Genetics and pathophysiology of granulomatosis with polyangiitis (GPA) and its main autoantigen proteinase 3.

Granulomatosis with polyangiitis (GPA) is a severe autoimmune disease and one of the small vessel anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Although its etiology and pathophysiology are still widely unknown, it is accepted that infections, environmental factors, epigenetic modifications, and a genetic predisposition provide the basis for this systemic disorder. GPA typically evolves into two phases: an initial phase characterized by ear, nose and throat (ENT) manifestations, such as chronic sinusitis and otitis, ulceration of the oral cavity and pharynx, as well as pulmonary nodules and a severe generalized phase, defined by the occurrence of rapidly progressive glomerulonephritis, pulmonary hemorrhage, and arthritis. ANCAs, directed against the neutrophilic enzymes proteinase 3 and myeloperoxidase, are present in up to 90% of the affected patients in the systemic phase. As the humoral immunity is predominantly directed against neutrophilic antigens, it is apparent that neutrophils play a critical role in GPA both as target and effector cells. Although GPA pathogenesis is not well known, some susceptibility genes and loci have been identified by candidate gene approaches, genome-wide association studies, and meta-analyses, as well as familial association studies. Such genes are CTLA4, PTPN22, COL11A2, SERPINA1, and the MHC class II gene cluster. This review highlights the clinical, pathophysiological, and genetic background of GPA and aims to give an overview of recent efforts to identify GPA susceptibility genes. We point out the genetic basis of the main autoantigen PR3 and why it is so difficult to establish a murine GPA model.

Mast cell-deficient Kit(W-sh) "Sash" mutant mice display aberrant myelopoiesis leading to the accumulation of splenocytes that act as myeloid-derived suppressor cells.

Mast cell-deficient Kit(W-sh) "sash" mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that Kit(W-sh) causes aberrant extramedullary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor-bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice, accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell-independent phenomenon. Thus, the Kit(W-sh) mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research.

Intronic promoters and their noncoding transcripts: a new source of cancer-associated genes.

Recent studies of mammalian genomes suggest that alternative promoters are associated with various disorders, including cancer. Here we present an intronic promoter of the murine proteinase 3 gene, which drives the expression of an alternative mRNA in intron 2 of the prtn3 gene. The proximal promoter sequences were identified and a series of promoter deletion constructs were used to identify the sequence elements that are required for basal promoter activity. Expression of the homeobox transcription factor CUX1 p75 isoform was found to suppress the activity of the alternative PR3 promoter. Data base analyses, multiple alignments and expression data showed that the intronic PR3 promoter is active in leukemia and other tumor cells as well as in mouse embryo, male mammary gland and bone marrow. In the spleen, the transcript is exclusively expressed by Gr-1(int) /CD11b(+) cells, which are also known as myeloid-derived suppressor cells (MDSCs). In humans, an alternative transcript of the PR3-gene could be detected in the bone marrow and in various cancer cell lines but not in primary leukemia cells, suggesting a species-overarching function of this kind of promoter. Therefore, the alternative PR3 promoter and its mRNA may be useful tools to investigate the fate of hematopoietic stem cells.

Mast cell-derived mediators promote murine neutrophil effector functions.

Mast cells are able to trigger life-saving immune responses in murine models for acute inflammation. In such settings, several lines of evidence indicate that the rapid and protective recruitment of neutrophils initiated by the release of mast cell-derived pro-inflammatory mediators is a key element of innate immunity. Herein, we investigate the impact of mast cells on critical parameters of neutrophil effector function. In the presence of activated murine bone marrow-derived mast cells, neutrophils freshly isolated from bone marrow rapidly lose expression of CD62L and up-regulate CD11b, the latter being partly driven by mast cell-derived TNF and GM-CSF. Mast cells also strongly enhance neutrophil phagocytosis and generation of reactive oxygen species. All these phenomena partly depend on mast cell-derived TNF and to a greater extend on GM-CSF. Furthermore, spontaneous apoptosis of neutrophils is greatly diminished due to the ability of mast cells to deliver antiapoptotic GM-CSF. Finally, we show in a murine model for acute lung inflammation that neutrophil phagocytosis is impaired in mast cell-deficient Kit (W-sh) /Kit (W-sh) mice but can be restored upon mast cell engraftment. Thus, a previously underrated feature of mast cells is their ability to boost neutrophil effector functions in immune responses.

