Th1/Th2 predominance and proinflammatory cytokines determine the clinicopathological severity of IgA nephropathy

Chun Soo Lim1, Shouhuan Zheng1, Yon Su Kim1, Curie Ahn1, Jin Suk Han1, Suhnggwon Kim,1, Jung Sang Lee1, Dong-Wan Chae2, Ja Ryong Koo2, Roh Won Chun2 and Jung Woo Noh2

1 Department of Internal Medicine, Seoul National University College of Medicine, Seoul, and 2 Department of Internal Medicine, Hallym University College of Medicine, Chunchon, Korea



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. IgA nephropathy is one of the most common forms of primary glomerulonephritis in adults. Its pathogenesis is complex. The nature of infiltrating and proliferating cells and of cellular mediators could contribute to the progression of IgA nephropathy towards end-stage renal failure.

Methods. To evaluate this hypothesis, we attempted to quantify the magnitude of intrarenal gene expression of various cytokines (IL-1ß, TNF-{alpha}, IL-6, IL-15, IL-2, IFN-{gamma}, IL-10) and chemokines (IL-8, RANTES, MCP-1) in 48 renal core biopsy specimens, diagnosed as IgA nephropathy by immunofluorescence microscopy. Semi-quantitative reverse-transcriptase polymerase chain reaction using internal competitors was used for the quantification of gene transcripts.

Results. The expression of intrarenal gene transcripts of various cytokines and chemokines was closely interrelated, but not associated with the pathological grading system. The IFN-{gamma}/IL-10 ratio was higher in patients with renal dysfunction than in those with normal renal function (P=0.0483). Gene transcript levels of proinflammatory cytokines were related to the amount of proteinuria. In patients with severe glomerular sclerosis, the ratio of IFN-{gamma}/IL-10 gene transcripts was high (P=0.04). IL-10 gene transcript level was related to the severity of tubulointerstitial damage. The levels of gene expression of IL-10 (P=0.009), IFN-{gamma} (P=0.03), and TNF-{alpha} (P=0.005) were related to the degree of mesangial matrix expansion and the extent of intrarenal arteriolar changes correlated with the expression of the IL-8 gene transcript (r=0.43, P=0.004).

Conclusions. We propose that Th1/Th2 predominance and the level of proinflammatory cytokines could determine the pathogenetic processes and the severity of the clinical manifestations of IgA nephropathy.

Keywords: cytokine; IgA nephropathy; interferon-{gamma}; interleukin-8; interleukin-10; semi-quantitative reversetranscriptase polymerase chain reaction



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Glomerulonephritis is an important cause of end-stage renal failure, yet the pathogenetic mechanisms of most forms of glomerulonephritis are not clear. This makes prevention of progression of glomerulonephritis difficult. In both adolescents and adults, IgA nephropathy (IgAN) is the most common type of primary glomerulonephritis worldwide, especially in eastern Asia, and 30–40% of patients with the disorder progress to dialysis or renal transplantation within 10–25 years [1,2]. IgA nephropathy is known to be an immune-complex-mediated glomerulonephritis defined morphologically by the constant presence of predominant or co-dominant mesangial deposits of IgA accompanied by a variety of histopathological lesions [1]. However, the aetiology is unknown in that no consistent environmental or infectious agent has been shown to be antigenic to the extent of inducing an IgA antibody response. Until recently, no therapeutic agent has been shown to influence renal function favourably and consistently, in spite of several positive reports on corticosteroids, fish oil, and angiotensin-converting enzyme inhibitors [1].

Cytokines are polypeptide regulatory hormones produced during the activation and effector phases of innate and specific immunity. They serve to mediate and regulate immune and inflammatory responses, and have many functional roles in glomerulonephritis [3]. There is evidence for Th1/Th2 polarization of nephritogenic immune responses in various forms of glomerulonephritis [46]. In IgAN, cell proliferation, cytostatic effects, and the regulation of matrix synthesis are dependent on target tissues, and the synergistic and antagonistic effects imposed by cytokines [3,7]. However, there is no clear evidence for either Th1 or Th2 predominance in IgAN, except in crescentic IgAN in which strong Th1 predominance is observed [4,5]. Until now the pattern of renal expression of cytokines suggests both Th1 and Th2 involvement [4].

