Interleukin-6 in peripheral blood and inflammatory sites in Behçet's disease

Interleukin-6, a potent pro-inflammatory cytokine, might be involved in Behçet's disease (BD) pathological pathways. We investigated IL-6 levels in sera and synovial fluids collected from BD patients. The IL-6 production was also studied in vivo, by measuring its activity in culture supernatants of PBMC and alveolar macrophages, stimulated or not with LPS. The patients with BD were compared to RA patients and healthy controls. High IL-6 levels were observed in sera, synovial fluid and LPS stimulated PBMC supernatants, from active BD patients, similar to those of RA patients. Alveolar macrophages production of IL-6 was significantly elevated in two active BD patients with an interstitial pneumonia, when compared to controls. These elevated levels of IL-6 suggest its involvement in the inflammatory sites of BD, which may be related to the progression of the acute lesions, at least in the joints and in the lungs.


Introduction
Behet's disease (BD) is a multisystemic disease, mainly observed in Mediterranean areas and in Japan; it is characterized by oral and genital ulcerations, associated with ocular and skin lesions, arthralgia, neurological involvement, and pulmonary manifestations.
Elevated levels of interleukin-1 (IL-1), tumour necrosis factor-(TNF-), soluble interleukin-2 receptor 2 and gamma-interferon 3-s have been observed in patients with active Behet's disease (BD), suggesting the involvement of these cytokines in the inflammatory process.
Among the immunological factors that have been recently involved in the inflammatory manifestations, interleukin-6 (IL-6) plays a prominent role. 6 IL-6 has pleitropic biological effects, including human T-cell activation, 7 and B-cell proliferation and differentiation.
The aim of our study was to evaluate in BD patients IL-6 level in sera and IL-6 production by blood mononuclear cells (PBMC) into culture supernatants. Moreover, IL-6 was quantified in two local inflammatory sites: synovial fluid and culture supernatants of alveolar macrophages obtained by bronchoalveolar lavage.

Materials and Methods
Patients'Thirty patients with BD, who fulfilled the criteria proposed by the International Study Group for Behet's Disease 9 were studied, 28 men and two women, with a mean (+ SEM) age of 34__+ 3 years. Twenty patients had active disease, and ten inactive disease. The clinical characteristics and the treatment of active BD patients are shown in Table 1. Two patients with active BD had also pulmonary manifestations" a chronic cough associated to interstitial shadows on the chest roentgenogram. 1 These patients received no treatment.
Six patients with inactive BD received thalidomide therapy for 6 to 38 months with an average of 20 months, l the other four patients with inactive disease received corticosteroids.
Ten patients with rheumatoid arthritis (RA), meeting the Arthritis Rheumatism Association criteria, were studied during acute flares of synovitis. All RA patients were on standard therapy including nonsteroidal anti-inflammatory drugs. Most patients were female, with a mean (+ SEM) age of 47 + 12 years.
Twenty healthy age-and-sex-matched subjects were studied as controls.
Serum and synovialfluid: Serum samples were collected from all patients and control subjects. Synovial fluid was obtained from the ten RA patients and ten active BD patients with arthritis. Freshly aspirated synovial fluids were collected into heparinized tubes (10 U ml-), treated with 1.5 U mlhyaluronidase for 30min at 37C, centrifuged at 1 200 x g to remove cellular debris and stored at -20C until use.
Peripheral blood mononuclear cells (PBMC) IL-6 production by PBMC was studied in active BD (ten cases), inactive BD (ten cases), RA patients (ten cases) and ten healthy controls. PBMC were separated by (C) 1992 Rapid Communications of Oxford Ltd  12 Hybridoma growth factor/IL-6 activity was expressed in U m1-1, defined as the dilution giving half the maximal proliferation of 7TD1 cells. One unit corresponds to approximately 5 pg mlof IL-6. 7TD1 cells respond neither to ILl, TNF0, IL2, IFN0 and 2, nor to any of the known colony-stimulating factors other than IL-6. Biological activity of IL-6 samples was completely neutralized by adding monospecific rabbit polyclonal anti-recombinant human IL-6 antibodies to test samples. To eliminate inhibitory effects present in undiluted sera and to determine the lower limit of detection of the assay, IL-6 activity in sera was measured as such and by adding 100U of recombinant cytokine. The standard used in these assays was r-IL-6 (Genzyme, Boston, MA). Results IL-6 in serum" Low levels of IL-6 activity could be detected in the serum from all healthy individuals IL-6 in Behet's disease tested (4.5 _-t-1.9 U ml-1). In patients with active BD, the values were significantly increased (19.8 -t-9 U ml-1) (Fig. 1)   to inactive BD and control subjects (p < 0.01).
There was no dierence in spontaneous IL-6 production between patients with inactive BD and healthy controls.
After LPS stimulation IL-6 production was significantly increased (p < 0.01) in patients with active BD (2980 518 U ml-1), in inactive BD patients (2390 __+ 310 U ml-1), and in RA patients (2950-t-873 U m1-1) (Fig. 2) as compared with healthy controls (1100 -t-117 U ml-1). IL-6 in synovialjquid: IL-6 was detected in all synovial fluid samples tested. IL-6 levels were similar in joint fluids obtained from patients with active BD (1835 +__ 703 U ml-1) and from RA patients (2010 -t-504 U ml-1). IL-6 secretion from alveolar macrophages from two patients with active BD" The total BAL cell yield in BD patients (9.9 x 106 and 11 x 106 ceils m1-1) was in the same range as that of the normal group (9.7 x 106 cells ml-1). BD patients had a greater percentage of lymphocytes in lavage (17% and 12%) than control population (6.8 q-1.4%). The percentages of alveolar macrophages in the BD patients (83.4% and 79.8%) were decreased when compared to the healthy controls (93.2 _ _ _ 1.6%; p < 0.01). Alveolar macrophages (AM) isolated from two patients with active BD and five controls, were assayed for spontaneous and after LPS-stimulation IL-6 production. The level of spontaneous IL-6 production was significantly increased (p < 0.01) in culture supernatants from the two patients with BD, 2893 U m1-1 and 2350 U m1-1, as compared to supernatants of AM from five control subjects (1200 -+-122.5 U ml-). After LPS-stimulation, AM from patients with BD produced 4540 U m1-1 and 4980 U m1-1, twice more than healthy controls (2240 _--k 194.9 U ml-1).
Thus, spontaneous and after LPS-stimulation IL-6 production by AM from active BD patients with interstitial pneumonia was significantly increased compared to controls (p < 0.01).

