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Gene expression profiling in nerve biopsy of vasculitic neuropathy
Journal of Neuroimmunology 225 (2010) 184–189
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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
Gene expression profiling in nerve biopsy of vasculitic neuropathy Jochen Kinter a,b, Laura Broglio a,b, Andreas J. Steck a,b,⁎, Markus Tolnay c, Peter Fuhr a,b, Norman Latov d, Daniel Kalbermatten e, Michael Sinnreich a,b, Nicole Schaeren-Wiemers a,b, Susanne Renaud a,b a
Department of Biomedicine, University Hospital Basel, Basel, Switzerland Department of Neurology, University Hospital Basel, Basel, Switzerland Institute of Pathology, University Hospital Basel, Basel, Switzerland d Department of Neurology and Neurosciences, Weill Medical College, Cornell University, New York, NY, USA e Division of Plastic Surgery, University Hospital Basel, Petersgraben 4,CH-4031 Basel, Switzerland b c
a r t i c l e
i n f o
Article history: Received 9 March 2010 Received in revised form 4 May 2010 Accepted 5 May 2010 Keywords: Neuropathy Vasculitis Gene expression
a b s t r a c t To investigate molecular mechanisms of peripheral nerve vasculitis, gene expression patterns in archived frozen sural nerve biopsies from patients with vasculitic neuropathy were compared to control nerves by DNA microarray technology. There was a striking upregulation of mRNA of genes involved in immune system processes. Of special interest was the activation of immunoglobulin genes, such as IGLJ3, IGHG3, IGKC, and IGL, and of several chemokines, such as CXCL9 or CCR2. Genes involved in vascular proliferation or remodelling such as CXC31 and AIF were also upregulated. Among the downregulated genes were the Krüppel-Like Transcription Factors KLF2, KLF4 and the nuclear orphan receptor NR4A1 genes known to be involved in endothelial cell activation. Thus, this gene expression profile analysis revealed that in peripheral nerve vasculitis a prominent activation of immune response related genes as well as genes involved in vascular proliferation is taken place, while genes inhibiting endothelial cell activation are down regulated. These data point to interesting mechanistic clues to the molecular pathogenesis of vasculitic neuropathies. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Peripheral neuropathy is a common complication of vasculitic disease (Said and Lacroix, 2005). Vasculitic neuropathy occurs as a primary or as a secondary phenomenon and can be restricted to the peripheral nervous system only. Various underlying pathogenic mechanisms, such as immune complex deposition, cell mediated interactions, and antineutrophil cytoplasmic antibody (ANCA) associated processes are discussed and have been extensively reviewed (Collins and Periquet-Collins, 2009; Jennette and Falk, 2007; Said and Lacroix, 2005). In most cases the initiating event is unknown, and a self-sustaining circuit attracts and activates inflammatory leucocytes in the wall of vessels. Recent studies have revealed homeostatic roles of vascular inflammation and have identified the action of humoral innate immunity, in particular injury-associated signals and acute phase proteins, on the activation of circulating leucocytes, platelets and endothelial cells (Maugeri et al., 2009). To elucidate pathogenetic mechanisms of vasculitis most studies rely on immunohistochemistry, but more recently DNA microarray analysis evolved as a powerful method for differential expression analysis of genes in normal and diseased tissues (Kinter et al., 2008; Schena et al., 1995). ⁎ Corresponding author. Department of Neurology, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland. E-mail address: [email protected] (A.J. Steck). 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.05.023
In a gene expression study of the human peripheral nerve using the microarray technology (Renaud et al., 2005) we reported and discussed differentially expressed genes in patients with chronic inflammatory demyelinating polyneuropathy (CIDP). One finding was the upregulation of Allograft Inflammatory Factor-1 (AIF-1) in three vasculitic neuropathy patients of the disease control group. In a subsequent study, we identified the expression of AIF-1 predominantly in vascular smooth cells of vasculitis nerves, suggesting that AIF-1 might play an important pathogenetic role in the proliferation and migration of vascular smooth muscle cells and subsequent narrowing of vessel lumen in peripheral nerves affected by vasculitis (Broglio et al., 2008). To follow up and extend our initial study, we performed a gene expression analysis to demonstrate distinct gene expression patterns in archived frozen nerve biopsies of patients with vasculitic neuropathies compared to control nerve by DNA microarray analysis. 2. Material and methods 2.1. Patients Nerve biopsies from 7 patients with vasculitic neuropathy were included in this study. The diagnosis was made according to established criteria (Said and Lacroix, 2005). Two patients were suffering from virus associated nerve vasculitis, another had non-systemic vasculitic neuropathy and the remainder were suffering from nerve vasculitis due to systemic disease. As controls, biopsy specimens were obtained
J. Kinter et al. / Journal of Neuroimmunology 225 (2010) 184–189
185
Table 1 Control and vasculitis patients. Subjects
Sex
Age
Controls 1 2 3 4
F M M M
5 85 18 37
Patients 5 6 7 8 9 10 11
M F F M F F F
72 72 68 78 69 39 85
Time to Biopsy
Cinical characteristics
Blood markers
Pathology
Treatment
ENA, ANA SS-A, SS-B
Necrotizing vasculitis Perivasculitis Vasculitis Vasculitis Necrotizing vasculitis Necrotizing vasculitis Necrotizing vasculitis
None NSAID NSAID None None Antiretroviral Prednisone
Dermatomyositis Alzheimer Neurinoma extirpation Trauma
3 weeks 3 months 4 months 3 months Unknown 2 months 4 weeks
Multiple mononeuropathy, Churg–Strauss Vasculitis Asymmetric sensorimotor neuropathy, Sjögren's Syndrome Multiple mononeuropathy Non-sysemic vasculitis Multiple mononeuropathy Hepatitis B associated Multiple mononeuropathy MALT lymphoma of the gut Multiple mononeuropathy HIV associated, Hepatitis C Multiple mononeuropathy Rheumatoid arthritis
cANCA ANA CCP-Ab, RF
Summary of control and vasculitis patient data. Abbreviations: MALT = Mucous membrane Associated Lymphoid Tissue, HIV = human immunodeficiency virus, ENA = extractable nuclear antigen, ANA = antinuclear antibody, SS-A = Sjögren's syndrome antigen A, SS-B = Sjögren's syndrome antigen B, cANCA = antineutrophil cytoplasmic antibodies, CCP-Ab = cyclic citrullinated peptide antibody, RF = rheumatoid factor, NSAID = non-steroidal anti-inflammatory drugs.
