STRUCTURES OF HUMAN HOST DEFENSE CATHELICIDIN LL-37 AND ITS SMALLEST ANTIMICROBIAL PEPTIDE KR-12 IN LIPID MICELLES.
Guangshun Wang. JBC Papers in Press. Published on September 25, 2008 as Manuscript M805533200
The cathelicidin anti-microbial peptide LL-37 is involved in re-epithelialization
of human skin wounds and is lacking in chronic ulcer epithelium
The
human cathelicidin anti-microbial protein, hCAP18 is a component
of the innate immune system and has broad anti-microbial activity
conferred by its C-terminal fragment LL-37. hCAP18 is constitutively
produced in leukocytes and is induced in barrier organs upon inflammation
and infection. We demonstrate here a novel role for this peptide
in re-epithelialization of skin wounds. We show that high levels
of hCAP18 are produced in skin in vivo upon wounding. The highest
hCAP18 levels are attained at 48 h post-injury, declining to pre-injury
levels upon wound closure. hCAP18 is detected in the inflammatory
infiltrate and in the epithelium migrating over the wound bed. In
chronic ulcers, however, hCAP18 levels are low and immunoreactivity
for hCAP18/LL-37 is absent in ulcer edge epithelium. Using a noninflammatory
ex vivo wound healing model, composed of organ-cultured human skin,
we show that hCAP18 is strongly expressed in healing skin epithelium,
and that treatment with antibodies raised and affinity purified
against LL-37, inhibits re-epithelialization in a concentration-dependent
manner. Immunoreactivity for the proliferation marker Ki67 is absent
in the epithelium of such inhibited wounds, suggesting that LL-37
may play a part in epithelial cell proliferation. Thus, we suggest
that, in addition to being an anti-microbial peptide, LL-37 also
plays a part in wound closure and that its reduction in chronic
wounds impairs re-epithelialization and may contribute to their
failure to heal.
Heilborn
JD, et al. J Invest Dermatol. 2003 Mar;120(3):379-89.
Endogenous antimicrobial peptides and skin infections in atopic
dermatitis
BACKGROUND:
The innate immune system of human skin contains antimicrobial peptides
known as cathelicidins (LL-37) and beta-defensins. In normal skin
these peptides are negligible, but they accumulate in skin affected
by inflammatory diseases such as psoriasis. We compared the levels
of expression of LL-37 and human beta-defensin 2 (HBD-2) in inflamed
skin from patients with atopic dermatitis and from those with psoriasis.
METHODS: The expression of LL-37 and HBD-2 protein in skin-biopsy
specimens from patients with psoriasis, patients with atopic dermatitis,
and normal subjects was determined by immunohistochemical analysis.
The amount of antimicrobial peptides in extracts of skin samples was
also analyzed by immunodot blot analysis (for LL-37) and Western blot
analysis (for HBD-2). Quantitative, real-time reverse-transcriptase-polymerase-chain-reaction
(RT-PCR) assays were used to confirm the relative expression of HBD-2
and LL-37 messenger RNA (mRNA) in the skin-biopsy specimens. These
peptides were also tested for antimicrobial activity against Staphylococcus
aureus with the use of a colony-forming assay. RESULTS: Immunohistochemical
analysis confirmed the presence of abundant LL-37 and HBD-2 in the
superficial epidermis of all patients with psoriasis. In comparison,
immunostaining for these peptides was significantly decreased in acute
and chronic lesions from patients with atopic dermatitis (P=0.006
and P=0.03, respectively). These results were confirmed by immunodot
blot and Western blot analyses. Real-time RT-PCR showed significantly
lower expression of HBD-2 mRNA and LL-37 mRNA in atopic lesions than
in psoriatic lesions (P=0.009 and P=0.02, respectively). The combination
of LL-37 and HBD-2 showed synergistic antimicrobial activity by effectively
killing S. aureus. CONCLUSIONS: A deficiency in the expression of
antimicrobial peptides may account for the susceptibility of patients
with atopic dermatitis to skin infection with S. aureus. Ong PY, et al. N Engl J Med 2002
Oct 10;347(15):1151-60
A cathelicidin family of human antibacterial peptide LL-37 induces
mast cell chemotaxis.
