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Clin Exp Immunol. Dec 2000; 122(3): 464–470.
PMCID: PMC1905797

Increased interferon-gamma (IFN-γ), IL-10 and decreased IL-4 mRNA expression in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE)

Abstract

Cytokines are important regulators of lymphocyte function in SLE. However, the profile of Th1 and Th2 cytokines produced by circulating lymphocytes in human SLE has not been clearly elucidated. The aim of the present study was to characterize the gene expressions of the Th1-type cytokine IFN-γ, and the Th2-type cytokines IL-10 and IL-4 in PBMC of 15 patients with SLE and 10 healthy individuals by a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Our results showed that expression of IFN-γ (P = 0·0004) and IL-10 (P = 0·002) transcripts were significantly increased in PBMC of patients with SLE compared with healthy controls. By contrast, expression of IL-4 transcripts in PBMC of patients with SLE was significantly decreased compared with the healthy controls (P = 0·0008). Primary sources of IL-10 were B cells and monocytes, with variable contribution of T cells as detected in various fractions of PBMC of patients with SLE (P = 0·049). These findings support the hypothesis that the enhanced production of IFN-γ by mononuclear cells may trigger inflammatory responses, together with the enhanced production of IL-10 resulting in autoantibody production by B cells in human SLE.

Keywords: systemic lupus erythematosus, interferon-gamma, IL-10, IL-4, cytokine

INTRODUCTION

SLE is characterized by polyclonal B cell hyperactivity and the production of a broad spectrum of autoantibodies against nuclear, cytoplasmic and cell surface antigens [1]. These autoantibodies reflect a breakdown of tolerance to self antigens and can be attributed to dysfunction of the immune system [1,2].

Cytokines produced by CD4+ helper T (Th) lymphocytes are thought to regulate the function of the immune system, including antibody production and cellular immune response. Th cells represent a functionally heterogeneous population, comprising distinct subsets termed Th1 and Th2 defined by their cytokine secretion profiles [3]. In general, cytokines produced by Th1 cells (e.g. IFN-γ, IL-2) promote both production of complement-fixing and opsonizing antibodies and macrophage activation. Cytokines produced by Th2 cells (e.g. IL-4, IL-5, IL-6, IL-10 and IL-13) stimulate antibody production and promote mast cell and eosinophil granulocyte differentiation and activation.

Recent studies in animal models of SLE suggested that in SLE there is an alteration in Th1 and/or Th2 lymphocyte function resulting in an enhanced production of cytokines that up-regulate autoantibody production by B cells. Accordingly, in murine models of SLE an altered production of both Th1 (such as IFN-γ and IL-2) and Th2 (such as IL-4 and IL-10) cytokines have been reported [4,5] and the disease could be significantly improved and/or the production of autoantibodies decreased by treatment of the animals with anti-IFN-γ, anti-IFN-γ receptor [6,7], and in contrast also with anti-IL-4 [8] or anti-IL-10 antibodies [9]. However, the Th1/Th2 profile of cytokines produced by circulating lymphocytes in human SLE has not been clearly elucidated because of the technical limitations of previously used methods [1014]. Recent studies suggest that quantitative determination of gene activation is a specific and sensitive method to study the active production of cytokines [15,16].

Therefore, in the present study we determined the quantity of IFN-γ, IL-10 and IL-4 transcripts in unstimulated PBMC of SLE patients by a highly sensitive competitive polymerase chain reaction (PCR) method. PBMC from healthy volunteers served as controls.

PATIENTS and METHODS

Patients and controls

All patients were treated at the Central Immunological Laboratory of Semmelweis University as out-patients, and were diagnosed as having SLE by established criteria. While of the 20 patients examined six had active disease (as determined by SLE Disease Activity Index (SLEDAI) score), 14 had inactive lupus (Table 1). Nineteen of the patients were female and their mean age was 39 years. Ten (nine female and one male) healthy controls (mean age 41 years) were also studied.

Table 1
Clinical and laboratory features of SLE patients

Cell separation and cellular RNA preparation

Blood samples were collected in sterile Vacutainer tubes containing 100 U/ml of heparin. PBMC were prepared by Ficoll–Uromiro cell density centrifugation. Separation of PBMC from five SLE patients (nos 16–20, Table 3) was followed by fractionation of the cells into monocytes, T, B lymphocytes and other cells. Monocytes were isolated by plastic adherence (30 min, 37°C), detached from the plastic by ice-cold 0·05% EDTA–PBS. T cells were separated from the monocyte-depleted cell population by E-rosette buoyant density centrifugation using aminoethylisothiouronium bromide–HBr (Sigma, St Louis, MO) as described elsewhere [17]. Erythrocytes were lysed with distilled water. Non-T (E-rosette-negative) cells were further fractionated into B cells and non-T, non-B cells (natural killer (NK) cells, etc.) by nylon vatta adherence.

