Genetically determined interferon-{gamma} production influences the histological phenotype of lupus nephritis

K. Miyake, H. Nakashima, M. Akahoshi, Y. Inoue, S. Nagano, Y. Tanaka, K. Masutani1, H. Hirakata1, H. Gondo, T. Otsuka and M. Harada

Department of Medicine and Biosystemic Science and
1 Department of Medicine and Clinical Science, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To clarify whether the interferon-{gamma} (IFN-{gamma}) gene (IFNG) is associated with the histological phenotype of lupus nephritis.

Method. We analysed microsatellite polymorphisms located within the first intron of the IFNG gene to determine the genotypes of patients with lupus nephritis WHO class IV (n=24), patients with WHO class V (n=12) and healthy controls (n=61). We used flow cytometric detection of intracellular cytokines to identify CD4+ T cells producing IFN-{gamma}. Production of IFN-{gamma} by peripheral blood mononuclear cells after stimulation with phytohaemagglutinin was evaluated with an enzyme-linked immunosorbent assay.

Result. The frequency of the IFNG allele 114 was significantly greater in WHO class V patients than in WHO class IV patients. Furthermore, the IFNG 114 +/+ genotype was more frequent in WHO class V than in WHO class IV patients. The level of IFN-{gamma} and the percentage of IFN-{gamma}-producing CD4+ T cells were lower in individuals with genotype 114 +/+ than in individuals with genotype 114 -/-.

Conclusion. The IFN-{gamma} gene is associated with the histological phenotype in lupus nephritis.

KEY WORDS: Lupus nephritis, WHO IV and V, Interferon-{gamma}, Th1/Th2 balance, Microsatellite polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by polyclonal B-cell activation, the production of antibodies to nuclear components and systemic inflammatory injury of multiple organs [1, 2]. Renal involvement in SLE, called lupus nephritis, is one of the most serious complications and demonstrates phenotypic and histological heterogeneity. Two polar morphological forms, diffuse proliferative glomerulonephritis [World Health Organization (WHO) class IV] and membranous glomerulonephritis (MGN; WHO class V), are representative of the histologies found in lupus nephritis [3, 4].

The ratio of Th1/Th2 cells, as demonstrated in an intracellular cytokine assay, differs greatly between WHO classes IV and V. Th1 cells, which secrete interferon-{gamma} (IFN-{gamma}), are predominant in WHO class IV, while Th2 cells, secreting interleukin (IL)-4, are predominant in WHO class V [5]. Immunohistological studies of nephritis identified large numbers of infiltrating macrophages, Th1 cells, CD40+ cells and osteopontin protein in WHO class IV, but not in WHO class V. In WHO class IV kidneys, we demonstrated a correlation between the Th1/Th2 ratio and the renal histological activity index [6]. These findings suggest that macrophages and Th1 cells play a central role in WHO class IV but not in not WHO class V.

IFN-{gamma}, a cytokine secreted by Th1 cells, promotes macrophage activation and Th1 differentiation, in addition to the up-regulation of the expression of MHC class I and II molecules [710] and adhesion molecules [11]. The gene for IFN-{gamma} (IFGN) is, therefore, likely to exert an effect on the Th1 immune response.

We analysed microsatellite polymorphisms contained within the first intron of the IFNG gene, which has been mapped to chromosome 12q24.1. An association between microsatellite polymorphisms in the IFNG gene and multiple immunological diseases has been suggested [1214]. Therefore, we examined the relationship of specific IFNG alleles with IFN-{gamma} production in order to clarify the association between this gene and the histological phenotype of lupus nephritis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and controls
All patients fulfilled the American College of Rheumatology criteria for the classification of SLE [15]. All patients had undergone renal biopsy and the histological analysis of lupus nephritis was based on the WHO classification [4]; 24 patients had class IV nephritis and 12 cases had class V nephritis. Renal biopsy was done prior to treatment in most cases. The mean age and sex ratio (M:F) were 34.2 (S.D. 9.57) yr and 1:20 respectively in WHO class IV patients, 32.58 (10.09) yr and 1:11 in class V patients and 33.42 (9.32) yr and 3:2 in normal controls. The analysis was carried out in three groups: SLE patients with WHO class IV nephritis (n=24); SLE patients with class V nephritis (n=12); and randomly selected healthy controls (n=61).

