From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
Interleukin-1 Induces Interleukin-6 Production in Peripheral Blood Monocytes
By Giovanna Tosato and Karen D. Jones
Interleukin-6 (IL-6). a multifunctional cytokine produced in
monocytes, fibroblasts, endothelial cells, and keratinocytes, is induced by a variety of stimulating signals,
including lipopolysaccharide (LPS), poly (I).poly IC), IL-1,
tumor necrosis factor (TNF), and platelet-derived growth
factor. Some of these signals induce IL-6 effectively only in
one cell type, and this selectivity of induction may explain
selectivity of biologic effects. In the present study, we
show that IL-18. previously known to be a potent inducer
of IL-6 in fibroblasts, endothelial cells, and keratinocytes,
but not in monocytes, is also a potent inducer of IL-6 in
peripheral blood monocytes. High level IL-6 activity that
could be neutralized by specific antibodies to IL-6 was
detected in supernatants of IL-I -stimulated monocytes.
Maximal induction required IL-1 concentrations of 10
ng/mL. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, IL-6
species of relative molecular mass of 19 to 2 6 Kd could be
specifically immunoprecipitatedfrom supernatants of IL-linduced monocytes. Size heterogeneity is a reported featurre of IL-6 produced in a variety of cell types, and
monocyte-derived IL-6 induced by either IL-I or LPS displayedsimilar size heterogeneity. The highly purified recombinant IL-18 preparation used contained little, if any, LPS.
In addition, monocyte production of IL-6. induced by IL-la,
was specifically neutralized by anti-IL-1 B antibodies, demonstrating that IL-I, rather than a contaminant in the IL-1
preparation, was responsible for IL-6 induction. A number
of biologic activities have been ascribed both to IL-1 and
IL-6. The finding that IL-I induced IL-6 in monocytes may
help in defining the spectrum of biologic activities of each
of these interactive cytokines.
This is a US government work. There are no restrictions on
its use.
M
conditions used, monocytes fail to secrete IL-6 spontaneously. Addition of purified preparations of recombinant,
LPS-free, IL- 1j3 to these cells causes high level IL-6 production. Thus, IL-1, previously known to induce IL-6 in fibroblasts, endothelial cells, and keratino~ytes,'~"~
is also a
potent inducer of IL-6 in human monocytes.
ULTIPLICITY OF targets and multiplicity of biologic effects are common features among cytokines.
Interleukin-6 (IL-6), one of the most pleiotropic and promiscuous of cytokines, has been reported to regulate the synthesis of many acute phase plasma proteins in liver cells,' to
promote growth and immunoglobulin (Ig) secretion in B cells
infected with Epstein-Barr virus (EBV):3 and to act as an
accessory signal for mitogen costimulated T cells!vs IL-6 has
also been reported to have antiviral
to support
colony formation by hematopoietic progenitor cells,' to act as
a growth factor for certain mouse B-cell hybridomas and
plasmacytomas,9-" to serve as a differentiation factor for the
development of cytolytic T cells,12to synergize with IL-1 in
the stimulation of thymocyte pr~liferation,'~
and to serve as a
growth factor for human plasmacytoma^.'^^'^
Many of the biologic activities attributed to IL-6 have also
been attributed to other cytokines, to hematopoietic colony
stimulating factors, to interferons, and to tumor necrosis
factor (TNF). In particular, IL-1 displays a number of the
biologic activities of IL-6, such as regulation of growth and
differentiation of cells in the immune and hematopoietic
systems, and modulation of acute phase responses.I6
To begin to separate the spectrum of activities of IL- 1
from those of IL-6, it is important to distinguish between
direct IL-1 effects, which derive from an interaction of this
ligand with a specific target, and indirect effects, which
derive from induction of secondary cytokines.
It is well-established that IL-1 is a potent inducer of IL-6
in fibroblasts, endothelial cells, and keratin~cytes.'~-'~
In
contrast, it is not clear whether IL-1 induces IL-6 production
in monocytes. Separate studies of this issue have reported
both that IL-1 is a poor inducer," a noninducer,2' or a good
inducer2' of IL-6 activity or IL-6 gene expression.
Peripheral blood monocytes are stimulated to produce
IL-6 by a variety of signals and are exquisitely sensitive to
bacterial lipopolysaccharide (for a review, see reference 21).
Therefore, it is possible that differences in culture conditions
and/or lipopolysaccharide (LPS) contamination of the IL- 1
preparation used might explain differences in results.
In the present study, we show that under the culture
Blood, Vol 75, No 6 (March 15). 1990: pp 1305-1310
MATERIALS AND METHODS
Preparation of cells and cell lines. Peripheral blood mononuclear cells (PBMC) were obtained by density centrifugation of
normal peripheral blood. Monocyte-enriched populations were obtained by plastic adherence of PBMC during 1.5-hour incubation at
37OC, and contained 78% to 82% monocytes as determined by
staining with nonspecific esterase.'' In selected experiments, monocytes were further purified by elutriation of PBMC, and contained
90% to 95% nonspecific esterase positive cells.24Lymphoblastoid cell
lines were obtained by EBV (B95-8) immortalization of peripheral
blood B cellsF5The IL-Mependent murine hybridoma cell line B926
was a gift of Dr R. Nordan (NIH, Bethesda, MD).
