Presence of autoantibodies against cytoplasmic proteins of neutrophil (c-ANCA) is a common finding in human diseases characterized by vasculitis. The main target for c-ANCA is proteinase-3 (PR3), a serine proteinase found in azurophili granules and secretory vesicles of the neutrophil (1). In patients with some autoimmune diseases like granulomatosis with polyangitis (Wegener's), PR3 expression on the neutrophil surface is high and correlates with disease severity (2). In healthy subjects expression of PR3 on resting neutrophil is low, medium or bimodal when both types of expression are present, but is inducible by exposure of neutrophil to proinflammatory cytokines, e.g. tumor necrosis factor (TNF-). This causes translocation of PR3 to the cell membrane, where it becomes available for c-ANCA binding (3). Neutrophil is well known as an important player in inflammation, but recent studies show that their role extends well beyond the traditional function as a professional phagocyte. It has been shown that under appropriate experimental conditions, human neutrophil can synthesize and secret a number of chemokines such as CXCL8, CXCL1, IP-10, MIG or MIP-1ß, which not only can modulate neutrophil behavior in an autocrine or paracrine action, but can also promote other immune cells activation and recruitment (4).

The classical model of ANCA-associated neutrophil activation assumes direct recognition of PR3 via Fab region of cANCA antibody and interaction of the Fc part of the antibody with immunoglobulin gamma receptors (FcRs) on neutrophil surface. This causes neutrophil activation, degranulation, generation of reactive oxygen intermediates and finally transmigration through the endothelial cell layer of the vessel (5). Although there is a lot of evidence supporting this mechanism, some important questions still remain unanswered. Development of new molecular biology techniques such as microarrays can contribute to better understanding of this processes. Despite the fact that some whole blood gene expression studies in patients with several types of ANCA-associated vasculitides have been already performed, none of them focused on specific neutrophil gene profile following c-ANCA stimulation (6). The disease is difficult to treat and may follow a rapidly progressing pulmonary-renal syndrome with alveolar haemorrhage and necrotizing glomerulonephritis (7). Any pharmacological targets in Wegener's granulomatosis, including molecules up-regulated by anti-PR3 autoantibodies in neutrophils, are of importance for development of new therapeutic strategies for the disease.


MATERIALS AND METHODS
Immunoglobulin G (IgG) purification

Antibodies were extracted from pooled serum samples stored at the collection of the local diagnostic laboratory as reference sera. Their originated from six well defined patients suffering granulomatosis with polyangiitis (anti-PR3 IgG>200 mU/L; anti-MPO<20 mU/L). The total IgG fraction was purified by ammonium sulfate precipitation followed by removal of other proteins using negative affinity adsorption (Melon Gel IgG Purification kits, Thermo Scientific, Rockford, USA). To remove a possible endotoxin contamination, samples were cleaned up using AffinityPak Endotoxin Removal Column (Pierce, Rockford, USA). Purity of IgG samples was assessed by SDS-PAGE electrophoresis. Concentration of total IgG following extraction and purification was determined by immunonephelometry (Siemens Dade Behring BN II Nephelometer, Munnich, Germany) and specific anti-PR3 IgG level was assessed by ELISA (anti-PR3 ELISA kit, EUROIMMUN Medizinische Labordiagnostika, Luebeck, Germany).

Neutrophil-enriched granulocyte isolation and stimulation

The study received ethical approval from the Bioethical Committee of Jagiellonian University. For enrollment of healthy blood donors, informed consent was obtained, and this non-interventional phase 1 in vitro study was performed in respect to Declaration of Helsinki considering confidentiality and lack of interest conflicts. Granulocytes were isolated from citrated blood of healthy donors (n=12, average age 30 years, 2 males and 10 females ) using dextran sedimentation and Histopaque (Sigma-Aldrich Chemical Co, St Louis, USA) centrifugation followed by hypotonic lysis of erythrocytes. Purity of neutrophil fraction was determinated by flow cytometry (>98%) and cells viability was verified by tryphan blue exclusion staining (>95%). Immediately after isolation, granulocytes were resuspended in Hanks balanced salts solution (HBSS) with calcium and magnesium containing 5% fetal bovine serum. Before stimulation experiments, neutrophils were primed with 2 ng/mL recombinant TNF-a (R&D Systems, Minneapolis, MN, USA) for 15 minute at 37°C. Primed neutrophils (3.5x106/well) were incubated with purified human IgG fraction containing native anti-PR3 (200 µg/ml) for 4 h at 37°C. To evaluate gene expression in neutrophils after anti-PR3 IgG stimulation, six independent experiments were performed. In each experiment neutrophils simulations were made in duplicates for two donors in parallel.

