BJA Advance Access originally published online on April 8, 2008
British Journal of Anaesthesia 2008 100(6):803-809; doi:10.1093/bja/aen074
Endogenous antimicrobial peptide LL-37 induces human vasodilatation 
Section of Anaesthesiology and Intensive Care, Department of Clinical Sciences, Lund University, SE-221 85 Lund, Sweden
* Corresponding author. E-mail: mikael.bodelsson{at}med.lu.se
Accepted for publication February 22, 2008.
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Abstract |
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Background: Septic shock includes blood vessel dilatation and activation of innate immunity, which in turn causes release of antimicrobial peptides such as LL-37. It has been shown that LL-37 can attract leucocytes via the lipoxin A4 receptor (ALX, FPRL1). ALX is also present in vascular endothelial cells. To explore possible ways of pharmacological intervention in septic shock, we investigated if LL-37 can affect vascular tone.
Methods: Human omental arteries and veins were obtained during abdominal surgery, and circular smooth muscle activity was studied in organ baths. Gene expression was studied using reverse transcriptase–polymerase chain reaction.
Results: LL-37, at micromolar concentrations, induced a concentration- and endothelium-dependent relaxation in vein but not in artery segments precontracted by endothelin-1. The relaxation was profoundly reduced by potassium chloride (30 mM) to inhibit endothelium-derived hyperpolarizing factor (EDHF), whereas it was less affected by the NOS inhibitor, L-NG-nitroarginine methyl ester, and not at all by indomethacin. The ALX agonist, WKYMVm, also induced a relaxation and both the relaxations induced by LL-37 and WKYMVm were inhibited by the ALX antagonist, WRWWWW. ALX was expressed in the vein endothelium.
Conclusions: We demonstrate, for the first time, that the human antimicrobial peptide, LL-37, induces endothelium-dependent relaxation in human omental veins mediated via an effect on endothelial ALX. The relaxation involves the release of nitric oxide and EDHF but not prostanoids. LL-37 released from white blood cells could contribute to blood vessel dilatation during sepsis and treatment with ALX antagonists might be successful.
Keywords: immune response; muscle vascular, responses; receptors, transmembrane
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Introduction |
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A systemic inflammatory response to an infection (sepsis) is often accompanied by blood vessel dilatation, resulting in an arterial pressure decrease and subsequent mismatch between the demand and delivery of oxygen to vital organs.1 Activation of innate immunity, as in sepsis, includes the release of antimicrobial peptides.2 3
The 37 amino acids human cathelicidin antimicrobial peptide, LL-37, is released from activated neutrophils and epithelial cells.4–7 LL-37 not only possesses extensive antimicrobial properties against gram-positive and gram-negative bacteria and fungi, but also binds and neutralizes lipopolysaccharides from the cell wall of gram-negative bacteria.4 8 9 Furthermore, LL-37 at micromolar concentrations has chemotactic activity: it attracts neutrophils, monocytes, and T-lymphocytes via activation of the lipoxin A4 receptor, ALX.10 11
ALX, which was initially cloned as a formyl peptide receptor (FPR) homologue,12 is a chemoattractant receptor belonging to the seven transmembrane G protein-coupled receptor family. ALX is also known as the formyl peptide-like 1 receptor (FPRL1).13 14 Several ALX agonists and antagonists have been identified, including the endogenous agonists lipoxin A4, LL-37, and the acute phase protein serum amyloid A.15 Useful synthetic selective ligands have been developed such as the hexapeptide agonist WKYMVm (WKY), which induces chemotaxis at subnanomolar concentrations.16 Furthermore, Bae and colleagues,17 screening hexapeptide libraries, discovered WRWWWW,W, which selectively antagonizes ALX-mediated chemotaxis.
Human umbilical vein endothelial cells express ALX. Activation of these receptors by LL-37 increases intracellular concentrations of cAMP and promotes angiogenesis.18 Results obtained in a mouse model suggest that ALX is involved in blood vessel function by reducing infiltration of polymorphonuclear leucocytes and vascular permeability.19 These results suggest the involvement of ALX in blood vessel function.
The present study was designed to test the hypothesis that the activation of endothelial ALX by LL-37 could affect smooth muscle contractility via release of endothelium-dependent factors. We demonstrate that LL-37 induces an endothelium-dependent relaxation mediated via ALX in isolated human omental vein.
