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Keywords:

  • gene expression;
  • iNOS activity;
  • l-NAME;
  • lymphocytes;
  • rats

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

We have shown previously that nitric oxide (NO) controls platelet endothelial cell adhesion molecule (PECAM-1) expression on both neutrophils and endothelial cells under physiological conditions. Here, the molecular mechanism by which NO regulates lipopolysaccharide (LPS)-induced endothelial PECAM-1 expression and the role of interleukin (IL)-10 on this control was investigated. For this purpose, N-(G)-nitro-l-arginine methyl ester (l-NAME; 20 mg/kg/day for 14 days dissolved in drinking water) was used to inhibit both constitutive (cNOS) and inducible nitric oxide (iNOS) synthase activities in LPS-stimulated Wistar rats (5 mg/kg, intraperitoneally). This treatment resulted in reduced levels of serum NO. Under this condition, circulating levels of IL-10 was enhanced, secreted mainly by circulating lymphocytes, dependent on transcriptional activation, and endothelial PECAM-1 expression was reduced independently on reduced gene synthesis. The connection between NO, IL-10 and PECAM-1 expression was examined by incubating LPS-stimulated (1 µg/ml) cultured endothelial cells obtained from naive rats with supernatant of LPS-stimulated lymphocytes, which were obtained from blood of control or l-NAME-treated rats. Supernatant of LPS-stimulated lymphocytes obtained from l-NAME-treated rats, which contained higher levels of IL-10, reduced LPS-induced PECAM-1 expression by endothelial cells, and this reduction was reversed by adding the anti-IL-10 monoclonal antibody. Therefore, an association between NO, IL-10 and PECAM-1 was found and may represent a novel mechanism by which NO controls endothelial cell functions.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Inflammation is a highly coordinated process, dependent upon a fine balance of pro- or anti-inflammatory molecules secreted by a diversity of circulating, migrating and resident cells. In this context, nitric oxide (NO) is one of the most versatile players. While NO produced by constitutive nitric oxide synthases (cNOS) is related mainly to the physiological control of vascular tonus, neurotransmission and maintenance of circulating cells into the vessel, NO produced by inducible NOS (iNOS) is involved in body defence and cytotoxicity [1,2]. In this latter context, NO modulates expression/activation of adhesion molecules involved in leucocyte recruitment into infectious and inflammatory sites [1,3].

We have demonstrated recently that chronic blockade of cNOS in rats, elicited by N-(G)-nitro-l-arginine methyl ester (l-NAME) treatment, impairs leucocyte–endothelial interactions which seems to be dependent, at least in part, on reduced PECAM-1 expression on neutrophils and microvascular endothelial cell membranes [4,5]. This control may be relevant in pathophysiological conditions, as PECAM-1 is involved in the modulation of cell adhesions, leucocyte transmigration, vascular remodelling, atherosclerotic lesion and angiogenesis [6–9]. The architecture of PECAM-1 structure provides a fundamental role in inside–out and outside–in signalling, as it is able to convert mechanical stimuli into intracellular signals [6,7,10–12]. In fact, PECAM-1 is a mechano-transducing molecule which reacts to fluid shear stress modification. Disturbed shear stress induces phosphorylation of tyrosine residues on the PECAM-1 cytoplasmic tail, which activates endothelial NOS (eNOS) resulting in NO synthesis. PECAM-1/eNOS co-localize on endothelial cells, and a correlation of PECAM-1/eNOS has been proposed [13,14]. In addition, activation of constitutive PECAM-1 triggers a proinflammatory response by activating transcription nuclear factor κB (NF-κB) and Akt pathways in endothelial cells [9,12].

Although NO mechanism of action on adhesion molecules expression has not been elucidated fully, it may be exerted by directly activating soluble guanylyl cyclase [15]. Nevertheless, it has been shown conclusively that NO controls the secretion of inflammatory chemical mediators and a NO indirect action on adhesion molecules expression may also be proposed [16–18]. In this context, recent evidence has shown an interplay between NO and interleukin (IL)-10 secretion, based on the findings that NO donors inhibited IL-10 secretion by activated human lymphocytes [19], and cultured cells obtained from the cartilage of patients with osteoarthritis treated with 1400 W, a specific inhibitor of iNOS, secreted augmented levels of IL-10 [20]. The negative control of IL-10 on adhesion molecule expression in several inflammatory and vascular diseases has been demonstrated [21–24]. Recently, it has been shown that PECAM-1 expression is controlled by IL-10 in hypertensive pregnant rats [25]. In vivo IL-10 administration at the beginning of gestation in deoxycorticosterone acetate (DOCA)/saline-treated rats inhibited PECAM-1 expression on endothelial cells from aortic and placental tissues [25].

