BJA Advance Access originally published online on April 2, 2008
British Journal of Anaesthesia 2008 100(5):612-621; doi:10.1093/bja/aen073
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Examination of the effect of procalcitonin on human leucocytes and the porcine isolated coronary artery
1 Department of Anaesthesia and Intensive Care Medicine
2 School of Biomedical Sciences and Centre for Integrated Systems Biology and Medicine, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2 UH, UK
* Corresponding author. E-mail: ravi.mahajan{at}nottingham.ac.uk
Accepted for publication January 29, 2008.
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Abstract |
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Background: The aim of this study was to investigate the effects of procalcitonin on the lipopolysaccharide (LPS)-induced changes in human leucocytes and porcine isolated coronary artery.
Methods: Using flow cytometry, changes in forward scatter and intracellular calcium in human neutrophils and monocytes were determined after exposure to procalcitonin, calcitonin gene-related peptide (CGRP), LPS, and the known chemoattractants formylated methionine-leucine-phenylalanine (fMLP) and interleukin-8 (IL-8). In porcine isolated coronary artery, the effects of procalcitonin were evaluated using the contractile function change and the release of TNF
.
Results: In human neutrophils and monocytes, procalcitonin (100 nM), but not CGRP, increased forward scatter and the expression of surface markers (CD16 and CD14, respectively) in a similar manner to 10 µg ml–1 LPS. Procalcitonin, but not CGRP, also increased the proportion of cells exhibiting an increase in intracellular calcium ions similar to that produced by fMLP and IL-8. Acute exposure of the coronary artery to procalcitonin produced a small, endothelium-independent relaxation (approximately 15% of constrictor tone), but failed to modify subsequent relaxations to CGRP. After 16 h exposure, procalcitonin (100 nM) increased TNF release from the coronary artery equivalent to 70% of that produced by LPS, but did not modify the inhibitory effect of LPS (100 µg ml–1) on contractile responses.
Conclusions: Procalcitonin has a proinflammatory effect on human leucocytes and porcine coronary artery, but it is not capable of modulating LPS-induced changes in vascular responsiveness in vitro.
Keywords: arteries, coronary; complications, endotoxaemia; cytokines, LPS; procalcitonin
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Introduction |
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Procalcitonin is a 116 amino acid prohormone that leads to the generation of a variety of biologically active peptides encoded by the Calc-1 gene.1 Under normal conditions, circulating procalcitonin levels are maintained at approximately 100 pg ml–1 or 0.01 nM. However, circulating levels of procalcitonin are known to increase dramatically in a variety of inflammatory states or surgical procedures independent of changes in the levels of calcitonin. In the case of septic shock, plasma procalcitonin levels can rise 30–1000-fold (100 mg ml–1 or 10 nM) and it is generally viewed as a better prognostic/severity indicator than C-reactive protein.1–3 There is evidence to suggest that procalcitonin should be primarily viewed as a proinflammatory hormone in septic shock.1 4 Immuno-neutralization of procalcitonin with a specific antibody added either before or 3 h after administration of endotoxin has been shown to arrest the progression of lethal porcine sepsis.5
The exact role of procalcitonin, and how it might exert its effects, in sepsis remain unclear. Monneret and colleagues6 7 reported that procalcitonin had no intrinsic activity on human neutrophil and monocyte expression of CD11b, but was capable of inhibiting the proinflammatory effect of lipopolysaccharide (LPS). This inhibitory effect on leucocyte function was similar to that noted for calcitonin-gene related peptide (CGRP), raising the possibility that both peptides act on similar targets. On the other hand, procalcitonin has been reported to promote the migration of human monocytes, yet impair activation of monocytes by formylated methionine-leucine-phenylalanine (fMLP).8 In cultured rat aortic smooth muscle cells, recombinant human procalcitonin has been reported to either inhibit9 or enhance10 mRNA for inducible nitric oxide synthase depending on whether procalcitonin was added before or 3 h after the proinflammatory insult, raising the possibility of timing of exposure being critical.
