T-cell contact-dependent regulation of CC and CXC chemokine production in monocytes through differential involvement of NFkappaB: implications for rheumatoid arthritis.

Abstract

We and others have reported that rheumatoid arthritis (RA) synovial T cells can activate human monocytes/macrophages in a contact-dependent manner to induce the expression of inflammatory cytokines, including tumour necrosis factor alpha (TNFalpha). In the present study we demonstrate that RA synovial T cells without further activation can also induce monocyte CC and CXC chemokine production in a contact-dependent manner. The transcription factor NFkappaB is differentially involved in this process as CXC chemokines but not CC chemokines are inhibited after overexpression of IkappaBalpha, the natural inhibitor of NFkappaB. This effector function of RA synovial T cells is also shared by T cells activated with a cytokine cocktail containing IL-2, IL-6 and TNFalpha, but not T cells activated by anti-CD3 cross-linking that mimics TCR engagement. This study demonstrates for the first time that RA synovial T cells as well as cytokine-activated T cells are able to induce monocyte chemokine production in a contact-dependent manner and through NFkappaB-dependent and NFkappaB-independent mechanisms, in a process influenced by the phosphatidyl-inositol-3-kinase pathway. Moreover, this study provides further evidence that cytokine-activated T cells share aspects of their effector function with RA synovial T cells and that their targeting in the clinic has therapeutic potential.

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Vol 8 No 6Research article
T-cell contact-dependent regulation of CC and CXC chemokine
production in monocytes through differential involvement of
NFκB: implications for rheumatoid arthritis
Jonathan T Beech1*, Evangelos Andreakos2*, Cathleen J Ciesielski1, Patricia Green1,
Brian MJ Foxwell1 and Fionula M Brennan1
1Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, 1 Aspenlea Road, Hammersmith, London W6 8LH, UK
2Foundation for Biomedical Research of the Academy of Athens, Center for Immunology and Transplantations, 4 Soranou tou Ephessiou, 11527
Athens, Greece
* Contributed equally
Corresponding author: Jonathan T Beech, j.beech@ic.ac.uk
Received: 7 Jun 2006 Revisions requested: 28 Jun 2006 Revisions received: 28 Sep 2006 Accepted: 13 Nov 2006 Published: 13 Nov 2006
Arthritis Research & Therapy 2006, 8:R168 (doi:10.1186/ar2077)
This article is online at: http://arthritis-research.com/content/8/6/R168
© 2006 Beech et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
We and others have reported that rheumatoid arthritis (RA)
synovial T cells can activate human monocytes/macrophages in
a contact-dependent manner to induce the expression of
inflammatory cytokines, including tumour necrosis factor alpha
(TNFα). In the present study we demonstrate that RA synovial T
cells without further activation can also induce monocyte CC
and CXC chemokine production in a contact-dependent
manner. The transcription factor NFκB is differentially involved in
this process as CXC chemokines but not CC chemokines are
inhibited after overexpression of IκBα, the natural inhibitor of
NFκB. This effector function of RA synovial T cells is also shared
by T cells activated with a cytokine cocktail containing IL-2, IL-6
and TNFα, but not T cells activated by anti-CD3 cross-linking
that mimics TCR engagement. This study demonstrates for the
first time that RA synovial T cells as well as cytokine-activated T
cells are able to induce monocyte chemokine production in a
contact-dependent manner and through NFκB-dependent and
NFκB-independent mechanisms, in a process influenced by the
phosphatidyl-inositol-3-kinase pathway. Moreover, this study
provides further evidence that cytokine-activated T cells share
aspects of their effector function with RA synovial T cells and
that their targeting in the clinic has therapeutic potential.
Introduction
A large and diverse range of proinflammatory cytokines and
chemokines have been detected in the synovium of patients
with rheumatoid arthritis (RA) (reviewed in [1,2]). This diversity
is not surprising, considering the heterogeneous mixture of
activated cells found at the sites of inflammation of RA syn-
ovium, which include macrophages, T cells, endothelial cells,
fibroblasts and plasma cells.
