A shift in the Th1/Th2 ratio accompanies the clinical remission of systemic lupus erythematosus in patients with end-stage renal disease

Gunnar Heine1, Urban Sester1, Martina Sester1, Jürgen E. Scherberich2, Matthias Girndt1 and Hans Köhler1,

1 Medical Department IV, Nephrology, University Homburg, Homburg and 2 Medical Department II, Nephrology, Städtisches Krankenhaus München-Harlaching, München, Germany



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Patients suffering from systemic lupus erythematosus (SLE) with renal involvement often show remission of systemic clinical activity after progression to end-stage renal disease (ESRD). SLE is characterized by predominantly humoral, T-helper (Th)2-mediated autoimmune responses. Since ESRD induces a state of immunodeficiency that affects the balance of Th cell subsets, we hypothesized that a Th1 shift induced by ESRD leads to clinical remission of SLE.

Methods. Using single-cell measurement of intracellular cytokines by flow cytometry after polyclonal stimulation with PMA/ionomycin, helper cell profiles were analysed in SLE patients with preserved renal function and in SLE patients with ESRD, from both isolated peripheral blood mononuclear cells (PBMC) and whole blood.

Results. Using the whole-blood assay, patients with SLE and preserved renal function showed a predominance of Th2 cells compared to healthy controls (patients, Th1/Th2 ratio 6.0±1.0 vs controls, 9.0±1.0; P<0.05). In contrast, SLE patients with ESRD have significantly more Th1 cells (36.8±5.0%) than those without ESRD (23.4±3.6%; P<0.05). This results in an enhancement of the Th1/Th2 ratio to 12.1±2.6, which is not significantly different from healthy controls. These data were confirmed using a PBMC-based assay.

Conclusions. SLE patients with preserved renal function show a bias in the differentiation of Th cells towards Th2. Once ESRD occurs, the Th1/Th2 ratio normalizes. This may contribute to the remission of Th2-mediated autoimmune diseases such as SLE.

Keywords: flow cytometry; haemodialysis; immunodeficiency; PBMC; systemic lupus erythematosus; Th1/Th2



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Ten to thirty per cent of patients with lupus nephritis progress to end-stage renal disease (ESRD). The onset of ESRD is often accompanied by a decrease in clinical and serological manifestations of systemic lupus erythematosus (SLE) [1]. The pathophysiology of this remission remains unclear.

In the 1990s, studies on the pathogenesis of various autoimmune and infectious disorders showed that these diseases were driven by T-helper cells that display either a Th1 or Th2 pattern of cytokine secretion [2]. For instance, T cells in patients with granulomatous vasculitis such as Wegener's granulomatosis or giant cell arteritis show a shift toward a Th1 pattern of helper cell differentiation, characterized by cytokines such as tumour necrosis factor {alpha} (TNF{alpha}) and interferon {gamma} (IFN{gamma}) [3]. In contrast, in patients suffering from systemic lupus erythematosus, T cells are characterized by Th2 cytokines such as interleukin (IL) 4 and IL10 [46] (see [7] for an in-depth-review).

In haemodialysis patients suffering from non-auto-immunological renal disease, circulating T-helper cells show a dysregulated cytokine differentiation profile that is characterized by a relative predominance of Th1 cells, as shown by us recently [8].

We thus hypothesized that the predominance of a Th2 pattern of T-helper cell differentiation in SLE patients is attenuated by progression of lupus nephritis to ESRD. As clinical and serological manifestations of SLE are driven by Th2 cells, a shift in Th cell differentiation after the onset of ESRD may clinically result in a quiescence of SLE.

The objective of this study was to analyse whether the clinical remission of SLE in patients with ESRD is associated with a shift in the T-helper cell response.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
Twenty-nine patients with SLE were studied. Ten of these patients had end-stage renal disease due to lupus nephritis (ESRD patients) and had been on renal replacement therapy for at least 7 months (range 7–208 months). Nine of these 10 patients were on haemodialysis and one patient was on peritoneal dialysis. In the remaining 19 patients, renal function was preserved (non-ESRD patients; serum creatinine <3 mg/dl). Disease duration of SLE did not differ significantly between the groups; it was 6.1±4.6 years in non-ESRD patients and 10.7±6.4 years in ESRD patients.

All patients except one in the ESRD group were female; all non-ESRD patients were female. SLE patients on dialysis treatment (ESRD patients) did not significantly differ in age from SLE patients with preserved renal function (53±5 years and 40±14 years respectively).