PR3 antibodies do not induce renal pathology in a novel PR3-humanized mouse model for Wegener's granulomatosis.

Different murine models have been used as basis for Proteinase 3 (PR3)-associated vasculitis models, but sufficient reproduction of the human clinical manifestation has failed to this date. As a reliable animal model is needed to further elucidate the pathological value of PR3-ANCA, we developed a PR3-humanized transgenic mouse model, in order to induce a glomerulonephritis. Our huPR3-transgenic mice were injected i.v. with our monoclonal antibodies, either unlabeled or directly labeled by fluorescein isothiocyanate. For a period of 5 days, proteinuria and erythrocyte count were measured with urine dip sticks. None of the mice exhibited proteinuria and/or an abnormal number of erythrocytes in the urine. Five days after antibody treatment, the mice were killed and different organs were fixed and immunohistochemically assessed. In the case of the kidney, we could detect a glomerulonephritis. Our study is able to show that although a direct renal target was given in transgenic human PR3 mice, no renal pathology was detectable. Multifactorial mechanisms for PR3-ANCA involvement in the development of Wegener's granulomatosis must be hypothesized.

Role of MHC-linked susceptibility genes in the pathogenesis of human and murine lupus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies against nuclear antigens and a systemic inflammation that can damage a broad spectrum of organs. SLE patients suffer from a wide variety of symptoms, which can affect virtually almost any tissue. As lupus is difficult to diagnose, the worldwide prevalence of SLE can only be roughly estimated to range from 10 and 200 cases per 100,000 individuals with dramatic differences depending on gender, ethnicity, and location. Although the treatment of this disease has been significantly ameliorated by new therapies, improved conventional drug therapy options, and a trained expert eye, the underlying pathogenesis of lupus still remain widely unknown. The complex etiology reflects the complex genetic background of the disease, which is also not well understood yet. However, in the past few years advances in lupus genetics have been made, notably with the publication of genome-wide association studies (GWAS) in humans and the identification of susceptibility genes and loci in mice. This paper reviews the role of MHC-linked susceptibility genes in the pathogenesis of systemic lupus erythematosus.

Targeting transcription factor Stat4 uncovers a role for interleukin-18 in the pathogenesis of severe lupus nephritis in mice.

Polymorphisms in the transcription factor Stat4 gene have been implicated as risk factors for systemic lupus erythematosus. Although some polymorphisms have a strong association with autoantibodies and nephritis, their impact on pathophysiology is still unknown. To explore this further we used signal transducers and activators of transcription 4 (Stat4) knockout MRL/MpJ-Fas(lpr)/Fas(lpr) (MRL-Fas(lpr)) mice and found that they did not differ in survival or renal function from Stat4-intact MRL-Fas(lpr) mice. Circulating interleukin (IL)-18 levels, however, were elevated in Stat4-deficient compared to Stat4-intact mice, suggesting that this interleukin might contribute to the progression of lupus nephritis independent of Stat4. In a second approach, Stat4 antisense or missense oligonucleotides or vehicle were given to MRL-Fas(lpr) mice with advanced nephritis. Each of these treatments temporarily ameliorated disease, although IL-18 was increased in each setting. Based on these findings, studies using gene transfer to overexpress IL-18 in MRL-Fas(lpr) and IL-12p40/IL-23 knockout MRL-Fas(lpr) mice reveal a critical role for IL-18 in mediating disease. Thus, the Stat4 and IL-12 (an activator of Stat4)-independent factor, IL-18, can drive autoimmune lupus nephritis in MRL-Fas(lpr) mice. Temporarily blocking Stat4 during advanced nephritis ameliorates disease, suggesting a time-dependent compensatory proinflammatory mechanism.

