 2
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.2.446-448.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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