ARTICLE
INTRODUCTION
Metabolic alterations following infection and endotoxins are known to
be mediated by cytokines. However, the importance of individual cytokines
is not clearly understood. In fact, the role of those cytokines involved
in LPS-induced metabolic changes and the pathway for these processes is
an area of controversy [1, 2], and the way they act to promote hypermetabolism
and/or anorexia requires further investigation [3, 4]. A number of different
cytokines such as TNF-alpha, IL-1 and IL-6 have been implicated in the
regulation of these two processes [3-7]. Some of which are thought to
act by intermediate factors such as corticotrophin-releasing factor [8,
9]. There is evidence indicating that infection and LPS hypermetabolism
and anorexia are induced by different cytokines. It must however be taken
into account that most of the cytokines have cross-regulatory capacities
to induce or inhibit each other, resulting in it being difficult to attribute
the physiological consequences of a single cytokine. A lower quantity
of cytokine is required when injected in centrally rather than peripherally
to obtain responses of the same intensity, suggesting the importance of
the brain in regulation of the physiological responses [5]. The use of
TNFR knockout (KO) [10], IL-1 KO [11] and IL-6 (KO) mice [12] shows that
the lack of these cytokines does not inhibit the anorectic responses to
LPS or endotoxic shock. In contrast, IL-1 and IL-6 KO models [11, 12]
have shown that these two cytokines are the mediators of anorexia in turpentine
model of sterile inflammation.
The present findings show that, following LPS administration, the hypermetabolic
response is dependent on TNF-alpha, LT-alpha, and IFN-gamma, but anorexia
rather involves IFN-gamma. Hypermetabolism was always associated with
increased cytokine expression in the central nervous system, which was
not the case for LPS-induced anorectic effect.
MATERIALS AND METHODS
Mice and diets. We used transgenic mice expressing
TNFR1-IgG3 of C57BL/6 background (generated as described [13]), and their
negative littermates were used as control. We also used TNF-alpha KO mice
[14], LT-alpha KO mice [15] and IFN-gamma R KO mice [16], control mice
were of C57BL/6 or Sv129 genetic background. A group of wild type mice
was treated with mouse anti-interferon-gamma antibody R4-6A2 (ATCC) (500
µg), 4 hours prior to LPS injection. Anti-asialo GM1 antibody (Cederlane
Labs, Ontario, Canada) was used to delete natural killer cells. Anti-asialo
GM1 antibody was injected i.p., one day prior LPS injection (100 µl).
This antibody batch had been tested for optimal in vivo depletion
of NK-like activity with a minimal effect on other cell types. The result
of this titration experiment is that at 1/2 the concentration used here,
the anti-asialo GM1 antibody resulted in abrogation of natural killer
cell activity to zero percent against the YAK tumor cells as tested in
vitro (Cerderlane). Rabbit antisera (Cedarlane) and mouse IgG2b (Sigma,
Buchs, Switzerland) were used as control for in vivo studies, in
the same concentrations as anti-asialo GM1 and anti-IFN-gamma.
Mice of both sexes, at 10 to 14 weeks of age, were used and kept at
room temperature with a 12:12 hours light-dark cycle. The mice were housed
in polycarbonate cages and had free access to tap water and standard laboratory
diet.
Food intake and energy expenditure in response to LPS in TNFR1-IgG
transgenic mice. Mice were kept in groups of four per cage
(n = 20-24), and food intake was determined at various periods of time
by calculating the difference between the amount of food given and that
removed from the cage. Energy expenditure (EE) was measured using open-circuit
indirect calorimetry with computerized equipment. The calorimeter includes
six chambers maintained at 29° C. The values for O2 consumption
were automatically and sequentially recorded every minute in each chamber
[17]. Mice were acclimatized to the metabolic chambers, two days before
the start of the experiment. Within each group, EE was measured in subgroups
of four mice per chamber, over ten hours. Lipopolysaccharide of Escherichia
coli 055:B5 (LPS, endotoxin; Difco Bacto) dissolved in phosphate-buffered
saline (PBS) (Boehringer Mannheim, Switzerland), 5 µg/g body weight
was injected intraperitoneally after the mice had spent 4 hours in the
calorimeter. The first 15 min after injection were not included in EE
determinations and the last 4 hours were used to compare the EE amongst
groups. EE were calculated off-line using Weir's equation. The values
were then averaged during 60 1-min sampling periods. Injection of the
vehicle buffer (PBS) under the same conditions had no detectable effect
on EE in the control or mutant mice.