All patients were diagnosed according to the criteria of the American College of Rheumatology for classification of SLE. As control group, 30 healthy individuals were studied. All patients and controls gave informed consent.

Cell preparation and stimulation
T-helper cell subpopulations were determined by single-cell measurement of intracellular cytokines by 3-colour flow cytometry, using (i) a whole blood assay and (ii) additionally, in a subgroup of 16 SLE patients and of seven healthy controls, Ficoll-isolated peripheral blood mononuclear cells (PBMC). Blood was drawn before the start of a haemodialysis session in HD patients, at least 48 h after the last haemodialysis had been performed. In patients with preserved renal function and with peritoneal dialysis, blood was drawn during an outpatient clinic appointment.

For the whole-blood assay, 150 µl of heparinized blood were incubated in 300 µl of complete medium (RPMI 1640, Biochrom, Berlin, Germany, supplemented with 5% fetal calf serum (FCS, PAA, Cölbe, Bettingen, Germany), 2 mmol/l glutamine and antibiotics (PAA)), using polypropylene tubes. Cells were stimulated polyclonally for 4 h (37°C, 6% CO2) using 10 ng/ml phorbol myristate acetate (PMA) and 2 µmol/l ionomycin (Sigma, Deisenhofen, Germany); 10 µg/ml of brefeldin A (Sigma) were added to inhibit intracellular transport of cytokines. As negative controls, blood cells were incubated with brefeldin A in the absence of ionomycin and PMA. Subsequently, 2 mmol/l EDTA was added for 15 min. The cells were incubated for 10 min with 4 ml lysing solution (BD, Heidelberg, Germany), containing diethylene glycol to lyse erythrocytes and formaldehyde to fix leukocytes. Cells were washed once with a FACS buffer (phosphate-buffered saline (PBS) containing 5% FCS, 0.5% bovine serum albumin (Sigma) and 0.07% NaN3).

Stimulation of Ficoll-isolated PBMC was done essentially as described previously [8].

Cytokine measurement by flow cytometry
For single-cell measurement of intracellular cytokines, cells from the whole-blood assay as well as PBMC were permeabilized with 2 ml FACS buffer containing 0.1% saponin (Sigma) for 10 min at room temperature. Subsequently, cells were stained for 30 min at room temperature in the dark, using mouse-anti-human antibodies conjugated to fluorescent dyes at saturating concentrations (FITC-labelled {alpha}IFNg (clone 4S.B3, Pharmingen, Hamburg, Germany), PE-labelled {alpha}IL4 (clone 8D3-8, Pharmingen) and PerCP-labelled {alpha}CD4 (clone SK3, BD, Heidelberg, Germany)). Cells were then washed with 3 ml of FACS buffer and fixed in 1% paraformaldehyde.

The samples were measured on a BD FACScan flow cytometer using Cellquest software. Data analysis was performed with the WinMDI 2.8 system provided by J. Trotter (Scripps Research Institute, La Jolla, USA) using a lymphocyte gate in the forward-scatter vs side-scatter dot plot. Subsequently, single-cell flow cytometric cytokine identification was performed within a CD4+ T-lymphocyte gate, analysing at least 10000 CD4+ cells. T-lymphocyte subsets were classified according to their intracellular cytokine profile as Th1 cells (IFN{gamma} positive) or Th2 cells (IL4 positive).

Statistics
Data management and statistical analysis were performed with the Prism V3.0 statistical software (Graphpad, San Diego, USA). All data are expressed as means±standard error of the mean unless indicated differently. Significance of differences in T-cell numbers between groups of patients was calculated using the Mann–Whitney test for unrelated, non-normally distributed samples. Serological activity and numbers of patients requiring any immunosuppressive medication, only low-dose corticosteroid therapy and other than low-dose corticosteroid therapy were compared in contingency tables, using Fisher's exact test. The level of significance was set at P<0.05.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Clinical activity of lupus erythematosus
The clinical activity of lupus erythematosus was assessed in a 2-fold manner: firstly, the need for immunosuppressive medication at the time point of blood sampling was documented; secondly, anti-double-stranded (ds) DNA antibodies were measured as markers of the serological activity of SLE, since among all autoantibodies in SLE, anti-ds-DNA correlate most closely with disease activity [9,10].

