Activated innate immunity and the involvement of CX3CR1–fractalkine in promoting hematuria in patients with IgA nephropathy

A hallmark of immunoglobulin A nephropathy (IgAN) is episodes of gross hematuria coinciding with mucosal infections that can represent the disease-triggering event. Here we performed a whole genomic screen of IgAN patients during gross hematuria to clarify the link between mucosal antigens and glomerular hematuria. Modulated genes showed a clear involvement of the intracellular interferon signaling, antigen-presenting pathway, and the immunoproteasome. The mRNA and protein level of the chemokine receptor characterizing cytotoxic effector lymphocytes, CX3CR1, was upregulated. In vitro antigenic stimulation of peripheral blood mononuclear cells from IgAN patients, healthy blood donors, and other nephropathies with microscopic hematuria showed that only in IgAN patients was CX3CR1 enhanced in a dose-dependent manner. A significantly higher amount of glomerular and urinary fractalkine, the only ligand of CX3CR1, was also found in IgAN patients with recurrent episodes of gross hematuria compared with other patients with microscopic or no hematuria. This suggests a predisposition for cytotoxic cell extravasation only in patients with recurrent gross hematuria. Thus, we found a defect in antigen handling in peripheral blood mononuclear cells of IgAN patients with a specific increase of CX3CR1. This constitutive upregulation of glomerular and urinary fractalkine suggests an involvement of the CX3CR1–fractalkine axis in the exacerbation of gross hematuria. A hallmark of immunoglobulin A nephropathy (IgAN) is episodes of gross hematuria coinciding with mucosal infections that can represent the disease-triggering event. Here we performed a whole genomic screen of IgAN patients during gross hematuria to clarify the link between mucosal antigens and glomerular hematuria. Modulated genes showed a clear involvement of the intracellular interferon signaling, antigen-presenting pathway, and the immunoproteasome. The mRNA and protein level of the chemokine receptor characterizing cytotoxic effector lymphocytes, CX3CR1, was upregulated. In vitro antigenic stimulation of peripheral blood mononuclear cells from IgAN patients, healthy blood donors, and other nephropathies with microscopic hematuria showed that only in IgAN patients was CX3CR1 enhanced in a dose-dependent manner. A significantly higher amount of glomerular and urinary fractalkine, the only ligand of CX3CR1, was also found in IgAN patients with recurrent episodes of gross hematuria compared with other patients with microscopic or no hematuria. This suggests a predisposition for cytotoxic cell extravasation only in patients with recurrent gross hematuria. Thus, we found a defect in antigen handling in peripheral blood mononuclear cells of IgAN patients with a specific increase of CX3CR1. This constitutive upregulation of glomerular and urinary fractalkine suggests an involvement of the CX3CR1–fractalkine axis in the exacerbation of gross hematuria. Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide among patients undergoing renal biopsy. Approximately 40% of IgAN patients, older than 30 years, develop end-stage renal disease after 20 years from the renal biopsy.1.Manno C. Strippoli G.F. D'Altri C. et al.A novel simpler histological classification for renal survival in IgA nephropathy: a retrospective study.Am J Kidney Dis. 2007; 49: 763-775Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,2.Schena F.P. Coppo R. IgA nephropathies.in: Davison A.M. Ritz E. Cameron J.S. Winearls C. Oxford Textbook of Clinical Nephrology. 3rd edn. Oxford University Press, Oxford, UK2005: 369-502Google Scholar IgAN is characterized by a dysregulation of the immune system, leading to an abnormal deglycosylated IgA1 synthesis, selective mesangial IgA1 deposition with ensuing mesangial cell proliferation, and extracellular matrix expansion, through poorly understood molecular mechanisms.3.Allen A.C. Bailey E.M. Brenchley P.E. et al.Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients.Kidney Int. 2001; 60: 673-969Abstract Full Text Full Text PDF Scopus (267) Google Scholar,4.Novak J. Tomana M. Matousovic K. et al.IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells.Kidney Int. 2005; 67: 504-513Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar Recurrence of IgA deposits in IgAN patients after transplantation suggests that the abnormality lies within the immune cells rather than local kidney abnormalities.5.van der Boog P.J. de Fijter J.W. Bruijn J.A. et al.Recurrence of IgA nephropathy after renal transplantation.Ann Med Interne. 1999; 150: 137-142PubMed Google Scholar,6.Ponticelli C. Traversi L. Feliciani A. et al.Kidney transplantation in patients with IgA mesangial glomerulonephritis.Kidney Int. 2001; 60: 1948-1954Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar Indeed, accumulating evidences suggest that peripheral blood mononuclear cells (PBMCs) from IgAN patients are hyperactivated7.Cox S.N. Sallustio F. Serino G. et al.Altered modulation of WNT-beta-catenin and PI3K/Akt pathways in IgA nephropathy.Kidney Int. 2010; 78: 396-407Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar,8.Coppo R. Camilla R. Amore A. et al.Toll-like receptor 4 expression is increased in circulating mononuclear cells of patients with immunoglobulin A nephropathy.Clin Exp Immunol. 2010; 159: 73-81Crossref PubMed Scopus (83) Google Scholar and show an upregulation of the cellular machinery involved in antigen processing.9.Coppo R. Camilla R. Alfarano A. et al.Upregulation of the immunoproteasome in peripheral blood mononuclear cells of patients with IgA nephropathy.Kidney Int. 2009; 75: 536-541Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar The most characteristic clinical feature of this disease is gross hematuria (GH) coinciding with or immediately following an upper respiratory or gastrointestinal tract infection; thus, a dysregulation of innate immunity has been extensively postulated in IgAN.10.Coppo R. Amore A. Peruzzi L. et al.Innate immunity and IgA nephropathy.J Nephrol. 2010; 23: 626-632PubMed Google Scholar A variety of common pathogens and alimentary components have been used in experimental models of IgAN, in which oral immunization was able to reproduce IgAN in mice.11.Sharmin S. Shimizu Y. Hagiwara M. et al.Staphylococcus aureus antigens induce IgA-type glomerulonephritis in Balb/c mice.J Nephrol. 2004; 17: 504-511PubMed Google Scholar,12.Amore A. Coppo R. Nedrud J.G. et al.The role of nasal tolerance in a model of IgA nephropathy induced in mice by Sendai virus.Clin Immunol. 2004; 113: 101-108Crossref PubMed Scopus (27) Google Scholar Furthermore, some of these exogenous antigens were also detected in renal tissue of IgAN patients.13.Suzuki S. Nakatomi Y. Sato H. et al.Haemophilus parainfluenzae antigen and antibody in renal biopsy samples and serum of patients with IgA nephropathy.Lancet. 1994; 343: 12-16Abstract PubMed Scopus (124) Google Scholar,14.Koyama A. Sharmin S. Sakurai H. et al.Staphylococcus aureus cell envelope antigen is a new candidate for the induction of IgA nephropathy.Kidney Int. 2004; 66: 121-132Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar The link between mucosal encountered antigens and the occurrence of glomerular hematuria has never been clarified. Increasing evidence suggests an altered cell homing in IgAN patients.15.Kennel-de March A. Bene M.C. Renoult E. et al.Enhanced expression of L-selectin on peripheral blood lymphocytes from patients with IgA nephropathy.Clin Exp Immunol. 1999; 115: 542-546Crossref PubMed Scopus (22) Google Scholar,16.Batra A. Smith A.C. Feehally J. et al.T-cell homing receptor expression in IgA nephropathy.Nephrol Dial Transplant. 2007; 22: 2540-2548Crossref PubMed Scopus (40) Google Scholar In particular, altered cell homing from mucosal to systemic sites has been demonstrated by Batra et al.16.Batra A. Smith A.C. Feehally J. et al.T-cell homing receptor expression in IgA nephropathy.Nephrol Dial Transplant. 