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Production of natural killer cell activity-augmenting factor interleukin-6 by human epiphyseal chondrocytes.

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Objective. We sought to determine the capacity of
human epiphyseal chondrocytes to modulate the cytotoxic activity of human natural killer (NK) cells by
determining whether they release interleukin-6 (IL-6), a
cytokine recently shown to stimulate NK cell activity.
Methods. Conditioned medium from human epiphyseal chondrocyte cultures (Ch-CM) was tested for
IL-6 activity using the B9 cell hybridoma assay. Its NK
cell-stimulating capacity in the presence of K562 (myelogenous leukemia) cells or human chondrocytes was
evaluated in a 4-hour 51Cr-release assay. Ch-CMderived ILd/NK cell-augmenting factor activity was
partially purified by high-performance liquid chromatography (HPLC) gel filtration and Western blot.
Results. Ch-CM contained an NK cell-augmenting factor (NKAF) which was blocked by IL-2 or IL-6
antibodies. Ch-CM did not contain detectable IL-2
activity, but it stimulated IL-2 production by human
From the Department of Histology and Embryology. Institute of Biostructure, and the Department of Dermatology, Warsaw
Medical School, Warsaw, Poland; the Department of Dermatology,
University of Miinster, Miinster, FRG; and the LBI-DVS. Laboratory of Cell Biology, University of Vienna, Vienna, Austria.
Supported by the Polish Ministry of Health and Welfare
(grant MZ-VU7.4) and by the Medizinisch-Wissenschaftlicher Fonds
des Biirgermeisters der Bundeshauptstadt Wien (grant 654).
Jacek Malejczyk, PhD: Assistant Professor, Department of
Histology and Embryology, Warsaw Medical School; Magdalena
Malejczyk, MSc: Research Assistant, Department of Dermatology,
Warsaw Medical School; Agatha Urbanski, MSc: Second Department of Dermatology, University of Vienna; Thomas A. Luger,
MD, DSc: Professor and Chairman, Department of Dermatology.
University of Miinster, and Head, Laboratory of Cell Biology,
University of Vienna.
Address reprint requests to Jacek Malejczyk, PhD. Department of Histology and Embryology, Institute of Biostructure, Warsaw Medical School, Chalubinskiego 5 , PL-02004 Warsaw, Poland.
Submitted for publication January 29, 1991; accepted in
revised form January 6, 1992.
Arthritis and Rheumatism, Vol. 35, No. 6 (June 1992)
peripheral blood lymphocytes (PBL). This IL-2inducing capacity was inhibited by IL-6 antibodies,
indicating that chondrocytes release an IL-&like activity. Ch-CM significantly enhanced the proliferation of
IL-Wependent B9 hybridoma cells, and Western blot
analysis of Ch-CM revealed specific bands corresponding to those of highly purified IL-6. Upon HPLC gel
filtration, chondrocyte NKAF copurified with chondrocyte IL-6. Pure IL-6 and chondrocyte IL-6 were tested
for their ability to stimulate the cytotoxic activity of
human PBL against chondrocytes. Both mediators significantly enhanced chondrocyte killing. Lysis of chondrocytes by PBL was mediated by NK cells, since
depletion of CD16+ cells resulted in inhibition of the
Conclusion. Thus, upon stimulation, chondrocytes produce IL-6 which, through IL-2 induction,
augments the activity of NK cells against K562 target
cells as well as against chondrocytes.
The role of chondrocytes in autoimmune destruction of cartilage is still poorly understood. Cartilage metabolism may be influenced by some immunoregulatory mediators such as interleukin-I (IL- 1) and
tumor necrosis factor Q (TNFa), which induce prostaglandin E,, collagenase, and proteoglycanase production by chondrocytes, as well as affect the synthesis of
cartilage proteoglycan and collagen (1-5). Although
the inflamed synovium appears to be a major source of
cytokines that participate in articular cartilage damage
(6), there is evidence that chondrocytes also release
immunoregulatory cytokines. Indeed, they have been
found to produce IL-1, which appears to act in an
autocrine manner in the destruction of cartilage (7).