Table 3
IFN-γ, IL-10 and IL-4 cytokine gene expression in PBMC from healthy adults and SLE patients

Cells were washed twice with physiological Hanks' solution (PBMC) or RPMI 1640 (cell fractions) and subsequently lysed by TRI reagent (Sigma) at the ratio of 1 ml TRI/107 cells. Total cellular RNA was extracted following the instructions of the manufacturer [18]. Genomic DNA contamination was then removed by RNase-free DNase (Boehringer, Mannheim, Germany) treatment (10 U of DNase/RNA of 5 × 106 cells, at 37°C for 10 min) in the presence of 2·5 U human placental RNase inhibitor (HPRI; Boehringer), followed by repurifying RNA by the protocol used for RNA isolation with TRI reagent. For reverse transcription 5 μg RNA were reprecipitated by acid/salt/ethanol (1 v RNA in aqueous solution:0·08 v 3 m Na-acetate pH 5·4:3·3 v 100% ethanol) at −20°C for 16 h, and dissolved in 1× reverse transcriptase buffer.

cDNA synthesis

cDNA was synthesized according to the method of Zou et al. [19]. Briefly, the reaction mixture containing 5 μg of cell-RNA, 0·5 μg of oligo(dT)12–18 (Boehringer), 10 mm DTT, 0·5 mm dNTP (Pharmacia Biotech, Inc., Piscataway, NJ), 200 U of Moloney murine leukaemia virus reverse transcriptase (Gibco-BRL Life Technologies, Inc., Gathersburg, MD) and 40 U of human placental RNase inhibitor in 20 μl of total volume was incubated at 42°C for 60 min, followed by denaturing cDNA at 94°C for 5 min (PTC 100, programmable thermal cycler; MJ Research, Watertown, MA). cDNA was kept at −80°C until DNA amplification was performed.

Competitive PCR and the quantification of PCR products

cDNA was quantified by competitive PCR, with minor modification of the method described by Zou et al. [19]. pQA1 and pQB3 plasmids containing multispecific competitor sequences of synthetic origin (kindly provided by Dr D. Shire, Sanofi Recherche, Labege, France) were used to compete IFN-γ, IL-4, and IL-10, as well as β-actin-specific cDNA amplification, respectively. pQA1, pQB3 and other members of the pQA and pQB family of plasmids have been successfully used for analysing cytokine gene expression by quantitative and semiquantitative reverse transcriptase (RT)-PCR [2022]. The sequences of sense and antisense primers used, lengths of the cDNA- and competitor-specific amplicons are listed in Table 2.

Table 2
Oligonucleotide primers

In short, β-actin cDNA (2 μl) was coamplified in the presence of a serial dilution of linearized pQB3 competitor DNA (0·01, 0·1, 1, 10 pmol). The PCR mixture (30 μl) contained 300 nm of both sense and antisense primers (Eurogentec S.A., Seraing, Belgium), 1·3 U of Taq polymerase (Zenon Biotechnology, Ltd, Szeged, Hungary), 50 μm of each dNTP in 1× Taq buffer supplemented with 2·5 mm MgCl2. Competitive PCR for IFN-γ, IL-10 and IL-4 was performed under the same conditions as for the β-actin except that an amount of cDNA corresponding to 106 β-actin cDNA molecules was coamplified with the competitor DNA. A 10-fold serial dilution of pQA1 and pQB3 competitor DNA ranging from 0·001 to 1 pmol and from 0·01 to 10 pmol, respectively, was used to compete for amplification. Amplification was performed with the following program: one cycle of 94°C for 5 min, 57°C for 1 min, 72°C 1·5 min, 31 cycles (β-actin PCR) or 35 cycles (IFN-γ, IL-4, IL-10 PCR) of 94°C for 1 min, 57°C for 1 min, 72°C for 1·5 min, one cycle of 94°C for 5 min, 57°C for 1 min, 72°C for 5 min. The temperature of annealing was 57°C during the amplification of β-actin-specific DNA.

