1 Departments of Nutrition and Medicine, University of Toronto, Toronto, Ontario M5S 1A8; and 2 Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
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ABSTRACT |
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Previous studies showed that weight-gaining rats
had greater retention and reduced turnover of
125I-labeled tumor necrosis factor
(TNF)- in the circulation compared with weight-losing
animals. We therefore tested the hypothesis that protein-energy
restriction with weight loss reduces the levels of soluble TNF-
receptor (sTNFR) and membrane TNFR (mTNFR) and the cellular expression
of TNF-
mRNA. Twenty-six male rats weighing 200-220 g were fed
a liquid formula diet for 10 days and divided equally into
weight-gaining rats meeting all nutritional requirements (WG rats) and
weight-losing rats with protein-energy restriction (WL rats).
125I-TNF-
binding was
demonstrated in plasma and plasma membrane to proteins of molecular
masses of 92 and 243 kDa, a finding identical to that seen with
purified human p55. Excess unlabeled TNF-
displaced the binding
showing its specificity. The degree of binding to plasma protein and
liver plasma membrane was markedly reduced in WL rats. Northern
analysis showed that the expression of p55 mRNA was increased in the
lungs and reduced in kidneys of WL compared with WG rats. The
expression of p75 mRNA was not influenced by the nutritional status. We
conclude that levels of sTNFR and mTNFR were reduced in WL rats.
Reduced sTNFR and liver mTNFR are not due to a reduction in the
expression of either p55 or p75 mRNA in WL rats. Reduced mTNFR,
together with reduced shedding of soluble receptors, may have a
protective role in WL rats.
electrophoresis; Northern blot; probes; plasma membranes
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INTRODUCTION |
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THE CRITICALLY SICK septic patient often exhibits
profound and accelerated signs of nutritional deficiency. Wasting,
hyperglycemia, hyperlipidemia, and intolerance to nutrients have all
been observed (5). However, the ability to nourish these patients is
often hindered because of an altered nutrient metabolism (18,
49). Cytokines (15), especially tumor necrosis factor
(TNF)- (7), interleukin-1, and interleukin-6, are associated with
either sepsis or the mediators of a number of metabolic phenomena
observed in sepsis (14, 31, 35, 43, 44). Furthermore, in sepsis and
other catabolic states such as heart failure, circulating TNF-
levels are increased (28). TNF-
is secreted by macrophages and
travels via the circulation to distant sites in the body, where it
binds to two receptors on the cell surface, the 55-kDa TNF-
receptor
(TNFR)-I and the 75-kDa TNFR-II (13, 39), exerting discrete metabolic
effects. The major tissue targets of TNF include the liver, skin,
kidneys, lungs, and gastrointestinal tract (8). TNFR-I and TNFR-II are
coexpressed on the surface of most cell types at comparable levels (11,
38), and both are proteolytically released as soluble molecules capable
of binding TNF. Experiments using receptor-specific
antibodies (51) and mice genetically deficient in either
p55 or p75 (17, 45) indicate that p55 is the primary signaling receptor
on most cell types through which the majority of inflammatory responses
classically attributed to TNF occur. In contrast, TNF-induced thymocyte
proliferation and apoptosis of activated mature T lymphocytes are
mediated by p75 (57, 62). A model was suggested whereby p75 acts by
"ligand passing" to provide increased local concentrations of
ligand for p55, which initiates intracellular signaling (58). The roles of TNFR and associated signaling pathways in mediating the diverse actions of TNF remain incompletely defined. There is recent evidence of
a mitochondrial-binding protein for an extracellular ligand and the
presence of a pathway capable of delivering TNF from the cell surface
to mitochondria (34). These findings suggest that TNF effects on cells
may be due in part to a direct effect on mitochondria.