The mast cell is one of the major effector cells in inflammatory reactions
and can be found in most tissues throughout the body. During inflammation,
an increase in the number of mast cells in the local milieu occurs,
and such accumulation requires directed migration of this cell population.
As it has previously been reported that the human cathelicidin-derived
antibacterial peptide, LL-37, stimulates the degranulation of mast
cells, we hypothesized that LL-37 could be a mast cell chemotaxin.
The present study shows that LL-37 is a potent chemotactic factor
for mast cells. The chemotactic response was dose-dependent and bell-shaped,
reaching an optimal concentration of 5 &mgr;g/ml. In addition,
checkerboard analysis showed that cell migration towards this peptide
was chemotactic rather than chemokinetic. Moreover, Scatchard analysis
using 125I-labelled LL-37-derived peptide revealed that LL-37 has
at least two classes of receptors, namely high- and low-affinity receptors,
on mast cells. Furthermore, the competitive binding assay suggested
that LL-37 is unlikely to utilize formyl peptide receptor-like 1 (FPRL1),
a functional LL-37 receptor for neutrophil and monocyte migration,
on mast cells. In addition, the treatment of cells with pertussis
toxin and phospholipase C inhibitor, U-73122, inhibited LL-37-mediated
migration, indicating that LL-37 induces mast cell chemotaxis through
a Gi protein-phospholipase C signalling pathway. These results show
that besides its antibacterial activities, LL-37 may have the potential
to recruit mast cells to inflammation foci. Niyonsaba F, et al. Immunology
2002 May;106(1):20-26
References:
Travis
S.M., et al. Infection and Immunity. 68, 2748~2755 (2000)
Niyonsaba F, Iwabuchi K, Someya A, Hirata M, Matsuda
H, Ogawa H, Nagaoka I. A cathelicidin family of human
antibacterial peptide LL-37 induces mast cell chemotaxis.
Immunology. 2002 May;106(1):20-26.
Le Y, Yang Y, Cui Y, Yazawa H, Gong W, Qiu C, Wang JM.
Receptors for chemotactic formyl peptides as pharmacological
targets. Int Immunopharmacol. 2002 Jan;2(1):1-13.
De
Yang, Chen Q, Schmidt AP, Anderson GM, Wang JM, Wooters
J, Oppenheim JJ, Chertov O. LL-37, the neutrophil granule-
and epithelial cell-derived cathelicidin, utilizes formyl
peptide receptor-like 1 (FPRL1) as a receptor to chemoattract
human peripheral blood neutrophils, monocytes, and T
cells. J Exp Med. 2000 Oct 2;192(7):1069-74.
Antimicrobial peptides in mucosal secretions: the
importance of local secretions in mitigating infection
The antimicrobial activity of the collective
molecules comprising human milk reflects an evolutionarily
successful paradigm for preventing and limiting microbial
infection. Understanding the molecules that participate
in this process and how they work can yield insight into
potentially new antimicrobial therapies. Upon proteolytic
processing, antimicrobial peptides can be derived from milk
proteins, such as lactoferrin, casein, and lysozyme. Similarly,
using the HIV-1 gp41 protein template, we have demonstrated
that the 28-residue C-terminus, when produced as an independent
peptide, exhibits selective toxicity for bacteria over eukaryotic
cells. Upon optimizing this sequence for cationic charge
and hydrophobic character presented as a alpha-helical structure,
we show improved capability of the parent LLP1 sequence
to selectively kill bacteria in the host environment and
that this activity is increased by the inclusion of Trp
residues on the hydrophobic face. We report that it is possible
to (i) design de novo antimicrobial peptides that demonstrate
optimal antimicrobial activity with minimal inflammatory
activity and (ii) design antimicrobial peptides to function
in a defined environment. In the end, we describe a de novo
designed antimicrobial peptide, WLBU2, which is selectively
toxic to microbial pathogens in complex environments and
does not stimulate a significant immunomodulatory response.