The total number of patients receiving any immunosuppressive drug at the time of blood analysis did not differ between SLE patient with preserved renal function (16 of 19 patients on immunosuppressive medication) and SLE patients with ESRD (6 of 10 patients respectively; Table 1Go). However, only one SLE patient with ESRD received immunosuppressive medication other than low-dose corticosteroid treatment, whereas nine out of 19 SLE patients with preserved renal function were on a more potent immunosuppressive drug regimen (P<0.01).


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Table 1.  Clinical activity of SLE

 
In addition, SLE patients with preserved renal function tended to have anti-ds-DNA antibody titres above normal ranges (7 of 19 patients) more frequently than SLE patients with ESRD (2 of 10 patients), although this difference did not reach statistical significance.

SLE patients with preserved renal function show a predominant Th2 pattern of T-helper cell proliferation
T-helper cell subpopulations were analysed by a whole-blood assay. Patients with SLE and preserved renal function show a biased T-helper cell differentiation in favour of Th2 cells when compared to healthy controls (Th1/Th2 ratio, SLE without ESRD 6.0±1.0; controls 9.0±1.0; P<0.05; Figure 1Go). This shift results from a tendency for a lower percentage of Th1 cells and for a higher percentage of Th2 cells in SLE patients with preserved renal function (Figure 2Go).



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Fig. 1.  Th1/Th2 ratio (ratio of IFN{gamma} single positive CD4+ lymphocytes and IL4 single positive CD4+ lymphocytes) in SLE patients (preserved renal function and ESRD) compared to healthy controls.

 


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Fig. 2.  Percentage of IFN{gamma} (A) and IL4 (B) single positive CD4+ lymphocytes in SLE patients (preserved renal function and ESRD) compared to healthy controls. Dots indicate the percentage of Th1 or Th2 cells among all CD4+T cells (whole-blood assay).

 

SLE patients with ESRD show a Th1/Th2 ratio that no longer differs from healthy controls
In SLE patients with ESRD, there is a shift in the Th1/Th2 ratio in favour of Th1 cells when compared to SLE patients with preserved renal function (Th1/Th2 ratio, SLE with ESRD 12.1±2.6; SLE without ESRD 6.0±1.0; P<0.05; Figure 1Go). This results from an increased number of Th1 cells in SLE patients with end-stage renal disease (SLE with ESRD 36.8±5.0%; SLE without ESRD 23.4±3.6%; P<0.05), whereas the numbers of Th2 cells do not change significantly. The Th1/Th2 ratio of SLE patients with ESRD does not differ from that of healthy controls.

Figure 3Go shows a representative example of this shift in the Th1/Th2 balance, showing the IFN{gamma}/IL4 dot-plots of an SLE patient with preserved renal function and an SLE patient with ESRD, exhibiting a marked shift towards IFN{gamma} positive Th1 cells.



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Fig. 3.  Example illustrating the shift in the Th1/Th2 balance, showing the IFN{gamma}/IL4 dot-plots of an SLE patient with preserved renal function (left dot-plot) and of an SLE patient with ESRD; (right dot-plot) (single-cell measurement of intracellular cytokines by three-colour flow cytometry; CD4+ lymphocyte gate). Right lower quadrant: IFN{gamma}+ T lymphocytes (Th1 cells), left upper quadrant: IL4+ T lymphocytes (Th2 cells).

 
The shift in T-helper cell differentiation in favour of Th2 cells in SLE patients with preserved renal function and its normalization in SLE patients with ESRD was confirmed when using polyclonally stimulated Ficoll-isolated PBMC (data not shown).

SLE patients with preserved renal function and those with end-stage renal disease did not differ in numbers of IFN{gamma}+IL4+ double-positive Th cells (‘Th0 cells’; SLE without ESRD 2.8±1.9%; SLE with ESRD 4.7±4.7) and of IFN{gamma}IL4 double-negative Th cells (SLE without ESRD 64.8±18.0%; SLE with ESRD 53.4±19.4).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The balance between two functionally different types of T-lymphocyte populations, characterized by their specific cytokine profiles, is reported to be of considerable significance in many human and animal diseases. Th1 cells participate in delayed type hypersensitivity reactions and cytotoxicity responses and, in addition to other cytokines, predominantly produce IFN{gamma}. Th2 cells, in addition to other cytokines, predominantly produce IL4. Their main function is to provide help for B cells to differentiate into specific immunoglobulin-producing cells.

Systemic lupus erythematosus is a humoral Th2-mediated autoimmune disease [57]. We were able to confirm that SLE patients, prior to the onset of ESRD, exhibit a shift in the Th1/Th2 balance in favour of Th2 cells.

