Cytokine balance in kidney tissue from lupus nephritis patients

W.-S. Uhm1,4,*, K. Na1,*, G.-W. Song3, S.-S. Jung1, T. Lee2, M.-H. Park3 and D.-H. Yoo1,

1 Hospital for Rheumatic Diseases,
2 Department of Urology, College of Medicine, Hanyang University and
3 Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea
4 Present address: Division of Rheumatology, Department of Internal Medicine, Soonchunhyang University Boocheon Hospital, Boocheon, Korea


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To identify the balance of Th1/Th2 cytokine expression in the kidney and evaluate the difference in cytokine balance between patients with lupus nephritis WHO classes IV and V.

Methods. The expression of the CD40 molecule on cultured human mesangial cells was assessed by flow cytometry after stimulation with interferon {gamma} (IFN-{gamma}) or other cytokines. Frozen sections of kidney tissue from 10 patients with lupus nephritis and two non-SLE patients (with minimal-change disease) were stained with monoclonal antibodies for interleukin (IL)-4, IL-10, IL-12, IFN-{gamma}, CD4, CD8, CD40, CD68 and CD40L.

Results. CD40 expression of cultured mesangial cells was up-regulated by IFN-{gamma}, but was not down-regulated in the presence of the Th2 cytokines IL-4 and IL-10. In the glomeruli, CD40 expression and the ratios of IFN-{gamma}-/IL-10-, IL-12-/IL-4- and (IFN-{gamma}+IL-12)/(IL-4+IL-10)-positive cells were significantly higher in class IV than in class V lupus nephritis (P < 0.05). Also CD40, IFN-{gamma} and the activity index derived from the renal biopsy were closely correlated.

Conclusion. IFN-{gamma} may contribute to the pathogenesis of proliferative glomerulonephritis by the up-regulation of CD40 and the activation of the cellular immune response in human lupus.

KEY WORDS: Systemic lupus erythematosus, Cytokine, Interferon-{gamma}, Lupus nephritis, CD40.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The balance between Th1 and Th2 cytokines plays an important role in the control of the immune response. Although systemic lupus erythematosus (SLE) is generally considered to be a prototype of dominant Th2 cytokine disease, recent reports of work in animal models and human lupus have stressed the role of Th1 cytokines, especially interferon {gamma} (IFN-{gamma}), in the pathogenesis [16]. Lupus nephritis may vary in pathogenesis, especially between WHO class IV (diffuse proliferative glomerulonephritis) and class V (membranous nephropathy).

IFN-{gamma} up-regulates CD40 expression [7], and the higher expression of CD40 in class III/IV lupus nephritis [8] suggests a direct relationship between level of CD40 expression and the Th1/Th2 cytokine balance in the renal microenvironment. We investigated whether IFN-{gamma} expression is dominant over that of Th2 cytokines, such as interleukin (IL)-4 and IL-10, in the kidney tissue of lupus patients, and whether the expression of the CD40 molecule represents the level of IFN-{gamma} in kidney tissue even in the presence of Th2 cytokines.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Regulation of mesangial cell CD40 expression by cytokines
Mesangial cells were isolated and cultured from nephrectomy specimens. Cells were stimulated for 48 h with IFN-{gamma}, IL-4 or IL-10, alone or in combination. Expression of CD40 was evaluated by flow cytometry after staining with anti-CD40 antibody, and then FITC (fluorescein isothiocyanate)-conjugated goat anti-mouse immunoglobulin (Ig) G (Biosource International, Camarillo, CA, USA). All reactions were performed at 4°C and washed three times with phosphate-buffered saline (PBS). Stained cells were analysed with a FACScan® (Becton Dickinson, San Jose, CA, USA) using CellQuest 3.0 (Becton Dickinson).

SLE patients and controls for immunohistochemistry
Ten lupus nephritis patients fulfilling the American College of Rheumatology (ACR) criteria for SLE and two non-SLE patients (minimal-change disease) were selected for this study. The SLE patients comprised two class II, four class IV and four class V patients. Profiles, including activity index, chronicity index, glomerular or tubulointerstitial immune deposits, were obtained from medical records and pathological specimens.

Monoclonal antibodies and cytokines
The following mouse anti-human monoclonal antibodies were used: CD4, CD8, CD40L and CD68 (Pharmingen, San Diego, CA, USA); CD40, CD54, CD106, IL-4, IL-12, IFN-{gamma} (R&D Systems, Minneapolis, MN); IL-10 (Biosource International); isotype control IgG1 (Pharmingen); and IgG2a and IgG2b (R&D Systems). IL-1ß, IL-2, IL-4, IL-6, IL-10, TNF-{alpha}, transforming growth factor ß (TGF-ß) and IFN-{gamma} were purchased from R&D Systems.

