The Transcriptional Activation Program of
Human Neutrophils in Skin Lesions Supports
Their Important Role in Wound Healing
This information is current as
of June 15, 2017.
Kim Theilgaard-Mönch, Steen Knudsen, Per Follin and Niels
Borregaard
J Immunol 2004; 172:7684-7693; ;
doi: 10.4049/jimmunol.172.12.7684
http://www.jimmunol.org/content/172/12/7684
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References
The Journal of Immunology
The Transcriptional Activation Program of Human
Neutrophils in Skin Lesions Supports Their Important Role in
Wound Healing1
Kim Theilgaard-Mönch,2* Steen Knudsen,† Per Follin,‡ and Niels Borregaard*
S
kin wounding elicits a cascade of repair processes involving
several types of cells. First, thrombocytes generate a clot,
which stops the bleeding, and serves as a temporary barrier
and a source of chemotactic substances. Subsequently, attracted leukocytes initiate an inflammatory response before fibroblasts and endothelial cells migrate to the wound to regenerate tissue that contracts
the wound margins. Finally, epithelial cells complete the repair process by covering the denuded wound surface (1).
Polymorphonuclear neutrophilic granulocytes (PMNs)3 are attracted to skin wounds within minutes of injury by chemotactic
*The Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet,
University of Copenhagen, Copenhagen, Denmark; †Center for Biological Sequence
Analysis, BioCentrum-Technical University of Denmark, Lyngby, Denmark; and ‡Division of Infectious Diseases, Department of Health and Environment, University of
Linköping, Linköping, Sweden
Received for publication February 3, 2004. Accepted for publication April 9, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by the Novo Nordisk Foundation, the Amalie
Jørgensens Memorial Foundation, the Danish Cancer Research Foundation, the Danish Medical Research Council, the Gangsted Foundation, the Danish National Research Foundation, and by the Lundbeck Foundation. K.T.-M. is the recipient of a
scholarship from the IMK Foundation and Rigshospitalet.
2
Address correspondence and reprint requests to Dr. Kim Theilgaard-Mönch, The
Granulocyte Research Laboratory, Department of Hematology-9322, Rigshospitalet,
University of Copenhagen, Blegdamsvej 9, DK 2100 Copenhagen, Denmark. E-mail
address: [email protected]
3
Abbreviations used in this paper: PMN, polymorphonuclear neutrophilic granulocyte; PB, peripheral blood; pb-PMN, PB PMN; sl-PMN, skin lesions; MIP, macrophage-inflammatory protein; uPA, urokinase plasminogen activator; MCP, monocyte
chemoacttractant protein; GRO, growth-related oncogene; GPR, G protein-coupled
receptor; IER3, immediate early response 3; BCL2A1, BCL2-related protein A1;
CASP8, caspase 8, apoptosis-related cystein protease 8; CXCL, CXC chemokine
ligand; TLR, Toll-like receptor; LAMB3, laminin 5 ␤3; VEGF, vascular endothelial
growth factor.
Copyright © 2004 by The American Association of Immunologists, Inc.
mediators released by thrombocytes and microorganisms. Upon
migration to sites of infection such as skin wounds, PMNs get
activated by microorganisms and their products, and by cytokines
generated by other leukocytes (monocytes and PMNs) and the
stromal environment (fibroblasts, endothelial and epidermal cells).
Following activation, PMNs immediately initiate a first line of
defense using a number of distinct mechanisms (2, 3). These defense mechanisms include the release of anti-microbial peptides,
phagocytosis, and the generation of reactive oxygen intermediates
for killing and degradation of microorganisms. De novo synthesis
of chemokines and cytokines, which are essential for the regulation
of the cellular immune response and the recruitment of additional
effector cells to the wound, constitutes another defense mechanism
of PMNs.
More recently, studies using genomic and proteomic approaches
have demonstrated a significant transcriptional response of human
PMNs upon in vitro activation by single agents such as bacteria,
LPS, and by phagocytosis of IgG- and complement-coated latex
beads (4 –7). However, at present, no genomic approaches have
been applied to investigate how PMNs respond in vivo to inflammatory mediators in skin wounds and whether their response contributes to healing of wounds.
To gain more insight into this complex process, we applied gene
array technology to compare changes of gene expression of highly
purified PMNs from peripheral blood (PB) and PMNs that had
transmigrated to inflammatory skin lesions in vivo. For the collection of PMNs, we used a model of skin wounding called skin
chamber technique. With this model, small areas of denuded dermis, termed “skin windows”, are generated and covered with skin
chambers containing a medium that attracts PMNs (8).
Our study demonstrates that migration of PMNs into skin lesions is associated with an extensive change in gene expression,
0022-1767/04/$02.00
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To investigate the cellular fate and function of polymorphonuclear neutrophilic granulocytes (PMNs) attracted to skin wounds,
we used a human skin-wounding model and microarray technology to define differentially expressed genes in PMNs from peripheral blood, and PMNs that had transmigrated to skin lesions. After migration to skin lesions, PMNs demonstrated a significant
transcriptional response including genes from several different functional categories. The up-regulation of anti-apoptotic genes
concomitant with the down-regulation of proapoptotic genes suggested a transient anti-apoptotic priming of PMNs. Among the
up-regulated genes were cytokines and chemokines critical for chemotaxis of macrophages, T cells, and PMNs, and for the modulation
of their inflammatory responses. PMNs in skin lesions down-regulated receptors mediating chemotaxis and anti-microbial activity, but
up-regulated other receptors involved in inflammatory responses. These findings indicate a change of responsiveness to chemotactic and
immunoregulatory mediators once PMNs have migrated to skin lesions and have been activated. Other effects of the up-regulated
cytokines/chemokines/enzymes were critical for wound healing. These included the breakdown of fibrin clots and degradation of
extracellular matrix, the promotion of angiogenesis, the migration and proliferation of keratinocytes and fibroblasts, the adhesion of
keratinocytes to the dermal layer, and finally, the induction of anti-microbial gene expression in keratinocytes. Notably, the upregulation of genes, which activate lysosomal proteases, indicate a priming of skin lesion-PMNs for degradation of phagocytosed
material. These findings demonstrate that migration of PMNs to skin lesions induces a transcriptional activation program, which
regulates cellular fate and function, and promotes wound healing. The Journal of Immunology, 2004, 172: 7684 –7693.
The Journal of Immunology
implicating that the cellular fate and function of PMNs attracted to
skin wounds is partially regulated at the transcriptional level.
Materials and Methods
Collection and purification of PMNs from PB and skin lesions
Total RNA and proteins for gene expression analysis and Western blot
analysis, respectively, were isolated from purified PMN preparations using
TRIzol (Invitrogen, Paisley, U.K.) according to the guidelines of the
manufacturer.
Gene expression analysis
For gene expression analysis, total RNA was biotinylated and hybridized to
Hu95A GeneChips (Affymetrix, Santa Clara, CA) according to instructions
of the manufacturer (www.affymetrix.com/pdf/expression_manual.pdf/).
Briefly, first-strand cDNA was generated by reverse transcription of 2–5
␮g of total RNA at 42°C for 1 h using a T7-oligo(dT)24 primer and Superscript II (Invitrogen). DNA second-strand synthesis was accomplished
using DNA polymerase I and RNase H (Invitrogen) at 16°C for 2 h. Biotinylated cRNA was subsequently generated by in vitro transcription of
dsDNA using T7 RNA polymerase at 37°C for 6 h in the presence of
biotinylated nucleotides (BioArray High Yield RNA transcript labeling kit;
Enzo Diagnostics, Farmingdale, NY). Finally, biotinylated cRNA was
fragmented and the quality was confirmed on a test GeneChip before hybridization to Hu95A GeneChips (Affymetrix).
