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-Helical Cationic Antimicrobial Peptide Selectively Modulates Macrophage Responses to Lipopolysaccharide and Directly Alters Macrophage Gene Expression1
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Department of Microbiology and Immunology, and
Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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-helical peptide, selectively modulates the transcriptional response
of macrophages to LPS and can alter gene expression in
macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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and other proinflammatory cytokines by macrophages. LPS is a potent
activator of macrophages and is responsible for sepsis caused by
Gram-negative bacteria. The activation of macrophages by LPS is
initiated when LPS-binding protein
(LBP)3 transfers LPS
to CD14 on the surface of macrophages. LPS-CD14 complexes then signal
via Toll-like receptors to activate NF-
B as well as the
extracellularly-regulated kinase, c-Jun N-terminal kinase, and p38
mitogen-activated protein kinases, all of which mediate the production
of inflammatory cytokines (5, 6, 7, 8).
Interfering with the ability of LPS to bind to macrophages is likely to
be an effective mechanism for preventing sepsis (9). We
have shown that a variety of cationic antimicrobial peptides bind LPS,
block the interaction of LPS with LBP, and suppress the ability of LPS
to stimulate the production of inflammatory cytokines by macrophages
(10, 11, 12). These cationic antimicrobial peptides are a
component of the innate host defenses of both vertebrates and
invertebrates and are found in all species of life (13).
For example, defensins are the most predominant protein species (15%
of total protein) in neutrophils. Defensins and other cationic peptides
are also found at mucosal and epithelial surfaces and in the gut,
lungs, kidneys, and skin. Cationic antimicrobial peptides have
broad-spectrum activity against bacteria, fungi, parasites, and
viruses. It is becoming increasingly clear that they play an important
role in the immune system (14). In addition to their
direct antimicrobial activities, they play an important early role in
the response to bacterial infections, and in many cases
(14) they are induced by the presence of LPS, lipoteichoic
acid, and bacteria (14, 15, 16, 17). In addition, both naturally
occurring cationic peptides as well as synthetic analogues may be
useful as therapeutics for suppressing inflammatory responses caused by
LPS. For example, CEMA, an -helical peptide derived from a hybrid of
the silk moth cecropin and bee melittin peptides, has been shown to
bind LPS, inhibit cytokine production by LPS-stimulated macrophages and
macrophage cell lines, and protect mice from lethal endotoxemia
(10). Thus, cationic antimicrobial peptides may be a
useful tool for preventing sepsis.
To gain a more complete understanding of how LPS activates macrophages
and how cationic peptides influence this process, we have used gene
array technology to profile gene expression patterns in RAW 264.7
macrophages treated with LPS in the presence or the absence of the
cationic antimicrobial peptide CEMA. We found that CEMA selectively
inhibited LPS-induced gene expression. For example, while CEMA strongly
inhibited LPS-induced expression of a variety of genes, including those
encoding the proinflammatory molecules IL-1ß, macrophage-inflammatory
protein-1 (MIP-1), MIP-1ß, and the CD40 ligand, it had little
or no effect on the ability of LPS to induce the expression of ICAM-1,
c-rel, and several other genes. In addition to selectively
inhibiting LPS-induced gene expression, we found that CEMA itself
induced the expression of a distinct set of genes. This suggests that
natural cationic peptides produced in response to bacterial infections
may directly regulate macrophage function in addition to selectively
modulating macrophage responses to LPS and directly killing
bacteria.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Salmonella typhimurium LPS was purchased from Sigma (St. Louis, MO). CEMA and LL-37 were synthesized at the Nucleic Acid/Protein Synthesis Unit at the University of British Columbia as described previously (10).
Cytokine production by RAW 264.7 cells
The murine macrophage cell line RAW 264.7 was obtained from
American Type Culture Collection (Manassas, VA) and maintained in DMEM
supplemented with 10% FCS. RAW 264.7 cells were plated in 24-well
dishes at 2.5 x 105 cells/well in the above
medium, except that DMEM was phenol red free to prevent interference
with the Griess reagent, incubated overnight, and then stimulated with
100 ng/ml S. typhimurium LPS alone, 50 µg/ml
CEMA alone, or 100 ng/ml S. typhimurium LPS and 50 µg/ml
CEMA added simultaneously to the cells. The cells were then incubated
for 24 h before measurement of IL-1ß and NO and for 4 h
before MIP-1 measurements. The cultures were assayed for IL-1ß by
ELISAs (R&D Systems, Minneapolis, MN) that could detect <10 pg/ml
IL-1ß. MIP-1 levels in the supernatant were also measured by ELISA
(R&D Systems) that could detect <31 pg/ml. These experiments were
performed a minimum of three times. MIP-1, IL-1ß, and TNF- were
also measured by ELISA (R&D Systems) in the supernatants of the cells
used for RNA isolation (see below).
