Semin Immunopathol DOI 10.1007/s00281-014-0435-7

REVIEW

Update on crescentic glomerulonephritis Carole Hénique & Christina Papista & Léa Guyonnet & Olivia Lenoir & Pierre-Louis Tharaux

Received: 3 March 2014 / Accepted: 27 May 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The recent years have seen a number of major progresses in the field of extracapillary glomerulonephritis. This entity is the final damage caused by unrelated immunological disorders such as immune complexes glomerular deposits or microvascular injury caused by proinflammatory cytokines, neutrophil extracellular traps (NET), and cell adhesion molecules in the context of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This review provides a summary of recent advances in the understanding of crescentic glomerulonephritis, focusing on interplays of local immune cells and on local mediators participating to crescent formation especially in anti-glomerular basement membrane (anti-GBM) antibody disease. The recent advances about AAV and lupus nephritis are covered by other chapters of this issue. Nevertheless, these considerations may apply to the general case of crescentic glomerulonephritis of all causes.

Introduction Crescentic rapidly progressive glomerulonephritis (RPGN) results from heterogeneous disease processes and has various

This article is a contribution to the special issue on Immunopathology of Glomerular Diseases - Guest Editors: P. Ronco and J. Floege C. Hénique : C. Papista : L. Guyonnet : O. Lenoir : P. 1st week)

Endocapillary and

Recruitment of antiinflammatory iNKT by immature dendritic cells (1st week)

Increase in neutrophil and monocyte dwell time (minutes-hours)

Lu/BCAM

PARs PARs

Endothelial cells Fibrin

Fibrin Thrombin ? Factor VIIa ?

GBM

PARs Switch in podocyte and PEC phenotype :

HIF-1α

Loss of polarity, micro-villous extensions, intercellular bridges formaon,

p-STAT3 p-EGFR

HB-EGF HB-EGF

PARs

Parietal cells

Crescent formation

mediators

local

switch in gene expression paern, proliferaon, cell death

EGFR

Extra - capillary

CXCR4

HB-EGF Switch in PEC phenotype

Fig. 2 Sequential involvement of multiple leukocyte subsets and endothelial damage contribute to failure of the glomerular epithelial cells to maintain a normal and functional phenotype. The duration of monocyte, neutrophil, and T cell attachment to the glomerular endothelium (dwell time) is constitutively high and is further increased early after deposition of anti-GBM Abs. Endothelial attachment is mediated through CD11b/ Mac-1 binding to a variety of ligands such as endothelial protein ICAM-1 and soluble factors such as fibrinogen, neutrophil elastase, iC3b, oligodeoxynucleotide, beta-glucan, heparin sulfate, carbohydrates, and denatured proteins. Constitutive expression of Lutheran/BCAM in the glomerular capillary further promotes adhesion of monocytes and T lymphocytes through alpha4/beta1 integrin binding. Dendritic cells (DCs) populate the kidney cortex in a CX3CR1-dependent fashion. CXCL16-producing immature DCs display anti-inflammatory actions early in the course of the experimental anti-GBM disease through

recruitment of CXCR6-expressing iNKT cells. Then, IL-23-producing maturing DCs recruit γδ T cells that also facilitate neutrophil influx. CX3CR1- and DC-SIGN-expressing DCs promote T cell-mediated delayed-type hypersensitivity (DTH) reactions. Next, activated TH1 cells recruit proinflammatory monocytes, and mannose receptor-dependent macrophages are recruited. Disruption of the glomerular capillary barrier leads to release and extravasation of proteins and coagulation zymogens, the latter activated by tissue factor (TF) induced on extravascular cells, leading to activation of their respective G protein-coupled receptors (GPCRs) such as PARs and subsequent transactivation of EGFR by HB-EGF synthesis and/or mobilization. EGFR activation promotes STAT3 activation in podocytes and synergizes with the HIF-1 pathway and HIF-dependent CXCR4 signaling. This, in turn, may contribute to glomerular inflammation, podocyte dedifferentiation induced by antiGBM antibodies or ICs