from individuals who did not suffer from polyneuropathy, but from muscle dystrophy, dermatomyositis or who received reconstructive surgery in their legs. One control specimen was a sural nerve from an autopsy case. The characteristics and clinical data of patients with vasculitis are listed in Table 1. Paraffin-embedded and fresh frozen biopsy specimens from the cutaneous branch of the superficial peroneal nerve were obtained after routine work-up. Permission to use these tissues was obtained from our institutional ethics committee. Results of routine histopathological analysis of nerve biopsy specimens were drawn from case file reports (Supplementary Figs. 1 and 2). 2.2. RNA sample processing Human nerve biopsies were embedded in Tissue-Tek medium (Sakura Finetek, USA) immediately after the biopsy and stored at −70 °C. The embedded tissue was cut with a cryostat (Microm HM-560) in 10 μm sections. Tissue homogenization was obtained with an electronic rotor stator homogenizer (Polytron, Kinematica, Switzerland). Total RNA extraction was obtained using the TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturers protocol. After DNAse treatment (DNAse I Ambion, Huntingdom, Cambridgeshire, UK) total RNA was purified using Rneasy Kit (Qiagen, Chatsworth, CA). RNA yields were measured by the NanoDrop spectrophotometer (NAME) and its quality was assessed by the microfluidics-based platform Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Wash and Stain Kit (Affymetrix, Cat# 900720). To increase the signal strength the antibody amplification protocol was used (FS450_0002). The GeneChips were processed with an Affymetrix GeneChip® Scanner 3000 7G (Affymetrix) DAT image files of the microarrays were generated using GeneChip Operating Software (GCOS 1.4; Affymetrix). 2.4. Data analysis Data were analyzed using the RACE software and the Bayes test for statistical significance (Psarros et al., 2005). Normalization was performed using robust GeneChip multi-array analysis (gcRMA). A fold change as well as a p-value threshold was set for generating subsets of differentially expressed genes (Table 2). 3. Results 3.1. Differentially regulated genes Microarray analysis revealed pronounced alterations in gene expression in vasculitis nerve tissues. Considering a cut-off point for Table 2 Number of differentially expressed genes sorted by p-value stringency and fold changes. Fold change
2.3. cRNA amplification Total
Biotin labelling of RNA was performed as described in the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, USA). cRNA was synthesized according to the GeneChip Expression 3′ Amplification Two-Cycle Target labelling and Control Reagents (Affymetrix, Cat# 900494), starting from 50 ng of total RNA. On average 112 μg of labelled cRNA from each reaction was obtained, and average size of the cRNA molecules was assessed on RNA Nano 6000 Chips (2100 Bioanalyzer, Agilent). The cRNA targets were incubated at 94 °C for 35 min in the provided “Fragmentation Buffer” and the resulting fragments of 50–150 nucleotides were again monitored using the Bioanalyzer. All synthesis reactions were carried out using a PCR machine (T1 Thermocycler; Biometra, Göttingen, Germany) to ensure the highest possible degree of temperature control. The hybridization cocktail (130 μl) containing fragmented biotin-labelled target cRNA at a final concentration of 0.05 μg/μl was transferred into Affymetrix Human Genome U133A 2.0 Arrays and incubated at 45 °C on a rotator in a hybridization oven 640 (Affymetrix) for 16 h at 60 rpm. The arrays were washed and stained on a Fluidics Station 450 (Affymetrix) by using the Hybridization
p-value
–
1.5
2
5
b 0.05 b 0.01 b 0.005 b 0.001
1469 392 227 64
622 236 149 47
327 135 90 26
45 33 26 12
b 0.05 b 0.01 b 0.005 b 0.001
556 169 105 33
346 140a) 94 29
239 105 75b) 22
41 30 25 12d)
b 0.05 b 0.01 b 0.005 b 0.001
913 223 122 31
276 96a) 55 18
88 30 15c) 4
4 3 1 0
Up
Down
Normalized hybridization signal intensities from vasculitis cases were compared with the average intensity of all control cases, providing fold changes (ratio). The average of all ratios and the statistical significance was calculated and sorted according to particular stringencies. This analysis revealed that 1469 gene sequences were differentially expressed with a p-value below 0.05. Applying a significance level with a p-value below 0.001, still 64 gene sequences were differentially expressed. a) The data list of these gene sequences are provided as Supplementary data. b) Data are shown in Table 3A. c) Data are shown in Table 3B.d) Genes upregulated more than five fold and a p-value lower than 0.001.
