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*Division of Rheumatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109; Division of Nephrology, Department of
Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109;
(Grant P30 CA46592), the Rheumatic Disease Core Center (Grant P30 AR48310),
and the Applied Systems Biology Core in the OBrien Renal Center (Grant P30
DK081943).
The sequences presented in this article have been submitted to the Gene Expression
Omnibus under accession number GSE26975.
E.V. and S.Y. contributed equally to the manuscript and share first authorship.
An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these lowdensity granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased
levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array
profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to
synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory proteins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial
cells and to stimulate IFN-a synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated
by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These
results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and
its subsequent responses may play a prominent deleterious role. The Journal of Immunology, 2011, 187: 538552.
Reagents
For LDG purification, biotinylated Abs recognizing CD3, CD7, CD19,
CD79b, CD56, MHC class II, CD86, and CD235a were obtained from
Ancell (Bayport, MN). For immunohistochemical staining, rabbit Abs
Purification of LDGs
Lupus LDGs were purified as described by our group (1). In brief, PBMCs
were isolated by Ficoll/Hypaque gradient, and cell pellets were incubated
with LDG isolation mixture (equal volumes of biotinylated Abs recognizing human CD3, CD7, CD19, CD79b, CD56, MHC class II, CD86, and
CD235a), followed by anti-biotin MACS beads (Miltenyi Biotec, Auburn,
CA). Cells were applied to an MACS-LS column, and nonimmobilized
cells were recovered by negative selection. The purity of the LDG fraction
was .95%, and cells were identified as CD15+/CD14lo or CD10+/CD14lo.
Isolation of neutrophils
Normal-density neutrophils were isolated by dextran sedimentation of RBC
pellets, as described (15). Purity was $95%. In previous studies, we had
compared the activation status of neutrophils obtained by dextran sedimentation versus double gradient and found no differences in their phenotype or function (1).
RNA isolation
Total RNA was isolated with Tripure (Roche, Indianapolis, IN), following
manufacturers recommendations. For microarray analysis, RNA was
further purified and concentrated using an RNeasy micro kit (Qiagen,
Valencia, CA). RNA samples were processed on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA) to assess integrity.
539
540
two resulting normalized files were corrected for batch effect (16) within
GenePattern. Of the 17,527 gene identification numbers (corresponding to
the 54,675 Affymetrix probesets), the number of genes expressed above
the Poly-A Affymetrix control expression baseline (negative controls) and
used for further analyses was 15,929. The Gene Expression Omnibus accession number for these arrays is GSE26975 (http://www.ncbi.nlm.nih.
gov/geo/).
Statistical paired analyses were performed using Significance Analysis of
Microarrays method implemented in MultiExperiment Viewer application
(17, 18) for comparing lupus neutrophils with lupus LDGs; unpaired
analyses were performed in the comparison of healthy control neutrophils
with lupus neutrophils and healthy control neutrophils with lupus LDGs.
The canonical pathways derived from the significantly regulated genes
between the groups (q , 0.05 depicting the false discovery rate) were
analyzed using the Ingenuity Pathway Analysis (IPA) Software (http://
www.ingenuity.com).
