EPILOGUE
Effect of nutrition on tumor necrosis factor receptors in weight-gaining and -losing rats

Nilima Raina1, Jonathan Lamarre2, Choong-Ching Liew1, Amir H. Lofti1, and Khursheed N. Jeejeebhoy1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies showed that weight-gaining rats had greater retention and reduced turnover of 125I-labeled tumor necrosis factor (TNF)-alpha 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-alpha receptor (sTNFR) and membrane TNFR (mTNFR) and the cellular expression of TNF-alpha 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-alpha 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-alpha 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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)-alpha (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-alpha levels are increased (28). TNF-alpha 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-alpha 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-alpha infusion in the rat model (48). Orally fed rats infused with TNF-alpha (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-alpha -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-alpha ; namely, the retention of TNF-alpha 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-alpha is partly bound to soluble TNFR and because the cellular effects of TNF-alpha 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.


    MATERIAL AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Composition of liquid defined-formula diet per 60 ml

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 at -70°C in small aliquots to be analyzed for cross-linked binding of labeled TNF-alpha with soluble receptors, as given in Measurement of Receptors. Organs (heart, lung, liver, and kidney) and muscle (diaphragm) from the rats were removed immediately after death, frozen quickly in liquid nitrogen, and stored until use for RNA isolation and Northern blotting to measure the cellular mRNA receptor expression (as described in RNA Separation and Northern Blot Analysis).

Isolation 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-alpha

Recombinant rat TNF-alpha (Endogen, Woburn, MA) was labeled with 125I-labeled sodium (Na125I) with the use of the chloramine-T procedure, as described previously (25). Briefly, 5 µg of rat TNF-alpha in 10 µl sodium phosphate buffer (pH 7.4) were incubated with freshly prepared chloramine-T (2 µg) for 5 min at room temperature in the presence of 0.5 mCi carrier-free Na125I. Free iodine was removed by gel filtration on a Sephadex G-25 column (PD-10; Pharmacia Biotech) equilibrated with PBS. The specific activity of the labeled rat TNF-alpha was ~60 µCi/µg. Human recombinant 125I-TNF-alpha was obtained from Amersham Canada (Oakville, ON).

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-alpha , as described in Analytic SDS-PAGE and validated in Preliminary Results in Determining Specificity of TNFR-Ligand Complex. Blood plasma or liver plasma membrane was incubated with recombinant human or rat 125I-TNF-alpha for 60 min at 37°C. Then, the cross-linking reagent BS3 (bis-sulfosuccinimidyl suberate; Pierce, Rockford, IL) was added to a final concentration of 0.125 mM and incubated for another 15 min at room temperature.

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-alpha 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-alpha and run concurrently. In addition, the positions of the bands demonstrating TNF binding were compared with those obtained by cross-linking 125I-TNF-alpha to purified human TNFR-I (p55).

After electrophoresis, gels to be stained were immediately fixed in a solution of 50% methanol-10% acetic acid. The plasma protein bands were visualized by staining with silver stain. The gels were dried between cellophane membranes, and the radioactivity of the labeled TNFR-ligand complex was visualized by exposing the dried gel to Kodak X-ray film at -70°C.

The X-ray films were scanned by a Bio-Rad model GS-670 imaging densitometer, and the band densities were analyzed by the Molecular Analyst software (version 2.1; Bio-Rad Laboratories, Molecular Biosciences Group, Hercules, CA). The ratios of the densities in the bound (92-243 kDa) region to those in the free (17-51 kDa) region were calculated. Expression of the data as a ratio prevented any slight difference in loading from influencing the conclusions of the finding, because the difference would equally affect the bound and free regions.

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
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha binding. Specificity of plasma TNFR-ligand complex was shown by comparing the binding of human 125I-TNF-alpha to plasma in the absence and presence of an excess of unlabeled TNF-alpha . 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-alpha , followed by SDS-PAGE. The first three TNF lanes, which had 125I-TNF-alpha 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-alpha 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-alpha 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-alpha .