The expression index for each gene was calculated using the Li-Wong
weighted average difference (10). Array signals on individual gene arrays
were normalized by the Qspline method developed by Workman et al. (11).
Qspline is a robust nonlinear method for normalization using array signal
distribution analysis and cubic splines. Qspline fits cubic splines to the
quantiles of the array signal distribution, and uses those splines to normalize signals dependent on their intensity.
The increase/decrease of gene expression in PMNs from skin lesions
relative to PMNs from PB was calculated as a log2-fold change to obtain
a symmetric distribution around zero (up-regulated genes have positive
log2 values and down-regulated genes have negative log2 values). For functional clustering, genes were annotated with Gene Ontologies (www.
geneontology.org/), which provides a unique identifier for genes having a
characterized biological function.
Western blot analysis
TRIzol (Invitrogen) purified cell lysates corresponding to 5 ⫻ 105 PBPMNs (pb-PMNs) and skin lesion-PMNs (sl-PMNs) were electrophoresed
on 10 –14% SDS polyacrylamide gels (BDH Laboratory Supplies, Poole,
U.K.) and transferred to nitrocellulose membranes (Amersham Bioscience)
by electroblotting. Subsequently, the membranes were incubated with primary Abs raised against IL-8, macrophage inflammatory protein (MIP)-1␣
(both from R&D Systems, Minneapolis, MN), and urokinase plasminogen
activator (uPA; a gift from Dr. J. Pass, The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark) followed by a secondary HRP-conjugated
swine anti-rabbit Ab or rabbit anti-mouse Ab (DAKO, Glostrup, Denmark). Binding of Abs was visualized by ECL (Amersham Bioscience,
Uppsala, Sweden). Loading of equal amounts of protein was assessed by
probing membranes with a monoclonal primary anti-␤-actin Ab (12).
Statistics
The statistical analysis was performed using the R statistics program environment available from www.r-project.org/. The variability between subjects was low as estimated by the correlation coefficient, which ranged
from 0.96 to 0.97 for pb-PMN genechips and 0.91 to 0.98 for sl-PMN
genechips. In contrast, the correlation coefficient between pb-PMN genechips and sl-PMN genechips ranged from 0.78 to 0.80.
A Student t test was applied to identify differentially expressed genes in
pb-PMNs and sl-PMNs. In the t test, the variance among individuals in the
two categories of pb-PMNs and sl-PMNs was calculated for each gene, and
differences between the two categories were only considered significant if
they far exceeded the variance. The p values calculated by the t test were
corrected for multiple testing (Benjamini-Hochberg, 13) to estimate the
false discovery rate for differentially regulated genes. The false discovery
rate for differentially regulated genes in the present study was 0.032.
Results
Purification of PMNs
Most critical for the comparison of gene expression in cell populations collected in vivo is the application of highly purified cell
preparations to minimize the false positive rate of differentially
regulated genes due to contamination with other cell types. Thus,
we reasoned to use a purification strategy based on density centrifugation and immunomagnetic depletion of nongranulocytic
cells to obtain highly purified PMN preparations from PB and skin
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PB and skin chamber samples were collected in parallel from four healthy
individuals. All samples were obtained following informed consent according to the guidelines established by the Ethics Committee of the Cities of
Copenhagen and Fredricksberg.
PMNs were isolated from PB by density centrifugation and subsequent
immunomagnetic depletion of nongranulocytic cells. Briefly, 60 – 80 ml of
anti-coagulated venous blood were mixed 1 ⫹ 1 with chilled saline/2%
dextran (Dextran 500; Amersham Bioscience, Uppsala, Sweden) and kept
on ice for 30 – 40 min to sediment erythrocytes. The resultant leukocyterich supernatant was centrifuged (200 ⫻ g, 4°C, 6 min), and the pellet was
gently resuspended in chilled saline. The leukocyte suspension was then
layered on 15 ml of Lymphoprep (1.077 g/ml; Nycomed, Oslo, Norway)
and centrifuged (400 ⫻ g, 4°C, 30 min). The supernatant including the
interphase containing mononuclear cells was discarded and the pellet
highly enriched for PMNs subjected to hypotonic lysis of erythrocytes
(resuspension in 5 ml of chilled H2O, gentle mixing for 30 s, and termination of lysis by addition 5 ml 1.8% NaCl). After lysis, cells were pelleted
(200 ⫻ g, 4°C, 6 min) and resuspended in PBS/0.5% BSA/2 mM EDTA
buffer. Subsequently, the PMN preparations were depleted of nongranulocytic cells by immunomagnetic sorting using the MACS system according
to the instructions of the manufacturer (MACS; Miltenyi Biotec, Bergisch
Gladbach, Germany). The mAbs used for depletion were raised against
Ags expressed by the following cell types: monocytes (anti-CD14), B cells
(anti-CD19), T cells (anti-CD3), platelets and megakaryocytes (antiCD61), NK cells (anti-CD56), erythroid cells (anti-glycophorin A), and
eosinophils (anti-CD49d; all mAbs were provided by BD Biosciences, San
Diego, CA). To minimize the activation of PMNs, all isolation procedures
were performed immediately after cell collection at ⱕ4°C, i.e., on ice, in
a cold room, or a cooled centrifuge, using nonpyrogenic reagents and
plasticware.
PMNs that had transmigrated from the blood circulation to skin lesions
generated by epidermal detachment and blister formation were collected by
the skin chamber technique as described previously (8, 9). Briefly, a cylindrical acrylic suction device containing 3 holes of 5 mm in diameter, was
placed on the volar surface of the nondominant forearm and negative pressure of 200 mm Hg was applied for 2 h by a vacuum pump resulting in the
formation of suction blisters. After detachment of the vacuum pump, the
blister roofs were removed with sterile tweezers and scissors, resulting in
3 uniform skin lesions, termed “skin windows”, of ⬃0.2 cm2. A collection
chamber with 3 collection compartments of 7 mm in diameter, was placed
on top of the skin window, filled with 0.5 ml of autologous serum, and
sealed with tightly fitting lids. The collection chamber was then fixed to the
forearm by tape and an elastic bandage. After 18 h, the collection compartments were emptied, washed, and refilled with autologous plasma. After an additional 6 h, exudated cells were collected from skin chambers and
depleted of nongranulocytic cells by immunomagnetic sorting as described
above. Notably, serum contains biological active components of the coagulation and complement system. When used in skin chambers, serum is a
documented strong chemoattractant, induces exocytosis of gelatinase granules and secretory vesicles by PMNs, and up-regulates Mac-1/CD11b on
exudated PMNs, whereas plasma has no such effects (9). Thus, serum itself
might activate PMNs. With the applied skin chamber protocol, PMNs were
collected in plasma rather than serum to minimize the influence of skin
chamber fluid on PMN activation. However, plasma alone is a poor chemoattractant and does not allow the collection of sufficient PMNs for gene
expression analysis. Moreover, with plasma, it is technically not possible to
extend the exudation period beyond 6 h to obtain more cells, since this will
activate coagulation and trap exudated cells in a clot (P. Follin, unpublished observations). Based on these findings the exudation process was
first initiated with serum for 18 h, followed by washing and refilling the
skin chambers with plasma, before collecting freshly exudated PMNs after
an additional 6 h (9). Since the applied skin chamber protocol is reproducible and uses an aseptic inflammation to attract cells to skin lesions, it
is in our opinion currently one of the most suitable techniques that mimics
the in vivo activation and resultant transcriptional response of PMNs in
skin lesions. However, the protocol did not discriminate to what extent the
migration process or the various stimuli (cytokines etc.) in the skin window
exudates contributed to the observed transcriptional response of PMNs.
The purity of PMN preparations was assessed by microscopy of WrightGiemsa-stained cytospins before and after immunomagnetic sorting. Cells
were enumerated using a Neubauer hemocytometer.