For the first time, the connection of NO/IL-10/PECAM-1 during in vivo inflammatory conditions elicited by LPS has been shown. The reduction in circulating levels of NO in LPS-stimulated rats, caused by in vivo blockade of iNOS activity, inhibited endothelial PECAM-1 expression dependent on enhanced IL-10 secretion by circulating lymphocytes.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Drugs, chemicals, reagents and other materials

Antibodies against adhesion molecules and anti-IL-10, as well as cytokine detection kits, were purchased from BD Pharmingen (San Diego, CA, USA). l-NAME, LPS (Escherichia coli; serotype 026 : B6), Percoll gradients and all reagents used to measure NOS activity and NO levels were obtained from Sigma (St Louis, MO, USA). Trizol reagent was purchased from Invitrogen (Grand Island, NY, USA), and the other reagents used in polymerase chain reaction (PCR) assays were purchased from Promega (Madison, WI, USA). SYBR Advantage qPCR Premix was obtained from Clontech (Mountain View, CA, USA). Calf bovine serum, Dulbecco's modified Eagle's medium (DMEM) and gentamycin were purchased from Gibco (Carlsbad, CA, USA). Sodium pentobarbital was obtained from Cristália (Itapira, Brazil).

Animals and l -NAME treatment

Male Wistar rats (180–220 g) were assigned randomly to either the treatment group or the control group. Animals in the treatment group received l-NAME (20 mg/kg/day) dissolved in drinking water for 2 weeks. Control animals received water without l-NAME. It is important to emphasize that the animals drank an average of 30 ml water/day and the calculation of l-NAME treatment was based on this information. Therefore, the animals consumed an average of 20 mg/kg/day of l-NAME, and the effectiveness of the treatment was confirmed by quantification of NOS activity. Animals were fed a standard pellet diet and water ad libitum. Before each experimental procedure, animals were anaesthetized with sodium pentobarbital [65 mg/kg, intraperitoneally (i.p.)] to minimize stress.

All procedures were performed according to protocols approved by the Brazilian Society of Science of Laboratory Animals (SBCAL) for the proper care and use of experimental animals.

LPS injection

On the last day of treatment, LPS (Escherichia coli, serotype 026 : B6) was injected into the peritoneal cavity of animals (5 mg/kg; 1 ml). Blood and other tissues were collected 4 h later.

cNOS and iNOS activity

Ex vivo NOS activity was evaluated in cremaster muscle homogenates, as described previously [26]. Protein content was determined using the Bradford colorimetric method, and NOS activity was expressed as pmol of l-citrulline produced per min per mg of protein.

Measurement of NO production

NO levels in blood were determined using an NO Analyzer (NOA™ 280; Sievers Inc., Boulder, CO, USA). Samples were deproteinized previously with cold ethanol (1:2 v/v), kept on ice for 30 min and centrifuged (100 g, 10 min). Next, each sample (10 µl) was injected into the equipment. Nitrite (NO2-) and nitrate (NO3-) were reduced to NO by vanadium chloride at 90°C. The NO produced was detected by gas phase chemiluminescence after reaction with ozone. A calibration curve was then created using a sodium nitrate standard solution.

Cell cultures

Leucocytes were isolated from blood obtained from the abdominal aorta of control and l-NAME-treated animals in the absence of LPS injection. Total leucocytes were recovered after lysis of erythrocytes using NH4Cl solution (0·13 M), washed with cold phosphate-buffered saline (PBS) solution and quantified in a Neubauer chamber. Lymphocytes were isolated using Percoll gradient. Briefly, blood was transferred to plastic tubes containing a Percoll gradient (56% in PBS) and centrifuged (600 g, 40 min, room temperature). Recovered lymphocytes were washed with cold PBS solution and counted in a Neubauer chamber. Total leucocytes and isolated lymphocytes (1 × 106/ml) were cultured in the presence or absence of LPS (5 µg/ml, 5% CO2, 37°C, humid atmosphere) for 18 h. The supernatant was kept at −20°C until quantification of IL-10 and incubation with endothelial cells.