Of particular interest are chemokines, which selectively recruit
haemopoietic cells from the blood into the inflamed synovium.
Several chemokines have been detected in RA synovium and
include IL-8 (CXCL8) [3], monocyte chemoattractant protein
1 (MCP-1; CCL2) [4], epithelial neutrophil activating peptide
78 [5], macrophage inflammatory protein 1 alpha (MIP-1α;
CCL3) [6], macrophage inflammatory protein 1 beta (MIP-1β;
CCL4) [7], RANTES (CCL5) [7] and growth-related gene
product alpha (GROα; CXCL1) [8] (reviewed in [9]). What
regulates chemokine gene expression in the RA synovium,
however, remains to be determined.
ELISA = enzyme-linked immunosorbent assay; FCS = foetal calf serum; GROα = growth-related gene product alpha; IL = interleukin; IP-10 = inter-
feron-gamma-inducible protein 10; LPS = lipopolysaccharide; mAb = monoclonal antibody; MCP-1 = monocyte chemoattractant protein 1; M-CSF
= macrophage-colony stimulating factor; MIP-1α = macrophage inflammatory protein 1 alpha; MIP-1β = macrophage inflammatory protein 1 beta;
MOI = multiplicity of infection; NFκB = nuclear factor kappa B; PBS = phosphate-buffered saline; PI3K = phosphatidyl-inositol-3-kinase; RA = rheu-
matoid arthritis; Tck cells = cytokine-activated T cells; TCR = T-cell receptor; Ttcr cells = anti-CD3-activated T cells; TNFα = tumour necrosis factor
alpha.

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T cells were recently shown to be essential for the production
of proinflammatory cytokines from macrophages in RA syno-
vial tissue [23]. Although synovial CD4+ T cells proliferate
poorly and produce low levels of IL-2 and interferon gamma
[10-12], they express cytokines and activation markers [13] –
and when put in contact with synovial fibroblasts or mono-
cytes/macrophages, synovial CD4+ T cells induce high levels
of inflammatory cytokines [14-16].
In vitro, we have shown that T cells activated by an anti-CD3
cross-linking antibody (that mimics TCR engagement (Ttcr)) or
stimulated with a 'cocktail' of cytokines (designated cytokine-
activated T cells (Tck)) also stimulate monocytes in a contact-
dependent manner to produce cytokines that include IL-1β,
TNFα, IL-12, IL-6 and IL-10 [15,17-21]. While the molecules
involved in this process have not been fully defined, a number
of T-cell-associated cell surface receptors/ligands, including
CD69 [17], CD40L [18], CD11b and CD2, have been sug-
gested of importance.
Histologically, T cells are often found in close contact with
macrophages in the interstitium of RA synovial tissue [22] and
T-cell depletion rapidly diminishes macrophage TNFα synthe-
sis in RA synovial cultures [23].
We previously reported that the contact-dependent effector
function of RA T cells in the joint is identical to that displayed
by bystander-activated T cells (Tck), which can be expanded
from normal blood with a cytokine cocktail containing TNFα,
IL-6 and IL-2 over an 8-day period [21,23]. RA synovial T cells
and Tck cells both induce TNFα production in resting mono-
cytes in a cell-contact dependent manner, which is abrogated
by blockade of the transcription factor NFκB but is augmented
if phosphatidyl-inositol-3-kinase (PI3K) is inhibited. Normal
blood T cells activated 'conventionally' via the TCR with cross-
linked anti-CD3 antibody result in TNFα production from
monocytes that is unaffected by NFκB blockade, but is inhib-
ited in the presence of PI3K blocking drugs [23].
In the present report we investigated whether chemokine pro-
duction from macrophages can also be induced in a contact-
dependent manner by activated blood T cells, or indeed by T
cells freshly isolated from rheumatoid tissue. We also exam-
ined which signalling pathways in macrophages are rate-limit-
ing for the expression of chemokines after T-cell contact, with
particular reference to the transcription factor NFκB, in order
to gain insight into the regulation of chemokines at sites of
inflammation.
