The influence of cathelicidin LL37 in human anti-neutrophils cytoplasmic antibody (ANCA)-associated vasculitis
Zhang et al. Arthritis Research & Therapy
The influence of cathelicidin LL37 in human anti-neutrophils cytoplasmic antibody (ANCA)-associated vasculitis
Ying Zhang 0
Weiwei Shi 0
Sha Tang 0
Jingyi Li 2
Shiwei Yin 0
Xuejing Gao 0
Li Wang 2
Liyun Zou 2
Jinghong Zhao 0
Yunjian Huang 0
Lianyu Shan 1
Abdelilah S Gounni 1
Yuzhang Wu 2
Fahuan Yuan 0
Jingbo Zhang 0
0 Department of Nephrology, Xinqiao Hospital, Third Military Medical University , Chongqing 400037 , China
1 Department of Immunology, University of Manitoba, Faculty of Medicine , Winnipeg, Manitoba R3E 0 T5 , Canada
2 Institute of Immunology of PLA, Third Military Medical University , Chongqing 400038 , China
Introduction: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterised by the autoinflammation and necrosis of blood vessel walls. The renal involvement is commonly characterised by a pauci-immune crescentic glomerulonephritis (PiCGN) with a very rapid decline in renal function. Cathelicidin LL37, an endogenous antimicrobial peptide, has recently been implicated in the pathogenesis of autoimmune diseases. To assess whether serum LL37 reflects renal crescentic formation, we measured the serum levels of LL37 in AAV patients with and without crescentic glomerulonephritis (crescentic GN) as compared to healthy controls (HCs). We also analysed the correlation of the serum levels of LL37 and interferon- (IFN-) with the clinical characteristics of the patients. Methods: The study population consisted of 85 AAV patients and 51 HCs. In 40 ANCA-positive patients, a parallel analysis was performed, including the assessment of LL37 and IFN- levels in the serum and renal biopsies. Of those studied, 15 AAV patients had biopsy-proven crescentic GN, and 25 AAV patients lacked crescent formation. The serum levels of cathelicidin LL37 and IFN- were both measured by ELISA, and the clinical and serological parameters were assessed according to routine procedures. Immunofluorescence staining was performed on frozen sections of kidney needle biopsies from AAV patients with crescentic GN. Results: The serum levels of LL37 and IFN- were significantly increased in AAV patients with crescentic GN compared to AAV patients without crescentic formation and HCs, and patients with high LL37 and IFN- levels were more likely to be in the crescentic GN group. The LL37 levels were positively correlated with the IFN- levels, and both LL37 and IFN- levels showed a positive correlation with serum creatinine and no correlation with complement C3. The renal tissue of crescentic GN patients showed expression of LL37 and IFN- at the Bowman's capsule and extracellular sites, suggesting the active release of LL37 and IFN-. Conclusions: Significantly higher levels of LL-37 and IFN- were observed in AAV patients, particularly those with crescentic formation, and LL37 and IFN- were expressed in the renal tissue of patients with crescentic GN. These data suggest that serum levels of LL37 and IFN- may reflect both local renal inflammation and systemic inflammation.
Anti-neutrophil cytoplasm antibody (ANCA)-associated
vasculitis (AAV) represents a group of systemic
autoimmune diseases including Wegeners granulomatosis,
microscopic polyangiitis, ChurgStrauss syndrome and
renal limited vasculitis . Renal involvement is
frequently manifested as focal segmental necrotising
glomerulonephritis (GN), typically pauci-immune crescentic
glomerulonephritis (PiCGN). Myeloperoxidase (MPO) and
proteinase-3 (PR3) have been identified as targets of
classical ANCA and have proved invaluable for the diagnosis
and monitoring of disease activity . In addition, the
presence of autoantibodies to lysosomal membrane protein-2
(LAMP-2) represents an additional ANCA subtype [2-4].
However, the mechanisms underlying the pathogenesis of
PiCGN remain elusive. As an endogenous antimicrobial
peptide, LL37 has recently been implicated in the
pathogenesis of autoimmune diseases . Autoinflammatory
conditions such as psoriasis and systemic lupus erythematosus
can be driven by plasmacytoid dendritic cells (pDCs), which
produce large amounts of interferon alpha (IFN) in the
presence of DNA and cathelicidin LL37 [6,7]. Recently,
DNA-containing LL37 was shown to be involved in the
renal damage in AAV, with increased concentrations of IFN
in serum samples from individuals with active AAV .
Cathelicidin LL37 is a member of an antimicrobial
peptide family found within the lysosomes of
macrophages, and polymorphonuclear leukocytes serve a
critical role in immune defence against invasive bacteria .
For example, neutrophil extracellular traps (NETs) are a
unique method by which neutrophils can cause cell death
via the release of a meshwork of chromatin fibres decorated
with granule-derived antimicrobial peptides; however, these
NETs are a potential source of auto-antigens and may
contribute to organ damage and vascular disease . These
NET-derived constituents stimulate pDCs to release IFN,
which establishes a positive feedback loop in which NETs
stimulate IFN release from pDCs, and this cytokine then
primes neutrophils for additional NET formation [6,10,11].