In the present study, we wished to address whether or not procalcitonin has effects similar to CRGP, and whether its effects are determined by the timing of exposure in relation to the inflammatory stimulus. Therefore, we have compared the effects of procalcitonin and CGRP on the human neutrophils and monocytes and the porcine isolated coronary artery. In addition, we aimed to study the effects of procalcitonin on LPS-induced changes in vascular responsiveness11 in relation to the timing of agent exposure, and compared the effect of procalcitonin and LPS on TNF production in the artery.
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Flow cytometry on human monocytes and neutrophils
Changes in neutrophil and monocyte shape were assessed in whole blood from healthy male and female volunteers (22–51 yr) based on the method of Buckland and colleagues.12 13 Aliquots of blood (196 µl) in polypropylene tubes were incubated at 37°C for 30 min with vehicle, procalcitonin 100 nM, GCRP 100 nM, or a combination of both agents. The action of procalcitonin 100 nM was also compared with that produced by LPS (Escherichia coli O111:b4)10 µg ml–1. Triplicate measurements were made for each condition with 20 µl aliquots taken into polypropylene flow cytometry tubes followed by the addition of 5 µl of a 1 in 20 dilution of fluorescein isothiocyanate anti-CD14 (anti-CD14-FITC) or anti-CD16-FITC antibodies (Caltag-MedSystems Ltd, Towcester, UK) in distilled water. All tubes were allowed to stand on ice for 20 min to allow for sample staining. The samples were lysed and fixed with Optilyse C (Beckman Coulter), which involved the addition of 100 µl of the reagent and immediate vortexing. Each sample was then allowed to stand for 20 min at room temperature, followed by the addition of a further 100 µl of phosphate buffered saline, vortexing, and a further 20 min incubation at room temperature. Forward scatter, side scatter, and fluorescence in each sample were measured by flow cytometry (Beckman Coulter Epics XL MCL).
Neutrophils and monocytes were gated on a plot of side scatter and fluorescence, neutrophils having a higher value for each parameter than monocytes. Data were acquired for 5000 events in the neutrophil gate. The data are expressed as the median forward scatter and fluorescence for each condition (average of three replicates) and shown as the mean (SEM) of observations on samples from different individuals.
In calcium experiments, blood obtained from volunteers was placed in 3 ml Sarstedt EDTA tubes followed by the addition of 0.3 ml of dextran 500 6% in NaCl 0.9%. Red blood cells were allowed to settle for approximately 30 min. The supernatant containing plasma and cells was removed with a pipette into a 15 ml polypropylene centrifuge tube and spun at 100g for 10 min at 4°C to remove platelets and excess plasma. The remaining cells were gently re-suspended in 0.2% ice-cold NaCl in a volume equivalent to the original blood volume (3 ml) followed by an equal volume of ice-cold 1.6% NaCl. The tube was gently inverted to ensure adequate mixing and then spun at 100g and 4°C for 10 min. The supernatant was removed and the cells gently re-suspended in 5 ml low potassium buffer (KLS: NaCl 118 mM, KCl 5 mM, MgSO4 0.8 mM, D-glucose 5.5 mM, Na2CO3 8.5 mM, BSA 0.1%, HEPES 20 mM, CaCl2 1.8 mM adjusted to pH 7.4). To each sample, 5 µl ml–1 FLUO-3 (1 M in DMSO) was added to give a final concentration of 5 mM FLUO-3 and the cells allowed to equilibrate for 30 min at 37°C with occasional gentle inversion of the tubes. Finally, the tube was placed on ice. Fifty microlitres of the cell suspension was added to 450 µl LKS buffer containing calcium, mixed on a vortex mixer, and assayed in the flow cytometer, with the run time set for 100 s. The machine was paused for 30 s and the agonist, procalcitonin, interleukin-8 (IL-8), calcitonin-gene related peptide (CGRP), LPS, or fMLP, added in 5–50 µl volumes. Control tubes with sample cells incorporating the appropriate volumes of vehicle were also included. All tubes were vortexed after each addition before the run was restarted. The flow cytometer was primed after each sample and washed with Isoton II solution (Beckman Coulter) for approximately 10 s.