Whole blood assay
Blood from three donors was collected by venipuncture into tubes (Becton Dickinson, Franklin Lakes, NJ) containing 14.3 USP units of heparin/ml blood. Whole blood was stimulated with 100 ng/ml LPS in the presence or the absence of peptide (50 µg/ml) in polypropylene tubes at 37°C for 6 h. The samples were centrifuged for 10 min at 2000 x g to separate the plasma and were stored at -20°C until analyzed for IL-1ß levels by ELISA (R&D Systems).
NO production
The RAW cells were cultured as described above, and the amount of NO in the supernatant was estimated from the accumulation of the stable NO metabolite nitrite with Griess reagent (Molecular Probes, Eugene, OR). Briefly 150-µl samples or standards and 130 µl of water were added to wells of a 96-well plate in duplicate. The Griess reagent (20 µl) was added to each well, the plate was incubated at room temperature for 30 min, and the OD450 was read with a spectrophotometer.
RNA isolation
RAW 264.7 cells were plated in 150-mm tissue culture dishes at 5.6 x 106 cells/dish, incubated overnight, and then stimulated with or without 100 ng/ml LPS in the presence or the absence of 50 µg/ml CEMA for 4 h. After stimulation, the supernatant was removed for the measurement of cytokine production, and the cells were washed once with diethyl pyrocarbonate-treated PBS, then detached from the dish using a cell scraper. Total RNA was isolated using TRIzol (Life Technologies, Gaithersburg, MD). The RNA pellet was resuspended in RNase-free water containing RNase inhibitor (Ambion, Austin, TX). The RNA was treated with DNase I (Clontech, Palo Alto, CA) for 1 h at 37°C. After adding termination mix (0.1 M EDTA (pH 8.0) and 1 mg/ml glycogen), the samples were extracted once with phenol/chloroform/isoamyl alcohol (25/24/1) and once with chloroform. The RNA was then precipitated by adding 2.5 vol of 100% ethanol and 0.1 vol of sodium acetate, pH 5.2. The RNA was resuspended in RNase-free water with RNase inhibitor (Ambion) and was stored at -70°C. The quality of the RNA was assessed by gel electrophoresis on a 1% agarose gel. Lack of genomic DNA contamination was assessed by using the isolated RNA as a template for PCR amplification with ß-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 (no. 7741-1), which consist of 588 selected mouse cDNAs spotted in duplicate on positively charged membranes, were purchased from Clontech. Details of the arrays and the methodology used can be found on the Clontech website: www.clontech.com. Briefly, 32P-radiolabeled cDNA probes were prepared from 5 µg of total RNA using the Moloney murine leukemia virus reverse transcriptase and pooled primers specific for the 588 genes. The 32P-labeled cDNA probes were separated from unincorporated nucleotides using ChromaSpin columns, and 1 x 106 cpm/ml of denatured probe in 5 ml of hybridization solution was used for hybridization. The gene array filters were prehybridized with ExpressHyb containing 0.5 mg/ml sheared salmon sperm DNA (Ambion) before incubating overnight at 71°C with the denatured cDNA probes in a hybridization oven at 5 rpm. The filters were washed extensively at low and high stringency conditions recommended by Clontech 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 PhosphorImager. The hybridization signals were analyzed using AtlasImage 1.0 Image Analysis software (Clontech) 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 between our stimulation conditions: ß-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 (18).
Northern blots
RNA was isolated as described above. Northern blots were performed using the NorthernMax-Gly kit (Ambion). The RNA was separated on glyoxal/DMSO gels and transferred to positively charged membranes (Ambion). The RNA was cross-linked to the filters using UV light, and the filters were then baked at 80°C for 15 min. DNA templates from which probes were produced were generated by PCR using macrophage cDNA and the following pairs of primers: IL-1ß, 5'-TCCAGGATGAGGACATGAGC-3' and 5'-CTTGTGCTCTGCTTGTGAGG-3'; cyclin D1, 5'-CAGCTTAATGTGCCCTCTCC-3' and 5'-GGTAATGCCATCATGGTTCC-3'; CD14, 5'-CTGATCTCAGCCCTCTGTCC-3' and 5'-CAGGAGGATGCAAATGTTCC-3'; and GAPDH, 5'-AGAACATCATCCCTGCATCC-3' and 5'-CTGGGATGGAAATTGTGAGG-3'.
