Nephrology and Inflammatory Vascular Diseases

Our group focuses on neutrophils in health and disease. The neutrophil is a major effector cell for both inflammation and innate immunity. Neutrophils mature in the bone marrow, circulate in the blood as terminally differentiated cells and migrate into tissues once they become activated. Neutrophils protect from infectious and non-infectious pathogens, but also have the capacity to harm the host organism if not tightly regulated.

We study several aspects of structural and functional neutrophil properties. Our clinical interest concerns patients with autoimmune systemic necrotizing vasculitis. This group of diseases can affect every organ and most frequently involve the kidney by causing glomerulonephritis (examples are given in figure 1). An anti-neutrophil cytoplasmic autoantibody (ANCA) that is directed against proteinase 3 (PR3) or myeloperoxidase (MPO) is detected in the patients’ plasma and causes the disease. ANCA recognize their target antigens on the cell membrane of neutrophils and monocytes and trigger signaling pathways that lead to cell activation. Our major goal is to translate clinical findings into experimental settings. This strategy will expand our knowledge with respect to disease mechanisms, but also allows testing of novel therapeutic targets.

Figure 1. Typical organmanifestations in patients with ANCA vasculitis


1) Presentation of the ANCA antigen PR3 on the neutrophil by the GPI-anchored NB1 receptor

ANCA are directed against either MPO or PR3. The majority of MPO and PR3 are stored in neutrophil granules, and are mobilized to the cell membrane during cytokine priming. ANCA bind to cell surface-expressed ANCA antigens resulting in neutrophil activation. PR3, in contrast to MPO, has a bimodal cell surface pattern. All neutrophils posses intracellular PR3, but only a subset of both resting and activated neutrophils present PR3 on the membrane (mPR3high) (figure 2). The percentage of mPR3high neutrophils ranges from 0 to 100%, is stable in a given individual, and significantly higher in ANCA patients compared to controls. In our patient cohort, a high PR3 expression percentage was associated with an increased risk for vasculitis and worse clinical outcome. We showed a strong genetic influence in a twin study, and found that the HLA region largely explains the genetic variance of the mPR3 phenotype. We previously identified neutrophil antigen B1 (NB1, CD177) as the receptor presenting PR3 on the neutrophil membrane. NB1 is a GPI-linked molecule that is expressed only on a neutrophil subset, and only the NB1-expressing subset is able to present PR3 on the membrane.

Figure 2. PR3 staining in human neutrophils. (left) permeabilized neutrophils show strong intracellular PR3 staining. (middle and right). Viable unfixed neutrophils can be distinguished in membrane PR3 positive and negative cells.


Our current research addresses the following issues:

a) To characterize the PR3-NB1 signaling complex allowing initiation of intracellular signaling. NB1 is a GPI-linked surface molecule lacking an intracellular domain. It is not clear how NB1 can initiate intracellular signaling.


b) To characterize mechanisms that control the NB1 expression. NB1 is expressed only on a neutrophil subset. Western blot analysis of whole cell lysates shows that that NB1 is present in the membrane-expressing subset and absent from the membrane-negative neutrophils. We want to elucidate the transcription, translation and epigenetic mechanisms controling NB1.


c) To characterize components that block the PR3-NB1 interaction. We will express recombinant CD177 and perform a compound screening to block the interaction between CD177 and PR3. We will then use selected compounds to block neutrophil activation by PR3-ANCA


d) To characterize the importance of NB1-mediated PR3 presentation for PR3-ANCA-induced neutrophil activation. In a first step we want to express NB1 and PR3 in myeloid cells using a lentiviral approach. We are currently working with cell lines, including estrogen-regulated Hoxb8 (ER-Hoxb8) progenitors that can execute normal differentiation and normal innate immune function upon ER-Hoxb8 inactivation.



2) Characterization of key mechanisms in ANCA vasculitis and testing their significance as novel treatment targets in vivo

Patients with ANCA vasculitis are currently treated with steroids and cytotoxic drugs. This protocol has dramatically improved patient survival. Unfortunately, the drugs are associated with serious adverse events causing morbidity and mortality. Optimized treatment strategies are needed. We will use an animal model of ANCA-induced glomerulonephritis to test novel treatments (Figure 3).

Figure 3. Animal model for MPO-ANCA-induced vasculitis. MPO-deficient mice were immuinized with murine MPO followed by irradiation and bone marrow transplantation from wild-type mice. Vasculitis examples after 8 weeks are shown.


a) We learned from in vitro studies that ANCA activate PI3 kinase that controls the subsequent generation of reactive oxygen species and degranulation of tissue-damaging granule proteins. We will elucidate the role of PI3 kinase isoforms, particularly the gamma isoform. Using an MPO-ANCA mouse model for glomerulonephritis, we will test the use of PI3 kinase gamma as a therapeutical target.


b) Plasma cells are responsible for the generation of antibodies to the ANCA antigens MPO and PR3. We will investigate the hypothesis that a MPO-ANCA mouse model allows the testing of the proteasome inhibitor bortezomib (BTZ). Anti-MPO IgG-induced NCGN will be established by transplanting wild-type bone marrow (BM) into irradiated MPO-deficient mice immunized with MPO. Four weeks after BM transplantation, actively treated mice will receive a steroid/cyclophosphamide (S/CYC) combination or BTZ, respectively. Urine and kidney tissue will be examined 4 weeks later.



3) Identification of new mechanisms participating ANCA vasculitis and glomerulonephritis

Pro-inflammatory cytokines are important in ANCA-mediated vasculitis. Interleukin-1ß (IL-1ß) is a key mediator of inflammatory and immune responses and monocytes are a major IL-1ß source. The significance of this cytokine in ANCA vasculitis is not known. The secretion of IL-1ß is a tightly regulated chain of events requiring proteolytic cleavage of inactive pro-IL-1ß to IL-1ß. This process is controlled by the “inflammasome”, promoting caspase 1 activation, the classical IL-1ß converting enzyme (ICE). We will test the hypothesis that IL-1ß participates in a mouse model of ANCA-induced necrotizing glomerulonephritis and that serine proteases promote IL-1ß generation and release. To address this issue we will use mice deficient in lysosomal protease dipeptidyl peptidase I (DPPI, also known as cathepsin C). These gene-deleted mice are not capable of executing the proteolytic processing of pro-IL-1ß to mature IL-1ß.