RNA isolation, reverse transcription and genes expression

Total cellular RNA was isolated using total RNA kit (A&A Biotechnology, Gdynia, Poland) as recommended by the manufacturer. Reverse transcription was done using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). cDNA from each experiment was pooled for two donors, two duplicates each, and relative expression of specific mRNA for genes studied was quantified using two low density expression arrays by 5'nuclease assay (TaqMan Low-density array inflammation panel; custom made panel - Applied Biosystems) using 7900HT fast real time PCR system (Applied Biosystems). Data were normalized to ribosomal 18S rRNA used as the endogenous control. Relative quantities were calculated with use of 2-Ct method, which reflects the ratio between abundance of transcripts after stimulation to the one at base. Two other transcripts, i.e. glyceraldehyde dehydrogenase-GAPDH and beta-actin - ACTB might be used as supplementary internal controls but showed greater variability than 18S rRNA control. Results were presented in comparison to TNF- primed but non anti-PR3 stimulated granulocytes.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 4.0 package (GraphPad Software Inc, San Diego, CA). Because of small size of the studied group and distribution of variables data which departed from the normal one, all comparisons were done using Wilcoxon's signed rank test for between the groups and Mann-Whitney's-U test for paired ones. For the same reason descriptive statistics was presented as medians and interquartile ranges (25th-75th percentile) of the fold-change in mRNA abundance. A conventional heat map of genes activated in cells by anti-PR3 was assembled using Matrix2png software, a freely available internet a tool for visualization of matrix data (8).


RESULTS
We stimulated TNF- primed neutrophil-enriched healthy donors granulocytes with anti-PR3. These was a purified IgG fraction obtained from clinically diagnosed patients affected by c-ANCA positive granulomatosis with vasculitides syndrome and verified for the presence of native anti-PR3 idiotype but negative for anti-MPO. Control experiments did not reveal any activity of the IgG fraction obtained by the same method from the sera of healthy donors, using the same experimental setup. Following exposure of the cells to specific c-ANCA for 4 h we used a commercial TaqMan low-density array to analyze expression of 147 genes involved in several pathways of inflammatory and immune response. We chose a single 4 hour time point on the basis of initial experiments (data not shown) and a literature queries on genes' expression profiles. Out of 147 measured transcripts, 128 genes expressed detectable levels of mRNA, while only 19 were not detectable or showed very low expression detected in some donors. By comparison of the expression profile between anti-PR3 IgG stimulated and non-stimulated cells, we observed up-regulation (>2 fold change in mRNA abundance) of 13 genes (CCL2, CXCL2, VCAM1, MMP9, PLCB4, PDE4C, PLA2G4C, RAC1, RHOA, IRAK1, CACNA1D, CACNB2, PTGDR), further 11 genes were up-regulated only in some donors (IL13, PF4, IL2RG, ITGB1, CD83, PLA2G7, ALOX12, AXNA1, AXNA5, LTA4H, MCR2) yet two others (HRH3 and PLA2G2D) were up-regulated in a few samples and undetectable in others (Fig. 1). Full list of analyzed genes and their corresponding protein products are presented in a Table 1 along with relative changes in their expression.

Fig. 1. Gene expression profile in neutrophil-enriched granulocytes stimulated with anti-PR3 IgG. Green color represents down-regulated genes, black color - genes with no change of expression and red color up-regulated genes. Undetectable genes were marked in white. To ensure that observed gene activation was specific for anti-PR3 neutrophil activation, similar experiments with IgG isolated using the same method from healthy volunteer were performed. No activation of neutrophils was observed following exposure to these IgG preparations (data not shown). Measurements were done on the cells pooled from two healthy, anti-PR3 negative donors.

Table 1. List of analyzed genes and their relative mRNA abundance changes following incubation of neutrophils with purified anti-PR3 (* significantly up-regulated genes, p<0.05, Wilcoxon signed rank test).