New perspectives on the renal slit diaphragm protein podocin.

Podocin is a critical component of the glomerular filtration barrier, its mutations causing recessive steroid-resistant nephrotic syndrome. A GenBank analysis of the human podocin (NPHS2) gene resulted in the possible existence of a new splice variant of podocin in the kidney, missing the in-frame of exon 5, encoding the prohibitin homology domain. Using RT-polymerase chain reaction and immunoblotting followed by sequence analysis, we are for the first time able to prove the expression of a novel podocin isoform (isoform 2), exclusively and constitutively expressed in human podocytes. Furthermore, we reveal singular extrarenal podocin expression in human and murine testis. Our data show the Sertoli cells of the seminiferous tubules to be the origin of testicular podocin. Confocal laser microscopy illustrates the co-localization of podocin with filamentous actin within Sertoli cells, suggesting a role of podocin in the blood/testis barrier. These results led to the rationale to examine podocin expression in testes of men with Sertoli cell-only syndrome, a disorder characterized by azoospermia. Interestingly, we observed a complete down-regulation of podocin mRNA in Sertoli cell-only syndrome, indicating a possible role of podocin in the pathogenesis of this germinal aplasia. Men with Sertoli cell-only syndrome show normal renal podocin expression, suggesting an alternate regulation of the testicular promoter. Our findings may change the perception of podocin and give new insights into the ultrastructure of glomerular slit diaphragm and the blood/testis barrier.

Comparative aspects of murine proteinase 3.

The neutrophilic granule protein proteinase 3 (PR3) is the preferred target antigen of anti-neutrophil cytoplasmic antibodies (ANCA) found in the serum of patients with Wegener's granulomatosis, a systemic small-vessel vasculitis. Due to the lack of data concerning the murine homologue of human PR3, we assessed the neutrophil marker polypeptide PR3 in mice by generating a murine-specific PR3 antibody. In contrast to humans, peripheral blood leukocytes are not the main resource of murine PR3. Interestingly, we could show that the mouse bone marrow is the main PR3 source, indicating that it is a large reservoir for functional neutrophils. This pool of neutrophils could be rapidly mobilized after injection of IL-8. The development of the new PR3 antibody provides a new tool for studying the maturation processes of the murine hematopoietic system and will also support the generation of infectious disease or vasculitis mouse models.

Downregulation of alpha-galactosidase A upregulates CD77: functional impact for Fabry nephropathy.

Anderson-Fabry disease, an inherited deficiency in the lysosomal enzyme alpha-galactosidase A, is characterized by the progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. We sought to clarify the pathogenesis of Fabry disease by establishing a cell model of this disorder. The expression of alpha-galactosidase A was transiently silenced by RNA interference in HK2 and primary human renal epithelial cells and stably silenced in HK2 cells by retroviral transfection with small hairpin RNA. All of the silenced cells had histological similarities to cells of patients with Fabry disease. The cells had reduced viability, significant accumulation of intracellular Gb3, and a modest but significant increase in membranous Gb3 expression compared to nonsilenced cells. When silenced HK2 cells were reconstituted with agalsidase-alpha, a protein used for enzyme replacement therapy, they decreased their membranous CD77 expression to levels indistinguishable from those of nonsilenced cells. Because plasma and urinary Gb3 levels are not reliable biomarkers for Fabry disease, our study suggests that membranous CD77 levels mirror Gb3 tissue load and that CD77 expression levels may be used to monitor the efficacy of enzyme replacement therapy.

Epigenetic Aspects of Systemic Lupus Erythematosus.

Autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis, autoimmune hepatitis, and inflammatory bowel disease have complex pathogeneses and the courses of events leading to these diseases are not well understood. The immune surveillance is a delicate balance between self and foreign as well as between tolerance and immune response. Exposure to certain environmental factors may impair this equilibrium, leading to autoimmune diseases, cancer, and the so-called "lifestyle diseases" such as atherosclerosis, heart attack, stroke, and obesity, among others. These external stimuli may also alter the epigenetic status quo and may trigger autoimmune diseases such as SLE in genetically susceptible individuals. This review aims to highlight the role of epigenetic (dys-)regulation in the pathogenesis of SLE.