In several studies on intrarenal cytokines gene expression in IgAN, the expression of specific cytokine genes in kidney were correlated with the degree of cellular proliferation, mesangial matrix expansion, and tubulointerstitial damage [3,7]. Interleukin-6 (IL-6) in urine, was reported to have prognostic implications [8]. However, the actions of cytokines are often redundant, and cytokines have unique characteristics, pleiotropism, and influence the synthesis of other cytokines. Therefore it is mandatory to integrate the degree of expression of various cytokines in order to elucidate the working network of cytokines in IgAN. Chemokines are also known to influence the nature of inflammation via their chemotactic properties, which augment neutrophil- and monocyte-mediated injury [9]. The intrarenal expression of chemokines such as monocyte chemotactic peptide-1 (MCP-1) and IL-8 in IgAN seemed to be associated with inflammatory cell reflux, but their unequivocal roles have to be demonstrated by further studies [9].

In this study, we quantified the intrarenal mRNA expression of various cytokines and chemokines by semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), using internal competitors, in 48 patients with IgAN, and tried to elucidate the relationships between the degree of cytokine transcript expression and the clinicopathological findings. Here we demonstrate that the levels of gene expression of various cytokines and chemokines are correlated with specific pathological or clinical parameters in IgAN.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
Experiments were performed on 48 patients who were admitted to our medical centres from January 1998 to December 1998 and had biopsy-proven IgAN (mean age 32 years; range 15–72; 21 male, 27 female); all were found to have a serum creatinine concentration <3.7 mg/dl. The histopathological findings were classified according to the grading system of Lee et al. [10]. The number of patients with grade II was nine, grade III 29, grade IV eight, and grade V two. For analysis, the patients were categorized into two groups and the clinicopathological parameters of grade II and III were compared to grade IV and V. The histopathological findings were graded as follows:

glomerular sclerosis, grade 1 (<=25% of glomeruli affected), grade 2 (up to 50% of glomeruli affected), grade 3 (up to 75% of glomeruli affected), grade 4 (>75% of glomeruli affected);
tubular atrophy, interstitial fibrosis, mesangial matrix expansion, increased cellularity, and cellular infiltration, grade 1 (mild), grade 2 (moderate), grade 3 (severe);
intrarenal arteriolar changes including hyaline arteriolosclerosis, intimal thickening, grade 0 (absent), grade 1 (present).

Renal dysfunction was defined as a serum creatinine concentration >1.4 mg/dl.

Total RNA extraction and reverse transcription
We extracted total RNA from renal core biopsy tissues as described previously [11]. The concentration of RNA was measured using a spectrophotometer. One microgram of RNA was reverse transcribed into cDNA with 1 unit/µl of M-MLV reverse transcriptase (Gibco BRL, MD, USA). The reaction mixture also contained 100 pmol/l of random hexamers, 1 mmol/l each of dATP, dCTP, dTTP, and dGTP, and 1 U/µl ribonuclease inhibitor (Promega, WI, USA). The reaction mixture was incubated at 37°C for 1 h, then heated to 65°C for 10 min to denaturate the RNA-cDNA hybrid and to inactivate the reverse transcriptase.

Generation of competitive mutant templates (competitors)
Competitive templates (CT) were generated using the long primer method except glyceraldehyde phosphate dehydrogenase (GAPDH), IL-10 and IL-15 to circumvent the problem of variable amplification efficiency. Briefly, the competitors were designed so that each was 35–100 base pairs (bp) shorter than the wild type (WT) gene segment of the gene targeted for amplification. The competitors were designed to have the same 5' and 3' ends as the corresponding WT gene segment. This caused both the WT and CT to compete for gene-specific sense and antisense oligonucleotide primers in the co-amplification PCR reaction. Synthesis of the CT was achieved using an additional gene-specific oligonucleotide primer (CT primer), which consisted of the 5-sense primer linked with a second primer designed ‘x' bp downstream, where ‘x’ was the desired length of CT shortening. PCR amplification using the CT and antisense primers was performed at an annealing temperature of 50°C. The PCR product was cloned into a TA cloning vector (Invitrogen, CA, USA), and the plasmid containing the mutant template DNA insert was purified and measured on a spectrophotometer. The GAPDH, IL-10 and IL-15 competitors were generated using restriction endonuclease, HpaII, SspI, and BstBI respectively, as described previously [12]. We cloned eleven competitors including constitutively expressed housekeeping gene (GAPDH), proinflammatory (IL-1ß, TNF-{alpha}, IL-6, IL-15) and T-cell-derived (IL-2, IFN-{gamma}, IL-10) cytokine, and chemokine (IL-8, RANTES, MCP-1) genes. The primer sequences, and the lengths of target gene segments and the CT are shown in Table 1Go.