View larger version (74K):
[in this window]
[in a new window]
 
Fig. 1.   A quantity of 3 µl of weight-gaining (WG) rat plasma was cross-linked with 125I-labeled tumor necrosis factor (TNF) with or without addition of 8 µl (2 µg/µl) of unlabeled TNF followed by 4-15% SDS-PAGE. First 3 lanes have 125I-TNF-alpha and cross-link, showing prominent band at 17 kDa and smaller bands at 34 and 51 kDa. Lanes 4-6 have plasma, 125I-TNF-alpha , and cross-link, showing 243- and 92-kDa bands in addition to above 3 bands. Lanes 7-9 show marked attenuation of bands when incubated with unlabeled TNF-alpha .

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.



View larger version (116K):
[in this window]
[in a new window]
 
Fig. 2.   A: quantities of 3, 4, 5, and 6 µl of plasma were cross-linked with 125I-TNF-alpha and subjected to SDS-PAGE. With increased plasma concentration, intensity of bands increased from lanes 2 to 5. B: result of X-ray film as described in A scanned by image densitometer, showing relationship of plasma added to 125I-TNF-alpha binding. Ratio of bound to unbound area, calculated as total area from 92- to 243-kDa region to that of 17- to 51-kDa region or area around 243-kDa band to area of 17- to 51-kDa region, showing linear increase with increasing addition of plasma.

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-alpha was cross-linked to pure human p55 receptor. Lane 1 shows the previously described features of only 125I-TNF-alpha . Human p55 (recombinant human sTNFR-I; R&D Systems, Minneapolis, MN) cross-linked with 125I-TNF-alpha 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-alpha [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.



View larger version (103K):
[in this window]
[in a new window]
 
Fig. 3.   A: lane 1 shows previously described features of cross-linked 125I-TNF (see Fig. 1). Human p55 [recombinant human soluble TNF-alpha receptor (TNFR)-I] was cross-linked with 125I-TNF, showing bands at ~92 and 243 kDa in lanes 2, 4, 6 and 8. Lanes 3, 5, and 7 show attenuation of same bands by addition of 8 µl (2 µg/µl) of unlabeled TNF-alpha . B: result of X-ray film as described in A scanned by image densitometer. Lane 7: human p55 receptor cross-linked with 125I-TNF-alpha and unlabeled TNF-alpha . Lane 8: human p55 receptor cross-linked with 125I-TNF-alpha .

Nutritional Effects on Rat TNF-alpha -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-alpha (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-alpha 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).



View larger version (141K):
[in this window]
[in a new window]
 
Fig. 4.   A and B: lane 1 shows previously described features of cross-linked 125I-TNF (see Fig. 1). Lanes 2-5, 11, and 12 show prominent binding with soluble receptors in 243-kDa region in WG rats. Lanes 6-9, 13, and 14 show negligible binding in 243-kDa region in weight-losing (WL) rats.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Result of X-ray film scanned by image densitometer showing ratio of area of 92- to 243-kDa region to area of 17- to 51-kDa region in soluble and membrane receptors. * Significant difference at P < 0.05.

Nutritional Effects on Rat TNF-alpha 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-alpha (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).



View larger version (138K):
[in this window]
[in a new window]
 
Fig. 6.   A and B: lane 1 shows previously described features of cross-linked 125I-TNF (see Fig. 1). Lanes 2-5, 10, 11, and 12 show prominent binding with membrane receptors in 243-kDa region in WG rats. Lanes 6-9, 13, 14, and 15 show negligible binding in 243-kDa region in WL rats.

When plasma was incubated with recombinant TNF for 30 min before the binding assay with 125I-TNF, there was no interference with binding unless the concentration exceeded 533 ng/µl, which is much greater than the levels observed previously in rats of the same strain and on a similar diet (40).

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.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 7.   A: expression of mRNA for p55 receptor in lungs of WG (lanes 1-4) and WL (lanes 5-8) rats by Northern blot analysis. B: result of X-ray film scanned by image densitometer, showing ratio of area of p55 to area of 18S mRNA. * Significant difference at P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

TNF-alpha 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-alpha 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-alpha 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-alpha (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-alpha .

