1  

 

Calcium-Binding Proteins S100A8 and S100A9 Initiate the Early Inflammatory Program in Injured Peripheral Nerve*

Andrei V. Chernov1, Jennifer Dolkas2,3, Khang Hoang2,3, Mila Angert2,3, Geetha Srikrishna1*, Thomas Vogl4, Svetlana Baranovskaya5, Alex Y. Strongin1 and Veronica I. Shubayev2,3,**

From the 1Sanford-Burnham Medical Research Institute, La Jolla, California, USA, the 2Department of Anesthesiology, University of California, San Diego, La Jolla, California, USA, the 3VA San Diego Healthcare System, La Jolla, California, USA, 4Institute of Immunology, University of Muenster, Muenster, Germany and the 5Agilent Technologies, La Jolla, California, USA.

**To whom correspondence should be addressed: Veronica I. Shubayev, University of California, San Diego, Mail Code 0629, 9500 Gilman Dr., La Jolla, CA, 92093-0629. Phone: (858) 534-5278; Fax: (858) 534-1445; E-mail: vshubayev@ucsd.edu §The work reported here was supported by RO1DE022757 from NIH (VIS and AYS) and VA Merit Review Award 5I01BX000638 from the Department of Veterans Affairs (VIS).

*Running Title: S100A8/A9 and immune circuitry in peripheral nerve

Keywords: Schwann cell, peripheral nerve, S100, nerve regeneration, neuropathic pain

Background: In peripheral nerve, initial immune response to injury influences regeneration.

Results: S100a8 and S100a9 are the top induced genes in nerve post-injury. S100A8/A9 activates the chemotactic genes and pathways in Schwann cell and stimulate myeloid cell infiltration into the nerve.

Conclusion: S100A8/A9 initiate immune cell transmigration into the nerve.

Significance. S100A8/A9 are novel modulators of peripheral nerve injury.

ABSTRACT

To shed light on the early immune response processes in severed peripheral nerve, we performed genome-wide transcriptional profiling and bioinformatics analyses of the proximal (P; regenerating) and distal (D; degenerating) nerve stumps at day 1 in the sciatic nerve axotomy model in rats. Multiple cell death-related pathways were activated in the degenerating D stump, whereas activation of the cytoskeletal motility and gluconeogenesis/glycolysis pathways was most prominent in the P stump of the axotomized nerve. Our bioinformatics analyses also identified the specific immunomodulatory genes of the chemokine, interleukin (IL), tumor necrosis factor (TNF), major histocompatibility complex (MHC), immunoglobulin-binding Fc receptor, calcium-binding S100, matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP), and ion channel families affected in both the P and D segments. S100a8 and S100a9 were the top up-regulated genes in both the P and D segments. Stimulation of cultured Schwann cells using the purified S100A8/A9 heterodimer recapitulated

activation of the myeloid cell and phagocyte chemotactic genes and pathways, which we initially observed in injured nerve. The S100A8/A9 heterodimer injection into the intact nerve stimulated macrophage infiltration. We conclude that following peripheral nerve injury an immediate acute immune response occurs both distally and proximally to the lesion site and that the rapid transcriptional activation of S100a8 and S100a9 genes results in the S100A8/A9 hetero and homodimers, which stimulate the release of chemokines and cytokines by the activated Schwann cells and generate the initial chemotactic gradient that guides transmigration of hematogenous immune cells into the injured nerve.

INTRODUCTION

In general, peripheral nerve displays a strong regenerative potential. Relative to other injury types, compete nerve transection (axotomy) severely damages the endoneurial tube and entails inefficient regenerative growth (1-4). Since the original findings of the nerve fiber breakdown distal to transection injury (5), substantial knowledge has been accumulated using sciatic nerve injury models in

http://www.jbc.org/cgi/doi/10.1074/jbc.M114.622316The latest version is at JBC Papers in Press. Published on March 19, 2015 as Manuscript M114.622316

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

2  

 

rodents. This knowledge recognizes that the dramatic endoneurial remodeling in the proximal (P, regenerating) and distal (D, degenerating) nerve stumps during the first hours post-transection sets the course for a long-term ‘staggered’ axonal growth (up to 4 weeks in rats) and consequently, an incomplete functional (motor and sensory) recovery (3,4,6).

Wallerian degeneration is a well-orchestrated process initiated by an instant influx of extracellular calcium, which activates the calpain- and ubiquitin-proteasome-dependent disintegration of the axonal cytoskeleton in the D stump (7). Concomitantly, the P axons die-back to the next node of Ranvier (4,8) and form axonal endbulbs (9). Though a rigorous, actin-supported process, an endbulb transforms into a regenerating P axonal sprout and forms a growth cone (4,10,11). Schwann cells, the main cell population in the peripheral nerve, carefully guide the growing axons towards the end-organ. However, disruption of endoneurial tubes caused by transection injury results in the disorganized extracellular matrix, which obscures the Schwann cell alignment into a column of proliferating cells, the bands of Büngner, and their ability to deposit the substrata favorable to axonal growth (4,6).

The denervated Schwann cells rapidly trans-differentiate into Büngner cells post-axotomy. This process involves the silencing of the genes coding for myelin proteins and the induction of glial fibrillary acidic protein, p75NGF, neuregulin and other genes that are required to support Schwann cell de-differentiation, mitosis and partnership with regenerating axons (7,12-14). Schwann cells also play a key role in secreting inflammatory chemokines, cytokines and matrix metalloproteinases (MMPs), which work in concert to stimulate the development of chemotactic gradients and the directed immune cell migration across the blood-nerve barrier and into the damaged nerve (12,15-17). Various hematogenous immune cell types, including granulocytes (neutrophils and mast cells) and agranulocytes (monocytes/macrophages and lymphocytes), infiltrate the nerve in the course of Wallerian degeneration (12,15-17).

Calprotectin (S100A8/A9) is a heterodimeric non-covalent complex of acidic calcium-binding S100A8 and S100A9. S100A8/A9 induces chemotaxis (18) 24), cytoskeletal reorganization (19), calcium signaling (20) and cytokine expression (21) in

immune cells, particularly phagocytes. In peripheral nerve, phagocytic clearance of axonal and myelin debris is initiated by Schwann cells, which extrude myelin sheaths to create ovoid “digestion chambers” (22,23). This clearance is completed by macrophages infiltrating the nerve at days 2-14 post-injury (16,17,22,24). Because the S100a8 and S100a9 genes were highly up-regulated at day 1 post-injury in murine nerves (25) we hypothesized that the initial positive chemotactic gradient is a result of Schwann cell stimulation by S100A8/A9 and that this gradient arises shortly after peripheral nerve injury and then stimulates the infiltration of the immune cells towards the trauma site.

Herein, we first confirmed and expanded the transcriptional profiling studies in peripheral nerve (25-33). Using the comparative genome-wide transcriptional profiling and subsequent bioinformatics (Ingenuity, NextBio) analyses in the P and D stumps at day 1 post-axotomy, we recorded the early gene expression changes in the severed sciatic nerve in rats. Stimulation of cultured Schwann cells by the purified S100A8/A9 heterodimer recapitulated those transcriptional events which supported chemotaxis of myeloid cells towards both the P and D segments post-injury in vivo. In addition, S100A8/A9 injection stimulated macrophage infiltration into the nerve in vivo. Our findings suggest that the rapid injury-induced up-regulation of S100A8/A9 in Schwann cells then stimulates the release of cytokines and chemokines, which, combined, provide the initial chemotactic gradient that attracts the hematogenous immune cells to the injured nerve.

MATERIALS AND METHODS

Reagents and Antibodies- Routine reagents were purchased from Sigma unless indicated otherwise. The following antibodies were used in our experiments: rabbit polyclonal anti-S100A9 (Novus, cat. #NB110-89726) and murine monoclonal anti-S100B (Sigma cat. #S2532) and anti-CD68 (Serotec, cat. #MCA341R). We also used the following antibodies from Cell Signaling Technologies: a rabbit polyclonal antibody to phospho (p)ERK (cat. #9101), to phospho pPI3K (cat. #4228), to phospho pJNK (cat. #4668), to ERK (cat. #9102), to PI3K (cat. #4292), and to JNK (cat. #9258). A murine monoclonal antibody to β-actin was from Sigma (cat. #A53166).

S100A8/A9- Purification of endotoxin-free murine S100A8/A9 proteins was performed as described for

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

3  

 

human S100A8/A9 in (34). The levels of homodimers were below 10%, as determined by size-exclusion chromatography and ELISAs employing all antibody combinations (A8/A8, A9/A9, A8/A9 and A9/A8, unpublished data). The levels of lipopolysaccharide (LPS) were below 2 pg/µg of the complex, as determined by the limulous amoebocyte lysate (LAL) test.

Animal Procedures- Adult female Sprague-Dawley rats weighing 200–225 g (Harlan) were maintained at 22°C on 12/12-h light/dark cycle. Animals had access to food and water ad libitum. Anesthesia was induced by a 5% isoflurane/air mixture (Forane, Butler-Schein) and maintained using a 2.5% mixture. The common sciatic nerve was exposed unilaterally at the mid-thigh level and (1) transected using surgical scissors or (2) injected into the fascicle with purified S100A8/A9 (10 µg in 5 µl PBS) or an equal volume of a vehicle (PBS alone), using a 33-gauge needle and a Hamilton syringe. At day 1 or week 1 after injury or injection, nerve segments (the proximal, distal and contralateral (normal) to the axotomy/injection site) were collected for the subsequent analysis. Animals were sacrificed using an overdose of a rodent anesthesia cocktail [50 mg/ml Nembutal (Abbott Labs) and 5 mg/ml diazepam (Steris Labs) in 0.9% PBS (Steris Labs)], followed by an i.p. injection of Beuthanasia (100-150 mg/ml; Schering-Plough Animal Health). All animal procedures were done in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and protocols approved by the Institutional Animal Care and Use Committee at the VA San Diego Healthcare System.