Antisense cDNA probes were prepared by incubating 50 ng of the PCR product with antisense primer and modified nucleotides that facilitate repeated stripping of blots (Strip-EZ PCR, Ambion). These single-stranded PCR products were purified using Qiagen spin columns and were biotinylated by incubating them with psoralen-biotin (Ambion) in the presence of 365 nm of UV light. After a prehybridization step, the filters were incubated with biotinylated probes (3 ng in 10 ml of UltraHyb or ZipHyb (Ambion)) at 45°C. Hybridization of the probes to the filter was visualized using the BrightStar nonisotopic detection kit (Ambion), and results were quantitated by densitometry, with GAPDH levels used for normalization.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We and others have previously shown that many cationic antimicrobial peptides potently inhibit the ability of LPS to stimulate the production of inflammatory cytokines by macrophages (10, 19, 20, 21, 22, 23, 24). However, the effects of these peptides on other macrophage functions have not been evaluated in detail. To gain a more complete understanding of how LPS activates macrophages and how cationic peptides inhibit this process, we used gene arrays to profile global patterns of gene expression in the RAW 264.7 murine macrophage cell line. Gene arrays allow high throughput analyses of diverse gene families that permit identification of previously unrecognized effects of LPS and cationic peptides on the host.
We used the RAW macrophage cell line, as it has been used extensively
as a model for macrophage responses to endotoxin. However, we confirmed
that CEMA inhibited LPS-induced cytokine (IL-6 and TNF-) production
in another macrophage cell line, J774.1, and in elicited mouse
peritoneal macrophages (10) as well as in human whole
blood. We chose to use CEMA, an -helical synthetic peptide, since we
have shown that it binds to LPS, potently inhibits cytokine production
by LPS-stimulated macrophages, and protects mice from lethal
endotoxemia (10). In addition to examining the effects of
CEMA on LPS-induced gene expression, we asked whether CEMA alone could
directly alter RAW 264.7 macrophage gene expression.
RNA was extracted from RAW 264.7 cells that were cultured for 4 h
with medium alone, 100 ng/ml S. typhimurium LPS, 100 ng/ml
LPS plus 50 µg/ml CEMA, or 50 µg/ml CEMA alone. After RT, cDNA
probes were hybridized to Clontech Atlas gene array filters. 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. 1
, and the
complete datasets representing the expression levels of all 588 genes
in the four different cell populations can be found on our web site
(http://www.cmdr.ubc.ca/arraydata1).
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and II column labeled Ratio
LPS: unstimulated). These included genes encoding inflammatory
cytokines such as IL-1ß and IL-15; inducible NO synthase (iNOS);
chemokines such as MIP-1, MIP-1ß, and MIP-2; cell surface
proteins such as Fas and CD40; and a variety of transcription factors,
including members of the pRb (retinoblastoma) family. Since many of
these genes had been previously reported to be LPS-regulated genes
(reviewed in Refs. 6, 7, 8), it confirmed the validity of our
array results. We also identified several novel LPS-regulated genes,
including a winged helix transcription factor called brain factor 1,
Brn-3.2 POU transcription factor, PD-1 (possible cell death inducer),
and HMG-14 chromosomal protein.
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shows
that when RAW 264.7 cells were cultured with LPS in the presence or the
absence of CEMA, CEMA significantly (30–86%) reduced the ability of
LPS to up-regulate the expression of 41 different genes. Interestingly,
there was a large variation in the inhibition of LPS-induced gene
expression. Notably, CEMA inhibited the LPS-induced up-regulation of
many of the inflammation-related genes on the arrays, including
IL-1ß, IL-15, MIP-1, and iNOS (Table II). In addition to inhibiting the
ability of LPS to increase the levels of cytokine mRNA, we found that
CEMA significantly reduced the ability of LPS to induce the expression
of a number of genes with other functions. In particular, LPS increased
the levels of mRNA for the pRb family retinoblastoma proteins p107 and
p130 by >30-fold, and CEMA inhibited these responses by 59% (p107)
and 77% (p130). CEMA also decreased the LPS-induced expression of
several transcription factors, including basic leucine zipper
transcription factor and Brn-3.2 POU transcription factor, by 80 and
70%, respectively. Many previous studies have focused on
peptide-mediated inhibition of the proinflammatory genes induced by
LPS. This is the first report of an antimicrobial peptide decreasing
LPS-stimulated induction of genes other than proinflammatory genes,
including genes involved in cell proliferation and apoptosis. Confirmation of selected array results
To assess the functional significance of these results, we
performed ELISAs on culture supernatants from the RAW 264.7 cells.
