Peiqi Hu

Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice

Antineutrophil cytoplasmic autoantibodies (ANCAs) are identified in the circulation of approximately 80% of patients with pauci-immune necrotizing and crescentic glomerulonephritis and systemic small vessel vasculitis, such as microscopic polyangiitis and Wegener granulomatosis. The most common antigen target for ANCAs is myeloperoxidase (MPO), which is found in neutrophils and monocytes. We report definitive experimental animal evidence that ANCAs are pathogenic. MPO knockout (Mpo(-/-)) mice were immunized with mouse MPO. Splenocytes from these mice or from control mice were injected intravenously into recombinase-activating gene-2-deficient (Rag2(-/-)) mice, which lack functioning B lymphocytes and T lymphocytes. All mice that received splenocytes developed mild to moderate glomerular immune deposits, but only mice that received 1 x 10(8) or 5 x 10(7) anti-MPO splenocytes developed severe necrotizing and crescentic glomerulonephritis, granulomatous inflammation, and systemic necrotizing vasculitis, including necrotizing arteritis and hemorrhagic pulmonary capillaritis. To test the pathogenic potential of antibodies alone, purified anti-MPO IgG or control IgG was injected intravenously into Rag2(-/-) mice and wild-type mice. Mice that received anti-MPO IgG but not mice that received control IgG developed focal necrotizing and crescentic glomerulonephritis with a paucity of glomerular Ig deposition. Thus, anti-MPO IgG alone was able to cause pauci-immune glomerular necrosis and crescent formation in the absence of functional T or B lymphocytes in Rag2(-/-) mice and in the presence of an intact immune system in wild-type C57BL/6J mice. This animal model offers strong support for a direct pathogenic role for ANCA IgG in human glomerulonephritis and vasculitis.

The Role of Neutrophils in the Induction of Glomerulonephritis by Anti-Myeloperoxidase Antibodies

In humans, circulating anti-neutrophil cytoplasm autoantibodies (ANCAs) with specificity for myeloperoxidase (MPO) are strongly associated with the development of pauci-immune necrotizing and crescentic glomerulonephritis (NCGN). In mice, we have demonstrated that intravenous injection of mouse antibodies specific for mouse MPO induces NCGN that closely mimics the human disease. We now report that the development of NCGN in this experimental model is accompanied by glomerular accumulation of neutrophils and macrophages. Neutrophil infiltration was most conspicuous at sites of glomerular necrosis and crescent formation, with macrophages also most numerous in crescents. Lymphocytes, however, were sparse in acute lesions. Importantly, mice that were depleted of circulating neutrophils with NIMP-R14 rat monoclonal antibodies were completely protected from anti-MPO IgG-induced NCGN. These findings provide direct evidence that neutrophils play a major role in the pathogenesis of anti-MPO-induced NCGN in this animal model and implicate neutrophils in the induction of human ANCA disease. This raises the possibility that therapeutic strategies to reduce circulating neutrophils could be beneficial to patients with ANCA-induced NCGN.

Desmosome Signaling INHIBITION OF p38MAPK PREVENTS PEMPHIGUS VULGARIS IgG-INDUCED CYTOSKELETON REORGANIZATION

In the human autoimmune blistering disease pemphigus vulgaris (PV) pathogenic antibodies bind the desmosomal cadherin desmoglein-3 (dsg3), causing epidermal cell-cell detachment (acantholysis). Pathogenic PV dsg3 autoantibodies were used to initiate desmosome signaling in human keratinocyte cell cultures. Heat shock protein 27 (HSP27) and p38MAPK were identified as proteins rapidly phosphorylated in response to PV IgG. Inhibition of p38MAPK activity prevented PV IgG-induced HSP27 phosphorylation, keratin filament retraction, and actin reorganization. These observations suggest that PV IgG binding to dsg3 activates desmosomal signal transduction cascades leading to (i) p38MAPK and HSP27 phosphorylation and (ii) cytoskeletal reorganization, supporting a mechanistic role for signaling in PV IgG-induced acantholysis. Targeting desmosome signaling via inhibition of p38MAPK and HSP27 phosphorylation may provide novel treatments for PV and other desmosome-associated blistering diseases.

