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*12-O-TETRADECANOYLPHORBOL-13-ACETATE
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*CALCIUM, ELEMENTAL
*NITRIC OXIDE
Endocrinology Vol. 145, No. 3 1106-1112
Copyright © 2004 by The Endocrine Society

Relaxin Inhibits the Activation of Human Neutrophils: Involvement of the Nitric Oxide Pathway

Emanuela Masini, Silvia Nistri, Alfredo Vannacci, Tatiana Bani Sacchi, Andrea Novelli and Daniele Bani

Departments of Preclinical and Clinical Pharmacology (E.M., A.V., A.N.) and Anatomy, Histology, and Forensic Medicine (S.N., T.B.S., D.B.), Section Histology; University of Florence, I-50139 Florence, Italy

Address all correspondence and requests for reprints to: Prof. Daniele Bani, Dipartimento di Anatomia, Istologia e Medicina Legale, Sezione di Istologia., Viale G. Pieraccini 6, I-50139 Firenze, Italy. E-mail: daniele.bani{at}unifi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In animal models of inflammation, the pregnancy hormone relaxin was shown to reduce the recruitment of leukocytes, especially neutrophils, in inflamed tissues. The current study was designed to clarify whether relaxin could inhibit activation of isolated human neutrophils and, if so, whether the nitric oxide (NO) biosynthetic pathway was involved, as occurs in other relaxin targets. Human neutrophils were preincubated with 1, 10, and 100 nmol/liter porcine relaxin for 1 h before activation with N-formyl-Met-Leu-Phe (10 µmol/liter) or phorbol-12-myristate-13-acetate (0.1 µmol/liter). In selected experiments, the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 100 µmol/liter) was added to the samples 30 min before relaxin. In other experiments, chemically inactivated relaxin (10 nmol/liter) was substituted for authentic relaxin. Untreated, nonactivated neutrophils were the controls. Relaxin reduced significantly and in a concentration-dependent fashion the expression of the surface activation marker CD11b, as well as the generation of superoxide anion, the rise of intracellular Ca2+, the release of cytoplasmic granules, and the chemotactic migration. These effects of relaxin were blunted by NG-monomethyl-L-arginine and could not be reproduced by inactivated relaxin. Relaxin also increased neutrophil inducible NO synthase expression and NO generation. This study provides evidence that relaxin inhibits the activation of human neutrophils stimulated by different proinflammatory agents. This novel property of relaxin could be of relevance in toning down maternal neutrophil activation during pregnancy, thereby counteracting the occurrence of pregnancy-related disorders such as preeclampsia, which is regarded as an excess maternal inflammatory response to pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUTROPHILS REPRESENT A crucial component of the nonspecific defense against bacterial infection and inflammatory response to tissue injury (1). Activated neutrophils adhere to the vascular endothelium, migrate into the inflamed tissue, release high amounts of reactive oxygen species (ROS) and lysosomal enzymes that are lethal to microbes, and eliminate bacteria and tissue debris by phagocytosis. Because the agents released by neutrophils are also toxic to host tissues, neutrophil response may be a critical step in inflammatory tissue damage, as for instance occurs in cardiac ischemia followed by reperfusion (2, 3). Thus, modulation of neutrophil activation is of key importance in determining the balance between host defense and host injury (4).

Recently, evidence has been provided that the peptide hormone relaxin (RLX), a member of the insulin/IGF superfamily (5), has a prompt, potent protective action against tissue injury in animal models of inflammatory diseases. Experiments in rats subjected to cardiac ischemia/reperfusion in vivo have shown that RLX protects against myocardial injury, an effect that is accompanied by a marked reduction of neutrophil extravasation into the tissue and of neutrophil-induced peroxidative damage due to generation of ROS (6). Similarly, RLX has been found to reduce the severity of asthma-like reaction in sensitized guinea pigs subjected to inhalation of the allergen, an effect that is paralleled by a clear-cut reduction of leukocyte infiltration in the lungs (7). On these grounds, it appears that RLX could exert an antiinflammatory effect by interfering with neutrophil activation and/or migration into the inflamed tissue.

The possibility that RLX can down-regulate neutrophil function is not completely unsustained, for RLX has been shown to depress the function of other bone marrow-derived cells, such as monocytic cells (8), mast cells (9), platelets (10), and basophils (11). Of note, in the above cells, the inhibitory action of RLX was mediated by the stimulation of endogenous nitric oxide (NO) generation. Indeed, the role of NO in neutrophil function is not completely understood, at least in humans (4). It has been shown that, like macrophages, activated neutrophils can express inducible NO synthase (iNOS) (12, 13), but there is still controversy as to whether neutrophils can produce functionally significant levels of NO (14). NO and its intracellular effector cGMP seem to mediate neutrophil chemotactic response (4), but paradoxically, NO donors can inhibit various aspects of neutrophil activation, including chemotaxis (15), ROS generation (16), and phagocytosis (17). This discrepancy may depend on the effects of different concentrations of NO on neutrophil behavior. In this view, NO donors could act as neutrophil inhibitors because they are able to release high NO amounts (4). The possibility that substances capable of up-regulating endogenous NO generation, such as RLX, may influence neutrophil function is currently unproven.