Primary cultures of microvascular endothelial cells were obtained from the cremaster muscle of naive rats using a previously described method [27,28]. The endothelial cells were identified routinely by a monoclonal antibody against PECAM-1 using confocal microscopy [28].

PECAM-1 expression

Vessels of microcirculation.  Testes of animals were removed surgically, frozen in nitrogen–hexan solution, cryosectioned (8-µm thickness) and fixed in cold acetone (10 min). Briefly, sections were incubated overnight with Superblock solution to avoid non-specific binding. Sections were then incubated overnight with phycoerythrin (PE)-labelled PECAM-1. Fluorescent-stained areas of vessel walls were selected and the fluorescence intensity was quantified using image analyser software (KS 300; Kontron Elektronik, Carl-Zeiss, Germany). The same procedures were also carried out in sections of testes incubated without antibody or using goat anti-mouse immunoglobulin G to evaluate the background reaction.

Endothelial cell culture.  Endothelial cells subcultured for up to two passages were seeded (104–105 cells) onto glass coverslips and allowed to grow for 48 h. Cells were incubated with LPS (1 µg/ml; 4 h) in the presence of supernatants of previously cultured lymphocytes obtained from control or l-NAME-treated rats. In another set of experiments, the supernatants of lymphocytes were incubated simultaneously with anti-IL-10 rat monoclonal antibody (20 µg/ml). In sequence, cells were washed with PBS solution, incubated for 20 min (37°C, 5% CO2) with PE-labelled anti-PECAM-1 (1:100 dilution) and washed with PBS solution. The cells were excited at 488 nm (Argon laser) and observed by confocal microscopy (LSM 500; Carl Zeiss) at 570–585 nm for detection of PECAM-1. The fluorescence intensity of experimental groups was subtracted from the values obtained from cells that were incubated without monoclonal antibody PE-labelled anti-PECAM-1, and the normalization was based on non-stimulated cells incubated with PE-labelled anti-PECAM-1. Three fields with 4–6 cells were chosen randomly and imaged, as described previously [29].

RNA isolation, reverse transcriptase and real-time PCR analysis

Total RNA was extracted from cremaster muscle homogenates using Trizol reagent following the manufacturer's instructions. RNA extraction was carried out in an RNAse free environment. RNA was quantified by measuring absorbance of the RNA solution at 260 nm.

cDNA was synthesized from total RNA (2 µg) using an oligo(dT)15 primer (20 µg/ml) after incubation (70°C, 5 min) in the presence of a deoxynucleotide triphosphate mixture (dNTP, 2 mM), ribonuclease inhibitor (20 U) and Moloney murine leukaemia virus reverse transcriptase (200 U) in reverse transcriptase buffer (25 µl final volume). The reverse transcription reaction occurred by incubating RNA at 42°C for 60 min. For qualitative PCR, the obtained cDNA was incubated with Taq DNA polymerase (2·5 U), 3′- and 5′- specific primers (0·4 µM) and dNTP mix (200 µM) in thermophilic DNA polymerase buffer containing MgCl2 (1·5 mM). To quantify PECAM-1 mRNA in endothelial cell culture, quantitative PCR reactions were carried out using the SYBR Advantage qPCR Premix (Clontech), according to the manufacturer's protocols. Control experiments revealed approximately equal efficiencies over different starting template concentrations for target gene and hypoxanthine phosphoribosyltransferase 1 (HPRT). For each animal, all samples were analysed in a single assay. A threshold cycle (ΔCt) value was obtained by subtracting HPRT Ct values from respective target gene Ct values. HPRT was used as a reference, and Ct values were subtracted from PECAM-1 Ct values to obtain a Δ-ΔCt value. The relative expression of each isoform was then calculated by 2-Δ-ΔCt.