RPGN could be induced (although with attenuated severity) in the absence of T lymphocytes in Rag2−/− mice [48] or B lymphocytes [19, 49], and depletion of macrophages attenuated injury with reduced glomerular crescents and improved functional and structural recovery [50]. Adoptive transfer of artificially generated macrophages (bone marrow derived macrophages and NR8383 macrophages) aggravated a model of accelerated nephrotoxic nephritis in rats after 3 days [51]. These macrophages subsequently release proinflammatory cytokines which result in cell proliferation and recruitment of more inflammatory cells (well reviewed in [52–54]). Meanwhile, the complexity of macrophage biology has increased in the recent years. We need more specific delimitation of the mechanisms and sequence of recruitment of distinct monocyte and macrophage subtypes.

Strategies that reduce monocyte infiltrates could be a promising avenue for complementary therapy of RPGN. Among those, strategies preventing leukocyte infiltration in the kidney could target endothelial molecules involved in cell adhesion. Among the candidates, Lutheran (Lu) blood group antigens and basal cell adhesion molecule (BCAM) antigen in endothelial cells are carried by Lu/BCAM (CD239) glycoproteins of the immunoglobulin superfamily. Lu/BCAM glycoproteins are receptors of laminin alpha-5 chains, a major component of the extracellular matrix. Another Lu/BCAM ligand is integrin α4β1 [55]. The α4β1 integrin or very late antigen (VLA-4) or CD49d/CD29 is expressed mainly on monocytes, lymphocytes, and eosinophils. High expression of Lu/BCAM in glomeruli of mice with crescentic RPGN suggests a potential role for the local expression of Lu/BCAM in nephritogenic recruitment of leukocytes. A novel pathophysiological

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interaction between leukocyte α4β1 integrin and endothelial Lu/BCAM proteins was described in vitro, and Lu/BCAM deficiency was sufficient to prevent severe anti-GBM nephrotoxic serum nephritis (NTN)-induced glomerular damage and renal failure in mice [56]. Another advance was recently made in better understanding endocapillary recruitment of myeloid cells. Studies of models of acute glomerulonephritis have revealed that neutrophil recruitment to the glomerulus follows a rapid, yet restricted, time course; neutrophils accumulate in glomeruli within hours of induction of inflammation, but are not present 4–8 h later [24, 57]. Anti-GBM Ab challenge of P-selectin-deficient mice and of neutrophil- and platelet-depleted wild-type animals was associated with milder increase in urinary protein excretion in acute experiments [58]. Leukocyte recruitment classically occurs predominantly via interactions in postcapillary venules. The peculiar situation of the glomerular capillary and challenging constraints for imaging have left us with few studies and little information about the duration and mechanism of leukocyte recruitments in glomeruli. A very significant advance using multiphoton microscopy in mouse hydronephrotic kidneys has been made by Devi et al. [59]. In the absence of inflammation, neutrophils and monocytes underwent unexpected retention and intravascular migration in glomerular capillaries, and the predominant leukocyte response in glomerular inflammation induced by anti-GBM antibodies was to increase the length of time that these intravascular cells were retained. Examination at 1–2 h after GBM antibody administration revealed that both crawling and static neutrophils were present. However, GBM antibody treatment did not induce a major increase in the number of neutrophils undergoing adhesion, with only the static population increasing significantly. The most striking change caused by anti-GBM Ab or by an antibody against myeloperoxidase (MPO) as well was a 3- to 4-fold increase in neutrophil dwell time (from 5 to 20–25 min), with these changes dissipating 4–5 h after initiation [59]. These early changes were Mac-1/CD11b-dependent. Likewise, GBM antibody treatment did not alter the number of adherent monocytes, but did induce a significant increase in CX3CR1+ monocyte dwell time. Furthermore, artificial routing of CD4+ ovalbuminspecific T cells in mice with localized antibody-OVA323–339 peptide in the glomerulus also cause significant increase in both the number and dwell time of adherent neutrophils and monocytes 4 and 24 h after OT-II cell transfer. To our knowledge, these data are the first to indicate that glomerular inflammation induced by T cell recognition of antigen also induces changes in the intraglomerular retention and migration of neutrophils and monocytes [59]. At a later time, during the autologous phase of the antiGBM disease, a role for macrophage polarization is suspected. The mannose receptor (MR) is a pattern recognition molecule belonging to the C-type lectin family that has established roles in macrophage phagocytosis of microorganisms and