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Table 1. The sequences of sense, antisense, and competitor (CT) primers, and the lengths of target gene segments (WT) and competitors

 

Semi-quantitative PCR
The expression of gene-specific transcripts identified within human renal biopsy tissues was quantified by competitive PCR (Perkin Elmer Applied Biosystems Thermal Cycler 2400). Competitive PCR was performed as described previously [13,14]. The PCRs involved co-amplification of a known amount of plasmid containing the gene-specific CT with a constant volume (1 µl) of stock-sample-derived RT-cDNA. Other constituents of the PCR mix included 100 ng each of sense and antisense primers, 160 µM dNTP, 2 mM magnesium chloride, 0.3 U of Taq polymerase (Takara, Japan), and buffer in a reaction volume of 50 µl. The thermal cycling parameters consisted of denaturing at 94°C for 1 min, annealing at 55–60°C for 1 min, and extension at 72°C for 1 min for 35–40 cycles. The PCR products were run in 1.8–2.2% agarose gels at 80–100 V for several hours to ensure adequate separation of the WT and CT products. The ethidium-bromide-stained gels were photographed and viewed under UV light (Gel Doc 1000 and Multi-Analyst Software, Bio-Rad Laboratories, CA, USA).

Data generation
The amount of WT was determined by calculating the ratio of the staining intensities (WT/CT), and multiplying by the known concentration of CT. The calculated amount of cDNA was normalized by the amount of GAPDH, to adjust the cDNA level in stock samples. Before performing the calculation, PCRs were performed to produce standard curves of each cytokine and GAPDH with several PCR thermal cycling parameters. Serial dilutions were used to produce a known range of CT concentration. PCRs were performed with diluted CT and constant amount of WT, and the band densities were measured. Log (CT/WT) band intensity values were plotted on the y-axis and the log of the various dilutions of the CT were plotted on the x-axis [15]. We chose the most appropriate PCR thermal cycling parameters and concentration of CT to be used in competitive PCR for each cytokine and GAPDH on the basis of the relationship linearity.

Statistics
The histopathological and clinical findings of patients involved in the study were analysed and compared according to their allocated grades using the Wilcoxon rank sum test or Student's t-test as appropriate. Relationships between the clinicopathological findings and cytokine gene expression were analysed using the Wilcoxon rank sum test. Correlations between cytokine gene expression and the clinicopathological characteristics were evaluated by Spearman's test. A P value <0.05 was considered to be statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Comparisons of the clinicopathological findings with the grading system
In the higher grade groups (grade IV and V), the serum creatinine concentrations and the amount of daily proteinuria at the time of biopsy were higher than those of the lower grade groups (grade II and III). In the histopathological findings, the degree of glomerular sclerosis, arteriolar changes, tubular atrophy, and interstitial fibrosis were more severe in the higher grade groups, but the degree of glomerular cellularity and the inflammatory cell infiltration were not significantly different (Table 2Go).


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Table 2. Comparisons of the clinicopathological findings with the grading system*

 

Expression of cytokine gene transcripts according to the grading system
The levels of cytokine and chemokine gene transcripts were not significantly different between the lower and higher grade groups (Table 3Go).


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Table 3. Expression of cytokine gene transcripts according to the grading system*

 

Comparisons of cytokine and chemokine gene expression with the clinicopathological parameters
The ratio of Th1/Th2 (IFN-{gamma}/IL-10) cytokine gene transcripts was higher in patients with renal dysfunction than that in normal patients. In patients with daily proteinuria >=1 g, the levels of proinflammatory cytokine (TNF-{alpha}, IL-1ß) gene transcripts were higher than those of patients with lower amounts of proteinuria. In patients with severe glomerular sclerosis, the ratio of IFN-{gamma}/IL-10 gene transcripts was high. The expression of the IL-10 gene transcript was high in renal tissues with severe pathological lesions in the tubulointerstitium. The degree of mesangial matrix expansion was related to the expression of TNF-{alpha}, IFN-{gamma} and IL-10 (Table 4Go).