In spite of these properties, WLBU2 activity against Pseudomonas
aeruginosa in human milk is inferior to the host peptide
LL37 with regard to antimicrobial potency. These studies
demonstrate that antimicrobial peptides can be engineered
for greater potency in one medium but may not be optimal
for working in a different medium such as human milk.
Phadke SM, et al. J Nutr. 2005 May;135(5):1289-93.
Dale BA, et al. BMC Oral Health.
2006 Jun 15;6 Suppl 1:S13.
Sequences of the WLBU. Hydrophobic residues are indicated
by bold type. Phadke SM, et al. J Nutr.
2005 May;135(5):1289-93
Peptide design. (A) The cationic amphipathic peptides were
designed as demonstrated in helical wheel diagrams. Arg,
Val, and Trp residues were arranged to form idealized amphipathic
alpha-helices, with the hydrophilic and hydrophobic faces
indicated in clear and shaded circles, respectively. The
12- and 48-mers LBU1 and WLBU4 are shown as representatives
of the LBU and WLBU series, respectively. (B) Primary sequences
of the LBU and WLBU peptides used in the present study.
The shortest peptide forms one lytic base unit (LBU1) of
12 amino acids, and the others were designed as multimers
(2, 3, and 4) of LBU1. The WLBU peptides were derived from
the LBU series by substituting Trp residues at the indicated
positions. Not shown here are the peptide hydrophobic moments
calculated according to the method of Eisenberg et al. (J.
Cell Biochem. 31:11-17.). Deslouches B, et al. Antimicrob
Agents Chemother. 2005 Jan;49(1):316-22.
Human bronchial epithelial (HBE) cell stimulation of IL-8
production. HBE cells grown in
polarized culture were stimulated on the apical side with
no peptide, LL37, or WLSA5. Phadke SM, et al. J Nutr.
2005 May;135(5):1289-93
AMP expression in submandibular salivary gland. Immunohistochemistry
for HNP1-3 and LL37 shows positive reactions in duct cells
of submandibular glands. (A) Hematoxylin and eosin stain.
(B) HNP1-3 immunostaining with monoclonal antibody D21 was
positive in salivary duct cells and neutrophils (inset).
(C) LL37 immunostaining was also positive in duct cells.
(D) Negative control without primary antibody. An isotype-specific
control for the HNP1-3 monoclonal antibody was also negative
(not shown). Original magnification, x40. Tao R.C. et al, Antimicrobial
Agents and Chemotherapy, September 2005, p. 3883-3888, Vol.
49, No. 9
Immunohistochemistry.
Formalin-fixed sections were evaluated for expression of
LL37 and HNP1-3 using the ABC technique. Briefly, sections
were deparaffinized, rehydrated, and treated with antigen-unmasking
solution (Vector Laboratories). Endogenous peroxide was
blocked using 1% hydrogen peroxide/Tris-buffered saline
for 30 min. Sections were blocked with appropriate sera
and incubated with the primary antibody overnight before
visualization with ABC reagents using 3,3'-diaminobenzidine
as the substrate. Methyl green counterstain was used to
visualize tissue morphology. The antibodies used were polyclonal
rabbit anti-LL37 (Catalog No.: H-075-06) (Phoenix Pharmaceuticals,
Inc., Belmont, CA) and monoclonal antibody
clone D21 anti-HNP1-3. Histological sections of minor salivary
glands were obtained from the Division of Oral Pathology,
School of Dentistry, University of Washington, in accordance
with Institutional Review Board procedures. Commercially
available histological sections of human submandibular glands
were from Spring Biosciences . Tao R.C. et al, Antimicrobial
Agents and Chemotherapy, September 2005, p. 3883-3888, Vol.
