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. 2001 May;124(2):330–336. doi: 10.1046/j.1365-2249.2001.01532.x

Imbalance between interleukin-6 and adrenomedullin mRNA levels in peripheral blood mononuclear cells of patients with lupus nephritis

Y Nishitani *, A Kubo *,†, M Iwano *, N Minamino ‡, K Hamano *, T Fujimoto *, T Nishino *, H Shiiki *, K Yonemasu †, K Dohi *
PMCID: PMC1906044  PMID: 11422212

Abstract

In this study, we measured the mRNA levels of adrenomedullin (AM), C-type natriuretic peptide, vascular endothelial growth factor, interleukin-1β (IL-1β) and interleukin-6 (IL-6) in peripheral blood mononuclear cells (PBMC) of 34 patients with lupus nephritis (LN) (15 active and 19 inactive) and 30 healthy volunteers. mRNA levels were measured using a real-time quantitative PCR method. Compared with healthy volunteers, IL-6 mRNA levels were elevated in LN patients (P < 0·005), while AM mRNA levels were decreased (P < 0·05). Also, IL-6 mRNA levels were higher and AM mRNA levels lower in active LN patients compared with inactive LN patients. In addition, IL-6 mRNA levels positively correlated and AM mRNA levels negatively correlated with SLE disease activity index and laboratory findings, such as blood urea nitrogen, serum creatinine, 50% haemolytic unit of complement and urinary excretion of protein over 24 h. Furthermore, IL-6 mRNA levels were negatively correlated with AM mRNA levels within the same LN patients. With regard to pathological findings, our results showed that IL-6 mRNA levels were higher, and AM mRNA levels significantly lower in patients with a high activity index compared to those with a low activity index. Following treatment with prednisolone, IL-6 mRNA levels in active LN patients decreased and AM mRNA levels increased to levels comparable to those in inactive LN and healthy volunteers. In vitro studies further demonstrated that elevated IL-6 mRNA levels in active LN patient PBMC were suppressed by the addition of adrenomedullin. Our results suggest that an imbalance between IL-6 and AM levels may play an important role in the progression of SLE, and that the mRNA levels of these genes in PBMC may be used as a disease activity index for SLE.

Keywords: adrenomedullin, interleukin-6, SLE

Introduction

Systemic lupus erythematosus (SLE) is characterized by several functional abnormalities of the immune system, including B cell hyperreactivity [1–4]. Previous studies have shown that cytokines have an important role in the immune dysregulation observed in SLE patients [1,2,5,6]. Abnormal regulation in the synthesis and expression of various cytokine genes in peripheral blood mononuclear cells (PBMC) of SLE patients has been observed in many studies [7–9]. Interleukin-6 (IL-6) is normally produced by monocytes, and mediates the late phase of B cell maturation [3,4,10]. In lupus nephritis (LN), the presence of interluekin-1 (IL-1), IL-6 and tumour necrosis factor-alpha (TNF-α) in diseased kidneys also appears to mediate local pathogenic effects such as mesangial proliferation [11,12]. Furthermore, the clinical course and renal involvement in SLE can be significantly altered by the addition of cytokines or cytokine inhibitors [2,5,6].

Vasoactive peptides, such as adrenomedullin (AM) and C-type natriuretic peptide (CNP), are produced and secreted from mesangial cells and PBMC, and suppress mesangial cell mitogenesis [13–16]. In a previous study, AM was shown to suppress the production of inflammatory cytokines such as IL-6 and TNF-α in a macrophage cell line [17]. These findings suggested that AM and CNP may function as suppressors of mesangial cell proliferation and cytokine production in patients with SLE. Vascular endothelial growth factor (VEGF) is also secreted from mesangial cells and PBMC, and participates in capillary repair in glomerulonephritis [18,19]. While serum VEGF levels of SLE patients are reported to be significantly higher than those of healthy volunteers [20], there has been no investigation to evaluate the level of VEGF mRNA in PBMC of LN patients.