A variety of inflammatory stimuli trigger the shedding of soluble p55 and p75 receptors from the cell surface into the circulation through proteolytic processing by a metalloproteinase that is located near the plasma membrane and is activated by protein kinase C (3, 36). So far, the exact function of soluble TNFR (sTNFR) is not known, although there are several speculations. Circulating sTNFR can function either as TNF antagonists, capable of neutralizing TNF bioactivity, or as TNF agonists, by prolonging the circulating half-life of TNF or by facilitating the interaction of TNF with membrane-bound receptors. An agonistic role of sTNFR is supported by the observation of Aderka et al. (1), who have suggested that sTNFR could prolong the biological effect of TNF by stabilization of the molecule and by formation of a "slow-release reservoir."
We have previously shown that nutritional manipulations
causing weight gain or loss altered the metabolic response to TNF- infusion in the rat model (48). Orally fed rats infused with TNF-
(23) developed anorexia and weight loss with the restriction of
protein-energy intake. Despite the weight loss, there was no mortality
or morbidity in these rats. In contrast, when weight gain was induced
in TNF-
-infused rats by parenteral or enteral nutrition without any
restriction of protein-energy intake, the rats became hyperglycemic and
azotemic, conditions which are associated with an increase
in mortality (40, 48). Correspondingly, in the same model, the
nutritional status altered the distribution of labeled TNF-
; namely,
the retention of TNF-
in the circulation was markedly increased in
weight-gaining but reduced in weight-losing animals (29). We
hypothesized that the difference was due to the effect of nutrition on
the concentration of membrane receptors and subsequently on the
concentration of circulating receptors. Because the circulating TNF-
is partly bound to soluble TNFR and because the cellular effects of
TNF-
are mediated by membrane TNFR, it was necessary to study the
effect of nutrition on TNFR expression. The present study was designed,
therefore, to test the hypothesis that nutritional status associated
with weight gain or loss altered the expression of membrane TNFR
(mTNFR) and sTNFR. In addition, we also attempted to determine whether
nutrition influenced the steady-state expression of mRNA for both the
p55 and p75 receptors.
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MATERIAL AND METHODS |
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Animals
Male Wistar rats (Charles River Canada) were housed in individual cages in a temperature-controlled room (22°C) with a 12:12-h light-dark cycle. On entry to the animal facility, rats weighed 200-220 g. They had free access to rat nonpurified diet (Purina Rodent Chow 5001; Ralston Purina, Strathroy, ON, Canada) for 1 wk.Diet Fed During Study
Seven days after acclimatization to the new environment, the rats were given the liquid formula diet (Table 1). To promote weight gain or loss, the rats were fed diets of different energy densities, keeping the volume and micronutrient (trace elements, vitamins, and electrolytes) intake constant. The energy density in the weight-loss group was reduced by decreasing all three of the major nutrients, namely, protein, carbohydrate, and fat contents, of the diet by 75%. The rats were randomly allocated to the following groups: weight-gaining rats (WG; n = 13) received orally a defined formula diet with an energy density of 0.35 MJ/60 ml ad libitum. Weight-losing rats (WL; n = 13) received orally a defined formula diet with a restricted energy density of 0.09 MJ/60 ml ad libitum.
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All animals were observed carefully, weighed daily, and allowed to take water freely throughout the experiment. A daily record of dietary intake was maintained for 10 days. The protocol was approved by the University of Toronto Animal Care Committee.
Blood and Organ Collection
After 10 days of being on the study diets, six rats from each group were anesthetized with pentobarbital sodium (6 mg intravenously), exsanguinated by abdominal aortic puncture, and killed by cervical dislocation. The blood was drawn into a heparinized syringe and centrifuged. The plasma was separated and stored atIsolation of Liver Plasma Membranes
Hepatocyte plasma membranes were isolated from the remaining seven rats from each group in accordance with the method of Hubbard et al. (24). The rats were anesthetized with pentobarbital sodium (50 mg/kg), and their livers were excised intact and perfused through the portal vein with ice-cold 0.154 M NaCl until blanched. All subsequent procedures using sucrose gradient were carried out at 0-4°C. Purity of plasma membranes was confirmed by measuring the 5'-nucleotidase activity by a kinetic method, using stabilized glutamate dehydrogenase (2). Touster et. al. (59) reported that the 5'-nucleotidase is the most specific marker for the plasma membrane. They observed that the enzymatic yields and relative specific activities of 5'-nucleotidase activity in the isolated fractions of plasma membranes were high compared with other enzymatic markers.Labeling of Recombinant Rat
TNF-
Protein Measurements
Total protein in plasma and plasma membranes was measured by the biuret method (19) with the use of BSA as a standard.Measurement of Receptors
Because reagents for radioimmunoassay in rats are not available, the level of TNFR in blood plasma and liver plasma membrane was estimated by cross-linked binding with recombinant human or rat 125I-TNF-Solubilization of TNFR. After cross-linking with BS3, the TNFR-ligand complex was suspended in 3 µl of solubilization buffer consisting of 50 mM Tris, pH 7.5, supplemented with aprotinin (0.2 mg/ml) and Triton X-100 detergent at a final concentration of 1% (vol/vol).