Stimulation of Cultured Schwann Cells Using S100A8/A9- Primary Schwann cells were isolated from sciatic nerves of postnatal day 1-3 Sprague-Dawley rats (24). Schwann cells were further purified as described previously (35). The purity of Schwann cell cultures was monitored by S100B immunostaining (24). Schwann cells (>99% purity) were then cultured in 75 cm2 flasks coated with 50 µg/ml poly-D-lysine (Chemicon) in DMEM containing 1 g/L glucose, 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin, 21 µg/ml bovine pituitary extract (Life Technologies) and 4 µM forskolin (Calbiotech) at 37°C and 5.0% CO2. Cells were used in experiments at passages 3-7. For stimulation, Schwann cells were plated in wells of a 6-well plate and allowed to reach a 75% confluence

level. Purified S100A8/A9 (5 µg/ml) was added to the cells for either 1 h or 24 h. Cells were then washed and harvested. Total RNA was isolated using Direct-zol RNA MiniPrep system (Zymo Research) and used in the follow-up transcription profiling experiments. The RNA purity was estimated by measuring the OD260/280 and the OD260/230 ratios. The RNA integrity was assessed using an Experion Automated Electrophoresis System (Bio-Rad). The purified RNA was quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific).

Genome-Wide Transcriptional Profiling- Nerve segments from three rats per group were isolated and stored at -20°C in RNA-later (Life Technologies). Total RNA was extracted using TRIzol and purified using an RNeasy column (Qiagen). The RNA purity, integrity and quantity were assessed as described above. Total RNA aliquots (50 ng each) were labeled using a LowInput QuickAmp Labeling Kit and Cy3-CTP (Agilent Technologies). The labeled samples (1,500 ng each) were hybridized for 18 h at 65°C to the SurePrint G3 Rat GE 8x60K microarray chips (Agilent Technologies), which featured over 30,000 individual transcripts. Microarray chips were washed, developed using Cy3-Streptavidin Fluor conjugates (GE Healthcare) and scanned using a C Scanner (Agilent Technologies). Raw data were processed using Feature Extraction software (Agilent Technologies). Normalization to the median was performed using GeneSpring GX software (Agilent Technologies). Differentially expressed mRNAs with the signal intensity over two-fold relative to the background standard deviation were identified by t-test. Statistically significant data (p<0.05) were analyzed further to calculate the gene expression levels. The gene expression data have been deposited to the GEO database (Accession GSE62282).

Systems Biology Analysis- To determine the affected cellular regulatory and signaling pathways, the individual genes, the expression of which differed at least 2-fold between the respective samples, were analyzed using Ingenuity Pathway Analyses (IPA) (Qiagen) and NextBio software (NextBio). The heatmap charts were generated using GenePattern software (Broad Institute, Cambridge, MA).

RT-PCR- Real-time RT-PCR was conducted using a Mx4000™ Multiplex Quantitative PCR System (Agilent Technologies) in 25 µl reactions containing Taqman Universal PCR Master Mix (Ambion),

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

4  

 

cDNA (50 ng), specific forward and reverse primers (900 nM each) and probes (200-300 nM). Primers and probes for Il6 and Il1β were from Roche (cat. #04686934001 and #04689011001) and for Timp1 they were from Applied Biosystems (cat. #Rn01430873_g1). Primers and probes for Mmp9, Tnfα and Gapdh were designed as previously described (35,36) and synthesized by Biosearch Technologies. Relative mRNA levels were quantified using the 2(-Delta Delta C(T)) method (37). Normalization to Gapdh and fold-change calculation were performed using MxPro software (Agilent Technologies).

Neuropathology- Nerve segments (3 animals per group) were isolated and post-fixed for 48 h at 4°C with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Specimens were washed with the phosphate buffer, post-fixed with 1% osmic acid (Ted Pella), dehydrated in graded (30-100%) Ethyl Alcohol and Propylene Oxide, and embedded in Araldite Resin (Alaldite 502 (cat. #18060), Eponate 12 Resin (cat. # 18005), dodecenyl succinic anhydride (cat. # 18022) and DMP-30 (cat. # 18042) (all obtained from Ted Pella). One µm thick sections were cut using a diamond knife in an automated RM2065 microtome (Leica Microsystems) and then stained using methylene blue/azure II solution, as described (35).

Immunohistochemistry- Three animals per group were perfused with 4% paraformaldehyde. Nerve segments were isolated, post-fixed in 4% paraformaldehyde, cryoprotected in graded sucrose solution and embedded in O.C.T. compound (Tissue-Tek) on dry ice (36). Sections (10 µm thick) were blocked for 16 h at 4°C in PBS containing 5% normal goat serum (Vector Labs) and 0.25% Triton X-100. Sections were incubated for 16 h at 4°C with respective primary antibodies, washed in PBS followed by incubation with the corresponding secondary Alexa Fluor 488 or Alexa Fluor 594 antibody conjugates (Invitrogen). Slides were mounted in SlowFade Gold Antifade media with DAPI (Life Technologies). Alternatively, sections were incubated with the respective biotin-conjugated secondary antibody and stained using Vectastain Elite ABC System and 3’-3-diaminobenzidine substrate (Vector Labs) and Methyl Green (Vector Labs) counterstain. Images were acquired using a DMR microscope (Leica Microsystems) and processed using Openlab 4 imaging software (Improvision).

Immunoblotting- Nerve segments (3 animals per group) were collected, snap-frozen in liquid nitrogen and then used for protein extraction. Nerve segments were homogenized in 50 mM Tris-HCl, pH 7.4, containing 1% Triton X-100, 150 mM NaCl, 10% glycerol, 0.1% SDS, 5 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, aprotinin and leupeptin (1 µg/ml each). Protein concentration was determined using bicinchoninic acid assay (Pierce). Proteins (50 µg) were separated using SDS-gel electrophoresis in 15% acrylamide gels (Bio-Rad) and transferred onto a nitrocellulose membrane using iBlot (Invitrogen). The membranes were blocked for 16 h at 4°C in 5% non-fat milk in TBS and incubated with the primary antibodies diluted with 5% BSA in TBS. Membranes were washed in TBS containing 0.1% Tween and incubated for 1 h at ambient temperature with respective HRP-conjugated secondary antibody (Cell Signaling Technologies) and Enhanced Chemiluminescence system (GE Healthcare). For a loading control, the membranes were re-probed for β-actin. The membranes were scanned and digitized.

Statistical Analyses- Statistical analyses were performed using a two-tailed, unpaired Student’s t-test and, when variances were unequal, by an unpaired t-test with Welch’s correction using either KaleidaGraph 4.03 (Synergy Software) or SPSS 16.0 (SPSS) software. Analyses of variance (ANOVA) for repeated measures were employed for comparing three or more groups, followed by Tukey-Kramer post-hoc test. The p-values below 0.05 were considered significant.

RESULTS

Early Post-Injury Response Genes in the Proximal and Distal Nerve Stumps- The early endoneurial changes in axotomized peripheral nerve are believed to predispose the regenerating axons for their long-term staggered growth (3,4,6). To identify genes the expression of which was affected by axotomy in the sciatic nerve, we performed comparative genome-wide transcriptional profiling of the rat sciatic nerve proximal (P, regenerating) and distal (D, degenerating) axotomized segments, and a contralateral normal (N) nerve as a control at day 1 post-transection. Over 3,831 individual affected genes (1,816 upregulated and 2,015 downregulated) in the P segment and over 4,540 individual genes (2,165 upregulated and 2,375 downregulated) in the D

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

5  

 

segment were identified relative to normal nerve. Heatmap of the 50 most affected up- and down-regulated genes in the P and D segments is shown in Fig. 1. In agreement with the earlier observations, the documented cell populations in sciatic nerve at day 1 post-injury included Schwann cells (~80% of the total cell count), fibroblasts, resident macrophages, mast cells, endothelial cells and infiltrating neutrophils (15,16,38).

In general, the early injury-induced transcriptional changes demonstrated a level of similarity in the P and D stumps. Thus, in both stumps we recorded the upregulation of the genes that are directly involved in the immune response (Fig. 1A), including interleukin (Il6), chemokine (C-X-C motif) ligand 3 (Cxcl3), chemokine (C-C motif) ligand 20 (Ccl20) and pentraxin-related protein Ptx3. Intriguingly, calcium-binding proteins S100a8 and S100a9 genes were highly upregulated in both the P and D stumps.

Further in-detail analyses of the data demonstrated a significant similarity in the immunomodulatory gene expression patterns between in the P and D stumps, albeit the expression levels of the certain genes were distinct (Fig. 2 and 3). Thus, the expression of Il6, Cxcl3 and Ccl20 was enhanced 390-fold, 300-fold and 160-fold in the P samples, respectively, and 105-fold, 50-fold and 88-fold in the D stumps, respectively (Fig. 2). Similarly, the expression of S100a8, S100a9 and Mmp9 (a typical pro-inflammatory MMP type) was enhanced 237-fold, 90-fold and 55-fold in the P stumps, and 161-fold, 73-fold and 24-fold in the D stumps, respectively (Fig. 2, 3A). On the other hand, the enhanced expression of toll-like receptors (TLRs), TNF superfamily, antibody-binding Fc receptor Fcgr1-3 genes and RT1 genes encoding the major histocompatibility complex (MHC) cell surface proteins in rats (Fig. 2), tissue inhibitor of metalloproteinase-1 (Timp1), Mmp12 (macrophage metalloelastase) and many ion channels was roughly similar in the P and D stumps (Fig. 3A,B). Using real-time RT-PCR, the induction of Il6, Il1β, Tnfα, Timp1 and Mmp9 was confirmed in the injured nerve segments (Fig. 3C). Taken together, these results indicate that nerve axotomy induced an early and robust transcriptional activity related to immune response genes in both the P and D segments.

