(CANCER RESEARCH 52, 4113-4116. August 1. 1992|
Interleukin 6 Reduces Lipoprotein Lipase Activity in Adipose Tissue of Mice in
Vivo and in 3T3-L1 Adipocytes: A Possible Role for Interleukin 6 in Cancer
Cachexia
Andrew S. Greenberg,1 Richard P. Nurdan, Joseph Mclntosh, Juan Carlos Calvo, Robert O. Scow, and
David Jablons2
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes, Digestive and Kidney Disease ¡A.S. G., R. O. S.J; Laboratory of Genetics
[R. P. N.I and Surgery Branch fJ. M., D. J.J, National Cancer Institute; and Laboratory of Theoretical and Physical Biology, National Institute of Child Health and
Development [J. C. C.J, NIH, Bethesda, Maryland 20892
ABSTRACT
To investigate whether interleukin 6 (11-6) might be a potential me
diator of the depleted fat reserves observed in malignancy-associated
cachexia, we measured lipoprotein lipase (LPL) activity in adipose tis
sue of mice after administration of IL-6 or tumor necrosis factor and in
cultured adipocytes after addition of these cytokines. Injection of IL-6
i.p. reduced adipose tissue LPL activity by 53% within 4.5 to 5.5 h.
Injection of tumor necrosis factor elevated serum IL-6 levels and re
duced adipose tissue LPL activity by 70%. Both human and murine IL-6
reduced heparin-releasable
LPL activity in 3T3-L1 adipocytes in a dosedependent
manner; half-maximal
inhibition of LPL activity was
achieved with 5000 hybridoma growth factor units/ml. Thus, IL-6 re
duces
stores
crosis
diated
adipose LPL activity and may contribute to the loss of body fat
associated with some cases of cancer cachexia. Since tumor ne
factor increases circulating IL-6, some of its effects may be me
or potentiated by IL-6.
INTRODUCTION
Animals and humans with chronic infections or cancer may
develop the syndrome of cachexia which is characterized by
severe wasting of both protein and fat stores. The syndrome is
often associated with elevated serum triglycérides(1-3) and
reduced serum LPL3 activity (4-6). LPL, produced in adipo
cytes and found at the endothelial cell surface, hydrolyzes cir
culating triglycéridesto fatty acids, which serve as the major
source of acyl groups used in fat storage in the adipocyte (7).
Cerami et al. (8, 9) discovered a factor produced by endotoxinstimulated macrophages that suppresses adipocyte LPL activ
ity. This factor, termed cachetin for its presumptive role in
lowering LPL activity and promoting the cachetic syndrome,
was later found to be identical to TNF (10). When injected into
animals, cachetin/TNF lowers adipose tissue LPL activity (11).
We recently reported that administration of recombinant
TNF to humans and mice induces the appearance of circulating
IL-6 (12, 13). IL-6 is a cytokine that appears to act as an
important systemic regulatory hormone in response to infection
and trauma (14); increased serum concentrations of IL-6 have
been measured in patients with sepsis (15), acquired immuno
deficiency syndrome (16), and plasma cell leukemia (17). In
creased concentrations of circulating IL-6 are detectable in an
imals bearing tumors of multiple different histologies (13, 18,
19), and serum IL-6 levels increase in direct proportion to the
amount of tumor burden (13, 19).
Since TNF increases serum IL-6 levels and since IL-6 activity
is elevated in the presence of malignancy and infections, we
investigated whether IL-6 might be a mediator of the depleted
fat stores of the cachetic state. In this paper, we demonstrate
that IL-6 significantly lowers adipose tissue LPL activity in vivo
and directly inhibits LPL activity in cultured adipocytes.