DISCUSSION
Polymorphonuclear leukocytes (neutrophils) are pivotal as an component of innate immune system. In this preliminary study we aimed to examine a molecular background of c-ANCA associated neutrophil activation. The majority of the studies addressing c-ANCAs induced neutrophil activation focused on reactive oxygen species production, chemotaxis and release of cytokines and chemokines. However, a little is still known about molecular mechanisms involved in regulation of neutrophils activation. Classical pathway of c-ANCA neutrophil activation assumes that both fragments of IgG, Fab binding to PR3 and Fc activating the immunoglobulin FcRs, are required for complete granulocyte activation. Neutrophil predominantly expresses two types of FcRs: RIIa (CD32) and RIIIb (CD16b), which have a different affinity for specific subclasses of IgG (9). Because PR3 is a protein not having a transmembrane domain (10), neutrophil activation caused by anti-PR3 Fab binding requires probably association with another, still unknown membrane docking protein. According to the current knowledge, the interaction between IgG and FcRs leads to activation of several pathways, like calcium signaling, phosphatidylinositol 3-kinase AKT and MAPK signaling pathways activation. Up-regulation of some genes like CACNA1D, CACNB2 or RAC1, RHOA and IRAK1 observed by us supports this findings, however up-regulation of PDE4C, PTGDR or PLCB4 suggests that G-protein signaling system may also contribute to the signal transduction in c-ANCA mediated neutrophil activation. In addition, we observed an increase in the expression of NF-B-dependent proinflammatory chemokines CCL2I and CXCL2 (11). It is of interest, that if confirmed by the chemokines measurements in serum of patients or the culture supernatant, this would strongly suggest, that activated neutrophil can participate in further recruitment of other inflammatory cells, like monocytes (12). A bioinformatic database search (13) on the transcipts consistently up-regulated in neutrophils by the native anti-PR3 IgG showed their clustering into five major functional pathways of: chemokine signaling (PLCB4, CCL2, CXCL2, RAC1, RHOA; p<0.001), leukocyte transendothelial migration (VCAM1, MMP9, RAC1, RHOA; p=0.0017), vascular smooth muscle contraction (PLCB4, RHOA, CACNA1D; p=0.023), neurotrophin signaling pathway (IRAK1, RAC1, RHOA, p=0.028) and Wnt signaling pathway (PLCB4, RAC1, RHOA; p=0.04). Among these, chemokine signaling, leukocyte transendothelial migration and vascular smooth muscle contraction are known to contribute in granulomatosis with polyangiitis. Out of the four transcripts, not linked to the pathways, cytosolic phospholipase A2 (PLA2G4C) and prostaglandin D2 receptor (PTGDR) are related to prostaglandin D2 mediated vasoconstriction and vascular leak, while voltage dependent calcium channel subunit 2 (CACNB2) and phosphodiesterase 4C (PDE4C) have direct impact on activation status of neutrophil by mediating calcium entry and decreasing cytosolic cAMP level. It was recently demonstrated, that superoxide anion radical mimics vasoconstriction induced by activated neutrophils (14). Oxidative burst is one of the main innate defense mechanism of neutrophils against bacterial pathogens. Thus, up-regulation of CACNB2 and PDE4C in parallel with pro-inflammatory signaling would suggest, that anti-PR3 can sensitize neutrophil to oxidative burst response.

Circulating neutrophils are the major source of matrix metaloproteases (MMPs), however, MMP9 gene is expressed rather during the early stage of neutrophil maturation, and synthesized MMP9 protein is stored in cytoplasmic granules (15). However, Nagaoka et al. showed that under specific inflammatory conditions, MMP9 expression level can increase in neutrophils due to de novo production following the gene activation (16). Our results seem to support these observation. In our experiments, anti-PR3 stimulated neutrophils showed increased levels of MMP9 mRNA.

In addition to up-regulation of genes involved into known FcRs-dependent signaling cascades, we detected an increased expression of vascular cell adhesion molecule-1 (VCAM-1) transcripts. Vascular cell adhesion molecule-1 is expressed on activated endothelial cells (17, 18) and further upregulated by proinflammatory stimuli, reactive oxygen species, or by anti-PR3 antibodies. By the interaction with integrin receptors present on neutrophil surface, VCAM-1 can initiate both rolling-type and a firm adhesion interactions between leukocytes and endothelia (19). Our observation, that neutrophils are also capable of expression of mRNA for VCAM-1 is surprising, and suggests that docking of granulocyte to vascular endothelial cells may be reinforced upon specific anti-PR3 activation of the granulocyte itself, allowing for cellular aggregates. It is probably of importance that we did not observe this transcript in non-stimulated granulocytes in most of analyzed donors (Fig. 2). mRNA for VCAM-1 was easy detectable following stimulation with anti-PR3, and two donors, whose VCAM-1 mRNA was detected in non-stimulated cells, responded with 10-fold up-regulation in stimulated neutrophils.

Fig. 2. Change in vascular cell adhesion molecule-1 (VCAM-1) transcripts abundance following anti-PR3 simulation of neutrophil-enriched granulocytes. Results are presented as real-time polymerase chain reaction threshold cycle standardized to 18S mRNA (VCAM1CT-18SCT). Median value of Ct VCAM-1 in non stimulated neutrophils is significantly higher than in anti-PR3 antibody stimulated cells (28.44 vs. 26.11; p<0.05).