7685
7686
TRANSCRIPTIONAL ACTIVATION OF NEUTROPHILS IN SKIN LESIONS
lesions for array analysis. In alignment with previous studies, density centrifugation of PB cells resulted in PMN preparations containing 95–97% PMNs, 2– 4% eosinophils, and ⬍1% mononuclear
cells (5, 7). Additional lineage-depletion increased the purity of
PMN preparations from PB to ⬎99.5% (n ⫽ 4, mean purity
99.7%). Cells collected from skin chambers contained 85–95%
PMNs and 5–15% contaminating monocytes/macrophages. After
depletion of nongranulocytic cells, the purity of PMN preparations
increased to ⬎99% (n ⫽ 4, mean purity 99.4%). Subsequent array
analysis revealed no detectable levels of lineage-specific transcripts for other relevant cell types such as eosinophils, basophils,
monocytes, T cells, endothelial cells, fibroblasts, and epidermal
cells in any of the purified PMN preparations (Table I; Affymetrix
absent call). These findings demonstrate that the applied purification protocol resulted in highly purified PMN preparations from
PB and skin lesions, and thus, minimized the false positive rate of
differentially expressed genes due to contamination of nongranulocytic cells.
FIGURE 1. Differentially regulated genes in PMNs collected from PB
and skin lesions were assigned to gene categories according to their biological functions using the Gene Ontology database. The numbers of upand down-regulated genes in PMNs from skin lesions compared with
PMNs from PB are shown for each gene category.
Microarray analysis was applied to compare differentially the expression of ⬇12,500 genes in highly purified PMNs from PB and
PMNs, which had transmigrated to skin lesions. Genes were defined as differentially expressed if they were among the 1000 most
significant differentially expressed genes (range of p values: 2.6 ⫻
10⫺3–3.6 ⫻ 10⫺9, estimated false discovery rate 0.032 (Benjamini-Hochberg)), and if they changed gene expression by ⱖ0.5
log2-fold. By these criteria, 314 differentially expressed genes assigned to various functional gene categories of the Gene Ontology
database were detected in PMNs upon migration to skin lesions
(Fig. 1). Almost no up- or down-regulated genes were detected in
categories critical for defense, cellular movement/transport or cell
structure indicating that functions such as the migration to sites of
infection, changes of cellular structure, and immediate antimicrobial defense are not regulated at the transcriptional level. In
contrast, the high numbers of differentially expressed genes in categories such as apoptosis regulators, signal transducers, and enzymes, indicated that PMN activity in skin lesions is partially regulated at the transcriptional level.
Genes involved in apoptosis
Detailed analysis of apoptosis regulators demonstrated the up-regulation of anti-apoptotic genes concomitant with the down-regulation of proapoptotic genes (Tables II and III). Up-regulated antiapoptotic genes included IEX1 (immediate early response (IER)3;
Table I. Detection of lineage-specific transcript in purified PMN
preparationsa
Gene Name
Cell Type Expressing
Lineage-Specific Gene
Detectable Transcripts
in Purified PMN
Preparations (n ⫽ 8)
G-CSFR
IL-5RA
M-CSFR (CSF1R)
TCR
FGFR1
FGFR2
EGFR
VEGFR
VECAM1
PMN
Eosinophil/basophil
Monocyte/macrophage
T cell
Fibroblast
Fibroblast/epidermal cell
Epidermal cell
Endothelial cell
Endothelial cell
Yes
No
No
No
No
No
No
No
No
a
The table demonstrates the absence of lineage-specific transcripts for nongranulocytic cells in highly purified PMN preparations collected from pb-PMNs (n ⫽ 4) and
sl-PMNs (n ⫽ 4) as detected by microarray analysis (Affymetrix; present/absent call).
protects cells from Fas-induced apoptosis) (14, 15), BCL2-related
protein A1 (BCL2A1; blocks mitochondrial release of cytochrome
c) (16), and FLIP (cFLAR; inhibits Fas-associated death domain
protein-mediated activation of CASP8) (17). Among the downregulated proapoptotic genes were Fas-associated death domain
protein (activates CASP8), CASP8 (activates downstream
caspases affecting apoptosis), APAF1 (activates CASP9 in a complex with cytochrome c), death-associated protein kinase 2 (18),
and TNFR (activates apoptosis pathway by ligand binding). This
change of gene expression among members of the apoptotic pathway suggests a transient anti-apoptotic priming of PMNs immediately after migration to skin lesions, regulated at the transcriptional level.
Genes involved in wound healing
The complex process of wound healing is orchestrated by signal
transduction through chemokines, cytokines, and their respective
receptors. Upon migration to skin lesions, PMNs up-regulated 26
and down-regulated 68 mediators of signal transduction (Tables II
and III). Up-regulated chemokines and cytokines were critical for
the recruitment of additional macrophages, T cells, and PMNs, and
for the modulation of inflammatory responses (IL-8, monocyte
chemoattractant protein (MCP)-1 (CC chemokine ligand 2),
MIP-1␣ (CC chemokine ligand 3), growth-related oncogene
(GRO)-␤ (CXCL2), GRO-␥ (CXCL3), IL-1␤ (IL1B), and TNF-␣
(TNF)) (19). Of interest, receptors mediating chemotaxis and cellular activation (IL-8RA (CXCR1), IL-8RB (CXCR2), G-CSFR,
Toll-like receptor (TLR)1, and TLR6) (19, 20) were down-regulated concomitant with the up-regulation of other receptors modulating inflammatory responses (IL-1R1, TGF-BR1, G proteincoupled receptors (GPR) 65, GPR18, and HM74). Hence, PMNs
might change their responsiveness to chemotactic and immunoregulatory mediators once activated in skin wounds.