49, No. 9
AMP levels in saliva as a function of caries score. (A, C,
and E) HNP1-3 (A), hBD3 (B), and LL37 (C) concentrations in
saliva, expressed as µg/ml; (B, D, and F) HNP1-3, hBD3,
and LL37 levels relative to salivary protein in µg/mg
protein. The caries-free group showed significantly higher
HNP1-3 concentration (A) than each of the groups with caries.(**,
P < 0.01) and significantly higher concentration
than the combined caries groups (P = 0.004). Also,
the HNP1-3 level relative to salivary protein (B) in the caries-free
group is significantly higher than each of the caries groups
(**, P < 0.01) and the combined caries groups
(P < 0.004). For LL37, even though no significant
difference was found among the groups with or without caries,
the LL37 level relative to salivary protein (F) shows a trend
of decreasing level in higher caries score groups. There is
no evidence of association of hBD3 with caries (C and D).
Box plots show the median and 25 to 75% range, with error
bars indicating 5% and 95% intervals. Tao R.C. et al, Antimicrobial
Agents and Chemotherapy, September 2005, p. 3883-3888, Vol.
49, No. 9
Salivary analysis of
LL37. Unstimulated saliva was collected (3 to 5 ml),
the detergent Nonidet P-40 (Sigma, St. Louis, MO) was added
to a final concentration of 0.1%, and the sample was frozen
for later analysis. Saliva was thawed and cleared by centrifugation
twice at 15,000 rpm for 10 min. Total protein concentration
was evaluated in the supernatant (cleared unfractionated saliva)
by bicinchoninic acid assay (Pierce Inc., Rockford, IL). Cleared
unfractionated saliva was also used for the HNP1-3 enzyme-linked
immunosorbent assay according to the manufacturer's instructions
(HyCult Biotechnology, Uden, The Netherlands). Aliquots (200
µl) of supernatant were acid extracted by the addition
of an equal volume of 1 M HCl/1% trifluoroacetic acid overnight
with mixing in the cold (23). The sample
was centrifuged, and the supernatant was concentrated by vacuum
evaporation and resuspended in distilled water equal to the
starting sample volume. Acid-extracted saliva was used for
immunoassay of LL37 and hBD3. LL37 was assayed by slot blot
using an assay kit (Catalog No.: WBK-075-06) (Phoenix Pharmaceuticals
Inc., Belmont, CA). Statistical analysis for association of
AMP levels with caries score was done using the Kruskal-Wallis
nonparametric test based on rank and designed for nonnormally
distributed data. Tao R.C. et al, Antimicrobial
Agents and Chemotherapy, September 2005, p. 3883-3888, Vol.
49, No. 9
Immunofluorescent staining of two sections (one section is
shown in a–d and the other is shown in e–h) from
the renal cortex of a NMRI mouse at 24 h of pyelonephritis.
Sections are stained with propidium iodide (a,e,
blue), neutrophil-specific antibody (b,f,
red), antibody to CRAMP (c,g,
green), and images showing these three sets of panels merged
(d,h). The upper panel represents
marginal areas of infection corresponding to the early stage
of inflammation. Bacteria are stained in the lumen of renal
tubulus by propidium iodide (a, arrowheads)
and stimulate the production and release of CRAMP (c,
green, arrowheads). A neutrophil with typical segmented nucleus
(b, starred arrowhead) and positive for a
neutrophil marker (c, starred arrowhead)
is also positive for CRAMP (c,d,
starred arrowhead). Cross indicates the lumen of the renal
tubulus. The lower panel shows an area of massive infection
with abundance of bacteria, destruction of normal parenchyma
and accumulation of neutrophils (f) that
exhibit CRAMP immunoreactivity (g,h).
After blocking of antibody with synthetic CRAMP (1–39)
peptide, we did not observe any staining, and, likewise, there
was no staining in CRAMP-deficient mice (data not shown).