Contractile studies
Porcine hearts were obtained from a local abattoir and transported to the laboratory in Krebs–Henseleit solution maintained at 4°C within 1 h. The anterior descending branch of the coronary artery was dissected from the hearts and cleaned of connective tissues as previously described.11 The artery was then divided into 5 mm long segments and placed in 2 ml Dulbecco's Modified Eagle Medium (DMEM) containing 60 µg ml–1 benzyl penicillin and 20 µg ml–1 streptomycin sulphate which was previously gassed with O2 95% and CO2 5% for 5 min and stored at either 4°C or 37°C for 16–18 h. In some experiments, segments stored at 37oC were co-incubated with either LPS 100 µg ml–1, procalcitonin 100 nM, procalcitonin 100 nM and LPS 100 µg ml–1, or dexamethasone 1 µM and LPS 100 µg ml–1 for 16–18 h. Dexamethasone was added 60 min before the addition of LPS, whereas procalcitonin was added either 30 min before or 3 h after the addition of LPS. In a further set of experiments, preparations were stored overnight at 37°C in DMEM (and a mixture benzyl penicillin 100 µg ml–1 and streptomycin 20 µg ml–1) with either LPS 100 µg ml–1 or a combination of LPS 100 µg ml–1 and nitro-L-arginine methyl ester (L-NAME) 100 µM, a non-selective inhibitor of nitric oxide synthase.14 The preparations were stored in sealed glass vials which had previously been autoclaved. All dissection instruments were washed immediately after use and stored in 70% industrial methylated spirit until required.
After overnight storage, segments were taken out of the incubation solution and prepared for isometric tension recording. The segments were suspended between two stainless steel wire (0.4 mm diameter) supports and placed in a 10 ml isolated organ bath containing Krebs–Henseleit solution (pH 7.4) gassed with O2 95% and CO2 5% and maintained at 37°C. In all cases, care was taken to ensure that the integrity of the endothelium was maintained. The lower support was fixed to a glass holder, whereas the upper support was connected to a Grass FT03 isometric force transducer by cotton thread. Some laxity in the suspended segment was maintained for approximately 40 min before the application of resting tension. The force transducer was connected to a MacLab Bridge amplifier and linked via a four-channel Maclab unit to a Macintosh LC II computer running chart 3.5.
An initial resting tension of 10 g wt was slowly applied to each segment at the end of the equilibration period and the recorded tension declined to 4–6 g wt over a further 40 min period. Each preparation was then exposed to KCl 60 mM for 15 min until a sustained response was obtained, followed by washout and further 15 min equilibration. A further two exposures to KCl 60 mM were performed until reproducible contractions were observed. The preparations were constricted with 5–50 nM U46619
[GenBank]
(a stable thromboxane mimetic analogue, 9,11-dideoxy-11a, 9a-epoxymethanoprostaglandin F2) in order to produce a degree of tone equivalent to approximately 60% (40–80%) of the response to KCl 60 mM. Once a stable response was achieved, preparations were exposed to substance P (10 nM) to evaluate the integrity of the endothelium. In some experiments, the tip of a fine forcep was inserted into the lumen and the segment gently rolled on moistened paper tissue to remove the endothelium. ‘Endothelium-denuded’ segments failed to respond to substance P, whereas only segments that responded with a rapid relaxation of greater than 40% of U46619
[GenBank]
-induced tone were considered ‘endothelium-intact’.
In initial experiments on segments stored overnight at 4°C, the acute effect of cumulatively increasing concentrations of procalcitonin against U46619 [GenBank] -induced tone revealed variable relaxations, so subsequent experiments involved an examination of the hormone using non-cumulative concentrations (10 and 100 nM) followed by cumulative exposure to CGRP. Cumulative concentration–response curves to U46619 [GenBank] were also constructed in the presence and absence of procalcitonin 100 nM. In segments stored at 37°C, overnight cumulative concentration–response curves to U46619 [GenBank] were generated followed by assessment of the vasodilator activity of sodium nitroprusside. In some experiments, preparations were exposed to 100 µM L-NAME for 30 min before the U46619 [GenBank] or sodium nitroprusside-induced concentration–response curves.