Determination of circulating cytokines
and tissue cytokine mRNA expression. Blood was obtained
by retro-orbital sinus puncture from mice at 0, 30, 90 and 240 min after
LPS or vehicle (PBS) injection to assess the circulating levels of TNF-alpha,
IL-1beta, IL-6, and IFN-gamma. Each group was injected ip with either
5 µg/g body weight of LPS in 500 µl of PBS or with the vehicle
PBS alone. After centrifugation, serum samples were immediately aliquoted
and frozen at - 20° C until analysis. Bioactive serum TNF-alpha was
determined colorimetrically by its toxicity to L929 cells in an in
vitro assay [18], and serum positive for TNF-alpha was confirmed by
repeating the assay using rabbit polyclonal antiserum against murine TNF-alpha
as a control. One cytotoxic unit was defined as the highest dilution of
test material that causes 50% death of L929 cells. This corresponded to
5 pg/ml of murine recombinant TNF (detection limit of this assay), and
the titer was expressed as picograms per ml of serum. Total serum TNF-alpha
and IL-1beta were measured by the murine IL-1beta and TNF-alpha Immunoassay
kits (Quantikine M from R&D Systems; UK) using recommended protocols.
All assays were run in duplicate. IL-6 was measured using the murine B-cell
hybridoma B9 cell line which requires IL-6 for survival and proliferation
[19]. Proliferation of B9 cells was determined by using (3H)
thymidine incorporation and scintillation counting. Positive samples were
confirmed by using anti-IL-6 antibody. Recombinant murine IL-6 was used
as control to calibrate the endpoint of titration.
To determine the amount of cytokine in tissues 90 min after LPS, brains
were aseptically removed and immediately placed in dry ice. They were
then put in CHAPS solution and homogenised [17], the supernatant was collected
and frozen at - 20° C. Cytokines were immunoassayed for TNF-alpha
and IFN-gamma using Quantikine Murine kits. Cytokine mRNA expression levels
in the spleen and brain were determined 90 min and 2 days after LPS by
dot blots as detailed previously [20].
Blood-brain barrier permeability. Thirty minutes
after LPS, mice were injected retro-orbitally with 100 µl of 1% Evans
blue in PBS and 90 min after LPS, they were sacrificed. Before excision
of the brain, the organs were washed by infusion of PBS into the left
heart ventricle. Following extraction of Evans blue from brain with 0.4%
NaSO4 (0.6 ml) and 1.4 ml of acetone, quantification was performed
by measurement of O.D. at 620 nm [21].
Statistical analysis. Data are presented as means ±
SE. Statistical analysis was performed using Student's t-test.
RESULTS
Metabolic changes following LPS injection. To determine
the role of TNF-alpha in the metabolic changes induced by endotoxin, we
studied LPS-induced hypermetabolic response by measuring the energy expenditure
(EE), and the anorectic response by monitoring the food intake and the
body weight. We used transgenic mice expressing high levels (100 µg/ml)
of soluble TNFR1-IgG chimaeric protein which is able to neutralize LPS-induced
TNF-alpha [13]. In control mice, LPS induced an increase in the absolute
level of EE which peaked at about 1 hour post-injection and remained elevated
over 4 hours as illustrated in Figure
1A. In the TNFR1-IgG transgenic mice, the increase in EE after LPS
injection was only 50% of that in controls and was statistically different
(P < 0.02 at 1 and 4 hours). However, at 2 days post-injection, there
was no statistically significant difference between the two groups (Figure
1B). Injection of the vehicle alone had no effect on EE (data not
shown).
One day after LPS administration, body weight and food intake were decreased
to similar extents in both groups of mice (Figure
1C and D). However, a more rapid recovery of food intake and body
weight, towards pre-LPS levels were observed in transgenic mice. At 2
and 4 days post-injection, body weight and food intake changes were significantly
different between control and TNFR1-IgG transgenic mice (P < 0.04).
These results suggest that TNF-alpha is required for maximal hypermetabolic
(EE) response, but that the maximal anorectic effect of LPS is not dependent
on TNF-alpha.
To discriminate between the differential effect of cytokines involved
in hypermetabolism and anorexia following endotoxin administration, TNF-alpha
KO and LT-alpha KO mice were used. It is to be noted that TNFR1-IgG also
binds to LT-alpha. Since IFN-gamma is an important mediator of septic
shock [22], we also used IFN-gamma R KO mice as well as mice treated with
anti-IFN-gamma antibodies. The EE was measured for 4 hours following LPS,
integrated over this period and expressed as percentage of the mean EE
value obtained 2 hours prior to LPS (Figure
2A). As can be seen, the increase in EE is blunted as expected in
TNF-alpha KO mice but, more surprisingly, in LT-alpha KO mice. IFN-gamma
is also shown to play a role in the EE response since in both anti-IFN-gamma
treatment and IFN-gamma R KO mice, this response was markedly decreased
compared to control mice. Food intake was measured on day 2 after LPS
and expressed as percentage of daily food intake prior to LPS (Figure
2B). Comparison between groups was done at day 2 of LPS administration,
since at this time period TNFR1-IgG transgenic mice started to eat again,
while control mice did not (Figure
1D). All groups of mice started to eat by day 2, and no striking difference
was observed between control, TNF-alpha KO and LT-alpha KO mice. More
marked was the significant resumption of food intake in IFN-gamma R KO
and anti-IFN-gamma treated mice compared to the other three groups. To
investigate if natural killer (NK) cells play a role in the LPS-induced
metabolic response, we treated mice with an anti-asialo GM1 antibody.
