Inflamm. Res. DOI 10.1007/s00011-014-0737-1
Inflammation Research
ORIGINAL RESEARCH PAPER
Methylene blue modulates adhesion molecule expression on microvascular endothelial cells Isabella Werner • Fengwei Guo • Ulrich A. Stock • Miche`le Lupinski Patrick Meybohm • Anton Moritz • Andres Beiras-Fernandez
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Received: 18 July 2013 / Revised: 17 January 2014 / Accepted: 15 April 2014 Ó Springer Basel 2014
Abstract Objective and design As methylene blue (MB) has been recently proposed to preserve blood pressure in case of vasoplegic syndrome and shock, an entity directly related to systemic inflammation, we aimed to elucidate the effect of MB on the expression of adhesion-molecules in endothelial-cells. Materials and treatment Human microvascular endothelialcells (HuMEC-1) were treated with 10, 30 or 60 lM MB for 30 min and 2 h each. Additionally, the treated HuMEC-1 were co-cultured with either human peripheral blood mononuclear cells (PBMCs) or Jurkat cells (human T-lymphocytes) for 2 h. Methods HuMEC-1 were analyzed after MB treatment and after co-culture experiments for expression of different adhesion-molecules (ICAM-1, VCAM-1, L-selectin, E-selectin) via FACS measurement and western blot analysis. The supernatants of the experiments were analyzed with regard to the soluble forms of the adhesion molecules. Results We found that MB is able to modulate the expression of adhesion-molecules on EC. Administration of MB increases the expression of E-selectin and VCAM-1 Responsible Editor: Ikuo Morita.
Electronic supplementary material The online version of this article (doi:10.1007/s00011-014-0737-1) contains supplementary material, which is available to authorized users. I. Werner (&) F. Guo U. A. Stock M. Lupinski A. Moritz A. Beiras-Fernandez Department of Thoracic and Cardiovascular Surgery, University Hospital Frankfurt, Goethe University, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany e-mail: [email protected] P. Meybohm Department of Anaesthesiology Intensive Care Medicine and Pain Therapy, University Hospital Frankfurt, Frankfurt/Main, Germany
depending on the dosage and time of exposure. ICAM-1 measurements provide evidence that different circulating blood cells can differently alter the adhesion-molecule expression on EC after MB exposure. Conclusion Our results provide evidence regarding the immunomodulatory effect of MB upon endothelial-cells after inflammation. Keywords Methylene blue Adhesion molecules Endothelial cells Peripheral blood mononuclear cells
Introduction Inflammatory responses after cardiac surgical interventions with cardiopulmonary by-pass are often related to high morbidity and mortality rate in patients, depending on the severity of the presentation. Refractory vasodilatory shock or vasoplegic syndrome is a severe complication of this inflammatory syndrome. Vasoplegia is frequently preceded by a profound vasodilatation occurring during surgery, refractory to conventional catecholamine treatment [1]. Leukocyte recruitment and the regulatory steps of capture, rolling, activation, and adhesion have been well studied and reviewed within the last decades [2, 3]. Selectins belong to the family of adhesion molecules that facilitate the primary capture of leukocytes from the bloodstream to the blood vessels at sites of tissue injury and inflammation [4]. Central characteristics of selectins are that they promote leukocyte attachment and rolling under shear stress and moreover force leucocytes to enter into tissues [5–7]. Rolling is mandatory for firm adhesion of leukocytes under flow conditions. Nonetheless, unless integrins start to interfere in this process, no firm adhesion and transmigration of the cells into the tissue can take
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place. Integrins are adhesion molecules with heterodimeric membrane proteins, which are transported to the cell surface [6, 8]. Immune cells start to change their shape and form bipolar characteristics of motile cells, after they adhered to the luminal endothelial surface [9, 10]. After adhesion, immune cells transmigrate through the endothelium and are directed to the site of inflammation [6]. Evidence is increasing that inhibitors of NO production greatly enhance the adhesion and emigration of polymorphonuclear leukocytes to venules, thus indicating that NO plays an important role in preventing leukocyte-endothelial cell adhesion [11], characteristic in inflammatory processes [2]. It has been proposed that methylene blue (MB) may have a direct inhibitory effect on eNOS, and probably iNOS [12], as well as a blocking effect on the formation of cGMP by inhibiting the GC enzyme [13]. As MB is known to be an NO/cGMP inhibitor and NO is preventing leukocyte adhesion, the modulation of adhesion molecules on endothelial cells due to MB treatment may be assumed. Vasoplegia is thought to be caused by the inflammationmediated endothelial dysfunction [14]. Some observational studies have shown that methylene blue (MB) can contribute to the preservation of blood pressure associated with endothelial dysfunction in vasoplegia [14–16]. An increase in inflammation has been observed associated to the administration of MB [17]. As MB is more and more often used in daily clinics, we were interested on the effect of MB on adhesion molecule expression on endothelial cells.
