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* Department of Microbiology and Immunology and
British Columbia Research Institute for Child and Family Health, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. LL-37 also up-regulated the chemokine
receptors CXCR-4, CCR2, and IL-8RB. These findings indicate that LL-37
may contribute to the immune response by limiting the damage caused by
bacterial products and by recruiting immune cells to the site of
infection so that they can clear the infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (11). Evidence for the key role of cationic
antimicrobial peptides in innate immunity includes mutations affecting
the induction of antibacterial peptides which reduce survival in
response to bacterial challenge. Indeed mutations of the Toll pathway
of Drosophila that lead to decreased antifungal peptide gene
expression result in increased susceptibility to lethal fungal
infections (12). Although there are multiple defensins in
mice, Wilson et al. (13) identified the single enzyme
necessary for processing the preprodefensins to the active mature form.
Genetic inactivation of this single gene (matrilysin) led to no
production of active defensin in the small intestine and a consequent
10-fold increase in the susceptibility to infection by orally
introduced virulent bacteria (13). Patients with specific
granule deficiency syndrome, completely lacking in defensins,
suffer from frequent and severe bacterial infections (2, 14). Similarly, a group of HIV patients with lower salivary
levels of histatin peptides showed a higher incidence of oral
candidiasis and fungal infection (2). Other evidence
includes the inducibility of some peptides by infectious agents and the
very high concentrations that have been recorded at sites of
inflammation (15, 16, 17). The single known human cathelicidin, hCAP-18, is a major protein of the specific granules in neutrophils (18) and is also present in monocytes and certain lymphocyte populations (19), testis (20), human keratinocytes during inflammatory disorders (21), and airway epithelium (22). The characteristic feature of cathelicidin peptides is a high level of sequence identity at the N terminus prepro regions (23), termed the cathelin domain (24). Cathelicidin peptides are stored as inactive propeptide precursors that, upon stimulation, are processed into active peptides. hCAP-18 was found to be cleaved extracellularly by proteinase 3 to generate the peptide LL-37 (25). Overexpression of this peptide, by adenovirus-mediated gene transfer, of LL-37 in the mouse airway results in the increased ability to reduce bacterial load from Pseudomonas aeruginosa challenge and improved survival after administration of lethal doses of LPS (26). It is of interest to determine whether the natural role of peptides in the body involves direct bacterial killing or a combination of killing and stimulation of other mechanisms of innate immunity.
Cationic antimicrobial peptides may also regulate cell migration to
promote the ability of leukocytes to combat bacterial infections. For
example, two human defensin peptides, HNP-1 and HNP-2, have been
indicated to have direct chemotactic activity for murine and human T
cells and monocytes (27, 28), and human 
defensins
appear to act as chemoattractants for immature dendritic cells and
memory T cells through interaction with CCR6 (29).
Similarly, the porcine cationic peptide PR-39 was found to be
chemotactic for neutrophils (30). LL-37 has been shown to
have chemotactic activity for monocytes, T cells, and neutrophils
(31, 32) as well as mast cells (33).
The aim of our current study was to gain knowledge of the roles that
the human peptide LL-37 may play in combating bacterial infection. We
performed gene arrays on macrophage cells stimulated with LL-37 and
discovered a large number of genes affected by the peptide. A number of
LL-37-induced gene expression changes were confirmed by
semiquantitative RT-PCR. We chose to follow-up on several genes
up-regulated by LL-37 that are involved in chemotaxis. We report here
the novel finding that LL-37 up-regulates the production of chemokines
and the surface expression of chemokine receptors, and thus could
promote cell migration, and that this occurs in vivo in the mouse lung.
These properties are in addition to the ability of LL-37 to reduce the
production of TNF- by macrophages stimulated with LPS, lipoteichoic
acid (LTA),3 and
Mycobacterium noncapped lipoarabinomannan (AraLAM)
suggesting that LL-37 has a multifaceted role in controlling bacterial
infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Salmonella typhimurium LPS and Escherichia coli 0111:B4 LPS were purchased from Sigma-Aldrich (St. Louis, MO). LTA (Sigma) from Staphylococcus aureus was resuspended in endotoxin-free water (Sigma-Aldrich). The Limulus amebocyte lysate assay (Sigma-Aldrich) was performed on LTA preparations to confirm that lots were not significantly contaminated by endotoxin. Endotoxin contamination was <1 ng/ml, a concentration that did not cause significant cytokine production in the RAW 264.7 cells (34). AraLAM was a gift from Dr. J. T. Belisle of Colorado State University (Fort Collins, CO). The AraLAM from Mycobacterium was filter sterilized and the endotoxin contamination was found to be 3.75 ng per 1.0 mg of LAM as determined by the Limulus amebocyte assay. LL-37 (amino acid sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was synthesized by F-moc chemistry at the Nucleic Acid/Protein Synthesis Unit at the University of British Columbia (UBC) as described previously (8).
Preparation and analysis of macrophages isolated from bone marrow of mice
Bone marrow macrophages were obtained from 8- to 10-wk-old BALB/c mice (Charles River Breeding Laboratories, Wilmington, MA) as previously described (35). The cells were cultured in 150-mm plates in DMEM (Life Technologies, Burlington, Ontario, Canada) supplemented with 20% FBS (Sigma-Aldrich) and 20% L cell-conditioned medium as a source of M-CSF. Once macrophages were 60–80% confluent, they were deprived of L cell-conditioned medium for 14–16 h to render the cells quiescent. The experiments were then conducted by adding 100 ng/ml E. coli O111:B4 LPS, LPS plus 20 µg/ml LL-37 (or CEMA), or medium alone (DMEM plus 20% FBS) to the cells for 24 h. The release of cytokines into the culture supernatant was determined by ELISA (R&D Systems, Minneapolis, MN).