Scale bars in a–d,
20 m; in e–h,
50 m. Chromek M., et al. Nature
Medicine 12, 636 - 641 (2006)
Immunohistochemical staining of sections from healthy human
renal cortical tissue (a–d), a piece of human renal
cortex incubated in cell culture medium for 24 h (e,f), and
a piece infected with uropathogenic E. coli for the
same time (g,h). The sections are stained using polyclonal
antibody to LL-37 (a,c–e,g)
and normal rabbit immunoglobulin (b,f,h).
In the healthy renal tissue, we found LL-37/hCAP-18 immunoreactivity
in the hyaline substance in the lumen of renal tubuli (a,c,
brown) and in neutrophils (d) indicated by
arrows. Parallel sections of the kidney (a,b)
are shown. We did not observe any staining in the hyaline
substance when we used normal rabbit immunoglobulin instead
of primary antibody (b). After 24 h of in
vitro incubation, tubular epithelial cells displayed
morphological signs of cell death (e, arrow).
Moreover, in the presence of bacteria, epithelial cells stained
positive for LL-37/hCAP-18 (g, brown, arrow).
Parallel sections of the kidney (e–h)
are shown. We did not detect any staining when we used normal
rabbit immunoglobulin instead of primary antibody (f,h).
Scale bars in a, b and d,
50 m; in c,
e, f, g
and h, 20 m.
Chromek M., et al. Nature Medicine12, 636 - 641
(2006)
Cathelicidins
defend the kidney from bacteria.
A wall of the urinary tract surrounding the lumen is depicted.
Bacteria ascend against the flow of urine. A thin film of
fluid covers the lumenal surface of the epithelium and represents
the 'battlefield' where microbe and cathelicidins meet.
From the bottom to the top of the figure: (1)
cathelicidin is constitutively expressed in the absence
of bacteria; (2) microbes ascend and approach
the epithelial surface anticipating attachment; (3)
cathelicidin mRNA levels rapidly rise, translation is activated,
and peptide secreted rapidly, killing nearby bacteria. In
this scenario, we permit some microbes to survive and continue
to ascend; thus, (4) neutrophils begin
to migrate toward the lumen; (5) neutrophils
secrete cathelicidins, complementing epithelial sources,
but viable microbes persist; (6) neutrophils
continue to invade the epithelium, resulting in cellular
damage. [by Katie Ris ] Michael Zasloff.
Nature Medicine12, 607 - 608
(2006)
Cathelicidin LL-37/hCAP-18 in vitro.
(a) mRNA encoding LL-37/hCAP-18 in uroepithelial
cells (UROtsa) without bacteria (continuous line) and after
infection with uropathogenic E. coli (dotted line).
All cell lines studied, both uroepithelial and renal epithelial,
reacted similarly after bacterial exposure. Data are presented
as median and range of CAMP/GAPDH X 1,000
mRNA ratios from two experiments in which each sample was
analyzed in triplicate. *P < 0.05. (b)
Western blot of supernatants from uroepithelial cells (UROtsa)
without bacteria (uninfected, u) and after 5, 15, 45 and
135 min infection with uropathogenic E. coli (infected,
inf). Similar results were obtained in all cell types studied,
both uroepithelial and renal epithelial. Synthetic LL-37
served as a positive control (+). Molecular weights are
in kDa. Discrepancy in the migration of the detected peptide
and the synthetic LL-37 is due to salt content in cell culture
medium (data not shown). (c) mRNA encoding
LL-37/hCAP-18 in renal epithelial cells (A498 and hPTC)
and uroepithelial cells (J82 and UROtsa) without bacteria
(open bars) and after 24-h infection with uropathogenic
E. coli (gray bars). mRNA levels from noninfected
cells are considered 1 and relative mRNA levels are calculated.
P < 0.01, compared with uninfected cells at
24 h. Data are presented as median and range of normalized
CAMP/GAPDH mRNA ratios from three independent
experiments. (d) Western blot of supernatants
from renal epithelial cells (A498 and hPTC) and uroepithelial
cells (J82 and UROtsa) without bacteria (uninfected, u)
and after 24-h infection with uropathogenic E. coli
(infected, i). Synthetic LL-37 served as a positive control
(+). Molecular weights are in kDa. Discrepancy in the migration
of the detected peptide and the synthetic LL-37 is due to
salt content in cell culture medium (data not shown).