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the p-value of p b 0.01 and a cut-off point for the fold change N1.5 (respectively b0.66 for downregulated genes), 140 genes were upregulated and 96 downregulated in vasculitis nerves (Table 2;a). Upregulated genes include genes involved in cytokine–cytokine receptor interaction, hematopoietic cell lineage, Toll-like receptor signalling pathway, cell adhesion pathway and natural killer cell mediated cytotoxicity (Supplementary data). More stringent conditions revealed 75 genes upregulated with more than two fold changes (p b 0.005, Table 2;b) and 15 genes downregulated (Table 2;c), while with an applied stringency with a statistical significance p b 0.001, still 12 genes showed an upregulation of more than 5 folds in vasculitis compared to control nerves (Table 2;d). The most striking observed alteration was the increased expression of genes involved in processes related to the immune system. Among the immune response genes there was an activation of immunoglobulin genes, the MHC II pathway, many immune receptors and their signalling pathways and the complement system (Table 3A). Among the most downregulated genes were the Krüppel-Like Transcription Factor (KLF), KLF4 and KLF2 (Table 3B). Also of interest is the decrease of the nuclear orphan receptor NR4A1 (Supplementary Table). 4. Discussion Our nerve biopsy samples reflect the spectrum of vasculitis encountered in clinical practice, including virus associated nerve vasculitis, non-systemic vasculitic neuropathy and nerve vasculitis due to systemic disease. In spite of this clinical heterogeneity, we believe that a pattern of differentially regulated gene emerges from the analysis of the data. This is in keeping with the hypothesis that there is a common pathogenic pathway in nerve vasculitis, even if the triggering mechanisms are not uniform (Maugeri et al., 2009). 4.1. Activation of cellular immune response genes Reflecting the strong cellular component of vasculitis there was activation of chemokines, such as CXCL9, a gamma interferon-induced monokine contributing to Th1-induced inflammation, and CCR2 a chemokine, which specifically mediates monocyte chemotaxis. Chemokines are a large family of more than 50 cytokines that bind to a smaller number of receptors. Their best known function is the regulation of cell migration, but they play also an important role in inflammation. They have been implicated in angiogenesis, vascular remodelling and mechanisms of pain (Keeley et al., 2008; Zlotnik et al., 2006). Other significantly upregulated genes in vascultis were: CPA3, a mast cell carboypeptidase that is released upon mast cell activation and degranulation. It is thought to play a key role in innate immunity (Stevens and Adachi, 2007). PTPRC is a protein tyrosine phosphatase, receptor type, which is an essential regulator of T- and B-cell antigen receptor signalling (Hermiston et al., 2009). CYBB has been proposed as a primary component of the microbicidal oxidase system of phagocytes (Heyworth et al., 2003). CSF1R is the receptor for colony stimulating factor 1 that is a cytokine which controls the production, differentiation, and function of macrophages (Hercus et al., 2009). IL10RA is the receptor for Il-10 which has been shown to mediate the immunosuppressive signal of interleukin 10, and thus inhibits the synthesis of proinflammatory cytokines. Taking these results together, a picture emerges of an interferongamma immune activation, monocyte migration and mast cell and macrophage activation in addition to innate phagocytic system involvement. IFN-gamma ranks among the most important endogenous regulators of immune responses (Billiau and Matthys, 2009). This Th-1 proinflammatory response could be, however, counteracted by the upregulation of the receptor for IL10, a strong anti-inflammatory cytokine. The exact role of these genes in nerve vasculitis and the
sequence involved in the activation of these genes remain to be investigated. 4.2. Upregulation of imunglobulin genes and complement There was a prominent activation of immunoglobulin genes, such as IGLJ3, IGHG3, IGKC, and IGL, which all function in B-cell selection or antigen recognition molecules of B-cells. C1QA a major constituent of the human complement subcomponent C1q was upregulated indicating that the classical pathway is activated. Our data are in line with the observation of deposits of immunoglobulin and complement, or the membrane attack complex, in the epineurial blood vessels as shown by immunohistochemistry (Engelhardt et al., 1993; Kissel et al., 1989), indicating the participation of humoral components to the vasculitic process. Our study strengthens the role of B cell immunity in the pathogenesis of vasculitic neuropathy. Still, it remains unclear, whether this is a secondary or a primary phenomenon. In some forms of vasculitis, as in Wegener Granulomatosis, the antineutrophil cytoplasmic autoantibodies (ANCA) probably play a pathogenetic role, and in mouse models of this particular disease a pathogenetic role of the alternative pathway of complement has been shown (Jennette and Falk, 2007). 