Reagents, cytokines, and antibodies. Highly purified, Escherichia coli-derived IL-lj3 (a gift of Dr Y. Hirai, Otsuka Pharmaceutical Co, Tokushima, Japan) had a specific activity of approximately
1 x lo7 Units/mg pr~tein.'~One unit of IL-1 is defined as the
amount required for half-maximal costimulation of mouse thymocytes. LPS from E coli 0127:BS was purchased from Sigma
Chemical Co, St Louis, MO. A previously characterized" rabbit
From the Division of Biochemistry and Biophysics, Center for
Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD.
Submitted July 17.1989: accepted November 29,1989.
Supported in part by Grant No. 87PP7855 from the Department
of the Army.
Address reprint requests to Giovanna Tosato. MD, FDA, NIH,
Bldg 29, Rm 505. Bethesda, MD 20892. Telephone (301) 496-3001.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
This is a US government work. There are no restrictions on its
use.
0006-4971/7506-O021$O.OO/O
1305
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
TOSATO AND JONES
1306
antiserum to purified E coli-derived IL-6 (a gift of Drs L. May and
P. Sehgal, Rockefeller University, NY) was purified using protein
A-Sepharose. A previously ~ h a r a c t e r i z e drabbit
~~
antiserum to
purified recombinant human IL-IS (R290; a gift of Drs R. Newton
and J. Giri, DuPont Glenolden Lab, Glenolden, PA) was purified
using protein A-Sepharose. Normal rabbit serum was purified using
protein A-Sepharose.
Cell cultures for IL-6 production and assay for IL-6 activity.
Culture medium for IL-6 production consisted of Opti-MEM
(GIBCO Laboratories, Grand Island, NY) supplemented with
mol/L 2ME and 5 pg/mL gentamicin (Sigma). PBMC, suspended
in Opti-MEM (15 x lo7 cells in 5 mL) were incubated in tissue
culture flasks (25 cm2; Costar, Cambridge, MA) for 1.5 hours at
37OC in a humidified atmosphere containing 5% CO,. After incubation, nonadherent cells were vigorously removed, 5 cc of culture
medium was added to the adherent cells, and flasks were incubated
for an additional 24 hours. At the end of culture, cell-free culture
supernatants were supplemented with Aprotinin (10 pg/mL, Sigma)
and frozen at - 2OOC.
Monocyte-enriched (90% to 95% nonspecific esterase positive
cells) cells, monocyte-depleted (less than 3% nonspecific esterase
positive cells) cells, and PBMC obtained by elutriation were incubated (30 x lo6cells in 5 mL Opti-MEM) for 24 hours at 37OC in a
humidified atmosphere containing 5% CO,. At the end of culture,
cell-free supernatants were harvested and stored as described above.
IL-6 in culture supernatants was quantitated by its ability to
support the growth of the IL-64ependent hybridoma cell line, B9.26
Briefly, 2 x lo3 exponentially growing B9 cells in 0.1 mL medium
consisting of RPMI 1640 (Biofluids, Rockville, MD) supplemented
with 20% fetal calf serum (FCS, Reheis, Armour Pharmaceutical
Co, Kankakee, IL), 2 mmol/L L-glutamine (GIBCO), 5 pg/mL
gentamicin (Sigma), and 2 x
mol/L 2ME were incubated in 96
well flat-bottom plates (Costar) for 3 days with the addition of 100
pL of either control medium or serial dilutions of test supernatants.
At the end of culture, cell proliferation was measured by
[3H]thymidine (net 027; New England Nuclear, Boston, MA;
specific activity 6.7 Ci/mmol/L) incorporation during an 18-hour
incubation at a final concentration of 0.5 pCi per culture. Proliferation was expressed as mean cpm of triplicate cultures. One unit of
IL-6 is defined as the amount required for induction of one-half
maximal stimulation of proliferation of B9 cells. IL-6 was also
measured by its ability to promote the growth of EBV-infected
lymphoblastoid cells cultured at low density, as described?
