Production of soluble tumor necrosis factor receptors by human
subcutaneous adipose tissue in vivo
Vidya
Mohamed-Ali1,
Steven
Goodrick1,
Karen
Bulmer1,
Jeffrey M. P.
Holly2,
John S.
Yudkin1, and
Simon W.
Coppack1
1 Centre for Diabetes and
Cardiovascular Risk, University College London Medical School,
London N19 3UA; and 2 Division of
Surgery, University Department of Medicine, Bristol Royal
Infirmary, Bristol BS2 8HW, United Kingdom
 |
ABSTRACT |
To investigate in
vivo adipose tissue production of tumor necrosis factor-
(TNF-
),
interleukin-6 (IL-6), and their soluble receptors: TNF receptor type I
(sTNFR-I), TNF receptor type II (sTNFR-II), and IL-6
receptor (sIL-6R), we determined arteriovenous differences in their
levels across abdominal subcutaneous adipose tissue in obese subjects.
Subjects had a median (interquartile range) age of 44.5 (27-51.3)
yr, body mass index (BMI) of 32.9 (26.0-46.6) kg/m2,
and %body fat of 42.5 (28.5-51.2) %. Although there was not a
significant difference in the arteriovenous concentrations of TNF-
(P = 0.073) or sTNFR-II
(P = 0.18), the levels of sTNFR-I (P = 0.002) were higher in the vein
compared with artery, suggesting adipose tissue production of this
soluble receptor. There was a significant arteriovenous difference in
IL-6 (P < 0.001) but not in its
soluble receptor (P = 0.18). There was
no relationship between TNF-
levels and adiposity indexes
(rs = 0.12-0.22, P = not significant);
however, levels of both its soluble receptor isomers correlated
significantly with BMI and %body fat (sTNFR-I rs = 0.42-0.72, P < 0.001; sTNFR-II
rs = 0.36-0.65, P < 0.05- <0.001).
IL-6 levels correlated significantly with both BMI and %body fat
(rs = 0.51, P = 0.004, and
rs = 0.63, P < 0.001), but sIL-6R did not. In
conclusion, 1) soluble TNFR-I is
produced by adipose tissue, and concentrations of both soluble isoforms
correlate with the degree of adiposity, and
2) IL-6, but not its soluble receptor, is produced by adipose tissue and relates to adiposity.
interleukin-6; tumor necrosis factor-
; soluble receptors; obesity
 |
INTRODUCTION |
TUMOR NECROSIS FACTOR-
(TNF-
) and interleukin-6
(IL-6) are proinflammatory cytokines with known potent effects in host
defense (14). Both of these cytokines have been implicated in the
regulation of lipid and glucose metabolism (5). Adipose tissue is a
significant source of endogenous TNF-
, with its expression in fat
tissue being elevated in obesity (11). Evidence for significant
circulating levels of this cytokine is variable, and it is thought to
operate mainly via autocrine/paracrine mechanisms in both adipose
tissue and skeletal muscle (8, 9). IL-6 is also expressed in and released by adipose tissue, and its levels increase with obesity (15,
16).
Two structurally distinct TNF receptors, TNFR-I and TNFR-II, have been
identified (4). The TNFR-I is thought to mediate most of the functions
of TNF-
, whereas the actions of TNFR-II are as yet unclear and
perhaps mainly cell specific. Both these receptors are expressed in
human adipose tissue, and the soluble forms are present in the
circulation (7). Although the nature of their physiological function is
still unclear, at least in some studies in vitro, they inhibit the
ligand- binding cell surface receptor, thereby acting as antagonists
(23).