Polymerase chain reaction (PCR) amplification and sequence analysis
Genomic DNA was extracted from peripheral blood of lupus nephritis patients and healthy controls after they had given informed consent. Molecular typing for IFNG microsatellite polymorphism was performed by DNA sequencing. Locus-specific amplification was done with the primers 5'-TGA TTT TAT TCT TAC AAC ACA-3' and 5'-CTT CCT GTA GGG TAT TAT TAT-3', using a high annealing temperature to enhance specificity. Genomic DNA (60–110 ng) was amplified with 10 pmol of each primer, 400 µmol/l of each dNTP, 1.5 µmol/l MgCl2, 50 µmol/l KCl, 10 µmol/l Tris–HCl (pH 8.3) and 0.5 U Taq polymerase in a Perkin-Elmer PCR cycler. Cycling conditions comprised a 3-min hot start at 96°C followed by 35 cycles of 96°C for 30 s, 50°C for 30 s and 72°C for 30 s with final extension for 7 min at 72°C in the last cycle. Purified PCR products (10–40 ng) were cycle-sequenced using the BigDye terminator system (Applied Biosystems, Foster City, CA, USA) and analysed with an ABI Prism 310 capillary sequencer (Applied Biosystems).

Phytohaemagglutinin-stimulated IFN-{gamma} and IL-4 production
Peripheral blood mononuclear cells (PBMNC; 8x105) were incubated for 72 h at 37°C in 5% CO2 in 0.2 ml RPMI 1640 medium (Nissui, Tokyo, Japan) containing 10% (v/v) foetal bovine serum with or without 5 µg/ml phytohaemagglutinin (PHA). Culture supernatant (0.3 ml) was assayed for IFN-{gamma} and IL-4 production by the use of specific cytokine enzyme-linked immunosorbent assays.

Intracellular cytokine detection
Flow cytometric determination of IFN-{gamma} and IL-4 in the cytoplasm of peripheral CD4+ T cells was performed as described previously [1618]. Briefly, aliquots (500 µl) of heparinized whole blood were first stimulated with a combination of 25 ng/ml phorbol myristate acetate and 1 µg/ml of ionomycin in the presence of 10 µg/ml of blefeldin A (Sigma, St Louis, MO, USA) then cultured for 4 h at 37°C in a humidified incubator containing 7% CO2. Activated cultures were stained with 20 µl peridinin chlorophyll protein-conjugated CD4-specific monoclonal antibody (mAb) (Becton Dickinson, San Jose, CA, USA) for 15 min at room temperature, and then treated with 2 ml fluorescein-activated cell sorter (FACS) lysing solution (Becton Dickinson). After 5 min of incubation, samples were centrifuged and combined with FACS permeabilization solution (Becton Dickinson) for 10 min at room temperature. The sample tubes were washed and incubated with mAb specific for fluorescein isothiocyanate (FITC)-conjugated IFN-{gamma} and mAb specific for phycoerythrin (PE)–conjugated IL-4 (Becton Dickinson) for 30 min at room temperature. FITC-conjugated mouse IgG2a and PE-conjugated mouse IgG1 were used as controls. After washing again, cells were resuspended in 1% paraformaldehyde and analysed by flow cytometry. Data were obtained with a FACScan flow cytometer (Becton Dickinson) and results were analysed using Cellquest software (Becton Dickinson). The percentages of cells positive for IFN-{gamma} and IL-4 were determined by FACS.

Statistical analysis
Differences in the means and standard deviations were analysed with the {chi}2 test, Welch's test and Student's paired t-test. A P value of <0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mean Th1/Th2 ratios in the 24 WHO class IV patients and 12 WHO class V patients
We studied 36 lupus nephritis patients, comprising 24 with WHO class IV and 12 with WHO class V. The mean (±S.D.) Th1/Th2 ratio in lupus nephritis patients with WHO class IV and V was 19.8±16.7 and 6.3±5.0 respectively (Fig. 1Go). The difference between WHO class IV and V was significant (P<0.005).



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FIG. 1.  Relationship between renal histological class and Th1/Th2 ratio (%) in individual patients with lupus nephritis WHO classes IV and V. Data are mean (X) and S.D. (bar). The mean ratio for WHO class V patients was significantly lower than that for WHO class IV patients. The P value was determined with Student's t-test.

 

Microsatellite polymorphisms in the IFNG gene
The analysis was carried out on three groups: 24 patients with lupus nephritis WHO class IV, 12 with WHO class V and 61 randomly selected healthy controls.

Ten alleles were documented at the microsatellite within the first intron of the IFNG gene, ranging from 106 to 124 bp in length. A single-nucleotide polymorphism (SNP) located at position +874 from the translation start site, T->A at the 5' end of the CA repeat region in the first intron of the human IFNG gene, has been detected previously [19, 20]. Alleles were identified according to the number of CA repeats, starting at position +875. These alleles contained a sequence ranging from nine dinucleotide repeats (the 106 bp allele) to 18 dinucleotide repeats (the 124 bp allele). Alleles were designated according to the number of amplified DNA base pairs. The allele with 13 CA repeats corresponded to the 114 bp allele, IFNG 114. We found complete linkage disequilibrium between SNPs at +874 and the 12-dinucleotide repeat (allele IFNG 112).

