Decreased Response to IL-12 and IL-18 of Peripheral Blood Cells in Rheumatoid Arthritis

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Decreased Response to IL-12 and IL-18 of Peripheral Blood Cells in

Rheumatoid Arthritis

Masanori Kawashima, Pierre Miossec

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Arthritis Res Ther 6(1):R39-R45, 2003. © 2003 BioMed Central, Ltd.

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Posted 12/10/2003

Abstract and Introduction

Abstract

Inflamed synovium of rheumatoid arthritis (RA) has been associated with

a T helper (Th)1 cytokine profile but the blood situation remains to be

clarified. We studied the differential IFN-? producing activity of

peripheral blood mononuclear cells (PBMCs) from RA patients (RA-PBMCs)

and from healthy controls (H-PBMCs) in response to IL-12 and IL-18.

RA-PBMCs had a decreased IFN-? production in response to IL-12 and IL-18

when compared with H-PBMCs. RA-PBMCs activated with phytohemagglutinin

and phorbol 12-myristate 13-acetate showed an increased sensitivity to

IL-12 and IL-18, but still the RA-PBMC response was lower. IL-18

increased IL-12-stimulated IFN-? production from RA synovium cells

obtained after collagenase digestion more effectively than that of RA-

or H-PBMCs. A specific inhibitor of IL-18 bioactivity, IL-18-binding

protein (IL-18BP), down-regulated IL-12-induced IFN-? production by RA-

or H-PBMCs and had a remarkable effect on RA synovium cells. In

conclusion, RA disease combines a polarized immune response with an

active Th1 in inflamed joints and a reduced Th1 pattern in peripheral

circulation.

Introduction

Rheumatoid arthritis (RA) is a chronic disorder of unknown aetiology

primarily affecting joints and leading to their progressive destruction.

The chronically inflamed synovium of RA is characterized by a massive

infiltration of lymphocytes and macrophages[1] and by an extensive

proliferation of fibroblast-like synoviocytes.[2] CD4+ CD45+memory T

cells are the major cellular component and show signs of activation,[3]

but their exact role in the pathogenesis of RA remains

controversial.[3,4] In particular, the important biologic mediators

produced by activated T cells, such as IL-2, IL-4, and IFN-?, have been

detected only at low levels in RA joints,[5-8] in contrast to the

abundance of cytokines from macrophages and synoviocytes, such as IL-1,

TNF-?, and IL-6.[3] However, some studies of T-cell cytokine patterns in

the RA joint at the mRNA level and others using T-cell clones indicate

the predominance of IFN-?-producing T helper (Th)1 cells.[9-11] By

intracellular cytokine staining of peripheral blood and synovial tissue

T cells from RA patients, we have confirmed the selective accumulation

of Th1 and Th0 cells in the synovium.[12] In addition, IL-12, which

plays a critical role in the differentiation of IFN-?-producing Th1

cells, is produced predominantly by macrophages localized adjacent to

lymphatic aggregates. IL-12 can potently and selectively stimulate IFN-?

production by RA synovial tissue, mainly by acting on synovial T cells.[13]

IL-18, initially described as an IFN-?-inducing factor, is a novel

cytokine of the IL-1 family.[14] IL-18 stimulates the synthesis of IFN-?

in T cells and natural killer (NK) cells, leading to the development of

Th1-type immune responses. In addition, IL-18 also activates the

proliferation of activated T cells and their production of IL-2 and

granulocyte/macrophage-colony-stimulating factor, and the cytotoxic

activity of NK cells through up-regulation of Fas ligand.[15] Early

studies suggested that the effects of IL-18 on Th1 differentiation were

independent of IL-12. However, later studies showed that exogenous IL-18

in the absence of IL-12 failed to drive the differentiation of naive T

cells to Th1 cells[16] and that IL-18 was a potent inducer of IFN-? from

established Th1 cells only in combination with IL-12 [15,17-19].

Accordingly, IL-18 may be involved in various immune-mediated

inflammatory conditions. Significant levels of its expression have been

demonstrated in the synovium of patients with RA.[20,21] It was

suggested that IL-18 in synergy with IL-12 and IL-15 could be involved

in both Th1 immune responses and macrophage production of inflammatory

cytokines such as TNF-?.[20] To control some of the potentially

deleterious properties of IL-18, IL-18-binding protein (IL-18BP) has

been identified as a specific inhibitor of its bioactivity. IL-18BP,

though it lacks significant homology with IL-18 receptor components, can

bind to IL-18 protein with high affinity, thereby acting as a soluble

decoy receptor.[22,23]

In the present study, we looked at the differential effects of IL-18

alone or in combination with IL-12 on RA cells from blood versus

synovium. Our results show that the PBMCs of RA patients produced less

IFN-? in response to IL-12 and IL-18 than those of healthy volunteers.