The biological activities of IL-6 are initiated by binding of the
ligand to a single receptor. The IL-6 receptor comprises two chains, a
ligand binding, predominantly extracytoplasmic chain (IL-6R; gp80), and
the signal-transducing gp130 chain (6, 12). The binding of IL-6 to
IL-6R is predominantly an extracellular process. This complex,
IL-6/IL-6R, can be formed with either soluble or membrane-bound IL-6R
(22). The binding of IL-6 to IL-6R, in the presence of gp130, leads to
the formation of high-affinity binding sites, gp130 dimerization, and
signal transduction (24). The signal-transducing gp130 is abundantly
expressed in most cell types, whereas IL-6R is expressed in a variety
of cells in extremely low quantity (12). Unlike the case of TNF-
, in
which the soluble receptors may function as inhibitors for the ligand
(6, 23), in vitro, both recombinant and naturally produced sIL-6R
appear to act as agonists in IL-6R-negative cells that express gp130 (6).
The source of these ligands and of their soluble receptors and their
role in obesity are still unclear. Both of these cytokines interact at
several levels, often regulating similar metabolic processes. To test
the hypothesis that the adipose tissue releases soluble TNF and IL-6
receptors, which may then act to localize and modulate the effects of
the cytokines, we investigated the in vivo release of these molecules
by measuring the arteriovenous balance in their levels across an
abdominal subcutaneous adipose tissue depot in nondiabetic, mainly
obese, subjects.
 |
EXPERIMENTAL SUBJECTS |
Arteriovenous differences for TNF-
and IL-6 were determined after an
overnight fast in 60 Caucasian subjects having a range of adiposity. In
a representative subset of 30 subjects (20 females and 10 males), their
respective soluble receptors were also assayed. The subset had a median
(interquartile range) age of 44.5 (27.0-51.3) yr, body mass index
(BMI) of 32.9 (26.0-46.6)
kg/m2, and a median percent body
fat of 42.5 (28.5-51.2) %.
All subjects gave written informed consent to these studies, which had
previously been approved by the local ethics committee.
 |
MATERIALS AND METHODS |
Cannulas were inserted, using local anesthesia, into a radial artery
and a superficial epigastric vein draining the subcutaneous adipose
tissue (3, 13). Lines were kept patent by a slow infusion of isotonic
saline. Blood samples were taken simultaneously from the two sites.
Previous work has shown that venous blood from superficial epigastric
veins approximates the effluent from an adipose tissue bed, and
arteriovenous differences across abdominal adipose tissue yield result
in good agreement with those of microdialysis studies (19).
Body composition was measured by electrical bioimpedance (Bodystat,
Douglas, Isle of Mann, UK).
Blood flow measurements and assays.
Abdominal subcutaneous adipose tissue blood flow was measured using the
133Xe washout technique on the
basis of the principle that the disappearance of
133Xe radioactivity is
proportional to adipose tissue blood flow (13). Blood flow measurements
were made twice during the postabsorptive blood sampling, and
133Xe washout was measured using a
Mediscint system (Oakfield Instruments, Witney, UK). Adipose tissue
blood flow was calculated as previously described, by use of a
partition coefficient of 10 ml/g for all subjects (13, 21).
Plasma glucose levels were determined with glucose oxidase reagent
(Beckman, Brea, CA), and specific insulin was assayed with a two-site
ELISA (Dako Diagnostics, Ely, UK). TNF-
and IL-6 were measured using
the high-sensitivity two-site ELISA from R & D Systems (Oxford, UK).
The limit of detection of the human TNF-
assay was 0.10 pg/ml, with
intra- and interassay coefficients of variation (CV) of 6.9 and 8.4%.
For human IL-6, the limit of detection was 0.09 pg/ml, and intra- and
interassay CVs were 5.3 and 9.2%. The assays for human sIL-6R,
sTNFR-I, and sTNFR-II were all sensitive to <3 pg/ml, with intra- and
interassay CVs <5% (R & D Systems).
All samples from one individual were always run on the same plate.
Calculations and statistics.
The local cytokine and soluble receptor production by subcutaneous
adipose tissue was calculated by the Fick principle, i.e., the product
of the arteriovenous difference and local plasma flow. All data are
presented as the median and interquartile range. Comparisons between
sites were made using Wilcoxon's paired tests, and Spearman's rank
correlations were used to determine the relationships between variables.
 |
RESULTS |
Arterial and abdominal venous cytokine and soluble receptor
concentrations.