In the present study, we measured the mRNA levels of IL-1β, IL-6, AM, CNP and VEGF in PBMC from LN patients to identify which factors have a close association with SLE disease activity.

Materials and methods

Patients and determination of clinical parameters

Thirty-four Japanese patients with LN were enrolled into the study. SLE was diagnosed according to the 1982 revised criteria from the American Rheumatism Association [21]. The mean age of patients was 40·5 ± 15·1 years (mean ±s.d.). Thirty healthy volunteers, aged from 21 to 59 (mean 36·9 ± 13·3) years, served as controls.

For the clinical parameters, the following data were determined using routine methods: blood urea nitrogen (BUN), serum creatinine (Scr), complement 3 (C3), C4, 50% haemolytic unit of complement (CH50), anti-DNA antibodies and urinary excretion of protein over 24 h. The SLE disease activity index (SLEDAI) was applied before and after prednisolone treatment [22]. LN patients were divided into two groups (15 active and 19 inactive) according to their disease activity. Patients with active LN had not received any prednisolone or immunosuppressive drugs before the study. All patients with active LN (n = 15) had clinical manifestations of lupus nephritis, such as proteinuria, and renal biopsy was performed. The SLEDAI score for all active LN patients was greater than 11 (mean 19·9 ± 1·5). Blood samples were obtained from patients with active LN before and after treatment with prednisolone for more than 3 months. Two active LN cases were also followed in more detail after treatment. Patients were grouped as inactive LN who had previously been treated and showed no exacerbation of disease, including fever, nephritis, arthritis, rash, serositis or central nerve system involvement accompanied by an increase of anti-DNA antibodies, a significant decrease in C3, C4 or CH50 levels and in other laboratory abnormalities such as proteinuria and leukopenia. Patients with inactive LN received an average of 11·5 ± 3·1 mg prednisolone on the day of blood sampling.

Patients with cardiac disease, hypertension (exceeding 150/90), renal dysfunction (Scr > 1·5 mg/dl) and diabetes mellitus were excluded. All patients provided informed consent prior to participation in this study.

Determination of light microscopic findings

Specimens for light microscopy were fixed in 10% neutral phosphate-buffered formalin, then embedded in paraffin and sectioned (3 µm) prior to staining with periodic acid-Schiff reagent. All biopsy specimens contained at least 10 glomeruli. Patient specimens were evaluated according to semiquantitative methods developed by Pirani et al. and modified by Austin et al. (total score 24) [23,24]. For the activity index, we calculated histological features such as cell proliferation, fibrinoid necrosis/karyorrhexis, cellular crescent formation, hyaline thrombi, wire loops, leucocyte infiltration in glomeruli and mononuclear cell infiltration in tubulointerstitium based on renal biopsy. According to these criteria, patients were divided into two groups based on lupus nephritis activity; Group I (score 0–7) and Group II (score 8–24). Evaluation of biopsy specimens was performed by two pathologists unaware of the purpose of this study.

Separation of peripheral blood mononuclear cells and RNA extraction

Blood samples (10 ml) were obtained by cubital venous puncture in standard sterile polystyrene vacuum tubes with heparin for RNA extraction. PBMC were isolated from blood samples by use of lymphocyte separating medium (ICN Pharmaceutical Inc. Costa Mesa, CA, USA).

Total RNA was extracted with RNAzol (Tel-Test Inc., Friendswood, TX, USA) from PBMC, and 2 µg total RNA reverse-transcribed into cDNA using the SuperScript preamplification system (Gibco BRL, Grand Island, NY, USA). The reaction mixture contained 1x PCR buffer, 2·5 mm MgCl2, 100 ng random hexamer, 0·5 mm each dNTP, 10 mm DTT and SuperScript II reverse-transcriptase. Reagents were incubated at 42°C for 50 min, then heated to 70°C for 15 min.