MATERIALS
AND METHODS
Cytokines. rHIL-6, a kind gift of Drs. Gordon Wong and Robert
Donahue of Genetics Institute (Cambridge, MA), had less than 5 endotoxin units/mg protein and an activity of 4-6 x IO6 TI 165 plasmacytoma units/mg. rHIL-6 was dissolved in HBSS containing 2% human
albumin, and the activity was assayed with the B9 hybridoma assay (see
below). rHTNF, a kind gift of the Cetus Corp. (Emeryville, CA), had an
activity of 2 x IO7 units/mg in the L929 assay (see below) and an
endotoxin content of <0.1 ng/mg protein. TNF was dissolved in HBSS
with 2% human serum albumin, and activity was confirmed using the
L929 assay (20).
Native murine IL-6 was purified by a modification of the procedure
of Nordan et al. (21). Briefly, serum-free supernatant from the P388D1
murine macrophage cell line was applied to an affinity column contain
ing the rat anti-murine IL-6 monoclonal antibody, D6906B4 (22), cou
pled to Sepharose 4B. After extensive washing with phosphate-buffered
saline, the murine IL-6 was eluted with 0.2 M glycine-HCI/0.02%
Tween 20, pH 2.O. The affinity-purified murine 1L-6 was applied to a
C-4 reverse phase HPLC column (214TP54, Vydac) and eluted with a
gradient of 40% to 50% acetonitrile/0.1% trifluoroacetic acid over 20
minutes. IL-6, which eluted as a single peak, was lyophilized and resuspended in phosphate-buffered saline/0.02% Tween 20 and stored at
4°C.Activity was determined with the B9 assay.
B9 Bioassay of IL-6 Activity. Recombinant, purified murine, and
serum IL-6 activities was assayed by a [3H]dThd uptake assay using the
IL-6-dependent murine hybridoma subclone B9 as described previously
(22, 23). Culture medium consisted of RPMI 1640 supplemented with
10% fetal calf serum, 5 x 10~5 M 2-mercaptoethanol, and 50 jig/ml
gentamicin. B9 cells were maintained in culture medium supplemented
with IL-6 at 100 HGF units/ml. For the assay, 2000 washed B9 cells
were cultured with 2-fold serial dilutions of the test sample in a final
volume of 200 u\ of culture medium. After an 84-h incubation at 37'C
in a humidifed atmosphere of 5% CO2-95% air, the cultures were pulsed
for 4 h with 0.5 ¿iCi[3H]dThd (6.7 Ci/mmol) and harvested over glass
fiber filters. The incorporation of [3H]dThd into DNA was determined
Received 1/14/92; accepted 5/12/92.
in a liquid scintillation counter. Preparations of known activity of
The costs of publication of this article were defrayed in part by the payment of
rHIL-6 were diluted in heat-inactivated bovine calf serum and tested
page charges. This article must therefore be hereby marked advertisement in accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
concurrently as standards. One hybridoma growth factor unit of IL-6 is
1To whom requests for reprints should be addressed, at LCDB. NIH. Building
defined as that amount which gives half-maximal stimulation of thy
6, Rm. BI-13, Bethesda. MD 20892.
2 Work done in partial fulfillment of Ph.D. requirements, Dept. of Genetics,
midine incorporation into B9 cells. The specificity of the assay was
George Washington University, Washington, DC.
verified by inhibition with a neutralizing rabbit anti-murine IL-6 anti3 The abbreviations used are: LPL, lipoprotein lipase: IL-6. interleukin 6; TNF,
serum (24).
tumor necrosis factor; HBSS, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acidAnimal Methods. Female C57BL/6 mice, 12 weeks of age, were
buffered saline solution: rl I, recombinant human: diluì,thymidine uptake; HGF,
hybridoma growth factor.
obtained from The Jackson Laboratory, Bar Harbor, ME, or from the
4113
INTERI.El KIN 6 REDITES
Small Animal Section, National Cancer Institute, Bethesda, MD. The
animals were fed aülibitum.
rHTNF and rHIL6 were prepared in HBSS and injected ¡.p.in
groups of six animals for each experiment. Animals received an injec
tion of either 1 ml of HBSS alone (vehicle), or 1 ml of HBSS containing
100 ¿<g
of rHIL-6, or 1 ml of HBSS containing 10 ng of rHTNF.