Analytic SDS-PAGE.
Before electrophoresis, the samples were mixed with the sample buffer
(SDS reducing buffer) for 5 min at 95°C. A total of 180 µg of
plasma and 60 µg of plasma membrane were loaded in each lane of the
electrophoresis gel. Samples were subjected to electrophoresis in the
presence of 10% SDS on 4- to 15%-acrylamide gradient gels with the
use of the method of Laemmli (32) at a constant current of 25 mA.
Concurrent with the plasma and plasma membrane samples, native and
cross-linked 125I-TNF- samples
without plasma or plasma membrane were run as controls. Molecular mass
standards were used to identify the approximate region of the gel where
the labeled receptor was located. To demonstrate that the binding was
specific, 180 µg of the same plasma samples were mixed with an excess
of unlabeled TNF-
and run concurrently. In addition, the positions
of the bands demonstrating TNF binding were compared with those
obtained by cross-linking
125I-TNF-
to purified human
TNFR-I (p55).
RNA Separation and Northern Blot Analysis
RNA isolation and Northern blotting. Total RNA was isolated from 300 mg of tissue by the method of Chomczynski and Sacchi (12). A value of 10 µg of RNA was subjected to electrophoresis in 1% formaldehyde-agarose gel. The separated RNA was transferred to a nitrocellulose membrane by capillary elution. The membrane was baked at 80°C for 1 h under vacuum, washed and prehybridized at 42°C for 4 h, and then hybridized for at least 24 h with labeled probes in 50% formamide, 4× Tris-phosphate buffer, 0.1% SDS, 0.5 M EDTA, and 100× Denhardt's solution plus 100 µg/ml salmon sperm DNA at 42°C. The blot was washed twice for 10 min at room temperature in 1× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) in 0.1% SDS and for 20 min in 0.1× SSC in 0.1% SDS. Further washes at 42-55°C were performed as required. The blots were subjected to autoradiography and image analysis as described in Analytic SDS-PAGE.
Plasmids. The specific probes used were 2.1- and 1.5-kb cDNA fragments encoding human p55 and p75, respectively, expressed in a pCMV plasmid, which was kindly donated by Dr. L. Moldawer (Univ. of Florida, Gainesville, FL). p55 and p75 were labeled by random-primer extension with [32P]dATP (Amersham). In addition, 18S cDNA was used as a loading control in a second hybridization of the same blots originally hybridized with p55 and p75 cDNA. Although our studies are in the rat, the human and murine nucleotide sequences are sufficiently homologous to produce a strong, specific signal (30).
Statistical Analysis
Data for dietary intake and body weight are expressed as means ± SE. The differences between WG and WL were compared with the use of the Student's unpaired t-test. The differences were considered significant at P < 0.05. ![]() |
RESULTS |
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Nutrient Intake
The cumulative dietary intake over 10 days was significantly higher (P < 0.01) in WG (3.3 ± 0.07 MJ) compared with WL (1.27 ± 0.10 MJ) rats.Body Weight
The initial weights of WG and WL rats were 264.8 ± 5.37 and 262.9 ± 3.56 g, and the final weights of WG and WL rats were 304.9 ± 6.92 and 193.0 ± 10.8 g, respectively. The changes in body weight were consistent with the dietary intake. There was a 15.25 ± 1.7% increase and a 26.81 ± 3.5% decrease in the body weights of WG and WL rats, respectively, from their initial body weight over a 10-day period. These differences were statistically significant (P < 0.01).Preliminary Results in Determining Specificity of TNFR-Ligand Complex
Specificity of
125I-TNF-
binding.