Immunohistochemistry
Biopsy specimens were snap-frozen in OCT compound (Miles, Elkhart, ID, USA) and fixed with cold acetone. Immunohistochemistry of frozen sections was performed with an LSAB kit (Dako, Carpinteria, CA, USA). Sections were incubated in 0.3% hydrogen peroxidase for 15 min, incubated with normal goat serum for 10 min and then with mouse monoclonal antibodies against IL-4, IL-10, IL-12, IFN-{gamma}, CD4, CD8, CD40, CD40L and CD68 in buffer containing 0.4% bovine serum albumin for 60 min. The secondary antibody, biotin-labelled goat anti-mouse IgG, was incubated with alkaline phosphatase-labelled streptavidin for 30 min. All steps were performed at room temperature and were followed by washing in PBS. Staining was completed after 10 min of incubation with freshly prepared 3,3'-diaminobenzidine in Tris-buffered saline, pH 7.6. The samples were finally counterstained in Mayer's haematoxylin (Sigma, St Louis, MO, USA). A pathologist analysed the stained specimens. Glomerular staining was assessed by calculating the overall percentage of cells stained with specific monoclonal antibodies, including infiltrated and renal resident cells in each glomerulus.

Statistical analysis
Differences in cytokine expression between class IV and class V lupus nephritis were tested with the Mann–Whitney test. The correlations between the frequency of CD40 expression, the frequency of positivity for IFN-{gamma} and the activity index and chronicity indexes derived from biopsy specimens were analysed. The four parameters were analysed for association by the use of Spearman's coefficient, using SPSS 9.0 for Windows (SPSS, Chicago, IL, USA).


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
CD40 expression was increased by IFN-{gamma} in cultured human mesangial cells
CD40 was expressed on cultured mesangial cells at a low level; it was up-regulated by IFN-{gamma} but not by the other cytokines tested, and INF-{gamma}-mediated CD40 expression was not down-regulated in the presence of IL-4 and IL-10 (Fig. 1Go).



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 1. Regulation of CD40 on human mesangial cells by IFN-{gamma} and Th2 cytokines. Human mesangial cells were stimulated with IFN-{gamma} (100 IU/ml), IL-10 (10 ng/ml), IL-4 (10 ng/ml) or combinations of these cytokines for 48 h. Mean fluorescence intensity of CD40 is indicated by filled histograms. The negative control is shown as the open histogram.

 

Immunohistochemistry of human kidney sections
CD4+ T lymphocytes were the dominant cells infiltrating the glomeruli of class IV patients (CD4 cells, 31.3±5.7%; CD8 cells, 17.8±4.1%; CD68 cells, 15.5±7.5%). CD40L expression was increased in class IV compared with class V (8.5±4.5 vs 0.7±0.7%, P=0.02). However, a few inflammatory cells were present in the glomeruli and interstitium of class V lesions. Inflammatory cells and the expression of cytokines were rarely found in the glomeruli of lupus nephritis class II patients (n=2), and in lesions of non-lupus (minimal change) patients (n=2).

When we counted the percentage of cells stained with anti-cytokine antibodies, irrespective of positive cell type, in the glomeruli in class IV vs V lesions, IFN-{gamma} was positive in 27.2±4.8 (mean±S.E.) vs 0.3±0.3%, IL-12 in 14.5±2.9% vs 1.0±0.6%, IL-4 in 11.0±0.7% vs 1.3±0.7%, and IL-10 in 12.2±2.6 vs 1.0±0.6%. The mean ratio of IFN-{gamma}-positive to IL-10-positive cells was 2.2±0.6 vs 0.5±0.5, the mean ratio of IL-12-positive to IL-4-positive cells was 1.2±0.2 vs 0.3±0.3, and the mean ratio of (IFN-{gamma}+IL-12)-positive cells to (IL-4+IL-10)-positive cells was 1.6±0.4 vs 0.3±0.0 in class IV vs V lupus nephritis (Table 1Go).


View this table:
[in this window]
[in a new window]
 
TABLE 1. Cytokine profiles in the glomeruli of the patients studied

 

Relationship between activity index, level of CD40 expression and IFN-{gamma} staining
The mean percentage of CD40 expression in the glomeruli of class IV lupus nephritis patients was 32.5%, whereas that of class V was 1.25% (P=0.02). The mean activity index of diffuse proliferative lupus nephritis was 13.5 and that of membranous nephritis was 3.25. There were close correlations between the levels of CD40 expression and IFN-{gamma} (r=0.798, P=0.006) and between IFN-{gamma} and activity index (r=0.829, P=0.003).