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The Research Ethics Committee of the University Hospital of Lund approved the study. After obtaining written informed consent, macroscopically normal segments of human omental arteries and veins (0.5–1.5 mm in diameter) were obtained from 21 female and 27 male patients aged 21–90 yr (median 68 yr) undergoing abdominal surgery. Exclusion criteria included patients with endocrine tumours and abdominal infections. The vessel segments were immediately dissected free from fat and connective tissue and then stored in aerated Krebs–Ringer solution (KRS, composition see below) at 4°C. Experiments were carried out within 24 h.
Measurement of smooth muscle force
The vessel segments were cut into 2–4 mm long ring segments and placed into 2 ml organ baths on two steel rods through the lumen. One of the rods was attached to a Grass FTO3C force displacement transducer for measurement of isometric force. The force was recorded on a Grass polygraph model 7D (Grass Medical Instruments, Quincy, MA, USA). Six segments in separate organ baths with KRS were run in parallel. The temperature in the baths was thermostatically maintained at 37°C and the KRS was continuously aerated with a gas mixture containing O2 92% and CO2 8% at a rate giving pH 7.4. The vessel segments were gradually stretched to a stable resting force of 6 mN during an equilibration period of 60–90 min to obtain optimal tension.20 Next, KCl (83 mM) was added and the resulting contraction was registered in order to obtain an internal contraction standard for each segment. Subsequently, the KCl was washed out by repetitive changes of the KRS during which the contraction was allowed to return to baseline.
Endothelin-1 (ET-1, 1 nM) was added and the resulting contraction was recorded. If required, the ET-1 concentration was increased stepwise until the contraction was equal to 70–130% of the contraction induced by 83 mM KCl. The ET-1 concentrations needed (1–10 nM) correspond to the EC50 values in these preparations.21 Within 10 min, the contraction reached a stable plateau (precontraction). Next substance P was added (100 nM) and the resulting relaxation recorded. In intact segments, substance P induces a relaxation corresponding to 80% of precontraction.22 In the present study, the endothelium was regarded as damaged, if substance P did not induce a relaxation and the segment was then excluded from the study.20
Effects of LL-37 on smooth muscle force
After thorough washout, ET-1 (1 nM) was added and the resulting contraction recorded. If required, the ET-1 concentration was increased stepwise as described. When a stable contraction was achieved, LL-37 was added cumulatively in 10x increments (1 nM–10 µM). The resulting relaxation was recorded. These experiments showed that the LL-37 induced relaxation in segments from some patients, at each concentration, required 5–10 min to reach maximum. Previous experiments have shown that ET-1, at these concentrations, induces a contraction that is constant without fade for at least 30 min, followed by a gradual decrease in tension.23 Thus, the relaxation induced by LL-37 in experiments extending for more than 30 min could be affected by a spontaneous decrease in ET-1-induced tension. Therefore, in all subsequent experiments, only two concentrations of LL-37 were used (100 nM and 1 µM), representing the mid part of the concentration–response curve. Thus, EC50 and maximum effect values could not be determined.
In a second series of experiments, the endothelium was removed in three of the six vein segments by gentle injection of the O2/CO2 gas mixture through the vessel lumen for 10 min during the equilibration period.24 To confirm that the endothelium had been removed successfully, substance P (100 nM) was added to the vessel segments precontracted with ET-1. If substance P induced a relaxation in gas-treated segments, after rinsing, the gas injection was repeated and the presence of endothelium was subsequently tested until substance P did not induce any relaxation. The organ baths were then rinsed several times and relaxations in response to LL-37 were assessed as described above.
In a third series of experiments, the relative contribution of prostanoids, nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF) to the LL-37-induced relaxation in vein segments was assessed. Relaxation experiments with LL-37 on ET-1 precontracted segments were repeated as above in the presence of the nitric oxide synthase inhibitor, L-NG-nitroarginine methyl ester (L-NAME, 0.3 mM), and KCl (30 mM), which prevents hyperpolarization of the vascular smooth muscle.25 L-NAME and KCl were added to the organ baths at least 10 min before the addition of LL-37. In separate experiments, the influence of prostanoids on the LL-37 induced relaxation was studied using the cyclooxygenase inhibitor, indomethacin (30 µM), in the same manner as above.