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-alpha 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-alpha , may contribute to this possibility. However, the same reduction may protect the individual from cell injury in situations in which TNF-alpha productions are excessive, as in severe sepsis and critical illness. The protective action of TNF-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha in circulation and augment its activity.

In a previous study (23), orally fed rats infused with TNF-alpha 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-alpha . 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-alpha in circulation, as suggested by Aderka et al. (1). The stabilization of TNF-alpha 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-alpha , 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-alpha . 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.


    ACKNOWLEDGEMENTS

This project was undertaken with the aid of Medical Research Council of Canada Grant MT-12238.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aderka, D., H. Engelmann, Y. Maor, C. Brakebusch, and D. Wallach. Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors. J. Exp. Med. 175: 323-329, 1992[Abstract].

2.   Arkesteijn, C. L. M. A kinetic method for serum 5'-nucleotidase using stabilized glutamate dehydrogenase. J. Clin. Chem. Clin. Biochem. 14: 155-158, 1976[Medline].

3.   Bazil, V. Physiological enzymatic cleavage of leukocyte membrane molecules. Immunol. Today 16: 135-140, 1995[Medline].

4.   Bazzoni, F., and B. Beutler. The tumor necrosis factor ligand and receptor families. N. Engl. J. Med. 334: 1717-1725, 1996[Free Full Text].

5.   Beisel, W. R. Nutrition, infection, specific immune responses and nonspecific host defences: a complex interaction. In: Nutrition, Disease Resistance, and Immune Function, edited by R. R. Matson. New York: Marcel Dekker, 1984, p. 3-34.

6.   Bemelmans, M. H. A., D. J. Gouma, and W. A. Buurman. Influence of nephrectomy on tumor necrosis factor clearance in a murine model. J. Immunol. 150: 2007-2017, 1993[Abstract/Free Full Text].

7.   Beutler, B., and A. Cerami. Cachectin and tumor necrosis factor as two sides of the same biological coin. Nature 320: 584-588, 1986[Medline].

8.   Beutler, B., W. I. Milsark, and A. Cerami. Cachectin/tumor necrosis factor: production, distribution and metabolic fate in vivo. J. Immunol. 135: 3972-3977, 1985[Abstract/Free Full Text].

9.   Björnberg, F., M. Lantz, and U. Gullberg. Metalloproteases and serineproteases are involved in the cleavage of the two tumor necrosis factor (TNF) receptors to soluble forms in the myeloid cell lines U-937 and THP-1. Scand. J. Immunol. 42: 418-424, 1995[Medline].

10.   Braxton, C. C., S. M. Coyle, R. N. Walton, W. J. Montegut, T. van der Poll, M. Roth, D. Pharm, S. E. Calvano, and S. F. Lowry. Parenteral nutrition alters monocyte TNF receptor activity. J. Surg. Res. 59: 23-28, 1995[Medline].

11.   Brockhaus, M., H. J. Schoenfeld, E. J. Schlaeger, W. Hunziker, W. Lesslauer, and H. Loetscher. Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 87: 3127-3131, 1990[Abstract].

12.   Chomczynski, P., and N. Sacchi. Single-step method for RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

13.   Dembic, Z., H. Loetscher, U. Gubler, Y. C. Pan, H. W. Lahm, R. Gentz, M. Brockhaus, and W. Lesslauer. Two human TNF receptors have similar extracellular, but distinct intracellular, domain sequences. Cytokine 2: 231-237, 1990[Medline].

14.   Dinarello, C. A., J. G. Cannon, S. M. Wolff, H. A. Bernheim, B. Beutler, A. Cerami, I. S. Figari, M. A. Palladino, Jr., and J. V. O'Connor. Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1. J. Exp. Med. 163: 1433-1450, 1986[Abstract].