The amplified cDNA-specific and competitor DNA-specific products was quantified by a colourimetric assay based on the method of Zou et al. [19]. Briefly, in the first step a digoxigenin-dUTP-labelled DNA strand was synthesized from the competitive PCR products (1·4 μl) in the presence of 5 μm dNTP, 250 nm digoxigenin-dUTP (Boehringer), 0·2 U Taq polymerase, 300 nm 5′-biotinylated oligonucleotide recognizing either the amplified cDNA or the amplified competitor (Genset, Inc., France; kindly provided by Dr D. Emilie, INSERM, Paris, France), 1× Taq buffer in 20 μl final volume using a single PCR cycle of 94°C for 5 min, 60°C for 1 min, and 72°C for 2 min, 94°C for 2 min. Sequences of 5′-biotinylated oligonucleotides are shown in Table 2. The labelled DNA (1·5 μl) was quantified using an ELISA-type assay after binding the DNA to streptavidin-coated microtitre plates (Boehringer) in 200 μl 0·2% Tween–PBS supplemented with 1% low-fat milk powder (3 h, 37°C), followed by incubation with 200 μl of 1:15 000-diluted peroxidase-conjugated anti-digoxigenin MoAb (Boehringer) at 37°C for 30 min. Enzymatic reaction was performed using ABTS substrate. The optical density (OD) was measured at 405 nm (Anthos HT II microplate reader; Anthos Labtec Instruments, Salzburg, Austria). Under these conditions a strict linearity was observed between the log ratio of amplified cDNA/competitor DNA molecules and log number of competitor DNAs in each case (r2 > 0·9). Numbers of amplified cDNA molecules were calculated from the equivalence point of linear regression curve.

Statistical analysis

The number of amplified cytokine cDNA molecules (corresponding to the numbers of mRNA transcripts) normalized by 1 × 106 β-actin cDNA molecules are reported as their median values, first and third quartiles. Comparisons between two sample populations were made with the non-parametric Mann–Whitney U-test. The non-parametric Kruskal–Wallis test was used to compare median level of gene expression in five cell fractions. P <0·05 was considered statistically significant.

RESULTS

We performed quantitative analysis of IFN-γ, IL-10 and IL-4 cytokine mRNA expression in PBMC from healthy adults (control) and SLE patients by competitive PCR assay. In order to perform statistical analysis of comparable data the number of cDNA molecules determined was normalized to the number of β-actin molecules in the same sample.

Our results show that the number of mRNA transcripts of IFN-γ was significantly (P < 0·001) increased in the SLE group (Fig. 1). IFN-γ transcripts could be detected only in two cases of controls.

Fig. 1
Quantitative analysis of IFN-γ expression in PBMC from healthy adults (control) and SLE patients. The results represent the number of IFN-γ cDNA molecules per 106 β-actin molecules as determined by competitive polymerase chain ...

The number of mRNA transcripts of IL-10 was also significantly (P < 0·001) increased in the SLE group (Fig. 2). In the control group IL-10 mRNA was determined only in two cases at low concentrations. In order to identify the cell population expressing IL-10 mRNA we determined IL-10 gene expression in purified monocytes, T, B lymphocytes and a fraction containing the rest of the cells (mostly NK cells) isolated from PBMC of five SLE patients. Our results show that B lymphocytes and monocytes appeared to be the primary source of IL-10 in SLE patients, while T cells contributed to the IL-10 expression to a varying extent (P < 0·049) (Fig. 3).

Fig. 2
Quantitative analysis of IL-10 expression in PBMC from healthy adults (control) and SLE patients. The results represent the number of IL-10 cDNA molecules per 106 β-actin molecules as determined by competitive polymerase chain reaction assay. ...
Fig. 3
Quantitative analysis of IL-10 expression in fractions of PBMC from SLE patients. The results represent the number of IL-10 cDNA molecules per 106 β-actin molecules as determined by competitive polymerase chain reaction assay (medians are indicated ...

In contrast, the number of mRNA transcripts of IL-4 was significantly (P < 0·001) decreased in the SLE group (Fig. 4.). In the group of patients IL-4 mRNA could be detected only in four cases at low concentrations. In the control group IL-4 transcripts could be measured in each case. Median values, 25th and 75th percentiles of the data are shown in Table 3.

Fig. 4
Quantitative analysis of IL-4 expression in PBMC from healthy adults (control) and SLE patients. The results represent the number of IL-4 cDNA molecules per 106 β-actin molecules as determined by competitive polymerase chain reaction assay. The ...

There was no significant difference in mRNA expression between patients with either inactive or active disease (Table 3).

