Elevation of plasma interleukin-18 concentration is correlated with disease activity in systemic lupus erythematosus
C. K. Wong,
E. K. Li1,
C. Y. Ho and
C. W. K. Lam
Departments of Chemical Pathology and
1 Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong, China
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Abstract
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Background. Previous studies have indicated that the autoimmune phenomenon might be caused by an imbalance of T-helper cell (Th) cytokines.
Methods. We investigated the plasma concentrations of a novel pro-inflammatory Th1 cytokine, interleukin (IL)-18, and its inducer, IL-12, in patients with systemic lupus erythematosus (SLE) and correlated them with the SLE disease activity index (SLEDAI). Plasma IL-18 and IL-12 concentrations of 40 SLE patients and 18 sex- and age-matched control subjects were measured by enzyme-linked immunosorbent assay.
Results. Plasma IL-18 and IL-12 concentrations were significantly higher in SLE patients than in control subjects [median (interquartile range): IL-18, 320.0 pg/ml (164.4475.6 pg/ml) vs 130.1 pg/ml (57.8202.4 pg/ml), P < 0.001; IL-12, 143.3 pg/ml (39.4247.2 pg/ml) vs 84.7 pg/ml (29.3140.1 pg/ml), P < 0.001]. Increases in IL-18 concentration correlated positively and significantly with SLEDAI score (r = 0.449, P = 0.004).
Conclusion. The novel cytokine IL-18 might play a crucial role in triggering the inflammatory processes in SLE.
KEY WORDS: Interleukin-18, Interleukin-12, Systemic lupus erythematosus, T-helper cell cytokines.
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Introduction
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Systemic lupus erythematosus (SLE) is a disorder of immune regulation manifested by the activation of T lymphocytes and polyclonal B lymphocytes, the production of autoantibodies and the formation of immune complexes causing tissue damage [1]. Complex networks of cytokines have been shown to regulate T-and B-cell growth, differentiation and effector functions. They can be grouped into the cyokines that are associated with T-helper cells of type 1 (Th1) [e.g. interleukin (IL)-2, IL-12, interferon
(IFN-
) and tumour necrosis factor
(TNF-
)], which induce cell-mediated immunity, and those that are associated with T-helper cells of type 2 (Th2) (e.g. IL-4 and IL-10), which stimulate antibody production [2]. There is recent evidence that abnormal Th1 and Th2 cytokines might be involved in the pathogenesis of autoimmune diseases. For example, peripheral blood mononuclear cells of SLE patients showed decreased production of the Th1 cytokines IL-2, IFN-
[3], TNF-
[4] and IL-12 [5, 6] and up-regulation of the Th2 cytokines IL-4 [3] and IL-10 [5]. Such imbalance of Th cytokines may account for the polyclonal B-cell activation observed in SLE, but the issue remains unsettled. Whereas an earlier report suggested that there was no relationship between the in vitro production of Th1 and Th2 cytokines and disease activity [7], other studies have demonstrated that concentrations of the serum Th1 cytokines IL-12 [8], TNF-
[9] and IFN-
[10] are significantly higher in SLE patients. These discrepancies imply that the Th cytokine response in SLE is very complex and requires further investigation.
Interleukin (IL)-18, formerly called IFN-
-inducing factor, is a novel Th1 cytokine produced by Kupffer cells, activated macrophages, keratinocytes, intestinal epithelial cells, osteoblasts and adrenal cortex cells [11]. It plays an important role in the Th1 response to toxic shock and shares functional similarities with IL-12 [11]. IL-12 can induce the production of IL-18 and has a synergistic effect with IL-18 on the activation of natural killer (NK) and cytotoxic T lymphocytes (CTL) [12]. The primary functions of IL-18 include the induction of IFN-
production in T cells and NK cells [11], the up-regulation of Th1 cytokines, including IL-2, granulocyte-macrophage colony stimulating factor (GM-CSF) and IFN-
[11], the stimulation of the proliferation of activated T cells [11], and the enhancement of Fas ligand expression in NK cells and CTL [13].