15.   Dinarello, C. A., and J. W. Mier. Current concepts: lymphokines. N. Engl. J. Med. 317: 940-945, 1987[Medline].

16.   Engelmann, H., D. Novick, and D. Wallach. Two tumor necrosis factor-binding proteins from human urine. Evidence for immunological cross-reactivity with cell surface tumor necrosis factor receptors. J. Biol. Chem. 265: 1531-1536, 1990[Abstract/Free Full Text].

17.   Erickson, S. L., F. J. de Sauvage, K. Kikly, K. Carver-Moore, S. Pitts-Meek, N. Gillett, K. C. Sheehan, R. D. Schreiber, D. V. Goeddel, and M. W. Moore. Decreased sensitivity to tumor-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature 372: 560-563, 1994[Medline].

18.   Frankel, W. L., N. J. Evans, and J. L. Rombeau. Scientific rationale and clinical application of parenteral nutrition in critically ill patients. In: Clinical Nutrition: Parenteral Nutrition, edited by J. L. Rombeau, and M. D. Caldwell. Philadelphia: W. B. Saunders, 1993, p. 597-616.

19.   Grant, G. H., and J. F. Kachnar. The proteins of body fluids. In: Fundamentals of Clinical Chemistry, edited by N. W. Tietz. Philadelphia: W. B. Saunders, 1976, p. 298-376.

20.   Hino, T., H. Nakamura, S. Abe, H. Saito, M. Inage, K. Terashita, S. Kato, and H. Tomoike. Hydrogen peroxide enhances shedding of type I soluble tumor necrosis factor receptor from pulmonary epithelial cells. Am. J. Respir. Cell Mol. Biol. 20: 122-128, 1999[Abstract/Free Full Text].

21.   Hoffman, C., K. Lorenz, S. S. Braithwaite, J. R. Colca, B. J. Palazuk, G. S. Hotamisligil, and B. M. Spiegelman. Altered gene expression for tumor necrosis factor-alpha and its receptors during drug and dietary modulation of insulin resistance. Endocrinology 134: 264-270, 1994[Abstract].

22.   Hohmann, H. P., R. Remy, M. Brockhaus, and P. G. M. van Loon. Two different cell types have different major receptors for human tumor necrosis factor (TNF-alpha ). J. Biol. Chem. 264: 14927-14934, 1989[Abstract/Free Full Text].

23.   Hoshino, E., C. Pichard, C. E. Greenwood, G. C. Kuo, R. G. Cameron, R. Kurian, J. P. Kearns, J. P. Allard, and K. N. Jeejeebhoy. Body composition and metabolic rate in rat during a continuous infusion of cachectin. Am. J. Physiol. 260 (Endocrinol. Metab. 23): E27-E36, 1991[Abstract/Free Full Text].

24.   Hubbard, A. L., D. A. Wall, and A. Ma. Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains. J. Cell Biol. 96: 217-229, 1983[Abstract].

25.   Hunter, W. M., and F. C. Greenwood. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194: 495-496, 1962.

26.   Jaattela, M. Biologic activities and mechanisms of action of tumor necrosis factor-alpha /cachectin. Lab. Invest. 64: 724-742, 1991[Medline].

27.   Katsura, K., M. Park, M. Gatanaga, E. C. Yu, K. Takishima, G. A. Granger, and T. Gatanaga. Identification of the proteolytic enzyme which cleaves human p75 TNF receptor in vitro. Biochem. Biophys. Res. Commun. 222: 298-302, 1996[Medline].

28.   Keith, M. E., A. Geranmayegan, M. J. Sole, R. Kurian, A. Robinson, A. S. Omran, and K. N. Jeejeebhoy. Increased oxidative stress in patients with congestive heart failure. J. Am. Coll. Cardiol. 31: 1352-1356, 1998[Medline].

29.   Keith, M. E., K. H. Norwich, and K. N. Jeejeebhoy. Nutrition support affects the distribution and organ uptake of cachectin/tumor necrosis factor in rats. J. Parenter. Enteral Nutr. 19: 341-350, 1995.[Abstract]

30.   Kemper, O., and D. Wallach. Cloning and partial characterization of the promoter for the human p55 tumor necrosis factor (TNF) receptor. Gene 134: 209-216, 1993[Medline].