Specificity of plasma TNFR-ligand complex was shown by comparing the
binding of human 125I-TNF-
to
plasma in the absence and presence of an excess of unlabeled TNF-
. A
quantity of 3 µl (60 µg/µl) of WG rat plasma was cross-linked
with 125I-TNF with or without the
addition of 8 µl (2 µg/µl) of unlabeled TNF-
, followed by
SDS-PAGE. The first three TNF lanes, which had
125I-TNF-
plus cross-link,
resolved into 1) a prominent band
corresponding to a molecular mass of 17 kDa,
2) the TNF monomer at 34 kDa
corresponding to the dimer, and 3) a
smaller band at 51 kDa corresponding to the trimer (Fig.
1). In lanes
4-6, in which
125I-TNF-
was cross-linked to
plasma, other bands were observed in addition to the above three bands:
at ~243 kDa, at 92 kDa, and a smaller band at 138 kDa. These bands
were markedly attenuated when incubated with unlabeled TNF-
in
lanes
7-9. This pattern of two prominent bands at 92 and 243 kDa and a smaller band between these masses was observed in all subsequent data in which plasma or
human p55 receptor was cross-linked to
125I-TNF-
.
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Relationship of quantity of TNFR-ligand complex
with increasing volume of plasma.
The quantitative nature of the observed density of the receptor-ligand
complex was demonstrated by either the ratio of density of the area
from 92 to 243 kDa to the area under the 17- to 51-kDa region or the
ratio of the area around the 243-kDa band to the area of the 17- to
51-kDa region (see Fig. 2B).
Quantities of 3, 4, 5, and 6 µl of plasma were cross-linked with
human 125I-TNF and subjected to
SDS-PAGE (Fig.
2A,
lanes
2-5). With
increased plasma concentration, the visual intensity of the bands
increased from lanes 2 to
5. The ratio of the bound to unbound
area, calculated as indicated above, also increased in a linear way
with an increasing amount of plasma (Fig.
2B). Similar results were obtained
when plasma was cross-linked with rat
125I-TNF (data not included).
Prominent bands at 92 and 243 kDa as well as a smaller band between the
masses were seen. The intensity of the bands increased with an
increasing amount of plasma.
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TNFR-ligand complex with p55 and displacement with
unlabeled TNF.
To determine whether the findings in rat plasma were similar to those
with pure receptor, 125I-TNF-
was cross-linked to pure human p55 receptor. Lane
1 shows the previously described features of only
125I-TNF-
. Human p55
(recombinant human sTNFR-I; R&D Systems, Minneapolis, MN) cross-linked
with 125I-TNF-
showed bands at
~92 and 243 kDa [Fig. 3,
A (lanes
2, 4, 6 and
8) and
B (lane
8)] that were attenuated by the addition of
unlabeled TNF-
[Fig. 3, A
(lanes 3,
5 and
7) and
B (lane
7)]. The pattern was similar to those seen with
rat plasma except for minor changes as seen with the binding of human
p55, which did not show a band at 138 kDa. This difference could be due
to species specificity.
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Nutritional Effects on Rat
TNF--Soluble Receptor-Ligand Complex
Rat TNFR-ligand complex in orally fed WG and WL
rats.
Prominent binding at 243 kDa was seen when plasma of WG rats was
cross-linked to rat 125I-TNF-
(Fig. 4, A
and B; lanes
2-5,
11, and
12). Plasma of WL rats showed faint
bands of binding at 243 kDa ( Fig. 4, lanes 6-9,
13, and
14). Comparison of Figs. 1 and
4A shows that the pattern and
intensity of binding of rat and human
125I-TNF-
to rat plasma are
similar; both show prominent bands at 92 and 243 kDa. Densitometer
analysis of the bands showed that the ratio of the bound (92- to
243-kDa region) to free (17- to 51-kDa region) area was significantly
(P < 0.05) reduced in the WL rats
compared with the WG rats (Fig. 5).