In this study, we hypothesised that the serum levels of
LL37 would reflect systemic and renal local
inflammation in patients with PiCGN. Therefore, we investigated
LL37 and IFN levels using enzyme-linked
immunosorbent assays (ELISAs) in AAV patients and correlated
these results to the serological parameters assessed.
Adult subjects with AAV, who were diagnosed according to
the Chapel Hill definition , were recruited from the
Department of Nephrology, Xinqiao Hospital, Third Military
Medical University. The patient characteristics are presented
in Table 1. Serum samples were collected from the patients
and healthy controls (HCs). Among the 85 patients, 40
patients underwent renal biopsies concomitantly obtained
with the serum samples, and all biopsies were reviewed and
classified by an experienced nephropathologist according to
the revised criteria for PiCGN. Criteria used in the study to
define PiCGN by immunofluorescence analysis for frozen
tissue sections presented deficiency of immunoglobulin and
complements. PR3-ANCA and MPO-ANCA were
evaluated using EUROBlot kits (DL-1200-6421-3G; Euroimmun;
Lbeck, Germany) and indirect immunofluorescence
(FA1200-2010; Euroimmun; Lbeck, Germany) according to
the protocol provided by the manufacturer. The levels of
creatinine, blood urea nitrogen and complement factor C3
in the sera were determined using routine techniques.
Crescentic GN was defined as crescents in >50% glomeruli.
Frozen sections of renal biopsy specimens obtained from 40
patients, among of them, 15 patients with crescentic GN
and 25 patients without crescentic GN were included in the
present study. The study protocols were approved by the
ethics committee board of Xinqiao Hospital, and all
subjects gave written informed consent.
Fluorescein isothiocyanate (FITC) anti-human CD16 was
purchased from Biolegend (San Diego, CA, USA). Anti-human
LAMP-2 mouse monoclonal antibody (H4B4), anti-human
LAMP-2 mouse monoclonal antibody-FITC, anti-human PR3
mouse monoclonal antibody-FITC (WGM2), anti-human
MPO mouse monoclonal antibody-FITC (266.6 K2),
antiLL37 (pAbC14), anti-IFN (pAbFL-189) and normal mouse
IgG1 were purchased from Santa Cruz (Heidelberg,
Germany). Anti-human histone H3 rabbit polyclonal antibody
was purchased from Abcam (Cambridge, UK). All secondary
antibodies conjugated with fluorescence were purchased from
ZSBio (Beijing, China). Phorbol 12-myristate 13-acetate was
purchased from Sigma-Aldrich (St Louis, MO, USA). HBSS
(without Ca2+ and Mg2+), RPMI-1640 medium (phenol
redfree) and penicillin/streptomycin solution were purchased
from Invitrogen (San Diego, CA, USA). The 12-mm round
glass cover slips were purchased from Thermo Fisher
Scientific (Waltham, MA, USA). Polymorphprep was purchased
from Axis-Shield (Oslo, Norway). The red blood cell lysis
buffer was purchased from Roche Diagnostics (Mannheim,
Germany). The immunostaining fix solution,
immunostaining blocking buffer, immunostaining primary
antibody dilution buffer, immunofluorescence staining
secondary antibody dilution buffer and anti-fade
mounting medium were purchased from Beyotime (Shanghai,
China). Anti-human BDCA2 mouse monoclonal
antibodyFITC (130-090-510) and anti-human BDCA2 mouse
monoclonal antibody-PE (130-090-511) were purchased
from Miltenyi Biotec (Bergisch Gladbach, Germany). The
ELISA kits for LL37 (HK321) and IFN (3423-1A-20)
were purchased from Hycult Biotech (Uden, Netherlands)
and MabTech (Nacka Strand, Sweden), respectively.
Table 1 Clinical characteristics of patients with sera included in the cohort
24-hour proteinuria (g/24 hours)
736.5 (147.6 to 1,316) ##,*
Data presented as number or median (range). AAV, anti-neutrophil cytoplasmic antibody-associated vasculitis; MPO, myeloperoxidase. ##Difference between the
crescentic-positive AAV patients and crescentic-negative AAV patients (P < 0.01). *Difference between the crescentic-positive AAV patients and AAV patients
without a renal biopsy (P < 0.05).
Note: Each group had one patient shown double positive MPO- and PR3- ANCA in crescentic-positive AAV and crescentic-negative AAV, respectively.
Isolation of neutrophils
Human neutrophils were isolated from HCs or patients with
PiCGN by density centrifugation using Polymorphprep
and red blood cell lysis buffer . Briefly, 5 ml blood
containing ethylenediamine tetraacetic acid was layered onto
5 ml Polymorphprep. After 35 minutes of centrifugation at
500 g, the neutrophils were separated from the
polymorphonuclear leukocyte-rich pellet. Residual erythrocytes were
eliminated by red blood cell lysis. The neutrophil
purity was routinely ~95%, as assessed by forward-scatter
and side-scatter flow cytometric analyses . Unless
otherwise stated, the cells were resuspended in RPMI
medium (phenol red-free) supplemented with 1%
penicillin/streptomycin. Then, 5106 neutrophils/ml
were seeded onto tissue culture plates for culture and
5105 neutrophils/ml were seeded onto glass
coverslips for immunofluorescence staining. The cells were
incubated at 37C in the presence of 5% CO2.