Measurement of TNF in the porcine coronary artery
Segments from the porcine isolated coronary artery were incubated in 1 ml of K-H solution containing penicillin 60 µg ml–1, streptomycin 20 µg ml–1, and 2% w/v ficoll. The solution was gassed with O2 95% and CO2 5% for 5 min before incubation and exposed to procalcitonin 100 nM overnight in the presence or absence of 100 µg ml–1 LPS. The samples were placed in capped sterile vials overnight in an air incubator at 37°C. The segments were then taken out of the medium and pressed using a 50 g wt three times on a piece of dry towel to remove the excess moisture. The wet weight of each tissue was determined, while the sample media were frozen at –20°C for measurement of TNF at a later date.
A competitive ELISA for porcine TNF/TNFSF1A (R&D systems, Abingdon, UK) was used to measure the TNF content of the medium. The minimum detectable concentration of porcine TNF for this assay ranged from 2.8 to 5.0 pg ml–1 with a mean of 3.7 pg ml–1. After defrosting the samples, 50 µl assay diluent was added into a 96-well microplate that was coated with monoclonal antibody specific for porcine TNF followed by the addition of 50 µl standard concentration solution or samples. The microplate was then sealed and incubated for 2 h and after this period 400 µl washing buffer was pipetted into each well five times to wash out unbounded antibodies. Then the microplate was reversed on pieces of tidy dry towels to remove excess buffer. Porcine TNF conjugate (100 µl) was added into each well and allowed to incubate for 2 h. The plate was washed as described previously before the addition of the substrate solution (100 µl). The plates were incubated in the dark for a further 30 min before the reaction was stopped using 100 µl Stop Solution and the optical density determined using a microplate reader at 450 nm. The whole process was performed at room temperature.
Solutions and drugs
The composition of Krebs–Henseleit solution is (mM): NaCl, 118; KCl, 4.8; MgSO4·7H2O, 1.2; CaCl2·2H2O, 1.3; NaHCO3, 25.0; and KH2PO4, 1.2. Recombinant human procalcitonin was provided as a gift by B.R.A.H.M.S. Diagnostica GmbH (Berlin, Germany). Dexamethasone, L-NAME, sodium nitroprusside, LPS (E. coli O111:b4), fMLP, and ficoll were all obtained from Sigma-Aldrich Company Ltd (Poole, Dorset, UK). Substance P, CGRP, and IL-8 were obtained from Bachem (UK) Chemical Company (Delphe Court, Merseyside, UK). U46619
[GenBank]
was obtained from Alexis Corporation (Nottingham, UK).
Data analysis
Differences in mean forward scatter values for neutrophils and monocytes or fold increase in individual median fluorescence values were assessed by Student's paired t-test (two-tailed) or, where there was more than one treatment condition, by ANOVA followed by a post hoc Dunnett's test. For the assessment of changes in intracellular calcium (a marker for cell activation), no distinction was made between neutrophils, basophils, or eosinophils and the cells were deemed to be granulocytes. Granulocytes were first gated on a plot of side scatter and forward scatter before exposure to drugs and the proportion of granulocytes responding to the agonists with an increase in intracellular calcium determined. As shown in Figure 3A (see later in Results section), time bins (100 s) D and E were identified on the fluorescence—time plot for granulocytes such that the majority of cells (>90%) reside in time bin E—taken as the normal unstimulated state. Time bins D and E correspond to time bins F and G, respectively, for monitoring post-agonist events. After addition of the agonists, the proportion of granulocytes exhibiting elevated intracellular calcium and, therefore, evidence of being stimulated (time bin F) was determined.