The present in vitro study was designed to clarify whether RLX can counteract the activation of human neutrophils stimulated by N-formyl-Met-Leu-Phe (fMLP) and, if so, to investigate whether the mechanism of action of RLX involves endogenous NO production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Unless otherwise specified, the reagents used were from Sigma Chemical Co. (St. Louis, MO). Sera for cell culture were from Life Technologies, Inc. (Paisley, UK). Cell culture plastic ware was from Falcon (Becton Dickinson Europe, Meylan, France). Highly purified porcine RLX (6 kDa, 2500–3000 U/mg), prepared according to Sherwood and O’Byrne (18), was generously provided by Dr. O. D. (Sherwood University of Illinois at Urbana-Champaign, Urbana, IL). Inactivated RLX (iRLX) was prepared by reaction with 1,2-cyclohexanedione and subsequent dialysis against distilled water, according to the method of Buellesbach and Schwabe (19). 1,2-Cyclohexanedione specifically binds to the arginines of the receptor-binding domain of the RLX B chain, thus preventing the modified hormone from binding to the RLX receptor.

Isolation of human neutrophils
Approximately 20 ml of venous blood anticoagulated with sodium heparin (500 IU/ml; Hepoxoclar Lab, Milan, Italy) was collected from 18 healthy adult male volunteers who did not suffer from inflammatory or infectious diseases and had not taken any drug in the previous 4 wk. They gave explicit informed consent to their enrolling in this study. Males were chosen to avoid possible influences due to endogenous luteal RLX on the experimental findings. The experimental protocol was designed in compliance with the guidelines of the Ethical Committee of the University of Florence, Italy. Neutrophils were obtained by dextran sedimentation followed by Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation and hypotonic lysis for the removal of residual erythrocytes. The final neutrophil pellet was suspended in a buffer solution of the following composition (mmol/liter): 138 NaCl, 2.7 KCl, 8.1 Na2HPO4, 1.5 KH2PO4, 1 MgCl2(6 H2O), and 1 CaCl2 to a final concentration of 2.5 x 105 cells/ml. Cell viability checked by trypan blue exclusion was >95%.

Treatments
Aliquots of the neutrophil suspension were incubated with RLX at concentrations of 1, 10, and 100 nmol/liter for 1 h at 37 C. These concentrations and exposure times were chosen because they are in the range of those previously found to be inhibitory for mast cells, platelets, and basophils (9, 10, 11). In selected experiments, the NOS competitive inhibitor NG-monomethyl-L-arginine (L-NMMA, 100 µmol/liter) was added to the samples 30 min before RLX to achieve a complete NOS inhibition at the moment of RLX addition. In other experiments, iRLX (10 nmol/liter) was substituted for authentic RLX. Some aliquots were not treated with any substance and were used as controls. Finally, neutrophils underwent activation before being processed for the analyses reported below. Receptor-mediated neutrophil activation was achieved by fMLP (Serva, Heidelberg, Germany) added to the neutrophil suspensions to a 10 µmol/liter final concentration and allowed to act for 10 min at 37 C. In preliminary dose-response experiments, a concentration of 10 µmol/liter fMLP was identified as that causing a maximal response of neutrophils, in terms of superoxide production (see below). The fMLP concentration was reduced to 1 µmol/liter in the chemotaxis assay, as high fMLP may inhibit chemotaxis per se. In some experiments, fMLP was replaced with phorbol-12-myristate-13-acetate (PMA, 0.1 µmol/liter) to induce receptor-independent activation. Untreated, nonactivated aliquots of neutrophils were also used, and they are referred to as unstimulated neutrophils.

Cytofluorimetric analysis
The neutrophil suspensions were treated as described above and labeled with anti-CD11b phycoerythrin-conjugated antibodies (Coulter, Hialeah, FL) at saturating concentrations as provided by the manufacturer. Surface expression of CD11b is considered a reliable marker to identify activated neutrophils (20). The fluorescent antibodies were incubated with the cell suspensions for 20 min. The samples were then centrifuged at 200 x g for 10 min at room temperature and the pellets resuspended in maintenance buffer and analyzed with a flow cytometer (EPICS XL, Coulter). The neutrophil-related events were sorted using appropriate electronic gates. The presence or absence of the fluorescent signal from phycoerythrin (emission peak at 575-nm wavelength) was used to characterize activated and nonactivated neutrophils, respectively.