Other effects of up-regulated chemokines/cytokines included the
promotion of angiogenesis (vascular endothelial growth factor
(VEGF), IL-8, GRO-␥, and MCP-1) (21), proliferation of keratinocytes and fibroblasts (IL-8, IL-1␤, and MCP-1) (19), and the induction of anti-microbial gene expression in keratinocytes (IL-1␤
and TNF-␣) (22). Additional up-regulated genes potentially involved in wound healing were laminin 5 ␤3 (LAMB3) (23), which
promotes adhesion of keratinocytes to the dermal layer, and uPA
(PLAU), which supports tissue remodelling by breakdown of fibrin
clots and degradation of extracellular matrix (24, 25). Other wound
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Differentially expressed genes are assigned to distinct functional
gene categories
The Journal of Immunology
7687
Table II. Genes up-regulated in PMNs upon migration to skin lesionsa
Gene Category
Apoptosis regulator
Binding
Cell adhesion molecule
Enzyme
Signal transducer
Structural molecule
Transcription regulator
Translation
Gene Symbol
Immediate early response 3
BCL2-related protein A1
Caspase 9
CASP8 and FADD-like apoptosis regulator
Aryl hydrocarbon receptor
Calumenin
Killer cell lectin-like receptor subfamily G, member 1
Laminin, ␤3
CD44 Ag
Plasminogen activator, urokinase
Omithine decarboxylase antizyme inhibitor
Uridine phosphorylase
Ceroid-lipofuscinosis, neuronal 2 (tripeptidyl-peptidase I)
Legumain (asparaginyl endopeptidase)
Phosphoprotein
Isopentenyl-diphosphate ␦ isomerase
Oxidative-stress responsive 1
Ubiquitin specific protease 14
Protease inhibitor 3
Omithine decarboxylase antizyme inhibitor
Protein phosphatase 2, regulatory subunit B, ␣ isoform
MIP-1␣ (chemokine (CC motif) ligand 3)
IL-8
Chemokine (CXC motif) ligand 2 (GRO␤)*
Vascular endothenal growth factor
IL-1, ␤
Pleckstrin
G protein coupled receptor 65
Putative chemokine receptor; GTP-binding protein
TGF-␤ receptor 1
Lymphocyte cytosolic protein 2
Adaptor protein with pleckstrin and src homology 2 domains
IL-1 receptor, type 1
TNF-␣*
Discs, large (Drosophila) homolog-associated protein 1
TNFR-associated factor 3
SKI-like
Mitogen-activated protein kinase kinase 3
Chemokine (CXC motif) ligand 3 (GRO␥)
G protein-coupled receptor 18
Protein tyrosine phosphatase, receptor type, E
Guanine nucleotide binding protein-like 1
Mannose-6-phosphate receptor (cation dependent)
Ore-B-cell colony-enhancing factor
TRAF family member-associated NFkB activator
Chemokine (CC motif) ligand 2 (MCP-1)
Tyrosine phosphatase, ␧
Myosin, light polypetide 4, alkali
TGF-B-inducible early growth response
Early growth response 3
V-ets erythroblastosis virus E26 oncogene homolog 2
NP-␬B1 (p105)
Nuclear receptor corepressor 2
Nuclear factor (erythroid-derived 2)-like 2
Chromodomain helicase DNA binding protein 1
Vitamin D (1,25-dihydroxyvitamin D3) receptor
Transcription factor EC
High mobility group AT-hook 1
Zinc finger protein 36
TAF6-like RNA polymerase II
PHD finger protein 1
Heterogeneous nuclear ribonucleoprotein C (C1/C2)
HIV-1 Tat interactive protein, 60kDa
Eukaryotic translation initiation factor 4A, isoform 1
Eukaryotic translation elongation factor 1 ␣ 1
Eukaryotic translation initiation factor 5
Ribosomal protein S16
Ribosomal protein S27 (metallopanstimulin 1)
Signal recognition particle 54kDa
IER3
BCL2A1
CASP9
CFLAR
AHR
CALU
KLRG1
LAMB3
CD44
PLAU
OAZIN
UP
CLN2
LGMN
C8FW
IDI1
OSR1
USP14
PI3
OAZIN
PPP2R2
CCL3
IL-8
CXCL2
VEGF
IL-1
PLEK
GPR65
HM74
TGF-R1
LCP2
APS
IL-1R1
TNF
DLGAP1
TRAF3
SKIL
MAP2K3
CXCL3
GPR18
PTPRE
GNL1
M6PR
PBEF
TANK
CCL2
PTPR
MYL4
TIEG
EGR3
ETS2
NFKB1
NCOR2
NFE2L2
CHD1
VDR
TFEC
HMGA1
ZFP36
TAF6L
PHF1
HNRPC
HTATIP
EIF4A1
EEF11
EIF5
RPS16
RPS27
SRP54
Affymetrix ID
Log2-Fold
Change
1237_at
2.2
2002_s_at
1.8
486_at
1.5
1867_at
0.9
40516_at
0.5
37345_at
0.5
34975_at
0.5
36929_at
1.9
40493_at
1.7
37310_at
4.5
33368_at
2.3
37351_at
2.1
32824_at
1
317_at
0.9
35597_at
0.8
36985_at
0.8
39136_at
0.8
36982_at
0.5
41469_at
3.2
1959_at
2.2
41167_at
1.2
36103_at
4.7
1369_s_at
3.4
37187_at
3.4
36100_at
3.3
39402_at
3.2
37328_at
1.8
34930_at
1.6
34951_at
1.5
32903_at
1.5
39319_at
1.5
37136_at
1.4
1368_at
1.4
1852_at
1.2
40388_at
1.1
37057_s_at
0.9
1866_g_at
0.9
1622_at
0.8
34022_at
0.8
252_at
0.7
32916_at
0.7
1162_g_at
0.7
32547_at
0.6
33849_at
0.6
39742_at
0.6
34375_at
0.5
1150_at
0.5
31421_at
0.8
224_at
4.2
40375_at
2.9
1519_at
2
1377_at
2
39358_at
1.7
853_at
1.5
39231_at
1.4
1388_g_at
1.1
34470_at
1.1
39704_s_at
1
40448_at
0.9
39908_at
0.8
40446_at
0.8
33666_at
0.7
465_at
0.6
1199_at
1.7
1288_s_at
1.1
167_at
1
38061_at
1
32748_at
0.9
36060_at
0.9
(Table continues)
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Enzyme regulator
Gene Name
7688
TRANSCRIPTIONAL ACTIVATION OF NEUTROPHILS IN SKIN LESIONS
Table II. Continued
Gene Category
Transporter
Gene Name
Gene Symbol
Solute carrier family 25, member 13 (citrin)
ATPase, H⫹ transporting, V1 subunit C, isoform 1
ATPase, Na⫹/K⫹ transporting, ␣ 1 polypeptide
RA81A, member RAS oncogene family
Chloride channel 7
Aquaporin 9
Phosphotidylinositol transfer protein
GABA(A) receptor-associated protein-like 2
Solute carrier family 25, member 6
Methylene tetrahydrofolate dehydrogenase
SLC25A13
ATP6V1C1
ATP11
RAB1A
CLCN7
AQP9
PITPN
GABARAL2
SLC25A6
MTHFD2
Affymetrix ID
Log2-Fold
Change
38328_at
37948_at
32225_at
1074_at
38069_at
34435_at
35251_at
35767_at
40435_at
40074_at
2.4
2
1.6
1.2
1.1
1
0.8
0.7
0.7
0.5
a
Genes up-regulated in PMNs upon migration to skin lesions including their gene category, gene name, gene symbol, Affymetrix ID, and the log2-fold change of gene
expression (range of p values: 2.6 ⫻ 10⫺3–3.6 ⫻ 10⫺9, estimated false discovery rate 0.032 (Benjamini-Hochberg)). Values of p for CXCL2 and TNF-␣ marked with an asterisk
were 0.025 and 0.026, respectively.
Transcriptionally highly induced genes are up-regulated at the
protein level
To investigate whether the transcriptional up-regulation of genes
in sl-PMNs detected by array analysis correlated with increased
protein levels, protein lysates were extracted from the same samples used for array analysis and subjected to Western blot analysis.
These analyses demonstrated that transcriptionally highly induced
genes in sl-PMNs including IL-8, MIP-1␣, and uPA were up-regulated at the protein level (Fig. 2).
Discussion
The present study demonstrates that the migration of PMNs to skin
lesions in man is accompanied with a substantial change in gene
expression. These findings are in line with in vitro studies showing
that human PMNs are capable of extensive changes in gene expression upon in vitro activation by single agents such as bacteria,
LPS, and by phagocytosis of IgG- and complement-coated latex
beads (4 –7). Not unexpectedly, the changes reported in those studies differ partially from those observed in the present study. For
example, of the top 5 up-regulated genes in the present study
(MIP-1␣, uPA, IL-8, VEGF, and IL-1␤) only IL-8 and IL-1␤ were
induced by bacteria and LPS (4, 5), only MIP-1␣ and VEGF were
induced by phagocytosis of IgG- and complement-coated latex
beads (7), and uPA was not induced by any of these agents. These
findings demonstrate that PMNs generate distinct transcriptional
responses depending on the type of stimuli and activated signaling
pathway. The different responses of PMNs stimulated in vitro and
in vivo further demonstrate that multiple, and not individual, stimuli and signaling pathways contribute to the transcriptional response of PMNs in skin lesions.
To define how migration of PMNs to skin lesions affects biological functions on a global level, differentially regulated genes
were assigned to categories according to their biological functions
using the Gene Ontology database. Functional clustering revealed
that genes critical for migration, cellular structure, and immediate
host defense were not activated, but were partially down-regulated,
whereas genes involved in apoptosis, wound healing, and other
distinct cellular functions were highly differentially regulated.