p38MAPK inhibition prevents disease in pemphigus vulgaris mice

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering skin disease characterized by detachment of keratinocytes (acantholysis). It has been proposed that PV IgG might trigger signaling and that this process may lead to acantholysis. Indeed, we recently identified a rapid and dose-dependent phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and heat shock protein (HSP) 27 after binding of PV antibodies to cultured keratinocytes. In human keratinocyte cultures, inhibitors of p38MAPK prevented PV IgG-induced phosphorylation of HSP27 and, more importantly, prevented the early cytoskeletal changes associated with loss of cell–cell adhesion. This study was undertaken to (i) determine whether p38MAPK and HSP25, the murine HSP27 homolog, were similarly phosphorylated in an in vivo model of PV and (ii) investigate the potential therapeutic use of p38MAPK inhibition to block blister formation in an animal model of PV. We now report that p38MAPK inhibitors prevented PV blistering disease in vivo. Targeting the end-organ by inhibiting keratinocyte desmosome signaling may be effective for treating desmosome autoimmune blistering disorders.

Pathogenesis of Antineutrophil Cytoplasmic Autoantibody–Associated Small-Vessel Vasculitis

Clinical, in vitro, and experimental animal observations indicate that antineutrophil cytoplasmic autoantibodies (ANCA) are pathogenic. The genesis of the ANCA autoimmune response is a multifactorial process that includes genetic predisposition, environmental adjuvant factors, an initiating antigen, and failure of T cell regulation. ANCA activate primed neutrophils (and monocytes) by binding to certain antigens expressed on the surface of neutrophils in specific inflammatory microenvironments. ANCA-activated neutrophils activate the alternative complement pathway, establishing an inflammatory amplification loop. The acute injury elicits an innate inflammatory response that recruits monocytes and T lymphocytes, which replace the neutrophils that have undergone karyorrhexis during acute inflammation. Extravascular granulomatous inflammation may be initiated by ANCA-induced activation of extravascular neutrophils, causing tissue necrosis and fibrin formation, which would elicit an influx of monocytes that transform into macrophages and multinucleated giant cells. Over time, the neutrophil-rich acute necrotizing lesions cause the accumulation of more lymphocytes, monocytes, and macrophages and produce typical granulomatous inflammation.

C5a Receptor (CD88) Blockade Protects against MPO-ANCA GN

Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO-induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO-induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.

A canine minidystrophin is functional and therapeutic in mdx mice

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder lacking a curative treatment. We wish to use the dystrophin-deficient golden retriever muscular dystrophy (GRMD) dog, a canine model of DMD, to investigate adeno-associated virus (AAV) vector-mediated minidystrophin gene therapy. The dog model is useful in evaluating vector dose requirement and immunological consequences owing to its large size and outbred nature. In this study, we have cloned and constructed a canine minidystrophin gene vector. Owing to limited availability of the GRMD dogs, here we first examined the functions and therapeutic effects of the canine minidystrophin in the mdx mouse model. We observed efficient minigene expression without cellular immune responses in mdx mice after AAV1-cMinidys vector intramuscular injection. We also observed restoration of the missing dystrophin-associated protein complex (DPC) onto the sarcolemma, including sarcoglycans and dystrobrevin, and a partial restoration of α-syntrophin and neural nitric oxide synthase (nNOS). In addition, minidystrophin treatment ameliorated dystrophic pathology, such as fibrosis and myofiber central nucleation (CN). CN remained minimal (<2%) after AAV injection in the neonatal mdx mice and was reduced from more than 75% to about 25% after AAV injection in adult mdx mice. Finally, in vivo cell membrane leakage test with Evans blue dye showed that the canine minidystrophin could effectively protect the myofiber plasma membrane integrity. Our results, thus, demonstrated the functionality and therapeutic potential of the canine minidystrophin and paved its way for further testing in the GRMD dog model.