Enzyme-linked immunosorbent assay
The levels of LL37 and IFN from sera or cell culture
supernatants were quantified by ELISA according to the
protocols provided by the manufacturers. The sensitivity
was 0.1 ng/ml for LL37 and 7.8 pg/ml for IFN.
Scanning electron microscopy
Freshly purified neutrophils were allowed to adhere to
glass coverslips in RPMI-1640 medium. After incubation
for 30 minutes at 37C in 5% CO2, the cells were
stimulated with 20 g/ml LAMP-2 antibody or left untreated.
The neutrophils were then fixed in 2.5% glutaraldehyde
for 2 hours at 4C. After washing with physiological saline
three times, the samples were dehydrated through a
graded ethanol series. The cover slips were then
transferred into a critical point dryer and dried. The surface of
the specimen was coated with a 5 nm platin/carbon layer
using a thin layer evaporator. The samples were then
viewed with a scanning electron microscope.
Immunofluorescence staining and detection by confocal
Immunofluorescence staining was performed on frozen
sections of kidney needle biopsies from patients with
PiCGN as described previously . Briefly, after
fixation in paraformaldehyde, the specimens were incubated
with anti-LL37 (pAbC14), anti-histone H3 (pAbab8284),
anti-IFN (pAbFL-189) and anti-BDCA-2 (mAbCD303)
antibodies or isotype control, followed by the
appropriate secondary antibodies. DNA was stained with
4,6diamidino-2-phenylindole. The neutrophils were seeded
onto lysinated glass slides in a 24-well cell culture plate
and incubated for 1 hour in a CO2 incubator at 37C. The
cells were left unstimulated or were stimulated with either
100 nM phorbol 12-myristate 13-acetate or 20 g/ml
LAMP-2 antibody for up to 180 minutes at 37C in 5%
CO2. Subsequently, the cells were fixed and permeabilised.
After rehydration with phosphate-buffered saline at room
temperature, the cells were incubated with blocking buffer
overnight at 4C, and then the specimens were incubated
with fluorescently labelled antibodies. The chromatin was
stained with an anti-histone H3 rabbit antibody. The
images were acquired as projections of a confocal stack.
The experiments were performed in triplicate at least three
separate times. Data are presented as the mean standard
error of the mean and were analysed using GraphPad Prism
software 5 (GraphPad Software, San Diego, CA, USA).
Where appropriate, either two-tailed Students t tests or the
KruskalWallis and MannWhitney U tests were used.
Differences were considered significant at P <0.05.
Serum LL37 levels are increased in AAV patients
To analyse the correlation between the serum levels of
LL37 and AAV, we first performed ELISAs to determine
the LL37 levels in 136 serum samples collected from 85
unrelated patients with documented AAV (50 females,
35 males; female/male ratio 10:7) (Table 1) and 51 HCs.
The AAV sera contained higher levels of LL37, with a
mean concentration of 100.3 ng/ml, as compared with
the HC sera, which showed a mean concentration of
28.53 ng/ml (U = 829.00, P <0.01; Figure 1A). Serum
levels of LL37 >100.3 ng/ml were arbitrarily considered
to represent high levels, and those <100.3 ng/ml were
considered to represent low levels. Among the 40
patients with renal biopsy, as shown in Figure 1B, the renal
tissue of patients with crescentic GN showed higher
levels of LL37 as compared with those from patients
with noncrescentic GN (P <0.01) and HCs (P <0.001).
The serum levels of LL37 in patients without crescentic
GN were also significantly higher than those in HCs
(P <0.01). Forty patients underwent renal biopsy and 22 of
them expressed high serum levels of LL37 (>100.3 ng/ml),
and 13 of these high-expression patients had crescentic
GN. In contrast, only two of the 18 patients expressing low
serum levels of LL37 (<100.3 ng/ml) had crescentic GN
(2 = 9.724, P = 0.002; Figure 1C). These results indicated
that patients with high LL37 expression had a greater risk
of having crescentic GN as compared with patients with
low LL37 expression.
Serum IFN levels are increased in AAV patients
We also performed ELISAs to analyse the serum IFN
levels in 136 serum samples collected from the
aforementioned 85 patients. We found that the AAV sera
contained higher levels of IFN, with a mean
concentration of 958.5 pg/ml, compared with the HC sera, which
showed a mean concentration of 252.1 pg/ml (U = 1438.00,
P = 0.000; Figure 2A).
Serum levels of IFN >958.5 pg/ml were arbitrarily
considered to represent high levels, whereas those
<958.5 pg/ml were considered to represent low levels.
Among the 40 patients with renal biopsy, as shown in
Figure 2B, 15 crescentic GN patients showed high-level
IFN expression as compared with 25 of the
noncrescentic GN patients (P <0.001) and the HCs (P <0.001).