Genetics and novel aspects of therapies in systemic lupus erythematosus.

Autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, autoimmune hepatitis and inflammatory bowel disease, have complex pathogeneses and the factors which cause these disorders are not well understood. But all have in common that they arise from a dysfunction of the immune system, interpreting self components as foreign antigens. Systemic lupus erythematosus (SLE) is one of these complex inflammatory disorders that mainly affects women and can lead to inflammation and severe damage of virtually any tissue and organ. Recently, the application of advanced techniques of genome-wide scanning revealed more genetic information about SLE than previously possible. These case-control or family-based studies have provided evidence that SLE susceptibility is based (with a few exceptions) on an individual accumulation of various risk alleles triggered by environmental factors and also help to explain the discrepancies in SLE susceptibility between different populations or ethnicities. Moreover, during the past years new therapies (autologous stem cell transplantation, B cell depletion) and improved conventional treatment options (corticosteroids, traditional and new immune-suppressants like mycophenolate mofetile) changed the perspective in SLE therapeutic approaches. Thus, this article reviews genetic aspects of this autoimmune disease, summarizes clinical aspects of SLE and provides a general overview of conventional and new therapeutic approaches in SLE.

Proteinase 3/myeloblastin as a growth factor in human kidney cells.

BACKGROUND: Numerous data support the possible role of myeloblastin/proteinase 3 (PR3) in growth and differentiation of neutrophil granulocytes and certain monocyte subtypes. However, whether PR3 is expressed in non-myeloid cells remains a matter of debate even though recent studies clearly demonstrated its expression in endothelium, kidney epithelial cells and epithelial tumor cell lines. METHODS: To survey PR3 transcript presence in human tissues, we analyzed different human tissues by dot blot and northern blot using a cloned PR3-cDNA probe. To examine the physiological function of PR3 expression in non-myeloid cells, we constructed different recombinant retroviral vectors containing human PR3-cDNA variants and expressed them in tubular epithelial cells (TEC). Using an MTT-based proliferation assay, we determined the proliferation rate of PR3-transduced kidney cells. RESULTS: The resulting expression pattern clearly indicated that PR3 transcripts are not only present in tissues known to harbor hematopoietic cells, but surprisingly PR3 was highly expressed in fetal organs including the kidney. The proliferation assay revealed that the growth rate of TEC transduced with native PR3 was significantly enhanced relative to non-transduced TEC. CONCLUSIONS: The results supported our theory that PR3 can act as a growth factor in non-hematopoietic cells, analogous to its role in hematopoietic cells. The cells, recombinant vectors and methods described here serve as a basis to investigate PR3 function in cellular differentiation and proliferation, as well as its role in autoimmune diseases.

Interleukin 6 (IL-6) deficiency delays lupus nephritis in MRL-Faslpr mice: the IL-6 pathway as a new therapeutic target in treatment of autoimmune kidney disease in systemic lupus erythematosus.