Materials and methods Ethics statement This study was approved by the Ethics Committee of the University of Frankfurt, Germany (Ref. Nr.: 189/13). All blood samples (n = 5) were obtained after informed consent and according to the declaration of Helsinki. All participants provided their written informed consent to participate in this study. Cell culture Human microvascular endothelial cells-1 (HuMEC-1) (kindly provided by Dr. V. Mirakaj, University Tu¨bingen, Department of Anesthesiology and Intensive Care Medicine) were cultured in MCDB-131 (Life Technologies, Darmstadt, Germany) supplemented with 10 % fetal calf serum (Gibco, Karlsruhe, Germany), 1 % glutamine (Gibco, Karlsruhe, Germany), 1 % pen/strep solution (Sigma Chemical Co. St. Louis, USA), 10 ng/ml Epidermal Growth Factor (Sigma Chemical Co. St. Louis, USA) and 1 lg/ml hydrocortisone (Sigma Chemical Co. St. Louis, USA).
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Jurkat cells (immortalized human T-lymphocytes) were cultured in RPMI-1640 medium (Sigma Chemical Co. St. Louis, USA) supplemented with 1 % pen/strep solution and 10 % fetal calf serum. All cells were cultivated at 37 °C and 5 % CO2 atmosphere. Isolation of peripheral blood mononuclear cells (PBMC) Blood was drawn into EDTA tubes from healthy volunteers. The probes were kept between 18 and 22 °C throughout the isolation process. The fresh blood was underlaid with 5 ml PolymorphprepÒ (Axis-Shield, Oslo, Norway), for density gradient centrifugation, and centrifuged at 450g for 35 min without break. The PBMC layer was washed twice with PBS (Gibco, Karlsruhe, Germany), counted and used for further experiments. HuMEC-1 treatment with MB and co-culturing of HuMEC-1 with PBMCs or Jurkat cells HuMEC-1 (1 9 106 cells/well) were plated into six well plates and cultured until 80 % confluence was reached. Consumed culture medium was removed and 10, 30 or 60 lM MB diluted in basal culture media was added for 30 min and 2 h each. Afterwards MB was removed and HuMEC-1 were used for either direct FACS analysis or coculture experiments. HuMEC-1 were co-cultured with either PBMCs or Jurkat cells. Therefore, 3 9 105 PBMCs or Jurkat cells were suspended in RPMI-1640 medium and co-cultured with MB treated HuMEC-1 monolayers. After 2 h at 37 °C, the supernatant was collected for ELISA experiments and cells were gently harvested and used for FACS analysis or western blot analysis. Untreated HuMEC-1 served as control. All experiments were performed in triplicates. FACS analysis of ICAM-1, VCAM-1, CD62E and CD62L To characterize the modulation of cell adhesion molecules of HuMEC-1 after MB treatment and followed co-culturing with PBMCs and Jurkat cells, we stained for ICAM-1 [fluorescein isothiocyanate (FITC)-conjugated CD54 antibody (BD, Heidelberg, Germany)], VCAM-1 [phycoerythrin (PE)-conjugated CD106 antibody (BD, Heidelberg, Germany)], CD62L [brilliant violet-421tm conjugated antibody (BioLegend, San Diego, USA)] and CD62E [PeridininChlorophyll (PerCP)-conjugated antibody (abcam, Cambridge, UK)]. Analysis was performed using a FACS Canto flow cytometer (BD, Heidelberg, Germany) and FLOW JO Software. Unstained cells served as controls. Expression levels of adhesion molecules are indicated as fluorescence intensity (FI).