Cytokine and chemokine production by RAW 264.7 macrophages and A549 epithelial cells
The murine macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (Manassas, VA) and the human epithelial cell line A549 was obtained from Dr. D. Speert (Department of Pediatrics, UBC). Both cell lines were maintained in DMEM supplemented with 10% FCS. RAW 264.7 cells were seeded in 24-well plates at a density of 106 cells/well in DMEM (see above) and A549 cells were seeded in 24-well plates at a density of 105 cells/well in DMEM (see above), and both were incubated at 37°C in 5% CO2 overnight. DMEM was aspirated from cells grown overnight and replaced with fresh medium. LPS or other bacterial products were incubated with the cells for 6–24 h at 37°C in 5% CO2. At the same time as LPS addition, cationic peptides were added at a range of concentrations. The supernatants were removed and tested for cytokine or chemokine production by ELISA (R&D Systems).
Chemokine production in whole blood
Blood from volunteer human donors was collected (according to
procedures accepted by UBC Clinical Research Ethics Board, certificate
C00-0537) by venipuncture into tubes (BD Labware, Franklin Lakes, NJ)
containing 14.3 USP units heparin/ml blood. The blood was mixed with
increasing concentrations of LL-37 in polypropylene tubes at 37°C for
6 h. The samples were centrifuged for 5 min at 2000 x
g, and the plasma was collected and then stored at -20°C
until analyzed for monocyte chemoattractant protein (MCP) 1, TNF-,
and IL-8 by ELISA (R&D Systems).
RNA isolation
RAW 264.7 cells were plated in 150-mm tissue culture dishes at
5.6 x 106 cells/dish, cultured overnight,
and then incubated with 50 µg/ml LL-37 or medium alone for 4 h.
After stimulation, the cells were washed once with diethyl
pyrocarbonate-treated PBS, and detached from the dish using a cell
scraper. Total RNA was isolated using TRIzol (Life Technologies) as
described previously (1, 36). The quality of the RNA was
assessed by gel electrophoresis on a 1% agarose gel. RNA samples were
treated with DNase according to the manufacturer’s instructions (DNA
free; Ambion, Austin, TX) to remove any contaminating genomic DNA. Lack
of genomic DNA contamination was confirmed by using the isolated RNA as
a template for PCR amplification with -actin-specific primers
(5'-GTCCCTGTATGCCTCTGGTC-3' and 5'-GATGTCACGCACGATTTCC-3'). Agarose
gel electrophoresis and ethidium bromide staining confirmed the absence
of an amplicon after 35 cycles.
Mouse cDNA expression arrays
Atlas cDNA Expression Arrays (Clontech Laboratories, Palo Alto,
CA), which consist of 588 selected mouse cDNAs spotted in duplicate on
positively charged membranes, were used for our gene array studies as
described previously (1). Briefly,
32P-radiolabeled cDNA probes were prepared from 5
µg total RNA that was incubated overnight at 71°C. The filters were
washed extensively and then exposed to a PhosphorImager screen
(Molecular Dynamics, Sunnyvale, CA) for 3 days at 4°C. The image was
captured using a Molecular Dynamics PSI phosphoimager. The
hybridization signals were analyzed using Atlas Image 1.0 Image
Analysis software (Clontech Laboratories) and Excel (Microsoft,
Redmond, WA). The intensities for each spot were corrected for
background levels and normalized for differences in probe labeling
using the average values for five genes observed to vary little
among our stimulation conditions: -actin, ubiquitin, ribosomal
protein S29, GAPDH, and Ca2+-binding protein.
When the normalized hybridization intensity for a given cDNA was <20,
it was assigned a value of 20 to calculate the ratios and relative
expression (1, 36).
Semiquantitative RT-PCR
RNA was prepared as described above. The 1-µg RNA samples were
incubated with 1 µl oligo(dT) (500 µg/ml) and 1 µl mixed dNTP
stock at 1 mM in a 12-µl volume with diethyl
pyrocarbonate-treated water at 65°C for 5 min in a
thermocycler. Briefly, 4 µl 5x first-strand buffer, 2 µl 0.1 M
DTT, and 1 µl RNaseOUT recombinant ribonuclease inhibitor (40 U/µl)
were added and incubated at 42°C for 2 min, followed by the addition
of 1 µl (200 U) of Superscript II (Invitrogen, Burlington, Ontario,
Canada). Negative controls for each RNA source were generated using
parallel reactions in the absence of Superscript II. cDNAs were
amplified in the presence of 5' and 3' primers (1.0 µM), 0.2 mM dNTP
mixture, 1.5 mM MgCl, 1 U Taq DNA polymerase (New England
Biolabs, Mississauga, Ontario, Canada), and 1x PCR buffer. Each PCR
was performed with a thermal cycler by using 30–40 cycles consisting
of 30 s of denaturation at 94°C, 30 s of annealing at
either 52 or 55°C and 40 s of extension at 72°C. The number of
cycles of PCR was optimized to lie in the linear phase of the reaction
for each primer and set of RNA samples. A housekeeping gene, -actin,
was amplified in each experiment to evaluate extraction procedure and
to estimate the amount of RNA. The reaction product was visualized by
electrophoresis and analyzed by densitometry, with relative starting
RNA concentrations calculated with reference to -actin
amplification. The primers used are shown in Table I
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To analyze cell surface expression of IL-8RB, CXCR-4, and CCR2, RAW 264.7 macrophage cells were stained with 10 µg/ml of the appropriate primary Ab (Santa Cruz Biotechnology, Santa Cruz, CA) followed by FITC-conjugated goat anti-rabbit IgG (IL-8RB and CXCR-4; Jackson ImmunoResearch Laboratories, West Grove, PA) or FITC-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology). The cells (10,000 live events were counted) were passed through a FACscan (BD Biosciences, Mountain View, CA) and forward and side scatter were used to gate on live cells.