2007; 22: 2540-2548Crossref PubMed Scopus (40) Google Scholar Chemokines and chemokine receptors have an important role in the homing and recruitment of specific immune cell populations in the correct effector sites. Among the chemokine receptors, chemokine (C-X3-C motif) receptor 1 (CX3CR1) clearly defines peripheral blood cytotoxic effector lymphocytes commonly armed with intracellular perforin and granzyme B. These cells include natural killer cells, γδ-T cells, and terminally differentiated CD8+ T cells.17.Nishimura M. Umehara H. Nakayama T. et al.Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression.J Immunol. 2002; 168: 6173-6180Crossref PubMed Scopus (250) Google Scholar Fractalkine (FKN) represents the only ligand for the CX3CR1 receptor. This is a unique chemokine that functions not only as a chemoattractant but also as a cell adhesion molecule for CX3CR1+ cells, and thus FKN and CX3CR1 represent a novel type of leukocyte trafficking regulator. FKN is expressed on endothelial cells and is activated by proinflammatory cytokines, such as IFN-γ, TNFα, IL-1, and MCP-1.18.Fraticelli P. Sironi M. Bianchi G. et al.Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses.J Clin Invest. 2001; 107: 1173-1181Crossref PubMed Scopus (286) Google Scholar The expression of FKN and the presence of CX3CR1-expressing cells have been demonstrated in patients with various types of nephropathies.19.Furuichi K. Wada T. Iwata Y. et al.Upregulation of fractalkine in human crescentic glomerulonephritis.Nephron. 2001; 87: 314-320Crossref PubMed Scopus (53) Google Scholar, 20.Ito Y. Kawachi H. Morioka Y. et al.Fractalkine expression and the recruitment of CX3CR1+ cells in the prolonged mesangial proliferative glomerulonephritis.Kidney Int. 2002; 61: 2044-2057Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 21.Koziolek M.J. Müller G.A. Zapf A. et al.Role of CX3C-chemokine CX3C-L/fractalkine expression in a model of slowly progressive renal failure.Nephrol Dial Transplant. 2010; 25: 684-698Crossref PubMed Scopus (33) Google Scholar, 22.Segerer S. Hughes E. Hudkins K.L. et al.Expression of the fractalkine receptor (CX3CR1) in human kidney diseases.Kidney Int. 2002; 62: 488-495Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar In particular, Segerer et al.22.Segerer S. Hughes E. Hudkins K.L. et al.Expression of the fractalkine receptor (CX3CR1) in human kidney diseases.Kidney Int. 2002; 62: 488-495Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar demonstrated that infiltrating inflammatory leukocytes in renal diseases express CX3CR1, confirming their role in the accumulation of intra-renal inflammatory cells. On the other hand, the treatment with anti-CX3CR1 antibody markedly blocks the infiltration of leukocytes into the glomeruli, prevents crescent formation, and improves renal function, suggesting a role for FKN in the pathogenesis of glomerulonephritis.23.Feng L. Chen S. Garcia G.E. et al.Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication.Kidney Int. 1999; 56: 612-620Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar FKN expressed on endothelial cells promotes adhesion and transmigration of leukocytes that bear the complementary receptor CX3CR1.24.Umehara H. Bloom E.T. Okazaki T. et al.Fractalkine in vascular biology: from basic research to clinical disease.Arterioscler Thromb Vasc Biol. 2004; 24: 34-40Crossref PubMed Scopus (187) Google Scholar The transmigration of CX3CR1+ cells into the tissue has been demonstrated to damage neighboring endothelial cells and to have a role in vascular injury.25.Yoneda O. Imai T. Gouda S. et al.NK cell-mediated vascular injury.J Immunol. 2000; 164: 4055-4062Crossref PubMed Scopus (181) Google Scholar, 26.Umehara H. Bloom E.T. Okazaki T. et al.Fractalkine and vascular injury.Trends Immunol. 