The IFN serum level of the patients without crescentic
GN were also significantly higher than those in HCs
(P <0.05). Of the 40 patients who provided a renal
biopsy, 22 showed high levels of IFN (>958.5 pg/ml),
and 13 of these 22 patients had crescentic GN. In
contrast, only two of the 18 patients expressing low serum
levels of IFN (<958.5 pg/ml) had crescentic GN (2 =
16.835, P = 0.000; Figure 2C). These results indicated that
patients expressing high IFN levels had a greater risk of
crescentic formation in the glomerulus in comparison
with patients expressing low levels of IFN.
Correlation of the serum LL37 and IFN levels with the
Given that the LL37 and IFN serum levels were
increased in AAV patients, particularly in those with
crescentic GN, we further investigated whether the serum
levels of LL37 were correlated with the IFN levels and
whether the levels of LL37 and IFN were correlated
with the serum levels of creatinine and complement C3.
We found a positive correlation between the serum
LL37 and IFN levels (R = 0.577, P = 0.000; Figure 3A)
as well as significant positive correlations between the
serum levels of LL37 and creatinine (R = 0.437, P = 0.000;
Figure 3B) and between the serum levels of IFN and
creatinine (R = 0.337, P = 0.004; Figure 3C). However,
neither LL37 nor IFN showed any correlation with
complement C3 (LL37: R = 0.020, P = 0.871; IFN: R = 0.12,
P = 0.922) (Figure 3D,E). To further investigate whether
LL37 and IFN reflect systemic inflammation, we analysed
the sera levels of LL-37 and IFN before and after
treatment (n = 7). We found that both LL-37 and IFN
levels had degraded after immunosuppressive therapy
(Additional file 1).
Figure 1 High serum LL37 levels in anti-neutrophil cytoplasmic antibody-associated vasculitis patients. (A) Anti-neutrophil cytoplasmic
antibody-associated vasculitis (AAV) sera contained higher levels of LL37 than the healthy control (HC) sera (U = 829.00, **P <0.01). (B) AAV
patients with crescentic glomerulonephritis (GN) showed higher levels of LL37 as compared with the noncrescentic GN AAV patients (**P <0.01)
and the HCs (***P <0.001). Serum levels of LL37 in the patients without crescentic GN were also significantly increased compared with those in
the HCs (##P <0.01). (C) Patients with high LL37 expression (>100.3 ng/ml) had a greater risk of having crescentic GN than patients with low LL37
expression (<100.3 ng/ml) (2 = 9.724, P = 0.002).
Figure 2 High serum IFN levels in anti-neutrophil cytoplasmic antibody-associated vasculitis patients. (A) Anti-neutrophil cytoplasmic
antibody-associated vasculitis (AAV) sera contained higher levels of interferon alpha (IFN) than the healthy control (HC) sera (U = 1,438.00,
***P = 0.000). (B) AAV patients with crescentic glomerulonephritis (GN) showed higher levels of IFN as compared with the noncrescentic GN
AAV patients (***P <0.001) and the HCs (***P <0.001). Serum levels of IFN in the patients without crescentic GN were also significantly higher
than those in the HCs (#P < 0.05). (C) Patients with high IFN expression (>958.5 pg/ml) had a greater risk of having crescentic GN than patients
with low IFN expression (<958.5 pg/ml) (2 = 16.835, P = 0.000).
Release of LL37 and IFN in the kidney
To assess the roles of LL37 and IFN in renal
inflammation, we evaluated the presence and localisation of
LL37 and IFN in renal biopsies from AAV patients
with crescentic GN. We found that the kidney tissues
from AAV patients showed strong expression of LL37,
especially in Bowmans capsule. We also found that LL37
expression co-localised with neutrophil and pDC (stained
for BDCA2) infiltrates in the affected glomeruli and
the interstitium (Figure 4A,B), suggesting that LL37
expression occurs predominantly during crescentic GN.
As autoinflammatory conditions such as psoriasis can be
driven by pDCs, which produce large amounts of IFN,
we next examined the expression of IFN in the
kidney tissues. Immunostaining revealed the co-localisation
of IFN and LL37 in the kidney tissues from AAV
patients (Figure 4C). However, there were only mild
immunofluorescence signals stained with IFN and LL37
in the renal tissues from patients without crescents
(Figure 4D), suggesting that LL37 may mediate pDC
activation to produce IFN in renal local
inflammatory in AAV.
Figure 3 Correlation of serum LL37 and IFN levels with serological parameters. (A) The serum LL37 levels were positively correlated with
the interferon alpha (IFN) levels (R = 0.577, P = 0.000). (B), (C) Both the serum LL37 (R = 0.437, P = 0.000) and IFN (R = 0.337, P = 0.004) levels
showed a positive correlation with the serum creatinine levels, (D), (E) but not with the complement C3 levels (LL37: R = 0.020, P = 0.871; IFN:
R = 0.12, P = 0.922). AAV, anti-neutrophil cytoplasmic antibody-associated vasculitis.