(a) Number of bacteria attached to urinary
bladders of CRAMP-producing (Camp+/+) and CRAMP-deficient
(Camp-/-) mice 1 h after bacterial inoculation.
CFU, colony-forming units. Data are presented as median
and range from one of two experiments with three to four
mice per group. P < 0.05. (b)
Infection rate of Camp+/+ and Camp-/-
mice with (Neu (+)) or without neutrophils (Neu (–)).
The infection rate was higher in Camp-/- mice,
irrespective of the presence of neutrophils (P
< 0.05). Data are presented as percentage of infected
mice versus mice challenged with bacteria from three independent
experiments using a total of 36 mice. (c)
Survival of bacteria attached to Camp+/+ and Camp-/-
mouse proximal tubular cells (Mouse PTC). Percentage of
viable bacteria attached to the cells at 30 min of incubation
in relation to the counts at 10 min is depicted. Data are
presented as mean + s.d. from two independent experiments
with nine samples per group. P < 0.05.
The course of experimental urinary tract infection in CRAMP-producing
Camp+/+ and CRAMP-deficient Camp-/- mice.
The number of bacteria in kidneys (a)
and the size of kidneys (b) at 48 h of
infection were higher in Camp-/- mice. P
< 0.05. Accordingly, systemic response to infection was
stronger in Camp-/- mice (data not shown). The
differences were observed only in the presence of neutrophils
and not after neutrophil depletion (data not shown). Three
independent experiments were performed with five to six
mice per group. Data are presented as medians with dot plots
(a) and mean + s.d. (b)
from representative experiments.
Sensitivity of uropathogenic E. coli to synthetic
LL-37 peptide expressed as a minimal inhibitory concentration
(MIC) of LL-37.Strains isolated from more invasive infection,
pyelonephritis (APN, n = 21), were more resistant
to LL-37 (P < 0.05) than strains isolated from
infection of the lower urinary tract, cystitis (n
= 14). Individual levels and means are shown.
Antipseudomonal
activity of LL37
EC50
(µM) against P. aeruginosa PAO1
25
mM NaCI
175
mM NaCI
LL37
0.81
?0.13
1.47
?0.33
Mean
?SE (n=3)
Relative
time for killing of P.aerginosa
PAO1 by LL37
Concentration
(µg/ml)
Half
time of killing (min)
LL37
36
62.0
?26.3
Mean
?SE (n=3)
Activity
of LL37 against gram-negative and
gram-positive bacteria
MIC
(µM) for:
E.coli
DH15a
E.coli
ML-35p
P.aeruginosa
PAO1
P.aeruginosa
MR3007
MRSA
ATCC33591
S.aureus
930918-3
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
LL37
0.19
0.44
1.20
0.58
0.54
1.03
0.53
1.54
5.45
30.8
3.58
2.58
Low-salt
(no NaCI) and High-salt (100mM NaCI)
LL-37 induces physiologic angiogenesis
in the CAM assay. (a) LL-37
(5 µg/pellet) induces the formation
of wheel spoke–like vessel formation
in comparison with the scrambled control
peptide sLL-37 or the solvent. bFGF
was used as positive control. Bars:
1 mm. (b) Cross sections of
CAMs. LL-37 increases the number of
erythrocyte-filled vessels (Masson-Goldner
stain of erythrocyte-filled vessels).
Bars: 250 µm or 62 µm in the x10 and
x40 micrographs, respectively. (c)
The numbers of erythrocyte-filled
vessels were counted in hot spots.
*P < 0.05 as compared with
the solvent group (n = 6/group;
three sections per CAM). Koczulla
R, J Clin Invest. 2003 Jun; 111 (11):
1665-72.