31.   Kettelhut, I. C., and A. L. Goldberg. Tumor necrosis factor can induce fever in rats without activating protein breakdown in muscle or lipolysis in adipose tissue. J. Clin. Invest. 81: 1384-1389, 1988[Medline].

32.   Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 227: 680-685, 1970[Medline].

33.   Lantz, M., S. Malik, M. L. Slevin, and I. Olson. Infusion of tumor necrosis factor (TNF) causes an increase in circulating TNF-binding protein in humans. Cytokine 2: 402-406, 1990[Medline].

34.   Ledgerwood, E. C., J. B. Prins, N. A. Bright, D. R. Johnson, K. Wolfreys, J. S. Pober, S. O'Rahilly, and J. R. Bradely. Tumor necrosis factor is delivered to mitochondria where a tumor necrosis factor-binding protein is localized. Lab. Invest. 78: 1583-1589, 1998[Medline].

35.   Lee, M. D., A. Zentella, P. H. Pekala, and A. Cerami. Effect of endotoxin-induced monokines on glucose metabolism in the muscle cell line L6. Proc. Natl. Acad. Sci. USA 84: 2590-2594, 1987[Abstract].

36.   Levine, S. J., C. Logun, D. P. Chopra, J. S. Rhim, and J. H. Shelhamer. Protein kinase C, interleukin-1, and corticosteroids regulate shedding of type I, 55 kD TNF receptor from human airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 14: 254-261, 1996[Abstract].

37.   Loetscher, H., Y. C. Pan, H. W. Lahm, R. Gentz, M. Brockhaus, H. Tabauchi, and W. Lesslauer. Molecular cloning and expression of human 55 kd tumor necrosis factor receptor. Cell 61: 351-359, 1990[Medline].

38.   Loetscher, H., E. J. Schlaeger, H. W. Lahm, Y. C. Pan, W. Lesslauer, and M. Brockhaus. Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells. J. Biol. Chem. 265: 20131-20138, 1990[Abstract/Free Full Text].

39.   Loetscher, H., M. Steinmetz, and W. Lesslauer. Tumor necrosis factor: receptors and inhibitors. Cancer Cells. 36: 221-226, 1991.

40.   Matsui, J., R. G. Cameron, R. Kurian, G. C. Kuo, and K. N. Jeejeebhoy. Nutritional, hepatic and metabolic effects of an infusion of cachectin/TNF in rats receiving TPN. Gastroenterology 104: 235-243, 1993[Medline].

41.   Müllberg, J., F. H. Durie, C. Otten-Evans, M. R. Alderson, S. Rose-John, D. Cosman, R. A. Black, and K. M. Mohler. A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor. J. Immunol. 155: 5198-5205, 1995[Abstract].

42.   Nakamura, T., M. Fukui, I. Ebihara, Y. Tomino, and H. Koide. Low protein diet blunts the rise in glomerular gene expression in focal glomerulosclerosis. Kidney Int. 45: 1593-1605, 1994[Medline].

43.   Oliff, A., D. Defeo-Jones, M. Boyer, D. Martinez, D. Kiefer, G. Vuocolo, A. Wolfe, and S. H. Socher. Tumors secreting human TNF/cachectin induce cachexia in mice. Cell 50: 555-563, 1987[Medline].

44.   Perimutter, D. H., C. A. Dinarello, P. I. Punsal, and H. R. Colten. Cachectin/tumor necrosis factor regulates hepatic acute-phase gene expression. J. Clin. Invest. 78: 1349-1354, 1986[Medline].

45.   Pfeffer, K., T. Matsuyama, T. M. Kündig, A. Wakeham, K. Kishihara, A. Shahinian, K. Wiegmann, P. S. Ohashi, M. Krönke, and T. W. Mak. Mice deficient for the 55 kD tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell 73: 457-467, 1993[Medline].

46.   Porteu, F., and C. Hieblot. Tumor necrosis factor induces a selective shedding of its p75 receptor from human neutrophils. J. Biol. Chem. 269: 2834-2840, 1994[Abstract/Free Full Text].