IFNG allele frequencies in patients with lupus nephritis and controls
IFNG 114 accounted for 16 of the 24 (67%) IFNG alleles in patients with WHO class V compared with 20 of the 48 (42%) IFNG alleles in 24 patients with WHO class IV and 55 of the 122 (45%) IFNG alleles in healthy controls (Table 1Go; Fig. 2Go). There was a significant difference in the frequency of the IFNG 114 allele between WHO class IV and WHO class V (P<0.05). There was no significant difference in the frequencies of other IFNG gene microsatellite polymorphisms between patients with WHO class IV, WHO class V and healthy controls.


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TABLE 1.  IFNG allele frequencies in patients with lupus nephritis and controls

 


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FIG. 2.  IFNG allele frequencies in patients with lupus nephritis and controls. The P value was determined by {chi}2 analysis.

 

Frequencies of IFNG genotypes in patients with lupus nephritis and healthy controls
Individual patients with WHO class IV and WHO class V and healthy controls were allocated to the genotypes +/+, +/- and -/- with respect to the IFNG 114 allele. Genotype +/+ was found in six of 12 (50%) patients with WHO class V compared with four of 24 (17%) patients with WHO class IV and 14 of 61 (23%) healthy controls (Table 2Go; Fig. 3Go). There was a significant difference in the frequency of the +/+ genotype between WHO class IV and WHO class V (P<0.05).


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TABLE 2.  Frequencies of IFNG genotypes in patients with lupus nephritis and normal controls

 


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FIG. 3.  Frequencies of IFNG 114 genotypes in patients with lupus nephritis and healthy controls. The P value was determined by {chi}2 analysis.

 

Production of IFN-{gamma} and IL-4 by PBMNC in healthy controls with different IFNG 114 genotypes
We evaluated the level of IFN-{gamma} secretion by the PBMNC after PHA stimulation in vitro. We studied 15 healthy controls, including five individuals with each genotype. After 48 h of incubation, mean IFN-{gamma} secretion from PBMNC of healthy individuals with genotypes +/+, +/- and -/- was 10 358±6066, 15 664±15 224 and 36 140±18 573 pg/ml respectively, and IFN-{gamma} secretion after 72 h of incubation was 13 776±11 192, 19 054±17 077 and 49 100± 12 161 pg/ml respectively (Fig. 4Go). There were significant differences in IFN-{gamma} secretion between individuals with genotypes +/+ and -/- (48 h, P=0.02; 72 h, P=0.007).



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FIG. 4.  Correlation between IFN-{gamma} cytokine production after 72 h of PBMNC culture in healthy controls with IFNG 114 genotypes +/+ (n=5), +/- (n=5) and -/- (n=5). Values are mean and S.E.M. The mean value for subjects with genotype +/+ was lower than that for -/- subjects (48 h, P<0.05; 72 h, P<0.005). P values were determined by Welch's test and Student's t-test.

 

Ratio of CD4+ T cells producing IFN-{gamma} to total CD4+ T cells in healthy controls with different IFNG 114 genotypes
To investigate the differences in genetic effects among IFNG 114 genotypes +/+, +/- and -/-, we measured the ratio of CD4+ T cells producing IFN-{gamma} in 27 healthy controls, comprising six with genotype +/+, 12 with genotype +/- and nine with genotype -/-. The mean values for the three genotypes were 16.66±4.57, 19.66±4.16 and 22.35±3.97% respectively (Fig. 5Go). There was a significant difference between genotypes +/+ and -/- (P<0.05).



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FIG. 5.  Percentage of CD4+ T cells producing IFN-{gamma} in individual healthy control subjects with IFNG 114 genotypes +/+, +/- and -/-. Data are mean (X) and S.D. (bar). The mean value for +/+ subjects was significantly lower than that for -/- subjects. The P value was determined by Student's t-test.

 


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The roles of Th1 and Th2 cytokines in the pathogenesis of SLE are still a matter of debate. In patients with SLE, Th2 cytokines, such as IL-4, IL-6 and IL-10, are increased [2123], whereas Th1 cytokines, including IFN-{gamma} and IL-2, are decreased [1]. Thus, SLE is considered to be a Th2-predominant disease. However, it has been reported previously that IFN-{gamma} levels in the sera of patients with SLE are significantly elevated and that there is a correlation between the severity of SLE and the amount of IFN-{gamma} secreted by stimulated PBMNC [24, 25]. These findings suggest that the Th1 and Th2 responses are both important in the pathogenesis of lupus-associated tissue injury. SLE is a disease involving a wide spectrum of cytokines.

Immune responses with the Th1 phenotype have been found in the kidneys of SLE mice. New Zealand BlackxNew Zealand White lupus mice treated with IFN-{gamma} showed accelerated development of glomerulonephritis [26]. Blocking the effect of IFN-{gamma} with anti-IFN-{gamma} antibody or soluble receptor inhibited the onset of glomerulonephritis [27, 28]. These results suggest that IFN-{gamma} plays a key regulatory role in the development of lupus nephritis.