4.3. Vascular remodelling CX3CR1 is the receptor for the CX3C chemokine fractalkine and mediates both its adhesive and migratory functions. The Fractalkine/ CX3CR1 axis induces inflammation and smooth muscle cell (SMC) proliferation (Chandrasekar et al., 2003; Liu et al., 2006; Zeiffer et al., 2004). In this context it is interesting to note the upregulation of AIF-1. The allograft inflammatory factor-1 (AIF-1) is a modulator of the immune response during macrophage activation (Utans et al., 1995). We recently identified that vascular smooth muscle cells in vasculitic nerve express AIF-1 at a much higher level compared to control nerves (Renaud et al., 2005), suggesting a direct role of AIF-1 in the proliferation and migration of SMC (Autieri, 2003; Autieri et al., 2000). AIF-1 is not expressed in unstimulated SMC but is rapidly expressed in response to injury and inflammatory cytokines (Tian and Autieri, 2007). 4.4. Down regulated genes Among the down regulated genes, are the Krüppel-Like Transcription Factors (KLFs) and the nuclear orphan receptor NR4A1. KLFs are transcription factors (Pearson et al., 2008) of which KLF 2 and KLF4 are critical regulator of vascular endothelial function (Hamik et al., 2007; SenBanerjee et al., 2004). KLF2 inhibits endothelial inflammation via multiple distinct mechanisms that inhibits NF-kB pathway activation (SenBanerjee et al., 2004). Many proinflammatory stimuli, such as TNFα, inhibit KLF2 expression in endothelial cells (Rao et al., 2007). Endothelial activation by proinflammatory cytokines is potentially mediated by reduction in KLF2 activity, leading to unopposed NF-kB activity. Since the functional role of KLF2 is to induce anti-thrombotic and anti-inflammatory pathways (Atkins and Jain, 2007), its inhibition will lead to thrombosis, a hallmark of vasculitis. Interestingly statin lead to upregulation of KLF2 (Tuomisto et al., 2008) and might be considered as a therapeutic option. The NR4A nuclear orphan receptors are a subfamily of the nuclear hormone receptor superfamily (Bonta et al., 2007; Pols et al., 2007). It comprises NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (NOR-1). Though at present the signalling pathways of NR4A nuclear receptor's function in disease are not fully unravelled, it has been shown that NR4As play key roles in macrophages and vascular cells, such as smooth muscle cells and endothelial cells (Bonta et al., 2007). Knockdown of Nur77 or NOR-1 in macrophages increases inflammatory cytokine expression and enhances oxidized LDL uptake in these cells, indicating that endogenous NR4A nuclear receptors may be
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Table 3 Genes with altered expression in vasculitis.
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Table 3 (continued)
Gene sequences with increased (A) or decreased (B) expression (fold change N 2 or b 0.5; pvalue b 0.005) in nerves from vasculitis patient compared to nerves from control patients are shown. For each patient the expression levels compared to the average of all control values are shown (red shaded: fold change >2; green shaded; fold change b 0.5).
involved in inhibitory feedback mechanisms that modulate macrophage activation (Pols et al., 2007). Nur77 plays an inhibitory role in SMC proliferation and modulates endothelial cell growth, survival and angiogenesis, and angiogenesis is decreased in Nur77-deficient mice (Zeng et al., 2006). It is interesting to note that azathioprine, a drug used in the treatment of vasculitis, enhances the transcriptional activity of NR4A nuclear receptors, and therefore, exerts a protective effect on endothelial cell as well as a growth inhibitory effect (Pols et al., 2007). 5. Conclusion Not unexpected, a marked activation of genes involved in immune system processes, genes involved in T and B-cell signalling as well as genes involved in vascular remodelling were detected in vasculitis nerves. The observed downregulation of genes involved in endothelial cell function can be interpreted as a response to the overexpression of inflammatory cytokines. Our study provides a molecular fingerprint of activated and downregulated genes. There emerges a complex pattern with several main key players, implicating a pathogenetic cascade possibly triggered by an interferon-gamma immune activation. Thus, gene expression profiling reveals mechanistic clues and possible targets for therapeutical interventions. Further more detailed analysis of particular mechanisms involved in vasculitic neuropathies is needed to better understand the molecular cascade of this complex condition. Acknowledgements This project was supported by a European Neurological Society (ENS) fellowship to LB. JK was supported by the National Multiple Sclerosis Society, the Novartis Stiftung and the Swiss Multiple Sclerosis Society (grants to NSW). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneuroim.2010.05.023. References Atkins, G.B., Jain, M.K., 2007. Role of Kruppel-like transcription factors in endothelial biology. Circ. Res. 100, 1686–1695.