Measurement of AM, CNP, VEGF, IL-1β and IL-6 mRNA by real-time quantitative PCR

To measure human AM, CNP, VEGF, IL-1β, IL-6 and β-actin mRNA levels, real-time quantitative PCR was performed using a Prism 7700 Sequence Detector (Applied Biosystems Japan, Tokyo, Japan) [25,26]. Primers were synthesized on a DNA synthesizer (Applied Biosystems) and PCR performed using an oligonucleotide hybridization probe labelled with 6-carboxyfluorescein as reporter fluorescence and 6-carboxy-tetramethyl-rhodamine as quencher fluorescence [27]. Known amounts of cDNA fragments of human AM, CNP, VEGF, IL-1β, IL-6 and β-actin were used to generate standard curves that were shown to be highly correlated (r > 0·99) over a wide range. β-actin mRNA levels were first determined by real-time quantitative PCR, and samples containing 100 fg of β-actin mRNA were used to assess the mRNA levels of AM, CNP, VEGF, IL-1β and IL-6 to account for differences in the efficiency of reverse-transcription between the samples. Next, AM, CNP, VEGF, IL-1β and IL-6 mRNA levels were evaluated in samples containing 100 fg of β-actin mRNA using the same method.

Evaluation of the effects of AM on IL-6 mRNA levels in cultured PBMC

PBMC were obtained from 3 patients with active LN and 3 healthy volunteers as mentioned above. PBMC were washed twice with DEPC-PBS, and cells (1·0 × 106 cells/well) placed in RPMI 1640 medium (Nakarai tesque, Kyoto, Japan) with or without AM (10−6 M). After 2 h culture at 37°C in a humidified atmosphere containing 5% CO2, RNA was isolated from each cultured PBMC sample as mentioned above. IL-6 mRNA levels were then measured by real-time quantitative PCR. RNA was also isolated from PBMC from each subject prior to culture, and IL-6 mRNA levels determined. Results are expressed as the mean of four individual experiments.

Statistical analysis

All values are expressed as mean ±s.e.m. Comparisons between two groups were evaluated by Student's t-test. Comparisons among more than three groups were evaluated using one-way analysis of variance (anova), followed by a multiple comparison test (Fisher's test). Linear regression analysis or Spearman's correlation coefficient by rank was used to assess the correlation between variables. The difference in median values was examined by the Wilcoxon signed-rank test for paired subjects (before and after treatment). A level of P < 0·05 was considered to be statistically significant.

Results

mRNA levels of AM, CNP, VEGF, IL-1β and IL-6 in PBMC of patients with LN and healthy volunteers

The mRNA levels of AM, CNP, VEGF, IL-1β and IL-6 in PBMC of LN patients and healthy volunteers were measured using real-time quantitative PCR. While IL-6 mRNA levels in LN patients were significantly higher than in healthy volunteers (P < 0·005), AM mRNA levels in LN patients were significantly lower than in healthy volunteers (P < 0·05) (Table 1). As shown in Table 1, there were no significant differences in CNP, VEGF and IL-1β mRNA levels between LN patients and healthy volunteers.

Table 1.

mRNA levels of AM, CNP, VEGF, IL-1β and IL-6 in PBMC of patients with LN

mRNA level (fg/pg β-actin)
Control (n = 30) LN (n = 34)
AM mRNA 0·161 ± 0·027 0·102 ± 0·012*
CNP mRNA 1·296 ± 0·320 1·171 ± 0·390
VEGF mRNA 1·904 ± 0·216 1·866 ± 0·253
IL-1β mRNA 5·723 ± 0·651 6·175 ± 0·837
IL-6 mRNA 0·402 ± 0·053 1·147 ± 0·217**
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Values are the mean ±s.e.m.

*

P < 0·05 versus control

**

P < 0·005 versus control.