endocytic clearance of host-derived glycoproteins [60]. The MR is typically expressed on IL-4 and IL-13 alternatively activated macrophages [61]. These macrophages have been associated with an anti-inflammatory profile with a role in clearance of apoptotic cells, immune regulation, and induction of tolerance [62]. Chavele et al. studied the role of the MR in the accelerated nephrotoxic model of glomerulonephritis in mice [63]. Where MR was deficient, the experimental RPGN was attenuated, macrophages had diminished Fc-mediated function, and the interaction of the macrophages with apoptotic mesangial cells in vitro led to a predominant less inflammatory macrophage phenotype, which may contribute to the disease protection found in Mr−/− mice. They further showed that the Fab portion of sheep immunoglobulins binds to MRbinding domains. Interestingly, the MR seems to interact with FcR-mediated cellular reactions. Indeed, IgG may bind to the Fc receptor with the Fc domain and to the MR with the Fab portion, leading to an increased oxygen burst. If the MR is absent, the oxygen burst is decreased and TNF-alpha production by LPS-stimulated macrophages is markedly reduced in the presence of apoptotic cells. A similar anti-inflammatory role for the MR had been also shown in immature monocytederived dendritic cells in which Ab-mediated engagement of the receptor caused a distinct profile of cytokines/chemokines with the ability to dampen inflammation and to inhibit the generation of Th1-polarized immune responses [64]. Meanwhile, Chavele et al. found no correlation between the mechanism of protection in Mr−/− mice with effector T cell function in terms of delayed-type hypersensitivity (DTH) responses, proliferation, or IL-17 production or with antibody generation [63].

Role of the “adaptive” immunity Dendritic cells (Table 2) In the immune-mediated renal injury model of nephrotoxic nephritis, inflammatory monocytes differentiate into both macrophages and DCs, but a much greater proportion develops into DCs [65, 66]. Dendritic cells are constituents of the mononuclear phagocytic system present in the kidney [65, 67]. DCs are restricted to the tubulointerstitium and are absent from the glomeruli. Depletion of immature DCs in the early stage of experimental crescentic GN accentuated the severity of the disease, likely through reduction of recruitment of iNKT cells [44], which attenuate crescentic GN [68]. The protective role of the renal DCs could be due to their ability to synthesize CXCL16 which may have led to recruitment of protective iNKT cells through activation of the cognate chemokine receptor CXCR6. Likewise, CXCR6-deficient mice exhibited less iNKT cell recruitment with more severe glomerulonephritis [44].

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Delayed depletion of DCs after the induction of nephritis aggravated the disease during the autologous phase, possibly through the loss of IL-10 production by infiltrating CD4+ Th1 cells [66]. Fcγ receptor engagement on kidney resident DCs can lead to priming of infiltrating T cells, which recruit macrophages and may also be involved in direct tissue injury [69]. Other recent studies have also demonstrated that DCs during the later stages of nephrotoxic nephritis activate the adaptive immune responses resulting in the production of proinflammatory cytokines that further mediate tubulointerstitial mononuclear infiltration and the progression of disease [70, 71]. This involves DC maturation that stimulates the intrarenal DTH that drives NTN [71]. The role of the C-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) [72] and the CX3CR1 pathways have been stressed. Perturbation of DC-SIGN, an immune-regulating molecule of the Ctype lectin family, led to impairment of DC maturation and alleviated experimental RPGN [73]. An interesting advance was made when a specific subtype of DCs that is prominent in the kidney was examined. Indeed, as the majority of kidney CD11c+ cells express high levels of CX3CR1 expression [74], the role of the CX3CR1 pathway in crescentic RPGN has been recently investigated. Flow cytometric analysis of kidney single-cell suspensions on day 10 after antiGBM disease induction revealed reduced numbers of IFN-γ+ CD4+ T cells and of TNF-producing DCs and macrophages in CX3CR1-deficient mice, suggesting an attenuated intrarenal DTH response [75]. Interestingly, the attenuated intrarenal DTH response in Cx3cr1−/− mice did not result from a failure to activate a nephritogenic T cell response, but from impaired DC density, primarily in the cortex. In Cx3cr1−/− mice, DC numbers were reduced 9-fold in the cortex, whereas medullary DCs were reduced 3.5-fold. Originally, CX3CR1 deficiency did not impair the immune defense against pyelonephritis [75]. Thus, targeting CX3CR1, for example, by systemic injection of blocking antibodies or recombinant CX3CL1 might represent a promising treatment for GN with fewer side effects than general immunosuppressive therapies (Fig. 2).