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
There is accumulating evidence for the role of IFN-{gamma} in the pathogenesis of lupus nephritis. (i) High levels of IFN-{gamma}, particularly in the late stage of the disease, in lupus-prone animal models [9] and increased expression of IL-12 in mononuclear cells and tubular epithelial cells in MRLlpr mice have been reported [10]. (ii) NZB x NZW lupus-prone mice treated with IFN-{gamma} show accelerated development of nephritis [1]. (iii) Treatment with IFN-{gamma}-specific monoclonal antibody or soluble IFN-{gamma} receptor (R) causes a significant delay in disease progression [2]. (iv) Mice with knockouts for IFN-{gamma} or IFN-{gamma} R show a significant reduction of renal pathology and prolonged survival [3, 4]. (v) IFN-{gamma} mediates crescent formation and cell-mediated immune injury [14]. (vi) Administration of IFN-{gamma} exacerbates SLE or develop SLE in RA patients [11]. (vii) Polymorphisms of IFN-{gamma} R1 and 2 are associated with lupus in some patients [12].

IFN-{gamma} is a typical Th1 cytokine; it enhances cell-mediated immunity and may accentuate lupus nephritis by augmentation of B-cell activity and the secretion of antibodies, leading finally to immune complex-mediated tissue injury [13]. In addition to its systemic effect, IFN-{gamma} may play a role in the perpetuation of local inflammatory processes in the kidney by the activation of monocytes, macrophages or renal resident cells.

In human lupus, Akahoshi et al. [5] observed skewing towards Th1 predominance in proliferative glomerulonephritis when they looked at the intracellular IFN-{gamma}/IL-4 ratio of peripheral CD4+ T cells after stimulation with phorbol-12-myristate 13-acetate (PMA) and ionomycin. They also observed higher expression of IFN-{gamma} in diffuse proliferative glomerulonephritis, especially in the interstitium rather than in the glomeruli, by staining paraffin-embedded kidney sections with polyclonal antibodies against IFN-{gamma} and IL-4 [6]. The results of our investigation indicated that the Th1/Th2 cytokine balance in glomeruli of the diffuse proliferative lupus nephritis showed a shift towards IFN-{gamma} and away from Th2 cytokines. Although the Th1/Th2 ratio used in this study does not directly reflect the entire cytokine balance in vivo, and we did not examine the peripheral blood cytokine profile from lymphocytes at the same time, it seems evident that the disruption of cytokine homeostasis towards Th1 cytokines, especially IFN-{gamma}, participates in the pathogenesis of diffuse proliferative lupus nephritis. There must be a significant difference in pathogenesis between the two classes of nephritis, at least in terms of the cytokine environment, though too few cells stained positively with monoclonal antibodies may suggest Th1/Th2 cytokine balance seemed to be less prominent in membranous nephropathy, due to rarity of positively stained cells with monoclonal antibodies for cytokines, than in proliferative glomerulonephritis.

CD40 expression was up-regulated in class III and class IV lupus nephritis but not in class V [8], and IFN-{gamma} up-regulated CD40 expression in vitro in a variety of cells [7]. According to our in vitro observation with mesangial cells, IFN-{gamma} seems to be a major cytokine involved in the up-regulation of CD40 expression in proliferative glomerulonephritis, even in the presence of the anti-inflammatory cytokines IL-4 and IL-10. We found a close relationship between activity index, the level of CD40 expression and the frequency of IFN-{gamma} staining in the glomeruli, and the high level of expression of CD40 in the kidney indirectly represents the high level of IFN-{gamma} in the renal microenvironment. In addition, IFN-{gamma} up-regulated CD54 and CD106, which are known to recruit inflammatory cells in glomerulonephritis associated with SLE [14]. In vitro studies have demonstrated that ligation of CD40 up-regulates the intercellular adhesion molecule (CD54) on endothelial cells and fibroblasts [15]. In the present study, we observed that proinflammatory cytokines, such as IFN-{gamma} and TNF-{alpha}, up-regulated CD54 and CD106 expression on cultured human mesangial cells even in the presence of IL-4 or IL-10 (data not shown).

In an animal model, the exacerbation of crescentic glomerulonephritis was mediated by IFN-{gamma} and IL-12 [16]. CD40–CD40L interaction induces or enhances local inflammatory processes by the up-regulation of adhesion molecules or the production of proinflammatory cytokines, including IL-12 [15] in the presence of prolonged expression of CD40L and of higher levels of sCD40L in lupus [17]. The high level of expression of IL-12 in glomeruli in class IV nephritis could result from a higher level of IFN-{gamma} or increased CD40–CD40L interaction (membrane bound or soluble form CD40L). It is possible that IFN-{gamma} from mononuclear cells in the kidney accentuates interaction between infiltrated cells and renal resident cells by means of up-regulation of CD40, CD40L, HLA class II molecules and other adhesion molecules, and IFN-{gamma} may augment the cellular immune response to weakly antigenic self-molecules that might not otherwise trigger T-cell activation [13].