In this study, we analysed microsatellite polymorphisms of the IFNG gene to determine the frequency of the IFNG 114 allele; the frequency of this gene was significantly greater in patients with WHO class V than in WHO class IV patients and in healthy controls. The frequency of IFNG genotype 114 +/+ in WHO class V was 0.5; this suggests that the INFG 114 allele is important in the pathogenesis of the disease.

A significant difference in the Th1/Th2 balance in peripheral blood exists between WHO class IV and V. Th1 cells are predominant in class IV but not in class V [5]. A strong correlation is also observed between the peripheral blood Th1/Th2 ratio and renal histological activity index in class IV, revealing an important role for macrophages and Th1 cells. In class V, the number of infiltrating cells was reduced, with a large percentage of CD4+ T cells producing IL-4 in the peripheral blood [6]. IL-4, a Th2 cytokine, induces the up-regulated production of IgE and IgG4 in humans [29]. In the MGN kidney, deposition of predominantly IgG4 in capillaries and basement membranes has been reported [3032]. These findings suggest that the Th1/Th2 balance in patients with WHO class V was a prominant shift to Th2. It is interesting that the same disease produces two distinct histological phenotypes with a unique Th1/Th2 balance in peripheral blood. A number of variables influence the type of response, including antigen dose and mode of delivery, the type of antigen-presenting cell, the nature of the stimulatory or costimulatory signals, and the specific cytokines present in the developmental microenvironment [33]. Although the determinants accounting for the type of immune response in lupus nephritis are unknown, our results suggest that the skewing of the immune response in each individual SLE patient towards Th1 or Th2 determines the phenotype of lupus nephritis. When the SLE patient reacts with a predominantly Th2 response, the probability of WHO V is high.

We also found that one of the two patients with WHO class V was homozygous for the IFNG 114 allele (114 +/+). We used cytokine assays of stimulated PBMNC to clarify the influences of genotype on IFN-{gamma} production and the peripheral blood Th1/Th2 balance. We obtained PBMNC from healthy volunteers of each of the IFNG 114 genotypes +/+, +/- and -/-. After stimulation with PHA, mean (±S.D.) IFN-{gamma} production was 13 776±11 192, 19 054±17 077 and 49 100±12 161 pg/ml for the three genotypes respectively (Fig. 4Go). Intracellular cytokine assays of stimulated CD4+ T cells revealed the following percentages of CD4+ T cells producing IFN-{gamma}, out of the total number of CD4+ T cells: 16.66±4.58, 19.66±4.16 and 22.35±3.97% respectively (Fig. 5Go). Both the quantity of IFN-{gamma} produced and the overall percentage of IFN-{gamma}-producing CD4+ T cells were significantly lower in individuals of genotype 114 +/+ than in 114 -/- individuals. However, IL-4 production and the overall percentages of IL-4-producing CD4+ T cells were not significantly different among the three genotypes (data not shown).

The maturation of naive CD4+ T cells to effector cells that produce lymphokines is critical for the development of the appropriate host immune response. Analysis of cells immediately after T-cell activation showed that IL-4 and IFN-{gamma} were both expressed in the early phase, and these were direct effects of T-cell receptor- and CD28-mediated signals. The cytokine signals mediated by the receptors were unnecessary for this initial cytokine expression, but were required for sustained expression [34]. IFN-{gamma} secreted by Th1 cells promotes Th1 differentiation, thereby down-regulating the reciprocal subset, Th2. Insufficient production of IFN-{gamma}, therefore, should gradually induce the development of a Th2 microenvironment and skew the Th1/Th2 balance in favour of Th2 cells. Once the immune response develops along one pathway, the response continues to become increasingly polarized in that direction. This suggests that the immune response of SLE patients bearing the IFNG 114 +/+ genotype tends to be predominantly skewed towards Th2, inducing IgG4 deposition in the capillaries and basement membrane of the glomerulus and the subsequent development of membranous glomerulonephritis. The genetic determination of IFN-{gamma} production influences the resulting histology of lupus nephritis.

Although the balance of Th1 and Th2 cells is affected by several cytokines, the loci for which are involved in the pathogenesis of lupus, the IFNG gene may be a principal genetic factor determining the histological phenotype of lupus nephritis.


    Acknowledgments
 
We thank Ms Yuko Furukawa for her skilful technical assistance. We are especially grateful to Dr Motosuke Hanada for his encouragement and helpful advice. This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare, Japan.


    Notes
 
Correspondence to: H. Nakashima, Department of Medicine and Biosystemic Science, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan, Maidashi 3-1-1 Higashi-ku, Fukuoka, 812-8582, Japan. Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 2 July 2001; Accepted 9 November 2001