Patients with active LN exhibited significantly higher IL-6 mRNA levels (2·070 ± 0·366 fg/pg β-actin) than healthy volunteers (0·402 ± 0·053 fg/pg β-actin) (P < 0·005) or patients with inactive LN (0·418 ± 0·072 fg/pg β-actin, P < 0·005) (Fig. 1a). AM mRNA levels in active LN patients (0·057 ± 0·012 fg/pg β-actin) were significantly lower than in healthy volunteers (0·161 ± 0·027 fg/pg β-actin) (P < 0·005) or patients with inactive LN (0·137 ± 0·015 fg/pg β-actin, P < 0·05) (Fig. 1b).

Fig. 1.

Fig. 1

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Levels of IL-6 and AM mRNA based on clinical activity in PBMC of patients with LN. SLE patients were divided into two groups (active and inactive) according to clinical activity. (a) IL-6 mRNA levels were significantly higher in patients with active LN than in healthy volunteers (control) or inactive LN. (b) AM mRNA levels were significantly lower in patients with active LN than in healthy volunteers (control) or inactive LN. *P < 0·05, **P < 0·005

Correlation between AM or IL-6 mRNA levels and clinical parameters

To clarify the potential role of PBMC-expressed IL-6 and AM on SLE progression, we examined the correlation of clinical parameters and pathological findings with IL-6 and AM mRNA levels. As shown in Table 2, IL-6 mRNA levels correlated positively with BUN, Scr and urinary excretion of protein over 24 h and negatively with CH50. In contrast, AM mRNA levels correlated negatively with BUN, Scr, anti-DNA antibodies and urinary excretion of protein over 24 h and positively with CH50 (Table 2). Furthermore, IL-6 mRNA levels correlated positively, and AM mRNA levels correlated negatively, with SLEDAI (Fig. 2).

Table 2.

Correlation between AM and IL-6 mRNA levels and clinical parameters

Clinical parameters IL-6 mRNA (fg/pg β-actin) r (fg/pg β-actin) P AM mRNA r P
BUN 0·71 < 0·0001 − 0·38 0·026
Scr 0·62 < 0·0001 − 0·38 0·026
C3 0·16 n.s. 0·26 n.s.
C4 0·11 n.s. 0·01 n.s.
CH50 − 0·71 < 0·0001 0·59 0·0003
Anti-DNA antibodies 0·33 ns − 0·46 0·006
Urinary excretion of protein for 24 h 0·54 0·010 − 0·48 0·004
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n.s.: not significant.

Fig. 2.

Fig. 2

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Correlation between IL-6 or AM mRNA levels and SLEDAI in PBMC from LN patients. (a) IL-6 mRNA levels correlated positively with SLEDAI (r = 0·72, P < 0·005). (b) AM mRNA levels correlated negatively with SLEDAI (r = − 0·55, P < 0·005).

Levels of IL-6 and AM mRNA in PBMC and pathological findings of patients with active LN

Patients were divided into two groups (Groups I or II) according to lupus nephritis activity as mentioned in Materials and methods. As shown in Fig. 3, AM mRNA levels were significantly lower in Group II (high activity) patients (0·025 ± 0·006 fg/pg β-actin, P < 0·05) than in Group I (low activity) patients (0·085 ± 0·018 fg/pg β-actin). While IL-6 mRNA levels tended to be higher in Group II patients (2·655 ± 0·638 fg/pg β-actin) compared with Group I (1·557 ± 0·342 fg/pg β-actin), this difference was not statistically significant (P = 0·14).

Fig. 3.

Fig. 3

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Levels of IL-6 and AM mRNA based on pathological findings of lupus nephritis in PBMC from LN patients. Patients with active LN were divided into two groups (Group I; low activity, Group II; high activity) according to the pathological findings. (a) IL-6 mRNA levels tended to be higher in Group II patients compared with Group I patients, although the difference was not statistically significant. (b) AM mRNA levels were significantly lower in Group II patients than in Group I patients. *P < 0·05

Correlation between AM, CNP, VEGF, IL-1 and IL-6 mRNA levels in PBMC from patients with LN

To evaluate peptide and cytokine regulation in LN patient PBMC, correlations between pairs of AM, CNP, VEGF, IL-1β and IL-6 mRNA levels were examined. Of these combinations, IL-6 mRNA levels were negatively correlated with AM mRNA levels (r = 0·43, P < 0·05) (Fig. 4).