2001; 22: 602-607Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 27.Umehara H. Bloom E.T. Okazaki T. et al.Fractalkine in vascular biology: from basic research to clinical disease.Arterioscler Thromb Vasc Biol. 2004; 24: 34-40Crossref PubMed Scopus (265) Google Scholar As GH can represent a triggering event in IgAN, we decided to study this clinical time point in order to uncover new mechanisms associated with the onset and development of IgAN. Genome-wide expression analysis followed by classical biomolecular approaches uncovered new pathways involved in the exacerbation of GH, and a central role for the CX3CR1–FKN axis in this clinical manifestation was revealed. Furthermore, we confirmed the predisposition of PBMCs of IgAN patients to respond in an aberrant and enhanced manner to antigen stimulation. The first step of the study was to perform an exploratory/hypothesis-generating analysis using microarray technology in order to identify genes/pathways modulated in the acute phase of the disease. For this aim, we analyzed the genomic profile of PBMCs from three IgAN patients at two different clinical time points; the first sample was analyzed during the GH episode and the second during the remission phase of the disease characterized by persistent microscopic hematuria (MH). During the GH episode, bioinformatic analysis revealed 150 upregulated (P<0.001) and 96 downregulated (P=0.043) genes compared with the same subjects in the MH status (Supplementary Tables S1, S2 online). Ingenuity pathway analysis (IPA) demonstrated that the identified genes were primarily involved in interferon signaling, antigen presentation, and cytotoxic T lymphocyte–mediated apoptosis of target cell canonical pathways (Table 1, Supplementary Figure S1 online). The most representative pathway selected by this analysis showed a clear involvement of the intracellular interferon signaling pathway. Reports conducted both in vivo and in vitro demonstrated the importance of our selected genes in the cellular machinery activated by interferons (Supplementary Table S3 online).28.Bennett L. Palucka A.K. Arce E. et al.Interferon and granulopoiesis signatures in systemic lupus erythematosus blood.J Exp Med. 2003; 197: 711-723Crossref PubMed Scopus (1571) Google Scholar,29.Baechler E.C. Batliwalla F.M. Karypis G. et al.Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.Proc Natl Acad Sci USA. 2003; 100: 2610-2615Crossref PubMed Scopus (1715) Google Scholar In addition, the top-ranked network included several genes encoding for regulators of these pathways (score 48, 54 associated genes, P<0.0001; Figure 1). Analysis of this network revealed new important insights related to the pathogenesis of the GH episode in IgAN. This network showed, for the first time, an involvement of the immunoproteasome pathway (e.g., PSMB8, PSMB9, and PSMB10), the central role of interferon signaling (e.g., STAT1), and the presence of a gene characterizing cytotoxic effector lymphocytes CX3CR1. Download .doc (1.52 MB) Help with doc files Supplementary Tables and FiguresTable 1The most representative canonical pathways dysregulated during the gross hematuria episodeIPA categoryPathway P-valueaP-value calculated with Fisher's exact test by IPA, this P-value determines the significance of the association between the uploded data set and the canonical pathway.Pathway corrected P-valuebP-value calculated with the Benjamini–Hochberg method by IPA; this corrected P-value takes into account the multiple canonical pathways tested by IPA.Gene symbolEntrez gene nameProbe set IDMean FCcThe mean FC was evaluated as the mean of the ratio between the actual probe values in the two experimental conditions, in the three subjects.Interferon signaling2.95E-63.59E-4IFIT3Interferon-induced protein with tetratricopeptide repeats 3204747_at8.81IFITM1Interferon-induced transmembrane protein 1214022_s_at2.51MX1Myxovirusresistance 1, interferon-inducible protein p78202086_at10.06OAS12′,5′-Oligoadenylate synthetase205552_s_at8.57PSMB8Proteasome subunit, β type, 8209040_s_at1.91STAT1Signal transducer and activator of transcription200887_s_at2.31Antigen presentation pathway1.14E-62.63E-3HLA-EMajor histocompatibility complex, class I, E200905_x_at1.54PSMB8Proteasome subunit, β type, 8209040_s_at1.91PSMB9Proteasome subunit, β type, 9204279_at1.74TAPBPTAP-binding protein208829_at1.47Cytotoxic T lymphocyte-mediated apoptosis of target cells6.02E-41.21E-2CD3ECD3e