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Table 4. Comparisons of cytokine and chemokine gene expression with the clinicopathological parameters*

 

Correlations between the expression of cytokine and chemokine gene transcripts
The levels of the gene expression of various cytokines and chemokines were closely interrelated. The gene expression of proinflammatory cytokines (IL-1ß, TNF-{alpha}, IL-6, IL-15) correlated with the gene expression of Th1 and Th2 cytokines and chemokines. The gene expression of the Th1 cytokines (IL-2 and IFN-{gamma}) correlated with chemokine (RANTES and IL-8) gene expression. The expression of the Th2 cytokine (IL-10) gene correlated with the expression of the IL-8 gene. As expected, the expression of the Th1 cytokine gene did not correlate with the expression of the Th2 cytokine gene (Table 5Go).


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Table 5. Correlations between the expression of cytokine and chemokine gen transcripts

 

Correlations of cytokine and chemokine gene expression with clinicopathological parameters
In Spearman's correlation tests, the degree of glomerular sclerosis and mesangial matrix expansion seemed to be correlated with the expression of proinflammatory (IL-15 and TNF-{alpha}) and Th1 (IL-2 and IFN-{gamma}) cytokine genes, but these correlations were not statistically significant. The degree of intrarenal arteriolar changes was correlated with the expression of the IL-8 gene (r=0.427, P<0.01) (Table 6Go).


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Table 6. Correlations of cytokine and chemokine gene expression with clinicopathological parameters

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Cytokines are polypeptide regulatory molecules that constitute a cellular signalling system and whose anatomical location is intermediate between the endocrine hormones and the neurotransmitters. They act in spaces between cells and within the confines of tissues, and if they overflow into the systemic circulation, they can exert toxic or pathological effects. Cytokines are bi- or multifunctional; that is, they can either stimulate or inhibit the same action, and they often influence the synthesis and actions of other cytokines [3,4]. Chemokines are a group of small cytokines that act as inflammatory mediators. In the kidney, several chemokines are present in the glomeruli or tubulointerstitium and they are considered to have active roles in renal lesions [9]. The stimuli that underlie the major forms of glomerular disease, including immunological factors in glomerulonephritis, all lead to a common pathway of cytokine release intended to protect the glomeruli by remodelling the cell–matrix interactions. When excessive or dysregulated, this remodelling can result in the pathological deposition of matrix or fibrogenesis. Therefore there has been a continued interest in the pathophysiological roles of cytokines and chemokines and in the possibility of therapeutic intervention using these molecules, for example in the case of renal injury [3].

The pathogenetic mechanisms and the causative agents have not yet been identified in IgAN. Cytokines are considered to have several roles in the pathogenesis and progression of IgAN. Proinflammatory cytokines (IL-6, TNF-{alpha}) and IFN-{gamma} induce Fc{alpha} receptor mRNA expression by human mesangial cells [16], and the interaction of IgA with Fc{alpha} receptors in mesangial cells activates transcription factor (NF{kappa}B) and induces the expression and synthesis of MCP-1, IL-8, and IFN-inducible protein 10 [17]. Moreover, IgA-containing immune complex leads to secretion of IL-1, IL-6 and platelet-activating factor [18,19]. The roles of IL-6 in IgAN have been widely investigated. It induces mesangial cell proliferation and matrix enlargement and has a positive correlation with both pathological score and prognosis [8,20,21]. Therefore it seems that the immune complex and proinflammatory and Th1 cytokines interact and lead to the perpetuation of inflammation and injury. Th2 cytokines have also been reported to be involved in the pathogenesis of glomerular injury in IgAN. The IL-4-secreting T lymphocytes in affected glomeruli seem to injure glomerular epithelium, and the responsiveness of glomerular epithelium to IL-4 may be pathologically enhanced in IgAN [22]. The expression of IL-10 was significantly upregulated in IgAN patients with marked glomerular proliferation, and upregulated in the tubulointerstitial region in IgAN patients with nephrotic-range proteinuria [23]. In spite of their well-known anti-inflammatory properties, Th2 cytokines also seem to be involved in the progressive injury of glomerulonephritis. The expression of monocyte attractant chemokine, MCP-1, was correlated with monocyte infiltrates and tubulointerstitial damage [9,24], and recently MCP-1 has been reported to have an atherogenic property [25]. It is recognized that cytokines and chemokines are involved in the pathogenesis and progression of IgAN, but their integrated roles still need to be elucidated.