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Nutritional Effects on Rat TNF-
Liver Membrane Receptor-Ligand Complex
Rat TNFR-ligand complex in orally fed WG and WL
rats.
Prominent binding at 243 kDa was seen when liver plasma membranes of WG
rats were cross-linked to rat
125I-TNF- (Fig.
6, A and
B; lanes
2-5,
10,
11, and
12). In contrast, liver plasma
membranes of WL rats showed faint bands of binding at 243 kDa (Fig. 6,
lanes
6-9,
13,
14, and
15). No bands were seen at the
92-kDa region in any of the rat plasma membrane. Densitometer analysis
of the bands showed that the ratio of the bound (92- to 243-kDa region)
to free (17- to 51-kDa region) area was significantly (P < 0.05) reduced in the WL rats
compared with the WG rats (Fig. 5).
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Northern Blot Analysis of mRNA for sTNFR-I (p55 Receptor) and sTNFR-II (p75 Receptor) in Liver, Lung, Kidney, Heart, and Diaphragm
The expression of p55 mRNA was higher in the lung of WL animals (Fig. 7A, lanes 5-8) compared with those animals gaining weight (Fig. 7A, lanes 1-4). On the contrary, the expression in the kidney was lower in WL animals compared with WG animals (Fig. 7B). Image analysis relating the changes to the expression of 18S mRNA confirmed the visual impression and showed that the higher expression in the lung and lower expression in the kidney were statistically significant. The expressions in the diaphragm, heart, and liver were not influenced by nutrition. The expression of p75 mRNA did not show any significant changes in lung, kidney, diaphragm, heart, or liver of the WG and WL rats.
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DISCUSSION |
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TNF- is the mediator of a number of metabolic phenomena observed in
sepsis, including anorexia (43), fever (14), acute-phase protein
synthesis (44), abnormalities of lipid (31), and glucose metabolism
(35). Wingfield et al. (60) demonstrated by equilibrium centrifugation
that TNF-
exists as a stable trimer in physiological solutions. The
TNF trimer exerts its effects by binding to specific cell surface
receptors, a process that probably involves cross-linking to two or
three molecules of receptors (56). Injection of labeled murine TNF-
into mice results in preferential binding to certain tissues (8). The
highest amounts are found in liver, kidneys, lung, and spleen, and
modest amounts are found in stomach, duodenum, small bowel, pancreas,
colon, and esophagus. The extracellular regions of the two TNFR contain
a repetitive pattern of cysteine-rich domains (37), which can be
liberated as soluble fragments in vitro (47) and in vivo (16). The
liberated soluble fragments circulate as sTNFR, especially after
endotoxin challenge (47, 54). sTNFR are biochemically and
immunologically distinct proteins (22) despite the high-sequence
homology within the extracellular domain (13).
In the present study, the TNFR-ligand complex in rat plasma and in
liver plasma membrane by cross-linking consisted of complexes ranging
from 92 to 243 kDa, indicating the cross-linking to two or three
molecules of receptors (4). This binding was inhibited by the addition
of excess of unlabeled TNF- (Fig. 1), showing the specific nature of
the binding (55). In addition, when purified human p55 receptor was
similarly cross-linked and subjected to electrophoresis, the complexes
migrated in the same region (Fig. 3A) and were similarly displaced by
the addition of unlabeled TNF-
.
Smith and Baglioni (52) have reported a cross-linked species of TNFR of HeLa cells with a relative molecular weight of 330,000, as characterized by gel filtration. The cross-linking of the cell-bound 125I-TNF in another study (61) yielded a complex of 105 kDa for HeLa S2 and THP-1 cells and a complex of 100 kDa for U937 cells. In both of these studies, the cross-linking was inhibited by unlabeled TNF. Smith et al. (53) demonstrated species specificity in the TNFR interaction on murine L cells and human HeLa S2 cells. In addition, the affinity-labeling studies in two species gave an identical pattern for the TNFR complexes with a relative molecular weight of 350,000. Stauber et al. (55) reported that the TNFR-ligand complex in the human histiocytic lymphoma cell line U937 has a subunit molecular weight of 100,000 when examined by SDS-PAGE; however, on gel filtration, the complex migrated with an apparent molecular weight of 480,000. These differences show that the exact pattern of the cross-linked bands varies with the species and may explain the minor differences seen between the rat and human cross-linked patterns as observed in the present study.