In summary, we demonstrated that c-ANCA mediated activation of neutrophils has a profound impact on mRNA expression of most genes involved into inflammatory response. Changes in mRNA suggest signal transduction not only via FcRs signaling system, but also with involvement of other pathways, like G-proteins. Neutrophil is a very sensitive cell, responding to many environment changes. Our results documented also a substantial variability of anti-PR3 responses across donors, despite the fact that experiments were done on total RNA pooled from two individuals. Nevertheless, an important finding of increased VCAM-1 mRNA following anti-PR3 stimulation seems constant for all replicates of our experiments. Whether this is reflected by expression of this adhesion molecule on the cell surface, will require flow cytometry confirmation. The role of VCAM-1 in endothelium and smooth muscle cells is studied extensively. It contributes to the pathomechanism of atheromatosis, promoting inflammatory cells infiltration to the vascular wall. It would be highly interesting to test, if a specific granulocyte vasculitis can develop due to presence of circulation anti-PR3 autoantibodies by a mechanism of mutual granulocyte activation and recruitment due to their expression of VCAM-1.

Conflict of interests: None declared.


REFERENCES

1. Hu N, Westra J, Kallenberg CG. Membrane-bound proteinase 3 and its receptors: relevance for the pathogenesis of Wegener's granulomatosis. Autoimmun Rev 2009; 8: 510-514.
2. Muller Kobold AC, Kallenberg CG, Tervaert JW. Leucocyte membrane expression of proteinase 3 correlates with disease activity in patients with Wegener's granulomatosis. Br J Rheumatol 1998; 37: 901-907.
3. Harper L, Radford D, Plant T, Drayson M, Adu D, Savage CO. IgG from myeloperoxidase-antineutrophil cytoplasmic antibody-positive patients stimulates greater activation of primed neutrophils than IgG from proteinase 3-antineutrophil cytosplasmic antibody-positive patients. Arthritis Rheum 2001; 44: 921-930.
4. Wright HL, Moots RJ, Bucknall RC, Edwards SW. Neutrophil function in inflammation and inflammatory diseases. Rheumatology (Oxford) 2010; 49: 1618-1631.
5. Rarok AA, Limburg PC, Kallenberg CG. Neutrophil-activating potential of antineutrophil cytoplasm autoantibodies. J Leukoc Biol 2003; 74: 3-15.
6. Alcorta DA, Barnes DA, Dooley MA, et al. Leukocyte gene expression signatures in antineutrophil cytoplasmic autoantibody and lupus glomerulonephritis. Kidney Int 2007; 72: 853-864.
7. Zycinska K, Wardyn KA, Zielonka TM, Otto M. The role of ANCA and anti-GBM antibodies in pulmonary-renal syndrome due to Wegener's granulomatosis. J Pharmacol Physiol 2007; 58(Suppl. 5): 839-846.
8. Pavlidis P, Noble WS. Matrix2png: a utility for visualizing matrix data. Bioinformatics 2003; 19: 295-296.
9. Dijstelbloem HM, van de Winkel JG, Kallenberg CG. Inflammation in autoimmunity: receptors for IgG revisited. Trends Immunol 2001; 22: 510-516.
10. van der Geld YM, Limburg PC, Kallenberg CG. Proteinase 3, Wegener's autoantigen: from gene to antigen. J Leukoc Biol 2001; 69: 177-190.
11. Thompson WL, Van Eldik LJ. Inflammatory cytokines stimulate the chemokines CCL2/MCP-1 and CCL7/MCP-3 through NFkB and MAPK dependent pathways in rat astrocytes. Brain Res 2009; 1287: 47-57.
12. Soehnlein O, Zernecke A, Weber C. Neutrophils launch monocyte extravasation by release of granule proteins. Thromb Haemost 2009; 102: 198-205.
13. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009; 4: 44-57.
14. Bauer V, Sotnikova R, Drabikova K. Effects of activated neutrophils on isolated rings of rat thoracic aorta. J Physiol Pharmacol 2011; 62: 513-520.
15. Atkinson JJ, Senior RM. Matrix metalloproteinase-9 in lung remodeling. Am J Respir Cell Mol Biol 2003; 28: 12-24.
16. Nagaoka I, Hirota S. Increased expression of matrix metalloproteinase-9 in neutrophils in glycogen-induced peritoneal inflammation of guinea pigs. Inflamm Res 2000; 49: 55-62.
17. Osborn L, Hession C, Tizard R, et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 1989; 59: 1203-1211.
18. Bryk D, Zapolska-Downar D, Malecki M, Hajdukiewicz K, Sitkiewicz D. Trans fatty acids induce a proinflammatory response in endothelial cells through ROS-dependent nuclear factor- B activation. J Physiol Pharmacol 2011; 62: 229-238.
19. Chen C, Mobley JL, Dwir O, et al. High affinity very late antigen-4 subsets expressed on T cells are mandatory for spontaneous adhesion strengthening but not for rolling on VCAM-1 in shear flow. J Immunol 1999; 162: 1084-1095.