Figure 4 Expression of LL37, IFN and BDCA2 in anti-neutrophil cytoplasmic antibody-associated vasculitis patients with crescentic
glomerulonephritis. (A), (B) Representative images showing the co-localisation of LL37 (red) and BDCA2, a marker of plasmacytoid dendritic cells
(green), in the glomeruli of frozen renal biopsy sections after immunofluorescence staining. (C) Co-localisation of interferon alpha (IFN; red) and
LL37 (green) in renal biopsy sections from anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) patients with crescentic
glomerulonephritis (GN). (D) Co-localisation of IFN (red) and LL37 (green) in renal biopsy sections from AAV patients without crescentic GN.
Release of auto-antigens and cathelicidin LL37 from
neutrophil extracellular traps
ANCA plays a critical role in the vascular damage
associated with AAV . Kessenbrock and colleagues
reported strong NET formation in patients with AAV ,
and the presence of anti-LAMP-2 antibodies has been
suggested to represent a new subtype of ANCA, with a
high prevalence in PiCGN [2,4,15]. Therefore, we asked
whether targeted auto-antigens and antimicrobial
peptides would be present in NETs stimulated by
antiLAMP-2-IgG. As expected, the immunofluorescent
analysis of NETs revealed that LAMP-2 co-localised with
extracellular chromatin fibres (Figure 5A). We also
observed that PR3 and MPO were expressed within the
NETs (Figure 5B,C), as reported previously , and we
found that LL37 expression was enriched in parts of the
extracellular chromatin fibres (Figure 5D). After culturing
for 180 minutes, neutrophils isolated from PiCGN
patients and HCs showed the typical features of NETosis,
including web-like structures, as visualised by scanning
electron microscopy (Figure 5E,F). To investigate the
release of LL37 in the NET supernatant, neutrophils isolated
from AAV patients or HCs were treated with
anti-LAMP2-IgG (AAV + H4B4, n = 5; HC + H4B4, n = 5), an isotype
control (HC + ISO, n = 5) or phorbol 12-myristate
13acetate as a positive control (n = 5) . After 180 minutes,
the supernatant was collected and measured by ELISA,
and the results indicated that the HC + H4B4 group
supernatant showed higher levels of LL37 as compared with the
HC or HC + ISO groups (P <0.001). Moreover, the AAV +
H4B4 supernatant showed higher levels of LL37 compared
with the HC + H4B4 supernatant (P <0.001) (Figure 5G).
No IFN was detected in the supernatant by ELISA. We
observed MPO, a marker of neutrophils, showing
coFigure 5 Anti-neutrophil cytoplasmic antibody-induced neutrophil extracellular traps release auto-antigens and LL37. Neutrophils
were incubated with anti-hLAMP2-IgG (H4B4, 20 g/ml) for 180 minutes with buffer from a healthy donor. Auto-antigens for (A) LAMP-2,
(B) myeloperoxidase (MPO), (C) proteinase-3 (PR3) and (D) LL37 were examined (green) in the neutrophil extracellular traps (NETs). Histones were
stained with an H3 rabbit polyclonal antibody (red), and DNA was stained with 4,6-diamidino-2-phenylindole (blue). Neutrophil features were
observed under scanning electron microscopy for (E) healthy controls (HCs) and (F) anti-neutrophil cytoplasmic antibody-associated vasculitis
(AAV) patients after incubation for 180 minutes in vitro. (G) Quantification of the supernatant levels of LL37 by enzyme-linked immunosorbent
assay: supernatant of neutrophils from healthy control incubated with anti-hLAMP2-IgG (HC+H4B4), neutrophils from AAV patients (AAV) and
neutrophils from AAV patients incubated with anti-hLAMP2-IgG (AAV+H4B4)groups showed higher levels of LL37 compared with HC group
(***P <0.001), respectively. Supernatant of neutrophils from AAV +H4B4 group showed higher levels of LL37 compared with the HC + H4B4 group
(###P <0.001). ISO; PMA, phorbol 12-myristate 13-acetate.
localisation with LL37, IFN and BDCA-2 in AAV patients
with crescentic GN (Additional file 2). To further clarify the
source IFN, pDCs were isolated by magnetically activated
cell sorting using the BDCA-4 dendritic cell isolation kit
(Miltenyi Biotec). pDCs (5 104/ml) were incubated with
supernatants from neutrophils treated as above mentioned,
with 3 g/ml ODN 2216 pDCs as positive control for
24 hours. Supernatants were harvested to detect the level of
IFN and LL-37 by ELISA. We found that IFN was
released by pDC incubation with supernatants of
anti-LAMP2 antibody-treated neutrophils and ODN2216 for 24 hours
(Additional file 3). No LL-37 was released from pDC.
The present study is the first to demonstrate that LL37
levels are significantly increased in AAV patients,
especially in those with PiCGN. The serum levels of LL37
and IFN were positively correlated with crescentic
formation and also positively correlated with the serum levels
of creatinine. Furthermore, we found evidence that LL37
and IFN expression were co-localised in inflammatory
kidney tissue. There were also higher levels of LL37
released in AAV patients as compared with HCs, as
determined by NET formation in vitro.