Tailbleeds were performed 4 h after i.p. injection to determine serum
cytokine activity. Blood was collected in serum separator tubes (SST,
Becton Dickinson), and the sera were assayed immediately for IL-6,
TNF, or both, or frozen at -70°C prior to analysis.
To measure in vivo adipose tissue LPL activity, animals were sacri
ficed by cervical dislocation 4.5 to 5.5 h after injection, and the entire
periovarian and periuterine fat pads were removed.
Adipose Tissue Lipoprotein Lipase Activity. Lipolytic activity in the
fat pads was determined by a modification of the methods of Peterson
et al. (25) and Langer et al. (26). The adipose tissue was weighed and
homogenized with a Polytron tissue grinder in 9 volumes (w/v) of
homogenization solution consisting of ice-cold 0.025 M ammonium
chloride buffer (pH 8.2) containing 5 lU/ml heparin, 10 Mg/rnl leupeptin, 1 jig/ml pepstatin, 25 KIE/ml Trasylol, 0.125% Triton X-IOO.
0.04% (w/v) sodium dodecyl sulfate, and 5 mM EDTA. The homogenates were centrifuged at 27.000 x g for 20 min at 4"C. The fat cake was
gently pierced, and 10 ¿il
of infranatant were removed and mixed with
10 ill of homogenization solution without Triton and sodium dodecyl
sulfate and assayed for LPL activity in duplicate.
A stock lipid emulsion containing 0.005 mmol of tri[9.10-'H|triolein
(l.OCi/mmol). 1.13 mmol of trioleoylglycerol. 60 mg of i-«-phosphatidylcholine, and 9 ml of glycerol was prepared according to the method
of Nilsson-Ehle and Schotz (27). Before assay an activated substrate
mixture was prepared by mixing 1 volume of the stock emulsion with 5
volumes of heat-inactivated fasted rat serum (heated at 60°Cfor 10
min) and 19 volumes of an assay solution that resulted in a final con
centration of 12% bovine serum albumin. 300 II) heparin/ml. 0.2 M
NaCI, and 0.2 MTris-HCl (pH 8.1) (25); the mixture was incubated at
37°Cfor 15 to 30 min. For assay, 100 n\ of the activated substrate
mixture, containing 2.0 ¿<Ci
of tripHjoleolyglycerol/ml and 23 nmol of
irioleoglycerol, were added to the adipose tissue extract, brought to a
total volume of 200 Mlwith 0.9% NaCI, and incubated at 37°Cfor 30
min. Radiolabeled fatty acids produced by lipolysis were extracted and
measured by the method of Vaughn and Belfrage (28). The LPL activity
was linear for duration of the assay. Lipolytic activity measured in the
assay was due to lipoprotein lipase since activity was inhibited by more
than 80% by the inclusion of l MNaCI or omission of fasted rat serum.
One milliunit of lipolytic activity represents release of 1 nmol of fatty
acid/min.
3T3-L1 Cell Culture. 3T3-L1 mouse embryo fibroblasts were ob
tained from American Type Culture Collection (Rockville, MD) and
cultured as described previously (29. 30). Briefly, cells were seeded at a
density of 2 to 4 x IO5 cells/60-mm dish (Costar) and cultured in 4 ml
of Dulbecco's modified Eagle's medium-25 mMglucose) (GIBCO), sup
COi-95% air. Fifty- and 100-Mlaliquots of the media were removed and
immediately assayed for LPL activity.
Heparin-releasable LPL activity in the culture medium was assayed
as decribed above with the following exceptions. The preactivated stock
mixture consisted of I volume of the stock emulsion, 19 volumes of 3%
bovine serum albumin in 0.2 M Tris-HCl (pH 8.1), and 5 volumes of
heat-inactivated fasted rat serum (heated at 60°Cfor 10 min); this
mixture was incubated at 37°Cfor 15 to 30 min. For assay, 100 ^1of the
activated substrate mixture were added to an aliquot of culture medium,
brought to a total volume of 200 n\ with 0.9% NaCI, and incubated at
37°Cfor 60 min. Fatty acids were recovered and enzyme activity was
defined as noted above.