To discriminate transcriptional changes in the P and D segments, we excluded the genes with the similar expression pattern changes in both segments

and then identified the genes that were significantly and selectively up-regulated in the single respective segment (Fig. 4A,B). Subsequent bioinformatics analysis revealed the specific pathways that were selectively affected in the P versus D samples. Specifically, multiple signaling pathways, including myosin, calcium, integrin-linked kinase (ILK), and gluconeogenesis/glycolysis signaling were selectively upregulated in the P stumps (Fig. 4C). The most dramatic difference was observed in the myosin cytoskeleton-related genes, which were 50-100-fold upregulated in the regenerating P segments and only several-fold enhanced in the D segment (Fig. 4D). In contrast, the signaling pathways that are largely focused on cell death, including, apoptosis, the tumor necrosis factor receptor 1 (TNFR1) signaling, natural killer cell and death receptor signaling, were selectively stimulated in the D stumps (Fig. 4C). Thus, transcriptional activation of the TNF-α signaling network in the degenerating segment was due to the selective upregulation of multiple pro-apoptotic genes, including MADD, FADD, BID and caspases (Fig. 4E).

Likewise, there was a bias in the P segment versus the D segment related to the affected protein types. The injury predominantly affected the genes coding for enzymes and ion channels in the P stumps, while the transmembrane G-coupled receptors were selectively up-regulated in the D segments (Fig. 4F). Overall, these findings support and extend the observations by others, focused mainly on either the individual P or D changes or on the later time points post-axotomy (25-33).

Schwann Cell S100A9 Expression and Differential Activation of Kinase Pathways in Nerve Post-Injury- Next, we assessed the levels and the source for S100A8/A9 protein in the injured nerve. The S100 protein levels in the normal nerve were low and increased dramatically at day 1 post-axotomy in both the P and D segments shown exemplarily for S100A9 (Fig. 5A).

Rapid gene induction post-injury is normally followed by the activation of the signaling kinase pathways. Specifically, ERK, JNK and PI3K regulate a wide variety functions relating to Schwann and immune cell functions in nerve post-injury (39-43) and S100A8/A9-induced cell signaling (18-21,44,45). In agreement with these earlier data, significant activation of pERK1/2 was observed in the P and D stumps at day 1 post-transection as compared with the

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

6  

 

N nerve (Fig. 5A). The main phosphorylated isoform in both the P and D samples was pPI3K 85 kDa, while the PI3K 110 kDa isoform was the dominant species in the total PI3K pool. In turn, pJNK1/2 was activated in the P stumps at day 1 post-transection, compared with the N nerve. There was no similar increase in pJNK in the D stump samples. The S100A9 protein predominantly localized to crescent-shaped Schwann cells of both the P and D segments (for simplicity, only D is shown), as confirmed by co-localization of S100A9 with a phenotypic Schwann cell marker, S100B (Fig. 5B,C). In addition, the insignificant S100A9 immunoreactivity was occasionally observed in non-S100B-reactive vessel endothelial and other endoneurial cells of axotomized nerves (Fig. 5C).

S100A8/A9 Stimulates Cultured Schwann Cells- Calprotectin (S100A8/A9) induces immune cell chemotaxis (18,46) and cytokine expression (21). Because S100a8 and S100a9 were among the top-induced genes in both the P and D segments, we hypothesized that the S100A8/A9 heterodimer was implicated in the initial positive chemotactic gradient in the denervated Schwann cells.

To test this hypothesis, we performed genome-wide transcriptional profiling of cultured Schwann cells co-incubated for 1 h and 24 h with the purified S100A8/A9 protein complex. As a result of a short-term, 1 h, co-incubation with S100A8/A9, a number of the genes, especially chemokine genes, including Ccl7, Ccl2 and Cxcl2, were multi-fold upregulated in Schwann cells. Our further IPA analysis suggested that S100A8/A9 affected multiple cell adhesion and movement, chemotaxis and signaling pathways in Schwann cells (Fig. 6A, C). In sharp contrast, only a few genes were up-regulated in Schwann cells after long-term, 24 h co-incubation with S100A8/A9 (Fig. 6B, D). S100A8/A9 did not significantly regulate the expression of many other inflammatory genes of the IL, TNF, MMP, TLR and S100 families in Schwann cells in either sample (Fig. 6A-D). Taken together, these results indicated that the affected pathways were largely dissimilar in the 1 h versus 24 h cell samples and the most significant effect of S100A8/A9 takes place in Schwann cells shortly after their stimulation with S100A8/A9.

Similarity of the Inflammatory Gene Network in Injured Nerve and S100A8/A9-Treated Schwann Cells- We next determined if the individual genes

upregulated in Schwann cells treated with S100A8/A9 for 1 h were similar to those affected in the P and D segments of the axotomized sciatic nerve (Fig. 7A). The follow-on IPA analysis pointed to these similarly affected biological functions, diseases and canonical pathways (Fig. 7B,C), which were characteristic for stimulating chemotaxis, adhesion and motility of immune cells, including neutrophils and phagocytes. Furthermore, in Schwann cells treated with S100A8/A9, adhesion of immune cells was directly related to upregulation of chemokine (Ccl2, Ccl7, Cxcl2), calcitonin CALCA, Fas, Il33 and urokinase-type plasminogen activator PLAU genes, and downregulation of a protease inhibitor and cytokine transporter A2m (alpha-2-macroglobulin) (Fig. 7D).

The role of the agranulocyte/granulocyte activation and adhesion pathways in injured nerve was recapitulated by S100A8/A9 stimulation of the cultured Schwann cells and confirmed by our additional bioinformatics analysis of the transcriptional profiling data. Thus, transcriptional activity of multiple genes, including P-, E- and L-selectins and Lfa1, was enhanced in both the P segment and in stimulated Schwann cells (Fig. 8).

Intriguingly, some key pro-inflammatory factors in the axotomized nerve, including Il1β, Tnfα, Mmp9 and Mmp12, were not similarly induced in the isolated Schwann cells stimulated with S100A8/A9 (Fig. 8A,C). These data imply the presence of the immune mechanisms in the injured nerve that are not fully recapitulated in the purified Schwann cell cultures or are independent of the S100A8/A9 activity. Conversely, multiple other biological functions and canonical pathways supporting myeloid, phagocyte and leukocyte cell movement, adhesion and chemotaxis, were induced in the P and D nerve segments in a similar way relative to the S100A8/A9-stimulated Schwann cells (Fig. 9A).

S100A8/A9 Stimulates Immune Cell Recruitment into the Nerves- Extravasation of myeloid cells into the nerve, specifically of hematogenous CD68+ macrophages, is a critical event of Wallerian degeneration between days 2 and 14 post-injury (12,15-17). Thus, we tested the IPA analyses prediction for the myeloid cell migration after S100A8/A9 stimulation (Fig. 9A), using direct intraneural injection of the purified S100A8/A9 heterodimer into the intact nerve, followed by

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

7  

 

ultrastructural and immunohistochemical CD68 analyses of the injection site at day 7 post-injection (Fig. 9B). The nerves exposed to S100A8/A9 displayed areas of endoneurial edema with clusters of infiltrating immune cells, including phagocytes (Fig. 9B). In contrast, the nerve bundles injected with control PBS maintained normal morphology, displaying uncompromised axons, surrounded by a compact rim of myelin sheath. The significant increase in the macrophage numbers after S100A8/A9 injection was confirmed using antibody to CD68 (Fig. 9B). These data confirmed the key role of S100A8/A9 in creating a functional chemotactic gradient that guides myeloid cell migration into peripheral nerve.

DISCUSSION

Peripheral nerve injury that involves complete transection/axotomy of the nerve trunk can be broadly characterized as a clearance Wallerian degeneration process within the distal (D) stump, and the regeneration of the surviving axons in the proximal (P) stump, which remain connected to the neuronal cell body in the ganglia. Although both the degenerative and regenerative processes generally begin immediately after most types of nerve injury, axotomy entails a prolonged lag period in the regenerative process and staggered neurite growth (for up to 4 weeks in rats) (3,4,6). Overall, the present data confirm and extend the findings by us and others of the early transcriptional response to peripheral nerve injury, characterized by disintegration of the axonal cytoskeleton, immune response and cell death unfolding in the D segment (26,29,31,32) and cell proliferation, migration, axon guidance, regeneration in the P segment proximal to nerve injury (33,47). Our results offer an additional comparative insight into the specific rapid cell responses and activation of gene families and pathways, which are favorable for chemotaxis, adhesion and extravasation of myeloid cells in the D and P segments within the first day of peripheral nerve axotomy.

Multiple immune response genes from the chemokine, IL, TNF, TLR, S100 and MMP families were induced in both the P and D stumps. These data indicate that the molecular programs facilitating the acute inflammatory or degenerative changes exist in both the D and P segments. Indeed, calpain-dependent acute axonal degeneration of the proximal axons occurs in the spinal cord within minutes to hours after injury (48). Likewise, an increase in the immediate-

early immune response genes (e.g., Mmp9, Ccl20, Cxcl2 and Il6) and the calcium, agranulocyte and granulocyte signaling pathways was observed in the P segment within day 1 post-axotomy in our present study and also by us and others earlier (33,47).