Materials and methods
Isolation of peripheral blood monocytes and
lymphocytes
Human monocytes were isolated from single-donor platelet
pheresis residues purchased from the North London Blood
Transfusion Service (Colindale, UK). Mononuclear cells were
isolated by Ficoll/Hypaque centrifugation (specific density
1.077 g/ml; Nycomed Pharma A.S., Oslo, Norway), prior to
cell separation in a Beckman JE6 elutriator (Torrence, CA,
USA). Elutriation was performed in culture medium containing
1% heat-inactivated FCS. The monocyte purity and lym-
phocyte purity were assessed by flow cytometry, and fractions
were typically >80% and 90% pure, respectively.
T-cell stimulation and fixation
Elutriation-enriched lymphocytes were resuspended in RPMI
1640 (containing 10% heat-inactivated AB+ human serum;
(Biowittaker, Wokingham, UK) at 1 × 106 cells/ml. The resus-
pended lymphocytes were then cultured in six-well cluster cul-
ture plates (Falcon, Bedford, MA, USA) at 37°C in a 5% CO2/
95% air-humidified incubator for 24 hours following stimula-
tion with immobilized anti-CD3 mAb (OKT3; ATCC, Rockville,
MD, USA), which had previously been coated onto the six-well
culture plates at 10 μg/ml overnight at 4°C.
Alternatively, T cells were presented with different saturating
concentrations of the following: 25 ng/ml TNFα (gift from Dr
W. Stec, Centre of Macromolecular Studies, Lodz, Poland),
100 ng/ml IL-6 (gift from Dr P. Ramage, Sandoz, Pharma Ltd.,
Basel, Switzerland) and 25 ng/ml IL-2 (gift from Dr U Gubler,
Hoffmann-LaRoche, Nutley, NJ) for 8 days in culture, prior to
fixation.
In all instances, control T cells were cultured in the absence of
any stimulus. Following stimulation, T cells were harvested and
washed three times in RPMI 1640 prior to fixation for 1 minute
in PBS containing 0.05% glutaraldehyde, and were than neu-
tralized with an equivalent volume of T-cell neutralizing buffer
containing 0.2 M glycine. Following a further three washes the
fixed T cells were resuspended in complete medium (RPMI
1640 containing 5% heat-inactivated FCS) at 2 × 106 cell/ml
and stored for up to 7 days at 4°C until use. The T cells were
washed twice in complete medium prior to use.
Isolation of CD3+ cells from synovial membrane tissue
Mononuclear cells were obtained from synovial tissue speci-
mens taken during joint replacement surgery, provided by the
Orthopedic/Plastic Surgery Department of Charing Cross
Hospital, London, UK. Tissue was teased into small pieces
and digested in medium containing 0.15 mg/ml DNAase type
I (Sigma, Gillingham, Dorset, UK) and 5 mg/ml collagenase
(Roche, Welwyn Garden City, Hertfordshire, UK) for 1–2
hours at 37°C. Cells are passed through a nylon mesh to
exclude cell debris, washed and resuspended in RPMI (sup-
plemented with 10% heat-inactivated FCS) at a density of 1 ×
106 cells/ml. Mononuclear cells were incubated with anti-CD3
monoclonal antibody-coated Dynabeads for 20 minutes at
4°C under constant rotation. Cells attached to beads were iso-
lated using a magnetic particle concentrator (Dynal, Mersey-
side, UK) and cultured for 6 hours at 37°C. Detached cells
were then removed from the magnetic beads and washed

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using the magnetic particle concentrator, which allows for iso-
lation of CD3+ cells yielding high purity (>99%) and high via-
bility (>95%). Cells were then fixed using the same protocol
described above.