Statistics. Differences between groups were assessed using Student's
unpaired t test. The calculations were performed using CL1NFO soft
ware (BBN Software Products. Cambridge. MA). All data are given as
means ±SE.
RESULTS
IL-6 Serum Levels. Previous studies demonstrated that i.p.
administration of IL-6 results in a more persistent elevation of
serum IL-6 concentration than i.v. administration (32). Since
animals with tumors have chronically elevated levels of circu
lating IL-6, the i.p. route of administration was used for the
studies in this paper. Circulating IL-6 concentrations (Table 1)
4 h after administration of either 10 ng of rHTNF or 100 Mgof
rHIL-6 were elevated to levels similar to those found in mice
with large tumor burdens (13, 19). The injection of HBSS ve
hicle in the control animals produced no detectable serum IL-6
activity. Representative sera from mice that received IL-6 were
analyzed for TNF activity by the L929 cytolytic assay, and none
was detected (data not shown). Thus, under our experimental
conditions, TNF injection increased circulating IL-6.
Adipose Tissue LPL Activity. Injection of rHIL-6 into mice
reduced adipose tissue LPL activity by 53% (Table 2), while in
mice treated with rHTNF, adipose tissue LPL activity was low
ered by 70%. The entire periuterine and periovarian fat pads
were carefully removed and used for the LPL assay. Since there
was no obvious difference between the fat pad weights of the
control and cytokine-treated groups, cytokine treatment did not
appear to reduce the triglycéridecontent of the tissues during
the brief time period examined here.
Table 1 Effect of TNF or II.-6 on serum IL-6 activity in mice
Mice were given i.p. injections in the morning of HBSS vehicle (control) or
cytokines rHIL-6 and rHTNF, at 100 and 10 Mg.respectively. Tailbleeds were
performed 4 h later and the serum IL-6 levels were determined using the B9/HGF
assay. Values represent the mean ±SE of six animals.
plemented with 10% fetal bovine serum (GIBCO or INOVAR) and 8.0
ng of pantothenic acid per ml, referred to as complete medium. Cultures
were maintained at 37°Cin an atmosphere of 5% CO;-95% air, and the
medium was changed every 2 days. Two days after reaching confluence,
3T3-L1 cells were stimulated to differentiate by the addition of 5 ^g/ml
of insulin and 0.39 Mg/ml of dexamethasone (30. 31). The medium was
removed after 48 h, and the cultures were incubated with 50 ng/ml of
insulin for an additional 48 h. Thereafter, the cells were maintained in
complete medium without any added hormones and used for assay
when greater than 95% of cells contained fat droplets (usually after at
least 10 days from the initial insulin and dexamethasone treatment).
3T3-L1 LPL Assay. Cells were assayed for heparin-releasablc LPL
activity using the method of Nilsson-Ehle and Schotz (27) as modified
by Kawakami et al. (1). The evening before an assay, cells received 4 ml
of complete media. The next day, cytokines at the concentrations noted
in Fig. 1 were added. After 8 h, the medium was removed and replaced
with 2 ml of complete medium supplemented with 10 units of heparin/
ml. The cells were incubated for 60 min at 37°Cin an atmosphere of 5%
LPL ACTIVITE
Injection
group
Time (h)
Scrum IL-6 activity
(HGF units/ml)
<20
452 ±113
54.1 ±204
Control
IL-6
TNF
Table 2 Effects of IL-6 and T\F in vivo on LPL activity in murine aJipóse
tissue
rHTNF (10 Mg) or rHIL-6 (100 Mg) in HBSS was administered i.p. in the
morning to mice. From 4.5 to 5.5 h later, the entire periovarian and perimetric fat
pads were removed, homogenized with a Polytron. and assayed for lipoprotein
lipase activity. Values are means ±SE of two experiments with six animals/
experiment.