The 60 Caucasian subjects had normal glucose and insulin concentrations
[5.0 (4.8-5.3) mmol/l and 57.9 (33.4-82.0) pmol/l, respectively]. In these subjects, fasting abdominal vein levels of IL-6, but not those of TNF-
, were significantly higher than in
the artery (P < 0.001 and
P = 0.073, respectively), suggesting release of IL-6 but not TNF-
(Fig. 1,
A and
B).

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Fig. 1.
Fasting arterial and superficial abdominal venous concentrations of
tumor necrosis factor- (TNF- ,
A) and interleukin-6 (IL-6,
B) in 60 subjects.
|
|
In the subset of 30 subjects, levels of sTNFR-II were higher than those
of sTNFR-I, as reported previously (9). Of the two TNF soluble
receptors studied, venous levels of sTNFR-I were significantly higher
than in the artery (P = 0.002),
demonstrating adipose tissue production of this soluble receptor.
sIL-6R levels were similar in the vein and the artery (Table
1).
Thus these data show adipose tissue release of IL-6 but not of its
soluble receptor and of soluble TNF receptor but not of its ligand.
Regulation of cytokines and their soluble receptors.
Levels of IL-6, both arterial and venous, were unrelated to those of
sIL-6R (rs = 0.03-0.17; P = 0.86-0.39).
No relationship was apparent between TNF-
and sTNFR-II
(rs =
0.05-0.14; P = 0.81-0.47) or between TNF-
and sTNFR-I
(rs = 0.17-0.34; P = 0.35-0.06).
There was a strong, positive correlation between the two soluble TNF
receptors (rs = 0.49-0.71; P = 0.005- P < 0.001).
The molar ratios of sTNFR-I and sTNFR-II to TNF-
and of sIL-6R to
IL-6 were calculated with the assumption of the following molecular
masses: 17 kDa for TNF-
(2), 30 kDa for sTNFR-I and
sTNFR-II (2), 26 kDa for IL-6 (10), and 50 kDa for sIL-6R (18). These
data show that both TNF and IL-6 soluble receptor concentrations are
far in excess of their respective ligands (Table 2).
Obesity, and cytokines and their soluble receptors.
Significant correlations were found between indexes of obesity (BMI and
%body fat) and concentrations of IL-6
(rs = 0.31-0.50; P = 0.02- P < 0.001; Table 3), sTNFR-I
(rs = 0.62-0.72; P < 0.001), and
sTNFR-II (rs = 0.39-0.65; P = 0.03- P < 0.001) (Figs. 2 and 3) but not with those of sIL-6R
(rs = 0.19-0.25; P = 0.32-0.18) or
TNF-
(rs = 0.15-0.22; P = 0.27-0.09).
There were no significant relationships between the molar ratios of the
sTNFRs to TNF-
and measures of obesity; however, significant
negative correlations were apparent between the sIL-6R-to-IL-6 ratio
and BMI and percent body fat (BMI and sIL-6R-to-IL-6 ratio, arterial
rs =
0.43,
P = 0.02; venous
rs =
0.38;
P = 0.04; percent body fat and
sIL-6R-to-IL-6 ratio, arterial
rs =
0.51, P = 0.006; venous
rs =
0.38;
P = 0.05).

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Fig. 3.
Correlations of venous concentrations of soluble TNF receptor I
(sTNFR-I; rs = 0.72, P < 0.001, A) and sTNFR-II
(rs = 0.65;
P < 0.001, B) to %body fat.
|
|
Adipose tissue IL-6 and sTNFR-I production rates.
Net subcutaneous abdominal adipose tissue IL-6 and sTNFR-I release in
vivo was determined from net arteriovenous balance and local plasma
flow and was 2.72 (0.68-5.19) pg · 100 g adipose tissue
1 · min
1
and 1,454.0 (777.09-1,941.03) pg · 100 g adipose
tissue
1 · min
1,
respectively, for the whole group.