In addition, differences were observed between blood and synovium RA

cells. Finally, differential regulatory effects of IL-18BP on these

cells were also observed.

Materials and Methods

Cytokines and Reagents

Recombinant human IL-12 was purchased from R & D Systems (Abingdon, UK).

Recombinant human IL-18 was from MBL (Nagoya, Japan). IL-18BP, kindly

provided by Dr Sims, Immunex/Amgen (Seattle, WA, USA), was produced

in COS cells as a fusion protein combining IL-18BP and the CH2 and CH3

domains of human IgG1 and purified by protein A affinity. RPMI (Roswell

Park Memorial Institute) 1640 culture media was purchased from

Invitrogen SARL (Cergy Pontoise, France) and supplemented with 100

units/ml penicillin, 100 ?g/ml streptomycin, and 10% fetal calf serum

(Invitrogen). Phytohemagglutinin (PHA) and phorbol 12-myristate

13-acetate (PMA) were purchased from Sigma-Aldrich SARL (St Quentin

Fallavier, France).

Preparation of Synovium and Cell Cultures

Peripheral blood samples were obtained from 14 patients (2 men and 12

women) with RA who fulfilled the 1987 revised criteria of the American

College of Rheumatology,[24] and 12 healthy volunteers (2 men and 10

women). The mean ages ± SEM of RA patients and healthy controls were

52.9 ± 4.8 and 49.3 ± 1.9 years, respectively. The mean disease duration

was 12.4 years (range 1–53 years). The majority of patients were being

treated with nonsteroidal anti-inflammatory drugs, prednisone (n = 7;

range 2–20 mg/day), methotrexate (n = 11) alone or combined with

cyclosporin (n = 1) or anti-TNF treatment (n = 1), and cyclophosphamide

(n = 2). PBMCs were isolated from heparinized blood by Ficoll

density-gradient centrifugation, washed twice with phosphate-buffered

serum, and re-suspended in RPMI 1640 medium. Synovium samples were

obtained from patients with RA who were undergoing wrist, elbow, or hip

synovectomy, or joint replacement.

For the isolation of synovium cells, samples were minced with scissors

and digested for 1 hour with collagenase (Sigma, St Louis, MO, USA) and

DNase (Invitrogen) in RPMI 1640 medium at 37°C. After removing tissue

debris through a cell strainer, the resulting cell suspensions were

washed twice with medium. The resultant single-cell suspensions and PBMC

suspensions were dispensed into the wells of 96-well plates (Nunc,

Roskilde, Denmark) at a density of 1 × 106 cells/ml in 200 ?l of RPMI

medium. Cultures were at 37°C in 5% CO2/95% humidified air.

Determination of IFN-? Levels by ELISA

IFN-? levels were measured by quantitative sandwich ELISA, using a

commercially available ELISA kit (DuoSet ELISA Development System human

IFN-?, R & D Systems). The detection limit of the assay was 20 pg/ml.

IFN-? values below this limit were regarded as 0.

Statistical Analysis

Results were expressed as mean ± SEM of the indicated number of

experiments. The statistical significance of differences between two

groups was determined by the Mann–Whitney U test. The Wilcoxon

signed-rank test was used to analyse matched pairs.

Results

Effect of IL-12 and/or IL-18 on IFN-? Production by Normal PBMCs and its

Modulation by IL-18BP

Peripheral blood mononuclear cells from healthy controls (H-PBMCs) were

cultured for 7 days for IFN-? production with or without IL-12 and IL-18

alone or in combination. As shown in Fig. 1a, IL-18 alone up to 50 ng/ml

had only a negligible effect on IFN-? production. In contrast, IL-12

alone (1 ng/ml) induced a high IFN-? production from peripheral blood

mononuclear cells (PBMCs). However, there was no clear difference

between 1 ng/ml vs 5 ng/ml of IL-12 on IFN-? production (data not

shown). IL-12-stimulated IFN-? production was increased by IL-18 dose

dependently in a synergistic fashion (Fig. 1b).