Consistent with the array findings, we found that the levels of the
chemokine MIP-1 secreted into the medium were greatly increased by
LPS stimulation (cytokine concentrations of 6.3–8 ng/ml compared with
<0.2 ng/ml for unstimulated cells) and that CEMA at 50 µg/ml
inhibited this response by 46%. Levels of IL-1ß (Fig. 2A) in the supernatant of RAW
macrophages incubated with LPS (100–130 pg/ml) were decreased by
53 ± 5% (inhibition ± SE) in the presence of 50 µg/ml
CEMA. In whole human blood incubated with LPS and LPS plus 50 µg/ml
CEMA for 4–6 h, there was similar inhibition of LPS-induced IL-1ß
production by CEMA. LPS alone resulted in serum levels of IL-1ß
ranging from 0.56–0.94 ng/ml, and CEMA inhibited this by 40 ±
3% (mean inhibition ± SE). This again is similar to the results
with the gene arrays. When the supernatants of the cells used for RNA
isolation were tested for the cytokine levels of TNF- and IL-6, CEMA
inhibited the LPS induction of these cytokines by 78 and 86%,
respectively, consistent with our previous studies and those with other
cell lines and primary macrophages (10, 11).
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B). The levels of NO were increased in
the presence of LPS (0–6.7 to 49.3–71.7 µM) and were inhibited an
average of 76 ± 2% by the addition of CEMA (Fig. 2B).
Similarly, the levels of iNOS on the gene arrays were up-regulated by
LPS (31-fold) and inhibited 86% by CEMA. It should be noted that
although these results demonstrated the same trends for the
transcriptional array and product assays, the measurement of iNOS and
IL-1ß was performed at 24 h to permit the development of
measurable amounts of product, whereas the gene arrays examined
transcriptional changes at 4 h. Evidence for a selective effect of the cationic peptide CEMA in suppressing endotoxin responses
CEMA varied widely in its ability to inhibit LPS-induced gene
expression; the transcription of some genes was inhibited by as much as
85% (IL-15), and that of other genes, such as Stat1 and NF-E2
transcription factor, was only partially inhibited (30–40%).
Furthermore, CEMA did not block the ability of LPS to increase the
expression of 16 other genes (Table II). These genes included several
that are strongly up-regulated by LPS such as c-rel,
mdm-2, and ICAM-1. This indicates that the peptide had a
selective effect on gene induction by LPS. This was surprising, since
we had previously shown that CEMA, like other cationic antimicrobial
peptides, binds LPS and inhibits its binding to LBP (12).
LBP catalyzes the transfer of LPS to CD14, and the binding of LPS to
CD14 is thought to be important for most responses to LPS. Based on
this model, one could predict that CEMA would globally suppress
responses to LPS. Several explanations are possible for why some LPS
responses are not blocked by CEMA. One possibility is that those
responses that are not blocked by CEMA do not involve the transfer of
LPS to CD14 by LBP. A second explanation is that different responses
have different thresholds for induction. Some genes may require a
stronger LPS signal to be induced than others. Inhibition of LPS
binding to CD14 by CEMA would reduce the ability of LPS to stimulate
intracellular signaling reactions. Therefore, genes that require very
strong LPS signals to be induced would be inhibited by CEMA, whereas
genes that require only small amounts of LPS for signaling may still be
induced maximally. A third possibility is that cationic peptides such
as CEMA also act directly on macrophages to regulate signaling
pathways, and that this differentially affects the ability of LPS to
up-regulate the expression of different genes.
Direct effect of the peptide CEMA on macrophage transcriptional responses
The possibility that CEMA acts directly on macrophages, as opposed
to merely neutralizing LPS, prompted us to determine whether treating
RAW 264.7 cells with CEMA alone caused any changes in gene expression.
Table III shows that CEMA treatment
up-regulated the expression of 35 different genes. The genes most
strongly induced by CEMA (by 2- to 35-fold) included ICAM-1,
cyclin-dependent kinases inhibitors, the anti-inflammatory cytokine
TGF-ß type I subunit (TGF-ß1) receptor, Jun-D; c-jun related
transcription factor, and Egr-1, which controls monocyte development
and also appears necessary for the maintenance of macrophage
differentiation (Table III). CEMA most notably affected the expression
of genes from three families with functions in cell proliferation,
apoptosis, and cell adhesion.