Adhesion molecule expression on microvascular endothelial cells
Enzyme-linked immunoadsorbent assay (ELISA) The soluble ICAM-1, VCAM-1, E-selectin and L-selectin levels in the supernatants after co-culture experiments with MB treated HuMEC-1 were determined using human sICAM-1/CD54 Quantikine ELISA Kit, human sVCAM-1/ CD106 Quantikine ELISA Kit, human sE-selectin/CD62E Quantikine ELISA Kit and human sL-selectin/CD62L ELISA Kit, all from R&D Systems (Minneapolis, USA) in accordance with the manufacturer’s protocol. Western blot analysis To analyze ICAM-1, VCAM-1, E-selectin and L-selectin in HuMEC-1, cell lysates were applied to a 7 % polyacrylamide gel and electrophoresed (90 min, 60 V followed by 100 V). The proteins were then transferred to nitrocellulose membranes (1 h, 100 V). The membranes were blocked with nonfat dry milk for 1 h, and then incubated overnight with monoclonal antibodies directed against human ICAM-1, VCAM-1, E-selectin and L-selectin (all Santa Cruz Biotechnology Inc., Heidelberg, Germany). HRP-conjugated goatanti-mouse IgG (Millipore, Temecula, CA, USA) served as the secondary antibody for ICAM-1, VCAM-1, L-selectin and b-actin. HRP-conjugated goat-anti-rabbit IgG (Millipore, Temecula, CA, USA) served as the secondary antibody for E-selectin. The membranes were briefly incubated with ECL detection reagent (ECLTM, Amersham/GE Healthcare, Mu¨nchen, Germany) to visualize the proteins and then analyzed by the Fusion FX7 system (Peqlab, Erlangen, Germany). b-actin (Sigma, Taufenkirchen, Germany) served as the internal control. The visualized protein bands were analyzed using ImageJ software. The relative amount of protein (RQ) was calculated in relation to b-actin expression. Statistical analysis Data represent mean ± SEM. Statistical analysis was performed with Prism 6 software (Graph Pad) using unpaired Students T test. Differences with p \ 0.05 were considered statistically significant.
Results Surface expression of adhesion molecules on endothelial cells
to control. In case of Jurkat cell co-culture, short time exposure of MB for 30 min lead to a significant drop of ICAM-1 surface expression compared to MB treatment alone (10 lM MB 30 min: 64 ± 2 FI vs. 10lM MB 30 min co-Jurkat: 50.3 ± 2.4 FI; 30 lM MB 30 min: 63.2 ± 0.4 FI vs. 30lM MB 30 min co-Jurkat: 50.85 ± 1.55 FI; 60 lM MB 30 min: 63.85 ± 0.15 FI vs. 60lM MB 30 min co-Jurkat: 53 ± 0.7 FI). This could be observed at 10, 30 and 60 lM MB treatment (Fig. 1a). On the other hand, PBMC co-culture following the same MB treatments did not cause a significant change in ICAM-1 expression compared to MB treatment alone, except at high MB dosage of 60 lM (10 lM MB 30 min: 64 ± 2 FI vs. 10lM MB 30 min co-PBMC: 61.7 ± 5.7 FI; 30 lM MB 30 min: 63.2 ± 0.4 FI vs. 30lM MB 30 min co-PBMC: 65.45 ± 0.95 FI; 60 lM MB 30 min: 63.85 ± 0.15 FI vs. 60lM MB 30 min co-PBMC: 66.65 ± 0.45 FI). PBMC and Jurkat cell co-culture differed significantly at 30 and 60 lM MB treatment for 30 min (30 lM MB 30 min co-Jurkat: 50.85 ± 1.55 FI vs. 30lM MB 30 min co-PBMC: 65.45 ± 0.95 FI; 60 lM MB 30 min co-Jurkat: 53 ± 0.7 FI vs. 60lM MB 30 min co-PBMC: 66.65 ± 0.45 FI). The long term exposure of MB for 2 h did not significantly change ICAM-1 expression independently of co-culture, whereas similar trend can be seen (Fig. 1a, for more details see supplementary information). Vcam-1/cd106 The evaluation of VCAM-1 (CD106) showed a dose and time dependent increase in surface expression after MB treatment and co-culture experiments (Fig. 1b). MB treatment alone caused a significant up-regulation of VCAM-1 from 10 to 60 lM MB with increasing exposure time (10 lM MB 30 min: 4.27 ± 1.91 FI vs. 60 lM MB 2 h: 22.67 ± 4.94 FI). This increase is also significant at 30 lM MB for 30 min compared to 60 lM MB for 2 h (30 lM MB 30 min: 5.34 ± 2.53 FI vs. 60 lM MB 2 h: 22.67 ± 4.94 FI) (Fig. 1b). Only co-culturing with PBMCs and not Jurkat cells caused significant changes in VCAM-1 surface expression. Short time (30 min) MB treatment for 30 min followed by PBMC co-culture significantly increased VCAM-1 on EC with increasing MB dosages (10 lM MB 30 min co-PBMC: 2.99 ± 0.32 FI vs. 60 lM MB 30 min co-PBMC: 5.81 ± 0.72 FI; 30 lM MB 30 min co-PBMC: 3.49 ± 0.25 FI vs. 60 lM MB 30 min co-PBMC: 5.81 ± 0.72 FI) (Fig. 1b). The contact with Jurkat cells did not cause any significant changes in VCAM-1 expression levels (Fig. 1b, for more details see supplementary information).
Icam-1/cd54 E-selectin/CD62-E In our experiments, treatment of HuMEC-1 with MB caused no significant changes in ICAM-1 (CD54) expression, irrespectively of dose and exposure time (Fig. 1a) in comparison
E-Selectin (CD62-E) was drastically modified by MB and followed co-culture with Jurkat cells and PBMCs (Fig. 1c).
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I. Werner et al. Fig. 1 Changes in adhesion molecule expression on HuMEC-1 after MB treatment, MB treatment followed by Jurkat cell co-culture and MB treatment followed by PBMC co-culture, each for 30 min and 2 h with either 10, 30 or 60 lM MB. a Changes in CD54 expression. b Changes in CD106 expression. c Changes in CD62-E expression. d Changes in CD62-L expression (n = 3, data represent mean ± SEM) (§ p \ 0.05 vs. control, * p \ 0.05)
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Adhesion molecule expression on microvascular endothelial cells
MB treatment alone caused a dose and time dependent increase of E-selectin expression (10 lM MB 30 min: 21.85 ± 4.35 FI vs. 60 lM MB 2 h: 84.5 ± 11.46 FI). The co-culturing of MB treated HuMEC-1 with Jurkat cells lead to a significant MB dose and exposure time dependent increase of E-selectin surface expression (10 lM MB 30 min co-Jurkat: 7.97 ± 2.23 FI vs. 30 lM MB 2 h coJurkat: 33.25 ± 1.55 FI, 60 lM MB 30 min co-Jurkat: 39.25 ± 5.15 FI and 60 lM MB 2 h co-Jurkat: 62.95 ± 11.05 FI; 10 lM MB 2 h co-Jurkat: 14.5 ± 3.1 FI and 30 lM MB 30 min co-Jurkat: 18.2 ± 2.4 FI vs. 30 lM MB 2 h co-Jurkat: 33.25 ± 1.55 FI). Similar significant results were observed after PBMC co-culture with MB treated HuMEC-1 (10 lM MB 30 min co-PBMC: 8.72 ± 1.78 FI and 10 lM MB 2 h co-PBMC: 16.15 ± 4.35 FI vs. 30 lM MB 2 h co-PBMC: 37.95 ± 2.45 FI, 60 lM MB 30 min co-PBMC: 42.55 ± 0.45 FI and 60 lM MB 2 h co-PBMC: 65.1 ± 9 FI; 30 lM MB 30 min co-PBMC: 22.55 ± 3.55 FI vs. 60 lM MB 30 min co-PBMC: 42.55 ± 0.45 FI and 60 lM MB 2 h co-PBMC: 65.1 ± 9 FI) (Fig. 1c). In comparison to ICAM-1 and VCAM-1, in this scenario no significant difference between MB treatment alone and the two coculture experiments could be observed. L-selectin/CD62-L In case of L-selectin (CD62-L) it seems that MB in contact to circulating blood cells does not influence the surface expression of this selectin. Even though a kind of modification can be seen