To study the regulation of IFN-? production by IL-18BP, IL-18 and

IL-18BP were pre-incubated together for 30 minutes at 37°C before

addition to the culture. IL-18BP could successfully neutralize IFN-?

production from PBMCs stimulated by IL-12 and IL-18. IFN-? production

induced by IL-12 (1 ng/ml) and IL-18 (5 ng/ml) was reduced by IL-18BP in

a dose-dependent manner (Fig. 1c). PBMCs were cultured for 3, 5, and 7

days with or without IL-12 (1 ng/ml), or IL-12 (1 ng/ml) + IL-18 (5

ng/ml). IFN-? production by spontaneous PBMC culture could not be

detected at day 3 or 5 (Fig. 1d). Accordingly, concentrations of 1 ng/ml

of IL-12, 5 ng/ml of IL-18, and 2 ?g/ml of IL-18BP for 7 days of culture

were selected for the following experiments, when these results were

extended to more individuals.

Unstimulated H-PBMCs (n = 12) were cultured for 7 days with or without

IL-12 and IL-18 alone and in combination with or without IL-18BP, and

IFN-? levels were compared by ELISA. IL-12 induced significant levels of

IFN-? production from H-PBMCs compared with medium alone (P < 0.001). As

shown in Fig. 2a (white bars), IL-12-stimulated IFN-? production from

H-PBMCs was significantly augmented by IL-18 (P < 0.01) but not by IL-18BP.

Effect of IL-12 and IL-18 on unstimulated PBMCs from RA patients

PBMCs from patients with RA (RA-PBMCs; n = 14) were tested in the same

experiments (Fig. 2a, black bars). In culture with medium alone (0.07 ±

0.05 vs 0.05 ± 0.02 ng/ml), H-PBMCs produced very low levels of IFN-?

similar to those produced by RA-PBMCs. IL-12-induced IFN-? production

was reduced in RA-PBMCs, being less than half that of H-PBMCs (P <

0.01). Exogenous IL-18 slightly augmented the IL-12-induced IFN-?

production from RA-PBMCs, but the effect was smaller than that seen in

H-PBMCs (RA-PBMCs, +22.6% vs H-PBMCs, +52.3%). In cultures with medium

alone, RA-PBMCs produced lower levels of IFN-? than did H-PBMCs, and

this was not corrected by stimulation with IL-12 or IL-18. Although the

difference in the percentage increase was not significant, RA-PBMCs

produced less IFN-? than H-PBMCs in response to IL-12 alone or combined

with IL-18 when results were expressed as concentrations.

Effect of IL-12 and IL-18 on PHA/PMA-stimulated PBMCs

Because IFN-? production is associated with T-cell activation, and to

further sensitize the cells to the action of IL-12 and IL-18, H- or

RA-PBMCs were cultured with the same conditions as above, but in the

presence of 200 ng/ml of PHA and 2 ng/ml of PMA. At these suboptimal

concentrations, additional PHA and PMA alone had only a small effect on

IFN-? production by PBMCs (Fig. 2b). Despite this stimulation, IL-18 by

itself did not induce either H- or RA-PBMCs to produce IFN-?. In

contrast, activated H- or RA-PBMCs (Fig. 2b) produced greater amounts of

IFN-? than resting ones (Fig. 2a) in response to IL-12. The synergistic

effect of IL-12 and IL-18 on IFN-? production by H- or RA-PBMCs was also

augmented by PHA and PMA treatment. These findings indicate that

activated cells are more sensitive to IL-12 and IL-18 than resting ones.

However, even with the stimulation of PHA and PMA, the IFN-? production

by RA-PBMCs in response to IL-12 and IL-18 was lower than that of

H-PBMCs (Fig. 2b).

Regulation of IFN-? Production From H- or RA-PBMCs by IL-18BP

While exogenous IL-18 showed a synergistic effect with IL-12 on IFN-?

production, levels of IL-12-stimulated IFN-? production from resting

PBMCs were not influenced by IL-18BP treatment (Fig. 2a). On the other

hand, IL-12-stimulated IFN-? production was decreased by IL-18BP in

PBMCs activated by PHA/PMA (reduction with IL-18BP was 47.2% in H-PBMCs

[P < 0.01] and 53.0% in RA-PBMCs [P < 0.01]). These observations

indicate that the stimulation of PHA/PMA induces PBMCs to produce

significant amounts of IL-18. Exogenous IL-18 increased IL-12-induced

IFN-? production by 47% for activated H-PBMCs versus 25% for activated

RA-PBMCs. This difference indicated that RA-PBMCs had a defective

response to IL-18 even when these cells were activated by PHA/PMA.