EC cytotoxicity assay
HUVECs were cultured in MCDB131 basal media (Life Technologies,
Carlsbad, CA) supplemented with EGM-2MV (without hydrocortisone)
bullet kit (Lonza, Walkersville, MD) on 0.2% gelatin-coated 24-well plates
(Fisher, Pittsburgh, PA). Lupus LDGs, their autologous lupus neutrophils,
and healthy control neutrophils were incubated with HUVECs at a 2:1 E
(neutrophil):T (HUVEC) ratio in MCDB131/EGM-2MV and 0.1 mg/ml
PMA in the presence or absence of MNase (10 U/ml) for 1620 h. Alternatively, cells were also cocultured with 0.1 mg/ml PMA, with or
without MNase, for 1 to 2 h, followed by replacement with fresh media
with or without nuclease. Following the incubation, nuclease reaction was
stopped by adding EDTA to final concentration of 2 mM. Adherent LDGs
and neutrophils were collected by gentle pipetting, and HUVECs were
harvested with 0.05% trypsin-EDTA (Life Technologies), followed by
centrifugation at 1600 rpm for 5 min. HUVECs were resuspended in 1%
horse serum/1% BSA in PBS, and 104 to 105 cells were incubated with
PEanti-human CD146, allophycocyanin-Annexin V, PE/Cy5anti-human
CD45 (BD Pharmingen, San Diego, CA), and/or PE/Cy5-anti-human
CD10 (Biolegend), followed by fixation with 4% paraformaldehyde. The
percentage of HUVEC cytotoxicity was measured in cells double-positive
for CD146 and Annexin V in the CD45- or CD10-negative gate.
Statistical analysis
For NET quantification, immunofluorescence was performed in neutrophils
isolated from individual patients (n $ 4 for both SLE and control patients),
and counts were averaged, represented as mean 6 SEM, and analyzed by
two-tailed Student t test, for which p # 0.05 was considered significant.
Comparison of NETosis within different areas of the skin was performed
by repeated-measures one-way ANOVA with a Tukeys multiple comparison posttest. For all other studies, paired or unpaired Student t tests were
performed. Microarray statistical analysis is detailed above.
Results
Lupus LDGs have a distinct gene expression profile from
autologous lupus neutrophils and control neutrophils
To determine whether LDGs represent a distinct pool of neutrophils, gene expression profiling of purified neutrophil fractions
of lupus LDGs, autologous lupus neutrophils, and gender-matched
control neutrophils was assessed using Affymetrix genechip microarrays. Samples were matched-paired in comparing lupus
LDGs with autologous lupus neutrophils. Although there were
no genes significantly differentially regulated when comparing
normal-density lupus neutrophils with healthy control neutrophils,
several genes were differentially expressed in LDGs relative to
either autologous lupus neutrophils or healthy control neutrophils.
In all, 302 genes were differentially expressed in lupus LDGs when
compared with control neutrophils, and 281 genes were identified
as altered by pairwise comparison of each patients LDGs to their
autologous lupus neutrophils (q value ,0.01, fold-change $1.5
and #0.7 for the upregulated and downregulated genes, respectively) (Fig. 1A, Supplemental Tables I, II). A total of 224 genes
were identified as upregulated in both comparisons (Fig. 1A). Con-
541
542
Table I. Affymetrix microarray expression data in lupus LDGs of genes corresponding to enzymes, bactericidal, and other molecules known to be
implicated in reactive oxygen species generation and/or NET formation
Lupus LDGs
Lupus LDGs
Compared with Compared with
Control
Lupus
Neutrophils
Neutrophils
Gene Symbol
Gene Name
RNASE2
ELANE (Elastase 2)
LCN2
CTSA
CTSG
MPO
DEFA4
CAMP (LL-37)
BPI
AZU1
4317
MMP8
4318
MMP9
6037
RNASE3
1991
3934
5476
1511
4353
Bactericidal molecule genes
1669
820
671
566
Other genes
1088
10321
634
4057
1191
Fold
q
Fold
q
Change Valuea Change Valuea
4.24 0.004*
3.06 0.066
1.37 0.047*
0.000*
0.000*
0.004*
0.000*
0.000*
10.92
19.02
1.54
17.73
14.66
0.000*
0.000*
0.043*
0.000*
0.000*
22.41
3.44
13.80
11.23
A q value ,0.05 was considered significant (in boldface and with asterisk).
Entrez Gene
Identification No.
543
Because NETs may be sources of autoantigens in other conditions (10), we assessed if various targeted autoantigens were
present in LDG NETs. During quantification, it became evident
that all NETs expose dsDNA regardless of the neutrophil source.
