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 lymphocyte purity were assessed by flow cytometry, and fractions were typically >80% and 90% pure, respectively.

Elutriation-enriched lymphocytes were resuspended in RPMI 1640 (containing 10% heat-inactivated AB⁺ human serum; (Biowittaker, Wokingham, UK) at 1 × 10⁶ cells/ml. The resuspended lymphocytes were then cultured in six-well cluster culture plates (Falcon, Bedford, MA, USA) at 37°C in a 5% CO₂/95% air-humidified incubator for 24 hours following stimulation 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 neutralized 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.

Mononuclear cells were obtained from synovial tissue specimens 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 (supplemented 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 isolated using a magnetic particle concentrator (Dynal, Merseyside, UK) and cultured for 6 hours at 37°C. Detached cells were then removed from the magnetic beads and washed using the magnetic particle concentrator, which allows for isolation of CD3⁺ cells yielding high purity (>99%) and high viability (>95%). Cells were then fixed using the same protocol described above.

Adenoviral gene transfer is a technique used for efficient gene transfer into dividing and nondividing cells, such as fibroblasts and monocytes 2425. 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 propagated 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 complete medium for 48–72 hours 27.

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 α_vβ₅ integrin, which acts as a cofactor for adenovirus infection 2829. 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 dissociation 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 complete medium. Monocytes were cultured for a further 2 days before stimulation to enable adenoviral production of IκBα to reach optimal levels.

In the assays for contact-dependent chemokine production, M-CSF-differentiated monocytes (with or without IκBα transduction) were replated at 1 × 10⁵ cells per well on a flat-bottom 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 chemokine assay.

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, Hillsborough, NJ, USA). Results are expressed as the mean concentration of triplicate cultures ± standard deviation.

Results were examined for statistical differences using Student's t test (two-tailed). P < 0.05 was considered significant, and such values are illustrated on the figures as appropriate.

We have previously reported that the production of proinflammatory cytokines by macrophages can be induced by cognate interaction with Ttcr cells or Tck cells 1921. 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 production of high levels of chemokines in M-CSF-differentiated human monocytes (macrophages). Levels of both CC chemokines (MCP-1, MIP-1α, MIP-1β and RANTES) and CXC chemokines (IL-8, GROα and IP-10) are all elevated in comparison with those found in cultures of M-CSF-differentiated monocytes alone (Figure 1a). This induction of chemokine production in monocytes can be significantly reduced if the monocytes 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 production by M-CSF-differentiated monocytes alone remains unchanged following coculture with unstimulated T cells (cultured for 24 hours prior to fixation).

Tck cells were also cultured with M-CSF-differentiated monocytes (Figure 1b). Tck cells, as seen with Ttcr cells, were able to induce significant production of all CC chemokines (MCP-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 cultured alone did not secrete any detectable levels of chemokines (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.

We have previously shown that the contact-dependent induction of TNFα production in resting monocytes by Tck cells or RA synovial T cells is abrogated by blockade of the transcription factor NFκB 23. As NFκB is a major transcription factor regulating the expression of numerous genes involved in immune and inflammatory responses 2831, we determined whether T-cell contact-dependent production of chemokines is also regulated by NFκB.

To inhibit NFκB with specificity we employed an efficient adenoviral 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 elevated even after LPS stimulation 30. As IκBα is a major inhibitory 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 manner, 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 further 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 production. 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 differential 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. Unexpectedly, we found that IκBα overexpression inhibited Tck-cell-dependent CXC chemokine production in M-CSF-differentiated monocytes, but had no effect in CC chemokine production. 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 contact-dependent induction of CC and CXC chemokines in monocytes is surprising and indicates that chemokine gene expression may be more complex than previously thought.

We next investigated whether RA synovial T cells enriched from dissociated RA synovial tissue could also induce monocyte chemokine secretion in a contact-dependent manner and whether this requires NFκB. RA synovial T cells were isolated from dissociated synovial membranes using anti-CD3 DynaBeads, as described in Materials and methods. We found that, like Ttcr and Tck cells, fixed RA synovial T cells were able to induce both CC and CXC chemokine production from M-CSF-differentiated human monocytes (Figure 3). Furthermore, overexpression of IκBα in these monocytes resulted in impaired RA synovial T cell-dependent CXC chemokine release, when compared with Ad0-infected monocytes. A significant reduction in IL-8 (54.1%, P ≤ 0.01), IP-10 (39.6%, P ≤ 0.05) and GROα (74.2%, P ≤ 0.01) production was therefore observed (Figure 3a). This effect was dose dependent, with increasing MOI of 40:1 and 80:1 inducing a reduction in GROα levels of 55.1% (P ≤ 0.01) and an optimal 74.2% (P ≤ 0.001), respectively (Figure 3b).

Similar dose-dependent profiles were observed for the other chemokines tested (data not shown). Interestingly, however, overexpression of IκBα had no significant effect on the expression of CC chemokines by M-CSF-differentiated monocytes, suggesting that RA synovial T cells possess similarities in their effector function to Tck cells, rather than Ttcr cells. It is noteworthy that RA T cells isolated based on CD2 expression have previously demonstrated an identical effector function to those isolated using anti-CD3 (data not shown), thus discounting the idea that CD3-based methods may influence the behaviour of RA T cells (through the potential for crosslinking) in this system.

Finally, we investigated what further cell signalling pathways (in addition to NFκB) could play a potential role in contact-dependent chemokine production. Ttcr cells and Tck cells were used to stimulate monocytes that had been pretreated with a chemical inhibitor of the PI3K pathway (LY294002), and the resulting effects on chemokine production were determined. We found that Ttcr-induced IP-10 (CXC chemokine) production (NFκB independent) was dose-dependently reduced in the presence of the inhibitor (Figure 4b). In contrast, Tck-induced MIP-1α (CC chemokine) production (also NFκB independent) could be dose-dependently enhanced in the presence of the PI3K inhibitor (Figure 4a), indicating the pathway plays a positive and negative regulatory role in each respective case. With NFκB-dependent Ttcr-induced MIP-1α production also displaying PI3K dependence, however, a role for this pathway in NFκB-dependent as well as NFκB-independent chemokine production cannot be ruled out.