Progresses in lymphocyte biology The presence of GBM antigen-specific T cells was first demonstrated 40 years ago [76, 77], and functional T cells are required for the development of anti-GBM RPGN in mice [78]. Nephritogenic GBM antigens can induce a T cell-mediated GN with crescents and proteinuria in the absence of anti-GBM

antibody as shown in a seminal study in chicken [79]. Transfer of mononuclear cells (primarily T cells) induced a disease in chicken [80]. Injection of Col4alpha3NC1-specific CD4+ lymphocytes was later shown to initiate glomerular injury in recipient rats in the absence of anti-GBM antibodies [81]. This has been nicely reviewed [52, 82]. The Th17 axis is central to the mediation of injury in antiGBM models [83], and recent progress has been made in identifying the cell types that produce IL-17A in the kidney, the mechanisms involved in its induction, and the IL-17Amediated effector functions that promote renal tissue injury. In the murine NTN model, CD4+ T cells, gamma-delta T cells, and a population of CD3+CD4−CD8− gamma-delta T cell receptor−NK1.1− T cells were found to produce IL-17A in the kidney [41]. Interestingly, the production of IL-17A by renal gamma-delta T cells depended on IL-23p19 signaling and retinoic acid-related orphan receptor-gamma T. This study provided additional insight about upstream stimulators of IL17A. IL-23 may originate in large part from DCs. In fact, depletion of dendritic cells (Table 2), which produce IL-23 in the kidney, reduced IL-17A production by renal gamma-delta T cells in this model [41]. The IL-23/IL-17 axis seems especially powerful in experimental RPGN because it links DC–lymphocytes interactions to recruitment of neutrophils. Indeed, the lack of IL-17A production in gamma-delta T cells, as well as the absence of all gamma-delta T cells, reduced neutrophil recruitment into the kidney and ameliorated renal injury [41]. Sustained Th17 response may rely on miR-155 activity [84]. Th1 subsets themselves exert a negative feedback action on Th17 subsets as expression of renal and splenic IL-17A, characteristically expressed by the Th17 subset of effector T cells, which have been implicated in the pathogenesis of autoimmune disease, was increased in Th1-specific T-box transcription factor-deficient (T-bet−/−) mice [85]. Although the role of CD8+ T lymphocytes in animal models of crescentic RPGN is generally thought to be minor or controversial [86–88], a pioneering work by Ken Smith and Paul Lyons identified prognostic transcriptomic signature in SLE and ANCA-associated vasculitis (AAV) patients. This team used microarray analysis to examine the transcription profile of purified CD8+ T cells from a total of 59 patients with AAV and 26 patients with SLE before treatment. The investigators observed an overlap in the prognostic CD8+ T cell gene-expression signature between the two diseases after follow-up after therapy initiation for up to 1,000 days. This signature included genes involved in the IL-7 pathway, T lymphocyte signaling, and memory T cells. Moreover, a predictive model that measured expression of only three genes (ITGA2, PTPN22, and NOTCH1) could identify the patients with a poor prognosis, but could not differentiate whether these patients had AAV or SLE [89]. The underlying disease pathogenesis that is associated with the CD8+ T cell transcription signature is still unknown.