However, as a higher percentage of LDGs undergo NETosis than
healthy controls or lupus neutrophils, they lead to overall enhancement in externalization of dsDNA (Fig. 4A, 4C). In contrast,
there was no evidence of expression of other common lupus
autoantigens (Ro, La, or Smith) within the NETs structure. Indeed,
the expression of Ro, La, and Smith was intracellular and equally
detected in LDGs, lupus, and control neutrophils (data not shown).
These observations were independent of whether the serum of the
patients was positive or negative for Abs against dsDNA, Smith,
Ro, or La (Table II and data not shown). These results indicate
that netting neutrophils externalize dsDNA and that LDGs may
represent an enhanced source of extracellular dsDNA through
heightened NET formation.
NET formation leads to enhanced externalization of IL-17 in
lupus LDGs
Innate IL-17producing cells are considered an integral part of IL17mediated immune responses (26). Neutrophils have previously
been identified as a source of IL-17, particularly in the context
of autoimmunity (27), although the signaling pathways by which
these cells synthesize and release this specific cytokine remain
uncharacterized. Neutrophils expressing IL-17 have been reported
in diseased tissues such as atherosclerotic plaques (28). Furthermore, neutrophils can externalize IL-17 through NETosis in skin
and peripheral blood from patients with psoriasis (29).
FIGURE 2. Circulating lupus LDGs undergo increased NETosis. A, Representative images of control
neutrophils, lupus neutrophils, and lupus LDGs isolated from peripheral blood and analyzed at baseline
(T0) or after stimulation for 2 h with DMSO or PMA.
Top panels show immunofluorescent merged images
of NETs, which were detected by neutrophil elastase
(green), and DNA was labeled with Hoechst 33342
(blue). Original magnification 340. Scale bar, 20 mm.
B, Quantification of the percentage of NETs (elastaselabeled cells over total number of cells) are plotted as
mean 6 SEM (n = 6 patients/group). *p # 0.05.
544
545
1N
2N
3N
4N
5N
6N
+
+
NA
+
+
+
dsDNA-Ab
Kidney
Diagnosis;
Activity/Chronicity
Patient
No.
dsDNA-Ab
+
+
+
+
+
2
NA
NA
NA
NA
2
Diagnosis
Patient
No.
+
+
+
2
2
+
+
2
+
2
+
2
+
+
+
2
2
2
+
2
DL
DL
Interface dermatitis
Lupus panniculitis
Lupus panniculitis
SCLE
DL
DL
DL
DL
Interface dermatitis
2
10
8
0
0
2
20
2
4
2
4
0
10
12
4
6
4
0
10
6
1B
2B
3B
4B
5B
6B
7B
8B
9B
10B
11B
12B
13B
14B
15B
16B
17B
18B
19B
20B
dsDNA-Ab
Low
Low
Low
Low
Normal
Low
C3, C4
Low
Low
Low
Low
Low
Normal
NA
NA
Normal
Normal
NA
C3, C4
Normal
Normal
Low
Normal
Normal
Normal
Low
Low
Normal
Normal
Normal
Normal
Low
Low
Low
Normal
Normal
Normal
Low
Normal
C3,C4
Sm, RNP
Sm
NA
Ro, La, Sm, RNP
Sm
Ro, Sm, RNP
ENA
Kidney
Negative
RNP, Ro
SmRNP
Negative
SmRNP, RNP
Ro
Negative
NA
NA
Negative
Ro, La
ENA
Skin
Negative
Negative
Sm, RNP
Ro
Negative
Sm, RNP
Sm, RNP
Sm, RNP
Negative
Negative
Ro
Negative
Ro, La
Ro, Sm, RNP
Sm, RNP
Sm, RNP, Ro
Negative
Sm, RNP
Ro
Negative
ENA
Peripheral Blood
1:160
1:320
1:640
1:1280
1:2560