molecule, epsilon γ polypeptide204747_at8.81FADDFas (TNFRSF6)-associated via death domain214022_s_at2.51FASTNF receptor superfamily (member 6)202086_at10.06FCER1GFc fragment of IgE, high affinity I, receptor205552_s_at8.57HLA-EMajor histocompatibility complex, class I, E209040_s_at1.91[email protected]T-cell receptor α locus200887_s_at2.31CX3CR1Chemokine (C-X3-C motif) receptor 1205898_at1.90Abbreviations: FC, fold change; Ig, immunoglobulin; IPA, ingenuity pathway analysis; TNF, tumor necrosis factor.a P-value calculated with Fisher's exact test by IPA, this P-value determines the significance of the association between the uploded data set and the canonical pathway.b P-value calculated with the Benjamini–Hochberg method by IPA; this corrected P-value takes into account the multiple canonical pathways tested by IPA.c The mean FC was evaluated as the mean of the ratio between the actual probe values in the two experimental conditions, in the three subjects. Open table in a new tab Download .doc (.06 MB) Help with doc files Supplementary Methods Abbreviations: FC, fold change; Ig, immunoglobulin; IPA, ingenuity pathway analysis; TNF, tumor necrosis factor. To further establish the validity of gene expression determined by microarray analysis, we performed quantitative real-time PCR (RT-PCR) on PBMCs from 12 IgAN patients from whom blood samples were collected at two clinical time points. We chose three representative genes that were found deregulated during the GH: CX3CR1, PSMB9, and FADD. Normalized gene expression levels for CX3CR1 and PSMB9 were significantly higher during the GH episode compared with the same subject in the MH phase (*P=0.0001, **P=0.006, respectively; Figure 2a and b). FADD-normalized gene expression level was significantly lower during the GH phase (***P=0.01, Figure 2c). Paired t test was applied. These results were in line with those obtained by the gene expression array. At this point, we focused our attention on the upregulation of CX3CR1. This receptor clearly defines peripheral blood cytotoxic effector lymphocytes involved in vascular injury and glomerulonephritis.17.Nishimura M. Umehara H. Nakayama T. et al.Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression.J Immunol. 2002; 168: 6173-6180Crossref PubMed Scopus (250) Google Scholar, 19.Furuichi K. Wada T. Iwata Y. et al.Upregulation of fractalkine in human crescentic glomerulonephritis.Nephron. 2001; 87: 314-320Crossref PubMed Scopus (53) Google Scholar, 20.Ito Y. Kawachi H. Morioka Y. et al.Fractalkine expression and the recruitment of CX3CR1+ cells in the prolonged mesangial proliferative glomerulonephritis.Kidney Int. 2002; 61: 2044-2057Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 21.Koziolek M.J. Müller G.A. Zapf A. et al.Role of CX3C-chemokine CX3C-L/fractalkine expression in a model of slowly progressive renal failure.Nephrol Dial Transplant. 2010; 25: 684-698Crossref PubMed Scopus (33) Google Scholar, 22.Segerer S. Hughes E. Hudkins K.L. et al.Expression of the fractalkine receptor (CX3CR1) in human kidney diseases.Kidney Int. 2002; 62: 488-495Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 25.Yoneda O. Imai T. Gouda S. et al.NK cell-mediated vascular injury.J Immunol. 2000; 164: 4055-4062Crossref PubMed Scopus (181) Google Scholar Flow cytometry and western blot were used to analyze the expression of CX3CR1 on freshly isolated PBMCs from IgAN patients at two different time points: during the GH episode and in the remission phase characterized by persistent MH. The surface expression of CX3CR1 was found significantly higher in PBMCs in the GH compared with the same patient during the MH phase (P=0.001; Figure 3a and b). The same results were confirmed by western blot analysis. In accordance with results obtained by flow cytometry, CX3CR1 protein levels were significantly higher during the GH compared with the same subject in the MH phase (P=0.001; Figure 3c and e). Concordant with genome-wide expression analysis, we also found that STAT1 protein levels were significantly higher during the GH compared with