in Fig. 1d but neither significant changes nor any trend could be observed. Protein expression of adhesion molecules in endothelial cells Western blot analysis did not reveal significant changes in CD54 expression comparing MB treatment and MB treatment followed by co-culture experiments. MB treatment alone significantly reduced CD54 expression compared to control after long-term treatment and higher dosages (ctr 1.51 ± 0.21 RQ vs. 30 lM MB 2 h 0.17 ± 0.01 RQ and 60 lM MB 2 h 0.16 ± 0.01 RQ). CD106 analysis has shown no appreciable significant differences comparing all experimental groups, except a dose-dependent increase from 30 lM MB 30 min coPBMCs to 60 lM MB 30 min co-PBMCs (30 lM MB 30 min co-PBMC 1.12 ± 0.05 RQ vs. 60 lM MB 30 min coPBMC 1.45 ± 0.01 RQ) and higher CD106 levels in 60 lM MB 30 min co-PBMCs compared to 60 lM MB treatment alone (60 lM MB 30 min co-PBMC 1.45 ± 0.01 RQ vs. 60 lM MB 30 min 0.38 ± 0.07 RQ). CD62-E was dosedependent reduced after short term MB treatment and dose dependent elevated after long term MB treatment (30 lM MB
30 min 1.11 ± 0.07 RQ vs. 60 lM MB 30 min 0.76 ± 0.00 RQ; 10 lM MB 2 h 0.85 ± 0.01 RQ vs. 60 lM MB 2 h 1.06 ± 0.01 RQ; p \ 0.05). High dose MB treatment caused a time-dependent up-regulation of CD62-E expression (60 lM MB 30 min 0.76 ± 0.00 RQ vs. 60 lM MB 2 h 1.06 ± 0.01 RQ; p \ 0.05). PBMC co-culture was able to significantly reduce CD62-E levels after 30 lM MB 30 min treatment (30 lM MB 30 min 1.11 ± 0.07 RQ vs. 30 lM MB 30 min co-PBMC 0.61 ± 0.04 RQ). CD62-L expression levels were only significantly modulated by Jurkat cell co-culture. Lowdose MB treatment caused less CD62-L expression compared to Jurkat cell co-culture (10 lM MB 30 min 1.33 ± 0.00 RQ vs. 10 lM MB 30 min co-Jurkat 1.73 ± 0.09 RQ). Furthermore, a dose and time dependent modification of CD62-L was observed after MB treatment followed by Jurkat cell co-culture (30 lM MB 30 min co-Jurkat 1.70 ± 0.00 RQ vs. 60 lM MB 30 min co-Jurkat 1.30 ± 0.0.06 RQ and 30 lM MB 30 min co-Jurkat 1.70 ± 0.00 RQ vs. 30 lM MB 2 h co-Jurkat 1.09 ± 0.13 RQ). Soluble forms of adhesion molecules sICAM-1, sVCAM-1, sE-selectin and sL-selectin released after MB treatment and co-culture sICAM-1 in the supernatant did not differ significantly in any group compared to control. The sICAM-1 levels after MB treatment compared to MB treatment followed by co-culture with Jurkat cells and PBMCs was significantly higher in the 10 lM MB 30 min group, 30 lM MB 2 h group and 60 lM MB 2 h group (Fig. 2a). MB treatment only has shown a dose dependent increase in the long-term treatment group (Fig. 2a). Soluble CD106 was not detectable in every experimental group. Low-dose MB treatment caused higher sCD106 release after PBMC co-culture than after Jurkat cell co-culture by trend. After high dose MB treatment the trend is reversed. Jurkat cell co-culture led to a time dependent elevation of sCD106 comparing 30 min vs. 2 h treatment (Fig. 2b) Soluble CD62-E was detected only in very low concentrations (Fig. 2c). PBMC co-culture showed higher levels of sCD62-E after low-dose MB compared to Jurkat cell co-culture and MB treatment only, by trend. Soluble CD62-L was only measurable in the PBMC co-culture groups and after short time and low-dose MB treatment (Fig. 2d). Longterm MB treatment combined with PBMC co-culture caused a significant dose dependent drop in sCD62-L levels.