LL-37 induces angiogenesis and arteriogenesis
in the rabbit hind-limb model. (a?b>d)
Application of the peptide in a
rabbit hind-limb model resulted
in increased collateral growth in
the LL-37–treated animals (a)
as compared with the sLL-37–treated
animals (b) or the buffer
control group (c). bFGF/VEGF
was used as positive control (d).
(e) Collateral growth was
significantly increased in the LL-37
group. (f) Tissue specimens
from the calf (gastrocnemius muscle)
revealed higher capillary density
in the treated group as compared
with the control group. (g)
Blood flow velocity as assessed
by cinedensitometry of the passage
time of the contrast agent between
the internal iliac artery and the
anterior tibial artery was significantly
augmented in the LL-37 group. *P
< 0.05 as compared with the buffer
control (n = 4/group). Koczulla
R, J Clin Invest. 2003 Jun; 111
(11): 1665-72.
Endothelial cells express FPRL1
in vitro and in vivo. (a)
Detection of FPRL1 transcripts in
cultivated HUVECs by RT-PCR. Control
is the BEAS-2B cell line. (b)
Detection of FPRL1 protein in HUVECs
by Western blot analysis using a
specific antiserum. (c) Immunohistochemistry
revealed expression in endothelial
cells (arrow) of sections of lung
tissue. (d) Control serum
revealed no positive staining. Bar:
60 µm. Koczulla
R, J Clin Invest. 2003 Jun; 111
(11): 1665-72.
LL-37 binds to endothelial FPRL1 and
induces cellular signaling. (a
and b) LL-37 induces Ca2+
flux in HUVECs. Fura-2–loaded HUVECs
were stimulated with LL-37, and relative
levels of intracellular Ca2+
were monitored using a calcium-imaging
system. Local application of LL-37
led to a concentration-dependent increase
of intracellular calcium. Preincubation
of the cells with pertussis toxin
(Ptx) resulted in partial inhibition
of the Ca2+ flux (a).
Preincubation with fMLP cross-desensitized
the LL-37–induced Ca2+
mobilization; sLL-37 had no effect
(b). (c) LL-37 activates
NF-B
in endothelial cells. This response
can be inhibited by GF109203X and
the addition of N-acetylcystein.
*P < 0.05 as compared with
the control group (n = 8).
NAC, N-acetylcystein. Koczulla
R, J Clin Invest. 2003 Jun; 111 (11):
1665-72.
Alignment
of RL-37, LL-37 and LL-37 pentamide
sequence
a
The five amides that were introduced
into the LL-37 pentamide sequence
are shown in boldface type. Residues
of RL-37 that are identical to
those in LL-37 are underlined.
cDNA sequence of the rhesus
monkey cathelicidin, RL-37. The precursor
has 170 residues, a mass of 18,861 Da,
and a pI of 10.06. The predicted
signal sequence (33 residues)
is underlined, and the expected mature
peptide is shown in bold face type.
The stop codon is indicated by asterisks,
and the polyadenylation site is indicated
by italics.
Comparison of rhesus and human
cathelicidin peptides. Both signal
peptides (underlined) have 30 residues,
of which 28 (93.3%) are identical.
Both cathelin domains have 101 residues,
of which 93 (92%) are identical.
The mature domains (dotted underlined)
of RL-37 and LL-37 both contain 37 residues,
of which 25 (67.6%) are identical.
Mature rhesus RL-37 has a mass of
4,100.9 Da, a theoretical pI
of 11.20, and a net charge of
+8. Mature human LL-37 has a mass
of 4527.34, a pI of 10.61, and
a net charge of +6. A vertical line
connects identical residues, and a
plus sign identifies conservative
substitutions. Residues that differ
in the human and rhesus peptides are
shown in boldface type.
Activity against E. coli,
P. aeruginosa, and L. monocytogenes.
Radial diffusion assays were performed
in underlay gels that contained different
amounts of NaCl (0 mM, 100 mM,
or 200 mM), in addition to their
common basic ingredients (10 mM
sodium phosphate buffer, pH 7.4; Trypticase
soy broth powder, 0.3 mg/ml;
and 1% [wt/vol] agarose).