Chondrocytes have been found to display other
immunologic properties. They constitutively express
class I1 major histocompatibility complex antigens
(8,9), and after allogeneic transplantation, they evoke
a strong immune response that leads to complete
destruction of cartilage (10). Chondrocytes may also
express CD4 antigen (9). Their immunostimulatory
functions have been further confirmed by their ability
to present antigens to T cells (8) and to trigger lymphocyte proliferation in mixed chondrocyte-lymphocyte cultures (8,ll-13). These findings suggest that
under certain pathologic conditions, when chondrocytes are exposed during advanced matrix degradation, they may acquire antigen-presenting cell (AX)like properties, present cartilage-associated autoantigens
to infiltrating lymphocytes, and thereby enhance local
and systemic autoimmune responses.
It has also been demonstrated that chondrocytes serve as targets for natural killer (NK) cells
(14,15), which are considered to be crucial to the
body’s immunosurveillance against neoplasia and infection (16). N K cells were shown to interact with
APC and t o decrease their stimulatory capacity
(17,18), suggesting that natural antichondrocyte cytotoxicity may represent a mechanism which downregulates or prevents potential APC functions of the
chondrocyte. N K cells, via chondrocyte killing, could
also participate in cartilage destruction. The present
study was therefore performed to determine whether
chondrocytes, by themselves, influence N K cell activity. We also investigated whether chondrocytes are
able to release the multifunctional cytokine IL-6,
which has recently been shown t o stimulate the activity of N K cells (19).
Cytokines and anticytokine antibodies. Ultrapure natural human IL-6, normal (preimmune) rabbit IgG, as well as
anti-human IL-2, anti-human TNFa, and anti-human 1L-1
afFmity-purified rabbit polyvalent antibodies (IgG fraction)
were purchased from Endogen (Boston, MA). Natural human IL-2 was obtained from Collaborative Research (Bedford, MA), and monoclonal anti-human interferon-y (IFNy)
and IFNP were obtained from Chemicon (Los Angeles, CA).
Affinity-purified rabbit anti-human IL-6 IgG and highly purified IL-6 derived from fibroblast cell line FS-4 were kindly
provided by Drs. P. B. Sehgal and L. T. May (Rockefeller
University, New York, NY).
Isolation of chondrocytes and generation of chondrocyte-conditioned medium (Ch-CM). Human epiphyseal chondrocytes were isolated enzymatically from the cartilaginous
portions of long bones from 10-12-week-old fetuses obtained
at the time of induced abortions (for nonmedical reasons) in
women attending the Department of Gynecology and Obstetrics, Warsaw Medical School (Warsaw, Poland), as described in detail elsewhere (14,15). Chondrocytes were
seeded on tissue culture flasks (Nunclon; Nunc, Roskilde,
Denmark) at a density of 1.5 x 105/cm2 in RPMI 1640
medium supplemented with 5% fetal bovine serum (Gibco,
Grand Island, NY), 10 mM HEPES and TES (Sigma, St.
Louis, MO), 2 mM glutamine (Sigma), and 1% antibioticantimycotic solution (Gibco) (medium hereafter referred to
as RPMI medium).
After overnight culture at 37°C in a humified atmosphere of 95% air and 5% C02, the medium was replaced
with serum-free medium, either alone or supplemented with
100 &ml of Escherichia coli O55:BS lipopolysaccharide
(LPS; Difco, Detroit, MI). Incubation continued for 3 hours,
and then the LPS-containing medium was removed and
discarded. Chondrocytes were cultured in serum-free RPMI
medium, and after 24 and 48 hours, supernatants were
harvested and stored frozen at -20°C until tested.
High-performance liquid chromatography (HPLC).
Ch-CM were concentrated 20 times by ultrafiltration on
Amicon YM-10 membranes (Amicon, Danvers, MA), and
15O-pl samples were subjected to gel-filtrationHPLC using a
Bio-Sil TSK 125 column (300 X 7.5 mm) and a Bio-Sil TSK
guard column (75 X 7.5 mm; Bio-Rad, Richmond, CA) (20).
Elution was carried out with phosphate buffered saline (pH
7.2) at a flow rate of 1 ml/minute at room temperature.