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Contents lists available at ScienceDirect
Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
Gene expression profiling in nerve biopsy of vasculitic neuropathy Jochen Kinter a,b, Laura Broglio a,b, Andreas J. Steck a,b,⁎, Markus Tolnay c, Peter Fuhr a,b, Norman Latov d, Daniel Kalbermatten e, Michael Sinnreich a,b, Nicole Schaeren-Wiemers a,b, Susanne Renaud a,b a
Department of Biomedicine, University Hospital Basel, Basel, Switzerland Department of Neurology, University Hospital Basel, Basel, Switzerland Institute of Pathology, University Hospital Basel, Basel, Switzerland d Department of Neurology and Neurosciences, Weill Medical College, Cornell University, New York, NY, USA e Division of Plastic Surgery, University Hospital Basel, Petersgraben 4,CH-4031 Basel, Switzerland b c
a r t i c l e
i n f o
Article history: Received 9 March 2010 Received in revised form 4 May 2010 Accepted 5 May 2010 Keywords: Neuropathy Vasculitis Gene expression
a b s t r a c t To investigate molecular mechanisms of peripheral nerve vasculitis, gene expression patterns in archived frozen sural nerve biopsies from patients with vasculitic neuropathy were compared to control nerves by DNA microarray technology. There was a striking upregulation of mRNA of genes involved in immune system processes. Of special interest was the activation of immunoglobulin genes, such as IGLJ3, IGHG3, IGKC, and IGL, and of several chemokines, such as CXCL9 or CCR2. Genes involved in vascular proliferation or remodelling such as CXC31 and AIF were also upregulated. Among the downregulated genes were the Krüppel-Like Transcription Factors KLF2, KLF4 and the nuclear orphan receptor NR4A1 genes known to be involved in endothelial cell activation. Thus, this gene expression profile analysis revealed that in peripheral nerve vasculitis a prominent activation of immune response related genes as well as genes involved in vascular proliferation is taken place, while genes inhibiting endothelial cell activation are down regulated. These data point to interesting mechanistic clues to the molecular pathogenesis of vasculitic neuropathies. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Peripheral neuropathy is a common complication of vasculitic disease (Said and Lacroix, 2005). Vasculitic neuropathy occurs as a primary or as a secondary phenomenon and can be restricted to the peripheral nervous system only. Various underlying pathogenic mechanisms, such as immune complex deposition, cell mediated interactions, and antineutrophil cytoplasmic antibody (ANCA) associated processes are discussed and have been extensively reviewed (Collins and Periquet-Collins, 2009; Jennette and Falk, 2007; Said and Lacroix, 2005). In most cases the initiating event is unknown, and a self-sustaining circuit attracts and activates inflammatory leucocytes in the wall of vessels. Recent studies have revealed homeostatic roles of vascular inflammation and have identified the action of humoral innate immunity, in particular injury-associated signals and acute phase proteins, on the activation of circulating leucocytes, platelets and endothelial cells (Maugeri et al., 2009). To elucidate pathogenetic mechanisms of vasculitis most studies rely on immunohistochemistry, but more recently DNA microarray analysis evolved as a powerful method for differential expression analysis of genes in normal and diseased tissues (Kinter et al., 2008; Schena et al., 1995). ⁎ Corresponding author. Department of Neurology, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland. E-mail address: [email protected] (A.J. Steck). 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.05.023
In a gene expression study of the human peripheral nerve using the microarray technology (Renaud et al., 2005) we reported and discussed differentially expressed genes in patients with chronic inflammatory demyelinating polyneuropathy (CIDP). One finding was the upregulation of Allograft Inflammatory Factor-1 (AIF-1) in three vasculitic neuropathy patients of the disease control group. In a subsequent study, we identified the expression of AIF-1 predominantly in vascular smooth cells of vasculitis nerves, suggesting that AIF-1 might play an important pathogenetic role in the proliferation and migration of vascular smooth muscle cells and subsequent narrowing of vessel lumen in peripheral nerves affected by vasculitis (Broglio et al., 2008). To follow up and extend our initial study, we performed a gene expression analysis to demonstrate distinct gene expression patterns in archived frozen nerve biopsies of patients with vasculitic neuropathies compared to control nerve by DNA microarray analysis. 2. Material and methods 2.1. Patients Nerve biopsies from 7 patients with vasculitic neuropathy were included in this study. The diagnosis was made according to established criteria (Said and Lacroix, 2005). Two patients were suffering from virus associated nerve vasculitis, another had non-systemic vasculitic neuropathy and the remainder were suffering from nerve vasculitis due to systemic disease. As controls, biopsy specimens were obtained
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Table 1 Control and vasculitis patients. Subjects
Sex
Age
Controls 1 2 3 4
F M M M
5 85 18 37
Patients 5 6 7 8 9 10 11
M F F M F F F
72 72 68 78 69 39 85
Time to Biopsy
Cinical characteristics
Blood markers
Pathology
Treatment
ENA, ANA SS-A, SS-B
Necrotizing vasculitis Perivasculitis Vasculitis Vasculitis Necrotizing vasculitis Necrotizing vasculitis Necrotizing vasculitis
None NSAID NSAID None None Antiretroviral Prednisone
Dermatomyositis Alzheimer Neurinoma extirpation Trauma
3 weeks 3 months 4 months 3 months Unknown 2 months 4 weeks
Multiple mononeuropathy, Churg–Strauss Vasculitis Asymmetric sensorimotor neuropathy, Sjögren's Syndrome Multiple mononeuropathy Non-sysemic vasculitis Multiple mononeuropathy Hepatitis B associated Multiple mononeuropathy MALT lymphoma of the gut Multiple mononeuropathy HIV associated, Hepatitis C Multiple mononeuropathy Rheumatoid arthritis
cANCA ANA CCP-Ab, RF
Summary of control and vasculitis patient data. Abbreviations: MALT = Mucous membrane Associated Lymphoid Tissue, HIV = human immunodeficiency virus, ENA = extractable nuclear antigen, ANA = antinuclear antibody, SS-A = Sjögren's syndrome antigen A, SS-B = Sjögren's syndrome antigen B, cANCA = antineutrophil cytoplasmic antibodies, CCP-Ab = cyclic citrullinated peptide antibody, RF = rheumatoid factor, NSAID = non-steroidal anti-inflammatory drugs.