Effects of ALX agonists and antagonists
In a fourth series of experiments, the relaxing effect of the ALX agonist, WKY, was studied in vein segments, in the same manner as for LL-37. Relaxation experiments with LL-37 and WKY were also performed in the presence of the ALX antagonist WRW4 or vehicle (dimethyl sulphoxide; see below) after 10 min contact time.
RNA isolation
Pieces of omental veins, about 40 mm long, were rinsed in sterile KRS and cut in half. One of the halves was maintained intact whereas the other was cut open and the luminal side gently wiped with a sterile cotton swab and rinsed in KRS. The vessel segments, with and without endothelium, were separately submerged in 1 ml TRIzol (Invitrogen, Carlsbad, CA, USA) on ice and homogenized mechanically. Isolation of RNA was carried out according to the manufacturer's instructions. The RNA yield was determined by spectrophotometry.
Reverse transcriptase–polymerase chain reaction
Synthesis of cDNA was performed using the iScript Select kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Briefly, total RNA (200 ng) and 2.5 µM primer [oligo(dT)20] in water were heated (65°C, 5 min), chilled on ice, and reverse transcribed in RT buffer (42°C, 60 min). After denaturation (5 min, 95°C), 2 µl of samples were amplified in 18 µl polymerase chain reaction (PCR) buffer containing 1.5 mM MgCl2, 0.2 mM dNTPs, 1 µM primer [ALX sense 5'-GAC CTT GGA TTC TTG CTC TAG TC-3'and ALX anti-sense 5'-CCA TCC TCA CAA TGC CTG TAA C-3' yielding a 620 bp product;18 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense 5'-CCA TGG AGA AGG CTG GG-3' and GAPDH anti-sense 5'-CGC CAC AGT TTC CCG GA-3' yielding a 280 bp product],26 and 0.05 U µl–1 TaqDNA polymerase (Qiagen, Venlo, The Netherlands) for 33 cycles with annealing at 58°C and extension at 72°C. The PCR products were separated on a 1.5% (w/v) agarose gel (Sigma-Aldrich, St Louis, MO, USA), containing 0.5 µg ml–1 ethidium bromide for UV detection.
Real-time PCR
For real-time PCR, 500 ng of total RNA was transcribed using a high-capacity cDNA reverse transcription kit. TaqMan gene expression assays for ALX, von Willebrand factor, and 18S rRNA provided primers and FAM-labelled TaqMan MGB probes. A two-step PCR amplification was performed using the TaqMan Universal PCR Master Mix, primers (900 nM), probes (250 nM; all from Applied Biosystems), and DNA (100 ng) in a real time PCR device (Applied Biosystems 7300). The amount of ALX and von Willebrand factor products was normalized to the 18S rRNA product. The von Willebrand factor is constitutively expressed in the endothelium and was used to assess the success of endothelial removal.
Drugs
The following compounds were used: substance P acetate, L-NAME hydrochloride, ET-1, and ethidium bromide (all obtained from Sigma-Aldrich), indomethacin (Confortid, Alpharma, Fort Lee, NJ, USA). LL-37, WKY, and WRW4 were synthesized by Innovagen AB, Lund, Sweden by Fmoc chemistry. The purity of these peptides (>95%) was confirmed by mass spectrometry. All substances were dissolved or diluted in distilled water, except for indomethacin and WRW4, which were dissolved in a phosphate buffer and dimethyl sulphoxide, respectively. The KRS contained (mM): Na+ 143, K+ 4.6, Cl– 126, Ca2+ 2.5, HCO3– 25.0, Mg2+ 0.79, SO42– 0.79, H2PO4– 1.2, glucose 5.5, and EDTA 0.024.
Analysis of data
Relaxations are expressed as percentage of the precontraction induced by ET-1. When values from more than one similar experiment were obtained from the same patient, the mean was calculated before further analysis and presentation and therefore the number of determinations (n) equals the number of patients. Two-way repeated measurement ANOVA for factors LL-37 or WKY concentration and treatment followed by Dunnett's post hoc test was used to compare the concentration–response data. Significance level: P<0.05.