Stabilization of Plakoglobin and Enhanced Keratinocyte Cell-Cell Adhesion by Intracellular O-Glycosylation

O-Glycosylation modifies and regulates a variety of intracellular proteins. Plakoglobin, which functions in both cell-cell adhesion and signal transduction, is modified by O-glycosylation; however, the significance is unknown. To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and overexpressed in keratinocytes murine O-GlcNAc transferase (mOGT). Over expression of mOGT in murine keratinocytes resulted in (i) glycosylation of plakoglobin and (ii) increased levels of plakoglobin due to post-translational stabilization of plakoglobin. Additionally, overexpression of mOGT in keratinocytes correlated with increased staining for cell-cell adhesion proteins and greater cell-cell adhesion. These observations suggest that O-glycosylation functions to regulate the post-translational stability of plakoglobin and keratinocyte cell-cell adhesion.

Complement in ANCA-Associated Vasculitis

Antineutrophil cytoplasmic autoantibodies (ANCA) are the likely cause for necrotizing small-vessel vasculitis and crescentic glomerulonephritis. Unlike other forms of crescentic glomerulonephritis induced by immune complexes or anti-glomerular basement membrane antibodies that have conspicuous vessel wall immunoglobulin and complement, there is a paucity, although usually not an absence, of vessel wall immunoglobulin and complement in ANCA-associated glomerulonephritis. Despite this comparatively lower level and more localized distribution of vessel wall complement, experimental and clinical observations strongly incriminate alternative complement pathway activation as critically important in the pathogenesis of ANCA disease. Experimental data in animal models and in vitro experiments has shown that primed neutrophils are activated by ANCA, which generates C5a, which engages C5a receptors on neutrophils. This attracts and in turn primes more neutrophils for activation by ANCA. In patients with ANCA disease, plasma levels of C3a, C5a, soluble C5b-9, and Bb have been reported to be higher in active disease than in remission, whereas no difference was reported in plasma C4d in active versus ANCA disease remission. Thus, experimental and clinical data support the hypothesis that ANCA-induced neutrophil activation activates the alternative complement pathway and generates C5a. C5a not only recruits additional neutrophils through chemotaxis but also primes neutrophils for activation by ANCA. This creates a self-fueling inflammatory amplification loop that results in the extremely destructive necrotizing vascular injury.

Overview of the Pathogenesis of ANCA-Associated Vasculitis

Background: Antineutrophil cytoplasmic autoantibodies (ANCA) are associated with a spectrum of necrotizing vasculitis including granulomatosis with polyangiitis, microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, and renal-limited necrotizing and crescentic glomerulonephritis. Clinical observations and in vitro and in vivo experimental evidence strongly indicate that ANCA are pathogenic. Summary: The etiology and pathogenesis of ANCA-associated vasculitis (AAV) are multifactorial, with contributions from genetic factors, environmental exposures, infections, characteristics of the innate and adaptive immune system, and the intensity and duration of the injury. Acute vascular inflammation is induced when resting neutrophils that have ANCA autoantigens sequestered in cytoplasmic granules are exposed to priming factors - for example, cytokines induced by infection or phlogogenic factors released by complement activation - that cause the release of ANCA antigens on the surface of neutrophils and in the microenvironment around the neutrophils. ANCA bind to these ANCA antigens, which activates neutrophils by Fcγ receptor engagement and F(ab′)2 binding at the neutrophil cell surface. ANCA-activated neutrophils release factors that activate the alternative complement pathway, which generates C5a, a chemoattractant for neutrophils; C5a also primes the arriving neutrophils for activation by ANCA. Activated neutrophils adhere to and penetrate vessel walls, and they release toxic oxygen radicals and destructive enzymes that cause apoptosis and necrosis of the neutrophils as well as of the adjacent vessel wall cells and matrix. Key Messages: Patients with active AAV have ongoing asynchronous onsets of countless acute lesions, with each lesion evolving through stereotypical phases within 1 or 2 weeks. Induction of remission results in termination of new waves of acute lesions and allows all lesions to progress to scarring or resolution.