from individuals who did not suffer from polyneuropathy, but from muscle dystrophy, dermatomyositis or who received reconstructive surgery in their legs. One control specimen was a sural nerve from an autopsy case. The characteristics and clinical data of patients with vasculitis are listed in Table 1. Paraffin-embedded and fresh frozen biopsy specimens from the cutaneous branch of the superficial peroneal nerve were obtained after routine work-up. Permission to use these tissues was obtained from our institutional ethics committee. Results of routine histopathological analysis of nerve biopsy specimens were drawn from case file reports (Supplementary Figs. 1 and 2). 2.2. RNA sample processing Human nerve biopsies were embedded in Tissue-Tek medium (Sakura Finetek, USA) immediately after the biopsy and stored at −70 °C. The embedded tissue was cut with a cryostat (Microm HM-560) in 10 μm sections. Tissue homogenization was obtained with an electronic rotor stator homogenizer (Polytron, Kinematica, Switzerland). Total RNA extraction was obtained using the TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturers protocol. After DNAse treatment (DNAse I Ambion, Huntingdom, Cambridgeshire, UK) total RNA was purified using Rneasy Kit (Qiagen, Chatsworth, CA). RNA yields were measured by the NanoDrop spectrophotometer (NAME) and its quality was assessed by the microfluidics-based platform Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Wash and Stain Kit (Affymetrix, Cat# 900720). To increase the signal strength the antibody amplification protocol was used (FS450_0002). The GeneChips were processed with an Affymetrix GeneChip® Scanner 3000 7G (Affymetrix) DAT image files of the microarrays were generated using GeneChip Operating Software (GCOS 1.4; Affymetrix). 2.4. Data analysis Data were analyzed using the RACE software and the Bayes test for statistical significance (Psarros et al., 2005). Normalization was performed using robust GeneChip multi-array analysis (gcRMA). A fold change as well as a p-value threshold was set for generating subsets of differentially expressed genes (Table 2). 3. Results 3.1. Differentially regulated genes Microarray analysis revealed pronounced alterations in gene expression in vasculitis nerve tissues. Considering a cut-off point for Table 2 Number of differentially expressed genes sorted by p-value stringency and fold changes. Fold change
2.3. cRNA amplification Total
Biotin labelling of RNA was performed as described in the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, USA). cRNA was synthesized according to the GeneChip Expression 3′ Amplification Two-Cycle Target labelling and Control Reagents (Affymetrix, Cat# 900494), starting from 50 ng of total RNA. On average 112 μg of labelled cRNA from each reaction was obtained, and average size of the cRNA molecules was assessed on RNA Nano 6000 Chips (2100 Bioanalyzer, Agilent). The cRNA targets were incubated at 94 °C for 35 min in the provided “Fragmentation Buffer” and the resulting fragments of 50–150 nucleotides were again monitored using the Bioanalyzer. All synthesis reactions were carried out using a PCR machine (T1 Thermocycler; Biometra, Göttingen, Germany) to ensure the highest possible degree of temperature control. The hybridization cocktail (130 μl) containing fragmented biotin-labelled target cRNA at a final concentration of 0.05 μg/μl was transferred into Affymetrix Human Genome U133A 2.0 Arrays and incubated at 45 °C on a rotator in a hybridization oven 640 (Affymetrix) for 16 h at 60 rpm. The arrays were washed and stained on a Fluidics Station 450 (Affymetrix) by using the Hybridization
p-value
–
1.5
2
5
b 0.05 b 0.01 b 0.005 b 0.001
1469 392 227 64
622 236 149 47
327 135 90 26
45 33 26 12
b 0.05 b 0.01 b 0.005 b 0.001
556 169 105 33
346 140a) 94 29
239 105 75b) 22
41 30 25 12d)
b 0.05 b 0.01 b 0.005 b 0.001
913 223 122 31
276 96a) 55 18
88 30 15c) 4
4 3 1 0
Up
Down
Normalized hybridization signal intensities from vasculitis cases were compared with the average intensity of all control cases, providing fold changes (ratio). The average of all ratios and the statistical significance was calculated and sorted according to particular stringencies. This analysis revealed that 1469 gene sequences were differentially expressed with a p-value below 0.05. Applying a significance level with a p-value below 0.001, still 64 gene sequences were differentially expressed. a) The data list of these gene sequences are provided as Supplementary data. b) Data are shown in Table 3A. c) Data are shown in Table 3B.d) Genes upregulated more than five fold and a p-value lower than 0.001.