DISCUSSION

The main findings of our study are that (i) IFN-γ and IL-10 mRNA transcripts are up-regulated, and (ii) IL-4 transcripts are decreased in freshly isolated PBMC from SLE patients compared with healthy controls.

In the present study we used freshly isolated PBMC and measured cytokine mRNA expression using a semiquantitative PCR technique. Until now, cytokine protein production by immunocompetent cells has usually been investigated by detection of the cytokine in the sera [11,2325], but in isolated PBMC very small amounts of cytokine protein are produced spontaneously. Thus, in previous studies mitogen- or calcium ionophore-stimulated PBMC were also used [12,23,25,26]. In the present study the high sensitivity of the method used allowed us to determine cytokine mRNA expression in isolated PBMC without any stimulation, which can reflect the in vivo situation. Since transcriptional control is probably the most important mechanism regulating cytokine production [15], direct measurement of cytokine mRNA expression is a physiologically relevant indicator of cytokine secretion [2729].

We found an elevated IFN-γ mRNA expression in unstimulated PBMC of SLE patients. Our results accord with previous findings demonstrating increased IFN-γ levels in the serum of SLE patients with lymphadenopathy or nephritic syndrome [24] and support the hypothesis that in patients with SLE IFN-γ up-regulates IgG production by mononuclear cells [30]. The role of the Th1 cytokine IFN-γ in the development of SLE was previously documented by the earlier appearance of the disease after repeated injections of recombinant IFN-γ into the murine model of SLE [6]. Treatment of these mice when young with anti-IFN-γ antibodies inhibited the progression toward SLE. In conventional immune responses, IFN-γ produced by regulatory T cells down-regulates autoreactive B cells [31]. Recent studies also demonstrated a significant correlation between the severity of SLE and the amounts of IFN-γ secreted by stimulated PBMC from SLE patients [26]. Interestingly, no increase in the ratio of IFN-γ-positive lymphocytes could be detected by flow cytometry in SLE patients [12].

We also found an elevated IL-10 mRNA expression in unstimulated PBMC of SLE patients. Our result is in accordance with previous findings in SLE patients demonstrating increased IL-10 levels in the sera with ELISA [11] and an increased rate of PBMC that secrete IL-10 [12,13]. The severity of tissue damage in SLE is also thought to correlate with an increased IL-10 serum level [25] and/or an increased ratio of IL-10 cytokine-producing cells [13]. IL-10 is a pleiotropic Th2 cytokine [32], which is thought to be primarily produced by CD14+ monocytes and B cells in SLE [33]. Our results confirm that primary sources of IL-10 are B lymphocytes and monocytes. Although IL-10 expression by T cells showed high individual variance, in most cases their contribution was similar to the monocytes.

Because IL-10 is a potent stimulator of B cells, it is likely to play a major role in the polyclonal B lymphocyte hyperactivity associated with this disease and in the development of autoimmunity. IL-10 production was also found to be enhanced in patients with rheumatoid arthritis or Sjögren's syndrome, two disorders characterized by prominent B lymphocyte hyperactivity which results in increased production of immunoglobulins and the synthesis of autoantibodies [34]. The over-expression of IL-10 may also affect the production of cytokines by T cells and monocytes [35,36]. The inhibitory effect of IL-10 on the production of IL-4 by Th2 clones, and its stimulatory effect on the production of IFN-γ by T cell clones after stimulation, has also been reported recently [37,38]. Enhanced expression of IL-10 accompanied by increased production of IFN-γ may indicate a dysfunction of cross-regulation of Th1 and Th2 cells.

In order to characterize the production of another prominent Th2 cytokine we examined the mRNA expression of IL-4. IL-4 was originally described as a cytokine that delivers early activation and class-switch signals to lymphocyte [39]. In the murine model of SLE, up-regulation of IL-4 expression has been demonstrated [4] and is thought to be essential for stimulating B cells. Therefore one could hypothesize that IL-4 levels are increased in human SLE as well. However, we found a significant decrease in IL-4 mRNA expression in unstimulated PBMC of SLE patients. This finding confirms the previous studies of Horwitz et al. [14], who demonstrated a decrease in the Th2 mRNA level in PBMC of patients with SLE. Our data suggest that IL-4 does not have a predominant role in human SLE. The latter hypothesis is supported by the findings that in human SLE the anti-dsDNA autoantibodies are predominantly of the complement-fixing IgG1 and IgG3 subclasses, whereas IL-4 probably provides B cell help for non-complement-fixing antibodies [40].