Physiologically, these findings are meaningful, as basic functions
such as migration and immediate host defense are inherent to circulating PMNs and do not require a prolonged phase of transcriptional activation. In contrast, the data demonstrated that PMNs
were capable to transcriptionally activate specific functions such as
the promotion of wound healing once they have migrated to sites
of infection.
The cellular fate of PMNs at sites of infection includes necrotic
death, immediate apoptosis, or the prolongation of life span by
inhibition of apoptosis. Upon necrotic death, PMNs release toxic
granule proteins resulting in tissue damage, whereas the phagocytosis of apoptotic PMNs by macrophages protects against such
damage (30, 31). When cultured in vitro, PMNs rapidly undergo
apoptosis, a process, which is delayed by addition of G-CSF and a
variety of inflammatory mediators to the medium (32, 33). Hence,
cytokines and inflammatory mediators present at sites of infection
might augment the inflammatory response of PMNs by prolongation of cellular life span. This statement is supported by the present
study showing that PMNs in skin wounds up-regulate antiapoptotic genes and down-regulate proapoptotic genes, and thus,
might acquire a transient “anti-apoptotic state”. Notably, two of
the up-regulated anti-apoptotic genes, i.e., IEX1 (IER3) and
BCL2A1, have been defined as target genes of NF-␬B, a transcription factor that was found to be up-regulated in sl-PMNs and,
which is activated through IL-1␤ and TNF-␣ signaling and binding
of pathogens to TLRs (34). Because macrophages and PMNs both
produce IL-1␤ and TNF-␣ at sites of infection (35, 36), the “antiapoptotic state” of PMNs in skin lesions might partially be regulated at the transcriptional level through NF-␬B activation
pathways.
The present study supports the notion that PMNs are a rich
source of cytokines and chemokines in skin wounds (36). Moreover, PMNs clearly outnumbered macrophages at 24 h in our skinwounding model, suggesting that PMNs are the major source of
inflammatory mediators during the initial phase of wound healing.
Some of the factors found to be up-regulated in the present study
including IL-1␤, TNF-␣, and IL-8, have been described as upregulated in PMNs 1 day after incisional wounding (36, 37). However, to the best of our knowledge, the up-regulation of VEGF,
MCP-1, MIP-1␣, and GRO-␥ has not been reported earlier in
PMNs upon migration to skin wounds. These findings demonstrate
that up-regulated chemokines not only recruit more PMNs (IL-8),
but also specifically attract macrophages (MCP-1 and MIP-1␣)
(38) and T cells (MCP-1), which have been reported to be the most
abundant leukocyte populations 2 days after incisional
wounding (37).
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
healing activities that are stimulated by uPA include the proliferation, migration, and adhesion of keratinocytes, fibroblasts, and
endothelial cells in skin wounds (25).
The up-regulation of tripeptidyl-peptidase I/ceroid-lipofuscinosis, neuronal 2, legumain/asparaginyl endopeptidase, and the mannose-6-phosphate receptor suggested a priming of lysosomal activity once PMNs have migrated to skin lesions (26 –29).
The Journal of Immunology
7689
Table III. Genes downregulated in PMNs upon migration to skin lesionsa
Gene Category
Apoptosis regulator
Binding
Cell adhesion molecule
Chaperone
Defense/immunity
Enzyme
Gene Symbol
Caspase 8, apoptosis-related cysteine protease
Ret finger protein
TIA1 cytotoxic granule-associated RNA binding protein-like 1
Topoisomerase (DNA) II-binding protein
HIV-1 Tat interactive protein 2, 30kDa
Apoptotic protease activating factor
Death-associated protein kinase 2
Fas (TNFRSF6)-associated via death domain
CASP2 and RIPK1 domain containing adaptor with death domain
Grancalcin, EF-hand calcium binding protein
Myc single-strand binding proteinretropseudogene 1c
Xeroderma pigmentosum, complementation group C
Folliststin-like 1
Zinc finger protein 185 (LIM domain)
Annexin A11
Platelet/endothelial cell adhesion molecule (CD31 Ag)
Flotillin 2
Pinin, desmosome associated protein
Ectonucleoside triphosphate diphosphohydrolase 1
Fasciculation and elongation protein ␨ 2 (zygin II)
Retinoblastoma-like 2 (p130)
Transducer of ERBB2, 1
Cyclin-dependent kinase inhibitor 2D (p19, inhibits CDK4)
SET translocation (myeloid leukemia-associated)
Tubulin-specific chaperone c
Protein leukocyte specific transcript 1
Transketolase (Wernicke-Korsakoff syndrome)
Ribosomal protein S6 kinase, 90 kDa, polypeptide 5
Sialyltransferase 8D (␣-2, 8-polysialyltransferase)
Serine/threonine kinase 24 (STE20 homolog, yeast)
Ubiquitin specific protease 1
Homo sapiens mRNA; cDNA DKFZp686D0521
Methyl-CpG binding domain protein 4
GNAS complex locus
Adrenergic, ␤, receptor kinase 1
Inositol polyphosphate-5-phosphatase, 145 kDa
Phospholipase C, ␤ 2
Protein phosphatase 1A, magnesium-dependent, ␣ isoform
Protein phosphatase 1, catalytic subunit, ␥ isoform
Ubiquitin-activating enzyme E1C (UBA3 homolog, yeast)
Protein phosphatase 1 ␣ catalytic subunit
UDP-galactosamine N-acetylgalactosaminyltransferase 3
Aminopeptidase puromycin sensitive
Phospholipase C, ␥ 2 (phosphatidylinositol-specific)
Prolylcarboxypeptidase (angiotensinase C)
␳-associated, coiled-coil containing protein kinase 1
Zinc metalloproteinase (STE24 homolog, yeast)
Phosphorylase kinase, ␤
Regulator of nonsense transcripts 1
Thiosulfate sulfurtransferase (rhodanese)
Unc-51-like kinase 1 (Caenorhabditis elegans)
Ubiquitin specific protease 15
Iduronate 2-sulfatase (Hunter syndrome)
Phosphorylase kinase, ␣ 2 (liver)
Serine/threonine kinase 38
Tyrosine kinase 2
X-ray complementing defective dsDNA repair in Chin, hamster cells 5
PTK9L protein tyrosine kinase 9-like (A6-related protein)
A disintegrin and metalloproteinase domain 10
MAP/microtubule affinity-regulating kinase 2
Tyrosylprotein sulfotransferase 2