1:1280
ANA
1:160
1:2560
1:2560
1:640
1:2560
Negative
Negative
Negative
Negative
1:640
Negative
ANA
1:160
1:320
1:2560
1:2560
1:320
1:320
1:2560
1:320
1:160
1:160
1:640
1:320
1:2560
1:2560
1:320
1:2560
1:2560
Negative
1:320
1:320
ANA
Medications
MMF, PDN
PDN, antimalarials
PDN, antimalarials
None
PDN, antimalarials
Antimalarials
None
None
None
None
None
CNS, nephritis
CNS, Raynauds
Nephritis
Arthritis
Sicca
Arthritis
Other Clinical
Manifestations
PDN, AZA
PDN, antimalarials
None
PDN, MMF
PDN
PDN, AZA
(Table continues)
MMF, antimalarials
None
PDN, antimalarials
Antimalarials
Antimalarials
PDN, antimalarials
PDN, antimalarials, MMF
PDN, antimalarials, MMF
Antimalarials, MMF
PDN, antimalarials, MMF
Antimalarials
Antimalarials
MMF, PDN
MMF, MTX, PDN
Antimalarials
PDN, antimalarials, AZA
Antimalarials
PDN, antimalarials
PDN, MMF, antimalarials
PDN, antimalarials
Serositis, arthritis,
Skin, serositis, arthritis, cytopenia
Cytopenia
Skin, joints, serositis
CNS
Medications
Clinical Manifestations
1S
2S
3S
4S
5S
6S
7S
8S
9S
10S
11S
SLEDAI
Score
Patient
No.
546
ABERRANT NETosis IN LUPUS GRANULOCYTES
Antimalarials
Antimalarials
PDN, antimalarials
Antimalarials
PDN, antimalarials
None
PDN, antimalarials
Antimalarials
None
Antimalarials
Skin
Skin
Skin, arthritis
Skin, arthritis, pleuritis
Arthritis, skin, fever
Cytopenias, arthritis
Skin, arthritis, serositis
Arthritis, CNS
Arthritis
Nephritis, skin, arthritis
RNP, Sm
Negative
Sm, RNP
Negative
Ro
Negative
La, RNP
Negative
Negative
Sm, RNP
Normal
Low
Low
Normal
Low
Normal
Low
Normal
Low
Low
4
2
4
2
10
4
14
4
8
10
1M
2M
3M
4M
5M
6M
7M
8M
9M
10M
2
2
+
+
2
2
+
2
+
+
SLEDAI
Score
Patient
No.
The dashes in the dsDNA Ab column mean negative. The dashes in the clinical manifestations column mean none.
+, positive; ANA, anti-nuclear Ab; AZA, azathioprine; DL, discoid lupus; ENA, extractable nuclear Ags; MMF, mycophenolate mofetil; MTX, methotrexate; NA, not available; PDN, prednisone; SCLE, subacute cutaneous lupus; SM, Smith;
SmRNP, Smith/RNP.
Medications
Clinical Manifestations
ANA
ENA
C3, C4
dsDNA-Ab
Microarray Analysis
Negative
1:160
1:2560
1:160
1:160
1:2560
1:2560
1:2560
1:640
1:2560
Discussion
Recent work from various groups indicates that NET formation
may be an important phenomenon in autoantigen modification and
exposure to the immune system, as well as in the induction of
tissue damage (9, 10). As such, aberrant NET formation may
play an important role in the development and perpetuation of
autoimmune diseases and organ damage observed in chronic inflammatory disorders. Our work now expands and reinforces this
concept by reporting that a distinct subset of neutrophils found in
SLE patients (LDGs) have enhanced capacity to form NETs and
upregulate expression of various neutrophil proteins and enzymes
implicated in NET formation and autoimmunity induction. These
NETs also expose dsDNA, an autoantigen considered key in lupus
pathogenesis. Furthermore, lupus neutrophils and, in particular,
LDGs elicit enhanced EC cytotoxicity through NET formation.