Columns were calibrated with a gel-filtration standard (BioRad) containing bovine thyroglobulin (620 kd), bovine
gamma globulin (158 kd), chicken ovalbumin (44 kd), horse
myoglobulin (17 kd), and cyanocobalamin (1.3 kd). Column
fractions (0.5 ml) were diluted with RPMI medium, filtersterilized (0.2 p m pore size; Flow, Irvine, Scotland), and
stored frozen until used.
Western blot analysis. Ch-CM were extensively dialyzed against double-distilled H,O for 24 hours, lyophilized,
and subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (12% total acrylamide containing 2.6%
methylene-bis-acrylamide) under reducing conditions, as de-
Table 1. Effect of chondrocyte-conditioned medium on NK cell
activity of human PBL*
culture duration,
LPS stimulation
24 hours
U nstimulated
48 hours
Dilution of chondrocyte-conditioned medium
224. I
* Peripheral blood lymphocytes (PBL) were incubated with different
dilutions of chondrocyte-conditioned medium (see Materials and
Methods) or in RPMI medium (control) at a final density of 5 X lo6
cells/ml for 24 or 48 hours, and then tested for natural killer (NK)
cell activity against K562 cells. Values are lytic units (LU) per lo7
effector cells, from 1 representative experiment. Control cultures
yielded NK cell activity of 34.5 LU/107 cells. LPS = lipopolysaccharide; NT = not tested.
scribed elsewhere (21). Proteins were transferred onto nitrocellulose paper and stained with anti-IL-6 antibody using
biotinylated goat anti-rabbit IgG antiserum and Streptavidin-alkaline phosphatase (BioMakor, Rehovot, Israel).
Rainbow molecular weight markers (Amersham, Buckinghamshire, UK) served as standard.
Isolation and incubation of human peripheral blood
lymphocytes (PBL). Human peripheral blood mononuclear
cells were isolated from buffy-coat using Lymphoprep (Nycomed, Oslo, Norway) density-gradient centrifugation. Cells
were rinsed twice in RPMI medium and enriched for PBL by
removing plastic-adherent cells as described previously (15).
PBL (5 x 10‘ cells/ml) were incubated in the presence of
Ch-CM, Ch-CM HPLC fractions, or cytokines in plastic
tubes (Falcon) at 37°C in a humidified atmosphere of 95% air,
5% COz for 24 hours. In neutralization experiments, test
samples or cytokines were preincubated with a given anti-
Table 2. Effect of chondrocyte-conditioned medium on IL-2 production by human PBL*
culture duration,
LPS stimulation
24 hours
48 hours
U nstimulated
Dilution of chondrocyte-conditioned medium
1 :320
I .6
* PBL were incubated with different dilutions of chondrocyteconditioned medium (see Materials and Methods) or in RPMI
medium (control) at a final density of 5 X lo6 cellslml for 24 or 48
hours. Supernatants were harvested and tested for interleukin-2
(1L-2) activity using CTLL cells. Values are unitslml, from 1
representative experiment. See Table 1 for explanations of other
NK activity increaae (%)
Figure 1. Effects of different cytokine antibodies (Ab) on chondrocyte-natural killer (NK) cell-augmenting factor activity. Chandrocyte-conditioned medium (Ch-CM) was obtained 48 hour5 after
stimulation with lipopolysaccharide and tested on K562 target cells.
Before assay, peripheral blood lymphocytes (PBL) were incubated
for 24 hours with crude Ch-CM (final dilution 1:40), either alone
(bar) or in the presence of different dilutions of antibody to interleukin-2 (IL-2) (*), IL-6 ( x ) . normal rabbit IgG (O),tumor necrosis
factor a (+), interferon-y (IFNy) (0).or IFNp (A). Anti-1L-2 and
anti-lL-6 were prediluted to 150. Values are the mean %increase in
NK activity versus control (PBL in RPMI medium alone, 42. I lytic
unitsll0’ cells; n = 3 experiments): SD values did not exceed 10%.