Radiolabeling, immunoprecipitation, and polyacrylamide gel
electrophoresis. Monocyte-enriched populations, obtained by plastic adherence (1.5 hour on 25 cm2 tissue culture flasks) of 15 x IO6
PBMC in 5 mL methionine-deficient MEM (GIBCO) with 1%
heat-inactivated dialyzed FCS (Reheis Armour Pharmaceutical),
and 2 mmol/L L-glutamine were cultured in the same medium ( 5
mL) with 100 pCi/mL %methionine (New England Nuclear;
1,000 Ci/mmol). At the end of culture, cell-free supernatants were
mixed with a saturated solution of ammonium sulfate (50% vol/vol)
and the resulting precipitates were suspended in 0.25 mL Trisbuffered saline (PBS; 25 mmol/L Tris, pH 7.4) containing 2
mmol/L phenylmethylsulfonyl fluoride (PMSF) and 150 pg/mL
L-1-tosylamido-2-phenylethyl-chloromethyl
ketone (TPCK). The
immunoprecipitation was performed essentially as described.24An
aliquot (100 pL) of labeled precipitate that had been precleared
using 100 pL of a 10% (wt/vol) suspension of heat-killed and
formalin-treated Staphylococcus aureus Cowan strain 1 (Calbiochem, La Jolla, CA), was incubated for 18 hours at 4OC with 1 pL or
either a control normal rabbit serum on an antiserum to purified E
coli-derived IL-6 (a gift of Drs L. May and P. Sehgal). A total of 100
pL of the 10%(wt/vol) S aureus preparation was then added, and
after 45 minutes incubation at 4OC, the bacteria were pelletted and
washed three times with a cold (4OC) immunoprecipitation buffer
(PBS, 25 mM Tris, pH 7.4, 1% Triton X-100,2 mmol/L PMSF, 150
pg/mL TPCK, and 2.5 mol/L KCI). After two additional washes
with isotonic immunoprecipitation buffer at 4OC, the pellets were
suspended in 40 pL of 2x gel sample buffer (0.125 mol/L Tris, pH
6.8, 4% sodium dodecyl sulfate (SDS), 10% 2ME, 20% glycerol)
boiled 2 minutes, centrifuged, and then electrophoresed through a
12.5% polyacrylamidegel containing 0.1%SDS." After electrophoresis, the gel was fixed, fluorographed by treating with Enlightening
(New England Nuclear), dried and autoradiographed at - 7OOC.
RESULTS
To investigate IL-1 induction of IL-6 in monocytes, we
selected a preparation of E coli-derived IL-lp (Otsuka
Pharmaceutical) that was found to have a very low content of
LPS. In three separate testings, this IL-1 preparation a t the
concentration of 1 mg/mL, contained equal to or less than
2.5 endotoxin Units/mL, as determined by the Limulus
assay.30 This corresponds to a content of LPS not greater
than 250 pg/mg of IL-1 and, in each determination, the
diluent in which IL-1 was suspended, 0-MEM, was found to
contain an equal or slightly higher amount of LPS.
As a source of monocytes we used plastic adherent PBMC
that contained 79% to 82% nonspecific esterase-positive cells.
Monocyte-enriched populations were incubated for 24 hours
in serum-free culture medium (0-MEM) alone or supplemented with either LPS (1 pg/mL) or recombinant IL-lp
(100 ng/mL). At the end of culture, the filtered (0.45 p )
supernatants were tested for IL-6 activity using the IL-6dependent hybridoma cell line, B9.26As shown in representative experiments (Table l), while unstimulated cell culture
supernatants contained low level IL-6 activity, both LPS and
IL-1 stimulated culture supernatants contained large and
comparable levels of IL-6 activity. It should be noted that
previous experiments had shown that the dose of LPS used, 1
pg/mL, induced maximal IL-6 release in plastic adherent
PBMC.24It should also be noted that control media supplemented with LPS (1 pg/mL) or IL-1 (100 ng/ml) run in
parallel had no detectable IL-6 activity (not shown).
Production of IL-6 activity in monocyte-enriched populations was dependent upon the dose of IL-1 used. As shown in
Fig 1, maximal IL-6 activity was induced by IL-1 at
concentrations of 10 ng/mL. This finding indicates that
Table 1. IL-la Induces IL-6 Activity in Monocyte-Enriched
Populations
11-6 Content (U/mL)
Stimuli
Exp 1
None
LPS (1 pglmL)
IL-lP ( 1 0 0 ng/mL)
15
9.472
10,240
Exp 2
Exp 3
6,656
5,120
1,824
7.1 14
8,004
Plastic adherent mononuclear cells (obtained by incubating 15 x lo7
PBMC in 5 mL 0-MEM for 1.5 hours in 25 cmz tissue culture flasks) were
cultured for 2 4 hours in 5 mL serum-free culture medium (0-MEM) alone
or supplemented with either LPS (Sigma), or IL-10 (Otsuka). Cell-free
supernatantsof plastic adherent PBMC were tested for IL-6 content using
the IL-6-dependent hybridoma cell line, 8 9 . Culture medium supplemented with IL-1 (100 ng/mL) or LPS (1 pg/mL) failed to stimulate
proliferation in B9 cells above background. Results are expressed as
units/mL of IL-6 in cell-free culture supernatant.
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
IL-1 INDUCES IL-6 PRODUCTION IN MONOCYTES
1307
Table 2. Mononuclear Cell Production of IL-6 Upon IL-1
Stimulation Is Due to Monocytes
1,000
IL-6 (U/mLl
400
8 200
r
-E
X
.