The primer sequences used in qualitative PCR were: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-TATGATGACATCAAGAAGGTGG-3′ (forward), 5′-CACCACCCTGTTGCTGTA-3′ (reverse); PECAM-1, 5′-TCTCCATCCTGTCGGGTAACG-3′ (forward), 5′-CTTGGGTGTCATTCACGGTTTC-3′ (reverse); and IL-10, 5′-GAAAACAGAGCTTCAGCATGCTTGG-3′ (forward), 5′-TTGAGTGTCACGTAGGCTTCTATGC-3′ (reverse). The primer sequences used in real-time PCR were: HTRP, 5′-AAGCTTGCTGGTGAAAAGGA-3′ (forward), 5′-TGATTCAAATCCCTGAAGTGC-3′ (reverse); PECAM-1, 5′-GAAGGTTTCTGAGCCCAGTC-3′ (forward), 5′-TCAAGGGAGGACACTTCCAC-3′ (reverse).

Quantification of IL-10

Levels of IL-10 were determined from the serum, supernatant of circulating leucocytes and isolated lymphocyte cultures. The cytokine levels were quantified using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol.

Data and statistical analyses

The mean and standard error of the mean [standard error of the mean (s.e.m.)] of all data presented here were compared by Student's t-tests or analysis of variance (anova). Tukey's multiple comparisons test was used to determine the significance of differences calculated between the values for the experimental conditions.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

In vivol -NAME treatment inhibits LPS-induced cNOS and iNOS activity and reduces levels of circulating NO

LPS injection into control animals did not modify cNOS activity, but elevated iNOS activity relative to non-inflammatory conditions. However, l-NAME treatment markedly inhibited cNOS and iNOS activity after LPS injection (Fig. 1a). In addition, levels of circulating NO metabolites were increased following LPS injection in control animals, but these levels were reduced markedly in l-NAME-treated rats (Fig. 1b).

image

Figure 1. Effects of N-(G)-nitro-l-arginine methyl ester (l-NAME) treatment on lipopolysaccharide (LPS)-induced nitric oxide (NOS) activities in the cremaster muscle and circulating levels of NO. Rats were treated previously with l-NAME (20 mg/kg/day; 14 days) or water (control) and then inflamed with LPS (5 mg/kg; intraperitoneally; 4 h). Constitutive NOS and induced NOS activities were quantified in cremaster muscle by enzymatic reaction (a), and sera samples were used to determine NO2-/NO3- by chemiluminescence (b). The dotted line represents NOS activity or NO levels under non-inflammatory conditions. Results are expressed as the mean ± standard error of the mean of data obtained from samples collected from four to six animals in each group.*P < 0·05 and ***P < 0·001 versus respective control, determined by analysis of variance or Student's t-test.

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In vivo LPS-induced endothelial PECAM-1 expression is reduced by l -NAME treatment

Animals treated with l-NAME presented reduction on PECAM-1 expression on microvascular endothelial cells from cremaster muscle after in vivo LPS administration compared to control animals (Fig. 2a). The effect was not dependent on gene synthesis, as PECAM-1 mRNA levels were not modified by the treatment (Fig. 2b). A representative image of PECAM-1 mRNA expression in agarose gel electrophoresis is shown in Fig. 2c.

image

Figure 2. Effects of N-(G)-nitro-l-arginine methyl ester (l-NAME) treatment on in vivo lipopolysaccharide (LPS)-induced platelet endothelial cell adhesion molecule (PECAM-1) expression in the cremaster muscle. Rats were treated previously with l-NAME (20 mg/kg/day; 14 days) or water (control) and then inflamed with LPS (5 mg/kg; intraperitoneally; 4 h). PECAM-1 expression was quantified in endothelial cells from vessels of cremaster muscle by immunohistochemistry (a); PECAM-1 mRNA expression was determined in endothelial cells from vessels of cremaster muscle by reverse transcription–polymerase chain reaction (RT–PCR) (b). A representative image of PECAM-1 mRNA expression in agarose gel electrophoresis is shown (c). Results are expressed as the mean ± standard error of the mean of samples collected from seven to 10 animals in each group.**P < 0·01 versus respective control, determined by Student's t-test.