body for 1-2 hours before addition of PBL. Following
incubation, cells were washed twice and resuspended in
RPMI medium to a concentration needed for the N K cell
activity assay. In some experiments, conditioned media
from PBL (PBL-CM) were stored and tested for cytokine
NK cell assay. NK cell activity of preincubated PBL
was tested against human K562 myelogenous leukemia target cells in a 4-hour “Cr-release assay, as described elsewhere (22). Briefly, PBL and lo4 K562 cells were mixed in
wells of round-bottom multiwell plates at effector-to-target
cell ratios ranging from 80: 1 to 10:1. After a 4-hour incubation, the radioactivity released into the supernatant was
measured using a gamma-counter. In some experiments, NK
cell activity of preincubated PBL was also tested against
human epiphyseal chondrocytes, according to the method
described in detail previously (15). NK cell activity was
expressed in lytic units (LU) per lo7 effector cells, where 1
LU = the number of PBL needed to lyse 30% of target cells,
calculated from the linear portion of the dose-response
Cytokine bioassays. IL-6 activity was measured using
an IL-6-dependent murine hybridoma cell line, B9, generously donated by Dr. L. Aarden (University of Amsterdam,
The Netherlands) (23). IL-2 activity was evaluated using a
murine IL-24ependent T cell line (CTLL16) (24). Cytokine
activity was expressed either as the mean counts per minute
(2SEM) of triplicate cultures or as units per milliliter, where
1 unit = the amount of mediator needed for half-maximal
stimulation of responder cells.
Ch-CM obtained after 24 and 48 hours were
tested for their effect on NK cell activity against KS62
target cells. Compared with cultures in RPMI medium,
Ch-CM from unstimulated chondrocytes contained no
NK cell-enhancing activity. Different concentrations of
Ch-CM from LPS-stimulated chondrocytes, however,
significantly augmented NK cell activity (Table 1).
Moreover, supernatants obtained after 48 hours con-
factor present in chondrocyte supernatants is related
to any other known NK cell-stimulating cytokine,
PBL were preincubated with Ch-CM in the presence
of various cytokine antibodies. A dose-dependent inhibition of NK cell activity was observed only in the
presence of IL-2 or IL-6 antibodies, whereas antibodies directed against IL-1, TNFa, IFNy, and IFNP, as
well as normal rabbit IgG, did not block Ch-CMmediated NK cell activation (Figure 1).
Because of the blocking effects of anti-IL-2 on
NK cell activity, Ch-CM were also tested for IL-2
activity. Neither LPS-stimulated nor unstimulated
Ch-CM contained any detectable IL-2 activity when
tested in the CTLL bioassay. However, significant levels
of IL-2 were found in supernatants from PBL incubated
with different concentrations of LPS-stimulated Ch-CM
(Table 2). The level of IL-2 activity in PBL-CM
IL-2 activity increase (U/rnl)
3i-l-TdR uptake (cpm x
No A b
A b dilution
Figure 2. Effect of 1L-2 (*) and IL-6 (+) antibodies on IL-2 activity
generated in 24-hour cultures of PBL stimulated with Ch-CM (final
dilution 1:40) obtained 48 hours after stimulation with lipopolysaccharide. Bar shows IL-2 activity in PBLconditioned medium from cultures treated with Ch-CM alone. Spontaneous release of IL-2 by
control PBL cultured in RPMI medium alone was 1.8 unitdml. Anti1L-6 did not affect proliferation of CTLL cells responding to exogenous
natlual human IL-2. Values are the mean of 3 experiments; SD values
did not exceed 10%. See Figure 1 for explanations of abbreviations.
tained higher levels of NK cell-augmenting activity
than those obtained after 24 hours.
To investigate whether the NK cell-activating
Table 3. Production of interleukin-6 (IL-6) by human epiphyseal
chondroc ytes*
Chondrocyte culture
duration, LPS stimulation
24 hours
48 hours
IL-6 activity
* Chondrocyte-conditioned medium was generated as described in
Materials and Methods and tested for IL-6 activity, using the B9
bioassay. Values are the mean of 3 independent experiments; SD
values did not exceed 10%. LPS = lipopolysaccharide.
No Ab
Ab dilution
Figure 3. Effect of anti-IL-6 antibodies (0)and normal rabbit LgG
(+) on the proliferation response of B9 cells to Ch-CM (final dilution
1:80) obtained 48 hours after stimulation with lipopolysaccharide.