Copine III
O-linked N-acetylglucosamine (GlcNAc) transferase
ATP citrate lyase
Cell division cycle 2-like 5 (cholinesterase-related cell division controller)
Peroxiredoxin 3
Glycogen synthase kinase 3 ␤
Myotubularin-related protein 3
Phosphatidylinositol glycan, class B
Peroxisome biogenesis factor 1
Serine palmitoyltransferase, long chain base subunit 1
CASP8
RFP
TIAL1
TOPBP1
HTATIP2
APAF1
DAPK2
FADD
CRADD
GCA
MSSP1
XPC
FSTL1
ZNF185
ANXA11
PECAM1
FLOT2
PNN
ENTPD1
FEZ2
RBL2
TOB1
CDKN2D
SET
TBCC
LST1
TKT
RPS6KA5
SIAT8D
STK24
USP1
MBD4
GNAS
ADRBK1
INPP5D
PLCB2
PPM1A
PPP1CC
UBE1C
GALNT3
NPEPPS
PLCG2
PRCP
ROCK1
ZMPSTE24
PHKB
RENT1
TST
ULK1
USP15
IDS
PHKA2
STK38
TYK2
XRCC5
PTK9L
ADAM10
MARK2
TPST2
CPNE3
OGT
ACLY
CDC2L5
PRDX3
GSK3B
MTMR3
PIGB
PEX1
SPTLC1
Affy ID
Log2-Fold
Change
33774_at
⫺2.1
40176_at
⫺1.4
41762_at
⫺1.1
38834_at
⫺1.1
38824_at
⫺1
37227_at
⫺0.9
34912_at
⫺0.8
38755_at
⫺0.7
1211_s_at
⫺0.6
37556_at
⫺1
31671_at
⫺0.8
1873_at
⫺0.7
40132_g_at
⫺0.6
32139_at
⫺0.6
36637_at
⫺0.5
37397_at
⫺1.5
32181_at
⫺1.2
33543_s_at
⫺1.1
32826_at
⫺0.8
38651_at
⫺0.6
32597_at
⫺1.6
40631_at
⫺1.2
1797_at
⫺1.2
40189_at
⫺0.7
36176_at
⫺1.9
37967_at
⫺0.8
38789_at
⫺2
41432_at
⫺1.8
33649_at
⫺1.5
40473_at
⫺1.5
34383_at
⫺1.5
38581_at
⫺1.5
34386_at
⫺1.4
37448_s_at
⫺1.3
38447_at
⫺1.2
172_at
⫺1.2
210_at
⫺1.2
857_at
⫺1.2
37725_at
⫺1.2
40066_at
⫺1.2
954_s_at
⫺1.2
36484_at
⫺1.1
39431_at
⫺1.1
37180_at
⫺1.1
36672_at
⫺1.1
34735_at
⫺1.1
33912_at
⫺1.1
37392_at
⫺1
39404_s_at
⫺1
36123_at
⫺1
34827_at
⫺1
34295_at
⫺1
40814_at
⫺0.9
36480_at
⫺0.9
36218_g_at
⫺0.9
993_at
⫺0.9
584_s_at
⫺0.9
35796_at
⫺0.8
40797_at
⫺0.8
965_at
⫺0.8
35172_at
⫺0.8
39706_at
⫺0.8
38614_s_at
⫺0.7
40881_at
⫺0.7
41821_at
⫺0.7
36631_at
⫺0.7
40645_at
⫺0.6
35739_at
⫺0.6
314_at
⫺0.6
38365_at
⫺0.5
38818_at
⫺0.5
(Table continues)
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Cell cycle
Gene Name
7690
TRANSCRIPTIONAL ACTIVATION OF NEUTROPHILS IN SKIN LESIONS
Table III. Continued
Gene Category
Enzyme regulator
Motor
Signal transducer
Gene Name
OAZ1
CSTA
KNS2
MYO9B
SMC1L1
RGS2
SELPLG
ARHGAP1
RALBP1
CAMK2G
CHC1L
ITGB2
PRKCB1
EDG6
GNAO1
HRMT1L1
ITGAL
LILRA2
ARHGDIB
GNAI2
AKT1
RAF1
ICAM3
IGF2R
IL-8RB
CALM2
PRKAR1A
IL-8RA
MCP
PPP1R12A
RGS14
RGS19
IQGAP1
RASGRP2
MAPKAPK3
PDE3B
PPP1R12B
CD97
TLR1
CALM1
JIK
CREBBP
MAP3K5
GDI2
LASP1
MAP3K3
ITPKB
CAMK2G
GNAT2
GNB2
JAK1
ARHGAP4
GPR19
MAPK3
TGF-1
TLR6
PPFIA 1
CSF3R
FKBP1A
LILRB5
LTB
PSCD1
TRN-SR
WAS
MAP2K4
DOCK2
TGFA
RIPK1
ACVR1B
RABIF
RASA1
Affy ID
Log2-Fold
Change
1315_at
⫺0.8
39581_at
⫺0.7
39057_at
⫺1.2
33816_at
⫺0.7
32849_at
⫺0.5
37701_at
⫺2.5
37541_at
⫺2.2
553_g_at
⫺1.9
36628_at
⫺1.8
32105_f_at
⫺1.7
35193_at
⫺1.7
37918_at
⫺1.7
1336_s_at
⫺1.7
33602_at
⫺1.6
34138_at
⫺1.6
39348_at
⫺1.6
38547_at
⫺1.6
34033_s_at
⫺1.6
1984_s_at
⫺1.5
37307_at
⫺1.4
1564_at
⫺1.4
1917_at
⫺1.4
402_s_at
⫺1.4
160027_s_at
⫺1.4
664_at
⫺1.4
911_s_at
⫺1.3
226_at
⫺1.3
1353_g_at
⫺1.3
38441_s_at
⫺1.3
40438_at
⫺1.3
38290_at
⫺1.3
34268_at
⫺1.3
1825_at
⫺1.2
38359_at
⫺1.2
1637_at
⫺1.2
35872_at
⫺1.2
41137_at
⫺1.2
35625_at
⫺1.2
36243_at
⫺1.2
41143_at
⫺1.1
41646_at
⫺1.1
33831_at
⫺1.1
1327_s_at
⫺1.1
955_at
⫺1
35307_at
⫺1
36181_at
⫺1
1330_at
⫺1
37272_at
⫺0.9
32104_i_at
⫺0.9
34571_at
⫺0.8
38831_f_at
⫺0.8
34877_at
⫺0.8
39649_at
⫺0.8
156_s_at
⫺0.8
1000_at
⫺0.8
1830_s_at
⫺0.8
34144_at
⫺0.8
41780_at
⫺0.7
34223_at
⫺0.7
880_at
⫺0.7
36789_f_at
⫺0.7
40729_s_at
⫺0.7
38666_at
⫺0.7
35813_at
⫺0.7
38964_r_at
⫺0.7
1845_at
⫺0.6
32704_at
⫺0.6
160025_at
⫺0.5
40696_at
⫺0.5
34056_g_at
⫺0.5
38264_at
⫺0.5
1675_at
⫺0.5
(Table continues)
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Omithine decarboxylase antizyme 1
Cystatin A (stefin A)
Kinesin 2 60/70 kDa
Myosin IXB
SMC1 structural maintenance of chromosomes 1-like 1 (yeast)
Regulator of G-protein signalling 2, 24 kDa
Selectin P ligand
␳ GTPase activating protein 1
RalA binding protein 1
Calcium/calmodulin-dependent protein kinase (CaM kinase) II ␥
Chromosome condensation 1-like
Integrin, ␤2 (CD18 (p95) alias mac-1 ␤ subunit)
Protein kinase C, ␤ 1
Endothelial differentiation, G protein-coupled receptor 6
G protein, ␣ activating activity polypeptide O
HMT1 hnRNP methyltransferase-like 1 (S. cerevisiae)
Integrin, ␣L (CD11A (p180))
Leukocyte immunoglobulin-like receptor subfamily A, member 2
␳ GDP dissociation inhibitor (GDI) ␤
G protein ␣ inhib. activity peptide 2
V-akt murine thymoma viral oncogene homolog 1
V-raf-1 murine leukemia viral oncogene homolog 1
Intercellular adhesion molecule 3
Insulin-like growth factor 2 receptor
IL-8R␤
Calmodulin 2 (phosphorylase kinase, ␦)
Protein kinase, cAMP-dependent, regulatory, type I, ␣
IL-8R␣
Membrane cofactor protein (CD46)
Protein phosphatase 1, regulatory (inhibitor) subunit 12A
Regulator of G protein signalling 14
Regulator of G protein signalling 19
IQ motif containing GTPase activating protein 1
RAS guanyl releasing protein 2 (calcium and DAG-regulated)
Mitogen-activated protein kinase-activated protein kinase 3
Phosphodiesterase 3B, cGMP-inhibited
Protein phosphatase 1, regulatory (inhibitor) subunit 12B
CD97 Ag
Toll-like receptor 1
Calmodulin 1 (phosphorylase kinase, ␦)
STE20-like kinase
CREB-binding protein (Rubinstein-Taybi syndrome)
Mitogen-activated protein kinase kinase kinase 5
Calmodulin 3 (phosphorylase delta)
GDP dissociation inhibitor 2
LIM and SH3 protein 1
Mitogen-activated protein kinase kinase kinase 3
Inositol 1,4,5-trisphosphate 3-kinase B
Calcium/calmodulin-dependent protein kinase (CaM kinase) II ␥
G protein, ␣-transducing activity polypeptide 2
G protein, ␤ polypeptide 2
Janus kinase 1 (a protein tyrosine kinase)
␳ GTPase activating protein 4
G protein-coupled receptor 19
Mitogen-activated protein kinase 3
Transforming growth factor, ␤ 1 (Camurati-Engelmann disease)
Toll-like receptor 6
Protein tyrosine phosphatase, f polypeptide, interacting protein, ␣ 1
Colony stimulating factor 3 receptor (granulocyte), G-CSFR
FK508 binding protein 1A, 12-kDa