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the p-value of p b 0.01 and a cut-off point for the fold change N1.5 (respectively b0.66 for downregulated genes), 140 genes were upregulated and 96 downregulated in vasculitis nerves (Table 2;a). Upregulated genes include genes involved in cytokine–cytokine receptor interaction, hematopoietic cell lineage, Toll-like receptor signalling pathway, cell adhesion pathway and natural killer cell mediated cytotoxicity (Supplementary data). More stringent conditions revealed 75 genes upregulated with more than two fold changes (p b 0.005, Table 2;b) and 15 genes downregulated (Table 2;c), while with an applied stringency with a statistical significance p b 0.001, still 12 genes showed an upregulation of more than 5 folds in vasculitis compared to control nerves (Table 2;d). The most striking observed alteration was the increased expression of genes involved in processes related to the immune system. Among the immune response genes there was an activation of immunoglobulin genes, the MHC II pathway, many immune receptors and their signalling pathways and the complement system (Table 3A). Among the most downregulated genes were the Krüppel-Like Transcription Factor (KLF), KLF4 and KLF2 (Table 3B). Also of interest is the decrease of the nuclear orphan receptor NR4A1 (Supplementary Table). 4. Discussion Our nerve biopsy samples reflect the spectrum of vasculitis encountered in clinical practice, including virus associated nerve vasculitis, non-systemic vasculitic neuropathy and nerve vasculitis due to systemic disease. In spite of this clinical heterogeneity, we believe that a pattern of differentially regulated gene emerges from the analysis of the data. This is in keeping with the hypothesis that there is a common pathogenic pathway in nerve vasculitis, even if the triggering mechanisms are not uniform (Maugeri et al., 2009). 4.1. Activation of cellular immune response genes Reflecting the strong cellular component of vasculitis there was activation of chemokines, such as CXCL9, a gamma interferon-induced monokine contributing to Th1-induced inflammation, and CCR2 a chemokine, which specifically mediates monocyte chemotaxis. Chemokines are a large family of more than 50 cytokines that bind to a smaller number of receptors. Their best known function is the regulation of cell migration, but they play also an important role in inflammation. They have been implicated in angiogenesis, vascular remodelling and mechanisms of pain (Keeley et al., 2008; Zlotnik et al., 2006). Other significantly upregulated genes in vascultis were: CPA3, a mast cell carboypeptidase that is released upon mast cell activation and degranulation. It is thought to play a key role in innate immunity (Stevens and Adachi, 2007). PTPRC is a protein tyrosine phosphatase, receptor type, which is an essential regulator of T- and B-cell antigen receptor signalling (Hermiston et al., 2009). CYBB has been proposed as a primary component of the microbicidal oxidase system of phagocytes (Heyworth et al., 2003). CSF1R is the receptor for colony stimulating factor 1 that is a cytokine which controls the production, differentiation, and function of macrophages (Hercus et al., 2009). IL10RA is the receptor for Il-10 which has been shown to mediate the immunosuppressive signal of interleukin 10, and thus inhibits the synthesis of proinflammatory cytokines. Taking these results together, a picture emerges of an interferongamma immune activation, monocyte migration and mast cell and macrophage activation in addition to innate phagocytic system involvement. IFN-gamma ranks among the most important endogenous regulators of immune responses (Billiau and Matthys, 2009). This Th-1 proinflammatory response could be, however, counteracted by the upregulation of the receptor for IL10, a strong anti-inflammatory cytokine. The exact role of these genes in nerve vasculitis and the
sequence involved in the activation of these genes remain to be investigated. 4.2. Upregulation of imunglobulin genes and complement There was a prominent activation of immunoglobulin genes, such as IGLJ3, IGHG3, IGKC, and IGL, which all function in B-cell selection or antigen recognition molecules of B-cells. C1QA a major constituent of the human complement subcomponent C1q was upregulated indicating that the classical pathway is activated. Our data are in line with the observation of deposits of immunoglobulin and complement, or the membrane attack complex, in the epineurial blood vessels as shown by immunohistochemistry (Engelhardt et al., 1993; Kissel et al., 1989), indicating the participation of humoral components to the vasculitic process. Our study strengthens the role of B cell immunity in the pathogenesis of vasculitic neuropathy. Still, it remains unclear, whether this is a secondary or a primary phenomenon. In some forms of vasculitis, as in Wegener Granulomatosis, the antineutrophil cytoplasmic autoantibodies (ANCA) probably play a pathogenetic role, and in mouse models of this particular disease a pathogenetic role of the alternative pathway of complement has been shown (Jennette and Falk, 2007). 