Our 125I-TNF- complexes are
consistent with binding to sTNFR in rat plasma and mTNFR in liver
plasma membranes. The technique is semiquantitative, as shown by the
linear increase in complex levels with the increase in the amount of
added plasma. Hence, changes in the degree of binding represent changes
in sTNFR levels in plasma and mTNFR levels in plasma membrane. In the
present study, we have observed decreased binding in weight-losing rats compared with weight-gaining rats in both blood plasma (Fig. 4, A and
B) and liver plasma membrane (Fig.
6, A and
B), indicating that weight loss and
gain were associated with a reduction and increase, respectively, in
the concentration of sTNFR and mTNFR.
To the best of our knowledge, these are the first observations of the effect of experimental protein-energy restriction not complicated by sepsis or by micronutrient deficiency on sTNFR, mTNFR, and TNFR mRNA expression.
Other studies on the effect of nutrition have been complicated by sepsis or the influence of endotoxin challenge. In children with kwashiorkor and edema, TNFR levels were found to be increased, but the observations are complicated by concurrent infection as shown by raised C reactive protein levels. Furthermore, children with marasmus with similar degrees of wasting as those with kwashiorkor had lower levels of TNFR (50). In human volunteers maintained on parenteral nutrition, sTNFR-II (p75) was three times higher after endotoxin administration compared with those fed enterally. Conversely, sTNFR-I (p55) was higher in the enterally fed group (10). However, the human study did not compare the effect of dietary restriction on sTNFR and mTNFR.
It is generally observed that malnourished individuals are more
susceptible to infections; low levels of TNFR, by reduction of the
stabilization of circulating TNF-, may contribute to this possibility. However, the same reduction may protect the
individual from cell injury in situations in which TNF-
productions
are excessive, as in severe sepsis and critical illness. The protective action of TNF-
in this situation becomes counterproductive,
especially when we now have the ability to control sepsis with
antibiotics. In support of this finding, the rats infused with TNF-
developed multisystem organ failure, equivalent to that of the
intensive care unit patients when fed enterally or
parenterally and made to grow normally (40, 48).
Possible Mechanisms of Effect of Nutrition on TNFR Levels
In contrast to the profound reduction in sTNFR and mTNFR in weight-losing animals, the expression of mRNA for the p55 receptor was significantly increased in the lung, decreased in the kidney, and unchanged in other organs. The difference observed among various organs, however, may arise because of the possibility that there may be cell-specific receptors with different functions. Hence, changes in steady-state mRNA expression cannot explain the increased and decreased sTNFR and mTNFR levels seen with increased and reduced nutrient intake. A comparison of liver mTNFR and liver TNFR mRNA for both p55 and p75 in protein-energy restricted rats suggests that the reduction in liver mTNFR cannot be due to reduced transcription. Therefore, the reduction in mTNFR in protein-energy restricted rats is due to reduced translation in the liver. On the other hand, the lower sTNFR levels seen with protein-energy restriction can also be due to reduced shedding of p55 and p75 TNFR.Shedding is a ubiquitous phenomenon that has been found for all human
and murine cells and cell lines tested. Although it is known that an
increase in TNF- influences shedding, there is no data about the
effect of nutrition on receptor shedding. Porteu and Hieblot (46) have
demonstrated selective shedding of TNF-induced TNFR-II (p75) from both
human neutrophils and mononuclear cells. The administration of TNF to
cancer patients is also followed by increased sTNFR levels (33).
Several lines of evidence demonstrate the involvement of
metalloproteases (9, 41, 27) and serine proteases (9) in TNFR shedding.