Contractions produced by U46619 [GenBank] were measured as g wt force. The effect of the vasodilator agents was determined as a percentage of U46619 [GenBank] -induced tone. Where possible, the sensitivity of the preparation to the constrictor or dilator agent has been determined as the negative logarithm of the concentration causing 50% of the maximum response (pEC50).
The amount of TNF in each preparation was expressed as pmol per mg wet weight tissue and shown as median values with inter-quartile range (IQR). All other responses have been expressed as mean (SEM). Differences between mean values were assessed by Student's paired t-test (two-tailed) or, where there was more than one treatment condition, by ANOVA followed by a post hoc Dunnett's test. For comparison of TNF production under different conditions, a Wilcoxon signed rank test was used to assess statistical significance as the data were not normally distributed.
In all experiments, P<0.05 was considered statistically significant.
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The acute effect of procalcitonin on immune cells and vascular smooth muscle
Exposure to LPS for 30 min caused alteration in cell shape and activation of human neutrophils and monocytes as assessed by changes in forward scatter (Fig. 1); 10 µg ml–1 LPS producing a larger increase in forward scatter in neutrophils than in monocytes. LPS-induced activation was associated with a 1.49 (SD 0.05)-fold (n=14) increase in the expression of CD16 on neutrophils and a 1.70 (0.13)-fold (n=14) increase in the expression of CD14 on monocytes. Procalcitonin (100 nM) caused a significant (P<0.05) increase in forward scatter in human neutrophils and monocytes equivalent to 63.7 (7.5)% (n=14) and 79.7 (7.7)% (n=14), respectively, of the response produced by LPS 10 µg ml–1 (Fig. 1). Although procalcitonin 10 nM also significantly increased forward scatter (data not shown, n=5), lower concentrations (1 nM) of procalcitonin were inactive (n=5).
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The increase in forward scatter produced by LPS 10 µg ml–1 in neutrophils and monocytes, or changes in the expression of CD 16 on neutrophils, were not significantly altered by the inclusion of procalcitonin 100 nM (Table 1); expression of CD 14 on monocytes was not measured in this experiment. In marked contrast to procalcitonin, 100 nM CGRP did not affect either forward scatter values of neutrophils and monocytes, nor did it alter the expression of CD16 on neutrophils and CD14 on monocytes (Fig. 2). In addition, procalcitonin-induced changes in forward scatter were not significantly affected by co-incubation with 100 nM CGRP and the expression of CD16 (neutrophils) and CD14 (monocytes) was reduced without reaching statistical significance. (0.1>P>0.05, ANOVA, post hoc Dunnett's test).
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IL-8 and fMLP caused an increase in intracellular calcium in approximately 60% of granulocytes identified from the forward scatter/side scatter plot (Fig. 3). In marked contrast, neither LPS 10 µg ml–1 (n=4), CGRP 0.1 µM (Fig. 3, n=6), nor CGRP 1 µM (n=2) affected intracellular calcium in granulocytes. Although procalcitonin 100 nM did not significantly increase intracellular calcium in granulocytes (n=6), higher concentrations of procalcitonin (300 nM and 1 µM) caused a concentration-dependent increase in the proportion of granulocytes with elevated intracellular calcium (Fig. 3). A characteristic feature of the responses to procalcitonin was that the number of cells exhibiting elevated intracellular calcium was not sustained, unlike that elicited by 0.1 µM fMLP which was stable for the period of observation (not shown).
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Figure 4 shows that non-cumulative addition of procalcitonin 10 and 100 nM caused a slow-developing relaxation of U46619 [GenBank] -induced tone similar to that produced by CGRP. However, the magnitude of the relaxations to procalcitonin 10 nM [9.8 (2.3)%, n=14] and 100 nM [15.6 (4.1)%, n=14] were significantly (P<0.01) less than that produced by GCRP 10 nM [98.3 (3.2)%, n=14]. Neither the potency of CGRP [pEC50 –8.72 (0.08), n=14] as a relaxant of U46619 [GenBank] -induced contractions nor the maximum response was significantly (P>0.05) affected by the presence of procalcitonin 100 nM [pEC50 –8.87 (0.05), n=14; maximum CGRP relaxation 89.3 (5.6)%, n=14]. In a separate series of experiments, procalcitonin 100 nM inhibited U46619 [GenBank] -induced contractions [18.1 (3.8)%, n=7] in endothelium-denuded segments of the coronary artery.