41 14
Group
Control
IL-6
TNF
" P < 0.005 relative to control.
h P < 0.001 relative to control; P
n
(milliunits/g wet wt)
12
12
12
492 ±64
2.10 ±26"
I50± 15*
< 0.05 relative to IL-6.
INTERLEl'KIN
6 REDUCES
3T3-L1 Adipocyte LPL Activity. Previous studies have indi
cated that the heparin-releasable fraction of LPL activity in
3T3-L1 adipocytes reflects whole cell LPL activity (8). Treat
ment of the 3T3-L1 adipocytes with IL-6 reduced LPL activity
in a dose-dependent manner (Fig. 1). Maximal reduction of
heparin-releasable LPL activity was seen with 50,000 HGF
units/ml of murine IL-6 (78% reduction), while 5000 HGF
units/ml of murine IL-6 reduced LPL activity by 50% (Fig. 1).
Recombinant human IL-6 gave similar results; 5000 HGF units
reduced heparin-releasable LPL activity by 43% and 50,000
HGF units/ml of rHIL-6 by 70%. As previously reported (31),
a maximally effective rHTNF concentration of 50 ng/ml low
ered LPL activity by 96%.
DISCUSSION
Lipoprotein lipase plays an important role in adipocyte me
tabolism by hydrolyzing circulating triglycéridesto fatty acids
for subsequent storage as fat in the adipocyte. Thus, a reduction
in the activity of LPL would contribute to a decrease in total
body fat stores. Reduced lipoprotein lipase activity has been
documented in patients and animals with the cachetic syn
drome (4-6). An intensive search for mediators of cachexia has
been conducted in a number of laboratories, with special em
phasis on the role of TNF/cachetin (12). However, elevated
TNF concentrations have generally not been found in cancer
patients with cachexia (33, 34). In addition, animals with tu
mors of different etiologies have circulating TNF concentra
tions that were undetectable or minimal (13, 35), but serum
IL-6 concentrations were elevated in parallel with tumor burden
(13, 19). Similarly, other illnesses which can result in the
cachectic syndrome, such as infectious disease, have been asso
ciated with elevated serum concentrations of IL-6 (15, 16, 36).
In this study, IL-6 was shown to reduce LPL activity in
adipose tissue in vivo when injected i.p. in mice. Thus, this
cytokine may be important in promoting the depletion of fat
stores observed in the cachetic syndrome associated with dis
ease states. Further support for this hypothesis stems from a
LPL
ACTIVITY
recent study in which the introduction of a retroviral expression
vector containing the IL-6 coding sequence into the bone mar
row of mice resulted in high serum concentrations of IL-6 and
mice with markedly decreased amounts of s.c. fat (37).
The evidence presented in this paper indicates that the
3T3-L1 adipocyte is a direct target for IL-6 action. IL-6, like
TNF, reduces LPL activity when added to the culture medium.
Although TNF decreases the rate of LPL gene transcription
(31, 38, 39), the mechanism of action for IL-6 is unknown. As
with other actions attributed to IL-6 (14), both murine and
recombinant human IL-6 were equipotent in their effects on the
adipocyte, indicating no species specificity in the murine re
sponse.
Many cytokines, including TNF, interleukin 1, interleukin 2,
and tt-interferon elevate serum IL-6 concentrations when in
jected into animals (12). Moreover, many types of cells, includ
ing fibroblasts, monocytes, and endothelial cells, elaborate
IL-6, and the production of this cytokine increases in response
to TNF as well as other cytokines (40). The 3T3-L1 preadipocyte cell line is a clonal population of cells derived from mouse
fibroblasts (41), raising the possiblity that the TNF effects on
differentiated adipocytes observed in this study might be medi
ated by the increased production of IL-6. Alternatively, the
presence of IL-6 might potentiate the direct actions of TNF on
differentiated adipocytes. Whether alone or in conjunction with
TNF, the data clearly suggest a role for IL-6 in the development
of the cachectic syndrome.
100 -,
ACKNOWLEDGMENTS
The authors thank Drs. C. Londos and A. Kimmel as well as Dr. M.