Schwann cells remain the main cell population in peripheral nerve at 1 day post-injury. Accordingly, the predominant changes described here mainly represent features of the injury-induced Schwann cell activation and trans-differentiation into the Büngner cell. In addition, resident reactive fibroblasts may regulate genes involved to the epineurial scarring, as endothelial cells of the nerve vasculature, and a small population of the resident macrophages and mast cells may contribute, albeit insignificantly, to the transcriptional profiles in nerve at day 1 post-injury.

Infiltrating cell populations in nerve within day 1 post-injury include neutrophils and patrolling lymphocytes (15,17,38,49). Consistently, activation of granulocyte and agranulocyte signaling is recorded at day 1 post-axotomy. Hematogenous macrophages generally infiltrate the D or P segments after day 2 post-injury (2) in order to complete the debris clearance processes (22,23).

Herein, we identified S100a8 and S100a9 among the top induced genes in peripheral nerve post-injury, and by employing the purified S100A8/A9 heterodimer (calprotectin) established its important role in stimulating the chemokine-cytokine network and the initial chemotactic gradient in Schwann cells. This gradient attracts the hematogenous immune cells to the nerve injury site. In addition, this gradient may control migration of resident cells, including Schwann cells. Schwann cells migrate from both the P and D stumps into the nerve gap resulting from transection (2). Specifically, stimulation of cultured Schwann cells with S100A8/A9 recapitulated a significant portion of the pro-inflammatory gene network activation we observed in the axotomized nerve, including chemokine (Ccl7, Ccl2, Cxcl2), inflammatory cytokine (Il1r1, Il33) and MMPs (Mmp3, Mmp7, Mmp13), and agranulocyte- and granulocyte activation signaling pathways.

S100A8/A9, also known as myeloid-related proteins MRP8/14, initiated myeloid (CD68+ macrophage) migration into the intact nerve. Schwann cells were the main cell source for S100A9 at day 1 post-axotomy. Future studies will need to decipher the S100A8 and S100A9 source and roles in the

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

8  

 

orchestration of complex immune cell migration patterns and functions in the course of Wallerian degeneration (15,17,38,49).

The S100A8 and S100A9 homodimers replicate, and in some circumstances exceed, activities of the S100a8/A9 heterodimer. The present study does not rule out the possibility that some effects observed here relate to homodimers formation in the heterodimer preparations or homodimers formation in the injured nerve, expressing high levels of both S100a8 and S100a9 transcripts. Future investigation is required to distinguish between the effects of hetero and homodimers in modulating the inflammatory program in peripheral nerve injury and repair.

S100A8/A9 are endogenous ligands of TLR4 (44) and RAGE (45) both of which are expressed in Schwann cells (50,51). The S100B protein, a phenotypic marker of Schwann cells, is distinct structurally and functionally from S100A8/A9. Interestingly, S100A8/A9 stimulated other immune response genes in Schwann cells, including Itga4 gene, coding for integrin alpha 4, which forms the VLA4/α4β1 lymphocyte/monocyte homing receptor. S100A8/A9 also induced the axonal guidance signaling pathway, Htr4 gene encoding G-protein-coupled serotonin 5-HT4 receptor and the inhibition of the MMP pathway in Schwann cells.

S100A8/A9 stimulation was, however, ineffective in regulating the Schwann cell expression of the top-

induced inflammatory genes expressed in nerve shortly after axotomy, including Il6, Il1β, Tnfα, Timp1 and Mmp9. These data corroborate the presence of the S100A8/A9-independent immune activation mechanisms in injured nerve in vivo. In addition, S100A8/A9 stimulation did not activate lymphocyte migration signaling, a late-response immune activation event in the damaged nerve (38). We conclude that S100A8/A9 may initiate the acute-phase response signaling and chemotactic gradient preceeding the major inflammatory response in the damaged nerve.

Worth noting are the differences in transcriptional programs observed between the murine and rat nerve injury models. Thus, Timp1 was among the top 10 up-regulated genes in the axotomized murine nerves (25). In turn, due to the high pre-existing expression of the Timp1 in normal rat sciatic nerve, only a 2-fold induction of Timp1 was recorded post-axotomy, suggesting the presence of the species-specific mechanisms of transcriptional regulation.

In sum, we determined that S100A8/A9 are potent initiators of the immune response in the stimulated Schwann cells of the injured peripheral nerve. Upregulation of S100A8/A9 in Schwann cells shortly post-injury contributes to the activation of the chemokine-cytokine network and the initial chemotactic gradient that guides the hematogenous immune cells towards the injury site.

REFERENCES:

1.   Gordon,   T.,   Sulaiman,   O.,   and   Boyd,   J.   G.   (2003)   Experimental   strategies   to   promote   functional  recovery  after  peripheral  nerve  injuries.  J  Peripher  Nerv  Syst  8,  236-­‐250  

2.   Zochodne,  D.  W.  (2012)  The  challenges  and  beauty  of  peripheral  nerve  regrowth.  J  Peripher  Nerv  Syst  17,  1-­‐18  

3.   McDonald,   D.,   Cheng,   C.,   Chen,   Y.,   and   Zochodne,   D.   (2006)   Early   events   of   peripheral   nerve  regeneration.  Neuron  Glia  Biol  2,  139-­‐147  

4.   Wood,  M.  D.,  Kemp,  S.  W.,  Weber,  C.,  Borschel,  G.  H.,  and  Gordon,  T.  (2011)  Outcome  measures  of  peripheral  nerve  regeneration.  Ann  Anat  193,  321-­‐333  

5.   Waller,  A.  (1850)  Experiments  on  the  section  of  the  glossopharyngeal  and  hypoglossal  nerves  of  the  frog  and  observations  of  the  alterations  produced  thereby  in  the  structure  of   their  primitive  fibers.  Philos  Trans  R  Soc  Lond  B  Biol  Sci  140,  423-­‐429  

6.   Gordon,  T.,  Tyreman,  N.,  and  Raji,  M.  A.  (2011)  The  basis  for  diminished  functional  recovery  after  delayed  peripheral  nerve  repair.  J  Neurosci  31,  5325-­‐5334  

7.   Vargas,  M.  E.,  and  Barres,  B.  A.   (2007)  Why   is  Wallerian  degeneration   in   the  CNS  so  slow?  Annu  Rev  Neurosci  30,  153-­‐179  

8.   Chen,  Y.  Y.,  McDonald,  D.,  Cheng,  C.,  Magnowski,  B.,  Durand,   J.,  and  Zochodne,  D.  W.  (2005)  Axon  and  Schwann  cell  partnership  during  nerve  regrowth.  J  Neuropathol  Exp  Neurol  64,  613-­‐622  

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

9  

 

9.   Ramon   y   Cajal,   S.   (1991)   Cajal's   Degeneration   &   Regeneration   of   the   Nervous   System,   Oxford  University  Press,  New  York  

10.   Nix,   P.,   Hisamoto,   N.,   Matsumoto,   K.,   and   Bastiani,   M.   (2011)   Axon   regeneration   requires  coordinate  activation  of  p38  and  JNK  MAPK  pathways.  Proc  Natl  Acad  Sci  U  S  A  108,  10738-­‐10743  

11.   Kenney,   A.   M.,   and   Kocsis,   J.   D.   (1998)   Peripheral   axotomy   induces   long-­‐term   c-­‐Jun   amino-­‐terminal  kinase-­‐1  activation  and  activator  protein-­‐1  binding  activity  by  c-­‐Jun  and  junD  in  adult  rat  dorsal  root  ganglia  In  vivo.  J  Neurosci  18,  1318-­‐1328  

12.   Chen,   Z.   L.,   Yu,  W.  M.,   and   Strickland,   S.   (2007)   Peripheral   regeneration.  Annu  Rev  Neurosci  30,  209-­‐233  

13.   Allodi,   I.,   Udina,   E.,   and   Navarro,   X.   (2012)   Specificity   of   peripheral   nerve   regeneration:  interactions  at  the  axon  level.  Prog  Neurobiol  98,  16-­‐37  

14.   Jessen,  K.  R.,  and  Mirsky,  R.  (2008)  Negative  regulation  of  myelination:  relevance  for  development,  injury,  and  demyelinating  disease.  Glia  56,  1552-­‐1565  

15.   Kieseier,  B.  C.,  Hartung,  H.  P.,  and  Wiendl,  H.  (2006)  Immune  circuitry   in  the  peripheral  nervous  system.  Curr  Opin  Neurol  19,  437-­‐445  

16.   Myers,   R.   R.,   Campana,   W.   M.,   and   Shubayev,   V.   I.   (2006)   The   role   of   neuroinflammation   in  neuropathic  pain:  mechanisms  and  therapeutic  targets.  Drug  Discov  Today  11,  8-­‐20  

17.   Thacker,  M.  A.,  Clark,  A.  K.,  Marchand,  F.,  and  McMahon,  S.  B.  (2007)  Pathophysiology  of  peripheral  neuropathic  pain:  immune  cells  and  molecules.  Anesth  Analg  105,  838-­‐847  

18.   Lackmann,  M.,  Rajasekariah,  P.,  Iismaa,  S.  E.,  Jones,  G.,  Cornish,  C.  J.,  Hu,  S.,  Simpson,  R.  J.,  Moritz,  R.  L.,   and  Geczy,  C.  L.   (1993)   Identification  of  a   chemotactic  domain  of   the  pro-­‐inflammatory  S100  protein  CP-­‐10.  J  Immunol  150,  2981-­‐2991  