Measurement of LL-37-induced chemokine production in the respiratory tract of mice
Animal studies were approved by the UBC Animal Care Committee
(UBC ACC no. A01-0008). BALB/c mice were purchased from Charles River
Breeding Laboratories and housed in standard animal facilities. Age-,
sex-, and weight-matched adult mice were anesthetized with an i.p.
injection of Avertin (4.4 mM 2,2,2-tribromoethanol, 2.5%
2-methyl-2-butanol, in distilled water) using 200 µl/10 g body
weight. The instillation was performed using a nonsurgical,
intratracheal instillation method adapted from Ho and Furst
(37). Briefly, the anesthetized mouse was placed with its
upper teeth hooked over a wire at the top of a support frame with its
jaw held open and a spring pushing the thorax forward to position the
pharynx, larynx, and trachea in a vertical straight line. The airway
was illuminated externally and an intubation catheter was inserted into
the clearly illuminated tracheal lumen. A 50-µg bolus of LL-37
suspended in 20 µl of sterile water, or sterile water alone, was
placed in a well at the proximal end of the catheter and gently
instilled into the trachea with 200 µl of air. The animal was
maintained in an upright position for 2 min after instillation to allow
the fluid to drain into the respiratory tree. After 4 h, the mice
were euthanized by i.p. injection of 300 mg/kg pentobarbital. The
trachea was exposed and an i.v. catheter was passed into the proximal
trachea and tied in place with suture thread. Lavage was performed by
introducing 0.75 ml sterile PBS into the lungs via the tracheal cannula
and then after a few seconds, withdrawing the fluid. This was repeated
three times with the same sample of PBS. The lavage fluid was placed in
a tube on ice and the total recovery volume per mouse was 
0.5 ml.
The bronchoalveolar lavage (BAL) fluid was centrifuged at 1200 rpm for
10 min and the clear supernatant removed and tested for TNF- and
MCP-3 by ELISA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cationic antimicrobial peptides have been shown to block many LPS-induced responses and are being considered as candidates for the treatment of sepsis. We have previously shown that CEMA (a synthetic peptide derived from the sequence of cecropin and melittin) blocks LPS-induced production of inflammatory cytokines and selectively inhibits the ability of LPS to alter gene expression in the RAW 264.7 murine macrophage cell line (8, 36). Although CEMA reduced LPS-induced up-regulation of >40 genes, including many genes encoding inflammatory mediators, the regulation of 16 other genes by LPS was unaffected by CEMA. Thus, we proposed that cationic antimicrobial peptides selectively down-regulate the proinflammatory responses of macrophages to LPS while leaving certain other responses intact. It appears possible that naturally occurring cationic antimicrobial peptides normally play such a role in either limiting or terminating inflammatory responses. Therefore, we wished to analyze the effects of a human cationic antimicrobial peptide, LL-37, on macrophage responses to LPS and other bacterial products.
The extensively studied RAW 264.7 macrophage cell line was used to
determine whether the LL-37 could inhibit the production of TNF- by
macrophages stimulated with bacterial products. The RAW 264.7 cells
were incubated with three types of LPS, with LTA, or with AraLAM and
then the release of TNF- into the culture supernatants was
quantitated by ELISA. We used these bacterial products to represent
products release by both Gram-negative and Gram-positive bacteria.
LL-37 was able to significantly inhibit TNF- production stimulated
by S. typhimurium, Burkholderia cepacia, and E.
coli O111:B4 LPS, with the former being affected to a somewhat
lesser extent (Fig. 1A). At
concentrations as low as 1 µg/ml LL-37 (0.25 nM), substantial
inhibition of TNF- production was observed in the latter two cases.
These results were confirmed in primary cells, in that LL-37 and the
positive control peptide CEMA significantly inhibited TNF-
production (>90%) by bone marrow-derived macrophages from BALB/c mice
that had been stimulated with 100 ng/ml E. coli 0111:B4 LPS
(Fig. 1B). These experiments were performed in the presence
of serum, which contains LPS-binding protein (LBP), a protein that can
mediate the rapid binding of LPS to CD14. Thus, we examined the
kinetics of antagonism of LPS induction of TNF- production (Fig. 1C). Delayed addition of LL-37 to the supernatants of
macrophages 1 h after stimulation with 100 ng/ml E.
coli LPS still resulted in substantial reduction of TNF-
production (70%).
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in vitro, LL-37 partially protected mice against lethal
endotoxic shock induced by a high concentration of endotoxin (LPS).
CD-1 mice were sensitized to endotoxin with a prior injection of
galactosamine. Mice that were injected with 3 µg of E.
coli 0111:B4 LPS were all killed within 4–6 h. When 200 µg of
LL-37 was injected 15 min after the LPS, 50% of the mice survived
(Table II).
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D).
Since LL-37 was able to inhibit LPS-induced TNF- production, we
asked whether it could inhibit the ability of other bacterial products
to induce TNF- release. We therefore examined whether LL-37 would
block the ability of Mycobacterium noncapped AraLAM and
S. aureus LTA to activate RAW 264.7 cells. LL-37 and CEMA
did indeed reduce induction of TNF- in RAW 264.7 cells by AraLAM
(Fig. 2). At a concentration of 1
µg/ml, LL-37 was able to substantially inhibit (>75%) the induction
of TNF- production by 1 µg/ml S. aureus LTA. At 20
µg/ml LL-37, there was >60% inhibition of AraLAM-induced TNF-.
Polymyxin B was included as a control to demonstrate that contaminating
endotoxin was not a significant factor in the inhibition of
AraLAM-induced TNF- by LL-37. These studies demonstrated that the
human peptide LL-37 can neutralize the effect of bacterial products on
the immune system and may aid the immune response to bacterial
infection by modulating the immune response to bacterial products
released upon infection.
