The present study identifies new players, molecular connections, and sequences of events involved in the development of IgAN. Our data demonstrate that besides IgA1, TfR1 can also interact with sCD89 and TGase2. Based on the present and previous data, we propose a model whereby polymeric IgA1 (because of abnormal glycosylation in humans or to physiological polymerization in mice) induces shedding of membrane CD89 (Grossetête et al., 1998
; Launay et al., 2000
) with the formation of circulating complexes containing sCD89 and IgA1. These complexes have the capacity to bind mesangial TfR1 through the ability of both sCD89 and IgA1 (Moura et al., 2001
) to interact with this receptor. Upon deposition on mesangial cells, sCD89, and thus IgA1–sCD89 complexes, induces surface expression of TGase2. The latter can bind TfR1 and control its cell surface expression, allowing binding of additional IgA1–sCD89 complexes enhancing mesangial deposition. The multimolecular complexes thus stabilized on mesangial cell surface that include TfR1, TGase2, sCD89, and IgA1 would induce chronic stimulation of the cells with release of proinflammatory mediators leading to kidney disease. The tissue TGase2, which thus appears as a new partner of mesangial TfR1, is proposed to be an essential molecular actor in IgAN pathogenesis in mice as it controls mesangial deposition of IgA1 complexes that lead to renal dysfunction. TGase2 would be the factor responsible for TfR1 overexpression in primary cells implicated in IgA-related diseases (i.e., IgAN [Haddad et al., 2003
] and celiac disease [Matysiak-Budnik et al., 2008
]) in the absence of alterations of iron metabolism.
Since our first description of the putative role of sCD89 in generating mouse IgA deposits in the mesangium (Launay et al., 2000
), there was a lack of formal demonstration that sCD89 actively participates in IgA1 mesangial deposition in mice and humans. The production of mice expressing both human IgA1 and human CD89 on monocytes/macrophages allowed us to readdress this question and to demonstrate that sCD89 is required for pathogenic mesangial IgA1 deposits leading to disease development. Interestingly, in the absence of CD89, IgA1 deposition occurring in α1KI mouse glomeruli did not lead to any detectable renal dysfunction, which is in line with observations in humans that not all subjects with IgA glomerulus deposition express overt signs of glomerulonephritis (Varis et al., 1993
; Glassock, 2011
). The spontaneous sCD89 production observed in mice that express human IgA1 and CD89 may be related to the high polymeric/monomeric ratio of IgA in the mouse. Indeed, although ~30% of circulating chimeric IgA1 (composed by a human α1 heavy chain and mouse λ and κ chains) were polymeric in both α1KI and α1KI-CD89Tg mice, in human normal serum, this form represents ~10% of total IgA (Mestecky, 1988
; Kerr, 1990
). Thus, small IgA1 complexes, mimicking complexes formed by underglycosylated IgA1 found in patients, may induce CD89 aggregation and shedding from myeloid cells in α1KI-CD89Tg mice as reported for human myeloid cells (Launay et al., 2000
). This would result in the formation of circulating complexes containing IgA1 and sCD89 that would deposit in kidney mesangium. However, so far, there was no published evidence for mesangial sCD89 deposition in patients. Using a new polyclonal antibody raised against sCD89, we clearly detected CD89 in the mesangium of α1KI-CD89Tg mice and of IgAN patients. Acid elution procedure, a standard method to isolate antibodies and complexes from glomeruli (Woodroffe and Wilson, 1977
), confirmed the presence of sCD89 in mesangial IgA1 deposits, further suggesting that the molecular mass of IgA1 complexes is a major factor promoting IgAN. In line with this observation, acidic elution of IgA1–sCD89 complexes from kidney eluates was predominantly of high molecular mass, confirming previous data obtained with IgAN patients (Monteiro et al., 1985
). Interestingly, sCD89 was also detected in serum as high molecular mass forms, suggesting that sCD89 could be covalently linked to IgA1. This is in agreement with previous data showing that sCD89 can be covalently linked to IgA in a polymeric form (van der Boog et al., 2002
). However, although in a previous study authors found a 30-kD sCD89 protein complexed with IgA in normal human sera (van der Boog et al., 2002
), we observed a 50–70-kD sCD89 form that is exclusively found in the serum of IgAN patients (Launay et al., 2000
). It is noteworthy that all sCD89 found in serum in α1KI-CD89Tg mice was associated with IgA1, demonstrating that no IgA-free sCD89 may exist in the circulation. It is also remarkable that in the absence of CD89, no high molecular mass IgA1 complexes were found in the circulation of α1KI mice. These IgA1–sCD89 complexes appeared nephrotoxic because only α1KI-CD89Tg mice developed renal failure as indicated by proteinuria, hematuria, and increased serum creatinine levels. Our data may support recent observations by others in which severity of renal dysfunction in IgAN patients correlates with the disappearance of IgA1–sCD89 complexes in the circulation, suggesting that this could be caused by their increased deposition in kidneys (Boyd and Barratt, 2010
; Vuong et al., 2010
). The large size of the deposited pathogenic aggregates observed in these mice that include IgA1, TfR1, TfR1-associated TGase2, and sCD89 and that may also include IgA1-associated MBL with their associated serine proteases (MASP; Roos et al., 2006
) may explain why deposited mesangial sCD89 has escaped detection so far. Yet, the role of sCD89 in pathogenic mesangial deposits in our IgAN murine model stresses the importance of a systematic evaluation of CD89 presence in IgAN patient biopsies. Generation of adapted molecular tools, such as our new polyclonal anti-sCD89 antibody, will be important in this regard.