19.   Vogl,  T.,  Ludwig,  S.,  Goebeler,  M.,  Strey,  A.,  Thorey,  I.  S.,  Reichelt,  R.,  Foell,  D.,  Gerke,  V.,  Manitz,  M.  P.,  Nacken,   W.,   Werner,   S.,   Sorg,   C.,   and   Roth,   J.   (2004)   MRP8   and   MRP14   control   microtubule  reorganization  during  transendothelial  migration  of  phagocytes.  Blood  104,  4260-­‐4268  

20.   Hobbs,  J.  A.,  May,  R.,  Tanousis,  K.,  McNeill,  E.,  Mathies,  M.,  Gebhardt,  C.,  Henderson,  R.,  Robinson,  M.  J.,  and  Hogg,  N.  (2003)  Myeloid  cell  function  in  MRP-­‐14  (S100A9)  null  mice.  Mol  Cell  Biol  23,  2564-­‐2576  

21.   Simard,   J.   C.,  Cesaro,  A.,  Chapeton-­‐Montes,   J.,  Tardif,  M.,  Antoine,  F.,  Girard,  D.,   and  Tessier,  P.  A.  (2013)  S100A8  and  S100A9   induce  cytokine  expression  and   regulate   the  NLRP3   inflammasome  via  ROS-­‐dependent  activation  of  NF-­‐kappaB(1.).  PLoS  One  8,  e72138  

22.   Fernandez-­‐Valle,   C.,   Bunge,   R.   P.,   and   Bunge,   M.   B.   (1995)   Schwann   cells   degrade   myelin   and  proliferate   in   the   absence   of   macrophages:   evidence   from   in   vitro   studies   of   Wallerian  degeneration.  J  Neurocytol  24,  667-­‐679  

23.   Perry,   V.   H.,   Brown,   M.   C.,   and   Gordon,   S.   (1987)   The   macrophage   response   to   central   and  peripheral  nerve   injury.  A  possible   role   for  macrophages   in   regeneration.   J  Exp  Med  165,   1218-­‐1223  

24.   Brockes,   J.   P.,   Fields,   K.   L.,   and   Raff,   M.   C.   (1979)   Studies   on   cultured   rat   Schwann   cells.   I.  Establishment  of  purified  populations  from  cultures  of  peripheral  nerve.  Brain  Res  165,  105-­‐118  

25.   Kim,   Y.,   Remacle,   A.   G.,   Chernov,   A.   V.,   Liu,   H.,   Shubayev,   I.,   Lai,   C.,   Dolkas,   J.,   Shiryaev,   S.   A.,  Golubkov,  V.  S.,  Mizisin,  A.  P.,  Strongin,  A.  Y.,  and  Shubayev,  V.  I.  (2012)  The  MMP-­‐9/TIMP-­‐1  axis  controls   the   status   of   differentiation   and   function   of   myelin-­‐forming   Schwann   cells   in   nerve  regeneration.  PLoS  One  7,  e33664  

26.   Nagarajan,  R.,  Le,  N.,  Mahoney,  H.,  Araki,  T.,  and  Milbrandt,  J.  (2002)  Deciphering  peripheral  nerve  myelination  by  using  Schwann  cell  expression  profiling.  Proc  Natl  Acad  Sci  U  S  A  99,  8998-­‐9003  

27.   Jiang,  N.,  Li,  H.,  Sun,  Y.,  Yin,  D.,  Zhao,  Q.,  Cui,  S.,  and  Yao,  D.  (2014)  Differential  gene  expression  in  proximal   and   distal   nerve   segments   of   rats   with   sciatic   nerve   injury   during   Wallerian  degeneration.  Neural  Regen  Res  9,  1186-­‐1194  

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

10  

 

28.   D'Antonio,  M.,  Michalovich,  D.,  Paterson,  M.,  Droggiti,  A.,  Woodhoo,  A.,  Mirsky,  R.,  and  Jessen,  K.  R.  (2006)   Gene   profiling   and   bioinformatic   analysis   of   Schwann   cell   embryonic   development   and  myelination.  Glia  53,  501-­‐515  

29.   Bosse,   F.,   Hasenpusch-­‐Theil,   K.,   Kury,   P.,   and   Muller,   H.   W.   (2006)   Gene   expression   profiling  reveals  that  peripheral  nerve  regeneration  is  a  consequence  of  both  novel   injury-­‐dependent  and  reactivated  developmental  processes.  Journal  of  neurochemistry  96,  1441-­‐1457  

30.   Kubo,  T.,  Yamashita,  T.,  Yamaguchi,  A.,  Hosokawa,  K.,  and  Tohyama,  M.   (2002)  Analysis  of  genes  induced  in  peripheral  nerve  after  axotomy  using  cDNA  microarrays.  J  Neurochem  82,  1129-­‐1136  

31.   Araki,   T.,   Sasaki,   Y.,   and   Milbrandt,   J.   (2004)   Increased   nuclear   NAD   biosynthesis   and   SIRT1  activation  prevent  axonal  degeneration.  Science  305,  1010-­‐1013  

32.   Yao,   D.,   Li,   M.,   Shen,   D.,   Ding,   F.,   Lu,   S.,   Zhao,   Q.,   and   Gu,   X.   (2013)   Expression   changes   and  bioinformatic  analysis  of  Wallerian  degeneration  after  sciatic  nerve  injury  in  rat.  Neurosci  Bull  29,  321-­‐332  

33.   Li,  S.,  Liu,  Q.,  Wang,  Y.,  Gu,  Y.,  Liu,  D.,  Wang,  C.,  Ding,  G.,  Chen,  J.,  Liu,  J.,  and  Gu,  X.  (2013)  Differential  gene  expression  profiling  and  biological  process  analysis  in  proximal  nerve  segments  after  sciatic  nerve  transection.  PLoS  One  8,  e57000  

34.   Vogl,   T.,   Leukert,  N.,   Barczyk,   K.,   Strupat,   K.,   and  Roth,   J.   (2006)  Biophysical   characterization   of  S100A8  and  S100A9  in  the  absence  and  presence  of  bivalent  cations.  Biochim  Biophys  Acta  1763,  1298-­‐1306  

35.   Shubayev,   V.   I.,   Angert,   M.,   Dolkas,   J.,   Campana,   W.   M.,   Palenscar,   K.,   and   Myers,   R.   R.   (2006)  TNFalpha-­‐induced  MMP-­‐9  promotes  macrophage  recruitment   into   injured  peripheral  nerve.  Mol  Cell  Neurosci  31,  407-­‐415  

36.   Chattopadhyay,   S.,   and   Shubayev,   V.   I.   (2009)   MMP-­‐9   controls   Schwann   cell   proliferation   and  phenotypic   remodeling   via   IGF-­‐1   and  ErbB   receptor-­‐mediated   activation  of  MEK/ERK  pathway.  Glia  57,  1316-­‐1325  

37.   Livak,  K.  J.,  and  Schmittgen,  T.  D.  (2001)  Analysis  of  relative  gene  expression  data  using  real-­‐time  quantitative  PCR  and  the  2(-­‐Delta  Delta  C(T))  Method.  Methods  25,  402-­‐408  

38.   Austin,   P.   J.,   and   Moalem-­‐Taylor,   G.   (2010)   The   neuro-­‐immune   balance   in   neuropathic   pain:  involvement  of  inflammatory  immune  cells,  immune-­‐like  glial  cells  and  cytokines.  J  Neuroimmunol  229,  26-­‐50  

39.   Harrisingh,  M.  C.,  Perez-­‐Nadales,  E.,  Parkinson,  D.  B.,  Malcolm,  D.  S.,  Mudge,  A.  W.,  and  Lloyd,  A.  C.  (2004)   The   Ras/Raf/ERK   signalling   pathway   drives   Schwann   cell   dedifferentiation.  Embo   J  23,  3061-­‐3071  

40.   Sheu,   J.   Y.,   Kulhanek,  D.   J.,   and   Eckenstein,   F.   P.   (2000)  Differential   patterns   of   ERK   and   STAT3  phosphorylation  after  sciatic  nerve  transection  in  the  rat.  Exp  Neurol  166,  392-­‐402  

41.   Maurel,  P.,  and  Salzer,  J.  L.  (2000)  Axonal  regulation  of  Schwann  cell  proliferation  and  survival  and  the  initial  events  of  myelination  requires  PI  3-­‐kinase  activity.  J  Neurosci  20,  4635-­‐4645  

42.   Zrouri,  H.,  Le  Goascogne,  C.,  Li,  W.  W.,  Pierre,  M.,  and  Courtin,  F.  (2004)  The  role  of  MAP  kinases  in  rapid  gene  induction  after  lesioning  of  the  rat  sciatic  nerve.  The  European  journal  of  neuroscience  20,  1811-­‐1818  

43.   Abe,  N.,  and  Cavalli,  V.  (2008)  Nerve  injury  signaling.  Curr  Opin  Neurobiol  18,  276-­‐283  44.   Vogl,  T.,  Tenbrock,  K.,  Ludwig,  S.,  Leukert,  N.,  Ehrhardt,  C.,  van  Zoelen,  M.  A.,  Nacken,  W.,  Foell,  D.,  

van  der  Poll,  T.,  Sorg,  C.,  and  Roth,  J.  (2007)  Mrp8  and  Mrp14  are  endogenous  activators  of  Toll-­‐like  receptor  4,  promoting  lethal,  endotoxin-­‐induced  shock.  Nature  medicine  13,  1042-­‐1049  

45.   Turovskaya,  O.,  Foell,  D.,  Sinha,  P.,  Vogl,  T.,  Newlin,  R.,  Nayak,  J.,  Nguyen,  M.,  Olsson,  A.,  Nawroth,  P.  P.,   Bierhaus,   A.,   Varki,   N.,   Kronenberg,   M.,   Freeze,   H.   H.,   and   Srikrishna,   G.   (2008)   RAGE,  carboxylated   glycans   and   S100A8/A9   play   essential   roles   in   colitis-­‐associated   carcinogenesis.  Carcinogenesis  29,  2035-­‐2043  