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Long-term effects of procalcitonin on LPS-induced changes in vascular reactivity and TNF
productionU46619 produced concentration-dependent contractions that were significantly reduced in magnitude and potency (1.5-fold) after prolonged exposure to 100 µg ml–1 LPS (Table 2). Similarly, concentration-dependent relaxations to sodium nitroprusside were reduced four-fold in potency by prior exposure to 100 µg ml–1 LPS (Table 2) and these effects were associated with a significant increase (P<0.005, Wilcoxon test) in the amount of TNF
detected on the incubation medium [control median value (IQR) 3.4 (0–16.5) ng mg–1 wet wt; LPS median value (IQR) 10.0 (2–41.7) ng mg–1 wet wt, n=14].
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Table 2 shows that the presence of dexamethasone 1 µM during overnight incubation prevented LPS-induced changes in potency to both U46619 [GenBank] and sodium nitroprusside. Exposure to L-NAME 100 µM before evaluation of the effect of U46619 [GenBank] and sodium nitroprusside was associated with a slow-developing contraction in control preparations [0.68 (0.25) g wt, n=8] that was not significantly different (P>0.05) from segments exposed to 100 µg ml–1 LPS [0.91 (0.31) g wt, n=8]. In the presence of L-NAME 100 µM responses to U46619 [GenBank] and sodium nitroprusside after exposure to 100 µg ml–1 LPS were similar to control preparations (Table 2).
Overnight exposure to 100 nM procalcitonin alone did not significantly (P>0.05) affect contractions to U46619
[GenBank]
[control pEC50 –7.18 (0.04); post-procalcitonin pEC50 –7.19 (0.05) (n=12), Student's paired t-test]. However, the presence of procalcitonin 100 nM alone significantly increased (P<0.001, Wilcoxon test) the amount of TNF in the incubation medium [control median (IQR) 0.3 (0–2.8) ng mg–1 wet wt; procalcitonin median value (IQR) 10.6 (3.2–29.3) ng mg–1 wet wt, n=12], whereas the combination of procalcitonin 100 nM and LPS 100 µg ml–1 was associated with a further significant increase (P<0.05; Wilcoxon test) in the amount of TNF detected [LPS median value (IQR) 16.8 (2.19–51.7) ng mg–1 wet wt; LPS and procalcitonin median value (IQR) 20.0 (0.9–92.8), n=19].
Figure 5A shows that the inclusion of procalcitonin 100 nM 30 min before overnight exposure to LPS 100 µg ml–1 did not significantly alter the subsequent change in the sensitivity of the porcine coronary artery to U46619. [GenBank] Similarly, LPS-induced changes in the contractions of the porcine coronary artery to U46619 [GenBank] were not significantly altered when procalcitonin 100 nM was added 3 h after exposure to the endotoxin (Fig. 5B and Table 3). Table 3 shows that the LPS-induced reduction in potency of sodium nitroprusside was not significantly modified by the inclusion of procalcitonin 100 nM added either 30 min before or 3 h after exposure to LPS.
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Discussion |
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Several studies have highlighted qualitative similarities between the action of N-procalcitonin and CGRP on immune cells,6 7 with the latter generally viewed as possessing anti-inflammatory activity,15 16 whereas procalcitonin has been reported to cause heterologous desensitization of chemotactic responses in human monocytes.8
In the present study, procalcitonin showed a proinflammatory action; the peptide increased forward scatter in both neutrophils and monocytes and elevated the surface expression of CD16 and CD14, respectively. All of these effects are similar to those produced by other proinflammatory agents, such as fMLP and LPS,17–19 which is consistent with cell activation. The acute increase in the expression of CD16 and CD14 in response to proinflammatory stimuli is thought to reflect the movement of pre-formed molecules in secretory vesicles to the cell surface.17–19 These unambiguous findings contrast with earlier studies where procalcitonin was reported to be devoid of intrinsic activity in these models.6 20 Although Monneret and colleagues7 used CD11b/CD18 as a marker of cell activation, it is unlikely that this factor accounts for the difference between the studies since it is also stored preformed in secretory vesicles.