Fig. 4.

Fig. 4

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Correlation between AM and IL-6 mRNA levels in LN patient PBMC. The correlation between IL-6 and AM mRNA levels in PBMC was examined in the same individuals. IL-6 mRNA levels correlated negatively with AM mRNA levels (r = − 0·43, P < 0·05).

IL-6 mRNA and AM mRNA levels after prednisolone treatment

All active LN patients were treated with prednisolone and were followed-up after therapy. After treatment, patients showed no exacerbation of clinical features, including fever, nephritis, arthritis, rash, serositis or central nerve system involvement, and laboratory examinations such as anti-DNA antibodies, C3, C4 and CH50 for at least 3 months.

IL-6 and AM mRNA levels were compared before and after treatment in the 15 active LN patients (Fig. 5). Increased IL-6 mRNA levels in active LN patient PBMC before treatment (2·070 ± 0·366 fg/pg β-actin) were significantly decreased after treatment (0·713 ± 0·097 fg/pg β-actin, P < 0·005) and were reduced to levels similar to those of healthy volunteers. Decreased AM mRNA levels in active LN patient PBMC before treatment (0·057 ± 0·012 fg/pg β-actin) increased significantly after treatment (0·141 ± 0·013 fg/pg β-actin, P < 0·05) similar to levels in healthy volunteers.

Fig. 5.

Fig. 5

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Levels of IL-6 and AM mRNA in PBMC of patients with active LN before and after prednisolone treatment. Patients with active LN were treated with prednisolone and followed-up for more than 3 months. (a) IL-6 mRNA levels in active LN patients significantly decreased after prednisolone treatment. (b) AM mRNA levels in active LN patients significantly increased after prednisolone treatment. *P < 0·05, **P < 0·005

Two active LN patients were evaluated in more detail during the follow-up period after therapy. PBMC were isolated 5 times from these patients, and AM and IL-6 mRNA levels were shown to change within 30 days after starting therapy (Fig. 6).

Fig. 6.

Fig. 6

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Time-course of IL-6 and AM mRNA levels after prednisolone treatment. Two active LN patients were treated with prednisolone and followed-up for more than 3 months. IL-6 and AM mRNA levels in PBMC of active LN patients started to change at the earliest time point after prednisolone treatment. Data represent mRNA levels of IL-6 (•) and AM (○) at each time point.

Modulation of IL-6 mRNA expression in PBMC of patients with active LN

To elucidate the connection between AM and IL-6 expression in PBMC, we examined the effect of AM on IL-6 gene transcription in PBMC from active LN patients. Elevated PBMC IL-6 mRNA levels in all 3 patients were markedly suppressed by the addition of AM for 2 h (Fig. 7).

Fig. 7.

Fig. 7

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Effect of AM on IL-6 mRNA levels in PBMC from patients with active LN. PBMC were obtained from 3 patients with active LN and 3 healthy volunteers and cultured for 2 h. PBMC were incubated in the presence (○) or absence (•) of AM (10−6 m) for 2 h. IL-6 mRNA levels were then measured by real-time quantitative PCR. Data represent the average from 4 individual experiments.