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

11  

 

46.   Lackmann,  M.,  Cornish,  C.   J.,  Simpson,  R.   J.,  Moritz,  R.  L.,  and  Geczy,  C.  L.   (1992)  Purification  and  structural   analysis   of   a  murine   chemotactic   cytokine   (CP-­‐10)  with   sequence   homology   to   S100  proteins.  The  Journal  of  biological  chemistry  267,  7499-­‐7504  

47.   Wang,  Y.,  Tang,  X.,  Yu,  B.,  Gu,  Y.,  Yuan,  Y.,  Yao,  D.,  Ding,  F.,  and  Gu,  X.  (2012)  Gene  network  revealed  involvements  of  Birc2,  Birc3  and  Tnfrsf1a  in  anti-­‐apoptosis  of  injured  peripheral  nerves.  PLoS  One  7,  e43436  

48.   Kerschensteiner,   M.,   Schwab,   M.   E.,   Lichtman,   J.   W.,   and   Misgeld,   T.   (2005)   In   vivo   imaging   of  axonal  degeneration  and  regeneration  in  the  injured  spinal  cord.  Nature  medicine  11,  572-­‐577  

49.   Ren,  K.,  and  Dubner,  R.  (2010)  Interactions  between  the  immune  and  nervous  systems  in  pain.  Nat  Med  16,  1267-­‐1276  

50.   Goethals,  S.,  Ydens,  E.,  Timmerman,  V.,  and  Janssens,  S.  (2010)  Toll-­‐like  receptor  expression  in  the  peripheral  nerve.  Glia  58,  1701-­‐1709  

51.   Perrone,  L.,  Peluso,  G.,  and  Melone,  M.  A.  (2008)  RAGE  recycles  at  the  plasma  membrane  in  S100B  secretory  vesicles  and  promotes  Schwann  cells  morphological  changes.  J  Cell  Physiol  217,  60-­‐71  

FIGURE LEGENDS

FIGURE 1. A-D, heatmaps of the genome-wide transcriptional profiling data of the axotomized (day 1) rat sciatic nerve. Red and blue correspond to the high and the low expression levels, respectively. Color map inset shows the signal intensity scale. Top 50 up-regulated (A-B) and 50 down-regulated (C-D) genes in the proximal and distal segments are shown. Only the statistically significant data (p<0.05) were used in our analysis. P, D and N are proximal, distal and normal contralateral nerve samples, respectively. S100a8 and S100a9 genes are highlighted with asterisks.

FIGURE 2. Immunomodulatory gene families affected in the proximal and distal segments at day 1 post-axotomy. Chemokine, S100, TLR, TNF, interleukin, Fc receptor and MHC families are shown. Fold-change values were calculated relative to the normal nerve using the normalized intensity values (p<0.05). Red and blue correspond to up- and downregulated genes, respectively. Heatmaps for the affected genes (p<0.05) are shown. The genes with changes over 2-fold (5-fold for chemokines and interleukins) in n=3/group were included in the analysis. Color map inset shows the signal intensity scale. P, D and N are proximal, distal and normal nerve samples, respectively.

FIGURE 3. A-C, specific gene families affected in the proximal and distal segments at day 1. MMP and TIMP (A), ion channel (B) gene families are shown. Fold-change values were calculated relative to the normal nerve using the normalized intensity values (p<0.05). The ion channel genes with fold-change over 3 are displayed. Red and blue correspond to up- and downregulated genes, respectively. Heatmaps of the affected genes (p<0.05) are shown. Color map inset shows the signal intensity scale. P, D and N are proximal, distal and normal nerve samples, respectively. C, real-time RT-PCR of Il6, Il1β, Tnfα, Timp1 and Mmp9 genes in rat sciatic nerves at day 1 post-axotomy in the D sample. The mean relative mRNA levels of n=4/group were normalized to Gapdh. Solid bars indicate fold-change relative to the normal nerve samples (*p<0.05, **p<0.01). Standard errors are indicated.

FIGURE 4. A-D, top 25 up-regulated genes that are selectively upregulated at day 1 post-axotomy in the proximal (A) or distal (B) nerve segments. Horizontal axis represents the signal intensity ratio (log2). C, canonical pathway analysis (IPA) of the differentially expressed genes in the P (orange) and D (blue) nerve segments. Up-regulated myosin (D) and pro-apoptotic (E) gene families affected in the proximal and distal segments at day 1. Fold-change values were calculated relative to the normal nerve using the normalized intensity values (p<0.05). Red and blue correspond to up- and downregulated genes, respectively. Heatmaps of the affected genes (p<0.05) are shown. Color map inset shows the signal intensity scale. P, D and N are proximal, distal and normal nerve samples, respectively. F, protein types up-regulated in the P (orange) and D (blue) nerve segments. The genes with fold-change over 3 were included in this analysis. The p values were

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

12  

 

calculated using the right-tailed Fisher exact test. P, D and N are proximal, distal and normal nerve samples, respectively.

FIGURE 5. A-C, S100A9 and activation of ERK, JNK and PI3K kinases. A, immunoblotting for S100A9, β-actin, phosphorylated and total ERK, PI3K, and JNK in the proximal and distal sciatic nerve segments at day 1 post-axotomy. Normal samples represent contralateral nerve control. The data from n=2/group are shown. P, D and N are proximal, distal and normal contralateral nerve samples, respectively. B, immunostaining of S100A9 (2’2-diaminobenzidine, brown) in the distal stump at day 1 post-axotomy. Schwann cells are indicated by arrows. Inset in the upper left corner shows a single Schwann cell. Scale bar correspond to 25 µm. C, immunostaining of S100B (green) and S100A9 (red) in the injured nerve. S100B/S100A9-positive Schwann cells are shown with asterisks. S100A9-positive, S100B-negative non-Schwann cells and vessel cells are shown with arrows and V, respectively. Nuclei were stained with DAPI (blue). Scale bars correspond to 15 µm.

FIGURE 6. A-D, top up- and downregulated genes in cultured Schwann cells stimulated with S100A8/A9 for 1 h (A) and 24 h (B). Fold-change values were calculated relative to the intact Schwann cell control (p<0.05). Red and blue, up- and downregulated genes, respectively. C-D, canonical pathway analysis (IPA) of the differentially expressed genes in Schwann cells following a 1 h (C) and 24 h (D) stimulation with S100A8/A9. The p values (green bars) were calculated using the right-tailed Fisher exact test.

FIGURE 7. A-D, up-regulated gene network overlap in S100A8/A9-stimulated cultured Schwann cells and injured nerve. A, up-regulated genes in Schwann cells after a 1 h stimulation with S100A8/A9 relative to the intact cell control and in the proximal (P/N) and distal (D/N) nerve segments relative to the normal nerve control. Bars correspond to fold change (log2) of normalized signal intensity (p<0.05). B, biological function and disease analysis (IPA) of the genes in panel A. C, canonical pathway analysis (IPA) of the genes in panel A. Green bars, p values, which were calculated using the right tailed Fisher exact test. D, Activation of the immune cell adhesion network following a 1 h stimulation of Schwann cells with S100A8/A9. Red and green correspond to up- and downregulated genes, respectively. Arrows indicate stimulation. P, D and N are proximal, distal and normal nerve samples, respectively.

FIGURE 8. A-C, agranulocyte/granulocyte activation and adhesion pathways in S100A8/A9-stimulated Schwann cells and the injured nerve. A, chemokine, claudin, integrin, interleukin, Mmp, myosine and other genes regulated in both proximal/distal nerve segments and in cultured Schwann cells after 1 h and 24 h stimulation with S100A8/A9. Signal intensity scale is shown. B, agranulocyte activation pathway indicates upregulated (red) and downregulated (green) genes observed in the proximal nerve segment. C, regulation of agranulocyte-specific genes in the injured nerve compared to cultured Schwann cells after 1 h and 24 h stimulation with S100A8/A9. P/N, fold-change (log2) of signal intensity in proximal nerve segment relative to the normal nerve (p<0.05). Red and blue correspond to up- and downregulated genes, respectively. Heatmap of the affected genes (p<0.05) in post-injury nerve segments and in cultured Schwann cells after 1 h and 24 h stimulation with S100A8/A9. P, D and N are proximal, distal and normal nerve samples, respectively.

FIGURE 9. A-C, S100A8/A9 stimulates macrophage migration into the nerve. A-B, IPA biological functions (A) and canonical pathways (B) in injured nerve and Schwann cells stimulated for 1 h and 24 h with S100A8/A9. Color intensity map inset shows the IPA prediction confidence Z-scores (biological functions) and -log(P) values (canonical pathways). The p values were calculated using the right-tailed Fisher exact test. P, D and N are proximal, distal and normal nerve samples, respectively. C, ultrastructure and immunostaining analyses of the naïve nerve after injection of S100A8/A9. Top left panel, methylene blue/azure II staining in 1-µm-thick sciatic nerve sections of S100A8/A9-injected nerves display the areas of endoneurial edema with infiltrating phagocytes (arrows). Top right panel, uncompromised axons surrounded by a compact rim of myelin sheath in the control nerve. Bottom panels, CD68 immunostaining of macrophages (green) in S100A8/A9-injected and control 10-µm-thick nerve sections. Scale bars correspond to 25 µm.