Significantly, CGRP did not mimic any of the proinflammatory effects of procalcitonin on either neutrophils or monocytes, nor did it significantly modify the action of the prohormone. These observations suggest that the acute proinflammatory actions of procalcitonin are not mediated by CGRP receptors. The difference between CGRP and procalcitonin is further underlined by the finding that high concentrations of procalcitonin increased intracellular calcium in granulocytes, while CGRP was inactive. This action is qualitatively similar to that produced by the chemoattractants IL-8 and fMLP and further emphasizes the proinflammatory nature of procalcitonin. Since this type of response is often associated with the activation of pertussis toxin-sensitive G protein-coupled receptors on granulocytes, we speculate that procalcitonin may also act at a specific receptor coupled in a similar manner, as suggested for the proinflammatory effects reported in human monocytes.21
In this study, acute exposure of the porcine isolated coronary artery to procalcitonin was associated with a small, endothelium-independent vasodilator response, equivalent to approximately 20% of the maximum effect elicited by either CGRP or sodium nitroprusside. This finding represents the first report of a direct effect of procalcitonin on vascular smooth muscle. Despite the qualitatively similar effects of procalcitonin and CGRP in the coronary artery, a high concentration of procalcitonin failed to modify responses to the more efficacious actions of CGRP (as might be expected for a partial agonist). Thus, the vasodilator effect of procalcitonin also appears to be mediated by a receptor distinct from that activated by CGRP. It remains to be determined whether procalcitonin produces similar responses in other vascular beds, or vessels associated with the microvasculature, but this is clearly an important area of investigation for understanding the role of this hormone in inflammatory processes.
The concentration of procalcitonin found to be biologically active in this study (10–100 nM) is higher than the plasma concentrations reported during septic shock and other disorders (1–10 nM),1–3 which suggests that the observations are of only pharmacological significance. However, it should be noted that activated monocytes, neutrophils, and adipocytes have the potential to synthesize procalcitonin,22 23 raising the possibility that the local concentration of the hormone, and that experienced by some vascular beds, may be far higher than that noted in the plasma. Thus, the present findings may have relevance to pathological conditions.
Prolonged exposure of the porcine isolated coronary artery to LPS caused a reduction in the potency of the vasoconstrictor U46619
[GenBank]
and the vasodilator sodium nitroprusside, as has been noted in other vessels, for example, porcine mesenteric24 and basilar arteries,25 the rat isolated aorta26 and human blood vessels.27 Furthermore, this effect of LPS was associated with a significant increase in basal levels of cyclic GMP (authors, unpublished observations) and TNF production. The latter finding is similar to that reported in both porcine and human vascular smooth muscle.28 29 The ability of dexamethasone and L-NAME, an inhibitor of nitric oxide synthase,14 to prevent/reverse LPS-induced changes in vascular responsiveness indicates that these effects can be attributed to the induction of nitric oxide synthase and excessive production of nitric oxide. Thus, suppression of U46619
[GenBank]
-induced contractions after exposure to LPS appears to involve elevated levels of cyclic GMP, whereas the impairment of relaxations to sodium nitroprusside may arise from the subsequent down-regulation of soluble guanylyl cyclase activity.26 30
In the present study, significantly, in the model of LPS-induced changes in vascular responsiveness, prolonged co-incubation with procalcitonin failed to modify the effect of either U46619
[GenBank]
or sodium nitroprusside. Furthermore, even when procalcitonin was added 3 h after LPS, to more closely mimic events in the initial stages of sepsis,1 neither the maximum response nor the potency of either U46619
[GenBank]