2
4
1
Stimuli
Monocyte-Enriched
Monocyte-Depleted
None
LPS (1 pglmL)
IL-lj3(10ng/mL)
48
3,176
3,615
4
28
14
Monocyte-enriched (greater than 95% nonspecific esterase-positive
cells) and monocyte-depleted (2% nonspecific esterase-positive cells)
PBMC obtained by elutriationof PBMC were cultured (30x 10' cells in 5
mL 0-MEM) for 24 hours. Cell-free supernatants of 24-hour monocyte
cultures were tested for IL-6 content using the IL-6-dependent cell line,
69. Results are expressed as 11-6 units/mL in cell-free culture supernatants.
80
40
20
10
$5
0
0.01
0.1
1.0
10
100
1,000
IL-1 (nglml)
Similar to LPS-induced monocyte supernatants, IL- 1induced monocyte supernatants promoted growth in EBVimmortalized B cells, a property previously attributed to
IL-6 (Table 3).'2*24 This B cell stimulatory function was also
neutralized by anti-IL-6 antibodies (data not shown).
To further establish that IL-6 is induced in IL-1stimulated monocytes, we tested whether it could be immunoprecipitated from these supernatants. As shown in Fig 3A,
Monocyte-enriched
Fig 1. Induction of IL-6 activity. by. IL-1s.
.
populations, obtained by plastic adherence of 15 x lo7 PBMC.
were incubated in tissue culture flasks (25 cm2)for 24 hours in 5
mL serum-free culture medium (0-MEMI alone or with the addition
of recombinant IL-lB at varying concentrations. At the end of
culture, cell-free supernatants were tested for IL-6 activity using
the IL-6-dependent hybridoma cell line, B9. The results represent
the geometric mean x / t SEM of four determinations.
optimal induction of IL-6 activity in monocyte-enriched
populations is observed at IL-1 concentrations similar to
those required for IL-6 induction in fibroblasts and endothelial cell^.'^^^'
Among PBMC, monocytes are known to represent the
major cellular source of IL-6.26It was possible, however, that
contaminating lymphocytes contributed to IL-6 activity
production. To address this possibility, we further purified
PBMC by elutriation to achieve a cell population containing
greater than 95% monocytes (nonspecific esterase positive
cells). A comparison between IL-6 activity produced in
mononuclear cells enriched and depleted of monocytes (containing 3% nonspecific esterase-positive cells) showed that
only negligible levels of IL-6 activity were induced by IL-lB
in monocyte-depleted populationswhen compared with monocyte-enriched populations (Table 2). This finding demonstrates that monocytes are the source of IL-6 activity upon
stimulation with IL-1p.
Although the hybridoma cell line B9 has been reported to
be IL-6-specific, and not to proliferate in response to other
known growth factors,26it is possible that molecules other
than IL-6 might induce it to proliferate. To ensure that IL-6,
induced by IL-1 in monocytes, was the molecule responsible
for growth of B9 cells, we performed neutralization experiments. As shown in Fig 2, unlike a control rabbit IgG, an
antibody to E coli-derived IL-628 neutralized the growth
promoting activity of an IL- 1-induced monocyte supernatant in a dose-dependent manner.
Supernatant
+ Control Antibody
Supernatant
+
Anti-IL-6
Antibody
1
None
2.5
5
10
20
Antibody Concentration (pglml)
Fig 2. Neutralization of IL-1-induced IL-6 activity by antibodies
to E coli-derived IL-6. B9 cells were cultured for 72 hours with or
without the addition of an IL-1-induced monocyte supernatant
(diluted 1:250) alone or in the presence of serial dilutions of either
control normal rabbit lgG or a rabbit antibody to IL-6. Appropriately diluted rabbit IgG (control and anti-IL-6) were incubated with
either medium alone or monocyte supernatant for 1 hour at 37°C
before the addition of B9 cells. Proliferation was measured by
[3H]thymidine incorporation during the last 18 hours of culture,
and the results are expressed as mean cpm of triplicate culture.
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
-
1308
TOSIT0 AND JONES
-
3. 8-COU6aw((l FrCar Aatkky h 11-1
1-
sw--t-t.
-w
I
'
M
-
kOdClBUd
VDSO
*dd"
~ - 3 ~ 4
2.1 11
410
715
1.317
1.925
2.714
2W7
-*
8.140
10.516
8.914
SuD(*Nlmtducdn"nkn
% m ~ t m t w m -
LPS
RY
1.478
1.1 ( 4
M.am*LPS
M
W + IL-1
-~
SJOO-U~~~-~-
TI
316
218
Mabwn
-
12.61a Ba125
~~v.-.lurd
B 0l.r 13 x i o ' p ~r v ~ ln 0.1 ml OMEM) ban
ttrw bmohobhtadcd km NDS.TI. AW n " w r d Qorwchphao
-0
CJ1w.d f~ 3 dm mrh 0 . 1 ml d ma
Q "OCYI.
wmotmt* (1:4 d*nm).R*"*vu
-dhl'H)tm
lrYXXOQnm
duln0 tho bt 'E
of CJrwo. Adcbtom Iotho
EBV-~.cc.dBo*rnclubdrclwnwamlO-MEM)-.arnam
-1.d
wvth LPS I 1 rJml) Q mrdvn -t.d
wlth IL- 1
I10 ne/mL): swwufmt d phatu o & m n r PBMC CJCwd for 24 ham
a,hr n
,O-MEM).lorw or nuckm ~
t with LPSd ( 1
---rJd).