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

Dow

n-re

gula

ted

gene

s

Figure 1

1 5 10 15

Signal intensity scale, log2

P D N DP N DP NP D N

P D N DP N DP NP D N

A ProximalU

p-re

gula

ted

gene

s

Up-

regu

late

d ge

nes

Dow

n-re

gula

ted

gene

s

Distal

Proximal Distal

B

C DUmodl1Olr1541Foxb1Olr1765Cyp2e1Olr205Gria2Rgd1565502Rgd1562011Zfp280bRgd1307461Rnase1Loc683422Slc35d3Shank1Rnase1l2Loc690352Best1Skint4Aldh1a7Atp2b4Loc689570Hoxa1Mast3Mrgprg

Rgd1311080Bin2aKcnip4Slc15a2Pck1Lrrn1Rgd1561551Rgd1564894Rgd1563815Rgd1311501Loc680226Rgd1565629Olr179Tex15Gpr1Gh1Loc64038Ube2ql1Rnf148Rgd1563975Ctsql2Adhfe1Sval1Ly6hRgd1307595

Umodl1Rgd1307461Olr1541Foxb1Loc683422Olr205Hsd3b5Loc500567Zfp280bRgd1562011Cyp2e1Rgd1563815Rgd1565502Gria2Rgd1563975Tex15Slc35d3Shank1Loc681453Loc680226MrgprgSkint4Rnf148Stk32bOprk1

Hoxa1Rgd1565629Loc691259Loc689570Loc680613Loc690352Olr1765Ly6hLrrn1Loc683891Loc500702Dleu7Ube2ql1Rgd1564894Atp2b4Olr179Vom1r8Kcnh8Cyp26b1DdnRgd1307595Ctsql2Aox4Cytl1Olr1486

Il6Ucn2Cxcl3S100a8TrhCcl20Reg3gPtx3Rgd1564937Myh4S100a9Myl1NppbMylpfCxcl2Mybpc2Olr1Clec4dMyf6Fosl1Stfa2l2MyotMyh8Kng1Es1

Art1Mmp9SiglechPram1Ampd1Clca4lTnnc2Wfdc15bGrxcr1Serpinb10Cox6a2Cdc6Kng1l1Clec4a2TrdnTtnHist2h3cAtp2a1Kbtbd10PbkDio3Slco4a1Ube2tIl1rnVgf

Ucn2S100a8Ptx3SiglechReg3gIl6Fosl1Ccl20Serpinb10PbkS100a9Clec4dSlco4a1Clec4a2Ube2tHist2h3cSpibMs4a12Cxcl3Stfa2l2Rrm2Cdc6Ska1Pram1Wfdc15b

Asf1bLilrb4Clca4lKng1Ccna2Mlf1ipHist1h1aIl1rl1Mybl2Gtsf1Hk3NppbFam111aLctlKng1l1MelkMpeg1Olr1Vom1r15TrhNs5atp9Hs3st3a1Gdf3TtkCol7a1

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

Figure 2

1 5 10 15

Signal intensity scale, log2

P/N

D/N

Chemokines P D NCxcl3Ccl20Cxcl2Cxcl1Ccl2Ccl7Cxcl6Cxcl14Ccl3Ccl12

100 200 300

296.0163.179.131.921.914.012.87.05.75.1

100

51.087.716.814.217.911.94.15.38.9

10.2

Interleukins P D NIl6Il1rnIl1r2Il1rl1Il8rbIl1bIl1f8Il21rIl1r1Il10

391.536.228.928.218.413.511.29.87.66.1

100 300

104.722.98.7

38.010.99.3

12.316.06.0

12.040 120

P/N

D/N

P/N

TNFs P D N

D/N

Tnfsf9Tnfrsf12aTnfaip6Tnfrsf10bTnfrsf18Tnfrsf1bTnfrsf14Tnfsf14TnfTnfaip8l2Tnfsf15Tnfsf10

-5 5 5

7.96.74.14.54.84.85.33.94.14.1

9.07.75.85.45.13.93.73.02.62.5

-5.2-4.5

-4.8-4.9

-5

P/N

TLRs P D N

D/N

5 5

Tlr2Tlr10Tlr6Tlr7Tlr12Tlr8Tlr4

7.76.05.82.82.22.11.5

7.68.07.95.32.82.92.5

P/N

P D N

D/N

Fc receptors

10 20 10

24.35.05.05.04.13.02.6

12.06.18.87.14.96.04.2

FcarFcgr1aFcgr3aFcgr2bFcgr2aFcrlaFcer1g

P D N

P/N

S100 genes

D/N

-5 100 100

236.990.4-2.4-2.4-3.1-3.1-3.3-3.7

-5

161.072.9-2.6-2.0-4.3-4.0-7.0-3.1

S100a8S100a9S100a1S100zS100bS100bS100a3S100a7a

P D N

D/N

MHC

2-2

RT1-N3RT1-DMbRT1-DMaRT1-BaRT1-N1RT1-HaRT1-CE6RT1-M2RT1-M6-1

2.72.12.31.82.52.12.1

-2.1-5.3

P/N

2-2

2.22.12.12.11.91.81.7

-1.7-2.4

fold changefold change

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

A

Figure 3

1 5 10 15

Signal intensity scale, log2

P/N

TIMPs P D N

D/N

Timp1Timp2Timp3Timp4

-3 3 6 -3 3

5.4 4.8-1.9-1.8-3.6

-1.8-2.7-3.7

P/N

D/N

MMPs P D NMmp9Mmp12Mmp3Mmp7Mmp13Mmp8Mmp10Mmp17Mmp19Mmp2Mmp20Mmp16Mmp1aMmp1bMmp25Mmp15Mmp21Mmp28Mmp14Mmp24Mmp11Mmp23Mmp27

-10 20 40 60

55.433.025.48.66.04.72.82.82.2

-1.0-1.0-1.1-1.1-1.2-1.2-1.3-1.3-1.4-1.6-1.8-2.6-2.8-4.3

-10 10 20

24.325.812.511.62.63.5

-1.42.82.5

-1.4-1.0-1.5-1.2-1.11.4

-2.1-1.2-1.5-1.7-1.7-3.4-3.1-5.8

C

0

20

40

60

80

100**

**

** *R

elat

ive

mR

NA

in d

ista

l ner

ve

day 1 post-injuryN Il6 Il1β Tnfα Timp1 Mmp9

B Ion channels

P/N

D/N

P D NClca4lCacng1Kcnn4Kcnj16Kcnn4Scn3bCacna1sScn4bCacng6Cacng1Kcnk10Kcna3Kcnh1Clcn3Kcnb1Kcnk16Clcn2Kcna5Kcnk1Cacna1hCacnb3Kcna2Scn2a1Clcn4-2Kcnk16Scn7aScn9aKcnv1Cacnb4Kcne2Scn7aKcnj13Kcnh2Cacnb4Kcnh8Kcnip4

20-20 40

51.39.58.27.76.25.85.75.55.04.64.23.93.3

-3.1-3.1-3.3-3.3-3.5-3.6-3.7-3.7-3.8-3.8-3.9-4.0-4.1-4.3-4.4-4.4-4.4-4.9-5.3-5.8-6.2-6.5-19.4

20-20 40

40.81.67.6

11.55.31.8

-1.0-1.05.11.01.32.51.9

-4.6-2.7-5.0-4.9-4.9-6.0-2.0-4.8-5.1-4.0-4.2-5.7-7.0-7.4-2.7-3.5-3.8-8.7-7.8-4.9-5.2-16.2-7.5

fold change

fold change

fold change

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

Figure 4

1 5 10 15

Signal intensity scale, log2

D/ND

D

P/NP

P

A B C

D

E

F

2

5

Cal

cium

Sig

nalin

gA

gran

uloc

yte

Adh

esio

n an

d D

iape

desi

sG

lyco

lysi

s I

Glu

cone

ogen

esis

IIL

K S

igna

ling

Epith

elia

l Adh

eren

s Ju

nctio

n Si

gnal

ing

His

tidin

e D

egra

datio

n VI

IL-6

Sig

nalin

gA

ctin

Cyt

oske

leto

n Si

gnal

ing

Rol

e of

IL-1

7A in

Art

hriti

s

2

5

TNFR

1 Si

gnal

ing

Tum

oric

idal

Fun

ctio

n of

Nat

ural

Kill

er C

ells

T Ly

mph

ocyt

e-m

edia

ted

Apo

ptos

is o

f Tar

get C

ells

Ret

inoi

c ac

id M

edia

ted

Apo

ptos

is S

igna

ling

TWEA

K S

igna

ling

Gra

nzym

e B

Sig

nalin

gN

atur

al K

iller

Cel

l Sig

nalin

gD

eath

Rec

epto

r Sig

nalin

gSp

hing

osin

e-1-

phos

phat

e Si

gnal

ing

PI3K

Sig

nalin

g in

B L

ymph

ocyt

es

3 5 7

C11orf1Myf6MyotArt1Trdn

Loc100361099Ajap1

Trim54Art5

Stac3Gimd1

AregAnkrd2

Sypl2Pdlim3Trim72

Xirp2Myoz1Ckmt2Cmya5

Loc691670Fbp2Myh2

HalCxcl6

fold change, Log2 fold change, Log2

-Log

(P)