Discussion

Previous studies have shown increased serum IL-6 levels in patients with active SLE [9,28]. Linker-Israeli et al. demonstrated that whole blood cells secreted IL-6 into cultured supernatant and IL-6 mRNA could be detected in active SLE patient PBMC [9]. In the present study, we demonstrated that PBMC IL-6 mRNA levels were increased in active LN patients compared to both patients with inactive LN and healthy volunteers (Table 1,Fig. 1a,Fig. 7). IL-6 mRNA levels were correlated positively with SLEDAI and negatively with CH50 levels (Table 2,Fig. 2a). Furthermore, following treatment with prednisolone, the increased IL-6 mRNA levels in active LN patient PBMC decreased significantly, reaching levels corresponding to that of healthy volunteers (Figs 5, 6). These findings suggested that PBMC may be one of the major sources for the elevated serum IL-6 levels, and that increased IL-6 mRNA levels reflected the disease activity of SLE.

We have reported previously that urinary levels of IL-6 correlated with lupus activity, and that these elevated levels decreased after treatment [29]. Herrera-Esparza et al. demonstrated IL-6 mRNA in infiltrating mononuclear cells of glomeruli and renal interstitium by in situ hybridization [12]. Furthermore, studies have also shown that cultured mesangial cells produce IL-6, and that IL-6 induced mesangial cell proliferation [11]. As shown in Fig. 3, IL-6 mRNA levels tended to be high in Group II patients compared with Group I, although this difference was not statistically significant. Thus, overproduction of IL-6 from PBMC may be associated with the progression of lupus nephritis.

Lymphocytes, monocytes and macrophages secrete AM into culture medium [17,30]. In the present study, we showed that AM mRNA levels in PBMC were decreased in patients with active LN, and was negatively correlated with SLEDAI (Table 1, Figs 1b, 2b). AM mRNA levels in PBMC also correlated negatively with levels of anti-DNA antibodies and positively with CH50, which are clinical parameters that reflect SLE disease activity (Table 2). The decreased AM mRNA levels in PBMC from active LN patients increased significantly after treatment to levels that corresponded to those observed in healthy volunteers (Fig. 5). In addition, we demonstrated in a previous study decreased AM mRNA levels in PBMC of IgA nephropathy patients with high disease activity [27]. Thus, these findings suggest that decreased AM mRNA levels in PBMC are involved in increased disease activities of SLE and IgA nephropathy. In the previous study, we showed that AM suppressed production of inflammatory cytokines, such as IL-6 and TNF-α from macrophages [17]. As shown in Fig. 7, the present study also demonstrated that AM suppressed IL-6 mRNA levels in cultured PBMC of patients with active LN, suggesting that AM may play a role as the suppressor of SLE progression through the inhibition of IL-6 production by PBMC.

As shown in Fig. 3, AM mRNA levels were significantly lower in patients with high disease activity than those with low activity based on the pathological evaluation of lupus nephritis. Chini et al. demonstrated that AM strongly inhibited mesangial cell mitogenesis via a cAMP-dependent signalling pathway, even when the cells were stimulated by potent mitogens such as PDGF [14,15]. They suggested that endogenous AM could act as a potent autocrine or paracrine regulator-suppressor of mesangial cell proliferation. Thus, the decreased AM production in PBMC may promote the proliferation of mesangial cells in LN.

In the present study, we have shown that PBMC IL-6 mRNA levels increased, while AM mRNA levels decreased in patients with LN (Table 1, Figs 1, 2). Furthermore, there was a negative correlation between IL-6 and AM mRNA levels in the same individuals (Fig. 4). Thus, our findings suggested that the imbalance between IL-6 and AM levels in PBMC is related to the disease activity of SLE, and may play an important role in the initiation and progression of SLE.

In conclusion, we have demonstrated that IL-6 mRNA levels increased and AM mRNA levels decreased in PBMC from active LN patients. These altered levels in PBMC were also shown to correlate with SLE disease activity. Thus, imbalance between IL-6 and AM in PBMC may participate in the pathophysiology of SLE.

Acknowledgments

This study was supported in part by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency (Encouragement System of COE) and by a research grant from the Ministry of Health and Welfare, and the Ministry of Education, Science and Culture of Japan. We thank Ms M. Kikui and Ms F. Kunda of the Nara Medical University for their technical assistance.

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