Aging of RA PBMCs and Sensitivity to Th1-inducing Cytokine

Since immune defects have been associated with age, we classified the

patients into two groups according to their age and compared the IFN-?

production by their PBMCs in response to IL-12 and IL-18. The mean age,

disease duration, and C-reactive-protein levels at the date of the blood

sampling are shown in Table 1. The values of C-reactive protein were

almost identical in the two groups. The older RA group showed a greater

decrease in response to IL-12 and IL-18 than the younger RA group,

indicating that this defect was disease related but increased with age,

although without reaching significance.

IFN-? Production by RA Synovium Cells in Response to IL-12 and IL-18

T cells from RA synovium are characterized by a reduced production of

IFN-?, contrasting with the large production of IFN-? by T-cell clones

isolated from RA synovium. To further explore this contrast, total RA

synovial cells (n = 7), obtained after collagenase digestion, were

exposed to IL-12 and IL-18 using the same conditions as were used with

the PBMCs (Fig. 3a). IL-12 alone but not IL-18 induced total RA synovium

cells to produce significant amounts of IFN-?. However, IL-18 increased

IL-12-stimulated IFN-? production from total RA synovium cells more

effectively (+78.4%, P < 0.05) than those of H- or RA-PBMCs. In cells

activated with PHA and PMA, IL-18 increased by 71.4% the

IL-12-stimulated IFN-? production from total RA synovium cells, and

IL-18BP decreased it by 38.5% (Fig. 3b). Thus, total RA synovium cells

showed a greater response to IL-18 than blood cells. Stimulation with

PHA and PMA further augmented the magnitude of IFN-? production, but the

percentage increase of IFN-? production by exogenous IL-18 was not

changed by PHA/PMA. Accordingly, total RA synovium cells were considered

to respond better to exogenous IL-18 than the blood cells did. IL-18BP

decreased IL-12-stimulated IFN-? production from total RA synovium cells

cultured with or without PHA/PMA (-38.5% and -46.2%, respectively).

RA synovium is known to produce significant amounts of IL-18

spontaneously.[21] Addition of IL-18BP could decrease the IL-12-induced

IFN-? production by these cells by neutralizing the endogenously

produced IL-18, indicating the importance of endogenous IL-18 in IFN-?

production by RA synovium.

Discussion

Several lines of evidence have indicated that IFN-?-producing Th1 cells

predominate at the site of chronic inflammation in RA. IL-12 is

considered to play a critical role in inducing Th1-cell-mediated

organ-specific autoimmune diseases, as shown in several animal

models.[25-28] In addition, we have already demonstrated the presence of

IL-12 as a contributory factor in inducing the IFN-?-dominant T-cell

cytokine response in joints of patients with longstanding RA.[13] IL-18

is a proinflammatory cytokine that plays an important role in the

Th1-type immune response through the induction of IFN-? synthesis in T

cells and NK cells, T-cell proliferation, and cytokine production.[15]

Significant levels of expression of IL-18 have been previously

demonstrated in the synovium of RA patients,[20,21] and the major effect

of IL-18 is to increase the IFN-?-dominant T-cell response induced by

IL-12.[21]

T cells and NK cells are the major source of IFN-? in PBMC cultures, and

IL-12 and IL-18 augment IFN-? production by these cells. Defects in

IFN-? production may result from changes in cell number and/or in cell

response. Although differing conclusions have been reached regarding the

possible changes in numbers of CD4+ T cells, NK cells, and NK T cells

between RA and healthy PBMCs,[29] the precise subpopulation with reduced

ability to produce IFN-? remains to be clarified.[29-31] In order to

assess overall differences in systemic response to the Th1-inducing

cytokines, IL-12 and IL-18, we compared the IFN-? production by PBMCs

from RA patients to that by cells from healthy controls. The IFN-?

production of RA-PBMCs in response to IL-12 and IL-18 was lower than

that of H-PBMCs, even with the activation of PHA and PMA. This was still

observed when the younger RA patients were compared with age-matched

controls. Furthermore, PBMCs from the older RA patients demonstrated a

greater decrease in response to IL-12 and IL-18 than that of the younger

patients. Indeed, a reduction of the systemic Th1 functions with aging

in normal individuals has been reported.[32] The same tendency was

observed for the response to Th1-inducing cytokines in RA. These results

indicate that RA-PBMCs are defective in their response to Th1-inducing

cytokines, with an additional effect related to age. When looking at a

possible effect of treatment on these defects, we found no difference

between patients treated or not treated with prednisone (data not

shown). The finding was similar for patients receiving methotrexate.

Preliminary results appear to indicate that PBMCs from RA patients

taking methotrexate produced greater amounts of IFN-? after successful

anti-TNF treatment. This result, if it proves to be correct, would

further indicate that the defective IFN-? production by RA-PBMCs might

be related to disease activity.