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In vivo LPS-induced IL-10 secretion is enhanced by l -NAME treatment

After LPS injection, circulating concentrations of IL-10 were significantly higher in the serum of l-NAME-treated rats relative to control rats (Fig. 3a). The elevated concentration of IL-10 in the circulation was due to enhanced secretion of IL-10 by circulating leucocytes, mainly lymphocytes, as cytokine levels were elevated in cultured blood leucocytes and lymphocytes obtained from l-NAME-treated rats and in vitro stimulated with LPS (Fig. 3b). Secretion of IL-10 by lymphocytes was dependent on transcriptional activations, because IL-10 mRNA levels were higher in lymphocytes obtained from l-NAME-treated rats than those obtained from control rats after LPS in vitro stimulation (Fig. 3c,d).

image

Figure 3. Effects of N-(G)-nitro-l-arginine methyl ester (l-NAME) treatment on interleukin (IL)-10 secretion. Rats were treated previously with l-NAME (20 mg/kg/day; 14 days) or water (control) and then inflamed with LPS (5 mg/kg; intraperitoneally; 4 h). Levels of IL-10 were quantified by enzyme-linked immunosorbent assay: (a) represents concentrations of IL-10 in the circulation; (b) correspond to levels of IL-10 in the supernatant of circulating leucocytes or isolated lymphocytes obtained in non-inflammatory condition and cultured in presence of LPS (5 µg/ml1; 18 h). IL-10 mRNA expression was determined in circulating lymphocytes by reverse transcription–polymerase chain reaction (RT–PCR) (c). (d) shows a representative image of IL-10 mRNA expression in agarose gel electrophoresis. The dotted line represents cytokine concentrations under non-inflammatory conditions. Results are expressed as the mean ± standard error of the mean of samples collected from six animals in each group.*P < 0·05, **P < 0·01 and ***P < 0·001 versus respective control, determined by analysis of variance or Student's t test.

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Association of NO and IL-10 on PECAM-1 expression by endothelial cells

Relationships between NO, IL-10 secretion by lymphocytes and PECAM-1 expression on endothelial cells were investigated in vitro using lymphocytes obtained from l-NAME-treated or control rats. In this context, cultured endothelial cells from naive animals were stimulated with LPS in order to express PECAM-1, and lymphocytes obtained from control or l-NAME-treated animals were also stimulated in vitro with LPS to induce the IL-10 secretion. Confocal microscopy studies demonstrated that incubation of endothelial cells (Fig. 4a) with supernatant obtained from lymphocytes of l-NAME-treated animals reduced LPS-induced PECAM-1 expression (Fig. 4b,c). Blockade of IL-10 activity, by adding monoclonal antibody to the lymphocyte supernatant, reversed the reduced PECAM-1 expression by endothelial cells (Fig. 4b,c). This effect suggests that IL-10 acts on PECAM-1 re-internalization or cleavage, as PECAM-1 mRNA levels were not altered (Fig. 4d). Notably, concentrations of NO in the supernatant of lymphocytes from control and l-NAME-treated rats were similar (Table 1), consistent with the finding that reduced PECAM-1 expression is not dependent on the direct action of NO.

image

Figure 4. Effects of interleukin (IL)-10 secreted by lymphocytes on platelet endothelial cell adhesion molecule (PECAM-1) expression. Rats were treated with N-(G)-nitro-l-arginine methyl ester (l-NAME) (20 mg/kg/day; 14 days) or water (control). PECAM-1 expression was evaluated in endothelial cells (a) incubated with lymphocyte supernatant of control or l-NAME-treated rats, stimulated in vitro with lipopolysaccharide (LPS) (1 µg/ml; 4 h) in the absence or presence of monoclonal antibody anti-IL-10 by confocal microscopy (b,c). Using reverse transcription–polymerase chain reaction (RT–PCR), PECAM-1 mRNA levels were quantified in endothelial cells incubated with lymphocyte supernatant of control or l-NAME-treated rats and stimulated with LPS (1 µg/ml; 4 h) (d). Results express the mean ± standard error of the mean of samples collected from six animals in each group.*P < 0·001 versus respective control, and **P < 0·001 versusl-NAME (–) anti-IL-10, determined by Student's t-test.

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Table 1.  Nitric oxide secretion by lymphocytes.
 BasalLipopolysaccharide
  1. l-NAME: N-(G)-nitro-l-arginine methyl ester.

Control (µm)5·06 ± 0·255·33 ± 0·30
l-NAME (µm)5·85 ± 0·686·01 ± 0·31

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Using long-term in vivol-NAME treatment, which blocks NOS activities and reduces levels of circulating NO, we found that the absence of NO induces secretion of the anti-inflammatory cytokine IL-10 by circulating lymphocytes, which impairs LPS-induced PECAM-1 expression on endothelial cells. To our knowledge, this is a novel mechanism of NO control on PECAM-1 expression contributing to the comprehension of NO actions on vascular functions under inflammatory conditions.