Lotze for many constructive conversations. Dr. S. Roberts for valuable
advice during preparation of the manuscript as well as assistance with
statistical analysis, and also Dr. S. Steinberg for assistance with the
statistical analysis.
Note Added in Proof
Further support for the role of IL-6 in cancer cachexia is found in the recent
paper by Strassman et al. (F.vidence for the involvement of interleukin 6 ¡n
experimental cancer cachexia, J. Clin. Invest., K9: 1681-1684. 1992). who found
that injection of anti-IL-6 antibody ¡ntumor-bearing mice significantly inhibited
the appearance of various parameters of cachexia.
01
§ t
Q- c
O o
O. «
REFERENCES
80 -
40 ^
LU LU
CONTROL
500
5000
50,000
MURINE INTERLEUKIN-6
(HGF units/ml)
50 NG/ML
TNF
Fig. I. The effect of IL-6 and TNF on LPL activity in cultured adipocytes.
3T3-L1 adipocytes were treated with the indicated concentrations of TNF and
IL-6. The cells were analyzed for heparin-releasable LPL activity 8 h after the
addition of cytokine. Data arc presented as a percentage of control LPL activity,
which was 104 milliunits/plate for IL-6 experiments and 72 milliunits/plate for
TNF experiments. Values for IL-6 experiments are means ±SE (hars) (n = 6) of
Ì
experiments and for TNF means ±SE (n = 5) of two experiments. *. signifi
cantly
different
from HGF
controlunits/ml.
P < 0.0001:
# significantly different from murine
IL-6 "value
at 50.000
P s 0.0001
4115
1. Kern. K. A., and Norton. J. A. Cancer cachexia. J. Parenteral Enterai Nutr..
12: 286-298. 1988.
2. Gossberg. S. F.. and O'Leary. W. M. Hyperlipaemia following viral infection
in the chicken embryo: a new syndrome. Nature (Lond.), 20H: 954-956. 1965.
3. Lees, R. S.. Beisel. W. R., and Bartelloni. P. J. Effects of an experimental
viral infection on plasma lipid and lipoprotein metabolism. Mctab. Clin.
Exp.. 21: 825-833, 1972.
4. Rouzcr. C. A., and Cerami, A. Hypertriglyccridemia associated with Trypanosonta brucei brucei infection ¡nrabbits: role of defective triglycéridere
moval. Mol. Biochem. Parásito!.. 2: 31-38. 1980.
5. Vlassara. H.. Speigel, R. J., San Doval. D.. and Cerami. A. Reduced lipo
protein lipase activity in patients with malignancy-associated weight loss.
Horm. Metab. Res.. 18: 698-703. 1986.
6. Lanza-Jacoby, S., Lansey, S. C., Miller, E. E., and Cleary, M. P. Sequential
changes in the activities of lipoprotein lipase and lipogenic en/ymes during
tumor growth in rats. Cancer Res.. 44: 5062-5067, 1984.
7. Eckel, R. H. Adipose tissue lipoprotein lipase. In: 3. Borens/tajn (ed.). Li
poprotein Lipase. pp. 149-185. Chicago: Evener. 1987.
8. Kawakami. M.. Pekala. P. H., Lane. M. D.. and Cerami, A. I.ipoprotcin
lipase suppression in 3T3-L1 cells by an endotoxin induced mediator from
exúdatecells. Proc. Nati. Acad. Sci. USA, 79: 912-916. 1982.
9. Beutler. B., Hamhoney. J.. Le Trang. N.. Pekala. P.. and Cerami, A. Purifi
cation of cachectin, a lipoprotein lipase-suppressing hormone secreted by
endotoxin-induced RAW 264.7 cells. J. Exp. Med., 161: 984-995. 1985.
H). Beutler, B. A.. Greenwald, D.. Hulmes, J. D., Chang. M., Pan, U-C. E.,
Mathison. J.. Ulevich, R., and Cerami, A. Identity of tumor necrosis factor
and the macrophage-secreted factor cachetin. Nature (Lond.). il fi: 552-554.