LPS binding. We used a chromogenic
Limulus amoebocyte assay to
obtain binding isotherms for E. coli
0111:B4 lipopolysaccharide. The peptides
examined included polymyxin B, LL-37,
LL-37 pentamide, RL-37, and rabbit
CAP-18 (another LPS binding cathelicidin).
The EC50 (i.e., the peptide
concentrations that bound 50% of the
LPS) are shown and provide an approximate
binding constant.
TABLE
3. Uffect of amidation on antimicrobial
activitya
a
Radial diffusion assays were performed with underlay
gels that contained 100 mM NaCl; 10 mM sodium
phosphate buffer, pH 7.4; and Trypticase soy broth
powder, 30 mg/ml. P values (for LL-37
versus LL-37 PAM) were calculated by the t
test. Abbreviations: NT, not tested; NS, not significant.
Induction by LL-37 of migration of (A) and Ca2+ flux
in (B) human monocytes. (A) The migration of monocytes (106
cells/ml) was assessed by chemotaxis assay using 5-µm
uncoated membranes. Spontaneous cell migration (without
LL-37) was 30–50 cells per HPF. The average C.I. (mean
± SD) of triplicate wells is shown. *P < 0.05
when compared with chemotaxis medium alone (open bar). (B)
Arrow indicates the time point at which LL-37 was applied
to the cells. Yang D., et al. The Journal
of Experimental Medicine, Volume 192, Number 7, October
2, 2000 1069-1074
Effect of PTX (A) and serum (B) on LL-37–induced chemotaxis
of monocytes. (A) Monocytes were incubated with (closed
bar) or without (hatched bar) PTX at a final concentration
of 200 ng/ml for 30 min at 37°C before performing chemotaxis
assay. To show that the spontaneous cell migration (C.M.)
was not affected by PTX pretreatment, the results are presented
as no./HPF. (B) Chemotaxis assay was performed in the absence
(hatched bar) or presence (closed bar) of 10% human AB serum,
which can completely block the antimicrobial activity of
LL-37 at 10-5 M. Yang D., et al. The Journal
of Experimental Medicine, Volume 192, Number 7, October
2, 2000 1069-1074
LL-37 uses FPRL1 as its receptor. (A) Selective induction
of FPRL1/293 cell chemotaxis by LL-37. The migration of
parental HEK293 (open bars), FPRL1/293 (dotted bars), or
ETFR (closed bars) cells was assessed by chemotaxis assay
with the use of collagen-coated 10-µm membranes. Cells
were used at a concentration of 106 cells/ml. The average
C.I. (mean ± SD) of triplicate wells is shown. Spontaneous
cell migration (without LL-37) was 10–20 cells per
HPF. *P < 0.05 when compared with chemotaxis medium alone.
(B) LL-37–induced Ca2+ flux in FPRL1/293 cells. Arrow
indicates the time point at which LL-37 was applied to the
cells. (C) Cross-desensitization of LL-37–induced
Ca2+ flux in monocytes by FPRL1-specific agonistic ligand.
Arrows indicate the time points at which LL-37 and Su peptides
were applied to the cells. Yang D., et al. The Journal
of Experimental Medicine, Volume 192, Number 7, October
2, 2000 1069-1074
Chemotaxis of human neutrophils (A) and CD4 T lymphocytes
(B) in response to LL-37. The cell migration was assessed
by chemotaxis assay with the use of uncoated (A) or fibronectin-coated
(B) 5-µm membranes. The results are presented as
the average C.I. (mean ± SD) of triplicate wells.
*P < 0.05 when compared with spontaneous cell migration
(chemotactic medium alone). Neutrophils and CD4 T cells
were used at a concentration of 106 cells/ml and 5 x 106
cells/ml, respectively. Spontaneous neutrophil and T cell
migration (without LL-37) was 30–50 and 30–40
cells per HPF, respectively. Yang D., et al. The Journal
of Experimental Medicine, Volume 192, Number 7, October
2, 2000 1069-1074