EE and food intake of these mice, in response to LPS, were similar to
those in IFN-gamma R KO mice, suggesting that these lymphocytes play an
important role in the metabolic response to LPS, and that cells depleted
by anti-Asialo GM1 antibody are an important source of IFN-gamma. These
results confirm that TNF-alpha, LT-alpha and IFN-gamma are required for
LPS-induced hypermetabolism, and indicate that TNF-alpha and LT-alpha
are not the major appetite-suppressing cytokines, a feature which is rather
attributed to IFN-gamma.
Serum and brain cytokines levels. The serum cytokine
kinetics induced by LPS were followed in control and in TNFR1-IgG transgenic
mice and are illustrated in Figure
3. Transgenic mice showed a complete lack of TNF-alpha bioactivity.
IL-6 bioactivity was only observed at 240 min, and was 18-fold decreased
when compared to control mice. IFN-gamma, was mainly elevated in control
mice at 240 min. In TNFR1-IgG transgenic mice, IFN-gamma was detected
at 240 min with a 5-fold reduction in serum levels when compared to control
mice. IL-1beta kinetics were similar in both groups of mice at 90 min
and reduced at 240 min in transgenic compared to control mice. These circulating
cytokine measurements demonstrate that TNF-alpha plays an important role
in their up-regulation following LPS injection (Figure
3).
We also investigated immunoreactive TNF-alpha by ELISA in serum and
brain of the transgenic mice, as well as in TNF-alpha KO, LT-alpha KO,
and IFN-gamma R KO mice. Following LPS injection, upregulation of TNF-alpha
was also observed in control mouse sera (6,235 pg/ml, 90 min after LPS).
In all other groups of mice the values were below 20 pg/ml. In the brain,
TNF-alpha was also increased in control mice (380 pg/ml, 90 min after
LPS) and IFN-gamma was only found in serum (35 pg/ml) of control mice
at 90 min after LPS injection. Together, these results indicate that TNF-alpha,
LT-alpha and IFN-gamma influence each other production.
Cytokine mRNA expression in tissues. Cytokine mRNA
expression was measured in brain and spleen 90 min after LPS, when the
EE was already at a maximal level in LPS-treated control mice. TNF-alpha,
IL-1 and IL-6 mRNA expressions were reduced in the spleen of TNFR1-IgG
transgenic mice when compared to that of control mice (Figure
4A). In contrast, these expressions were only detected in the brain
of control and not in transgenic mice (Figure
4B). On day 2 after LPS, no cytokine mRNA expression was detected
in the two groups of mice. Therefore, an increase in EE is associated
with expression of these cytokines in the brain, whereas peripheral cytokine
expression is associated with the anorectic effect of LPS in TNFR1-IgG
transgenic mice.
Blood-brain barrier permeability. As can be seen
in Figure 5, only wild type mice
showed a marked Evans blue accumulation in their brains 90 min after LPS,
thereby indicating that the permeability of the blood brain barrier of
these mice was clearly increased. Apart from the already mentioned differences
in cytokine expression, between control and TNFR1-IgG transgenic mice,
there is an increased blood-brain barrier permeability. This is associated
with the enhanced EE found mainly in control mice, rather than with the
anorectic response induced by endotoxins.
DISCUSSION
The pathways involving metabolic responses to LPS, from the onset of
induced cytokine expression to the final physiological response, are complex,
but some aspects can be elucidated from this study. Ninety minutes after
LPS injection, control mice are hypermetabolic and anorectic, they express
cytokines peripherally and centrally. This is in marked contrast to TNFR1-IgG
transgenic mice, which show low or undetectable levels of cytokines associated
with the lower EE response. These results suggest that higher levels of
cytokines are associated with the onset of hypermetabolism, whilst lower
levels or their absence induce anorexia, which is similar to the different
dose response to induction of fever or anorexia [23]. The results of our
study also support the view that peripheral cytokine expression precedes
central cytokine expression and the latter is associated with hypermetabolism.