1985.
INTERLEUKIN
6 REDUCES LPL ACTIVITY
11. Semb, H., Peterson, J., Tavernier, J., and Olivecrona, T. Multiple effects of
tumor necrosis factor on lipoprotein lipase in vivo. J. Biol. Chem., 261:
8390-8394, 1987.
12. Jablons, D. M., Mulé,J. J., Mclntosh, J. K., Sehgal, P. B., May, L. T.,
Huang. C. M., Rosenberg, S. A., and Lotze, M. T. IL-6/IFN-/3-2 as a circu
lating hormone. Induction by cytokine administration in humans. J. Immunol., 142: 1542-1547, 1989.
13. Mclntosh, J. K., Jablons, D. M., Mulé,J. J., Nordan, R. P., Rudikoff, S.,
Lotze, M. T., and Rosenberg, S. A. In vivo induction of IL-6 by administra
tion of exogenous cytokines and detection of de novo serum levels of IL-6 in
tumor-bearing mice. J. Immunol., ¡43:162-167, 1989.
14. J. Van Snick. Interleukin-6: an overview. Annu. Rev. Immunol., 8: 253-278,
1990.
15. Helfgott, D. C., Tatter, S. B., Santhanam, U., Clarick, R. H., Bhardwaj, N.,
May, L. T., and Sehgal, P. B. Multiple forms of Ifn-/32/H-6 in serum and body
fluids during acute bacterial infection. J. Immunol., 142: 948-953, 1989.
16. Breen, E. C., Rezai, A. R., Nakajima, K., Beali, G. N., Mitsuasu, R. T.,
Hirano, T., Kishimoto, T., and Martinez-Maza, O. Infection with HIV is
associated with elevated II-6 levels and production. J. Immunol., 144: 480484, 1990.
17. Bataille, R., Jourdan, M., Zhang, X-G.. and Klein, B. Serum levels of IL-6,
a potent myeloma cell growth factor, as reflect of disease severity in plasma
cell dyscrasias. J. Clin. Invest., 84: 2008-2011, 1989.
18. Utsumi, K., Takai, Y., Tada, T., Ohzeki, S., Fujiwara, H.. and Hamaoka, T.
Enhanced production of IL-6 in tumor-bearing mice and determination of
cells reponsible for its augmented production. J. Immunol., 145: 397-403,
1990.
19. Gelin, J„Moldawer, L. L., Lonnroth, C.. deMan, P., Svanborg-Eden, C.,
Lowry, S. F., and Lundholm, K. G. Appearance of hybridoma growth factor/
interleukin-6 in the serum of mice bearing a methylcholanthrene-induced
sarcoma. Biochem. Biophys. Res. Commun., 157: 575-579, 1988.
20. Flick, D. A., and Gifford, G. E. Comparison of in vitro cell cytotoxic assays
for tumor necrosis factor. J. Immunol. Methods, 68: 167-175, 1984.
21. Nordan, R. P., Pumphrey, J. G., and Rudikoff, S. Purification and NH2terminal amino acid sequence of a plasmacytoma growth factor derived from
the murine macrophage cell line P388D1. J. Immunol., 139: 813-817. 1987.
22. Vink, A.. Coulie, P. G., Wauters, P., Nordan, R. P., and Van Snick, J. B. Cell
growth and differentiation activity of interleukin-HPl and related murine
plasmacytoma growth factors. Synergy with interleukin-1. Eur. J. Immunol.,
/«:607-612, 1988.
23. Aarden, L. A., Degroot, E. R.. Schaap, O. L., and Lansdorf, P. M. Produc
tion of hybridoma growth factor by human monocytes. Eur. J. Immunol., 17:
1411-1416,1987.
24. Hubert, D. M., Cancro, M. P., Sberle, P. A., Nordan, R. P., Van Snick, J.,
Gerhardt, J., and Rudikoff, S. T cell derived IL-6 is differentially required for
antigen specific antibody secretion in primary versus secondary B cell. J.