PiCGN is a serious manifestation in patients with AAV
and can rapidly progress to end-stage renal failure. Despite
the large number of studies, the pathogenesis of PiCGN
has not been fully clarified. Recently, the constituents
derived from NETs, such as high-mobility group box-1
protein, have been shown to play an important role in the
renal pathology of systemic lupus erythematosus patients,
potentially reflecting both local renal inflammation and
systemic inflammation [16,17]. The role of cathelicidin LL37
released from these NETs has been demonstrated in
autoimmune and chronic inflammatory diseases, especially
those with renal manifestations such as systemic lupus
erythematosus and psoriasis [6,7]. However, no studies have
been performed to evaluate the serum levels of LL37 to
determine whether LL37 expression is a reflection of systemic
and/or renal local inflammation in AAV patients. We found
that the serum levels of LL37 were significantly increased
in AAV patients, especially in those with PiCGN compared
with patients without crescentic formation and HCs. In line
with a previous study, the serum levels of IFN were
significantly increased in patients with active AAV , and we
also found that the serum levels of IFN were increased
similarly in AAV patients with PiCGN. However, the origin
of LL37 that is, whether it is produced outside the kidney
or locally within the inflamed kidney remains unresolved.
The precise events leading to glomerular inflammation
and damage in ANCA-related PiCGN remain poorly
understood. pDCs comprise a DC population that is
highly specialised to sense viral and certain microbial
infections. LL37 plays a key role in converting self-DNA
into a stimulatory ligand for pDCs , and the LL37
expression in renal biopsy tissues from PiCGN patients
was strong. BDCA-2 is a pDC-specific marker , and
we found that BDCA2 co-localised with LL37 in the
glomeruli and interstitium. Furthermore, a previous
report indicating that pDCs produce large amounts of
IFN in the presence of DNA and LL37  suggests that
IFN may be released from pDCs in renal tissue. We
also found that LL37 co-localised with IFN in PiCGN
renal biopsies, and these results indicate that pDCs are
activated and participate in the inflammatory response
in the kidney. As kidney injury coupled with the
expression of LL37 may elicit the sustained accumulation and
activation of pDCs in the kidney, LL37 antagonists may
potentially be developed as therapies for PiCGN and
other chronic inflammatory diseases, whereas LL37 itself
may potentially serve as a vaccine adjuvant .
Neutrophils are considered the mainstay of the cellular
innate immune response. Many data have shown that
neutrophils are not only basic players and mediators of
innate immunity but are also involved in the activation,
regulation and effector functions of adaptive immune
cells, such as dendritic cells, B cells and T cells [19,20].
In addition, there has been increased attention on
extracellular neutrophil traps in recent years with regard to the
pathogenesis of diverse inflammatory and autoimmune
diseases [21-26]. Summers and colleagues reported that
neutrophil recruitment may play an important role in
experimental anti-MPO crescentic GN , and recent
research has reported that autoantibodies against human
LAMP-2 represent a new ANCA subtype that can be
induced by infection with fimbriated bacteria, which occurs
at a high prevalence in PiCGN cases [2,3]. We found that
anti-LAMP-2-IgG triggered NET formation and that
targeted auto-antigens for LAMP-2, PR3, MPO and LL37
were present within those NETs. There were also higher
levels of LL37 released in AAV patients compared with
HCs, as determined by NET formation in vitro. This result
demonstrates that ANCA can activate neutrophil-released
auto-antigens through NET formation. This process
results in the expression of LAMP-2, MPO, PR3 and LL37
or high-mobility group box-1 protein, which all have the
characteristic dual capacity to mobilise and activate
antigen-presenting cells, thereby further inducing the
activation of immune cells . These observations indicate
that ANCA may perpetuate a vicious circle of NET
production that maintains the delivery of endogenous danger
signals to the immune system.
The present study provides evidence that LL37
expression is increased not only in the sera but also at the site of
local renal inflammation in AAV. The serum levels of LL37
could thus reflect both local and systemic inflammation.
However, further studies are needed to evaluate the clinical
significance of LL37 in a larger sample as well as its value
as a biomarker in AAV patients with renal involvement.
Together, our findings indicated that the serum levels of
LL37 and IFN levels were increased in AAV patients,
particularly those with crescentic GN. These increases in
the serum LL37 and IFN levels were correlated with
crescentic formation. Accordingly, the crescentic
patients showed evidence of LL37 and IFN expression in
their local inflammatory renal tissue. Taken together,
these data suggest that LL37 may play an important role
in the renal pathology of AAV patients.
Additional file 1: Figure S1. Showing the levels of serum LL-37 and
IFN before and after treatment. Serum levels of LL37 (A) and IFN (B)
were significantly decreased after immunosuppressive therapy than
before treatment (n = 7, *P < 0.05).