Recent studies have demonstrated that IL-18 can elicit an anti-vaccinia virus effect by activation of NK cells and CTL [14], and can elicit anti-tumour activity by activation of the Fas-dependent pathway [15]. Elevation of plasma IL-18 has been shown in haemophagocytic lymphohistiocytosis [16], Crohn's disease [17] and leukaemia [18]. IL-18 mRNA was found to be increased in the spinal cord in experimental autoimmune encephalomyelitis, a model of multiple sclerosis [19], but levels of IL-18 in SLE have not yet been studied. Since IL-12 can induce IL-18 production and has been shown to be increased in concentration in SLE patients [8], we measured the plasma concentrations of IL-18 and IL-12 in SLE patients to investigate their possible causative role in SLE.
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Patients and methods
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SLE patients, control subjects and blood samples
Forty Chinese SLE patients [36 females and four males, mean age of 35.5 yr (S.D. 8.8), range 1755 yr] were recruited at the Rheumatology Out-patient Clinic of the Prince of Wales Hospital, Hong Kong. Diagnosis of SLE was established according to the 1982 revised American Rheumatism Association criteria [20], and disease activity was evaluated by the SLE Disease Activity Index (SLEDAI) [21]. The mean interval since the diagnosis of SLE at the time when the patients were evaluated for this study was 6.8 ± 5.9 yr (range 0.221.6 yr). Patients were being treated with prednisolone 8.9 ± 6.4 mg daily, hydroxychloroquine 200 mg daily, azathioprine 77 ± 30 mg daily, cyclosporin A 129 ± 82 mg daily, or combinations of these drugs. Eighteen sex- and age-matched healthy Chinese volunteers (16 females and two males, aged 35.2 ± 7.5 yr, range 2352 yr) were recruited as controls. Twenty millilitres of heparinized blood was collected from each patient and control subject. Plasma samples were preserved at -70°C for cytokine assays.
IL-18 and IL-12 assays
Plasma IL-18 and IL-12 concentrations of SLE patients and control subjects were measured by enzyme-linked immunosorbent assay (ELISA) using the human bioactive IL-18 ELISA kit of Medical & Biological Laboratories (Nagoya, Japan) and the human IL-12 p40 ELISA kit of R & D Systems (Minneapolis, Minnesota, USA), respectively.
Statistical analysis
Since plasma IL-18 and IL-12 concentrations, SLEDAI and the prednisolone dose were not distributed in a Gaussian manner, the MannWhitney rank sum test was used to assess differences in the concentrations of cytokines between patients and controls and the effects of drug treatment. Spearman's rank test was used to ascertain the correlation between plasma IL-18, IL-12, SLEDAI and prednisolone dose. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) statistical software for Windows, Version 9.0 (SPSS, Chicago, IL, USA). A probability (P) value of <0.05 was considered as indicating a significant difference.
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Results
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The concentrations of plasma bioactive IL-18 in SLE patients were significantly higher than those in control subjects [median (interquartile range) 320.0 pg/ml (164.4475.6 pg/ml) vs 130.1 pg/ml (57.8202.4 pg/ml), P < 0.001] (Fig. 1
). The plasma levels of IL-12 in SLE patients were also significantly higher than in controls [143.3 pg/ml (39.4247.2 pg/ml) vs 84.7 pg/ml (29.3140.1 pg/ml), P < 0.001]. As shown in Fig. 2
, there was a significant positive correlation between IL-18 concentration and SLEDAI score in SLE patients (r = 0.449, P = 0.004). Moreover, a significant correlation was found between IL-18 concentration and the dose of prednisolone (r = 0.55, P < 0.01). No significant correlation was found between IL-18 and IL-12 concentrations (r = 0.168, P = 0.300). There were also no significant differences in IL-12 and IL-18 levels between SLE patients treated and not treated with azathioprine, hydroxychloroquine or cyclosporin A.
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Discussion
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Recent studies have suggested that changes in Th1 and Th2 cytokines might be involved in the pathogenesis of autoimmune diseases [22]. The development of experimental SLE in mice was found to involve two stages: (i) increases in Th1 cytokines, and (ii) the subsequent elevation of Th2 cytokines. The pro-inflammatory cytokines TNF-
and IL-1 were found to remain elevated throughout the course of disease [23]. An in vitro study has also indicated that the production ratios of IL-10/IL-2 and IL-10/IFN-
in stimulated blood mononuclear cells were positively correlated with SLEDAI [24]. Thus, SLE is characterized by elevated production of the Th2 cytokine IL-10, reflecting the basal state of activation of the immune system regardless of the severity of SLE. The elevation of IL-2 or IFN-
levels causes further inflammation and tissue injury, as observed in exacerbations of SLE [24].