Discussion These data show that MB may have an impact on adhesion molecule expression of endothelial cells. E- and L-selectin are both critical for leukocyte capture and rolling in the circulation [18]. In our study, expression of E-selectin
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I. Werner et al. Fig. 2 Changes in soluble adhesion molecules in the supernatant after MB treatment, MB treatment followed by Jurkat cell co-culture and MB treatment followed by PBMC co-culture, each for 30 min and 2 h with either 10 lM, 30 lM or 60 lM MB. a Changes in sCD54 levels. b Changes in sCD106 levels. c Changes in sCD62-E levels. d Changes in sCD62-L levels (n = 3, data represent mean ± SEM) (§ p \ 0.05 vs. control, * p \ 0.05)
significantly increased in a dose dependent manner following MB. ICAM-1 and VCAM-1 were also modulated by MB. In this respect, MB may potentially affect the firm
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adhesion and transmigration of immune cells into inflammed tissue. The fact, that E-selectin is highly expressed on activated inflamed endothelial cells [19] indicates that MB
Adhesion molecule expression on microvascular endothelial cells
potentially induces endothelial cell activation at high doses or prolonged intervals of exposure. Given that ICAM-1 and VCAM-1, known to be highly expressed on activated endothelial cells [20], are in part also higher expressed due to MB and thus would support this theory. As the interaction between activated endothelial cells and circulating blood cells is essential in inflammatory processes, we co-incubated HuMEC-1 with human circulating blood cells after MB treatment. To get a deeper insight, if different circulating blood cell types may affect the adhesion molecule expression on endothelial cells with contact to MB, two different cell types were used for coculturing experiments, human PBMCs and human T-lymphocytes. Comparing the effects of PBMC and Jurkat cell co-culture we found significant differences in ICAM-1 surface expression. It seems that in case of short term MB exposure the change of ICAM-1 on endothelial cells is predominantly T-lymphocyte dependent. The change in adhesion molecule expression observed in the co-culture experiments seems not to be a result of the removal of MB but the interaction of circulating blood cells and endothelial cells after MB exposure. Would the modification of adhesion molecules just be a result of the displacement of MB from the endothelial cells, no significant differences between PBMC and Jurkat cell co-cultures should be observed. Furthermore, the increase of soluble ICAM-1 after isolated MB treatment may be related to an increased apoptosis of endothelial cells after MB, as previously described [21]. Co-incubation with PBMCs or T-lymphocytes would have a protective effect in our hypothesis. Interestingly, this fact could not be demonstrated for VCAM-1. We showed increased expression of VCAM-1 in co-cultures with both PBMCs and T-lymphocytes. The effect on these types of cells seems to be time and dose dependent, confirming our flow-cytometric results. However, we observed an increased expression of VCAM-1 in the supernatants of co-cultures with T-lymphocytes after high dosed MB, probably related to an increased cellular damage. The changes of L- and E-Selectin were not significant, probably related to the surface endothelial nature of these adhesion molecules. The deviating results of our western blot analysis may be related to total cell protein isolation, thus reflecting the amount of intra and extracellular protein levels. A recent study showed that MB treatment in high concentrations of 100 lM inhibited cytokine and chemokine secretion from microglia in an in vitro model of inflammation [22]. Other studies suggested that MB administered to tumor-bearing mice was able to decrease TNF production. [23]. Our results confirm the immunomodulatory effect of MB, although in our experiments, the expression of adhesion molecules on endothelial cells was mainly increased after administration. As some adhesion
molecules are lesser expressed in our experimental set up, it could be hypothesized that MB has a slight anti-inflammatory effect on endothelial cells in contact with circulating blood cells. However, the overall time/dose dependent increased expression of adhesion molecules confirm some clinical reports, related to increased extravasation and toxic effects of MB [17, 24]. Even though MB is more often used in daily clinics, our research is not focused on reproducing the clinic setup of MB application, but on the elementary effect of MB on adhesion molecules and the potential accompanied influence on inflammatory processes. Thus, our study may have some limitations regarding the translation to the clinical use of MB. In our experimental model we applied a constant volume and concentration of MB to the endothelial cells. In real life, MB is distributed in the body via blood flow when injected intravenously (IV). We applied MB to the endothelial cells once in a static setup and thus cannot mimic the distribution. Animal studies have demonstrated that depending on administering MB IV or oral, substantially higher concentrations of MB can be found in some organs than in blood [25]. MB is eliminated in the bile, feces and urine as leucomethylene blue, the metabolized form of MB [16]. Consequently, the MB concentration is usually not stable throughout the body. Our in vitro study does not mimic this scenario and therefore our data should be food for thought when further investigating MB in vitro as well as in vivo. In daily clinical routine, MB is often used as a rescue therapeutical option in patients with refractory vasoplegic shock. Patients suffering from this condition show an increased inflammatory response. From our data, MB may modulate adhesion molecule expression on endothelial cells and thus may lead to increased inflammatory processes, including infiltration and capillary leakage. Therefore, more clinical data are needed to confirm the possible pro-inflammatory effects of MB in critically-ill patients. Acknowledgments We thank Maryam Tabib and Falko Seyffarth for excellent technical support and Dr. V. Mirakaj, University Tu¨bingen, Department of Anesthesiology and Intensive Care Medicine for providing the HuMEC-1 cell line.