This phenomenon also appears to play a role in the induction of
IFN-a synthesis by pDCs. We have also identified that enhanced
NETosis occurs in vivo in SLE in affected skin and kidney. Furthermore, neutrophils from blood and skin from SLE patients
frequently externalize IL-17 as part of the NETosis process, which
547
Cytopenias, arthritis
Arthritis, cytopenias
Arthritis, lung
Skin, arthritis
548
may contribute to tissue damage and immune dysregulation. Overall, these observations further support a pathogenic role for neutrophils in organ damage in SLE.
One of the findings from our study is that no differences in gene
expression were found when normal density lupus neutrophils were
compared with gender-matched healthy control neutrophils. Thus,
previous reports examining alterations in lupus neutrophils may
in part reflect responses specifically elicited in the LDG pool. In
contrast, lupus LDGs obtained from the same patients from whom
the normal-density neutrophils were obtained showed significant
differences in gene expression when compared with healthy control
and lupus neutrophils. The pairwise comparison of gene expression
in LDGs and autologous neutrophils in SLE patients provides
a control for many potential sources of variability such as medications, disease activity, clinical manifestations, and exposure to
FIGURE 7. IL-17positive neutrophils infiltrate SLE skin. Direct immunofluorescence staining of IL-17 (green) and MPO (red) in affected
lupus dermis. A, Arrows highlight intact IL-17+ neutrophils in a blood
vessel. Scale bar, 200 mm. B, IL-17 present in a NET (arrow). Scale bar, 10
mm. C, Frequency of IL-17 expression in intact neutrophils and NETs in
SLE skin. Percentages of: 1) IL-17+ neutrophils of all IL-17+ cells (d); 2)
IL-17+ neutrophils of all intact neutrophils (n); and 3) IL-17+ NETs of all
NETs (:) are reported in the graph.
549
550
proteinase may play a key role in vascular damage in other conditions (51).
Lupus patients develop accelerated atherosclerosis, leading to
premature cardiovascular disease. We had previously found that
ECs from lupus patients undergo accelerated apoptosis in vivo and
that this phenomenon may be pathogenic, as it correlates with
endothelial dysfunction development (36). LDGs are capable of
killing ECs, but the mechanism involved was unclear (1). The
current study shows that, through enhanced NET formation, LDGs
acquire a heightened capability to damage the endothelium, as
cytotoxicity was abrogated with MNAse treatment. As such, accelerated NETosis may represent an important mechanism of
premature vascular damage in SLE.
Death by NETosis may also represent an important immunostimulatory event with regards to activation of innate immunity in
SLE, given the enhanced capacity of netting lupus neutrophils to
activate pDCs. This could promote chronic activation of the immune system and perpetuation of type I IFN synthesis characteristic
of this disease (52). Enhanced NETosis in SLE may be one of the
mechanisms explaining why infections may trigger flares in this
disease (53), with NET induction by microorganisms that in turn
leads to autoantigen externalization and immune system activation. The observation that both lupus neutrophils and LDGs induced comparable induction of IFN-a synthesis by pDCs may
indicate that this process cannot be fully explained by pure enhancement of NETosis in LDGs and that other variables are involved in this phenomenon. Future studies are needed to better
understand how this process occurs.
The skin is an important target organ of the immune system in
SLE, and a significant proportion of lupus patients have cutane-
Acknowledgments
We thank Dr. Michelle Kahlenberg for critical review of the manuscript.
Disclosures
D.S. is an employee of Centocor Research & Development and owns stock
in Johnson & Johnson. The other authors have no financial conflicts of
interest.
References
1. Denny, M. F., S. Yalavarthi, W. Zhao, S. G. Thacker, M. Anderson, A. R. Sandy,
W. J. McCune, and M. J. Kaplan. 2010. A distinct subset of proinflammatory
neutrophils isolated from patients with systemic lupus erythematosus induces
vascular damage and synthesizes type I IFNs. J. Immunol. 184: 32843297.