2 4

Vom1r10Cenpw

Rgd2301395HellsLrr1

Olr991Tas2r39Kcnj16Ckap2l

Jakmip1Foxm1

AgxtNcapgBaiap3

Kiaa1524Primpol

Dna2Ly49i2Daw1MndaEme1Kctd4CenpeUhrf1

Igfbp2

20Number of up-regulated genes0 10

enzymeion channel

phosphatasecytokine

peptidasetransporter

growth factortranscription regulator

kinaseG-protein coupled receptor

transmembrane receptorP/N

Pro-apoptotic P D N

2 4

D/N

2 4 6

6.72.42.32.22.22.3

4.31.91.81.81.91.9

BidMaddTraddFaddCasp9Casp8

fold change

P/N

Myosins P D NMyh4Myl1MylpfMybpc2Myh8Myh2Myl3Myh1Myo18b

20 60 100 10

5.84.66.93.24.90.61.40.64.7

109.188.882.173.859.713.212.211.59.6

D/N

fold change

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

A B

C

Figure 5

N D P Schwann cells

S100B S100A9 merge/DAPI

kDa16S100A9

pERK

ERK

pPI3K

PI3K

pJNK

JNK

β-actin 40

44424442

11085

11085

5446

5446

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

A B

C

D

1h

1h

24h

24h

Fold change

Fold change

-log(P)

Ccl7Ccl2Htr4

Casp1Cxcl2Calca

Adamts1Six1

Aldh1a1Ccnd1

Etv5Gjb2

EpcamFosl1Shc3Il1rl1Arl4c

Il33Akap12Rbfox1

Sgk1DdcEln

Olr1Pdgfc

Tnfaip8Gprc5a

Plk3Dyrk3Areg

Nr1h4Plau

Cntn6Robo2Myh8Ltbp1

Slc26a1Igf1

Bhlhe40Usp18Spry1MgmtMyh1

Ccne2Hells

FasGzmbCdh3Postn

5 15

18.714.911.27.66.26.14.74.43.83.83.53.53.23.12.92.92.72.72.72.62.62.62.52.52.52.52.42.42.32.32.32.32.32.32.22.22.22.22.22.22.12.12.12.12.12.12.12.12.0 -2

Entpd2H19Cxcl14Gfra3AlplAsmtTktl1Mmp17MagPer1Epb41l4bCdkn1cPrss12Ugt1a6BmfScube1Wisp2Atf3Gpr37l1Sema3bCox6a2Pik3ip1Itgb4C1sCpWt1AplnNgfrEnpp2Fblim1RxrgKlf15Atf2Bcl3Tyrp1TxnipNtrk3Efna1Plxnb3A2mHey2Acss1Cstf1Ddit4C1qbFxyd1ProdhSlc6a12

-10

-9.3-4.8-4.3-4.2-4.1-4.0-3.7-3.7-3.2-3.0-3.0-3.0-3.0-2.9-2.8-2.8-2.8-2.8-2.7-2.6-2.6-2.6-2.5-2.5-2.5-2.4-2.4-2.4-2.3-2.3-2.3-2.3-2.3-2.2-2.2-2.2-2.2-2.2-2.2-2.1-2.1-2.1-2.1-2.1-2.1-2.1-2.0-2.0

Up-

regu

late

d

Dow

n-re

gula

ted

-6 -4 -2 3

2.82.52.42.32.12.1

-6.2-2.7-2.6-2.6-2.3-2.2-2.2-2.0

PtprvpBchePtproMcf2lPtgesUnc5aAsmtKcnma1Cstf1Atf2ChdhSlc6a12Socs7Traf3ip1

2.0 4.0

3.83.33.33.03.02.12.01.81.71.61.51.51.41.41.41.41.41.3

Axonal Guidance SignalingAgranulocyte Adhesion and Diapedesis

LXR/RXR ActivationRole of IL-17F in Allergic InflammationGranulocyte Adhesion and Diapedesis

ILK SignalingTREM1 Signaling

VDR/RXR ActivationHistamine Biosynthesis

Coagulation SystemERK5 Signaling

Tryptophan DegradationProline Degradation

Neurotrophin/TRK SignalingAryl Hydrocarbon Receptor Signaling

Calcium SignalingEphrin Receptor Signaling

LPS/IL-1 Inhibition of RXR Function

2.0

2.272.061.931.871.801.781.761.691.621.601.541.521.501.481.391.361.33

Eicosanoid SignalingcAMP-mediated signaling

Choline Degradation IFGF Signaling

Circadian Rhythm SignalingG-Protein Coupled Receptor Signaling

Calcium SignalingProtein Kinase A Signaling

Role of IL-17F in Allergic InflammationGαs Signaling

Serotonin and Melatonin Biosynthesisp38 MAPK Signaling

P2Y Purigenic Receptor Signaling PathwayGαi Signaling

PhospholipasesPhospholipase C Signaling

ATM Signaling

Figure 6

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

A B

C

D

P/N

Schwann cells stimulated with S100A8/A9

adhesion of immune cells

D/N1h

CCL2

CCL7

A2M

CALCA

CORO1A

CXCL2

FASIL33

PLAU

Figure 7

Ccl7Ccl2

Csf2rbCxcl2

Loc691670Rnase6

Adamts1Clec2aSpink2

MroGjb2

Clec9aArl4c

EpcamFosl1Arl4cIl1rl1Vcan

Clec2aPhlda1

Arl4cMyct1

Rbfox1Vcan

Nr4a3Olr1

Gprc5aPlk3

AregbStac2

PlauRab20Myh8

Sdcbp2Coro1a

Nrg1Bhlhe40

Pcdhgb5Vgf

Fcgr2aMyh1

Ccne2Hells

Tuba8Cpm

Isg15

fold change, Log2

0 1 2 3

0 1 2

2 24 4 8 2 4 8

3

4 5

shape change of blood cellscell movement of neutrophils

adhesion of neutrophilsproliferation of cells

formation of capillary vesselshape change of granulocytes

Rheumatic Diseasecell spreading of blood cells

chemotaxis of phagocytesInflammatory Disease

chemotaxis of neutrophilsocclusion of artery

arthritisphagocytosis of neutrophils

adhesion of immune cellsinvasion of epithelial cell lines

chemotaxis

Agranulocyte Adhesion and DiapedesisEpithelial Adherens Junction SignalingGranulocyte Adhesion and Diapedesis

TREM1 SignalingIL-10 Signaling

LXR/RXR ActivationGADD45 Signaling

-log(P)

-log(P)

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

B

C

Proximal nerveINJURY

AP DN C

1h24h

|

che

mok

ines

|

|cla

udin

s |

| int

erle

ukin

s|

M

MPs

|

myo

sins

|

oth

ers

Cxcl1Ccl20Cxcl3Cxcl2Ccl2Cxcr2Ccl7Cxcl6Cxcl14Ccl3l1Cxcr4Ccl12Ccl19Cxcl10Cldn4Cldn1Cldn22Cldn24Itgb2Itgb7Itga4Il1rnIl1bIl36bIl1r1Il18Il1aMmp9Mmp12Mmp3Mmp7Mmp13Mmp27Myh4Myl1Myh8Myh2Myl3Myh1Acta1Actg2SellSelpSelplgSeleTnf

integrins

P-SelectinPSGL1

IL1RIL1

TNF TNF-RL-Selectin

E-Selectin

Chemokines

CD34

CXCR1/2 CXCR4 VLA5

CXCL2

ITGα5

G-αi

ITGβ1/2

LFA1

VLA4

Figure 8

endothelial or

Schwann cells

Activatedagranulocyte

Fold change, log2

PP/N DN C1h

24h

5.795.044.673.513.323.113.082.652.592.472.472.442.362.282.241.931.771.651.501.421.271.131.101.061.04

-3 -1 1 5

-1.10-1.37-1.39-1.45-1.49-2.00-2.10

Mmp9Mmp12Mmp3ItgalActa1Mmp7Actg2Pik3cgMmp13Cxcr4Ncf2Timp1Cldn4Itgb2Arhgap8SelplgNcf4Cd44Ncf1RhohItga4Mmp19Actc1Mapk10Itga5Jam3Mmp11Itga1Timp3Mmp23Timp4Mmp27

15

1015

Sign

al in

tens

ity s

cale

, log

2

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

A

C ControlS100A8/A9

Bbiological functions

P D1h 24h

cell movement of myeloid cellscell movement of phagocytesadhesion of immune cellsinflammatory responsechemotaxis of myeloid cellschemotaxis of phagocytescell movement of leukocyteschemotaxis of leukocytescell movement of granulocytesleukocyte migrationproliferation of cellscell movement of neutrophilshoming of blood cellschemotaxis of cellscell movement of blood cellschemotaxisproliferation of tumor cell linesimmune response of phagocytesactivation of myeloid cellscell movement of leukocytes

canonical pathways

P D1h 24h

Agranulocyte Adhesion and DiapedesisGranulocyte Adhesion and DiapedesisLeukocyte Extravasation SignalingIL-10 SignalingAryl Hydrocarbon Receptor SignalingRole of IL-17A in PsoriasisTREM1 SignalingLXR/RXR ActivationInhibition of Matrix MetalloproteasesMSP-RON Signaling PathwayLPS/IL-1 Mediated Inhibition of RXR FunctionHepatic CholestasisRole of Macrophages in Rheumatoid ArthritisDendritic Cell MaturationdTMP De Novo BiosynthesisEndothelin-1 SignalingCell Cycle Control of Chromosomal ReplicationFcγ Receptor-mediated PhagocytosisEstrogen-mediated S-phase EntryRole of Hypercytokinemia

z-sc

ore

5.04.03.02.01.00.0

10.08.06.04.02.00.0

z-score -Log(P)

-Log

(P)

Figure 9

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from

Thomas Vogl, Svetlana Baranovskaya, Alex Y. Strongin and Veronica I. ShubayevAndrei V. Chernov, Jennifer Dolkas, Khang Hoang, Mila Angert, Geetha Srikrishna,

program in injured peripheral nerveCalcium-binding proteins S100A8 and S100A9 initiate the early inflammatory

published online March 19, 2015J. Biol. Chem. 

  10.1074/jbc.M114.622316Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on June 14, 2020http://w

ww

.jbc.org/D

ownloaded from