-.
"
Q
b1 6.715
for
"m0
c~t-:
"
wml,.
Au
. h,
SO m t 10%
~ d tb
w,h IL-l llO
-nd
"S-labeled supernatants of IL-1 ( 5 ng/mL)-induced m o m
cytes contained at least four IL-6 species ranging in size
between 19 and 26 Kd. as judged by SI)S-polyacrvlamidegel
elcctrophomis (PAGE) under reducing conditions after
immunoprecipitation (Fig 3A. lane 3). Ry contrast. control
supernatants of monocytes cultured in medium alone contained little immunoprccipitable 11.4 (Fig 3A. lane I).
Taken together. these mults confirm that 11.-1 induces IL-6
secretion in monocytes. and indicate that the induccd lb-6 is
heterogenous in s i x . Heterogeneity is a reported property of
human 11.4 produced in a variety of cell t y p . ' ' Indeed.
supernatants of 1.W-stimulated monocytes. rated in paralIcl. also revealed the pmenceof distinct 11.-6 species. similar
in site to those detected in IL-I-induced supernatants (Fig
3A. lane 2).
As described earlier. the recombinant IL-I8 used in thesc
experiments had little. if any. LK contamination and was
highly purified." To further establish that IL-I. rather than
a contaminant in the IL-l preparation. was inducing IL-6
production in monocytes. a specific neutralizing antibody to
recombinant human Il,-lS.- was u . 4 together with II.-Id
during monocyte stimulation. As shown in Fig 3h. significantly lower amountsof IL-6 were immunoprecipitated from
"S-labeled supernatantsof monocytes incubated with IL-IB
( 5 ng/ml.) plus anti-IL-lfl antibody (20 aglml.) when
compared with supernatants of monocytes incubated with
IL-ld(5 ng/mL) alone (Iancs6and 3. respectively). Indeed.
comprable amounts of immunoreactive 11.4 were detected
in supernatants of monocytes incubated r i t h anti-IL-l
antibody alone (lane 5 ) or together with IL-I6 (lane 6).In
contrast to the results obtaincd with anti-lL-It? antibody.
control rabbit IKJ(20rg/ml.) failed to inhibit IL-6production in IL-I-stimulated monocytes ( f i g 3C. lane 5 ) . These
results further prove that 11.-Id indum IL-6 production in
peripheral blood monocytes.
DISCUSSION
In this report. we show that IL-119 indum IL-6 production
in peripheral blood monocytes. Supernatants of IL-113induced monocyte populations supported the growth of an
IL-6-dependent murine hybridoma cell line. €39. and enhanced the proliferation of ERV-infected R cells. Roth of
these biologic activities. previously ascribed to human IL-6.
were neutralired by antibodies r a i d against purified E
coliderived IL-6. In addition. 11.4 species could be specifia l l y immunoprecipitated from '%-lablcd supernatants of
Il.-IB-induccd monocytes.
It is unlikely that a contaminant prescnt in the highly
purified I I . - I d prepamtion used was responsible for inducing
-
Fb 3. IC-1 lnductlm d I C 4 k ryvhJI.d by
mi-N-16. m w r ~ l d w d
fd.r M by -(e
.
d
h
.
r
md 16 x 10' PSMC)
-0
Incubocod (a 24 h a r m h 6 n-d. rodiol~boting
madkm (mothmdno-dofockmM M 0 u p p b " t . d
m t h 1% dulyzod CCS. 2 " d l L L-gknomtno. and
100 rCilmL "S.nwthmdno). At tho md of cukwo.
coll4roo *up.rnotontm wora immunoproeiptotd
m r h o i t h on onti-IL-0 robbh onth.rum (A. hnoa
1 throvgh 7: ond C. Ionom 4 through 0 ) 01 eontrd
normol robbh u ~ u m
18. hnoa 1 through 7: ond C.
Ionom 1 through 3). A d d h e " w e om o)or*.:A
and B: hno 1. d k m aton.: imo 2. LPS 1 rglmC:
hno 3. n - 1 8 6 ng/mL: bno 4. n-1B 1 nglmL: brim
6. ant~-tL-18ontibodylOrglmL:L.cwO.mti-L-16
ontibody 10 rglmL pkn IL-1B 6 nglmL: L.no 7.
tL.14 2 nglmC. C: tanom 1 and 4. normol r A b h IgO
10 rglmL: lonu 2 and 4. ngrnul robbit IgG 10
(rglmL pha n - 1 6 6 nglml: md Ionom 3 and 6.
m w m o l r o ~ I g O l O ~ g l m L p k n I L - 1l B
nglmL.
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
1309
IL-1 INDUCES IL-6 PRODUCTION IN MONOCYTES
IL-6 in monocyte preparations. LPS, an excellent inducer of
IL-6 in human monocytes that could have contaminated E
coli-derived material, was either absent or present in concentrations too low to account for the results. In addition, an
antibody to highly purified IL-18, but not a control antibody,
inhibited the capacity of this IL-18 preparation to induce
IL-6 secretion in monocyte preparations.