Superoxide production assay
Aliquots of the neutrophil suspensions, each containing 105 cells, were treated as described above. The cells were then treated with cytochalasin B (5 µg/ml) before exposure to fMLP or PMA to inhibit cell motility and spontaneous degranulation and to potentiate superoxide anion production (21). Superoxide anion was monitored by the method reported by Babior et al. (22), which allows a continuos spectrophotometrical measurement of the superoxide dismutase-induced inhibition of cytochrome c reduction (both reagents were from Boehringer, Mannheim, Germany). None of the drugs used in the experiments affected superoxide anion production in a cell-free, xanthine-xanthine oxidase system.

Intracellular Ca2+ assay
Aliquots of the neutrophil suspension were incubated with RLX (10 nmol/liter), RLX plus L-NMMA, or iRLX for 1 h. Aliquots of untreated neutrophils were the controls. Then, neutrophils were centrifuged and resuspended in a medium containing (mmol/liter) 10 HEPES, 140 NaCl, 3 KCl, 0.1 MgCl2, and 1 CaCl2 plus 0.1% glucose, 0.1% human serum albumin, and Sörensen phosphate buffer to pH 7.4. Neutrophils were loaded with fura-2 AM (3 µmol/liter) for 30 min into a shaking water bath at 37 C, centrifuged at 200 x g, washed twice, resuspended in the same medium to a concentration of 2 x 105 cells/ml, and placed in quartz cuvettes at 37 C. Neutrophils were then activated with fMLP (10 µmol/liter), except for an unstimulated aliquot that was used to evaluate basal intracellular Ca2+ levels. Cytosolic free Ca2+ concentrations were determined fluorimetrically using a Shimadzu DR 15 spectrofluorimeter (Osaka, Japan). In a preliminary experiment, the fluorescence excitation spectrum of fura-2 AM-loaded neutrophils activated by fMLP was scanned for wavelengths ranging from 300–420 nm with an emission wavelength fixed at 510 nm to identify the peak excitation wavelength. This wavelength (usually 380 nm) was then used for experimental measurement. The values of cytosolic Ca2+ concentration were estimated by specific software, as described previously (10).

Electron microscopy
Neutrophil suspensions treated as reported above were pelleted by centrifugation, fixed in cold 4% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h at room temperature and postfixed in cold 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 h at room temperature. The pellets were then dehydrated in graded acetone, passed through propylene-oxide, and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and alkaline bismuth subnitrate and examined under a JEM 1010 electron microscope (Jeol, Tokyo, Japan) at 80 kV.

Chemotaxis assay
The chemotaxis assay was carried out by the under-agarose migration assay, as reported previously (23). Briefly, six-well multiplates were filled with 3 ml of a 1% agarose solution containing 50% H2CO3-buffered Hanks’ balanced salt solution and 50% RPMI 1640 culture medium with 20% fetal calf serum. After the agarose solidified, three wells, 3 mm in diameter and 3.5 mm apart, were cut into a straight line in the gel. The gels were allowed to equilibrate for 1 h at 37 C. The center well was loaded with the suspension of neutrophils, not treated or treated with RLX or iRLX at the noted concentrations, the outer left well was loaded with fMLP (1 µmol/liter) as chemoattractant, and the outer right well was loaded with phosphate buffer and served as control for random migration. Once loaded, the gels were incubated for 2 h at 37 C in a cell culture incubator with a 5% CO2/95% O2 atmosphere, which allowed sufficient time for neutrophils to migrate toward the outer wells. At the end of the incubation, agarose was removed and the cells were fixed in methanol and stained with Giemsa. Results were recorded using a video camera (model Colcam OS-75D1, WPI, Sarasota, FL) applied to a Nikon TMS microscope. The under-agarose assay allows quantification of both random migration (RM) and directional chemotactic migration (CM). To determine whether the migration we observed was directional, two test areas were analyzed. Area A was a segment 500 x 2000 µm between the neutrophil-containing well and the fMLP-containing well. Area B was of the same size and extended from the neutrophil-containing well to the buffer-containing well. The number of cells within the two test areas was counted; thus, the number of cells in area A accounted for CM and that in area B accounted for RM. The chemotaxis index was calculated as the ratio between CM and RM.