2. Hakkim, A., B. G. Furnrohr, K. Amann, B. Laube, U. A. Abed, V. Brinkmann,
M. Herrmann, R. E. Voll, and A. Zychlinsky. 2010. Impairment of neutrophil
extracellular trap degradation is associated with lupus nephritis. Proc. Natl.
Acad. Sci. USA 107: 98139818.
3. Bennett, L., A. K. Palucka, E. Arce, V. Cantrell, J. Borvak, J. Banchereau, and
V. Pascual. 2003. Interferon and granulopoiesis signatures in systemic lupus
erythematosus blood. J. Exp. Med. 197: 711723.
4. Hacbarth, E., and A. Kajdacsy-Balla. 1986. Low density neutrophils in patients
with systemic lupus erythematosus, rheumatoid arthritis, and acute rheumatic
fever. Arthritis Rheum. 29: 13341342.
5. Brinkmann, V., U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, D. S. Weiss,
Y. Weinrauch, and A. Zychlinsky. 2004. Neutrophil extracellular traps kill
bacteria. Science 303: 15321535.
6. Fuchs, T. A., U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn,
Y. Weinrauch, V. Brinkmann, and A. Zychlinsky. 2007. Novel cell death program
leads to neutrophil extracellular traps. J. Cell Biol. 176: 231241.
7. Gupta, A. K., M. B. Joshi, M. Philippova, P. Erne, P. Hasler, S. Hahn, and
T. J. Resink. 2010. Activated endothelial cells induce neutrophil extracellular
traps and are susceptible to NETosis-mediated cell death. FEBS Lett. 584: 3193
3197.
8. Neeli, I., S. N. Khan, and M. Radic. 2008. Histone deimination as a response to
inflammatory stimuli in neutrophils. J. Immunol. 180: 18951902.
9. Papayannopoulos, V., K. D. Metzler, A. Hakkim, and A. Zychlinsky. 2010.
Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil
extracellular traps. J. Cell Biol. 191: 677691.
10. Kessenbrock, K., M. Krumbholz, U. Schonermarck, W. Back, W. L. Gross,
Z. Werb, H. J. Grone, V. Brinkmann, and D. E. Jenne. 2009. Netting neutrophils
in autoimmune small-vessel vasculitis. Nat. Med. 191: 677691.
11. Caielli, S., G. Garcia.-Romo, B. Vega, J. Connolly, F. Xu, Z. Allantaz,
M. Punaro, J. Baisch, C. Guiducci, F. Barrat, et al. 2010. Netting neutrophils are
major inducers of type 1 IFN production in SLE (PO1.M.11 Abstract of Poster
Session). Lupus 19(1 Suppl): 117118.
12. Fuchs, T. A., A. Brill, D. Duerschmied, D. Schatzberg, M. Monestier,
D. D. Myers, Jr., S. K. Wrobleski, T. W. Wakefield, J. H. Hartwig, and
D. D. Wagner. 2010. Extracellular DNA traps promote thrombosis. Proc. Natl.
Acad. Sci. USA 107: 1588015885.
13. Hochberg, M. C. 1997. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis
Rheum. 40: 1725.
14. Bombardier, C., D. D. Gladman, M. B. Urowitz, D. Caron, C. H. Chang; The
Committee on Prognosis Studies in SLE. 1992. Derivation of the SLEDAI. A
disease activity index for lupus patients. Arthritis Rheum. 35: 630640.
15. Clark, R. A., and W. M. Nauseef. 2005. Isolation and functional analysis of
neutrophils, Unit 7.23. In: Current Protocols in Immunology. J. E. Coligan,
A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds. W. John
Wiley & Sons, Hoboken, NJ.