TfR1, a multiligand receptor, participates in several cellular functions (Lebrón et al., 1998
; Radoshitzky et al., 2007
; Schmidt et al., 2008
). TfR1 is a receptor for IgA1 whose binding depends on the size and the glycosylation of IgA1 (Moura et al., 2004a
). TfR1–IgA1 interaction plays a crucial role in physiology where polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia (Coulon et al., 2011
). In pathology, cell surface overexpression of TfR1 is a major characteristic of IgAN and celiac disease (Moura et al., 2001
; Haddad et al., 2003
; Matysiak-Budnik et al., 2008
). However, molecular mechanisms involved in TfR1 overexpression in primary cells are not yet identified. Several factors have been shown to up-regulate TfR1 expression, including iron deprivation and human hemochromatosis protein, as well as polymeric IgA1 from patients with IgAN (Moura et al., 2005
). In this study, we show that sCD89 is a new ligand for TfR1 able to up-regulate its expression on mesangial cells and induce secretion of proinflammatory cytokines such as IL-8, IL-6, and TNF. sCD89–TfR1 interaction has a marked effect on cell stimulation, suggesting that IgA1–sCD89 complexes might be responsible for a local mesangial cell activation and the development of IgAN.
Our study demonstrates that sCD89 plays a pivotal role in IgAN in mice by (a) the formation of IgA1 circulating complexes allowing their deposition in mesangium, (b) the induction of increased mesangial expression of TfR1, and (c) the induction of surface expression of TGase2. TGase2, a calcium-dependent multifunctional protein, is ubiquitously expressed in almost all cells and tissues (Lorand and Graham, 2003
). It acts predominantly as a cytosolic enzyme but can be externalized from cells by an unknown secretory pathway, after which it cross-links proteins of the extracellular matrix and induces renal fibrosis development (Fesus and Piacentini, 2002
; Shweke et al., 2008
). Our data reveal that TGase2 surface expression is induced by sCD89 and is up-regulated in the mesangium of α1KI-CD89Tg mice and in IgAN patients. The latter observation is in agreement with data reported by others showing increased mesangial TGase2 expression in IgAN patients (Ikee et al., 2007
). Moreover, the correlation of TGase2 mesangial expression with deterioration of renal function (Ikee et al., 2007
) supports our observations that only α1KI-CD89Tg mice that overexpress TGase2 develop proteinuria, whereas α1KI mice alone show no proteinuria and mild TGase2 overexpression, further suggesting that TGase2 controls the triggering of IgAN pathology. The mechanism by which TGase2 surface expression increases in turn surface expression of TfR1 remains unresolved whether it involves increased TfR1 gene transcription or interference in TfR1 cycle by favoring its recycling to, and stabilization at, the plasma membrane. Mesangial fibronectin expression is also up-regulated in α1KI-CD89Tg mice and colocalizes with TGase2 and IgA1. Interestingly, it has been shown that TGase2 binds to fibronectin and acts as an integrin coreceptor (Lorand and Graham, 2003
), emphasizing that TGase2 could intervene at multiple levels in mesangial cell activation for inflammation and fibrosis in IgAN.
In conclusion, our study demonstrates that IgA1–sCD89 complexes could initiate a process of autoamplification involving hyperexpression of TfR1 and TGase2, allowing increased mesangial deposition of pathogenic IgA1 complexes and chronic mesangial cell activation. The critical role played by TGase2 revealed by our humanized mouse model opens new perspectives for pharmacological modulation of excessive TGase2 expression as a promising strategy for therapeutic intervention in IgAN.