There was a significant relationship between production of sTNFR-I and
venous TNF-
(rs = 0.40;
P = 0.03).
 |
DISCUSSION |
Previous studies have shown a relationship between the expression of
TNF-
in fat tissue and measures of obesity, as well as a reduction
in this expression on weight loss (20). The relationship between
circulating TNF-
and obesity and insulin resistance is less clear.
The reason for this discrepancy may be that TNF-
functions mainly
via paracrine/autocrine mechanisms. More recently, it has been shown
that both TNFR-I and TNFR-II are expressed in human adipose tissue and
that levels of soluble TNFR-II in the systemic circulation correlate
positively with obesity (7). This study suggests that the source of the
soluble TNF receptors may, at least in part, be adipose tissue. Our
results show, for the first time, that there is in vivo release of
sTNFR-I by human subcutaneous adipose tissue. The significant
correlation between net adipose tissue release of sTNFR-I and
circulating TNF-
concentrations suggests that this production may be
regulated by the ligand itself. Alternatively, sTNFR-I production by
adipose tissue could increase the circulatory half-life of TNF-
and,
hence, its circulating concentration.
Therefore, these results support the suggestion put forward by
Hotamisligil et al. (7) of adipose tissue-derived soluble TNF receptors
in obesity. Furthermore, we also show a strong correlation between the
two soluble receptors, perhaps suggesting coregulation of receptors,
with the levels of both soluble receptors being strongly related to
measures of obesity. The relationship between sTNFR-II and obesity,
even though we had no clear evidence for net production of this
molecule by the adipose tissue, may suggest an indirect effect via
sTNFR-I or TNF-
. Alternatively, although the production rate for
sTNFR-II may be very similar to that of sTNFR-I, because its absolute
concentration is much higher, the arteriovenous difference is perhaps
more difficult to detect. We also confirm higher circulating
concentration of sTNFR-II, perhaps due to slower clearance of this
molecule, compared with that of sTNFR-I.
We have previously reported in vivo production of IL-6 by adipose
tissue, and we confirm this result on a larger number of subjects in
this study (15). Unlike the inhibitory effects of soluble receptors on
the activity of TNF-
, most of the data show that the sIL-6R
facilitates and enhances IL-6 activity (6, 17). We show that sIL-6R
does not appear to be released by this adipose tissue depot and does
not show a relationship between obesity and its levels.
The physiological significance of these soluble receptors is unclear.
The adipose tissue release of the inhibitory sTNFRs may localize the
effects of TNF-
within the tissue, enabling it to function as an
autocrine/paracrine factor. Furthermore, it has also been shown that
IL-6 inhibits TNF-
expression in primary rat astrocytes, although
this effect has not been reported in adipocytes (1). Thus, in adipose
tissue, TNF-
may induce the release of IL-6, which may then act as
the endocrine signal emanating from adipose tissue, but with TNF-
playing an autocrine/paracrine role.
It is interesting to note the vast excess of the soluble receptors for
both the cytokines compared with ligand. The assays employed in this
study are ELISAs and determine all the immunologically active protein;
this therefore raises the question as to whether all the peptides
measured are also functionally active. Furthermore, because most of the
evidence suggests that sTNFRs are inhibitory but that sIL-6R enhances
the ligand activity, the excess circulating binding proteins would have
very different physiological implications.
Our findings support the concept that IL-6 is an endocrine factor
released from adipose tissue, whereas the same tissue is an active
producer of sTNFR-I, the prime function of which is to ensure that
TNF-
acts principally as an autocrine/paracrine factor in this
tissue. In conclusion, these novel results suggest a mechanism for the
regulation of IL-6 and TNF-
by abdominal subcutaneous adipose tissue.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Gokhan S. Hotamisligil for helpful comments in the
preparation of this manuscript.
 |
FOOTNOTES |
This work was supported by a grant from the Sir Jules Thorn Charitable
Trust (97/18A).
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 correspondence and reprint requests: V. Mohamed-Ali, Centre
for Diabetes and Cardiovascular Risk, University College London Medical
School, London N19 3UA, UK (E-mail:
rmhavma{at}ucl.ac.uk).
Received 24 May 1999; accepted in final form 27 July 1999.
 |
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