There is evidence that IL-18 acts on monocytes rather
than on contaminant cell populations to induce IL-6 production. First, among PBMC, monocytes represent the principal
source of IL-6.24v26s32
Second, we have found that highly
purified blood monocytes, but not monocyte-depleted mononuclear cells, produce abundant IL-6 activity upon IL-1
stimulation.
A variety of stimuli are known to induce IL-6 production.
Endotoxin is a strong inducer of IL-6 in human fibroblast and
monocyte
Infection with several RNA- and
DNA-containing viruses is associated with IL-6 production
in
TNF, platelet-derived growth factor, and
81-interferon also induce IL-6 in
IL- 1 promotes IL-6 production in human fibroblasts, endothelial
cells, and keratin~cytes.l~-’~
However, the role of IL-1 as an inducer of IL-6 in
monocytes has been
A previous study
reported that natural IL-18 was a poor inducer of IL-6 in
human monocytes that were found to constitutively secrete
significant levels of IL-6 activity.” When cultured in serumfree medium, as shown in this report, monocytes secrete very
little IL-6. This suggests that “constitutive” IL-6 production
in monocyte cultures derives from in vitro exposure to
IL-6-inducing signals; monocytes already activated by these
signals may be refractory to further stimulation with IL-1. It
is also possible that the failure to detect IL-18 induction of
IL-6 may reflect the state of maturation of the target cells,
because, unlike monocytes, macrophages have been reported
not to secrete IL-6 in response to IL-18.37 When compared
with LPS a t optimal concentrations, we found that human
IL-18 a t 10 to 100 ng/mL is an equally potent inducer of
IL-6. In addition, as judged by SDS-PAGE in reducing
conditions, IL-6 induced by IL- 1 in monocytes has a similar
degree of heterogeneity to that of IL-6 induced by LPS in the
same cells.
IL-la, not used in the present experiments, was reported in
one study not to induce IL-6 gene expression in peripheral
blood monocytes,21and, in a contrasting study, was reported
to be a good inducer of IL-6 gene in the same cell population.22
While I L - l a and 8 have been reported to use a single type of
plasma membrane re~eptor,~’
further studies will be necessary to clarify the role of I L - l a as an inducer of IL-6 in
monocytes.
Over the last 2 years, we have learned a great deal about
the structure and biologic activity of IL-6 in experimental
conditions, and we are beginning to understand its role in
Nonetheless, we know little about IL-6 interactions
with other cytokines also involved in the regulation of acute
responses and in the growth and differentiation of cells of the
immune and hematopoietic systems. Within this cytokine
network, we now know that IL-1 and TNF act as inducers of
IL-6 gene expression in a variety of cell type^.^,^,'',^^ W e also
know that IL-6 cooperates with IL-1 for stimulation of T
cells and thymocyte^.'^.^^.^^ Alone, IL-6 can act as a cytokine
inducer promoting secretion of IL-2 and perhaps of other
autocrine T-cell growth f a ~ t o r s . ’ ~ . ~ ~
Future studies will amplify our knowledge of how IL-6 fits
in the cytokine network. The finding reported here, that IL-1
is a potent inducer of IL-6 in monocytes, provides the basis
for a better understanding of how these interactive cytokines
act individually and in concert to mediate monocyte-derived
functions.
ACKNOWLEDGMENT
We thank Dr T. Gerrard for providing monocytes, Drs L. May
and P. Sehgal for providing an anti-IL-6 antiserum, Drs R. Newton
and J. Giri for providing an anti-IL-lP antiserum; Dr R. Nordan for
providing the B9 cell line, Dr Y. Hirai for providing human IL-1j3,
Dr H.D. Hochstein for performing Limulus assays, Dr S. Epstein for
critically reading the manuscript, and E. Caruso for expert preparation of the manuscript.
