Idiopathic membranous nephropathy (IMN) is a rare immune disease and also the most common cause of nephrotic syndrome in adults. It is caused by the accumulation of immune complexes in the basal glomerular membrane, which in turn damages the podocytes through complement-dependent processes. The major role of podocytes is to synthesize the components of glomerular filtration barrier [1] . The damaged podocytes cause a decrease in the filtration capacity of the glomerulus, resulting in marked proteinuria. The immune complexes deposited in the subepithelial glomerulus in IMN patients are mainly IgG4 [2] - [4] .

IgG4 is a subclass of antibodies that is produced in response to prolonged and recurrent antigenic stimulations [5] , or to helminthic infections [6] . These antibodies are monovalent and have low avidity. IgG4 antibodies are unable to bind the complement, but they form small non-precipitating complexes [7] [8] . The serum levels of these antibodies are often increased in subjects affected by IMN [2] [3] .

IMN is a Th2 nephritogenic immune disorder. T helper lymphocytes, which are often increased in these patients, produce significantly more IL-4 [9] , whereas B lymphocytes generate more IgG4, than healthy individuals, when cultured in presence of IL-4 [10] . IL-4 is a major Th2 cytokine that induces the isotype switching towards IgG4 and IgE, this induction is potentiated by IL-10, another Th2 cytokine [11] - [13] .

The polymorphisms of IL-4 and IL-13 are already associated to the minimal change nephrotic syndrome (MCNS), a disorder of pediatric age characterized by an increase in IgE levels [14] . Moreover, genetic variants of STAT6, which transduces the signal through the IL-4 receptor alpha (IL-4Ralpha), are also linked to MCNS’s clinical course [15] . Nevertheless, a direct correlation between IL-4 polymorphisms and IMN was never established.

Some authors indicated that the impaired immune response in IMN could be also related to the B cell compartment. In fact, there are also large infiltrates of B CD20 positive lymphocytes in tubule-interstitial compartment [16] . The role of these B cells in pathogenesis of the IMN is still unknown. B cells probably act as antigen presenting cells (APCs) rather than antibody producing cells. In fact Rituximab, an anti-CD20 antibody, is currently used in the treatment of the Membranous Nephropathy [17] [18] .

The cytokines are key factors in modulating the immune response and class switching of antibodies. Cytokines modulate the activity of T and B lymphocytes, regulate cellular activities and the response to pathogens. The polymorphisms in promoter regions of cytokine genes can influence the transcriptional and expression levels of these cytokines [19] - [23] .

Many research groups have established the link between these polymorphisms and various pathologies. For instance, the variant -572C of IL-6 gene and the polymorphism -308A of TNF-α and TNF d2 allele were associated to IMN in different ethnic groups [24] [25] . Moreover, it is demonstrated that TNF-α-308A can also cause Non Idiopathic Membranous Nephropathy [26] .

In this study we evaluated the association between polymorphisms in cytokine genes and IMN. We also evaluated the amount of Th1/Th2 cytokines in the serum and the urine collected before the therapeutic treatment. Finally, on the basis of the ratio of CD4/CD8 T lymphocytes, we analyzed whether there were differences in the frequencies of polymorphisms between patients and control group.

In this study, we revealed the association between polymorphisms in IL-4 (T-590C, T-33C) and in TNF-α (G-308A) and the development of IMN syndrome. In addition, in a subgroup of patients with increased T-helper lymphocytes, an association is shown between IL-10 polymorphisms G-1082A and C-819T and the disease. Furthermore, we showed that the cytokine levels of IL-4 in serum and IL-5 and IL-10 in urine are often increased in IMN patients.

These data are in agreement with the hypothesis that IMN is a Th2 type immune-mediated pathology. Some authors already described that T-helper lymphocytes of patients affected by IMN have increased production of IL-4 [9] and B lymphocytes, stimulated with IL-4, generate more IgG4 compared to healthy individuals [10] . In the latter study, the authors showed the increased expression of IL-10 and IL-13 in PBMCs, as well as an increase in CD4/CD8 ratio in patients.

The increased CD4/CD8 ratio in IMN represents the altered immune response associated to the pathogenesis of the disease [10] , [28] - [31] . We found that CD4/CD8 ratio was greater than two only in twenty four patients out of forty five. Nevertheless, in this subgroup, we observed the presence of two predominant polymorphisms of IL-10. IL-10 is one of the major Th2 cytokines, which acts on B lymphocytes stimulating the production of antibodies. In particular, this effect is further enhanced on B lymphocytes that are already IgG4 committed. In fact, in PBMCs stimulated with anti CD40 and IL-4, the addition of IL-10 increases IL-4 induced γ4 transcription and IgG4 production [11] - [13] . The most frequent allele of IL-10 in group of patients with CD4/CD8 ratio more than two was -1082G, which is associated to the increased production of IL-10 [20] [32] , in fact in this subgroup of patients the levels of IL-10, in the urine, were higher compared to patients with CD4/CD8 ratio less than two.

Therefore, in the group of patients with increased CD4/CD8 ratio, IL-10 can be related to the pathogenesis of IMN and it might represent a new insight in the pathogenesis of this disease, as well as its role has been shown in other kidney diseases [33] .

Moreover, IL-10 is a strong anti-inflammatory cytokine, which down regulates the production of TNF-α and the activity of APCs [34] - [36] . IL-10, as for TNF-α, is not only produced by Th2 lymphocytes but also by mo-

nocytes and macrophages. It has been observed that inflammatory cells recruited to glomerulus and the visceral epithelial cells produce TNF-α in IMN [37] . In a similar way, IL-10 can also be produced in loco by the inflammatory cells recruited to the damaged glomerulus. Therefore, the presence of IL-10 in urine, but not in se- rum, can be partly explained as an effect of inflammatory response triggered by the presence of TNF-α. In agree- ment with this observation some authors described the presence of IL-10 in glomerular lesions, in patients affected with glomerulonephritis [38] .