In this study, the intrarenal mRNA expression of Th1 and proinflammatory cytokines were high in patients with severe glomerular lesions and poor clinical features. The gene expression of the Th2 cytokine, IL-10, was related to the pathological lesions of the tubulointerstitium. The gene expression of chemokine, IL-8, correlated with vascular lesions. The gene expression of proinflammatory cytokines were closely correlated with the gene expression of Th1 and Th2 cytokines and chemokines. The gene expression of Th1 cytokines was correlated with the gene expression of chemokines, and the gene expression of the Th2 cytokine (IL-10) with the expression of chemokine (IL-8) gene. These results imply that in IgAN, whatever the initiating stimuli, the cytokine and chemokine networks are closely interactive, and that the Th1 cytokines change the pathogenetic processes in the glomeruli, resulting in glomerular sclerosis and mesangial matrix expansion. We could theorize that the Th2 cytokines are involved in the damage of the tubulointerstitium rather than the glomeruli, and the chemokine, IL-8, induces intrarenal arteriolar damage, probably by the chemoattraction of polymorphonuclear cells. In the correlation analyses, the expression of IL-8 was positively related to the expression of the Th1 and Th2 cytokines. We are tempted to believe that any predominance of Th1 or Th2 cytokines induces vascular lesions.

This study has several limitations. We used semi-quantitative PCR to measure the expression of intrarenal cytokine and chemokine mRNAs in patients with IgAN. This procedure uses a competitive mutant template with the same sequence as the target except for the deletion of nucleotides, which allowed us to discriminate between the wild type template and the mutant template. To circumvent the problem of RNA quantification, we first quantified the amount of GAPDH cDNA, and normalized the amount of cytokine cDNA using the amount of GAPDH cDNA [11,14]. However, this could only reflect the relative amount of gene transcript rather than its absolute level. Cytokine secretion is a brief and self-limited event, and most of the cellular responses to cytokine require new mRNA and protein synthesis [3]. Quantifying the expression of mRNA might provide information about protein levels and thus, biological activity, but there is no direct or quantitative relationship between the expression of mRNA and the biological activity at the protein level. We did not perform any experiments designed to resolve the intrarenal expression of cytokines and chemokines at the protein level. In the PCR experiments, we extracted total RNA from snap-frozen renal tissues instead of using isolated glomeruli from the tubulointerstitial region. Therefore, we could not discriminate between the cellular and compartmental origins of each cytokine or chemokine. Notwithstanding these limitations, this study proved to be worthwhile because it demonstrated something of the working interrelationships of cytokine and chemokine in IgAN, in which the pathogenetic processes are diverse and the clinical course protracted.

In conclusion, the increased intrarenal gene expression of proinflammatory and Th1 cytokines was associated with glomerular lesions, whereas Th2 cytokine expression was associated with tubulointerstitial lesions, and IL-8 expression with vascular lesions in IgAN. Moreover, the gene expression of Th1 and proinflammatory cytokines was high in patients with poor clinical features. We propose that Th1/Th2 predominance and proinflammatory cytokines determine the pathogenetic process and the severity of the clinical manifestations of IgAN. The cytokines and chemokines are linked by networks and are influenced by each other. In order to derive intervention strategies implying these molecules further studies are needed, including transgenic or knockout gene experiments in animals for each specific molecule.



   Acknowledgments
 
This work was supported in part by research grants from Ministry of Health and Welfare of Korea (HMP-98-M-2-0023).



   Notes
 
Correspondence and offprint requests to: Suhnggwon Kim MD, Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Korea. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 13. 7.99
Revision received 13. 6.00.