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We hypothesized that LL-37 could affect other macrophage functions
as we had previously observed with CEMA (1). We therefore
performed gene array studies to determine the transcriptional responses
of macrophages to LL-37. RNA was extracted from RAW 264.7 cells that
were cultured for 4 h with medium alone or 50 µg/ml LL-37 alone
and converted into 32P-labeled cDNA. The
hybridization of the cDNA probes to each immobilized DNA was visualized
by autoradiography and quantified using a
PhosphorImager. Representative
autoradiographic images of the gene arrays are shown in Fig. 3. Table III shows that LL-37 treatment
of RAW 264.7 cells up-regulated the expression of at least 30 different
genes. The genes up-regulated by LL-37 were mainly from two categories:
one that includes receptors (growth, chemokine, IL, IFN, hormone,
neurotransmitter), cell surface Ags, and cell adhesion and another one
that includes cell-cell communication (growth factors, cytokines,
chemokines, IL, IFN, hormones), cytoskeleton, motility, and protein
turnover. The specific genes up-regulated included those
encoding chemokine MCP-3, the anti-inflammatory cytokine IL-10,
M-CSF, and receptors such as IL-1R2 (a putative antagonist of
productive IL-1 binding to IL-1R1), platelet-derived growth
factor receptor B, NOTCH4, LIF receptor, LFA-1, TGF- receptor 1,
G-CSF receptor, and IFN-
receptor. Our gene array data suggested
that LL-37 up-regulates the expression of the chemokine receptors
IL-8RB, CXCR-4, and CCR2 by 10-, 4-, and 1.4-fold above unstimulated
cells, respectively. To confirm our gene array data, we examined, using
flow cytometry, the surface expression of these receptors on RAW cells
stimulated with peptide for 4 h. When 50 µg/ml LL-37 was
incubated with RAW cells for 4 h, IL-8RB was up-regulated an
average of 2.4-fold above unstimulated cells, CXCR-4 was up-regulated
an average of 1.6-fold above unstimulated cells, and CCR2 was
up-regulated 1.8-fold above unstimulated cells (data not shown). As a
control we demonstrated that CEMA caused similar up-regulation. LL-37
also up-regulated genes encoding several metalloproteinases and
inhibitors thereof, including the bone morphogenetic proteins bone
morphogenetic protein (BMP) 1, BMP-2, BMP-8a, tissue inhibitor
of matrix metalloproteinase 2, and tissue inhibitor of matrix
metalloproteinase 3. As well, LL-37 up-regulated specific transcription
factors, including JunD, and the YY and LIM-1 transcription factors,
and kinases such as Etk1 and Csk, demonstrating its widespread effects.
We also discovered from our gene array studies that LL-37
down-regulated at least 20 genes in RAW macrophage cells (Table IV). The genes down-regulated by LL-37
included DNA repair proteins and several inflammatory mediators such as
macrophage-inflammatory protein (MIP) 1, oncostatin M, and
IL-12.
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The changes in gene expression in RAW 264.7 cells in response to
LL-37 compared with medium-only controls were also confirmed for a
number of genes by semiquantitative RT-PCR (Fig. 4). These included genes that were up-
and down-regulated or unchanged. The chemokine receptors CXCR-4 and
IL-8RB were up-regulated to similar extents by gene arrays and RT-PCR.
CXCR-4 was up-regulated by LL-37 relative to medium-only controls,
4 ± 1.7-fold by gene arrays, and 4.1 ± 0.9-fold by RT-PCR,
and IL-8RB was up-regulated 9.5 ± 7.6-fold (average ± SE)
by gene arrays and 7.1 ± 1.4-fold by RT-PCR. The relative
expression of CD14 was 0.9 ± 0.1-fold and 0.8 ± 0.3-fold by
gene arrays and RT-PCR, respectively. MCP-1, which was not on the gene
arrays but whose expression was followed up at the protein level (Fig. 5), was found to be up-regulated 3.5
± 1.4-fold by RT-PCR. However, there was a disparity in the recorded
levels of expression of IL-10, MCP-3, cyclin D1, and MIP-1 (Fig. 4)
after LL-37 treatment relative to medium control in gene array and
RT-PCR experiments indicating that the array methodology provides
qualitative rather than quantitative data. Although the changes in RNA
levels for a number of genes in RAW 264.7 cells treated with LL-37 were
confirmed, it should be noted that this could be the result of either
an increase/decrease in transcription or change in RNA stability.
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Our gene array studies indicated that LL-37 increased the
expression of chemokine genes in RAW 264.7 cells. This suggests that
LL-37 may induce macrophages to produce chemokines, which could in turn
recruit additional immune cells to the sites of infection. We went on
to confirm the up-regulation of chemokines in several different
systems. First, we examined the effect of LL-37 on RAW 264.7 cell
production of MCP-1 (MCP-1 was not represented on our arrays but has
similar activities to MCP-3 for which no ELISA is available). The
murine MCP-1, a homologue of the human MCP-1, is a member of the
(C-C) chemokine family. MCP-1 has been demonstrated to recruit
monocytes (38), NK cells (39), and some T
lymphocytes (40). When RAW macrophages were stimulated
with increasing concentrations of LL-37, they produced significant
levels of MCP-1 in their supernatant, as judged by ELISA (Fig. 4).
RAW264.7 cells stimulated with peptide concentrations ranging from 20
to 50 µg/ml for 24 h produced significant levels of MCP-1
(200–400 pg/ml above background). When the cells were stimulated with
100 µg/ml LL-37, very high levels of MCP-1 (>1000 pg/ml above
background) were produced. The ability of LL-37 to induce human MCP-1
in blood was also tested. Human blood from three separate donors was
incubated with LL-37 for 4 h, the samples were centrifuged, and
the serum was removed and tested for human MCP-1 by ELISA. Although
there was significant production of human MCP-1 in response to LL-37 by
all three donors, there was substantial variation of donor response to
the peptide, as indicated by the large SE in Fig. 5.
We also examined the effect of LL-37 on chemokine induction in a
completely different cell system, A549 human epithelial cells.
Interestingly, although these cells produce MCP-1 in response to LPS
(Fig. 1D) and this response could be antagonized by LL-37,
there was no production of MCP-1 in direct response to LL-37. LL-37 did
however induce production of IL-8, a neutrophil-specific chemokine
(Fig. 6A). Thus, LL-37 can
induce a different spectrum of responses from different cell types.
Significant but low levels of IL-8 were produced at LL-37
concentrations of 20 µg/ml. At 100 µg/ml LL-37, there were high
levels of IL-8 produced (>1 ng/ml). This is in contrast to another
cationic helical peptide that was tested and found not to cause
induction of IL-8 (data not shown), suggesting that the effect of LL-37
is specific. LL-37 also induced significant levels of IL-8 in whole
human blood (Fig. 6A). Since LPS is a known potent stimulus
of IL-8 production and LL-37 neutralized the responses of LPS (Fig. 1D), we studied the direct effect of LL-37 on LPS-induced
IL-8 production in A549 cells. We confirmed that at low concentrations
of LL-37 (1–20 µg/ml), the peptide inhibited the LPS-induced IL-8
production, but that at high concentrations of LL-37 (50–100 µg/ml),
there was stimulation of IL-8 production independent of LPS. This
indicates that LL-37 may have differential roles in the immune system
depending on the concentration found at the site of infection.