Western blot analysis for iNOS
Neutrophil preparations, either untreated or treated with RLX (10 nmol/liter) for 1 h at 37 C, were pelleted and resuspended in cold lysis buffer of the following composition: 20 mM Tris/HCl (pH 7.4), 10 mM NaCl, 1.5 mM MgCl2, 5 mM EGTA, 2 mM Na2EDTA, 1 mM phenylmethylsulfonyl fluoride, 1%Triton X-100, 20 µg/ml leupeptin, 1 µg/ml pepstatin, 500 µg/ml Pefabloc, and 2.5 µg/ml aprotinin. The supernatant was collected for nitrite assay, as described below. Upon centrifugation at 17,000 x g at 4 C, the supernatants were collected and the total protein content was measured spectrophotometrically using the bicinchoninic acid assay (Pierce, Rockford, IL). The samples, each containing 80 µg of proteins, were electrophoresed by SDS-PAGE (200 V for 1 h) using a denaturating 7.6% polyacrylamide gel with proper molecular weight markers (Bio-Rad, Hercules, CA) and blotted onto nitrocellulose membranes (Amersham, Milan, Italy) (150 V for 1 h). After thorough washes in PBS added with 0.1% Tween (T-PBS), the membranes were treated with 5% albumin in T-PBS and incubated overnight at 4 C under stirring with rabbit polyclonal antibodies against iNOS (Alexis, Laeufelingen, Switzerland), diluted 1:50,000 in T-PBS added with 1% BSA. The membranes were also immunostained with antiactin antibodies (Zymed, San Francisco, CA; diluted 1:20,000), assuming actin as internal control protein. Immune reaction was revealed by peroxidase-labeled goat antirabbit antibodies (Vector, Burlingame, CA), diluted 1:10,000 in T-PBS with 1% BSA and applied to the membranes for 1 h at room temperature under stirring, followed by 1-min incubation with the chemiluminescent substrate ECL (Amersham) and exposure to high-sensitivity photographic film (Biomax ML, Kodak, Rochester, NY).

Nitrite assay
This step was performed by measuring the accumulation of nitrite, a stable end product of NO metabolism, in the supernatant of untreated and RLX-treated (10 nmol/liter) neutrophils. The amount of nitrite was determined spectrophotometrically by the Griess reaction adapted for a 96-well plate reader. In brief, 100 µl of sample was added to 100 µl of Griess reagent (1% sulfanilamide and 0,1% N-[1-naphtyl]ethylendiamine in 5% phosphoric acid). The optical density at a wavelength of 546 nm was measured with a Bio-Rad 550 microplate reader. Nitrite concentrations in the supernatants were calculated by comparison with standard concentrations of NaNO2 dissolved in culture medium. The reported values are the mean (±SEM) of three separate experiments.

Statistical analysis
Unless otherwise stated, the reported data are expressed as mean ± SEM of at least four independent experiments. Statistical comparison of differences between the different groups was carried out using one-way ANOVA test followed by Student-Newman-Keuls multiple comparison test. A P value <= 0.05 was considered significant. Calculations were done using a GraphPad Prism 2.0 statistical program (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytofluorimetric analysis (Fig. 1Go) demonstrated that, upon treatment with fMLP (10 µmol/liter) or PMA (0.1 µmol/liter), the percentage of cells expressing the activation marker CD11b at their surface was increased compared with the unstimulated neutrophil preparations. Preincubation with RLX before the addition of activators resulted in a concentration-dependent reduction of the percentage of activated neutrophils, which reached statistical significance with 10 nmol/liter RLX (P < 0.05) and dropped even to the basal levels with 100 nmol/liter RLX (P < 0.001). Conversely, neither RLX (10 and 100 nmol/liter) given in combination with the NOS inhibitor L-NMMA (100 µmol/liter) nor iRLX (10 nmol/liter) gave any significant reduction.



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  		FIG. 1. A, Representative flow cytometry of human neutrophils labeled with anti-CD11b: unstimulated cells (left panel), cells challenged with 10 µmol/liter fMLP for 10 min (center panel), and cells pretreated with 10 nmol/liter RLX for 1 h before fMLP challenge (right panel). The number of CD11b-expressing neutrophils increases upon fMLP stimulation and is reduced by RLX pretreatment. Each point represents a cluster of individual cell events. The total cell number in each plot is indicated by n. B, The expression of CD11b by activated neutrophils is reduced by RLX in a dose-related fashion. The effect of RLX is reverted by L-NMMA (100 µmol/liter) and is not reproduced by iRLX (10 nmol/liter). White column, unstimulated neutrophils; gray columns, fMLP-treated neutrophils; striped columns, PMA-treated neutrophils. Each column is the mean (±SEM) of four separate experiments. Significance of differences (one-way ANOVA): a, P < 0.001 vs. basal; b, P < 0.05 vs. activated neutrophils; c, P < 0.001 vs. activated neutrophils; d, P < 0.05 vs. 10 nmol/liter RLX; e, P < 0.001 vs. 10 nmol/liter RLX; f, P < 0.001 vs. 100 nmol/liter RLX; g, P > 0.05 vs. 10 nmol/liter RLX; h, P > 0.001 vs. 10 nmol/liter RLX.