Because the human IL-18 ELISA kit that we used detects only bioactive IL-18 and not the immature form pro-IL-18, our present results demonstrate that the plasma concentration of the novel Th1 cytokine mature IL-18 was significantly elevated in SLE patients compared with controls, and that this elevation was positively correlated with SLEDAI. This may be because IL-18 can enhance the expression of Fas ligand in NK cells and CTL, causing Fas-mediated apoptosis in epithelial cells and tissue damage. In combination with other pro-inflammatory cytokines, including IL-1 and TNF-
, IL-18 must be an important cytokine for initiating and progressing the catabolic response and fever in SLE [25, 26]. IL-18 has been shown to account at least partly for the hepatocytotoxic pathways in endotoxin-induced liver injury [27], probably through the Fas-mediated apoptosis of liver cells. Moreover, recombinant IL-18 has been found to enhance the in vitro cytotoxicity of murine splenic and hepatic mononuclear cells via Fas/Fas ligand interaction [15]. The elevation of IL-18 might therefore be crucial for organ damage in the exacerbation of SLE. Of interest is that the plasma concentration of IL-18 correlated with the dose of prednisolone but was not affected by therapy with other drugs, such as azathioprine, hydroxychloroquine and cyclosporin A. We cannot be certain whether this was a direct effect of corticosteroid treatment or simply a reflection of the increased SLEDAI justifying a higher prednisolone dose.
Both the study of Tokano et al. [8] and our study showed that plasma IL-12 concentrations in SLE patients were significantly higher than those of normal subjects. IL-12 is similar to IL-18 in that both are synthesized by macrophages in response to infection, and promote the activation of cell-mediated immunity [25]. IL-12 was shown to induce the production of IL-18 in primates [28]. The elevation of IL-12 might therefore induce the release of IL-18; plasma concentrations of IL-12 and IL-18 were elevated simultaneously in SLE patients in this study. The combination of IL-12 and IL-18 is critical for the induction of the innate immune response and inflammatory reaction [12].
However, there are some important technical considerations in this study. First, cytokines in the plasma may not reflect the locally high concentrations produced in lymphoid tissues. Secondly, cytokines in the serum may be bound by proteins or complexed to soluble receptors or autoantibodies, which may affect their clearance [22]. This may account for the lack of correlation between the plasma concentrations of IL-12 and IL-18. We did not find any significant difference between IL-18 levels in the culture supernatant of peripheral blood mononuclear cells isolated from normal controls and SLE patients after overnight incubation. However, in vitro production of IL-18 from peripheral blood mononuclear cells upon activation by different mitogens (e.g. phytohaemagglutinin) was not investigated. As Liu and Jones [5] have demonstrated impaired in vitro production of IL-12, the mechanism of in vitro production of IL-18 by peripheral blood mononuclear cells from SLE patients should be investigated extensively to elucidate the production mechanism and pathogenic role of IL-18.
Concentrations of cytokines other than IL-18, including the Th1 cytokine IL-16 [29] and the Th2 cytokine IL-10 [30], have also shown significant correlation with SLEDAI. We found that the plasma concentrations of the Th1 cytokine IL-17 and the Th2 cytokine IL-4 were also significantly higher in SLE patients than in control subjects (data not shown). The increased production of the Th1 cytokines IL-18, IL-12 and IL-17 should exert a synergistic effect in inflammatory reactions, leading to increased production of a panel of proinflammatory cytokines including IFN-
, IL-2 and GM-CSF in SLE. The significant elevation of the Th2 cytokine IL-4 in SLE patients confirmed its putative role in the activation of autoreactive B lymphocytes [31]. Collectively, the activation of both Th1 and Th2 cytokines appears to be important in the pathogenesis of SLE.
In conclusion, the circulating IL-18 concentration is significantly elevated in SLE patients and correlates with the SLEDAI score as well as with the prednisolone dose, but not with the doses of other drugs. This may be related to the initiation of the inflammation, fever and organ damage that is commonly observed in SLE patients.
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Acknowledgments
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This study was supported by a donation from Zindart (De Zhen) Foundation Ltd, Hong Kong.
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Notes
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Correspondence to: C. W. K. Lam. 
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Submitted 14 October 1999;
revised version accepted 17 March 2000.