During the course of inflammation, temporal kinetics determines expression and activity of different isoforms of NOS mediated by endogenous and exogenous substances [30,31]. In this study, we found that in vivo LPS administration did not alter cNOS activity, but elevated iNOS activity in control animals. However, l-NAME treatment modified this pattern, as activities of both cNOS and iNOS were noticeably reduced following LPS administration in l-NAME-treated rats, resulting in very low levels of circulating NO. These data indicated that l-NAME treatment employed here was an efficient experimental approach to study in vivo actions of NO in the inflammatory course.

An interplay between NO and IL-10 secretion has been proposed recently [19,20]. IL-10 is secreted by macrophages, monocytes, B and T cells during inflammation and acts to limit the process [19,24,32]. LPS activates Toll-like receptor 4 (TLR-4) and induces lymphocyte secretion of IL-10 in a signal transducer and activator of transcription (STAT)-dependent mechanism [33,34]. IL-10 binds to two distinct receptors (IL-10R1 and IL-10R2) and activates a complex of intracellular components, which leads to phosphorylation and translocation of transcription factors [35]. Here, we show that circulating levels of IL-10 are augmented in l-NAME-treated animals after LPS challenge and this increment is due to transcriptional induction in circulating lymphocytes. It is noteworthy that this effect is not dependent on direct action of l-NAME, as in vitrol-NAME incubation did not alter IL-10 secretion by lymphocytes under LPS stimulation (data not shown).

Although it has been demonstrated recently that B1 lymphocytes secretes NO [36], the ability of other lymphocyte subtypes to produce NO remains uncertain. Conversely, it has been demonstrated clearly that lymphocytes are an important target for NO actions [37,38]. Accordingly, it has been demonstrated herein that circulating lymphocytes do not produce high levels of NO, corroborating data presented by [39] Shuler et al. (1995), but NO controls lymphocyte functions involved in the immune response.

It has been shown that PECAM-1 activates eNOS during altered shear stress, which in turns produces NO [13,14], and we have demonstrated previously that physiological NO modulates endothelial PECAM-1 expression [5]. Here we corroborated an action of NO on PECAM-1 expression by endothelial cells in an LPS-stimulated condition. Considering that NO controls IL-10 secretion and the inhibitory role of this cytokine on endothelial adhesion molecule expressions during inflammation [40–42], we hypothesized an indirect effect of NO on PECAM-1 expression via IL-10. This association was demonstrated by reduced PECAM-1 expression on LPS-stimulated endothelial cells incubated with the supernatant of LPS-stimulated lymphocytes obtained from l-NAME-treated rats. Moreover, it was supported further by the finding that an IL-10 monoclonal antibody reversed the effect. In this context, recent data demonstrated that therapeutic administration of recombinant IL-10 inhibited PECAM-1 expression on the endothelium of aortic and placental vessels of hypertension pregnant rats after normalizing blood pressure [25]. We hypothesize that this mechanism is not involved in the NO/IL-10/PECAM-1 connection proposed here, as l-NAME treatment does not evoke alterations in systemic arterial pressure and in microvascular venules and arteriole haemodynamic parameters [4].

The membrane pool of PECAM-1 also depends on gene expression, internalization and enzymatic cleavage [10,43]. Our data suggest strongly that the control exerted by the NO/IL-10 connection is based on a post-transcriptional event. LPS-induced PECAM-1 mRNA levels were not modified in endothelium from l-NAME-treated animals or in endothelial cells incubated with the supernatant of lymphocytes collected from l-NAME-treated rats. Therefore, we consider that NO controls IL-10 gene synthesis by lymphocytes which, in turn, controls mechanisms related to the PECAM-1 re-internalization or cleavage on endothelial membranes.

Taken together, the results presented here highlight NO, IL-10 and PECAM-1 connections, which may be relevant in immune or vascular diseases presenting altered blood flow, such as vascular inflammatory diseases and hypertension.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The authors thank FAPESP for financial support (grant no. 05/60329-0). Sandra H. P. Farsky and Regina P. Markus are fellows of the Conselho Nacional de Pesquisa e Tecnologia (CNPq), and Cristina B. Hebeda is a Coordenação de Aperfeiçoamento de Nível Superior (CAPES) postdoctoral fellow.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References