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in the
BAL fluid of mice given LL-37 by intratracheal instillation
To examine the effects of LL-37 in an in vivo situation, BALB/c
mice were given LL-37 or endotoxin-free water by intratracheal
instillation and the levels of MCP-1 and TNF- were examined in the
BAL fluid after 3–4 h. We found that the mice treated with an
intratracheal bolus of LL-37 produced significantly increased levels of
MCP-1 over mice given water or anesthetic alone (Fig. 7). This was not a general
proinflammatory response to LL-37 since no significant difference in
BAL TNF- was observed in peptide-treated mice when compared with
mice given water or anesthetic alone. Furthermore, no significant
induction of TNF- production was observed in RAW 264.7 cells or bone
marrow-derived macrophages treated with LL-37 (100 µg/ml LL-37
resulted in 0.033 ± 0.001 ng/ml TNF-, medium alone resulted in
0.038 ± 0.008 ng/ml TNF-). No decrease in the viability of RAW
264.7 cells was observed in the presence of serum with up to 125
µg/ml LL-37 as measured by the MTT assay (data not shown). Thus,
LL-37 selectively induces the production of chemokines without inducing
the production of inflammatory mediators such as TNF-. This
illustrates the dual role of LL-37 as a factor that can block bacterial
product-induced inflammation while helping to recruit phagocytes that
can augment clearance of bacterial infections.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(8). The antiendotoxin
activity of CEMA could be partially attributed to its ability to block
the interaction of LPS with LBP (41) and its ability to
selectively suppress LPS-induced macrophage gene expression
(36). We found here that the human peptide LL-37, when
added to macrophages stimulated with bacterial products, was able to
reduce the production of the proinflammatory cytokine TNF-. By
binding bacterial products and reducing their ability to stimulate
macrophages, LL-37 could reduce the development of sepsis in moderate
infections. We have previously shown that other cationic antimicrobial
peptides bind to LPS and inhibit its binding to LBP (41).
Thus, cationic peptides provide not only a natural response to
bacterial infection, as seen with LL-37, but also a possible
therapeutic intervention. LL-37 could help not only limit bacterial
infection but also prevent an overwhelming immune response that can
lead to sepsis and even death. Their ability to block macrophage
activation suggests that they have a role in terminating immune
responses. The results presented here indicate that before acting as feedback inhibitors of the immune response, cationic peptides may also have a role in promoting the ability of leukocytes to combat bacterial infections. This could be done in part by up-regulating the chemokines IL-8 and MCP-1 and possibly also by up-regulating the surface expression of chemokine receptors such as IL-8RB, CXCR-4, and CCR2. The function of LL-37 could depend on the concentration of the peptide. For instance, at low peptide concentrations (i.e., 2 µg/ml LL-37; Ref. 42) found in normal human epithelia, LL-37 could function as an immune watchdog and, at high concentrations found when LL-37 is induced by bacteria or bacterial products, LL-37 could function to promote migration of immune cells to help control the infection. This feature appears to involve the regulation of a large number of genes, some of which are known to have anti-inflammatory and some proinflammatory roles. Importantly, we have discovered from our studies the novel finding that LL-37 induces chemokine production and surface expression of chemokine receptors and appears to do this when instilled into the lungs of mice. Thus, LL-37 may act indirectly to promote the migration of immune cells, and this may be partially responsible for its ability to protect against infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert E. W. Hancock, Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3. E-mail address: bob{at}cmdr.ubc.ca
3 Abbreviations used in this paper: LTA, lipoteichoic acid; AraLAM, lipoarabinomannan; MCP, monocyte chemoattractant protein; BAL, bronchoalveolar lavage; LBP, LPS-binding protein; BMP, bone morphogenetic protein; MIP, macrophage-inflammatory protein.
Received for publication July 24, 2002. Accepted for publication July 24, 2002.
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References |
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  G. Bergsson, E. P. Reeves, P. McNally, S. H. Chotirmall, C. M. Greene, P. Greally, P. Murphy, S. J. O'Neill, and N. G. McElvaney LL-37 Complexation with Glycosaminoglycans in Cystic Fibrosis Lungs Inhibits Antimicrobial Activity, Which Can Be Restored by Hypertonic Saline J. Immunol., July 1, 2009; 183(1): 543 - 551. [Abstract] [Full Text] [PDF]
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 A. Jones, M. Georg, L. Maudsdotter, and A.-B. Jonsson Endotoxin, Capsule, and Bacterial Attachment Contribute to Neisseria meningitidis Resistance to the Human Antimicrobial Peptide LL-37 J. Bacteriol., June 15, 2009; 191(12): 3861 - 3868. [Abstract] [Full Text] [PDF]
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 Y. Okuyama-Nishida, N. Akiyama, G. Sugimori, K. Nomura, K. Ogawa, K. J. Homma, K. Sekimizu, M. Tsujimoto, and S. Natori Prevention of Death in Bacterium-Infected Mice by a Synthetic Antimicrobial Peptide, L5, through Activation of Host Immunity Antimicrob. Agents Chemother., June 1, 2009; 53(6): 2510 - 2516. [Abstract] [Full Text] [PDF]