Additional file 2: Figure S2. Showing expression of MPO, LL37, IFN
and BDCA2 in AAV patients with crescentic GN. (A) Representative
images showing the co-localisation of MPO (green) and LL37 (red) in
the glomeruli of frozen renal biopsy sections after immunofluorescence
staining. (B) Co-localisation of MPO (green) and IFN (red) in renal biopsy
sections from AAV patients with crescentic GN. (C) Co-localisation of MPO
(green) and BDCA2 (red) in renal biopsy sections from AAV patients
without crescentic GN.
Additional file 3: Figure S3. Showing IFN released from pDC. None
IFN were detected in the supernatants from neutrophils treated with or
without anti-LAMP-2 antibodies (H4B4) for 3 hours. The mean level in the
supernatants of IFN was 7,357 pg/ml from pDCs incubated with
ODN2216 groups, and was 866 pg/ml from pDCs incubated with
supernatants of anti-LAMP-2 antibody-treated neutrophils. The level was
under-detectible in supernatants from pDCs treated with medium.
AAV: Anti-neutrophil cytoplasm antibody-associated vasculitis; ANCA:
Anti-neutrophil cytoplasm antibody; C3: Complement 3; ELISA:
Enzyme-linked immunosorbent assay; FITC: Fluorescein isothiocyanate;
GN: Glomerulonephritis; HC: Healthy control; IFN: Interferon alpha;
LAMP-2: Lysosomal membrane protein-2; MPO: Myeloperoxidase;
NET: Neutrophil extracellular trap; pDC: Plasmacytoid dendritic cell;
PiCGN: Pauci-immune crescentic glomerulonephritis; PR3: Proteinase-3.
JBZ, ASG, YZW and FHY were involved in all aspects of the study conception,
design and direction. JBZ, ASG, YZW, YZ, FHY, WWS, ST, LS, LW, and SWY
were involved in the data acquisition, the analysis and interpretation of the
results and drafted the manuscript. YZ, WWS, ST, LS, LW and XJG performed
the cell isolation, YZ, WWS, XJG, JYL and LYZ carried out the measurements of
NET, LL37 and IFN. YZ, ST, WWS, JHZ and YJH carried out immunofluorescence
staining and assays. All authors read and approved the final manuscript.
The authors thank associate Prof. Changjun Cai for providing statistical
assistance. They also thank Ms Xiaolan Fu for her assistance in FACS analyses.
This research was supported by the National Science Foundation of China
(30971366), the International Cooperation Projects of Chongqing Science
& Technology Committee (CSTC201110004) and the Clinical Research Project
of the Third Military Medical University (2011XLC37).
1. Jennette JC , Falk RJ : Small-vessel vasculitis . N Engl J Med 1997 , 337 : 1512 - 1523 .
2. Kain R , Tadema H , McKinney EF , Benharkou A , Brandes R , Peschel A , Hubert V , Feenstra T , Sengolge G , Stegeman C , Heeringa P , Lyons PA , Smith KG , Kallenberg C , Rees AJ : High prevalence of autoantibodies to hLAMP-2 in anti-neutrophil cytoplasmic antibody-associated vasculitis . J Am Soc Nephrol 2012 , 23 : 556 - 566 .
3. Kain R , Exner M , Brandes R , Ziebermayr R , Cunningham D , Alderson CA , Davidovits A , Raab I , Jahn R , Ashour O , Spitzauer S , Sunder-Plassmann G , Fukuda M , Klemm P , Rees AJ , Kerjaschki D : Molecular mimicry in pauciimmune focal necrotizing glomerulonephritis . Nat Med 2008 , 14 : 1088 - 1096 .
4. Salama AD , Pusey CD : Shining a LAMP on pauci-immune focal segmental glomerulonephritis . Kidney Int 2009 , 76 : 15 - 17 .
5. Frasca L , Lande R : Role of defensins and cathelicidin LL37 in autoimmune and auto-inflammatory diseases . Curr Pharm Biotechnol 2012 , 13 : 1882 - 1897 .
6. Garcia-Romo GS , Caielli S , Vega B , Connolly J , Allantaz F , Xu Z , Punaro M , Baisch J , Guiducci C , Coffman RL , Barrat FJ , Banchereau J , Pascual V : Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus . Sci Transl Med 2011 , 3 : 73ra20 .
7. Lande R , Gregorio J , Facchinetti V , Chatterjee B , Wang YH , Homey B , Cao W , Su B , Nestle FO , Zal T , Mellman I , Schroder JM , Liu YJ , Gilliet M : Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide . Nature 2007 , 449 : 564 - 569 .
8. Kessenbrock K , Krumbholz M , Schonermarck U , Back W , Gross WL , Werb Z , Grone HJ , Brinkmann V , Jenne DE : Netting neutrophils in autoimmune small-vessel vasculitis . Nat Med 2009 , 15 : 623 - 625 .
9. Nizet V , Ohtake T , Lauth X , Trowbridge J , Rudisill J , Dorschner RA , Pestonjamasp V , Piraino J , Huttner K , Gallo RL : Innate antimicrobial peptide protects the skin from invasive bacterial infection . Nature 2001 , 414 : 454 - 457 .