Likewise, IL-4 and IL-5 are both produced by Th2 lymphocytes. IL-4 is up regulated by peripheral T-helper cells in IMN [9] , demonstrating its’ systemic origin rather than produced in loco after glomerular damage. However in some cases, it has been demonstrated that there are increased mRNA levels of IL-4 and IL-5 in renal biopsies from MN patients, giving an evidence of local origin of these cytokines at the site of inflammation [39] . So, our results for the presence of IL-4 in serum are in agreement with Masutani et al. [9] , while the presence of IL-5 in urine can be related to the immunological phenomena in glomerulus, as partly stated by Ifuku et al. [39] Nevertheless, these results confirm an injurious immune response due to T lymphocytes dysregulation in IMN pathogenesis.

The association between polymorphisms G-308A in TNF-α and IMN has already been described [25] [26] . Bantis C et al. [26] showed that the allele −308A was more frequent in patients affected by membranous glomerulonephritis. Likewise, the genotype distribution for genotypes GG, GA, and AA were also different. Similar results were shown by Tibaudin et al. [25] in IMN, where frequency of -308A allele was increased in patients. Nevertheless they did not suggest any significant and independent influence of different genotypes on diseases’ progression. The increased levels of TNF-α in urine [40] - [42] and its expression in kidney cells [37] [40] - [42] of IMN affected patients, suggest a role of TNF-α in this syndrome. Hence, the presence of TNF-α together with IL-10 in urine emphasizes the significance of inflammatory processes and the role of TNF-α in the pathogenesis of IMN.

This role is more evident considering that the allele -308A of TNF-α is associated with a high expression of this cytokine [22] - [23] . TNF-α plays a role in the pathogenesis by contributing to the alterations of glomerular filtration barrier [43] . Besides, -308A allele of TNF-α is in linkage with HLA-B8/DR3 haplotype [44] , which is also associated to the development of IMN [45] . Recent studies have further shown that TNF-α polymorphisms increase susceptibility to develop other forms of kidney disease [46] .

The relevance of IL-4 (which is not in linkage disequilibrium with the MHC complex genes) and other Th2 cytokines is already described in the context of Minimal Change Nephrotic Syndrome, where the polymorphism T-590C of IL-4 [14] were more frequent in patients. Additionally, in some cases, the polymorphism T-590C of IL-4 is linked to the up regulation of this cytokine [19] . The potential role of this “high producer” genotype is also described in other Th2 mediated pathologies like atopic dermatitis, allergies and asthma. For example, our group described the association of T-590C polymorphism with asthma in the Italian population [27] and Kawashima et al. associated the same polymorphism with atopic dermatitis in the Japanese population [19] .

Furthermore, we found an increase in two polymorphisms of IL-4 in our cohort of patients, namely T-590C and T-33C, moreover, we showed that T-590 allele is also associated with a proteinuria increment.

Moreover, we observed increased levels of IL-4 in serum of patients with IMN. This observation was in agreement with Masutani et al. [9] who described an increase of Th2 lymphocytes in peripheral blood of IMN patients. The key role of IL-4 might be the induction of isotype switching towards IgG4 [10] , which is the main antibody subtype to accumulate in glomerular epithelium, causing the alterations and pathogenesis of this disease. The relevance of IL-4 has also been shown in other nephropathies. For example, Acharya B et al. described the association between minimal change nephrotic syndrome (MCNS) and C-590T polymorphism of IL-4 [14] . MCNS is the most common nephrotic syndrome in children and it is associated with proteinuria, hypogammaglobinemia, hypercholesterolemia and increased levels of IgE in serum. MCNS is also characterized by an aberrant immune response of T lymphocytes. Furthermore, factors derived from T cells are responsible for the alterations at glomerular barrier observed in this syndrome [47] - [50] . So, the same mechanisms can also be considered in the pathogenesis of IMN.

However, the association between the allele T-590 and the production of IL-4 remains questionable.

Li et al. have recently confirmed, through a study of meta-analysis, that the genotype -590TT is associated with an increase in the risk of developing allergic rhinitis. However, they have not shown a correlation between the serum level of IL-4 and allergic rhinitis [51] . While, Amirzargar et al. have shown that T-590 allele is associated with increased levels of IgE in vivo, as well as to the predisposition to asthma development [52] . The same correlation was observed in the cohort of asthmatic subjects by Smolnikova et al. In addition, here, the author shows that the T-590 allele of IL-4 is responsible for an increase in serum levels of IL-4 [53] , confirming what we have observed in our group of patients.

In conclusion, our results suggest that polymorphisms in cytokine genes, mainly IL-4, IL-10 and TNF-α can be associated to IMN, suggesting that this pathology can be considered as a complex disease and that multiple mechanisms might be involved in its pathogenesis. Moreover, to our knowledge, this is the first study to associate IL-4 polymorphisms in Italian population to the Idiopathic Membranous Nephropathy.

We are grateful to the unit of Nephrology of Ospedali Riuniti of Bergamo for their contribution to this work.

MauroGiacomelli,11,RajeshKumar,11,GiacomoTampella,11,SimonaCeffa,MarioBontempelli, (2015) IL-4, IL-10 and TNF-α Polymorphisms in Idiopathic Membranous Nephropathy (IMN). Open Journal of Immunology,05,233-243. doi: 10.4236/oji.2015.55019