16. Johnson, W. E., C. Li, and A. Rabinovic. 2007. Adjusting batch effects in
microarray expression data using empirical Bayes methods. Biostatistics 8: 118
127.
17. Saeed, A. I., N. K. Bhagabati, J. C. Braisted, W. Liang, V. Sharov, E. A. Howe,
J. Li, M. Thiagarajan, J. A. White, and J. Quackenbush. 2006. TM4 microarray
software suite. Methods Enzymol. 411: 134193.
18. Saeed, A. I., V. Sharov, J. White, J. Li, W. Liang, N. Bhagabati, J. Braisted,
M. Klapa, T. Currier, M. Thiagarajan, et al. 2003. TM4: a free, open-source
system for microarray data management and analysis. Biotechniques 34: 374
378.
19. Chaperot, L., A. Blum, O. Manches, G. Lui, J. Angel, J. P. Molens, and
J. Plumas. 2006. Virus or TLR agonists induce TRAIL-mediated cytotoxic activity of plasmacytoid dendritic cells. J. Immunol. 176: 248255.
20. Weening, J. J., V. D. DAgati, M. M. Schwartz, S. V. Seshan, C. E. Alpers,
G. B. Appel, J. E. Balow, J. A. Bruijn, T. Cook, F. Ferrario, et al; International
Society of Nephrology Working Group on the Classification of Lupus Nephritis;
Renal Pathology Society Working Group on the Classification of Lupus Nephritis. 2004. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int. 65: 521530.
551
552
21. Papayannopoulos, V., and A. Zychlinsky. 2009. NETs: a new strategy for using
old weapons. Trends Immunol. 30: 513521.
22. Ganguly, D., G. Chamilos, R. Lande, J. Gregorio, S. Meller, V. Facchinetti,
B. Homey, F. J. Barrat, T. Zal, and M. Gilliet. 2009. Self-RNA-antimicrobial
peptide complexes activate human dendritic cells through TLR7 and TLR8. J.
Exp. Med. 206: 19831994.
23. Lande, R., J. Gregorio, V. Facchinetti, B. Chatterjee, Y. H. Wang, B. Homey,
W. Cao, Y. H. Wang, B. Su, F. O. Nestle, et al. 2007. Plasmacytoid dendritic cells
sense self-DNA coupled with antimicrobial peptide. Nature 449: 564569.
24. Nakou, M., N. Knowlton, M. B. Frank, G. Bertsias, J. Osban, C. E. Sandel,
H. Papadaki, A. Raptopoulou, P. Sidiropoulos, I. Kritikos, et al. 2008. Gene
expression in systemic lupus erythematosus: bone marrow analysis differentiates
active from inactive disease and reveals apoptosis and granulopoiesis signatures.
Arthritis Rheum. 58: 35413549.
25. Pham, C. T. 2006. Neutrophil serine proteases: specific regulators of inflammation. Nat. Rev. Immunol. 6: 541550.
26. Cua, D. J., and C. M. Tato. 2010. Innate IL-17-producing cells: the sentinels of
the immune system. Nat. Rev. Immunol. 10: 479489.
27. Hoshino, A., T. Nagao, N. Nagi-Miura, N. Ohno, M. Yasuhara, K. Yamamoto,
T. Nakayama, and K. Suzuki. 2008. MPO-ANCA induces IL-17 production by
activated neutrophils in vitro via classical complement pathway-dependent
manner. J. Autoimmun. 31: 7989.
28. de Boer, O. J., J. J. van der Meer, P. Teeling, C. M. van der Loos, M. M. Idu,
F. van Maldegem, J. Aten, and A. C. van der Wal. 2010. Differential expression
of interleukin-17 family cytokines in intact and complicated human atherosclerotic plaques. J. Pathol. 220: 499508.
29. Lin, A. M., C. J. Rubin, R. Khandpur, J. Y. Wang, M. B. Riblett, S. Yalavarthi,
E. C. Villanueva, P. Shah, M. J. Kaplan, and A. T. Bruce. 2011. Mast cells and
neutrophils release IL-17 through extracellular trap formation in psoriasis.