Reduction in T-cell Reduction in renal γδ T numbers in the cells at day 3 [41]. No tubulointerstitium assessment of glomerular on day 10 damage and Reduction function tubulointerstitial and glomerular Macrophages on day 10 No change in albuminuria and creatinine clearance on day 10 Reduction in crescent numbers on day 10

Equal glomerular deposition of sheep Ig in DCdepleted and nondepleted animals on day 5 No influence on intrarenal macrophages and lymphocytes (NK, CD4+, and CD8+ T cells) on day 5 but reduced number of renal macrophages, CD4+, CD8+ T cells and B cells on day 14 No significant changes in the scores for glomerular sclerosis and chronic tubulointerstitial damage on day 11 More severe acute tubulointerstitial damage (caused by DC death?) on day 11 No effect on scores for extracapillary proliferation/necrosis, glomerular sclerosis, and chronic tubulointerstitial damage on day 14 No effect on albuminuria on day 14

Outcome

Non-accelerated NTN model in mice depletion in CD11c-DTR mice on day 2 after disease induction [41]

Non-accelerated NTN model in mice, DC depletion in CD11c-DTR mice on day 7 after disease induction [71]

RPGN model/ Non-accelerated anti-GBM model of DC RPGN (NTN) model manipulation/ in mice, DC depletion time of in CD11c-DTR mice depletion on day 4 and 10 after disease induction [66]

Table 2 Summary of studies using depletion or manipulation of dendritic cells in vivo DCs were depleted on day 3 after induction of nonaccelerated NTN in mice by injecting CD11c.LuciDTR mice with diphtheria toxin (DTx) [44] Reduction in renal invariant natural killer T (iNKT) cells (defined as CD45+ CD1dTetramer+) at day 4 with no change in CD3+ T cells and macrophages No assessment of glomerular damage and function Increased abundance of CXCL16 mRNA in periglomerular areas colocalized with DCs CXCR6 deficiency aggravated renal injury 8 days after induction of nephritis (glomerular crescent formation, BUN levels) with no change in albuminto-creatinine ratio DCs treated in vitro with PsL-EGFmAb displayed low expression of costimulatory molecules and an impaired capability to stimulate CD4+ T cells PsL-EGFmAb significantly downregulated DCSIGN and CD80 expression and slightly decreased MHC class II expression in renal DCs from nephritic rats on day 14 Diminished expression of IFN-γ, TNF-α, IL -6, and reversion of IFN-γ/IL-4 ratio in nephritic kidneys treated with PsLEGFmAb Promotion of Tregs priming Significant reduction in levels of urinary 24-h proteins, BUN and serum creatinine on day 14

Non-accelerated NTN model in Wistar-Kyoto rats/infusion of anti-lectinEGF domain monoclonal antibody (PsL-EGFmAb) at 0 and 2 h after nephrotoxic serum injection [73]

Reduction of kidney DCs in nephritic Cx3cr1−/− mice on day 10 Impaired renal recruitment of CD11c+ and MHC-II+ Cx3cr1−/− DCs on days 3 and 5 of NTN (days 2 and 4 after transfer) Reduced number of reduced numbers of IFN-γ+ CD4+ T cells and of T NF-alpha-producing DCs and macrophages on day 10 Fewer infiltrating F4/80+ cells, proportion of crescentic glomeruli and albuminuria in Cx3cr1−/− mice on day 15

Non-accelerated NTN model in mice with constitutive CX3CR1 deficiency [75]