Björnberg et al. (9), in addition to demonstrating that TNFR
shedding is induced by Zn2+,
indicated the possible involvement of a
Zn2+-dependent metalloprotease.
Recent evidence suggests that extracellular reactive-oxygen
intermediates such as hydrogen peroxide are involved in the activation
of metalloproteinase and protein kinase C, which are responsible for
the shedding of sTNFR-I (20). In another report (42),
metalloproteinase expression in the kidney is reduced by a low-protein
intake, which could reduce receptor shedding from the kidney. It was
our observation that rats subjected to protein-energy restriction had
reduced p55 mRNA expression; this may reduce the availability of
receptor to be shed. Therefore, it is tempting to
speculate that low protein-energy intake may reduce plasma sTNFR by a
combination of reduced mRNA expression, reduced translation, and
reduced shedding, depending on the source of the shed receptor.
Significance of Effect of Nutrition on sTNFR Levels
sTNFR may, in some situations, inhibit the effect of TNF; in others, sTNFR serves as carriers for TNF, in some cases even augmenting the effects of TNF by prolonging its function. The differences in these effects depend on concentration at the site of TNF action, the relation of concentration to the local concentration of TNF, and the rates at which sTNFR and TNF are cleared from the site of TNF action in relation to the rate of decay of TNF activity (1, 6).The demonstration that administration of TNF- or lipopolysaccharide
to humans increases the concentration of sTNFR in the serum suggests
that they may be part of a negative-feedback mechanism to inhibit the
biological effects of TNF (33). Jaattela (26) has shown that sTNFR
competes with cell surface receptors for the binding of free TNF-
and may therefore be important for the physiological regulation of TNF
bioactivity. On the other hand, Aderka et. al. (1) have demonstrated
that sTNFR does not block and may even stabilize TNF-
in circulation
and augment its activity.
In a previous study (23), orally fed rats infused with TNF-
developed anorexia and weight loss. Despite the weight loss, there was
no mortality or morbidity in these rats. To avoid the effects of
anorexia-induced malnutrition, rats were fed a diet parenterally and
enterally (gastrostomy) with nutrients identical in composition to the
oral diet, in quantities permitting normal growth (40, 48). The rats
developed hyperglycemia, azotemia, increased plasma triglyceride
levels, and mortality as well as increased levels of TNF-
. The data
suggest that weight loss is associated with a reduction in the
biological effect of TNF (23), which could be a result of decreased
sTNFR and mTNFR, as observed in the present study.
In addition, insulin resistance was observed in the total parenteral
nutrition enterally fed weight-gaining animals (40, 48). Insulin
resistance seen with TNF infusion is also observed in obese, diabetic
mice, in which it is associated with increased expression of TNFR mRNA
in adipose tissue and muscle (21). In our model, the increased sTNFR in
weight-gaining rats, together with a larger circulating pool of TNF
(29), suggests stabilization of TNF- in circulation, as suggested by
Aderka et al. (1). The stabilization of TNF-
in the circulation will
augment its activity and prolong its action, resulting in a greater
degree of metabolic abnormalities, as seen in previous studies (40, 48). Conversely, the markedly reduced binding seen in weight-losing rats could result in rapid clearance of TNF-
, thereby protecting the
animal from metabolic effects such as the hyperglycemia, azotemia, and
liver injury seen in weight-gaining rats fed enterally or parenterally
(40, 48).
The data presented herein suggest that nutritional manipulations in
critically ill and septic patients may be used to attenuate the
undesirable effects of TNF-. Furthermore, an understanding of these
interrelationships is likely to be important in designing a suitable
nutrient regimen for septic patients in the intensive care unit.
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ACKNOWLEDGEMENTS |
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This project was undertaken with the aid of Medical Research Council of Canada Grant MT-12238.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. N. Jeejeebhoy, Rm. 6352, Med. Sci. Bldg., Univ. of Toronto, Toronto, ON, Canada M5S 1A8 (E-mail: khush.jeejeebhoy{at}utoronto.ca).
Received 18 December 1998; accepted in final form 20 May 1999.
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