Leukocyte immunoglobulin-like receptor, subfamily B, member 5
Lymphotoxin ␤ (TNF superfamily, member 3)
Pleckstrin homology, Sec7 and coiled/coil domains 1(cytohesin 1)
Transportin-SR
Wiskott-Aldrich syndrome (eczema-thrombocytopenia)
Mitogen-activated protein kinase kinase 4
Dedicator of cyto-kinesis 2
Transforming growth factor, ␣
Receptor (TNFRSF)-interacting serine-threonine kinase 1
Activin A receptor, type IB
RAB-interacting factor
RAS p21 protein activator (GTPase activating protein) 1
Gene Symbol
The Journal of Immunology
7691
Table III. Continued
Gene Name
Structural molecule
Coronin, actin-binding protein, IA
Adducin 3 (␥)
Tubulin, ␣, ubiquitous
Tubulin, ␣ 2
Tubulin, ␣ 1 (testis specific)
Actinin, ␣ 4
Spastic paraplegia 4 (autosomal dominant; spastin)
Flightless I homolog (Drosophila)
Lymphocyte-specific protein 1
Actin related protein 2/3 complex, subunit 2, 34 kDa
Actinin, ␣ 1
Actin related protein 2/3 complex, subunit 1B, 41 kDa
Actin related protein 2/3 complex, subunit 3, 21 kDa
Capping protein (actin filament) muscle Z-line, ␤
Titin-cap (telethonin)
␦ sleep inducing peptide, immunoreactor
Zinc finger protein 36, C3H type-like 2
Nuclear factor (erythroid-derived 2), 45 kDa
General transcription factor II, i
Arginine-glutamic acid dipeptide (RE) repeats
Lamin B receptor
Friend leukemia virus integration 1
Ubinuclein 1
Hematopoietically expressed homeobox
Polyhomeotic-like 2 (Drosophila)
BarH-like homeobox 2
Leucine rich repeat (in FLII) interacting protein 1
Chromobox homolog 1 (HP1 ␤ homolog Drosophila)
Zinc finger protein 217
Nuclear receptor coactivator 4
YY1 transcription factor
High-mobility group box 2
Nuclear receptor coactivator 1
Signal transducer and activator of transcription 6, IL-4 induced
Sjogren syndrome Ag A2 (ribonucleoprotein autoantigen SS-A/Ro)
Cbp/p300-interacting transactivator, carboxyl-terminal domain, 2
COP9 constitutive photomorphogenic homolog subunit 5 (Arabidopsis)
Heat shock factor binding protein 1
␣ thalassemia/mental retardation syndrome X-linked
Forkhead box O1A (rhabdomyosarcoma)
SWI/SNF related, matrix associated, regulator of chromatin, member 2
Meis1, myeloid ecotropic viral integration site 1 homolog 2 (mouse)
TAF4 RNA polymerase II, TATA box binding protein-associated factor
Histone deacetylase 5
Retinoblastoma binding protein 1
Polymerase (RNA) II (DNA directed) polypeptide J, 13.3 kDa
Heat shock transcription factor 1
Retinoic acid receptor, ␣
Transcriptional adaptor 3 (NGG1 homolog, yeast)-like
Hematopoietic cell-specific Lyn substrate 1
Myeloid/lymphoid leukemia (trithorax homol., Dros.); translocated to 7
TBP-like 1
Core-binding factor, runt domain, ␣ subunit 2; translocated to, 3
Growth arrest-specific 7
General transcription factor IIB
General transcription factor IIE, polypeptide 1, ␣ 56 kDa
General transcription factor IIIC, polypeptide 1, ␣ 220 kDa
C-myc binding protein
SFRS protein kinase 2
RNA-binding motif protein 5
Ribosomal protein, large P2
Eukaryotic translation initiation factor 4 ␥, 2
RNA binding protein (hnRNP-associated with lethal yellow)
Splicing factor, arginine/serine-rich 1
Signal recognition particle 9 kDa
Ribosomal protein S6 kinase, 90 kDa, polypeptide 1
CUG triplet repeat, RNA-binding protein 2
Ribosomal protein L26
Splicing factor, arginine/serine-rich 4
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 9 (DNA helicase)
Prp28, U5 snRNP 100 kDa protein
Transcription regulator
Translation
Gene Symbol
CORO1A
ADD3
K-␣-1
TUBA2
TUBA1
ACTN4
SPG4
FLII
LSP1
ARPC2
ACTN1
ARPC1B
ARPC3
CAPZB
TCAP
DSIPI
ZFP36L2
NFE2
GTF2I
RERE
LBR
FLI1
UBN1
HHEX
PHC2
BARX2
LRRFIP1
CBX1
ZNF217
NCOA4
YY1
HMGB2
NCOA1
STAT6
SSA2
CITED2
COPS5
HSBP1
ATRX
FOXO1A
SMARCA2
MEIS2
TAF4
HDAC5
RBBP1
POLR2J
HSF1
RAR
TADA3L
HCLS1
MLLT7
TBPL1
CBFA2T3
GAS7
GTF2B
GTF2E1
GTF3C1
MYCBP
SRPK2
RBM5
RPLP2
EIF4G2
RALY
SFRS1
SRP9
RPS6KA1
CUGBP2
RPL26
SFRS4
DDX9
U5–100K
Affy ID
Log2-Fold
Change
38976_at
⫺2.1
33102_at
⫺2
32272_at
⫺1.7
38350_f_at
⫺1.7
36591_at
⫺1.6
41753_at
⫺1.4
35171_at
⫺1.4
33133_at
⫺1.2
36493_at
⫺0.9
38445_at
⫺0.9
39330_s_at
⫺0.9
39043_at
⫺0.9
35810_at
⫺0.7
37012_at
⫺0.5
39002_at
⫺0.5
36629_at
⫺2.8
32587_at
⫺2.7
37179_at
⫺2.6
35450_s_at
⫺2.5
32253_at
⫺2.3
288_s_at
⫺2.1
41425_at
⫺2
32858_at
⫺2
37497_at
⫺1.9
36960_at
⫺1.8
35425_at
⫺1.8
41320_s_at
⫺1.7
37304_at
⫺1.6
32034_at
⫺1.6
39174_at
⫺1.5
891_at
⫺1.3
38065_at
⫺1.2
36118_at
⫺1.1
41222_at
⫺1.1
35294_at
⫺1
33113_at
⫺1
1789_at
⫺1
31906_at
⫺1
39147_g_at
⫺0.9
40570_at
⫺0.9
40962_s_at
⫺0.8
41388_at
⫺0.8
142_at
⫺0.7
38810_at
⫺0.7
1849_s_at
⫺0.6
38055_at
⫺0.6
244_at
⫺0.6
1337_s_at
⫺0.6
35749_at
⫺0.6
31820_at
⫺0.5
36238_at
⫺0.5
31797_at
⫺0.5
41442_at
⫺0.5
33387_at
⫺0.5
37380_at
⫺0.5
37882_at
⫺0.5
35671_at
⫺0.5
37250_at
⫺0.5
1213_at
⫺1.3
1556_at
⫺1.2
34091_s_at
⫺1
41785_at
⫺1
36125_s_at
⫺0.8
36098_at
⫺0.8
36981_at
⫺0.8
1127_at
⫺0.8
32851_at
⫺0.6
32444_at
⫺0.5
36991_at
⫺0.5
662_at
⫺0.5
40465_at
⫺0.5
(Table continues)
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Gene Category
7692
TRANSCRIPTIONAL ACTIVATION OF NEUTROPHILS IN SKIN LESIONS
Table III. Continued
Gene Category
Gene Name
Transporter
Nucleoporin 214 kDa
SEC14-like 1 (S. cerevisiae)
Neutrophil cytosolic factor 4, 40 kDa
Nucleoporin 153 kDa
Synaptosomal-associated protein, 23 kDa
ATP-binding cassette sub-family G (WHITE), member 1
Malic enzyme 2, NAD⫹-dependent, mitochondrial
Aminopeptidase-like 1
Solute carrier family 31 (copper transporters), member 2
Phosphatidylinositol transfer protein, membrane-associated
Solute carrier family 25 (mitochondrial phosphate carrier), member 3
Ubiquinol-cytochrome c reductase (6.4kD) subunit
CDP-diacylglycerol-inositol 3-phosphatidyltransferase
Cytochrome c oxidase subunit Vb
Solute carrier family 19 (folate transporter), member 1
Solute carrier family 23 (nucleobase transporters), member 1
E1B-55 kDa-associated protein 5
Gene Symbol
Affy ID
Log2-Fold
Change
NUP214
SEC14L1
NCF4
NUP153
SNAP23
ABCG1
ME2
NPEPL1