 
When assaying production of superoxide anion by neutrophils (Fig. 2Go), we found that this free radical was barely detectable in the unstimulated preparations, whereas it rose significantly upon challenge with fMLP (P < 0.01) or PMA (P < 0.01). RLX pretreatment caused a dose-dependent decrease in the amount of superoxide anion that reached statistical significance at 10 and 100 nmol/liter RLX (P < 0.001). The effect of RLX (10 and 100 nmol/liter) was abolished when the hormone was given in combination with L-NMMA (100 µmol/liter). On the other hand, iRLX (10 nmol/liter) had no significant effect.



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  		FIG. 2. Production of superoxide anion by human neutrophils under different experimental conditions. Pretreatment of the cells with RLX reduces the rise in superoxide anion generation induced by fMLP (10 µmol/liter) or PMA (0.1 µmol/liter) in a concentration-dependent fashion. The effect of RLX is blunted by L-NMMA (100 µmol/liter) and is not reproduced by iRLX (10 nmol/liter). White column, unstimulated neutrophils; gray columns, fMLP-treated neutrophils; striped columns, PMA-treated neutrophils. Each column is the mean (±SEM) of six separate experiments. Significance of differences (one-way ANOVA): a, P < 0.01 vs. basal; b, P < 0.001 vs. activated neutrophils; c, P < 0.01 vs. 10 nmol/liter RLX; d, P < 0.001 vs. 10 nmol/liter RLX; e, P < 0.001 vs. 100 nmol/liter RLX.

 
Assay of intracellular Ca2+ (Fig. 3Go) revealed very low levels of this ion in the untreated neutrophils, whereas it rose markedly in the samples activated with fMLP (10 µmol/liter). On the other hand, the fMLP-induced intracellular Ca2+ rise underwent a clear-cut, significant reduction in the neutrophils preincubated with 10 nmol/liter RLX (P < 0.001). Of note, either RLX (10 nmol/liter) given in combination with L-NMMA (100 µmol/liter) or iRLX (10 nmol/liter) were ineffective in reducing the fMLP-induced Ca2+ elevation.



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  		FIG. 3. Intracellular Ca2+ levels in human neutrophils under different experimental conditions. Pretreatment of the cells with RLX (10 nmol/liter) reduces the rise in Ca2+ induced by fMLP (10 µmol/liter). The effect of RLX is blunted by L-NMMA (100 µmol/liter) and is not reproduced by iRLX (10 nmol/liter). White column, unstimulated neutrophils; gray columns, fMLP-treated neutrophils. Each column is the mean (±SEM) of four separate experiments. Significance of differences (one-way ANOVA): a, P < 0.001 vs. basal; b, P < 0.001 vs. fMLP alone; c, P < 0.001 vs. RLX.

 
By electron microscopic examination (Fig. 4Go), untreated neutrophils were characterized by many specific and nonspecific cytoplasmic granules (Fig. 4AGo). In the samples challenged with fMLP (10 µmol/liter), neutrophils had mostly the features of activated cells, with very few cytoplasmic granules (Fig. 4BGo). Conversely, in the samples preincubated with RLX (10 nmol/liter) before challenge with fMLP, most of the neutrophils contained appreciable amounts of cytoplasmic granules (Fig. 4CGo), and in the samples treated with L-NMMA (100 µmol/liter) and RLX (10 nmol/liter) before fMLP challenge, neutrophils were similar to those challenged with fMLP alone, with a marked reduction of cytoplasmic granules (Fig. 4DGo).



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  		FIG. 4. Representative electron micrographs of human neutrophils in different experimental conditions. A, Untreated controls, with the cell showing many specific and nonspecific cytoplasmic granules of varying electron density and inconspicuous synthetic organelles; B, challenge with fMLP (10 µmol/liter), with the cell showing very few cytoplasmic granules; C, pretreatment with RLX (10 nmol/liter) before fMLP challenge, with the cell containing appreciable amounts of cytoplasmic granules; D, pretreatment with L-NMMA (100 µmol/liter) and RLX before fMLP challenge, with the cell showing a marked reduction of cytoplasmic granules. Magnification, x7500; bar, 1 µm.

 
Evaluation of chemotaxis by under-agarose migration assay (Fig. 5Go) showed that RLX, at any concentration assayed, markedly reduced the chemotactic response of neutrophils to fMLP (1 µmol/liter). Conversely, iRLX had little or no effect.