Bar shows proliferative response of B9 cells treated with Ch-CM
alone. Proliferation of B9 cells was measured by 3H-thymidine
(3H-TdR) incorporation (see Materials and Methods). Background
incorporation was 563 2 120 (mean 2 SD). Values are the mean of
3 experiments; SD values did not exceed 10%. See Figure 1 for
explanations of other abbreviations.
correlated with the concentration of Ch-CM used to
stimulate PBL, and was increased when PBL were
incubated with 48-hour-stimulated Ch-CM. Addition
of anti-IL-2 inhibited IL-2 activity in PBL-CM in a
dose-dependent manner (Figure 2). Furthermore, ChCM-mediated induction of IL-2 production by PBL
was blocked by antibodies directed against IL-6.
Ch-CM were therefore also tested for IL-6
activity using the B9 cell proliferation assay. We found
that unstimulated Ch-CM contained only minimal
amounts of IL-6, whereas LPS-stimulated chondrocytes produced significant levels of IL-6 activity (Table 3). The specificity of the IL-&like activity in
Ch-CM was tested in neutralization experiments using
an anti-IL-6 antibody. Incubation of Ch-CM with
anti-IL-6 antibody resulted in a specific, dosedependent inhibition of B9 cell proliferation (Figure 3).
The presence of IL-6 in Ch-CM was further
confirmed by Western blot analysis. Specific bands in
the molecular weight range of 2&30 kd, similar to the
profile of highly purified fibroblast IL-6, were seen
(Figure 4). Upon gel filtration of Ch-CM, the NK
cell-stimulatory activity eluted at a molecular weight
range of 20-30 kd and copurified with IL-6 activity
m.w. (kD)
- 160 0
100 -
2 3 4
g 10
11 12 13 14 15 10
Fraction number
Figure 5. NK cell-augmenting activity and IL-6 activity in fractions
from high-performance liquid chromatography (HPLC) gel filtration
of Ch-CM obtained 48 hours after stimulation with lipopolysaccharide. Ch-CM were concentrated 20 times, subjected to HPLC gel
filtration, and each fraction was tested for stimulation of NK cell
activity and for IL-6 activity. NK cell activity is the % increase
versus control (PBL in RPMI medium alone, 38 lytic units/lO' cells).
m.w. = molecular weight; see Figure I for explanations of other
(Figure 5). NK cell stimulation by fractions enriched
for NK-augmenting factor/IL-6 (fractions 7-9) could
also be inhibited by anti-IL-2 as well as anti-IL-6
antibodies (data not shown).
To investigate whether IL-6 would also specifically stimulate NK cell activity against chondrocytes,
a "Cr-release assay was performed using chondrocytes as target cells. Compared with untreated cells,
PBL stimulated with Ch-CM as well as with pure IL-6
significantly enhanced NK cell activity against chondrocytes (Figure 6).
1 2 3 4 5
Figure 4. Western blot of IL-6 present in Ch-CM. Ch-CM from
unstimulated or lipopolysaccharide (LPS)-stimulated cultures were
harvested after 24 and 48 hours, subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose paper, and immunostained for IL-6 (see Materials and
Methods). Lanes I and 3, 24- and 48-hour Ch-CM, respectively,
from unstimulated cultures; lanes 2 and 4, 24- and 48-hour Ch-CM.
respectively, from LPS-stimulated cultures; lane 5 , highly purified
FS-4 fibroblast-derived 1L-6. Molecular weight markers are shown
at the right. See Figure 1 for explanations of other abbreviations.
The results of the present study show that
human epiphyseal chondrocytes, upon stimulation
with LPS, produce a cytokine which significantly
enhances NK cell activity of human PBL. NK cell
activity is known to be regulated by various factors,
and the most remarkable stimulatory effect is exerted
by interferons and IL-2 (25-28). In some systems, NK
cells may also be influenced by TNFa (29) and IL-1
(30). Chondrocyte-derived NK cell-augmenting factor
(Ch-NKAF), however, appears not to be related to
these cytokines, since addition of antibodies specific
for IFNP, IFNy, TNFa, or IL-1 did not block Ch-
NK cell activity ( ~ ~ 1 1 0 7
tinct cytokines, the possibility that other mediators
present in Ch-CM may also cause B9 cell proliferation
must be considered. This, however, seems unlikely,
since B9 cell stimulation by Ch-CM was specifically
inhibited by anti-IL-6 antibodies, and upon Western
blot analysis and HPLC gel-filtration, chondrocytederived IL-6 exhibited molecular weight heterogeneity
in the 2G30-kd range typical of macrophage or fibroblast IL-6 (38). Chondrocyte-natural killer cellaugmenting factor copurified with chondrocytic IL-6
activity on HPLC gel filtration, and both activities
were inhibited by anti-lL-6 antibodies. This suggests
that Ch-NKAF and IL-6 are the same cytokine. This is
also confirmed by previous findings that both natural
and recombinant IL-6 increase NK cell activity (19).