Clinically, patients with SLE usually lose their disease activity once ESRD occurs [1], as reflected in our study by the less frequent need for immunosuppressive medication other than maintenance therapy (low-dose corticosteroids) and by a tendency towards a lower serological activity. In contrast, patients with Wegener's granulomatosis, representing a Th1-driven disease, suffer more frequently from ongoing or relapsing autoimmune activity despite the need for renal replacement therapy [11]. Thus, our hypothesis was that ESRD differentially influences Th1- and Th2-driven diseases [8].

In general, patients with ESRD show a state of immunodeficiency clinically characterized by a high risk for infectious complications and by an impaired response to vaccinations [12].

The pathogenesis of the immunodeficiency in patients with ESRD involves both uraemia per se and the dialysis treatment procedure. These factors influence the function of antigen-presenting cells (APC) such as monocytes. On the one hand, APC have an impaired capacity to present co-stimulatory signals for T-cell activation [13], resulting in a decreased T-cell proliferation. On the other hand, APC produce enhanced levels of pro-inflammatory monokines such as TNF{alpha}, IL1, IL6 or IL12 [14].

A pattern of cytokines characterized by a high concentration of IL2, IL12 or IFN{gamma} and a low concentration of IL4 and IL10 induces a Th1 pattern of T-helper cell development [15]. In line with increased levels of IL12, we recently showed that CD4 T-cell differentiation in haemodialysis patients is skewed in a Th1 direction [8].

Thus, the aim of the study was to analyse whether a shift in the Th1/Th2 ratio accompanies the remission of clinical and serological activity of SLE after the onset of ESRD. Therefore we first polyclonally stimulated Ficoll-isolated PBMC in a subgroup of 16 SLE patients and of seven healthy controls. We then established a whole-blood analysis of T-cell subpopulations, which is a less time-consuming assay for the discrimination of Th1 and Th2 T-helper cell subsets.

In both assay systems, SLE patients with preserved kidney function showed a shift in T-helper cell differentiation with a relative predominance of Th2 cells. After the onset of ESRD this difference levels out, which results in a Th1/Th2 balance similar to that seen in healthy controls.

Since we could show that HD patients have an increased synthesis of IL12 [8], it is conceivable that the uraemic immunodeficiency similarly induces an enhanced synthesis of IL12 in SLE patients during the progression towards ESRD. This enhanced synthesis of IL12 induces a shift in T-helper cell development in favour of Th1 cells and thereby suppresses humoral autoimmune activity, resulting in a remission of the clinical activity of SLE. In support of this concept, the administration of DNA plasmids encoding IL12 in MRL/MP-lpr/lpr mice, which are prone to a lupus-like autoimmune disease, was recently shown to skew the cytokine balance towards Th1 and to have a beneficial impact on immunological and clinical disease manifestations [16].

In patients with ESRD, Th2-mediated immune responses are additionally suppressed because of a co-stimulatory defect that affects both Th1 and Th2 cells. This might cause a further decrease in the activity of humoral Th2-mediated autoimmune diseases such as SLE and may additionally contribute to the remission of SLE (‘burn out of SLE’).

It may be argued that the lesser use of immunosuppressive medication in SLE patients with ESRD might have influenced the data presented. However, no patient used any immunosuppressive agent that selectively influences the Th1/Th2 ratio. In addition, a recent study by Horwitz et al. [4], which included only patients with a recent onset of SLE not yet under immunosuppressive medication, showed that patients suffering from SLE with clinical activity have a biased T-helper cell development toward Th2 cells, very similar to the bias seen in our SLE patients. Therefore we suggest that the biased Th1/Th2 ratio in SLE patients cannot exclusively be explained by the immunosuppressive treatment used.



   Acknowledgments
 
We thank Andrea Müller, Silke Maas-Omlor, and Candida Guckelmus for excellent technical assistance. We would further like to thank Dr Hümpfner (Saarbrücken, Germany), Dr Hartmann (Saarbrücken, Germany), and Dr Krämer (Pirmasens, Germany) for recruitment of patients.



   Notes
 
Correspondence and offprint requests to: Hans Köhler, MD, Medical Department, Nephrology, Kirrberger Straße, University Homburg, D-66421 Homburg, Germany. Email: inhkoe{at}uniklinik\|[hyphen]\|saarland.de Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 2. 7.01
Accepted in revised form: 5. 6.02





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