10. Knight JS , Kaplan MJ : Lupus neutrophils: 'NET' gain in understanding lupus pathogenesis . Curr Opin Rheumatol 2012 , 24 : 441 - 450 .
11. Chamilos G , Gregorio J , Meller S , Lande R , Kontoyiannis DP , Modlin RL , Gilliet M : Cytosolic sensing of extracellular self-DNA transported into monocytes by the antimicrobial peptide LL37 . Blood 2012 , 120 : 3699 - 3707 .
12. Jennette JC , Falk RJ , Andrassy K , Bacon PA , Churg J , Gross WL , Hagen EC , Hoffman GS , Hunder GG , Kallenberg CG , et al: Nomenclature of systemic vasculitides, Proposal of an international consensus conference . Arthritis Rheum 1994 , 37 : 187 - 192 .
13. Brinkmann V , Laube B , Abu Abed U , Goosmann C , Zychlinsky A : Neutrophil extracellular traps: how to generate and visualize them . J Vis Exp 2010 , 36 : 1724 .
14. Bosch X : LAMPs and NETs in the pathogenesis of ANCA vasculitis . J Am Soc Nephrol 2009 , 20 : 1654 - 1656 .
15. Bosch X , Mirapeix E : Vasculitis syndromes: LAMP-2 illuminates pathogenesis of ANCA glomerulonephritis . Nat Rev Nephrol 2009 , 5 : 247 - 249 .
16. Abdulahad DA , Westra J , Bijzet J , Dolff S , van Dijk MC , Limburg PC , Kallenberg CG , Bijl M : Urine levels of HMGB1 in systemic lupus erythematosus patients with and without renal manifestations . Arthritis Res Ther 2012 , 14 : R184 .
17. Abdulahad DA , Westra J , Bijzet J , Limburg PC , Kallenberg CG , Bijl M : High mobility group box 1 (HMGB1) and anti-HMGB1 antibodies and their relation to disease characteristics in systemic lupus erythematosus . Arthritis Res Ther 2011 , 13 : R71 .
18. Riboldi E , Daniele R , Cassatella MA , Sozzani S , Bosisio D : Engagement of BDCA-2 blocks TRAIL-mediated cytotoxic activity of plasmacytoid dendritic cells . Immunobiology 2009 , 214 : 868 - 876 .
19. Mantovani A , Cassatella MA , Costantini C , Jaillon S : Neutrophils in the activation and regulation of innate and adaptive immunity . Nat Rev Immunol 2011 , 11 : 519 - 531 .
20. Tillack K , Breiden P , Martin R , Sospedra M : T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses . J Immunol 2012 , 188 : 3150 - 3159 .
21. Brill A , Fuchs TA , Savchenko AS , Thomas GM , Martinod K , De Meyer SF , Bhandari AA , Wagner DD : Neutrophil extracellular traps promote deep vein thrombosis in mice . J Thromb Haemost 2012 , 10 : 136 - 144 .
22. Kambas K , Mitroulis I , Apostolidou E , Girod A , Chrysanthopoulou A , Pneumatikos I , Skendros P , Kourtzelis I , Koffa M , Kotsianidis I , Ritis K : Autophagy mediates the delivery of thrombogenic tissue factor to neutrophil extracellular traps in human sepsis . PLoS One 2012 , 7 : e45427 .
23. Leffler J , Martin M , Gullstrand B , Tyden H , Lood C , Truedsson L , Bengtsson AA , Blom AM : Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease . J Immunol 2012 , 188 : 3522 - 3531 .
24. Megens RT , Vijayan S , Lievens D , Doring Y , van Zandvoort MA , Grommes J , Weber C , Soehnlein O : Presence of luminal neutrophil extracellular traps in atherosclerosis . Thromb Haemost 2012 , 107 : 597 - 598 .
25. Sangaletti S , Tripodo C , Chiodoni C , Guarnotta C , Cappetti B , Casalini P , Piconese S , Parenza M , Guiducci C , Vitali C , Colombo MP : Neutrophil extracellular traps mediate transfer of cytoplasmic neutrophil antigens to myeloid dendritic cells toward ANCA induction and associated autoimmunity . Blood 2012 , 120 : 3007 - 3018 .
26. Thornton RB , Wiertsema SP , Kirkham LA , Rigby PJ , Vijayasekaran S , Coates HL , Richmond PC : Neutrophil extracellular traps and bacterial biofilms in middle ear effusion of children with recurrent acute otitis media - a potential treatment target . PLoS One 2013 , 8 : e53837 .
27. Summers SA , van der Veen BS , O'Sullivan KM , Gan PY , Ooi JD , Heeringa P , Satchell SC , Mathieson PW , Saleem MA , Visvanathan K , Holdsworth SR , Kitching AR : Intrinsic renal cell and leukocyte-derived TLR4 aggravate experimental anti-MPO glomerulonephritis . Kidney Int 2010 , 78 : 1263 - 1274 .
28. Yang D , de la Rosa G , Tewary P , Oppenheim JJ : Alarmins link neutrophils and dendritic cells . Trends Immunol 2009 , 30 : 531 - 537 .