IL-6, which is also known as IFNP, hybridomd
plasmacytoma growth factor, hepatocyte-stimulating
factor, or B cell stimulatory factor-2 according to its
multiple biological activities, has been shown to be
produced by a variety of cells, including monocyte/
macrophages, fibroblasts, keratinocytes, and endothelial cells, as well as some neoplastic cell lines (38-41).
Most recently, production of IL-6 by human chondrocytes from osteoarthritic joints and from normal joints
has also been reported (42). In general, 1L-6 is not
produced constitutively and is released only upon
stimulation. Similarly, chondrocytes released IL-6 on
stimulation with LPS, which is known to be a potent
inducer of this cytokine (43).
Aside from its stirnulatory effect on NK cell
activity against K562 target cells, chondrocyte-derived
IL-6 was also able to stimulate natural killing of
isolated chondrocytes. This feature suggests that IL-6
production by activated chondrocytes may possibly
prevent their stimulatory antigen-presenting cell functions via enhanced chondrocyte killing. On the other
hand, 1L-6 has been shown to stimulate chondrocyte
proliferation (42), and thus act as an autocrine regulator of cartilage growth.
There is additional evidence of a possible role
of IL-6 in inflammatory joint diseases. It has been
shown to be released by rheumatoid synovium, and
high levels of IL-6 have been found in synovial fluid
derived from patients with rheumatoid arthritis (6).
Thus, chondrocyte-derived and synovium-derived
IL-6 may be responsible for local as well as systemic
activation of autoimmune reactivity, and especially,
for T cell and B cell stimulation.
Thus the findings of our study provide more
evidence of immunostimulatory/immunoregulatory
properties of chondrocytes and support a view that
Figure 6. Effect of Ch-CM and 1L-6 on N K cell activity against
normal human epiphyseal chondrocytes. Effector PBL were preincubated for 24 hours in the presence of Ch-CM (final dilution 1:40),
highly purified natural human IL-6 ( I unithl), and RPMl medium
alone (control [C]), and then tested for antichondrocyte cytotoxicity
in an 18-hour "Cr-release assay. Values are lytic units (LU)per lo7
cells (n = 3 experiments); SD values did not exceed 10%. See Figure
1 for explanations of other abbreviations.
NKAF activity. In contrast, a strong dose-dependent
inhibition of Ch-NKAF activity was observed in the
presence of anti-IL-2 or anti-IL-6 antibodies, indicating that the final stimulatory effect may depend on 1L-2
and IL-6. Ch-CM did not contain any IL-2 activity;
however, Ch-CM stimulated 1L-2 production by PBL.
Since the release of IL-2 by PBL was blocked by
anti-IL-6 antibodies and this effect correlated with
decreased stimulation of NK cells, the increase in NK
cell activity appears to be due to the IL-2-inducing
capacity of chondrocyte-derived 1L-&like activity.
The finding that IL-6 is a potent inducer of IL-2 is
consistent with results of other studies using murine
thymocytes or lymphocytes (31,32), which suggests a
possible role of this cytokine in T cell activation and
cytotoxic cell maturation (33-37).
Since chondrocytes could produce several dis-
71 1
chondrocytes could be actively involved in cartilage
immunopathology .
We thank Dr. L. T. May for his generous gift of the
purified IL-6 preparation and Dr. L. A. Aarden for providing
us with B9 cells. We are also grateful to Marie Luke
Hiilsmann for her excellent preparation of the manuscript.
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production, factors, natural, killer, activity, interleukin, augmenting, human, epiphyseal, chondrocyte, cells
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