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C.-S. Yang, D.-M. Shin, K.-H. Kim, Z.-W. Lee, C.-H. Lee, S. G. Park, Y. S. Bae, and E.-K. Jo NADPH Oxidase 2 Interaction with TLR2 Is Required for Efficient Innate Immune Responses to Mycobacteria via Cathelicidin Expression J. Immunol., March 15, 2009; 182(6): 3696 - 3705. [Abstract] [Full Text] [PDF]
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S. Hansdottir, M. M. Monick, S. L. Hinde, N. Lovan, D. C. Look, and G. W. Hunninghake Respiratory Epithelial Cells Convert Inactive Vitamin D to Its Active Form: Potential Effects on Host Defense J. Immunol., November 15, 2008; 181(10): 7090 - 7099. [Abstract] [Full Text] [PDF]
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 J. L. Boehmer, D. D. Bannerman, K. Shefcheck, and J. L. Ward Proteomic Analysis of Differentially Expressed Proteins in Bovine Milk During Experimentally Induced Escherichia coli Mastitis J Dairy Sci, November 1, 2008; 91(11): 4206 - 4218. [Abstract] [Full Text] [PDF]
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 G. Diamond, N. Beckloff, and L.K. Ryan Host Defense Peptides in the Oral Cavity and the Lung: Similarities and Differences Journal of Dental Research, October 1, 2008; 87(10): 915 - 927. [Abstract] [Full Text] [PDF]
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R. Bucki, F. J. Byfield, A. Kulakowska, M. E. McCormick, W. Drozdowski, Z. Namiot, T. Hartung, and P. A. Janmey Extracellular Gelsolin Binds Lipoteichoic Acid and Modulates Cellular Response to Proinflammatory Bacterial Wall Components J. Immunol., October 1, 2008; 181(7): 4936 - 4944. [Abstract] [Full Text] [PDF]
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H. Y. Lee, S. D. Kim, J. W. Shim, S. Y. Lee, H. Lee, K.-H. Cho, J. Yun, and Y.-S. Bae Serum Amyloid A Induces CCL2 Production via Formyl Peptide Receptor-Like 1-Mediated Signaling in Human Monocytes J. Immunol., September 15, 2008; 181(6): 4332 - 4339. [Abstract] [Full Text] [PDF]
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 Z. Zhang, G. Cherryholmes, and J. E. Shively Neutrophil secondary necrosis is induced by LL-37 derived from cathelicidin J. Leukoc. Biol., September 1, 2008; 84(3): 780 - 788. [Abstract] [Full Text] [PDF]
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 M. L. Mangoni, R. F. Epand, Y. Rosenfeld, A. Peleg, D. Barra, R. M. Epand, and Y. Shai Lipopolysaccharide, a Key Molecule Involved in the Synergism between Temporins in Inhibiting Bacterial Growth and in Endotoxin Neutralization J. Biol. Chem., August 22, 2008; 283(34): 22907 - 22917. [Abstract] [Full Text] [PDF]
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 B. Rivas-Santiago, R. Hernandez-Pando, C. Carranza, E. Juarez, J. L. Contreras, D. Aguilar-Leon, M. Torres, and E. Sada Expression of Cathelicidin LL-37 during Mycobacterium tuberculosis Infection in Human Alveolar Macrophages, Monocytes, Neutrophils, and Epithelial Cells Infect. Immun., March 1, 2008; 76(3): 935 - 941. [Abstract] [Full Text] [PDF]
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J. Yu, N. Mookherjee, K. Wee, D. M. E. Bowdish, J. Pistolic, Y. Li, L. Rehaume, and R. E. W. Hancock Host Defense Peptide LL-37, in Synergy with Inflammatory Mediator IL-1beta, Augments Immune Responses by Multiple Pathways J. Immunol., December 1, 2007; 179(11): 7684 - 7691. [Abstract] [Full Text] [PDF]
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A. R. Martineau, K. A. Wilkinson, S. M. Newton, R. A. Floto, A. W. Norman, K. Skolimowska, R. N. Davidson, O. E. Sorensen, B. Kampmann, C. J. Griffiths, et al. IFN-{gamma}- and TNF-Independent Vitamin D-Inducible Human Suppression of Mycobacteria: The Role of Cathelicidin LL-37 J. Immunol., June 1, 2007; 178(11): 7190 - 7198. [Abstract] [Full Text] [PDF]
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N. Mookherjee, H. L. Wilson, S. Doria, Y. Popowych, R. Falsafi, J. Yu, Y. Li, S. Veatch, F. M. Roche, K. L. Brown, et al. Bovine and human cathelicidin cationic host defense peptides similarly suppress transcriptional responses to bacterial lipopolysaccharide J. Leukoc. Biol., December 1, 2006; 80(6): 1563 - 1574. [Abstract] [Full Text] [PDF]
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 M. Ding, S. Cui, C. Li, S. Jothy, V. Haase, B. Steer, P. Marsden, J. Pippin, S. Shankland, M. Rastaldi, et al. Faulty Podocyte Hypoxia Sensing--A Novel Pathway for Rapidly Progressive Glomerulonephritis: Loss of the Tumor Suppressor Vhlh Leads to Upregulation of Cxcr4 and Rapidly Progressive Glomerulonephritis. Nat Med 12: 1081-1087, 2006 J. Am. Soc. Nephrol., December 1, 2006; 17(12): 3267 - 3272. [Full Text] [PDF]
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 K. Kandler, R. Shaykhiev, P. Kleemann, F. Klescz, M. Lohoff, C. Vogelmeier, and R. Bals The anti-microbial peptide LL-37 inhibits the activation of dendritic cells by TLR ligands Int. Immunol., December 1, 2006; 18(12): 1729 - 1736. [Abstract] [Full Text] [PDF]
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P.T. Liu, S. Stenger, H. Li, L. Wenzel, B.H. Tan, S.R. Krutzik, M.T. Ochoa, J. Schauber, K. Wu, C. Meinken, et al. Vitamin D3-Triggered Antimicrobial Response--Another Pleiotropic Effect beyond Mineral and Bone Metabolism: Toll-Like Receptor Triggering of a Vitamin D-Mediated Human Antimicrobial Response. Science 311: 1770-1773, 2006 J. Am. Soc. Nephrol., November 1, 2006; 17(11): 2949 - 2953. [Full Text] [PDF]