SLC31A2
PITPNM
SLC25A3
UQCR
CDIPT
COX5B
SLC19A1
SLC23A1
E1B-AP5
40768_s_at
36207_at
38895_i_at
32850_at
32178_r_at
41362_at
36599_at
41121_at
34749_at
38297_at
37675_at
38451_at
33397_at
39443_s_at
33135_at
38122_at
40106_at
⫺1.8
⫺1.2
⫺1.2
⫺1.2
⫺1.2
⫺1
⫺1
⫺0.8
⫺0.8
⫺0.7
⫺0.7
⫺0.7
⫺0.6
⫺0.6
⫺0.6
⫺0.6
⫺0.5
The cellular response to cytokines and chemokines highly depends on the profile of receptors expressed on the cell membrane.
The up-regulation of the IL-1R1 concomitant with its own ligand,
IL-1␤ in sl-PMNs, suggests an autoregulatory enforcement of their
inflammatory response through the NF-␬B activation pathway.
Up-regulation of the TGF-␤R indicates an increased responsiveness to TGF-␤, a cytokine that is secreted by activated macrophages and perhaps is the most potent endogenous negative regulator of hemopoietic cells (39, 40). Hence, one might speculate
that once PMN-chemokines have attracted sufficient macrophages,
the macrophages will decrease the activity of PMNs through
TGF-␤ signaling, and thus, take over and initiate the next step in
wound healing. Indeed, this hypothesis is supported by in vivo
experiments showing that leukocyte infiltrates in skin wounds are
initially dominated by PMNs, which decline in numbers concomitant with the increase of macrophage numbers 2 days after
injury (37).
Other up-regulated receptors in sl-PMNs that might modulate
cellular activity in skin lesions included the GPR18/65 and HM74.
Whereas induction of GPR18/65 in PMNs has not been described
so far, the induction of HM74 has been reported upon stimulation
of PMNs by LPS and bacteria in vitro (4, 5). HM74 has been
defined as a receptor for nicotinic acid, which mediates decrease in
cAMP levels in adipose tissue when binding to its ligand (41).
Down-regulated receptor transcripts included IL-8R␣, IL8-R␤, GCSFR, and the TLRs 1 and 6, which mediate chemotaxis and cellular activation (19, 20). Overall, the altered expression of receptor
FIGURE 2. Comparison of mRNA an d protein levels in sl-PMNs and
pb-PMNs. The upper row depicts the protein expression detected by Western blotting and the lower row depicts the mean relative mRNA expression
(n ⫽ 4) detected by microarray analysis.
transcripts suggests a change in responsiveness to chemotactic and
immunoregulatory mediators once PMNs have migrated into skin
lesions and have been activated.
The healing of skin wounds is a multistep process where cytokines and chemokines orchestrate the collaboration of various cell
types. VEGF, probably the most important angiogenic cytokine,
stimulates both proliferation and migration of endothelial cells
(42). The present study demonstrates for the first time the upregulation of VEGF by PMNs in a human skin wounding model.
Moreover, sl-PMNs up-regulated the chemokines IL-8, GRO-␥,
and MCP-1, which have been reported to stimulate growth of endothelial cells, keratinocytes, and fibroblasts (19, 21). Other genes
that affect wound healing and were up-regulated by sl-PMNs included uPA and LAMB3. This was somewhat surprising as uPA is
not up-regulated by PMNs when activated in vitro by various stimuli (4, 5, 7). However, incubation of plasma with PMNs has been
shown to generate thrombolytic uPA activity (24). Hence, activated PMNs in skin wounds might generate uPA resulting in plasminogen activation and subsequent breakdown of fibrin clots and
extracellular matrix (25). Moreover, uPA has been reported to promote the proliferation, migration, and adhesion of keratinocytes,
fibroblasts, and endothelial cells in skin wounds (25). LAMB3 is
an important adhesion molecule of the basal membrane and the
deficiency of LAMB3 results in a blistering skin disease (junctional epidermolysis bullosa) due to the disruption of epidermaldermal coadhesion (23). The up-regulation of LAMB3 by PMNs in
skin lesions might therefore support adhesion of keratinocytes at
wound margins, and thus, promote epitheliazation.
An important function of PMNs at sites of infection is the release of antimicrobial proteins as well as the phagocytosis and
subsequent lysosomal degradation of microorganisms and cellular
debris. Importantly, PMNs in skin lesions demonstrated no transcriptional regulation of antimicrobial peptides and lysosomal enzymes.
However, the up-regulation of tripeptidyl-peptidase I/ceroid-lipofuscinosis, neuronal 2 and legumain/asparaginyl endopeptidase, which
activate lysosomal enzymes by cleavage (26 –28), and the up-regulation of the mannose-6-phosphate receptor (29), which target proteins
to the lysosome, suggested a priming of PMNs for degradation of
phagocytosed material upon migration to skin lesions.
We conclude that PMNs generate a substantial transcriptional
response upon migration to skin lesions. This response fits with a
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
a
Genes downregualted in PMNs upon migration to skin lesions including their gene category, gene name, gene symbol, Affymetrix ID, and the log2-fold change of gene
expression (range of p values: 2.6 ⫻ 10⫺3–3.6 ⫻ 10⫺9, estimated false discovery rate 0.032 (Benjamini-Hochberg)).
The Journal of Immunology
7693
model where PMNs change from a state primed for chemotaxis
and activation by immunoregulatory mediators and microorganisms toward a state promoting wound healing, and the recruitment
and activation of additional inflammatory effector cells.
19.
Acknowledgments
21.
We thank Katja Nielsen for technical assistance, and our highly appreciated
colleagues Pia Klausen, Lene Udby, Jack Cowland, Bo Porse,
Carsten Niemann, Lars Jacobsen, and Mikkel Faurschou for critical review
of the manuscript. We also thank Dr. Jesper Pass (The Finsen Laboratory,
Rigshospitalet, Copenhagen, DK) for the generous gifts of the anti-uPA Ab
and human uPA protein.
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38.
39.
40.
41.
42.
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