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  		FIG. 5. Chemotaxis index of human neutrophils under different experimental conditions, as shown by under-agarose assay. Pretreatment of the cells with RLX reduces the chemotactic migration induced by fMLP (1 µmol/liter) at any concentration assayed. The effect of RLX is not reproduced by iRLX (10 nmol/liter). Each column is the mean (±SEM) of four separate experiments. Significance of differences (one-way ANOVA): a, P < 0.001 vs. fMLP alone; b, P < 0.01 vs. 10 nmol/liter RLX.

 
Determination of iNOS expression by Western blot (Fig. 6AGo) revealed that a 1-h treatment with RLX (10 nmol/liter) caused an increase in iNOS protein as compared with the untreated cells. In keeping with this finding, the amount of nitrites, the end products of NO, in the neutrophil supernatant (Fig. 6BGo) also increased significantly upon RLX treatment (P < 0.001). This effect of RLX was reverted by L-NMMA (100 µmol/liter).



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  		FIG. 6. A, Western blot analysis of iNOS protein expression by neutrophils in basal conditions and upon treatment with RLX (10 nmol/liter for 1 h). RLX causes an increase in iNOS expression. On the other hand, actin assumed as control invariant protein is unchanged. B, Nitrite amounts in the supernatants of neutrophils. RLX (10 nmol/liter for 1 h) causes a marked increase in nitrite, which was abolished by coincubation with L-NMMA (100 µmol/liter). Significance of differences (one-way ANOVA): a, P < 0.001 vs. basal; b, P < 0.001 vs. RLX alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides direct evidence that RLX prevents the inflammatory activation of human neutrophils. A 1-h in vitro preincubation of isolated neutrophils with RLX at concentrations ranging from 1–100 nmol/liter before exposure to fMLP or PMA caused a concentration-related, significant decrease of key parameters of neutrophil activation, such as surface CD11b expression and superoxide anion generation. RLX also caused a clear-cut reduction of the fMLP-induced rise of intracellular Ca2+ and release of cytoplasmic granules and blunted markedly fMLP-induced chemotactic migration in vitro. Human neutrophils appear to be specific targets for RLX, as judged by the dose dependency of the inhibitory effect of RLX on the assayed parameters of neutrophil activation and by the fact that iRLX given in the place of the authentic hormone was inactive. These findings indirectly account for a receptor-mediated mechanism of action of RLX and suggest that these cells could express the specific RLX receptors LGR7 and/or LGR8 (24). The inhibitory effects of RLX do not appear to involve a mere competition for fMLP receptors; in fact, RLX is able to depress neutrophil activation even when induced by PMA through a receptor-independent pathway.

RLX also acts on neutrophils by increasing the expression of iNOS protein and the release of NO, as shown by the rise of nitrites, the stable end products of NO, in the cell supernatants. Because iNOS is known to yield high amounts of NO, it is conceivable that the inhibition by RLX of neutrophil activation may depend, at least in part, on the stimulation of the generation of high levels of endogenous NO by these cells, as high NO concentrations are deemed able to inhibit neutrophil function (4). This is also supported by the observation that the inhibitory action of RLX on several parameters of neutrophil activation was blunted when this hormone was given in combination with the NOS competitive inhibitor L-NMMA. This finding is in keeping with previous observations on the effects of RLX on other target cells, including vascular cells (25, 26) and bone marrow-derived cells (8, 9, 10, 11), in which RLX was also found to increase NO generation by up-regulating iNOS.

The possible pathophysiological consequences of the RLX-induced inhibition of neutrophil activation are a matter of speculation. Likely, this property of RLX can account for the reduction of neutrophil infiltration into the inflamed tissue observed in hearts of rats subjected to experimental myocardial infarction (6) and lungs of guinea pigs undergoing an allergic asthma-like reaction (7). This view is further supported by the chemotaxis experiments, in which RLX has also been shown to blunt neutrophil chemotactic migration. Going a step further, it is possible that RLX, which is mainly produced during pregnancy (27), might be involved in toning down maternal neutrophil activation during gestation to prevent the onset of pregnancy disorders such as preeclampsia and the consequent intrauterine fetal growth restriction. Recently, evidence has been provided by independent research groups that preeclampsia is characterized by elevated cytokine production and leukocyte activation (28, 29, 30, 31), and it has been suggested that preeclampsia may represent an excess maternal inflammatory response to pregnancy (32). In particular, it has been shown that maternal neutrophils are activated during their passage in the decidua of women with preeclampsia (30) and that neutrophil activation can be achieved by the conditioned medium of placental villous culture taken from preeclamptic pregnancies (33). Both these data suggest that decidual/placental factors are involved in this phenomenon. In humans, besides the circulating RLX of luteal origin, RLX is also produced by the decidua and the trophoblast (34, 35 ; also reviewed in Ref. 5). This decidual RLX isoform is thought to play a local, paracrine role in endometrial tissue differentiation and remodeling and in embryo accommodation (36). The results of the present study suggest an additional possible role for decidual RLX, as in normal pregnancies it could counterbalance the local activating factors for neutrophils. Thus, preeclampsia could result from an imbalance between activating and inhibitory agents for neutrophils at the placental/decidual site, which might include an impaired local production of RLX.


