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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. The expression of the CD40 molecule on cultured human mesangial cells was assessed by flow cytometry after stimulation with interferon (IFN-
) 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-
, CD4, CD8, CD40, CD68 and CD40L.
Results. CD40 expression of cultured mesangial cells was up-regulated by IFN-, 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-
-/IL-10-, IL-12-/IL-4- and (IFN-
+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-
and the activity index derived from the renal biopsy were closely correlated.
Conclusion. IFN- 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-, Lupus nephritis, CD40.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IFN- 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-
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-
in kidney tissue even in the presence of Th2 cytokines.
![]() |
Patients and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- (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-
, transforming growth factor ß (TGF-ß) and IFN-
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-, 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 MannWhitney test. The correlations between the frequency of CD40 expression, the frequency of positivity for IFN- 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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- 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-
-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-
+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 1
).
|
Relationship between activity index, level of CD40 expression and IFN- 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- (r=0.798, P=0.006) and between IFN-
and activity index (r=0.829, P=0.003).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IFN- 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-
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-/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-
in diffuse proliferative glomerulonephritis, especially in the interstitium rather than in the glomeruli, by staining paraffin-embedded kidney sections with polyclonal antibodies against IFN-
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-
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-
, 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- up-regulated CD40 expression in vitro in a variety of cells [7]. According to our in vitro observation with mesangial cells, IFN-
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-
staining in the glomeruli, and the high level of expression of CD40 in the kidney indirectly represents the high level of IFN-
in the renal microenvironment. In addition, IFN-
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-
and TNF-
, 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- and IL-12 [16]. CD40CD40L 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-
or increased CD40CD40L interaction (membrane bound or soluble form CD40L). It is possible that IFN-
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-
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-, although Th2 cytokines should not be considered as without effect in this disease [11].
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
Correspondence to: D.-H. Yoo, Hospital for Rheumatic Diseases, Hanyang University, Seoul 133-792, Korea. E-mail: dhyoo{at}hanyang.ac.kr
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|