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Measurement of smooth muscle force
KCl (83 mM) induced a contraction amounting to 13 (SD 4.9) mN (n=4) and 8.1 (0.88) mN (n=26) in artery and vein segments, respectively. After washout, ET-1 was cumulatively added as described as to achieve a precontraction. LL-37, at the concentrations tested, did not induce or induced only weak relaxations in artery segments precontracted by ET-1 (Fig. 1, n=4). LL-37 did, however, induce a concentration-dependent relaxation of the smooth muscle of ET-1 precontracted vein segments (Fig. 1). The LL-37-induced relaxations, in the vein segments, were markedly reduced after removal of the endothelium (Figs 1 and 2). Owing to the lack of effect of LL-37 in artery segments, the subsequent experiments were performed on vein segments only.
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The relaxation induced by LL-37 was statistically significantly reduced by about 30% in the presence of the nitric oxide synthase inhibitor, L-NAME (Fig. 3). Addition of KCl (30 mM) to inhibit hyperpolarization induced a contraction amounting to 88 (4.9)% (n=8) of the contraction previously induced by 83 mM KCl. The ET-1-induced precontraction in the presence of KCl (30 mM) was, however, lower than that in the absence of KCl (30 mM). Thus, the total precontraction was similar in the presence and absence of KCl (30 mM). The LL-37-induced relaxation was profoundly inhibited in the presence of KCl (30 mM) to inhibit hyperpolarization and absent in the presence of both L-NAME and KCl (Fig. 3). The inhibitor of prostanoid synthesis, indomethacin, did not affect the relaxation induced by LL-37 (n=3; not shown). This indicates that the LL-37-induced relaxation is mainly mediated by endothelial release of EDHF with a smaller contribution of nitric oxide. A similar pattern has been found for the relaxation induced by substance P in this preparation.20 27
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-nitro-L-arginine methyl ester (L-NAME, 0.3 mM), potassium chloride (30 mM), or both to inhibit hyperpolarization. L-NAME inhibited the LL-37-induced relaxation by about 30% whereas KCl nearly abolished the LL-37-induced relaxation. Values are means+SEM, n=8–11. Asterisk indicates statistically significant difference from control (two-way repeated measurement ANOVA for the factors LL-37 concentration and L-NAME, KCl or both treatment followed by Dunnett's post hoc test).The ALX agonist, WKY, at concentrations in the nanomolar range, which has previously been shown to induce migration of cells transfected by ALX,16 induced a relaxation in segments of human omental vein (Fig. 4). The relaxations in response to WKY and LL-37 were not affected by the selective ALX antagonist WRW4 at 1 µM (not shown) but were markedly inhibited by WRW4 at 10 µM (Figs 4 and 5). WRW4 (10 µM) did not affect the endothelium-dependent relaxation induced by substance P [100 nM; 78 (5.6)% and 80 (3.1)% of the ET-1-induced precontraction for WRW treated and control segments, respectively; n=5; Fig. 5] indicating that the effect of WRW4 was not because of a non-specific effect on endothelium-dependent relaxation.
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Expression of ALX
ALX was expressed in veins (Fig. 6). The amount of RT–PCR product was considerably lower in arteries and in the endothelium-denuded vein segments. This was reflected by the need for an average of 1.5 more cycles in real time RT–PCR to obtain an equal amount of product in endothelium-denuded compared with intact vein segments (n=3, not shown). These results confirm that ALX is mainly expressed in the vein endothelium.
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Human omental vessels constitute a well-characterized tissue for physiological and pharmacological investigations.23 28 29 The present results show that the human antimicrobial peptide, LL-37, induces endothelium-dependent relaxation in isolated human omental veins. The relaxation involves endothelial release of nitric oxide and EDHF. The ALX agonist WKY mimicked the effects of LL-37 and the ALX antagonist WRW4 inhibited the effects of both LL-37 and WKY. The potency of WRW4 for inhibiting WKY and LL-37 induced relaxation in the present experiments was similar to that previously found for WRW4 inhibiting ALX-mediated Ca2+ mobilization and cell migration.17 This indicates that the LL-37-mediated relaxation in the isolated human omental vein is mediated via ALX, which is further supported by the fact that the relaxation was induced by LL-37 in the same concentration range that induces ALX-mediated leucocyte chemotaxis.11 This is, to our knowledge, the first report on cardiovascular actions of LL-37, which thus could be a link between innate immunity and cardiovascular control.