or sodium nitroprusside was altered. No attempt was made to measure basal nitric oxide levels in the porcine coronary artery or changes in the activity of inducible nitric oxide synthase, as described by Hoffman and colleagues9 10 for cultured rat aortic smooth muscle cells, so it is not possible to directly compare findings. However, the most likely explanation for the discrepancy between the two studies is the use of a functional marker (contraction) in the present study that reflects the sum of all events in the isolated blood vessel. For example, prolonged exposure to LPS is known to up-regulate numerous vasoactive substances in smooth muscle, for example, COX-231 32 or endothelin-converting enzyme,33 that may mask any effect of procalcitonin on inducible nitric oxide synthase. Alternatively, the isolation procedure used for culturing the rat aorta9 10 may have exaggerated the effects of LPS and procalcitonin. Irrespective of the explanation for the discrepancy, this study emphasizes the importance of establishing a physiological correlate for changes in any molecular or biochemical marker. This point is underlined by the finding that both procalcitionin and LPS stimulated TNF production in this tissue and the combination of the two agents further increased cytokine production. However, only LPS produced changes in the responsiveness of the artery consistent with the induction of nitric oxide synthase. This finding clearly suggests that the production of TNF per se is not involved in LPS-induced changes in the vascular response.
In keeping with the results of Wiedermann and colleagues8 and Kaneider and colleagues,21 this study places the primary proinflammatory action of procalcitonin at the level of the immune system, particularly in terms of the multiplicity of effects on monocytes and neutrophils. Although immuno-neutralization of procalcitonin in pigs reversed the major cardiovascular complications (most notably hypotension) associated with sepsis,4 it seems likely that this is a secondary effect subsequent to activation of immune cells. Further studies are warranted to investigate the interaction between leucocytes and vascular smooth muscle in inflammatory conditions, as suggested for other disorders.34
In summary, we have established that the acute action of procalcitonin on human neutrophils and monocytes is primarily proinflammatory in nature. Procalcitonin also caused a small vasodilator response and stimulated TNF production in the porcine coronary artery, but did not directly modify LPS-induced suppression of vascular responses. The action of procalcitonin on both the vasculature and immune cells appears to involve a receptor distinct from that for CGRP.
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A.V. was supported by the European Society of Anaesthesiology (Grant no. RJ 2339).
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We wish to thank Woods Ltd, Clipstone, Nottinghamshire, for the supply of porcine hearts. The authors are indebted to Dr Sarah Rayment for helpful comments during preparation of the manuscript.
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1 Becker KL, Nylen ES, White JC, et al. Procalcitonin and the calcitonin gene family of peptide in inflammation, infection, and sepsis: a journey from calcitonin back to its precursors. J Clin Endocrinol (2004) 89:1512–25.
2 Giamarellos-Bourboulis EJ, Giannapoulou P, Grecka P. Should procalcitonin be introduced in the diagnostic criteria for the systemic inflammatory response syndrome and sepsis. J Crit Care (2004) 19:152–7.[CrossRef][Web of Science][Medline]
3 Mokart D, Merlin M, Sannini A, et al. Procalcitonin, interleukin 6 and systemic inflammatory response syndrome (SIRS): early markers of post-operative sepsis after major surgery. Br J Anaesth (2005) 94:767–37.
4 Wagner KE, Martinez JM, Vath SD, et al. Early immuno-neutralization of calcitonin precursors attenuates the adverse physiologic response to sepsis in pigs. Crit Care Med (2002) 30:2313–21.[CrossRef][Web of Science][Medline]
5 Martinez JM, Wagner K, Snider RH, et al. Late immuno-neutralization of procalcitonin arrests the progression of lethal porcine sepsis. Surg Infect (2001) 2:193–202.[CrossRef]
6 Monneret G, Pachot A, Laroche B, et al. Procalcitonin and calcitonin gene related peptide decrease LPS-induced TNF production by human circulating blood cells. Cytokine (2000) 6:762–4.
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