Adenoviral vectors and their propagation
Adenoviral gene transfer is a technique used for efficient gene
transfer into dividing and nondividing cells, such as fibroblasts
and monocytes [24,25]. Recombinant replication-deficient
adenoviral vector containing no insert (Adv0) was provided by
M. Wood (University of Oxford, UK), and the adenovirus
encoding porcine IκBα with a cytomegalovirus promoter and
nuclear localization sequence (AdvIκBα) [26] was provided by
Dr R. deMartin (Vienna, Austria). Briefly, viruses were propa-
gated in the 293 human embryonic kidney cell line and purified
by ultracentrifugation through two caesium chloride gradients.
Titres of viral stocks were determined by plaque assay in 293
cells after exposure to virus for 2 hours in serum-free RPMI
1640, followed by washing and re-culturing the cells in com-
plete medium for 48–72 hours [27].
Gene transfer into macrophage-colony stimulating
factor-treated monocytes with adenovirus
Prior to adenoviral infection, freshly elutriated monocytes were
cultured in a 175 cm3 culture flask (Falcon) for 2 days in RPMI
1640 supplemented with 5% heat-inactivated FCS (complete
medium) with 50 ng/ml macrophage-colony stimulating factor
(M-CSF). This process upregulates the αvβ5 integrin, which
acts as a cofactor for adenovirus infection [28,29]. Following
culture, M-CSF-differentiated monocytes were washed once
with PBS to remove nonadherent cells and the remaining
adherent monocytes were incubated with 10 ml cell dissocia-
tion solution (Sigma) for 30–45 minutes until removed from
the plastic. The cell suspension was washed three times in
complete medium and the cell viability was assessed by trypan
blue exclusion (>90%). Cells were plated at 2 × 105/ml in 96-
well flat-bottomed culture plates (Falcon) and were allowed to
adhere for 1 hour prior to infection with adenovirus. The media
and nonadherent cells were removed from each well and
replaced with serum-free RPMI 1640 and adenovirus at the
required multiplicity of infection (MOI) for 2 hours. Following
incubation, the medium was removed and replaced with com-
plete medium. Monocytes were cultured for a further 2 days
before stimulation to enable adenoviral production of IκBα to
reach optimal levels.
Coculture of M-CSF-differentiated macrophages and
lymphocytes
In the assays for contact-dependent chemokine production,
M-CSF-differentiated monocytes (with or without IκBα trans-
duction) were replated at 1 × 105 cells per well on a flat-bot-
tom 96-well plate. Fixed lymphocytes were then added to the
wells to give a final T cell:monocyte ratio of 7:1 and a final
assay volume of 200 μl. Cultures containing monocytes alone
and cultures containing lymphocytes alone were also included
as experimental controls. Further controls included cocultures
containing a porous membrane insert to physically separate
the two populations, while allowing the transition of soluble
mediators (0.2 μm Anopore® Membrane Nunc Tissue Culture
Inserts; Nunc, Roskilde, Denmark). After 18 hours of culture at
37°C (5% CO2, humidified atmosphere), the supernatants
were harvested and stored at -70°C for subsequent chemok-
ine assay.
Measurement of chemokines by sandwich ELISA
Concentrations of IL-8 (CXCL8) (PharMingen, San Diego, CA,
USA), GROα (CXCL1), interferon-gamma-inducible protein
10 (IP-10) (CXCL10), MCP-1 (CCL2), MIP-1α (CCL3), MIP-
1β (CCL4) and RANTES (CCL5) were determined by ELISA
(R&D Systems, Oxford, UK), following the manufacturer's
instructions. The absorbance was read and analysed at 450
nm on a spectrophotometric ELISA plate reader (Labsystems
Multiskan Biochromic, Labsystems, Uxbridge, UK) using the
Delta soft II.4 software programme (DeltaSoft Inc, Hillsbor-
ough, NJ, USA). Results are expressed as the mean concen-
tration of triplicate cultures ± standard deviation.
Statistical analysis
Results were examined for statistical differences using Stu-
dent's t test (two-tailed). P < 0.05 was considered significant,
and such values are illustrated on the figures as appropriate.