Next, we compared the response to TH1-inducing cytokines of blood versus

synovium cells from RA patients. Judging from the effect on IFN-?

production, total RA synovium cells showed a greater response to

exogenous IL-18 in the presence of IL-12 than resting PBMCs. The pattern

of response to IL-12 and IL-18 of total RA synovium cells was similar to

that of activated H-PBMCs, suggesting that these cells had been

activated and were more sensitive to TH1-inducing cytokines.

IL-18BP has been identified as a specific inhibitor of IL-18.[22,23]

There is no significant similarity between IL-18BP and IL-18 receptor

components. Since it lacks a transmembrane domain, IL-18BP appears to

exist only as a soluble, circulating protein. Its major function is to

regulate the inflammatory activity of IL-18 by acting as its soluble

decoy receptor.[22,23] Our results showed that exogenous IL-18 alone did

not induce total RA synovium cells to produce detectable levels of

IFN-?, but the neutralization of endogenous IL-18 by IL-18BP reduced by

about 50% the IL-12-induced IFN-? production by these cells, suggesting

a role for endogenously produced IL-18 in IFN-? production. The effect

of IL-18BP was much higher in RA synovium cells than in PBMCs, probably

because RA synovium produced more endogenous IL-18 and responsed more

strongly to IL-18. Although an IL-18-neutralizing activity was found in

RA synovial fluid samples, IL-18 bioactivity was still detectable.[21]

These findings indicate that endogenous IL-18 is an important

contributory factor to IL-12-induced IFN-? production in RA synovium.

In a previous study, we used flow cytometry to examine the ability of

CD4+ T cells of blood and synovium samples from RA patients to produce

IFN-? and/or IL-4. Total RA synovium cells showed a higher Th1 and a

lower Th2 frequency than peripheral blood cells.[12] In addition, Haddad

and colleagues reported that activated whole blood cells from RA

patients produced higher levels of IL-4 and lower IFN-? than did cells

from healthy controls.[33] Accordingly, the IL-4 : IFN-? ratio, which

reflects the Th2 : Th1 cytokine balance in blood, was higher in RA

patients. These findings suggest that in RA the Th1 : Th2 ratio in the

blood and that in the synovium are different.

Accumulation of Th2 vs Th1 cells in blood may result from changes in

T-cell migration. Indeed, IFN-? producing T cells were significantly

increased in the peripheral blood of RA patients shortly after

anti-TNF-? treatment, resulting in a shift of the Th1 : Th2 ratio in

favor of Th1 in peripheral blood.[34] Adhesion molecules, such as P- and

E-selectin or VCAM-1 (vascular cell adhesion molecule-1), are considered

to be important for the selective homing of Th1 cells and not Th2

cells.[35] Expression of E-selectin and VCAM-1 was significantly reduced

by anti-TNF therapy.[36] Thus, anti-TNF treatment may suppress the

selective migration of Th1 cells into RA synovium through the rapid

down-regulation of adhesion molecules. It remains to be clarified

whether this is associated with an increased migration of Th2 cells with

anti-inflammatory properties.

Recently, cases of severe tuberculosis have been reported in patients

with Crohn's disease and RA receiving anti-TNF treatment.[37]

Tuberculosis was the most common serious opportunistic infection

reported in those patients. Similar observations were made in an HIV

population where a secondary cell-mediated immune defect is present, as

demonstrated by a defect in IFN-? production. It is noteworthy that

tuberculosis developed in RA patients shortly after the beginning of

anti-TNF treatment. Our results showed a decreased response of RA

peripheral blood to IL-12 and IL-18, leading to a reduced production of

IFN-? compared with that of healthy controls. These findings could

explain to some extent the occurrence of tuberculosis during anti-TNF

treatment.

Conclusion

Our data demonstrate that compared with H-PBMCs, RA-PBMCs have a lower

response to IL-12 and IL-18, which are both important in Th1 development

via T-bet expression. These results are in contrast with the finding

that synovium cells in RA show an increased response to IL-12 and IL-18.

Thus, RA patients have a polarized immune response, Th1 being

overexpressed in inflamed joints and Th1 defects in peripheral circulation.

Competing Interests

None declared.

Correspondence Address

Prof Pierre Miossec, 5 place d'Arsonval, Department of Immunology and

Rheumatology and INSERM U-403, Pavillon F, Hospital Edouard Herriot,

69437 Lyon Cedex 03, France. Tel: +33 472 117487; fax: +33 472 117429;

e-mail pierre.miossec@...

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