OBJECTIVE: To investigate the pathophysiological effect of interleukin 6 (IL-6) on lupus nephritis in MRL-Fas(lpr) mice. METHODS: We generated IL-6-deficient MRL-Fas(lpr) mice using a backcross/intercross breeding scheme. Renal pathology was evaluated using immunohistochemistry detection for macrophages, lymphocytes, vascular cell adhesion molecule-1 (VCAM-1), and TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end-labeling) for apoptotic cells, and renal IgG and C3 deposition by immunofluorescence staining. Expression of inflammatory markers in the spleen was analyzed by quantitative real-time reverse transcription-polymerase chain reaction. Serum cytokine concentrations were detected by FACS analysis. RESULTS: IL-6 deficiency was highly effective in prolonging survival and ameliorating the clinical, immunological, and histological indicators of murine systemic lupus erythematosus. During the study period of 6 months, MRL-Fas(lpr) IL-6 -/- mice showed delayed onset of proteinuria and hematuria compared to IL-6-intact control mice. Survival rate was 100% in IL-6-deficient MRL-Fas(lpr) mice and 25% in the control group at 6 months of age. The absence of IL-6 resulted in significant reduction of infiltrating macrophages in the kidney (p < 0.05), a decrease in renal IgG and C3 deposition, and a reduction of CD4+ and CD8+ lymphocytes. The parenchymal adhesion molecule VCAM-1 was found to be downregulated in kidneys of MRL-Fas(lpr) IL-6 -/- compared to IL-6-intact mice. We found elevated serum levels of IL-10 and interferon-gamma in IL-6-deficient mice, while splenic mRNA showed an overall downregulation of immunoregulatory genes. CONCLUSION: IL-6 is a strong promoter of lupus nephritis and may be a promising new therapeutic target in the treatment of human lupus nephritis.

[Effect of enzyme replacement therapy (ERT) on renal function of patients with Fabry's disease]. / Wirkung der Enzymersatztherapie (ERT) auf die Nierenfunktion von Patienten mit Morbus Fabry.

Fabry's disease is an inherited lysosomal storage disorder characterized by the lack of enzyme alpha-galactosidase A (alpha-Gal A) which degrades globotriaosylceramides (Gb3) into products with lower molecular weight. The accumulation of Gb3 in different cell types is responsible for the variety of clinical manifestations. The renal function, estimated via proteinuria, hematuria and reduction of glomerular filtration rate (GRF), is heavily affected. Currently, substitution of alpha-Gal A remains the only therapeutic option for patients with Fabry's disease. Two products are approved for the treatment of Fabry's disease: agalsidase alfa and agalsidase beta. Both of these enzymes have shown a stabilization of renal function in various studies when evaluated by the creatinine clearance, estimated GFR, and serum creatinine. The pro gnosis has proven to be significantly better in cases of mild or moderate renal insufficiency from the baseline. For this reason, an early substitution of the lacking enzyme is necessary. Furthermore, enzyme replacement therapy (ERT) has proven efficient in reducing the amount of intracellular Gb3 and Gb3 in urine. Without treatment, an eGFR reduction of approximately 12 ml/min/year has been reported. After diverse studies of ERT, no significant correlation between enzyme substitution and improvement of patients' proteinuria could be shown. Furthermore, renoprotective drugs have not been consistently applied so far in the ERT trials. In any case, further studies to evaluate the long-term effect of ERT on the morbidity and mortality of patients with Fabry's disease are necessary.

Interferon-beta: a therapeutic for autoimmune lupus in MRL-Faslpr mice.

Type I interferons are associated with lupus. Genes that are regulated by IFN-alpha are upregulated in pediatric lupus patients. Gene deletion of the IFN-alpha/beta receptor in experimental lupus-like NZB mice results in reduced disease activity. Conversely, IFN-beta is a well-established treatment in multiple sclerosis, another autoimmune disease. For determining whether IFN-beta treatment is harmful or beneficial in lupus, MRL-Fas(lpr) mice were injected with this type I IFN. Treatment was initiated in MRL-Fas(lpr) mice with mild and advanced disease. IFN-beta was highly effective in prolonging survival and ameliorating the clinical (renal function, proteinuria, splenomegaly, and skin lesions), serologic (autoantibodies and cytokines), and histologic parameters of the lupus-like disease in mice that had mild and advanced disease. Several underlying mechanisms of IFN-beta therapy involving cellular (decreased T cell proliferation and infiltration of leukocytes into the kidney) and humoral (decrease in IgG3 isotypes) immune responses and a reduction in nephrogenic cytokines were identified. In conclusion, IFN-beta treatment of lupus nephritis in MRL-Fas(lpr) mice is remarkably beneficial and suggests that IFN-beta may be an appealing therapeutic candidate for subtypes of human lupus.