Taken together, our results indicate that the Th1/Th2 balance among SLE patients with diffuse proliferative lupus nephritis reveals a shift towards the production of Th1 cytokines, especially IFN-{gamma}, although Th2 cytokines should not be considered as without effect in this disease [11].


    Acknowledgments
 
We thank Dr Mary K. Crow for fruitful discussion and critical review of this manuscript.


    Notes
 
* These two authors contributed equally to this manuscript Back

Correspondence to: D.-H. Yoo, Hospital for Rheumatic Diseases, Hanyang University, Seoul 133-792, Korea. E-mail: dhyoo{at}hanyang.ac.kr Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Jacob CO, van der Meide PH, McDevitt HO. In vivo treatment of (NZBxNZW)F1 lupus-like nephritis with monoclonal antibody to gamma interferon. J Exp Med 1987;166:798–803.[Abstract]
  2. Ozmen L, Roman D, Fountoulakis M, Schmid G, Ryffel B, Garotta G. Experimental therapy of systemic lupus erythematosus: the treatment of NZB/W mice with mouse soluble interferon-gamma receptor inhibits the onset of glomerulonephritis. Eur J Immunol 1995;25:6–12.[ISI][Medline]
  3. Balomenos D, Rumold R, Theofilopoulos AN. Interferon-gamma is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice. J Clin Invest 1998;101:364–71.[Abstract/Free Full Text]
  4. Schwarting A, Wada T, Kinoshita K, Tesch G, Kelley VR. IFN-gamma receptor signaling is essential for the initiation, acceleration, and destruction of autoimmune kidney disease in MRL-Fas(Ipr)mice. J Immunol 1998;161:494–503.[Abstract/Free Full Text]
  5. Akahoshi M, Nakashima H, Tanaka Y et al. Th1/Th2 balance of peripheral T helper cells in systemic lupus erythematosus. Arthritis Rheum 1999;42:1644–8.[CrossRef][ISI][Medline]
  6. Masutani K, Akahoshi M, Tsuruya K et al. Predominance of Th1 immune response in diffuse proliferative lupus nephritis. Arthritis Rheum 2001;44:2097–106.[CrossRef][ISI][Medline]
  7. Banchereau J, Bazan F, Blanchard D et al. The CD40 antigen and its ligand. Annu Rev Immunol 1994;12:881–922.[CrossRef][ISI][Medline]
  8. Yellin MJ, D'Agati V, Parkinson G et al. Immunohistologic analysis of renal CD40 and CD40L expression in lupus nephritis and other glomerulonephritides. Arthritis Rheum 1997;40:124–34.[ISI][Medline]
  9. Fan X, Wuthrich RP. Upregulation of lymphoid and renal interferon gamma mRNA in autoimmune MRL-Fas(lpr) mice with lupus nephritis. Inflammation 1997;21:105–12.[ISI][Medline]
  10. Fan X, Oertli B, Wuthrich RP. Up-regulation of tubular epithelial interleukin-12 in autoimmune MRL-Fas(lpr) mice with renal injury. Kidney Int 1997;51:79–86.[ISI][Medline]
  11. Theofilopoulos AN, Koundouris S, Kono DH, Lawson BR. The role of IFN-{gamma} in systemic lupus erythematosus: a challenge to the Th1/Th2 paradigm in autoimmunity. Arthritis Research 2001;3:136–41.[CrossRef][ISI][Medline]
  12. Nakashima H, Inoue H, Akahoshi M et al. The combination of polymorphisms within interferon-gamma receptor 1 and receptor 2 associated with the risk of systemic lupus erythematosus. FEBS Lett 1999;453:187–90.[CrossRef][ISI][Medline]
  13. Kirou KA, Crow MK. new pieces to the SLE cytokine puzzle. Clin Immunol 1999;91:1–5.[CrossRef][ISI][Medline]
  14. Brady HR. Leukocyte adhesion molecules and kidney diseases. Kidney Int 1994;45:1285–300.[ISI][Medline]
  15. Grewal IS, Flavell RA. CD40 and CD154 in cell mediated immunity. Annu Rev Immunol 1998;16:111–35.[CrossRef][ISI][Medline]
  16. Kitching AR, Holdsworth SR, Tipping PG. IFN-gamma mediates crescent formation and cell-mediated immune injury in murine glomerulonephritis. J Am Soc Nephrol 1999;10:752–9.[Abstract/Free Full Text]
  17. Vakkalanka RK, Woo C, Kirou KA, Koshy M, Berger D, Crow MK. Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera. Arthritis Rheum 1999;42:871–81.[CrossRef][ISI][Medline]
Submitted 16 May 2002; Accepted 20 December 2002