47.   Porteu, F., and C. F. Nathan. Shedding of tumor necrosis factor receptors by activated human neutrophils. J. Exp. Med. 172: 599-607, 1990[Abstract].

48.   Raina, N., R. G. Cameron, and K. N. Jeejeebhoy. Gastrointestinal, hepatic, and metabolic effects of enteral and parenteral nutrition in rats infused with tumor necrosis factor. JPEN J. Parenter. Enteral Nutr. 21: 7-13, 1997[Abstract].

49.   Rolandelli, R. H., J. A. DePaula, P. Guenter, and J. L. Rombeau. Critical illness and sepsis. In: Clinical Nutrition: Enteral and Tube Feeding, edited by J. L. Rombeau, and M. D. Caldwell. Philadelphia: W. B. Saunders, 1990, p. 288-305.

50.   Sauerwein, R. W., J. A. Mulder, L. Mulder, B. Lowe, N. Reshu, P. N. M. Demacker, J. W. M. van der Meer, and K. Marsh. Inflammatory mediators in children with protein-energy malnutrition. Am. J. Clin. Nutr. 65: 1534-1539, 1997[Abstract].

51.   Shalaby, M. R., A. Sudan, H. Loetscher, M. Brockhaus, W. Lesslauer, and T. Espevik. Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors. J. Exp. Med. 172: 1517-1520, 1990[Abstract].

52.   Smith, R. A., and C. Baglioni. Multimeric structure of the tumor necrosis factor receptor of HeLa cells. J. Biol. Chem. 264: 14646-14652, 1989[Abstract/Free Full Text].

53.   Smith, R. A., M. Kirstein, W. Fiers, and C. Baglioni. Species specificity of human and murine tumor necrosis factor. J. Biol. Chem. 261: 14871-14874, 1986[Abstract/Free Full Text].

54.   Spinas, G. A., U. Keller, and M. Brockhaus. Release of soluble receptors for tumor necrosis factor (TNF) in relation to circulating TNF during experimental endotoxemia. J. Clin. Invest. 90: 533-536, 1992[Medline].

55.   Stauber, G. B., R. A. Aiyer, and B. B Aggarwal. Human tumor necrosis factor-alpha receptor. Purification by immunoaffinity chromatography and initial characterization. J. Biol. Chem. 263: 19098-19104, 1988[Abstract/Free Full Text].

56.   Tartaglia, L. A., and D. V. Goeddel. Two TNF receptors. Immunol. Today 13: 151-153, 1992[Medline].

57.   Tartaglia, L. A., D. V. Goeddel, C. Reynolds, I. S. Figari, R. F. Weber, B. M. Fendly, and M. A. Paladino, Jr. Stimulation of human T-cell proliferation by specific activation of the 75-kDa tumor necrosis factor receptor. J. Immunol. 151: 4637-4641, 1993[Abstract/Free Full Text].

58.   Tartaglia, L. A., D. Pennica, and D. V. Goeddel. Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signalling by the 55-kDa TNF receptor. J. Biol. Chem. 268: 18542-18548, 1993[Abstract/Free Full Text].

59.   Touster, O., N. N. Aronson, Jr., J. T. Dulaney, and H. Hendrickson. Isolation of rat liver plasma membranes. J. Cell Biol. 47: 604-618, 1970[Abstract/Free Full Text].

60.   Wingfield, P., R. H. Pain, and S. Craig. Tumor necrosis factor is a compact trimer. FEBS Lett. 211: 179-184, 1987[Medline].

61.   Yoshie, O., K. Tada, and N. Ishida. Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors. J. Biochem. (Tokyo) 100: 531-541, 1986[Abstract].

62.   Zheng, L., G. Fisher, R. E. Miller, J. Peschon, D. H. Lynch, and M. J. Lenardo. Induction of apoptosis in mature T cells by tumor necrosis factor. Nature 377: 348-345, 1995[Medline].


Am J Physiol Endocrinol Metab 277(3):E464-E473
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society