Results
Both Tck cells and Ttcr cells induce contact-dependent
chemokine production by M-CSF-differentiated human
monocytes
We have previously reported that the production of proinflam-
matory cytokines by macrophages can be induced by cognate
interaction with Ttcr cells or Tck cells [19,21]. In the present
paper we investigated whether activated T cells can also
induce macrophage CC or CXC chemokine secretion in a
contact-dependent manner. We found that, upon coculture, T
cells activated with anti-CD3 antibody are able to induce pro-
duction of high levels of chemokines in M-CSF-differentiated
human monocytes (macrophages). Levels of both CC chem-
okines (MCP-1, MIP-1α, MIP-1β and RANTES) and CXC
chemokines (IL-8, GROα and IP-10) are all elevated in com-
parison with those found in cultures of M-CSF-differentiated
monocytes alone (Figure 1a). This induction of chemokine pro-
duction in monocytes can be significantly reduced if the mono-
cytes and T cells are physically separated using a porous
membrane insert, demonstrating the importance of cell-cell
contact in the induction process. In contrast, chemokine pro-
duction by M-CSF-differentiated monocytes alone remains
unchanged following coculture with unstimulated T cells (cul-
tured for 24 hours prior to fixation).
Tck cells were also cultured with M-CSF-differentiated mono-
cytes (Figure 1b). Tck cells, as seen with Ttcr cells, were able
to induce significant production of all CC chemokines (MCP-

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1, MIP-1α, MIP 1β and RANTES) and CXC chemokines (IL-8,
GROα and IP-10) assayed to similar levels, again in a contact-
dependent manner. As expected, fixed Ttcr and Tck cells cul-
tured alone did not secrete any detectable levels of chemok-
ines (data not shown). Moreover, macrophages cultured in the
presence of the insert and stimulated with lipopolysaccharide
(LPS) secreted high levels of chemokines (data not shown) as
previously described [30], indicating that the presence of the
membrane insert does not influence macrophage function.
Differential utilization of NFκB in the Ttcr-cell and Tck-
cell contact-dependent induction of CC and CXC
chemokines in M-CSF-differentiated monocytes
We have previously shown that the contact-dependent induc-
tion of TNFα production in resting monocytes by Tck cells or
Figure 1
Activated T cells induce contact-dependent chemokine production by human macrophages Activated T cells induce contact-dependent chemokine production by human macrophages. Lymphocytes were left unstimulated or were stimulated
with either anti-CD3 for 48 hours (Ttcr cells) or a 'cocktail' of inflammatory cytokines (tumour necrosis factor alpha (TNFα), IL-2, IL-6) (Tck cells) for
8 days, before fixation. The unstimulated, Ttcr and Tck populations were then cultured with macrophage-colony stimulating factor-differentiated
monocytes (ratio 7:1) for 18 hours. Culture supernatants were then isolated and levels of CC chemokines (monocyte chemoattractant protein 1
(MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), RANTES) and CXC chemokines
(IL-8, growth-related gene product alpha (GROα) and interferon-gamma-inducible protein (IP-10)) measured by ELISA. In some cases, a porous
membrane insert was used to physically separate the two populations, while allowing the transition of soluble mediators. Results are shown from (a)
Ttcr-cell lymphocyte cultures and (b) Tck-cell lymphocyte cultures. Data represent a mean of triplicate cultures ± standard deviation and are repre-
sentative of at least three experiments. Statistically significant differences in chemokine detection are indicated.

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RA synovial T cells is abrogated by blockade of the transcrip-
tion factor NFκB [23]. As NFκB is a major transcription factor
regulating the expression of numerous genes involved in
immune and inflammatory responses [28,31], we determined
whether T-cell contact-dependent production of chemokines
is also regulated by NFκB.
To inhibit NFκB with specificity we employed an efficient ade-
noviral gene transfer method to overexpress IκBα in human
macrophages. We have previously shown that high levels of
IκBα are achieved by AdvIκBα transduction that remain ele-
vated even after LPS stimulation [30]. As IκBα is a major inhib-
itory component of the NFκB pathway, increased expression
of IκBα blocks NFκB nuclear translocation and DNA binding
induced by LPS.