the same subject in the MH phase (P=0.002; Figure 3d and e). Next, we decided to extend our characterization of CX3CR1 surface expression in lymphocyte subpopulations from freshly isolated PBMCs of five IgAN patients at two clinical time points, in order to evaluate whether the differences in the receptor expression affect a specific subset. We found, during the GH episode, a significant increase of CX3CR1 in cell subsets with well-known cytotoxic effector functions, such as natural killer cells (CD16+CD56+CD3−, P=0.004), natural killer T cells (CD16+CD56+CD3+, P=0.004), CD3+CD8+ T cells (P=0.025), and γ/δ-T cells (CD4−CD8−CD3+, P=0.001) (Figure 4a and b). We noted a significant increase also in CD3+CD4+ T cells (P=0.006) in the GH even when the percentage expression of CX3CR1 in this population was lower compared with CD8+ T cells. Interestingly, we observed that all cytotoxic cell subsets during the GH episode compared with the MH tend to expand, except for CD4+ cells, which show an opposite trend (43 vs. 27%, Figure 4a), but these changes do not reach statistical significance (data not shown). Paired t test was applied. Further, we decided to enroll an independent set of 12 IgAN patients and 10 healthy blood donors (HBDs) in order to evaluate whether CX3CR1 expression on PBMCs could be upregulated specifically in IgAN patients following antigenic stimulation. We performed PBMC in vitro stimulation with increasing concentrations of lipopolysaccharide (LPS; 0.01μg/ml, 0.1μg/ml, and 1μg/ml). LPS exposure resulted in a dose-dependent upregulation of CX3CR1 and STAT1 proteins in IgAN patients and not in HBDs, reaching a statistically significant 2.8- and 1.9-fold increase, respectively, with the dose of 1μg/ml (n=3, *P<0.01, **P<0.05; Figure 5a). Furthermore, we selected this dosage to evaluate CX3CR1 and STAT1 protein levels and found, respectively, a significant 1.8- and 1.6-fold increase in protein expression in IgAN patients compared with HBDs (P<0.01; Figure 5c and d). The same group was then divided into two clinical subgroups: IgAN patients who had never manifested GH and were characterized by permanent asymptomatic MH (pMH, n=6) and IgAN patients who have had at least three episodes of GH during the follow-up (recurrent GH, rGH, n=6). We found no statistically significant differences between the two clinical subgroups (P=0.8357; Supplementary Figure S2 online). These results suggest that the upregulation of this receptor is a specific characteristic of cells in IgAN patients following endotoxin challenge, and that the enhanced expression is not sufficient to cause GH. In fact, we evaluated whether the abnormalities in CX3CR1 and STAT1 were also found in IgAN patients without hematuria and in other glomerulonephritis characterized by hematuria,30.Cahen R. Francois B. Trolliet P. et al.Aetiology of membranous glomerulonephritis: a prospective study of 82 adult patients.Nephrol Dial Transplant. 1989; 4: 172-180PubMed Google Scholar,31.Zeng C.H. Chen H.M. Wang R.S. et al.Etiology and clinical characteristics of membranous nephropathy in Chinese patients.Am J Kidney Dis. 2008; 52: 691-698Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar i.e., membranous nephropathy (MN) and membranoproliferative glomerulonephritis (MPGN). CX3CR1 and STAT1 protein levels in five IgAN patients without hematuria increased 1.9- and 1.5-fold compared with MN and MPGN patients after PBMC in vitro stimulation with LPS (1μg/ml, Supplementary Figure S3 online). These results confirm that the deregulation of interferon signaling pathway through STAT1 and the enhanced expression of the receptor during endotoxin challenge are specific to IgAN patients and do not occur in other glomerulonephritis characterized by microhematuria. It is well established that FKN functions as an adhesion molecule to CX3CR1-positive cells and is capable of capturing leukocytes under static and physiological flow conditions.32.Imai T. Hieshima K. Haskell C. et al.Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1160) Google Scholar, 33.Goda S. Imai T. Yoshie O. et al.CX3C-chemokine, fractalkine enhanced adhesion of THP-1 cells to endothelial cells through integrin dependent and independent mechanisms.J Immunol. 2000; 164: 4313-4320Crossref PubMed Scopus (184) Google Scholar, 34.Fong A.M. Robinson L.A. Steeber D.A. et al.Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow.J Exp Med. 1998; 188: 1413-1419Crossref PubMed Scopus (592) Google Scholar Thus, we investigated in vivo, by means of immunohistochemical techniques, whether FKN glomerular expression was different in rGH compared with pMH patients. We found that FKN expression was significantly higher in IgAN patients with a history of rGH compared with pMH (n=10, P<0.0001) and normal glomeruli (n=10, P=0.0002; Figure 6a–e), in which the expression was virtually absent. To strengthen our hypothesis, we measured the urinary FKN level in IgAN patients with rGH, pMH, without hematuria, in MN patients with pMH, and in MPGN patients with pMH. We found a significantly higher FKN level only in IgAN patients with rGH compared with the other groups (P<0.05, Figure 6f). Moreover, we assessed the localization of this expression by immunofluorescent experiments. We found a perfect colocalization of FKN with glomerular endothelial cells and podocytes as shown by dual labeling, respectively, with anti-CD31 (Figure 7a–f) and anti-synaptopodin (Figure 7a–l). Altogether, these results suggest that the upregulated expression of FKN within the glomerulus of rGH could predispose these patients to the GH episode.Figure 7Glomerular expression of fractalkine (FKN) in IgA nephropathy (IgAN) patients characterized by recurrent gross hematuria episodes. Immunofluorescent labeling of FKN in biopsy specimens, with histological diagnosis of IgA nephropathy. We found a perfect colocalization of FKN within glomerular endothelial cells, as shown by (a–f) dual labeling with anti-CD31 and within podocytes as shown by (g–l) dual labeling with anti-synaptopodin.View Large Image Figure ViewerDownload (PPT) In this study, a whole-genome expression analysis followed by classical biomolecular approaches revealed new mechanisms underlying GH episode, which is considered the hallmark of IgAN.35.Coppo R. Amore A. Peruzzi L. et al.Innate immunity and IgA nephropathy.J Nephrol. 2010; 23: 626-632PubMed Google Scholar The response to an antigenic challenge clearly emerges from pathway analysis in IgAN patients during GH. In particular, upregulated genes intervene in the machinery involved in antigen presentation and in the conversion of protein antigens into antigenic epitopes. These processes are controlled by the immunoproteasome P28 (PSMB8, PSMB9, and PSMB10); subsequently, the processed epitopes are loaded onto the cell surface in a complex with major histocompatibility complex (MHC) class I (TAPBP, HLA-E). Once displayed on the cell surface, the antigen-loaded class I MHC complex is ready for inspection by the T-cell receptor ([email protected]) on circulating cytotoxic effector lymphocytes (CX3CR1). Surprisingly, a recent genome-wide association study for IgAN localized the strongest signal in the genomic area that encodes for TAPBP and PSMB8, and PSMB9.36.Gharavi A.G. Kiryluk K. Choi M. et al.Genome-wide association study identifies susceptibility loci for IgA nephropathy.Nat Genet. 2011; 43: 321-327Crossref PubMed Scopus (443) Google Scholar The switch from proteasome to immunoproteasome has already been described in IgAN patients at basal levels;9.Coppo R. Camilla R. Alfarano A. et al.Upregulation of the immunoproteasome in peripheral blood mononuclear cells of patients with IgA nephropathy.Kidney Int. 2009; 75: 536-541Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar however, our data support the hypothesis that these upregulated genes reflect a response to an infectious challenge during the active phase of the disease. IFN-γ is a key pleiotropic regulatory cytokine in this context,37.Zhou F. Molecular mechanisms of IFN-gamma to up-regulate MHC class I antigen processing and presentation.Int Rev Immunol. 2009; 28: 239-260Crossref PubMed Scopus (2