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The effect of CEMA on cell proliferation could also be related to the
anti-cancer properties observed with some cationic peptides,
including CEMA (26, 27). Such peptides are selectively
more toxic toward tumor cells than toward nonmalignant cells, although
the mechanism of their activity is not fully understood
(26, 27, 28). CEMA was shown here to have effects on a number
of genes involved in apoptosis. For example, CEMA up-regulated PD-1
(Table III), and CEMA down-regulated the expression of a number of
apoptosis-related genes (http://www.cmdr.ubc.ca/arraydata1), including
the apoptosis inhibitors BAG-1, Bcl-2 (both with a ratio of CEMA to
medium of 0.4), and A20 zinc finger protein (ratio of CEMA to medium of
0.1). These data might help explain the results of a previous study,
which found a cecropin-melittin hybrid peptide to have an apoptotic
effect on a murine macrophage cell line (29).
Cell migration is controlled by multistep processes that includes
chemoattraction, cell-cell adhesion, and, in some cases, transmigration
through cell layers (30). It has been reported that two
human -defensin peptides, human neutrophil peptide HNP-1 and -2,
have chemotactic activity for murine and human T cells and monocytes
(31, 32), while human ß-defensins are chemotactic for
immature dendritic cells and memory T cells through interaction with
CCR6 (33). LL-37, a human neutrophil -helical peptide
(34), has also been suggested to have chemotactic activity
for T cells and neutrophils (35), and the porcine peptide,
PR-39, has chemotactic activity for neutrophils (36). CEMA
up-regulated the expression of the urokinase plasminogen activator
receptor, which is widely expressed on different cell types, including
hemopoietic cells, and has been shown to involved in cell adhesion,
chemotaxis, receptor clustering, and changes in cell shape
(37). CEMA also up-regulated a number genes involved in
cell adhesion, including ICAM-1, 6 integrin
and MAC-1 (Table II) and, to a lesser extent,
5 integrin, CD44, and CD45 (data not
shown).
There have been a number of reports of the roles of cationic peptides in the immune system (14). It is becoming increasing clear that their effects on innate immunity are wide ranging and much more involved than their antimicrobial activity. This is the first report demonstrating that a cationic peptide, CEMA, has global effects on macrophage gene expression. There have been some reports that demonstrate that cationic peptides permeabilize eukaryote cells (28). Risso et al. found that two antimicrobial peptides, BMAP-27 and -28, permeabilized eukaryote cell membranes and possibly interacted with negatively charged sialyl residues on the membrane, causing Ca2+ flux into the cytosol (28). This could be a potential mechanism of how cationic peptides could alter macrophage signaling or gene expression. While the mechanism warrants further investigation, this report clearly shows for the first time that cationic antimicrobial peptides directly influence gene expression in macrophages of a large number of diverse genes.
Confirmation of selected array data by Northern analysis
Although the array data were reproducible, and we had confirmed
some of our findings with ELISAs, we also wanted to directly confirm
that LPS and CEMA affected mRNA levels similarly to the ways indicated
by the gene arrays. We chose to perform Northern blots to analyze the
expression of IL-1ß, CD14, and cyclin D1, since these genes represent
the three different scenarios we had observed. According to the gene
array results, IL-1ß mRNA levels were strongly up-regulated by LPS,
and this response was reduced by CEMA (Table I). Conversely, CD14 mRNA
levels were modestly up-regulated by LPS, and this response was not
blocked by CEMA (Table II), while cyclin D1 mRNA levels were not
induced by LPS, but were modestly up-regulated by CEMA. All these
results were confirmed by the Northern blots, and the quantification of
these results is shown in Fig. 3. We
conclude that the gene arrays successfully identified multiple patterns
of gene expression and demonstrated trends similar to those observed by
Northern blot analysis. To demonstrate that these results were not
confined to the synthetic antimicrobial peptide CEMA, LL-37, a human
neutrophil -helical peptide (34), was tested alongside
CEMA and was also found to inhibit LPS-induced gene expression of
IL-1ß (Fig. 4) and MIP-2 (data not
shown) in the RAW macrophages to an extent similar to that observed
with CEMA. Furthermore, preliminary studies indicated that LL37 was
also able to up-regulate a variety of genes in RAW cells.
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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.
3 Abbreviations used in this paper: LBP, LPS-binding protein; MIP, macrophage-inflammatory protein; iNOS, inducible NO synthase; CEMA, cecropin-melittin hybrid.
Received for publication March 13, 2000. Accepted for publication June 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-helical cationic antimicrobial peptides. Infect. Immun. 67:2005.. Infect. Immun. 64:1565.[Abstract]
, ß, or 
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),
LPS-stimulated, CEMA-treated to unstimulated cells (
), and
CEMA-treated to unstimulated cells (
). A ratio of 1 therefore
indicates no stimulation.