We then examined whether IκBα overexpression inhibits
monocyte chemokine production induced by Ttcr cells. We
found that AdIκBα inhibits the production of CC chemokines
induced by contact with Ttcr cells but has no effect on CXC
chemokine induction. MIP-1α production induced by Ttcr cells
was therefore profoundly reduced, in a dose-dependent man-
ner, in M-CSF-differentiated monocytes infected with AdIκBα
but not with Ad0, a control virus without insert. At MOI of 40:1
and 80:1, the inhibition of MIP-1α expression was 54% (P ≤
0.005) and 78% (P ≤ 0.005), respectively – which was not fur-
ther increased at higher MOI (Figure 2a).
Similar significant inhibition of the production of the other CC
chemokines MIP1-β (73.9%, P ≤ 0.005), RANTES (70.2%, P
≤ 0.005) and MCP-1 (67%, P ≤ 0.005) was also observed in
AdIκBα-infected monocytes (Figure 2b). In contrast, there
was no effect of IκBα overexpression on CXC chemokine pro-
duction. We found that there was no significant inhibition of
the chemokines GROα, I,L-8 or IP-10 in AdIκBα-infected
monocytes activated by Ttcr cells, suggesting that there is dif-
ferential utilization of NFκB for the expression of CC and CXC
chemokines in this system.
We also examined the role of NFκB in the Tck-cell contact-
dependent production of chemokines in monocytes. Unex-
pectedly, we found that IκBα overexpression inhibited Tck-
cell-dependent CXC chemokine production in M-CSF-differ-
entiated monocytes, but had no effect in CC chemokine pro-
duction. Thus, although contact-dependent induction of
GROα, IL-8 and IP-10 was significantly inhibited in AdIκBα-
infected macrophages by 78.7% (P ≤ 0.01), 63.2% (P ≤ 0.01)
and 52.1% (P ≤ 0.05), respectively, the induction of MIP-1α,
MIP-1β, RANTES and MCP-1 was unaffected (Figure 2c).
This inverted pattern of utilization of NFκB for the T-cell con-
tact-dependent induction of CC and CXC chemokines in
monocytes is surprising and indicates that chemokine gene
expression may be more complex than previously thought.
Figure 2
Differential utilization of NFκB in activated-T-cell contact-dependent chemokine production by human macrophagesDifferential utilization of NFκB in activated-T-cell contact-dependent
chemokine production by human macrophages. Macrophage-colony
stimulating factor-differentiated monocytes were infected with AdIκBα
or Ad0, an empty control virus. After a further 2 days of culture and
replating, anti-CD3-activated T cells (Ttcr cells) and cytokine-activated
T cells (Tck cells) were added at a lymphocyte:monocyte ratio of 7:1.
After 18 hours, culture supernatants were isolated and levels of CC
chemokines (monocyte chemoattractant protein 1 (MCP-1), macro-
phage inflammatory protein 1 alpha (MIP-1α), macrophage inflamma-
tory protein 1 beta (MIP-1β), RANTES) and CXC chemokines (IL-8,
growth-related gene product alpha (GROα) and interferon-gamma-
inducible protein (IP-10)) were measured simultaneously by ELISA. (a)
MIP-1α levels in uninfected, Ad0-infected (multiplicity of infection (MOI)
200:1) and AdIκBα-infected (MOI 40:1, 80:1 and 200:1) monocyte
cultures when stimulated with Ttcr-cells or Tck-cells. (b) and (c) Levels
of CC and CXC chemokines in Ad0-infected and AdIκBα-infected
monocytes (MOI 80:1) following stimulation with (b) Ttcr cells and (c)
Tck cells. Data represent the mean of triplicate cultures ± standard
deviation and are representative of at least three experiments. Statisti-
cally significant reduction in chemokine levels in AdvIκBα-infected (as
compared with Ad0-infected) cultures is indicated.