REFERENCES
1. Gauldie J, Richards C, Harnish D, Landsdorp P, Baumann H:
Interferon @2/BSF-2shares identity with monocyte-derived hepatocyte stimulating factor 9HSF and regulates the major acute phase
response in liver cells. Proc Natl Acad Sci USA 84:7251,1987
2. Tosato G, Seamon KB, Goldman ND, Sehgal PB, May LT,
Washington GC, Jones KD, Pike S E Identificationof a monocytederived human B cell growth factor as interferon-j32 (BSF-2, IL-6),
Science 239502,1988
3. Hirano T, Taga T, Nakano N, Yasukawa K, Kashiwamura S,
Shimizu K, Nakajima K, Pyun KH, Kishimoto T: Purification to
homogeneity and characterization of human B cell differentiation
factor (BCDF or BSPp-2). Proc Natl Acad USA 825490, 1985
4. Garman RD, Jacobs KA, Clark SC, Raulet DH: B-cellstimulatory factor 2 (82 interferon) functions as a second signal for
interleukin 2 production by mature murine T cells. Proc Natl Acad
Sci USA 84:7629,1987
5. Lotz M, Jirik F, Kabouridis P, Tsoukas C, Hirano T, Kishimot0 T, Carson D: B-cell stimulating factor 2/interleukin 6 is a
costimulant for human thymocytes and T lymphocytes. J Exp Med
expression of cDNA and genes from human interferon 82: A distinct
species inducible by growth stimulatory lymphokines. EMBO J
5:2529, 1986
7. May LT, Helfgott DC, Sehgal PB: Anti-&interferon antibodies inhibit the increased expression of HLA-B7 mRNA in tumor
necrosis factor-treated human fibroblasts: Structural studies of the
82 interferon involved. Proc Natl Acad Sci USA 833957, 1986
8. Ikebuchi K, Wong GG, Clark SC: Interleukin 6 enhancement
of interleukin 3-dependent proliferationof multipotential hemopoietic progenitors. Proc Natl Acad Sci USA 84:9035, 1987
9. van Damme J, Opdenakker G, Simpson RJ, Rubira MR,
Gayphas S, Vink A, Billiau A, van Snick J: Identification of the
human 26 kD protein interferon 82 (IND-P2) as a B cell hybridoma/
plasmacytoma growth factor induced by interleukin 1 and tumor
necrosis factor. J Exp Med 165:914, 1987
10. Nordan RP, Pumphrey JG, Rudikoff S: Purification and
NH,-terminal sequence of a plasmacytoma growth factor derived
from the murine macrophage of cell line P388D1. J Immunol
167:1253,1988
6. Zilberstein A, Ruggieri R, Korn JH, Revel M: Structure and
139313, 1987
11. van Snick J, Vink A, Cayphas S,Uyttenhove C: Interleukin-
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
1310
HPI, a T cell-derived hybridoma growth factor that supports the in
vitro growth of murine plasmacytoma. J Exp Med 165:641,1987
12. Takai Y, Wong GG, Clark SC: B-cell-stimulatory factor-2 is
involved in the differentiation of cytotoxic T lymphocytes. J Immuno1 140508, 1988
13. Elias JA, Trinchieri G, Beck JM, Simon PL, Sehgal PB, May
LT, Kern J A A synergistic interaction of IL-6 and IL-1 mediates
the thymocyte-stimulating activity produced by recombinant IL- 1stimulated fibroblasts. J Immunol 142509, 1989
14. Kawano M, Hirano T, Matsuda T, Taga T, Horii Y, Iwato K,
Asaoku H, Tang B, Tanabe 0, Tanaka H, Kuramoto A, Kishimoto
T: Autocrine generation and requirement of BSF-2/IL-6 for human
multiple myelomas. Nature 332:83,1988
15. Klein B, Zhang X-G, Jourdan M, Content J, Houssian F,
Aarden L, Piechaczyk M, Bataille R: Paracrine rather than autocrine regulation of myeloma cell growth and differentiation by
interleukin 6. Blood 73517, 1989
16. Dinarello CA, Mier JW: Current concepts. Lymphokines. N
Engl J Med 317:941,1987
17. Kohase M, May LT, Tamm I, Vilcek J, Sehgal PB: A
cytokine network in human diploid fibroblasts: Interaction of betainterferons, tumor necrosis factor, platelet derived growth factor and
interleukin 1. Mol Cell Biol7:273,1987
18. Sironi M, Brevario F, Proserpio P, Biondi A, Vecchi A, van
Damme J, Dejana E, Mantovani A: IL-1 stimulates IL-6 production
in endothelial cells. J Immunol 142549, 1989
19. Kirnbower R, Kock A, Schwarz T, Urbanski A, Krutmann J,
Borth W, Damm D, Shipley G, Ansel JC, Luger TA: IFN-j32, B cell
differentiation factor 2, or hybridoma growth factor (IL-6) is
expressed and released by human epidermal cells and epidermoid
carcinoma cell lines. J Immunol 142:1922, 1989
20. van Damme J, van Beeumen J, DeCook B, van Snick J, De
Ley M, Billiau A: Separation and comparison of two monokines with
lymphocyte-activating factor activity: IL-Ij3 and hybridoma growth
factor (HGF). Identification of leukocyte-derived HGF as IL-6. J
Immunol140:1534,1988
21. Ray A, Tatter SB, Santhanam U, Helfott DC, May LT,
Sehgal PB: Regulation of expression of interleukin 6. Molecular and
clinical studies. Ann NY Acad Sci 557:353, 1989
22. Navarro S, Debibi N, Bernaudin J-F, Vainchenker W, Doly
J: Regulation of the expression of IL-6 in human monocytes. J
Immunol142:4339,1989