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Local mediators are key in switching glomerular epithelial phenotype and cause crescent formation The cellular and molecular mechanisms that lead to crescent formation are incompletely known. The cells in evolved crescents are a mixture of infiltrating inflammatory cells, predominantly macrophages and proliferating resident cells. Recent works using cell lineage-tracing experiments indicate that podocytes [2, 5, 90] and parietal epithelial cells (PEC) [91, 92] participate significantly to crescent formation. However, sequence and mechanisms of recruitment of these hyperplasic epithelial cells in RPGN are still barely known. The recent demonstrations of the important role of glomerular epithelial pathways in protecting the glomerular filtration barrier have unraveled the crucial homeostatic importance of local factors such as hypoxia-inducible factor (HIF) alpha in podocytes [93–95]. Recent findings that non-immune cells play a role in crescent formation highlight the need to identify alternate and complementary therapeutic strategies. Artificial podocytespecific platelet-derived growth factor-D (PDGF-D) overexpression led to extracapillary and endocapillary proliferation, also nailing the concept that high local concentration of a mitogenic factor in glomerular cells can cause crescentic RPGN [96]. Indeed, the pathophysiological relevance of such concept was illustrated by Bollee and Flamant et al. who found de novo expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in glomerular epithelial cells (podocytes and parietal cells) from both mice and humans with crescentic RPGN, eliciting EGFR activation primarily in these cells [97]. In mice with HB-EGF-deficient kidney cells, the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in leading to loss of specific markers and proliferation. Conditional deletion of the Egfr gene from podocytes in mice was sufficient to alleviate the severity of RPGN. Likewise, pharmacological blockade of EGFR also markedly blunted the severity of RPGN, even if started when heavy albuminuria and glomerular inflammation are already present. Immune labeling of human kidney biopsies suggests that the same pathway may be active in human crescentic disease, but not in non-proliferative glomerular diseases. Interestingly, HB-EGF expression in the crescent is present whatever the etiology of the extracapillary glomerulonephritis. Furthermore, this condition might represent the first molecular system mediating a pathophysiological cross talk between PECs and podocytes as both cell types were shown to express HB-EGF [97]. In fact, HB-EGF mRNA expression was first found in PECs throughout the course of the disease. Both cell types express HB-EGF cognate receptor EGFR/ErbB1. The fact that podocyte-specific deletion of the Egfr gene was sufficient to partially but significantly limit glomerular injury and crescent formation suggest that PECderived HB-EGF may stimulate EGFR in an autocrine fashion as well as in a paracrine fashion in podocytes [97, 98].

The elucidation of the multiple actions of the HBEGF/EGFR cascade in glomerular epithelial cells is in process with recent evidence that the signal transducer and activator of transcription (STAT) family member STAT3 is activated in a EGFR-dependent manner in podocytes. Again, deletion of Stat3 alleles in podocytes protected mice against renal destruction during RPGN induced by injection of anti-GBM nephrotoxic serum (NTS) [99]. This indicates that the HB-EGF/EGFR/ STAT3 pathway could play a pivotal role in RPGN and opens therapeutic perspectives.

Conclusion and perspectives In summary, peculiar propensity of neutrophils and monocytes to slowly transit and patrol the glomerular endothelium has been unraveled with marked transient MAC-1-dependent increase in leukocyte dwell time upon experimental anti-GBM disease. At later time points, a role for the constitutive Lu/BCAM endothelial molecule was shown to promote monocyte and macrophage renal influx with progression of RPGN. The manipulation of DCs may prove to cause complex, mild, and uncontrolled effects as opposite effects of depletion of DCs on experimental crescentic GN have been reported depending on time of depletion. Nevertheless, targeting the CX3CL1/CX3CR1 pathway may prove safe for manipulating adaptive immune response by cortical DCs in RPGN without altering medullary DCs that are specialized at regulating innate immune responses. Blocking IL-23 production and/or subsequent recruitment of IL-17A-producing effector gamma-delta T cells and CD3 CD4 CD8 gamma-delta T cell receptor NK1.1 T cells may also prove beneficial in limiting glomerular damages, in part through limitation of myeloid cell recruitment (Fig. 2). It was initially felt that podocytes were not involved in the pathogenesis of crescent formation because terminally differentiated podocytes do not proliferate. For still obscure reasons, crescentic GN is a notable exception. In fact, establishing a causal link between immune stress of the glomerulus and pathophysiological switch of the epithelial phenotype (e.g., how immune-mediated glomerular stress leads to pathophysiological recruitment of the HB-EGF/EGFR/STAT3 pathway in podocytes) represents a challenge for the coming years. Targeting the abovementioned pathways may promote tolerance of glomerular cells to inflammation in the context of immune vasculitis and thus open for new therapeutic perspectives that extend beyond the specific case of anti-GBM disease. Acknowledgments We apologize to those authors whose important publications could not be quoted in this review owing to space limitations. We thank Pr. Pierre Ronco for critical and useful suggestions.

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