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Inflammation Research
ORIGINAL RESEARCH PAPER
Methylene blue modulates adhesion molecule expression on microvascular endothelial cells Isabella Werner • Fengwei Guo • Ulrich A. Stock • Miche`le Lupinski Patrick Meybohm • Anton Moritz • Andres Beiras-Fernandez
•
Received: 18 July 2013 / Revised: 17 January 2014 / Accepted: 15 April 2014 Ó Springer Basel 2014
Abstract Objective and design As methylene blue (MB) has been recently proposed to preserve blood pressure in case of vasoplegic syndrome and shock, an entity directly related to systemic inflammation, we aimed to elucidate the effect of MB on the expression of adhesion-molecules in endothelial-cells. Materials and treatment Human microvascular endothelialcells (HuMEC-1) were treated with 10, 30 or 60 lM MB for 30 min and 2 h each. Additionally, the treated HuMEC-1 were co-cultured with either human peripheral blood mononuclear cells (PBMCs) or Jurkat cells (human T-lymphocytes) for 2 h. Methods HuMEC-1 were analyzed after MB treatment and after co-culture experiments for expression of different adhesion-molecules (ICAM-1, VCAM-1, L-selectin, E-selectin) via FACS measurement and western blot analysis. The supernatants of the experiments were analyzed with regard to the soluble forms of the adhesion molecules. Results We found that MB is able to modulate the expression of adhesion-molecules on EC. Administration of MB increases the expression of E-selectin and VCAM-1 Responsible Editor: Ikuo Morita.
Electronic supplementary material The online version of this article (doi:10.1007/s00011-014-0737-1) contains supplementary material, which is available to authorized users. I. Werner (&) F. Guo U. A. Stock M. Lupinski A. Moritz A. Beiras-Fernandez Department of Thoracic and Cardiovascular Surgery, University Hospital Frankfurt, Goethe University, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany e-mail: [email protected] P. Meybohm Department of Anaesthesiology Intensive Care Medicine and Pain Therapy, University Hospital Frankfurt, Frankfurt/Main, Germany
depending on the dosage and time of exposure. ICAM-1 measurements provide evidence that different circulating blood cells can differently alter the adhesion-molecule expression on EC after MB exposure. Conclusion Our results provide evidence regarding the immunomodulatory effect of MB upon endothelial-cells after inflammation. Keywords Methylene blue Adhesion molecules Endothelial cells Peripheral blood mononuclear cells
Introduction Inflammatory responses after cardiac surgical interventions with cardiopulmonary by-pass are often related to high morbidity and mortality rate in patients, depending on the severity of the presentation. Refractory vasodilatory shock or vasoplegic syndrome is a severe complication of this inflammatory syndrome. Vasoplegia is frequently preceded by a profound vasodilatation occurring during surgery, refractory to conventional catecholamine treatment [1]. Leukocyte recruitment and the regulatory steps of capture, rolling, activation, and adhesion have been well studied and reviewed within the last decades [2, 3]. Selectins belong to the family of adhesion molecules that facilitate the primary capture of leukocytes from the bloodstream to the blood vessels at sites of tissue injury and inflammation [4]. Central characteristics of selectins are that they promote leukocyte attachment and rolling under shear stress and moreover force leucocytes to enter into tissues [5–7]. Rolling is mandatory for firm adhesion of leukocytes under flow conditions. Nonetheless, unless integrins start to interfere in this process, no firm adhesion and transmigration of the cells into the tissue can take