Lipoxin A4 is another naturally occurring ligand for ALX. The reports on the cardiovascular effects of this arachidonic acid metabolite in research animals are scarce and contradictory. Furthermore, whether these responses are mediated via the respective research animal counterpart to human ALX has not been determined. Thus, the present results are the first indications of a role for this receptor in the regulation of human vascular tone.
The physiological, pathophysiological, or both relevance of the present findings is unclear. The LL-37 propeptide, hCAP-18, is released from activated leucocytes8 and is then cleaved by simultaneously secreted proteinase 3 generating LL-37.7 Plasma concentrations of hCAP-18/LL-37 immunoreactivity in healthy individuals have been reported to be around 70 nM, which corresponds to the threshold concentration for LL-37 inducing relaxation found in the present study.30 This means that only a moderate increase in LL-37 concentration would suffice to induce a vascular response. Sepsis includes the degranulation of leucocytes31 but whether this results in increased concentrations of LL-37 in the circulation remains to be elucidated.
There is also the possibility that blood vessels could respond to a local increase in LL-37. During an inflammatory response, granulocytes interact with the vascular endothelium, first rolling along in the vessel lumen. This is followed by granulocyte activation and degranulation, which could lead to a local increase in LL-37 concentrations allowing for the activation of endothelial ALX.32 A subsequent relaxation of underlying smooth muscle, vasodilatation and reduction of blood flow velocity and sheer stress could facilitate leucocyte adhesion and transmigration through the vessel wall.
Previous reports on the effects of leucocytes on isolated vascular preparations contain conflicting results, which partly seems to be dependent on the species, the vascular bed, or both studied. For example, Rimele and colleagues33 demonstrated that rat neutrophils release a substance, probably nitric oxide, which relaxes rat aorta regardless of the state of the endothelium. A similar observation was made with human neutrophils and human internal mammary artery.34 On the other hand, human neutrophils contract the isolated human umbilical vein, at least partly via the release of leucotrienes.35 This finding is somewhat contradictory to the present findings demonstrating vascular relaxation to a neutrophil-derived substance, LL-37. Human umbilical vein endothelial cells express ALX, and an LL-37 induced relaxation should thus be possible.18 The human umbilical vein is, however, an atypical vessel as it does not exhibit any endothelium-dependent relaxation to any vasoactive substance tested.35 Collectively, the action of leucocytes on the motor response of the blood vessel wall is complex. The present results suggest that LL-37 could be an additional mediator of leucocyte-induced vasomotion.
We could only demonstrate LL-37 induced relaxation in vein segments. The reason for this is not clear but it cannot be caused by a loss of viability of the arterial specimens as both potassium chloride and ET-1 contracted and substance P relaxed the smooth muscle. There are numerous studies demonstrating differential receptor expression in human omental arteries and veins.36 37 A plausible reason is that human omental arteries do not express ALX but this remains to be confirmed. It should be noted, however, that leucocyte–vascular wall interactions mainly take place on the venous side of the circulation.38 On the other hand, Eastwood and colleagues demonstrated that human omental arteries become hyporesponsive to vasopressin after incubation in plasma from patients with severe sepsis.39 Their experimental approach included, however, a prolonged exposure to the septic plasma, which probably contained proinflammatory mediators, while our experiments aimed at elucidating the vascular response to LL-37 over a narrower time frame.
In conclusion, we have demonstrated, for the first time, that the human antimicrobial peptide, LL-37, induces an endothelium-dependent relaxation in human omental veins mediated via an effect on endothelial ALX. Relaxation involves the release of nitric oxide and EDHF but not prostanoids. LL-37 released from white blood cells could contribute to blood vessel dilatation during systemic inflammatory disorders, and treatment with ALX antagonists like WRW4 might be successful.
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The Swedish Research Council, Grant no. 2004-3874; the Lund University Hospital Research Funds; the Region Skåne Research Council; the Crafoord Foundation; the LPS Medical Foundation; the Royal Physiographic Society in Lund and the Scandinavian Society for Antimicrobial Chemotherapy.
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A preliminary account of these results has been presented to the 29th Congress of the Scandinavian Society of Anaesthesiology and Intensive Care, Gothenburg, September 5–8, 2007. 
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