The consistent detection of IL-4 mRNA in normal PBMC can be due to selective representation of the female gender among the healthy control subjects that corresponds to the prevalence of SLE in females [41]. Th1/Th2 cytokine production is under the control of sex hormones [42,43]. Healthy females tend to have a predominant Th2-like cytokine profile compared with males [44], also in multiparous, fertile women expression of IL-4 and IL-6 mRNA could be detected in the peri-implantation endometrium, while expression of IL-2, IFN-γ and IL-12 could not [45]. These observations can explain the higher background level of IL-4 in comparison with that of IFN-γ in normal females.

Besides its effects on B cells, IL-4 down-regulates the production of IL-1 and IL-6 by monocytes [46,47]. In SLE there is evidence of increased production of IL-1 and IL-6 [23].

The observed increase in IFN-γ gene expressions in PBMC from SLE patients is unlikely to be caused by the methylprednisolone (MP) treatment received, as MP has been shown to decrease IFN-γ protein/mRNA expression in mitogen-stimulated PBMC of healthy subjects and under pathological conditions associated with elevated IFN-γ production, e.g. in multiple sclerosis [4851]. Although inhibition by prednisolone of concanavalin A-induced IL-4 gene expression in normal donors' PBMC and inhibition of spontaneous IL-4 mRNA expression by PBMC from prednisolone-treated patients with an excessive Th2-type response, e.g. asthma, have been documented [52,53], one can argue against the role of MP therapy in diminished IL-4 expression in SLE, because patients suffering from, for example, the Th1-type cell-mediated autoimmune disease, multiple sclerosis, treated with MP showed an increased level of IL-4 production by PBMC compared with untreated ones [54].

However, we cannot exclude that in our study MP contributed to the increase of IL-10 gene expression in the patients with SLE. The effect of MP therapy on IL-10 mRNA production in SLE patients is not clear. Stimulatory and inhibitory effects of in vitro applied MP on lipopolysaccharide (LPS)-induced IL-10 production/mRNA level have been recently demonstrated in monocytes or PBMC from healthy subjects and multiple sclerosis patients with acute relapse, respectively [49,50]. In contrast, serum IL-10 concentration and IL-10 gene expression in PBMC from relapsed multiple sclerosis patients increased after receiving MP therapy, and spontaneous IL-10 secretion by normal PBMC was also up-regulated by MP in vitro [50]. MP did not induce changes of serum IL-10 levels when applied in vivo for treatment of patients during cardiac surgery [55], and it diminished LPS-induced IL-10 production/mRNA message level in PBMC from patients with asthma [56]. In our study monocytes have proved to be one of the major sources of IL-10, thus we cannot exclude the possibility that MP therapy contributes to the elevated level of IL-10 expression in SLE patients. However, the identification of B lymphocytes as the predominant source of IL-10, and to a lesser extent also T cells, argues against the influence of MP therapy on IL-10 gene expression.

We could not detect characteristic differences in IFN-γ, IL-4 and IL-10 gene expression between patients with active and inactive SLE that could point to a need to search for other soluble factors useful for monitoring the disease activity. In this respect, longitudinal studies could be especially helpful in establishing associations between individual changes in cytokine expression and disease activity or therapy. Cytokine production can be genetically determined (e.g. in the cases of tumour necrosis factor-alpha (TNF-α), IL-10) and small individual changes can be more noticeable in longitudinal analysis. However, large variation among the patients would make it difficult to take practical advantage of such results. So far, in terms of cytokines or their soluble receptors, disease activity in SLE has been characterised by increases in serum IL-6, TNF-α, soluble TNF-α receptors (soluble p55, soluble p75), soluble IL-2Rα [57], urinary MCP-1 levels [58], impaired IgG-stimulated IL-1Ra and LPS-induced IL-8 production by PBMC [59] and polymorphonuclear neutrophils [60], respectively.

In conclusion, our results indicate that while expression of IFN-γ and IL-10 increases, expression of IL-4 decreases in PBMC of patients with SLE. This finding supports the hypothesis that IFN-γ and IL-10 contribute to the B cell hyperactivity, polyclonal hypergammaglobulinaemia and autoantibody production observed in SLE, and highlight these cytokines as potential targets for immunotherapy.

Acknowledgments

The excellent technical support of Beáta Haraszti is acknowledged. We thank Zoltán Ungvári for his help in preparation of the manuscript. This work was supported by Grants from the Hungarian National Science Research Foundation OTKA T 026525, the Health Science Council ETT 502/96 and 215/999605.

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