Discussion
In this study, a high seric IL-6 level from active BD patients similar to that from RA patients was shown, while seric IL-6 from inactive BD patients was in the same range as healthy controls. Moreover spontaneous and after LPS stimulation IL-6 production by PMBC from active BD patients, was as high as that from RA patients. Spontaneous IL-6 production from inactive BD patients was similar to that of control subjects. However after LPS stimulation, IL-6 production was as high as in active BD and RA patients.
Thus, seric IL-6 levels seemed to be correlated to the clinical activity in BD, suggesting that in vivo, IL-6 is produced only during the clinical exacerbations. Corticosteroid treatment may influence directly IL-6 production. It seems to downregulate in vivo IL-6 production in active BD, since the patients receiving steroid therapy had lower seric values. This effect was not observed with in vitro production of II2-6 by PMBC, since IL-6 production was high in all active BD patients, receiving or not corticosteroid therapy.
A moderate level of seric IL-6 may also be due 13 to the presence of a circulating IL-6 receptor, which diminishes IL-6 detectable activity, acting as an immunoregulator. In the synovial fluids, IL-6 was detected at similar levels in active BD and RA patients. High IL-6 levels have been reported in several autoimmune diseases such as RA14 and systemic lupus erythematosus. 15 IL-6 in synovial fluid from active BD was at a lower level than in RA synovial fluid. It has been reported that IL-6 production was found in inflammatory synovium from RA 14'16 and BD patients, iv IL-6 is an inflammatory cytokine with multiple effects including the ability to stimulate or to enhance the differentiation and the proliferation of cytotoxic T-cells, and the differentiation of B-cells into plasma cells 18 as it was shown in systemic lupus erythematosus (SLE), where the overproduction of IL-6 contributes to the B-cell hyperactivity. 15 We have recently reported high cytotoxic T-cell activity against Herpes Simplex virus 19 and increased immunoglobulin production in active BD. 2 However, our data were obtained in vitro, and we have no evidence about the direct role of IL-6 on B-cell function.
Alveolar macrophages obtained from two BD patients with pulmonary manifestations, produced higher levels of I12-6 spontaneously and after LPS-stimulation than controls. Furthermore, AM from BD patients, in contrast to controls, expressed ICAM-1 antigen (data not shown), that could be related with the presence of IL-6. This cytokine may serve as an intermediate trigger regulating the expression of accessory surface molecules such as antigens of the MHC system and leukocyte adhesion molecules on the monocytes/macrophages. 21 The increased IL-6 levels in serum and LPS-stimulated PBMC supernatants, did not argue against the normal levels of TNF-0 found in vivo. Increased concentrations of IL-1 and TNF-0 may be found in vivo only in extreme circumstances, 22 whereas IL-6 is normally detected in the circulation, and rises rapidly after even minor trauma and inflammation. 23 IFN-2 and IL-6 are known to interact either synergistically or antagonistically. In active BD, the release of gamma-interferon (IFN-),) is increased. 4'5 It may be responsible for the enhancement of IL-6 production. 24 On the other hand, IL-6 may accelerate the inflammatory reaction by stimulating Tand B-cells, leading to further IFN-) production. 24 Our aim in investigating in vitro and in vivo IL-6 production, was to study the cell-cell interactions that are influenced in a positive or a negative manner by the in vivo release of various mediators. An exact knowledge of the role played by each cytokine in BD, may allow to inhibit damaging cytokine pathways or amplify regulatory pathways and thereby, have a therapeutic value. Various micro-organisms have been implicated as aetiological agents of BD such as Herpes Simplex virus as and streptococcal antigens. 2 We are currently investigating the IL-6 and other cytokines production after stimulation with different microorganisms.