   Acknowledgments
 
We are grateful to Dr. O. D. Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign (Urbana, IL), for his generous gift of purified porcine relaxin. Many thanks are also due to Dr. Laura Chiappini, Dr. Giovanni Zagli, Dr. Stefania Fallani, Dr. Maria Iris Cassetta, Mr. Patrizio Guasti, Mr. Daniele Guasti, and Mrs. Laura Calosi for skillful technical assistance. The administrative support of Mrs. Rita Scantimburgo is also acknowledged.


   Footnotes
 
This work was supported by a grant from the Italian Ministry for Education, University, and Research (MIUR), Co-funding of Research Programs of National Interest (PRIN), years 2000–2001, and by funds from the University of Florence.

Abbreviations: CM, Chemotactic migration; fMLP, N-formyl-Met-Leu-Phe; iNOS, inducible NO synthase; iRLX, inactivated relaxin; RM, random migration; L-NMMA, NG-monomethyl-L-arginine; PMA, phorbol-12-myristate-13-acetate; ROS, reactive oxygen species; T-PBS, PBS with Tween.

Received July 3, 2003.

Accepted for publication November 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Anderson BO, Brown JM, Harken AH 1991 Mechanisms of neutrophil-mediated tissue injury. J Surg Res 51:170–179[CrossRef][Medline]
  2. McCord JM 1985 Oxygen-derived free radicals in post-ischemic tissue injury. N Engl J Med 312:159–163[Abstract]
  3. Engler RL, Dahlgren MD, Morris D, Peterson MA, Schmid-Schönbein GW 1986 Role of leukocytes in the response to acute myocardial ischemia and reflow in dogs. Am J Physiol 251:H314–H323
  4. Armstrong R 2001 The physiological role and pharmacological potential of nitric oxide in neutrophil activation. Int Immunopharmacol 1:1501–1512[CrossRef][Medline]
  5. Bani D 1997 Relaxin: a pleiotropic hormone. Gen Pharmacol 28:13–22[Medline]
  6. Bani D, Masini E, Bello MG, Bigazzi M, Bani Sacchi T 1998 Relaxin protects against myocardial injury caused by ischemia and reperfusion in rat heart. Am J Pathol 152:1367–1376[Abstract]
  7. Bani D, Ballati L, Masini E, Bigazzi M, Bani Sacchi T 1997 Relaxin counteracts asthma-like reaction induced by inhaled antigen in sensitized guinea pigs. Endocrinology 138:1909–1915[Abstract/Free Full Text]
  8. Parsell DA, Mak JY, Amento EP, Unemori EN 1996 Relaxin binds to and elicits a response from cells of the human monocytic cell line, THP-1. J Biol Chem 271:27936–27941[Abstract/Free Full Text]
  9. Masini E, Bani D, Bigazzi M, Mannaioni PF, Bani Sacchi T 1994 Effects of relaxin on mast cells. In vitro and in vivo studies in rats and guinea pigs. J Clin Invest 94:1974–1980
  10. Bani D, Bigazzi M, Masini E, Bani G, Bani Sacchi T 1995 Relaxin depresses platelet aggregation. In vitro studies on isolated human and rabbit platelets. Lab Invest 73:709–713[Medline]
  11. Bani D, Baronti R, Vannacci A, Bigazzi M, Bani Sacchi T, Mannaioni PF, Masini E 2002 Inhibitory effects of relaxin on human basophils activated by stimulation of the FC{epsilon} receptor. The role of nitric oxide. Int Immunopharmacol 2:1195–1204[CrossRef][Medline]
  12. Laffi G, Foschi M, Masini E, Simoni A, Mugnai L, La Villa G, Barletta G, Mannaioni PF, Gentilini P 1995 Increased production of nitric oxide by neutrophils and monocytes from cirrhotic patients with ascites and hyperdynamic circulation. Hepatology 22:1666–1673[CrossRef][Medline]
  13. Evans TJ, Buttery LD, Carpenter A, Springall DR, Polak JM, Cohen J 1996 Cytokine-treated human neutrophils contain inducible nitric oxide synthase that produces nitration of ingestion bacteria. Proc Natl Acad Sci USA 93:9553–9558[Abstract/Free Full Text]
  14. Yan L, Vandivier RW, Suffredini AF, Danner RL 1994 Human polymorphonuclear leukocytes lack detectable nitric oxide synthase activity. J Immunol 153:1825–1834[Abstract]
  15. Wanikiat P, Woodward DF, Armstrong RA 1997 Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils. Br J Pharmacol 122:1135–1145[CrossRef][Medline]