4.3. Vascular remodelling CX3CR1 is the receptor for the CX3C chemokine fractalkine and mediates both its adhesive and migratory functions. The Fractalkine/ CX3CR1 axis induces inflammation and smooth muscle cell (SMC) proliferation (Chandrasekar et al., 2003; Liu et al., 2006; Zeiffer et al., 2004). In this context it is interesting to note the upregulation of AIF-1. The allograft inflammatory factor-1 (AIF-1) is a modulator of the immune response during macrophage activation (Utans et al., 1995). We recently identified that vascular smooth muscle cells in vasculitic nerve express AIF-1 at a much higher level compared to control nerves (Renaud et al., 2005), suggesting a direct role of AIF-1 in the proliferation and migration of SMC (Autieri, 2003; Autieri et al., 2000). AIF-1 is not expressed in unstimulated SMC but is rapidly expressed in response to injury and inflammatory cytokines (Tian and Autieri, 2007). 4.4. Down regulated genes Among the down regulated genes, are the Krüppel-Like Transcription Factors (KLFs) and the nuclear orphan receptor NR4A1. KLFs are transcription factors (Pearson et al., 2008) of which KLF 2 and KLF4 are critical regulator of vascular endothelial function (Hamik et al., 2007; SenBanerjee et al., 2004). KLF2 inhibits endothelial inflammation via multiple distinct mechanisms that inhibits NF-kB pathway activation (SenBanerjee et al., 2004). Many proinflammatory stimuli, such as TNFα, inhibit KLF2 expression in endothelial cells (Rao et al., 2007). Endothelial activation by proinflammatory cytokines is potentially mediated by reduction in KLF2 activity, leading to unopposed NF-kB activity. Since the functional role of KLF2 is to induce anti-thrombotic and anti-inflammatory pathways (Atkins and Jain, 2007), its inhibition will lead to thrombosis, a hallmark of vasculitis. Interestingly statin lead to upregulation of KLF2 (Tuomisto et al., 2008) and might be considered as a therapeutic option. The NR4A nuclear orphan receptors are a subfamily of the nuclear hormone receptor superfamily (Bonta et al., 2007; Pols et al., 2007). It comprises NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (NOR-1). Though at present the signalling pathways of NR4A nuclear receptor's function in disease are not fully unravelled, it has been shown that NR4As play key roles in macrophages and vascular cells, such as smooth muscle cells and endothelial cells (Bonta et al., 2007). Knockdown of Nur77 or NOR-1 in macrophages increases inflammatory cytokine expression and enhances oxidized LDL uptake in these cells, indicating that endogenous NR4A nuclear receptors may be
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Table 3 Genes with altered expression in vasculitis.
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Table 3 (continued)
Gene sequences with increased (A) or decreased (B) expression (fold change N 2 or b 0.5; pvalue b 0.005) in nerves from vasculitis patient compared to nerves from control patients are shown. For each patient the expression levels compared to the average of all control values are shown (red shaded: fold change >2; green shaded; fold change b 0.5).
involved in inhibitory feedback mechanisms that modulate macrophage activation (Pols et al., 2007). Nur77 plays an inhibitory role in SMC proliferation and modulates endothelial cell growth, survival and angiogenesis, and angiogenesis is decreased in Nur77-deficient mice (Zeng et al., 2006). It is interesting to note that azathioprine, a drug used in the treatment of vasculitis, enhances the transcriptional activity of NR4A nuclear receptors, and therefore, exerts a protective effect on endothelial cell as well as a growth inhibitory effect (Pols et al., 2007). 5. Conclusion Not unexpected, a marked activation of genes involved in immune system processes, genes involved in T and B-cell signalling as well as genes involved in vascular remodelling were detected in vasculitis nerves. The observed downregulation of genes involved in endothelial cell function can be interpreted as a response to the overexpression of inflammatory cytokines. Our study provides a molecular fingerprint of activated and downregulated genes. There emerges a complex pattern with several main key players, implicating a pathogenetic cascade possibly triggered by an interferon-gamma immune activation. Thus, gene expression profiling reveals mechanistic clues and possible targets for therapeutical interventions. Further more detailed analysis of particular mechanisms involved in vasculitic neuropathies is needed to better understand the molecular cascade of this complex condition. Acknowledgements This project was supported by a European Neurological Society (ENS) fellowship to LB. JK was supported by the National Multiple Sclerosis Society, the Novartis Stiftung and the Swiss Multiple Sclerosis Society (grants to NSW). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneuroim.2010.05.023. References Atkins, G.B., Jain, M.K., 2007. Role of Kruppel-like transcription factors in endothelial biology. Circ. Res. 100, 1686–1695.
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