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S. Dudal, C. Turriere, S. Bessoles, P. Fontes, F. Sanchez, J. Liautard, J.-P. Liautard, and V. Lafont Release of LL-37 by Activated Human V{gamma}9V{delta}2 T Cells: A Microbicidal Weapon against Brucella suis J. Immunol., October 15, 2006; 177(8): 5533 - 5539. [Abstract] [Full Text] [PDF]
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P. G. Barlow, Y. Li, T. S. Wilkinson, D. M. E. Bowdish, Y. E. Lau, C. Cosseau, C. Haslett, A. J. Simpson, R. E. W. Hancock, and D. J. Davidson The human cationic host defense peptide LL-37 mediates contrasting effects on apoptotic pathways in different primary cells of the innate immune system J. Leukoc. Biol., September 1, 2006; 80(3): 509 - 520. [Abstract] [Full Text] [PDF]
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 Y. H. Yang, W. K. K. Wu, E. K. K. Tai, H. P. S. Wong, E. K. Y. Lam, W. H. L. So, V. Y. Shin, and C. H. Cho The Cationic Host Defense Peptide rCRAMP Promotes Gastric Ulcer Healing in Rats J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 547 - 554. [Abstract] [Full Text] [PDF]
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 H. Jenssen, P. Hamill, and R. E. W. Hancock Peptide Antimicrobial Agents Clin. Microbiol. Rev., July 1, 2006; 19(3): 491 - 511. [Abstract] [Full Text] [PDF]
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J. E. Thwaite, S. Hibbs, R. W. Titball, and T. P. Atkins Proteolytic Degradation of Human Antimicrobial Peptide LL-37 by Bacillus anthracis May Contribute to Virulence. Antimicrob. Agents Chemother., July 1, 2006; 50(7): 2316 - 2322. [Abstract] [Full Text] [PDF]
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 K. Edfeldt, B. Agerberth, M. E. Rottenberg, G. H. Gudmundsson, X.-B. Wang, K. Mandal, Q. Xu, and Z.-q. Yan Involvement of the Antimicrobial Peptide LL-37 in Human Atherosclerosis Arterioscler. Thromb. Vasc. Biol., July 1, 2006; 26(7): 1551 - 1557. [Abstract] [Full Text] [PDF]
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 A. Macagno, M. Molteni, A. Rinaldi, F. Bertoni, A. Lanzavecchia, C. Rossetti, and F. Sallusto A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression J. Exp. Med., June 12, 2006; 203(6): 1481 - 1492. [Abstract] [Full Text] [PDF]
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 L. C. Huang, T. D. Petkova, R. Y. Reins, R. J. Proske, and A. M. McDermott Multifunctional Roles of Human Cathelicidin (LL-37) at the Ocular Surface. Invest. Ophthalmol. Vis. Sci., June 1, 2006; 47(6): 2369 - 2380. [Abstract] [Full Text] [PDF]
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 Y. E. Lau, D. M. E. Bowdish, C. Cosseau, R. E. W. Hancock, and D. J. Davidson Apoptosis of Airway Epithelial Cells: Human Serum Sensitive Induction by the Cathelicidin LL-37 Am. J. Respir. Cell Mol. Biol., April 1, 2006; 34(4): 399 - 409. [Abstract] [Full Text] [PDF]
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N. Mookherjee, K. L. Brown, D. M. E. Bowdish, S. Doria, R. Falsafi, K. Hokamp, F. M. Roche, R. Mu, G. H. Doho, J. Pistolic, et al. Modulation of the TLR-Mediated Inflammatory Response by the Endogenous Human Host Defense Peptide LL-37 J. Immunol., February 15, 2006; 176(4): 2455 - 2464. [Abstract] [Full Text] [PDF]
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 W. Xiao, Y.-P. Hsu, A. Ishizaka, T. Kirikae, and R. B. Moss Sputum Cathelicidin, Urokinase Plasminogen Activation System Components, and Cytokines Discriminate Cystic Fibrosis, COPD, and Asthma Inflammation Chest, October 1, 2005; 128(4): 2316 - 2326. [Abstract] [Full Text] [PDF]
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B. Deslouches, K. Islam, J. K. Craigo, S. M. Paranjape, R. C. Montelaro, and T. A. Mietzner Activity of the De Novo Engineered Antimicrobial Peptide WLBU2 against Pseudomonas aeruginosa in Human Serum and Whole Blood: Implications for Systemic Applications Antimicrob. Agents Chemother., August 1, 2005; 49(8): 3208 - 3216. [Abstract] [Full Text] [PDF]
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 D. M.E. Bowdish and R. E.W. Hancock Anti-endotoxin properties of cationic host defence peptides and proteins Innate Immunity, August 1, 2005; 11(4): 230 - 236. [Abstract] [PDF]
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C. D. Ciornei, T. Sigurdardottir, A. Schmidtchen, and M. Bodelsson Antimicrobial and Chemoattractant Activity, Lipopolysaccharide Neutralization, Cytotoxicity, and Inhibition by Serum of Analogs of Human Cathelicidin LL-37 Antimicrob. Agents Chemother., July 1, 2005; 49(7): 2845 - 2850. [Abstract] [Full Text] [PDF]
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 A. F. Gombart, N. Borregaard, and H. P. Koeffler Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3 FASEB J, July 1, 2005; 19(9): 1067 - 1077. [Abstract] [Full Text] [PDF]
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K. Kurosaka, Q. Chen, F. Yarovinsky, J. J. Oppenheim, and D. Yang Mouse Cathelin-Related Antimicrobial Peptide Chemoattracts Leukocytes Using Formyl Peptide Receptor-Like 1/Mouse Formyl Peptide Receptor-Like 2 as the Receptor and Acts as an Immune Adjuvant J. Immunol., May 15, 2005; 174(10): 6257 - 6265. [Abstract] [Full Text] [PDF]
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D. M. E. Bowdish, D. J. Davidson, M. G. Scott, and R. E. W. Hancock Immunomodulatory Activities of Small Host Defense Peptides Antimicrob. Agents Chemother., May 1, 2005; 49(5): 1727 - 1732. [Abstract] [Full Text] [PDF]
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J. Oliaro, S. Dudal, J. Liautard, J.-B. Andrault, J.-P. Liautard, and V. Lafont V{gamma}9V{delta}2 T cells use a combination of mechanisms to limit the spread of the pathogenic bacteria Brucella J. Leukoc. Biol., May 1, 2005; 77(5): 652 - 660. [Abstract] [Full Text] [PDF]