  16. Moilanen E, Vuorinen P, Kankaanranta H, Metsa-Ketela T, Vapaatalo H 1999 Inhibition by nitric oxide-donors of human polymorphonuclear leukocyte functions. Br J Pharmacol 109:852–858
  17. Forslund T, Sundqvist T 1997 Nitric oxide-releasing particles inhibit phagocytosis in human neutrophils. Biochem Biophys Res Commun 233:492–495[CrossRef][Medline]
  18. Sherwood OD, O’Byrne EM 1974 Purification and characterization of porcine relaxin. Arch Biochem Biophys 60:185–196
  19. Büllesbach EE, Schwabe C 1988 On the receptor binding sites of relaxins. Int J Pept Protein Res 32:361–367[Medline]
  20. Ross GD 2000 Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/M2-integrin glycoprotein. Crit Rev Immunol 20:197–222[Medline]
  21. Fantozzi R, Brunelleschi S, Cambi S, Blandina P, Masini E, Mannaioni PF 1984 Autacoid and ß-adrenergic agonist modulation of N-formylmethionyl-leucyl-phenylalanine evoked lysosomal enzyme release from human neutrophils. Agents Actions 14:441–450[CrossRef][Medline]
  22. Babior BM, Kipnes RS, Curnutte JT 1973 Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 52:741–744
  23. Heit B, Tavener S, Raharjo E, Kubes P 2002 An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients. J Cell Biol 159:91–102[Abstract/Free Full Text]
  24. Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD, Hsueh AJW 2002 Activation of orphan receptors by the hormone relaxin. Science 295:671–673[Abstract/Free Full Text]
  25. Bani D, Failli P, Bello MG, Thiemermann C, Bani Sacchi T, Bigazzi M, Masini E 1998 Relaxin activates the L-arginine-nitric oxide pathway in vascular smooth muscle cells in culture. Hypertension 31:1240–1247[Abstract/Free Full Text]
  26. Failli P, Nistri S, Quattrone S, Mazzetti L, Bigazzi M, Bani Sacchi T, Bani D 2002 Relaxin up-regulates inducible nitric oxide synthase expression and nitric oxide generation in rat coronary endothelial cells. FASEB J 16:252–254[Free Full Text]
  27. Bell RJ, Eddie LW, Lester AR, Wood EC, Johnston PD, Niall HD 1987 Relaxin in human pregnancy serum measured with an homologous radioimmunoassay. Obstet Gynecol 69:585–589[Abstract/Free Full Text]
  28. Greer IA, Haddad NG, Dawes J, Johnstone FD, Calder AA 1989 Neutrophil activation in pregnancy-induced hypertension. Br J Obstet Gynecol 96:978–982[Medline]
  29. Crocker IP, Wellings RP, Fletcher J, Baker PN 1999 Neutrophil function in women with pre-eclampsia. Br J Obstet Gynecol 106:822–828[Medline]
  30. Sabatier F, Bretelle F, D’Ercole C, Boubli L, Sampol J, Dignat-George F 2000 Neutrophil activation on preeclampsia and isolated intrauterine growth restriction. Am J Obstet Gynecol 183:1558–1563[CrossRef][Medline]
  31. Mellembakken JR, Aukrust P, Olafsen MK, Ueland T, Hestdal K, Videm V 2002 Activation of leukocytes during the uteroplacental passage in preeclampsia. Hypertension 39:155–160[Abstract/Free Full Text]
  32. Redman CW, Sacks GP, Sargent IL 1999 Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 180:499–506[CrossRef][Medline]
  33. Wang Y, Gu Y, Philibert L, Lucas MJ 2001 Neutrophil activation induced by placental factors in normal and pre-eclamptic pregnancies in vitro. Placenta 22:560–565[CrossRef][Medline]
  34. Bigazzi M, Nardi E, Bruni P, Petrucci F 1980 Relaxin in human decidua. J Clin Endocrinol Metab 51:939–941[Abstract]
  35. Yamamoto S, Kwok SCM, Greenwood FC, Bryant-Greenwood GD 1981 Relaxin purification from human placental basal plates. J Clin Endocrinol Metab 52:601–604[Abstract]
  36. Weiss G, Palejwala S, Tseng L, Goldsmith LT 2001 Synthesis and function of relaxin in human endometrium. In: Tregear GW, Ivell R, Bathgate RA, Wade JD, eds. Relaxin 2000. Dordrecht, The Netherlands: Kluwer Academic Publishers; 41–46



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