Central cytokine involvement appears of greater importance in LPS-induced
hypermetabolism than in anorexia. However, the role of other circulating
factors influencing the onset of hypermetabolism cannot be disregarded
since the blood-brain barrier is significantly modified when mice are
hypermetabolic.
Our data show that TNF-alpha and IFN-gamma play differential but pivotal
roles in anorexia and hypermetabolism. TNF-alpha and IFN-gamma up-regulate
each other, offering a possible explanation as to why no hypermetabolism
is seen in IFN-gamma R KO mice and anti-IFN-gamma treated mice. Indeed,
a strong reduction of TNF was previously reported in IFN-gamma R KO mice
after LPS administration [22]. The importance of IFN-gamma in the regulation
of LPS-induced anorexia is in line with other studies involving its role
in metabolic changes such as wasting [26-28], as well as in LPS-mediated
cachexia [22]. The results obtained ruled out a direct and major role
for TNF-alpha and LT-alpha in appetite suppression, however they highlight
the importance of TNF-alpha and more surprisingly of LT-alpha in the stimulation
of energy expenditure. Our results indicate that LPS activates lymphocytes
to produce LT-alpha, which may act in synergy with TNF-alpha in inducing
energy expenditure. This work also discerns a specific association between
IFN-gamma and TNF-alpha with hypermetabolism, but only IFN-gamma appears
to be involved in anorexia.
These studies show that IFN-gamma is not expressed in the brain of control
mice and hence its effects are mediated by peripheral expression. On the
other hand, TNF-alpha (and IL-1beta and IL-6) are expressed both peripherally
and centrally, hence they may mediate their effects in either compartment.
Since LPS increased blood brain barrier permeability in a TNF-alpha IFN-gamma
dependent manner, it cannot be ruled out that this response may be the
result of other synergistic cytokines [27] and/or blood-plasma borne products
that enter the brain.
The anti-IFN-gamma treatment experiment confirmed results in IFN-gamma
R KO mice showing that EE and anorexia are IFN-gamma dependent mechanisms.
Comparative results were obtained using the anti-asialo GM1 antibody,
removing most of the NK cell population and suggesting that these cells
are involved in LPS-induced IFN-gamma production. An essential role for
NK cells in lethal, LPS-induced Shwartzman-like reaction associated a
marked reduction of IFN-gamma has been reported (28). The effect observed
on NK cells can be direct or indirect. For instance, indirect effects
due to missing mediators required for the synthesis of IFN-gamma such
as IL-12, IL-15, or IL-18 can be implicated [29-31]. It has to be noted
that anti-asialo GM1 antibody can react with other cell types such as
cytotoxyc T cells and activated macrophages, the former of these activities
was observed at high antibody concentrations [32]. Indeed, we cannot exclude
the contribution of these cells to LPS-induced cytokine production. In
conclusion, our studies show that IFN-gamma plays a pivotal role in LPS-induced
anorexia, and although the exact cellular subset source remains to be
identified, natural killer cells appear to be a candidate.
Whether it is the IFN-gamma or natural killer cell product that directly
induces these effects remains to be elucidated, however GM-CSF KO mice
[33] have been shown to be resistant to LPS by a mechanism implicating
IFN-gamma. Although this is not proof that IFN-gamma is directly involved
in hypermetabolism or anorexia, a recent investigation in humans showed
that a low dose of IFN-gamma is associated with hypermetabolism but not
anorexia [34], similar to what was observed in our model of toxoplasmosis
[20]. This suggests dose-dependent effects of IFN-gamma on host metabolism.
The complexity of cytokines and LPS-induced metabolic responses is further
emphasized. Increasing LPS doses may actually change the mechanisms that
regulate the response, since at low doses IFN-gamma R KO mice show marked
differences in anorexia and body weight compared to wild type mice in
response to LPS, whereas at higher doses, differences between the two
groups are lost [22]. One must therefore consider the possible role of
(i) other mechanisms that are LPS dose-dependent that induce differential
cytokines from different cell sources, (ii) other mediators that are produced
late in response to LPS such as MIF [35], IL-18 [36], HMG-1 [37] and (iii)
non LPS-receptor mediated mechanisms can also be implicated since CD14
KO and TLR4 KO mice, respond to LPS but only at very high doses [38, 39].
CONCLUSION
In summary, the present work shows that LPS-induced hypermetabolic response
is dependent on TNF-alpha, LT-alpha, and IFN-gamma. Hypermetabolism was
shown to be associated with increased cytokine expression in the central
nervous system, whereas anorexia, which was mainly dependent on IFN-gamma,
was rather associated with peripheral cytokine expression.
Accepted for publication: 19/06/00
Acknowledgements. This work was supported by Grants 31-47211.96
and 3200-054401.98 from the Swiss National Fund and from the Société
Académique de Genève (to I.G.).
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