J. Immunol. 187: 490500.
30. Crispn, J. C., and G. C. Tsokos. 2010. IL-17 in systemic lupus erythematosus. J.
Biomed. Biotechnol. 2010: 943254.
31. Chen, X. Q., Y. C. Yu, H. H. Deng, J. Z. Sun, Z. Dai, Y. W. Wu, and M. Yang.
2010. Plasma IL-17A is increased in new-onset SLE patients and associated with
disease activity. J. Clin. Immunol. 30: 221225.
32. Edgerton, C., J. C. Crispn, C. M. Moratz, E. Bettelli, M. Oukka, M. Simovic,
A. Zacharia, R. Egan, J. Chen, J. J. Dalle Lucca, et al. 2009. IL-17 producing
CD4+ T cells mediate accelerated ischemia/reperfusion-induced injury in
autoimmunity-prone mice. Clin. Immunol. 130: 313321.
33. Camussi, G., F. C. Cappio, M. Messina, R. Coppo, P. Stratta, and A. Vercellone.
1980. The polymorphonuclear neutrophil (PMN) immunohistological technique:
detection of immune complexes bound to the PMN membrane in acute poststreptococcal and lupus nephritis. Clin. Nephrol. 14: 280287.
34. Gilliet, M., and R. Lande. 2008. Antimicrobial peptides and self-DNA in autoimmune skin inflammation. Curr. Opin. Immunol. 20: 401407.
35. Martinelli, S., M. Urosevic, A. Daryadel, P. A. Oberholzer, C. Baumann,
M. F. Fey, R. Dummer, H. U. Simon, and S. Yousefi. 2004. Induction of genes
mediating interferon-dependent extracellular trap formation during neutrophil
differentiation. J. Biol. Chem. 279: 4412344132.
36. Rajagopalan, S., E. C. Somers, R. D. Brook, C. Kehrer, D. Pfenninger, E. Lewis,
A. Chakrabarti, B. C. Richardson, E. Shelden, W. J. McCune, and M. J. Kaplan.
2004. Endothelial cell apoptosis in systemic lupus erythematosus: a common
pathway for abnormal vascular function and thrombosis propensity. Blood 103:
36773683.
37. Cowland, J. B., and N. Borregaard. 1999. The individual regulation of granule
protein mRNA levels during neutrophil maturation explains the heterogeneity of
neutrophil granules. J. Leukoc. Biol. 66: 989995.
38. Kessenbrock, K., L. Frohlich, M. Sixt, T. Lammermann, H. Pfister, A. Bateman,
A. Belaaouaj, J. Ring, M. Ollert, R. Fassler, and D. E. Jenne. 2008. Proteinase 3
and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin. J. Clin. Invest. 118: 24382447.
39. Adkison, A. M., S. Z. Raptis, D. G. Kelley, and C. T. Pham. 2002. Dipeptidyl
peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis. J. Clin. Invest. 109: 363371.
40. Meyer-Hoffert, U. 2009. Neutrophil-derived serine proteases modulate innate
immune responses. Front. Biosci. 14: 34093418.
41. Devaney, J. M., C. M. Greene, C. C. Taggart, T. P. Carroll, S. J. ONeill, and
N. G. McElvaney. 2003. Neutrophil elastase up-regulates interleukin-8 via tolllike receptor 4. FEBS Lett. 544: 129132.
42. Ding, D., H. Mehta, W. J. McCune, and M. J. Kaplan. 2006. Aberrant phenotype
and function of myeloid dendritic cells in systemic lupus erythematosus. J.
Immunol. 177: 58785889.
43. Bals, R., and J. M. Wilson. 2003. Cathelicidinsa family of multifunctional
antimicrobial peptides. Cell. Mol. Life Sci. 60: 711720.