23. Miller GA, Monahan PS: Use of non-specific esterase strain,
in Adams DO, Edelson PJ, Koren H S (eds): Methods for Studying
Mononuclear Phagocytes. New York, NY, Academic, 1981, p 367
24. Tosato G, Gerrard TL, Goldman NG, Pike SE: Stimulation
of EBV-activated human B cells by monocytes and monocyte
products. Role of IFN-j32/B cell stimulatory factor 2/IL-6. J
Immunol140:4329, f988
25. Tosato G, Marti GE, Yarchoan R, Hellman CA, Wang F,
Pike SE, Korsmeyer SJ, Siminovitch K: Epstein-Barr virus immortalization of normal cells of B cell lineage with non-productive,
rearranged immunoglobulin genes. J Immunol 137:12037, 1986
26. Aarden LA, DeGroot ER, Schaap DL, Landsorp PJ: Production of hybridoma growth factor by human monocytes. Eur J
Immunol 17:1411,1987
27. Nakari S, Mizuno K, Kaneta M, Hirai Y: A simple, sensitive
bioassay for the detection of interleukin 1, using human melanoma
A375 cell line. Biochem Biophys Res Commun 154:1189,1988
28. May LT, Gharayeb J, Santhanam U, Tatter SB, Sthoeger Z,
Helfgott DC, Chiorazzi N, Grieninger G, Sehgal PB: Synthesis and
TOSATO AND JONES
secretion of multiple forms of P2-interferon/B cell differentiation
factor 2/hepatocyte stimulatory factor by human fibroblasts and
monocytes. J Biol Chem 263:7760, 1988
29. Uhl J, Newton RC: Quantitation of related proteins by
Western blot analysis. J Immunol Methods 110:79, 1988
30. Hochstein HD, Seligmann EB, Marquina RE, Rivera E:
Limulus amebocyte lysate testing of normal serum albumin (human) released in the United States since 1975. Dev Biol Stand 44:35,
1979
31. van Damme J, Cayphas S, Opdenakker G, Billiau A, van
Snick J: Interleukin 1 and poly (r1)-poly (rC) induce production of a
hybridoma growth factor by human fibroblasts. Eur J Immunol17: 1,
1987
32. Horii Y, Muraguchi A, Suematsu S, Matsuda T, Yoshizaki
K, Hirano T, Kishimoto T: Regulation of BSF-2/IL-6 production by
human mononuclear cells. Macrophage-dependent synthesis of BSF2/IL-6 by Tcells. J Immunol 141:1529, 1988
33. Helfgott DC, May LT, Sthoeger Z, Tamm I, Sehgal PB:
Bacterial lipopolysaccharide (endotoxin) enhances expression and
secretion of j32 interferon by human fibroblasts. J Exp Med 166:
1300,1987
34. Sehgal PB, Helfgott DC, Santhanam U, Tatter SB, Clarick
RH, Ghrayeb J, May L T Regulation of the acute phase and
immune responses in viral disease: Enhanced expression of the
@2-interferon/hepatocyte simulating factor/interleukin 6 gene in
virus-infected human fibroblasts. J Exp Med 167:1951, 1988
35. Nakajima K, Martinez-Maza 0, Hirano T, Breen EC,
Nishamian P, Salazer-Gonzalez JF, Fahey JL, Kishimoto T: Induction of IL-6/B cell stimulatory factor 2/IFN-j32 production by HIV.
J Immunol 142:531,1989
36. Kohase M, Henriksen-DeStefano D, May LT, Vilcek J,
Sehgal PB: Induction of j32-interferon by tumor necrosis factor: A
homeostatic mechanism in the control of cell proliferation. Cell
45:659,1986
37. Bauer J, Canter U, Geiger T, Jacobshagen U, Hirano T,
Matsuda T, Kishimoto T, Audus T, Acs G, Gerok W, Ciliberto G:
Regulation of interleukin 6 expression in cultured human blood
monocytes and monocyte-derived macrophages. Blood 72: 1134,
1988
38. Dower SK, Kronheim SR, Hopp TP, Cantrell M, Deeley M,
Gillis S, Henney CS, Urdal DL: The cell surface receptors for
interleukin a and @ are identical. Nature 324:266, 1986
39. Nijsten MWN, De Groot ER, ten Duis HJ, Klasen HJ, Hack
CE, Aarden LA: Serum levels of interleukin-6 and acute phase
responses. Lancet 2:921, 1987
40. Helfgott DC, Tatter SB, Santhanam U, Clarick RC, Bhardnaj N, May LT, Sehgal PB: Multiple forms of IFN-j32/IL-6 in
serum and body fluids during acute bacterial infection. J Immunol
142:948,1989
41. Fong Y, Moldawer LL, Marano M, Wei H, Tatter SB,
Clarick RH, Santhanam U, Sherris D, May LT, Sehgal PB, Lowry
SF: Endotoxemia elicits increased circulating j32-interferonl
interleukin-6 in man. J Immunol 142:2321, 1989
42. Houssiau FA, Coulie PG, Olive D, van Snick J: Synergistic
activation of human T cells by interleukin 1 and interleukin 6. Eur J
Immunol 18:653,1988
43. Ceuppens JL, Baroja ML, Lorre K, van Damme J, Billiau A:
Human T cell activation with phytohemagglutinin. The function of
IL-6 as an accessory signal. J Immunol 141:3868, 1988
44. Tosato G, Pike S E Interferon j32/interleukin 6 is a costimulant for human T lymphocytes. J Immunol 141:1556
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
1990 75: 1305-1310
Interleukin-1 induces interleukin-6 production in peripheral blood
monocytes
G Tosato and KD Jones
Updated information and services can be found at:
http://www.bloodjournal.org/content/75/6/1305.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.