Immunol.. 143: 4010-4024, 1989.
25. Peterson, J., Olivecrona, T., and Bentsson-Olivecrona, G. Distribution of
4116
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
lipopoprotein lipase and hepatic lipase between plasma and tissues: effect of
hypertriglyceridemia. Biochim. Biophys. Acta, 837: 262-270, 1985.
Langer, C. A., Birkenmeier, E. H., Ben-Zeev, O., Schotz, M. C., Sweet, H.
O., Davisson, M. T., and Gordon, J. I. The fatty liver dystrophy (fid) muta
tion. J. Biol. Chem., 264: 7994-8003. 1989.
Nilsson-Ehle, P., and Schotz, M. C. A stable, radioactive substrate emulsion
for assay of lipoprotein lipase. J. Lipid Res., 17: 536-541, 1970.
Belfrage, P., and Vaughn, M. Simple lipid-liquid partition system for isola
tion of labeled oleic acid from mixture with glycerides. J. Lipid Res., 10:
341-344, 1969.
Mackall, J. C., Student, A. K., Polakis, S. E., and Lane, M. D. Induction of
lipogenesis during differentiation in a "pre-adipocyte" cell line. J. Biol.
Chem., 251: 6462-6464, 1976.
Rubin, C. S., Hirsch, A, Fung, C., and Rosen. O. M. Development of hor
mone receptors and hormonal responsiveness in vitro. 3. Biol. Chem., 253:
7570-7578.1978.
Zechner, R., Newman, T. C., Sherry, B., Cerami, A., and Breslow, J. L.
Recombinant human cachetin/tumor necrosis factor but not interleukin-la
downregulates lipoprotein lipase gene expression at the transcriptional level
in mouse 3T3-L1 adipocytes. Mol. Cell. Biol., 8: 2394-2401, 1988.
Mulé,
J. J., Mclntosh, J. K., Jablons, D. M., and Rosenberg, S. A. Antitumor
activity of recombinant interleukin-6 in mice. J. Exp. Med., ///; 629-636,
1990.
Socher, S. H., Martinez, D., Craig, J. B., Kühn.J. G.. and Oliff, A. Tumour
necrosis factor not detectable in patients with clinical cancer cachexia. J.
Nati. Cancer Inst., «ft-595-598, 1988.
Waage. A., Espevik, T., and Lamvik, J. Detection of tumour necrosis factorlike cytotoxicity in serum from patients with septicaemia but not from un
treated cancer patients. Scand. J. Immunol., 24: 739-743, 1986.
Moldawer, L. L., Gelin, J., Schersten, T., and Lundholm, K. G. Circulating
interleukin-1 and tumor necrosis factor during inflammation. Am. J. l'In si
ol., 253: R922-R928, 1987.
Waage, A, Brandtzaeg, P., Halstensen. A., Kierulf, P.. and Expevik, T. The
complex pattern of cytokines in serum from patients with meningococcal
septic shock. Association between interleukin-6, interleukin-1, and fatal out
come. J. Exp. Med.. 769: 333-338, 1989.
Brandt. S. J., Bodine, D. M., Umiliar. C. E., and Nienhuis, A. W. Dysregulated interleukin 6 expression produces a syndrome resembling Casleman's
disease in mice. J. Clin. Invest., 86: 592-599, 1990.
Fried, S. K., and Zechner, R. Cachectin/tumor necrosis factor decreases
human adipose tissue lipoprotein lipase mRNA levels, synthesis, and activity.
J. Lipid Res., 30: 1917-1923, 1989.
Cornelius, P., Enerback, S., Bjursell, G., Olivecrona, T., and Pekala, P. H.
Regulation of lipoprotein lipase mRNA in 3T3-L1 cells by tumour necrosis
factor. Biochem. J., 249: 765-769. 1988.
Kishimoto, T. The biology of interleukin-6. Blood, 74: 1-10, 1989.
Green, H., and Kehinde, O. Sublines of mouse 3T3 cells that accumulate
lipid. Cell, /: 113-116, 1974.