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M. Iimura, R. L. Gallo, K. Hase, Y. Miyamoto, L. Eckmann, and M. F. Kagnoff Cathelicidin Mediates Innate Intestinal Defense against Colonization with Epithelial Adherent Bacterial Pathogens J. Immunol., April 15, 2005; 174(8): 4901 - 4907. [Abstract] [Full Text] [PDF]
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D. M. E. Bowdish, D. J. Davidson, Y. E. Lau, K. Lee, M. G. Scott, and R. E. W. Hancock Impact of LL-37 on anti-infective immunity J. Leukoc. Biol., April 1, 2005; 77(4): 451 - 459. [Abstract] [Full Text] [PDF]
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N. Borregaard, K. Theilgaard-Monch, J. B. Cowland, M. Stahle, and O. E. Sorensen Neutrophils and keratinocytes in innate immunity--cooperative actions to provide antimicrobial defense at the right time and place J. Leukoc. Biol., April 1, 2005; 77(4): 439 - 443. [Abstract] [Full Text] [PDF]
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S. van Wetering, G. S. Tjabringa, and P. S. Hiemstra Interactions between neutrophil-derived antimicrobial peptides and airway epithelial cells J. Leukoc. Biol., April 1, 2005; 77(4): 444 - 450. [Abstract] [Full Text] [PDF]
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M. H. Braff, M. A. Hawkins, A. D. Nardo, B. Lopez-Garcia, M. D. Howell, C. Wong, K. Lin, J. E. Streib, R. Dorschner, D. Y. M. Leung, et al. Structure-Function Relationships among Human Cathelicidin Peptides: Dissociation of Antimicrobial Properties from Host Immunostimulatory Activities J. Immunol., April 1, 2005; 174(7): 4271 - 4278. [Abstract] [Full Text] [PDF]
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T. D. Starner, B. Agerberth, G. H. Gudmundsson, and P. B. McCray Jr. Expression and Activity of {beta}-Defensins and LL-37 in the Developing Human Lung J. Immunol., February 1, 2005; 174(3): 1608 - 1615. [Abstract] [Full Text] [PDF]
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Y. E. Lau, A. Rozek, M. G. Scott, D. L. Goosney, D. J. Davidson, and R. E. W. Hancock Interaction and Cellular Localization of the Human Host Defense Peptide LL-37 with Lung Epithelial Cells Infect. Immun., January 1, 2005; 73(1): 583 - 591. [Abstract] [Full Text] [PDF]
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D. Yang, Q. Chen, H. F. Rosenberg, S. M. Rybak, D. L. Newton, Z. Y. Wang, Q. Fu, V. T. Tchernev, M. Wang, B. Schweitzer, et al. Human Ribonuclease A Superfamily Members, Eosinophil-Derived Neurotoxin and Pancreatic Ribonuclease, Induce Dendritic Cell Maturation and Activation J. Immunol., November 15, 2004; 173(10): 6134 - 6142. [Abstract] [Full Text] [PDF]
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O. Levy Antimicrobial proteins and peptides: anti-infective molecules of mammalian leukocytes J. Leukoc. Biol., November 1, 2004; 76(5): 909 - 925. [Abstract] [Full Text] [PDF]
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T.-T. Wang, F. P. Nestel, V. Bourdeau, Y. Nagai, Q. Wang, J. Liao, L. Tavera-Mendoza, R. Lin, J. H. Hanrahan, S. Mader, et al. Cutting Edge: 1,25-Dihydroxyvitamin D3 Is a Direct Inducer of Antimicrobial Peptide Gene Expression J. Immunol., September 1, 2004; 173(5): 2909 - 2912. [Abstract] [Full Text] [PDF]
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D. M. E. Bowdish, D. J. Davidson, D. P. Speert, and R. E. W. Hancock The Human Cationic Peptide LL-37 Induces Activation of the Extracellular Signal-Regulated Kinase and p38 Kinase Pathways in Primary Human Monocytes J. Immunol., March 15, 2004; 172(6): 3758 - 3765. [Abstract] [Full Text] [PDF]
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 D. M. Shasby and P. McCray Sepsis and Innate Immunity Am. J. Respir. Crit. Care Med., January 15, 2004; 169(2): 144 - 145. [Full Text] [PDF]
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D. J. Davidson, A. J. Currie, G. S. D. Reid, D. M. E. Bowdish, K. L. MacDonald, R. C. Ma, R. E. W. Hancock, and D. P. Speert The Cationic Antimicrobial Peptide LL-37 Modulates Dendritic Cell Differentiation and Dendritic Cell-Induced T Cell Polarization J. Immunol., January 15, 2004; 172(2): 1146 - 1156. [Abstract] [Full Text] [PDF]
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M. Zanetti Cathelicidins, multifunctional peptides of the innate immunity J. Leukoc. Biol., January 1, 2004; 75(1): 39 - 48. [Abstract] [Full Text] [PDF]
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G. S. Tjabringa, J. Aarbiou, D. K. Ninaber, J. W. Drijfhout, O. E. Sorensen, N. Borregaard, K. F. Rabe, and P. S. Hiemstra The Antimicrobial Peptide LL-37 Activates Innate Immunity at the Airway Epithelial Surface by Transactivation of the Epidermal Growth Factor Receptor J. Immunol., December 15, 2003; 171(12): 6690 - 6696. [Abstract] [Full Text] [PDF]
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A. Di Nardo, A. Vitiello, and R. L. Gallo Cutting Edge: Mast Cell Antimicrobial Activity Is Mediated by Expression of Cathelicidin Antimicrobial Peptide J. Immunol., March 1, 2003; 170(5): 2274 - 2278. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) 100 ng/ml S.
typhimurium LPS, (
) 100 ng/ml B. cepacia LPS,
and (•) 100 ng/ml E. coli 0111:B4 LPS in the presence
of the indicated concentrations of LL-37 for 6 h. The
concentrations of TNF-
, results from
RT-PCR experiments. The data are presented as the mean ± SE of
three experiments.