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place. Integrins are adhesion molecules with heterodimeric membrane proteins, which are transported to the cell surface [6, 8]. Immune cells start to change their shape and form bipolar characteristics of motile cells, after they adhered to the luminal endothelial surface [9, 10]. After adhesion, immune cells transmigrate through the endothelium and are directed to the site of inflammation [6]. Evidence is increasing that inhibitors of NO production greatly enhance the adhesion and emigration of polymorphonuclear leukocytes to venules, thus indicating that NO plays an important role in preventing leukocyte-endothelial cell adhesion [11], characteristic in inflammatory processes [2]. It has been proposed that methylene blue (MB) may have a direct inhibitory effect on eNOS, and probably iNOS [12], as well as a blocking effect on the formation of cGMP by inhibiting the GC enzyme [13]. As MB is known to be an NO/cGMP inhibitor and NO is preventing leukocyte adhesion, the modulation of adhesion molecules on endothelial cells due to MB treatment may be assumed. Vasoplegia is thought to be caused by the inflammationmediated endothelial dysfunction [14]. Some observational studies have shown that methylene blue (MB) can contribute to the preservation of blood pressure associated with endothelial dysfunction in vasoplegia [14–16]. An increase in inflammation has been observed associated to the administration of MB [17]. As MB is more and more often used in daily clinics, we were interested on the effect of MB on adhesion molecule expression on endothelial cells.
Materials and methods Ethics statement This study was approved by the Ethics Committee of the University of Frankfurt, Germany (Ref. Nr.: 189/13). All blood samples (n = 5) were obtained after informed consent and according to the declaration of Helsinki. All participants provided their written informed consent to participate in this study. Cell culture Human microvascular endothelial cells-1 (HuMEC-1) (kindly provided by Dr. V. Mirakaj, University Tu¨bingen, Department of Anesthesiology and Intensive Care Medicine) were cultured in MCDB-131 (Life Technologies, Darmstadt, Germany) supplemented with 10 % fetal calf serum (Gibco, Karlsruhe, Germany), 1 % glutamine (Gibco, Karlsruhe, Germany), 1 % pen/strep solution (Sigma Chemical Co. St. Louis, USA), 10 ng/ml Epidermal Growth Factor (Sigma Chemical Co. St. Louis, USA) and 1 lg/ml hydrocortisone (Sigma Chemical Co. St. Louis, USA).
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Jurkat cells (immortalized human T-lymphocytes) were cultured in RPMI-1640 medium (Sigma Chemical Co. St. Louis, USA) supplemented with 1 % pen/strep solution and 10 % fetal calf serum. All cells were cultivated at 37 °C and 5 % CO2 atmosphere. Isolation of peripheral blood mononuclear cells (PBMC) Blood was drawn into EDTA tubes from healthy volunteers. The probes were kept between 18 and 22 °C throughout the isolation process. The fresh blood was underlaid with 5 ml PolymorphprepÒ (Axis-Shield, Oslo, Norway), for density gradient centrifugation, and centrifuged at 450g for 35 min without break. The PBMC layer was washed twice with PBS (Gibco, Karlsruhe, Germany), counted and used for further experiments. HuMEC-1 treatment with MB and co-culturing of HuMEC-1 with PBMCs or Jurkat cells HuMEC-1 (1 9 106 cells/well) were plated into six well plates and cultured until 80 % confluence was reached. Consumed culture medium was removed and 10, 30 or 60 lM MB diluted in basal culture media was added for 30 min and 2 h each. Afterwards MB was removed and HuMEC-1 were used for either direct FACS analysis or coculture experiments. HuMEC-1 were co-cultured with either PBMCs or Jurkat cells. Therefore, 3 9 105 PBMCs or Jurkat cells were suspended in RPMI-1640 medium and co-cultured with MB treated HuMEC-1 monolayers. After 2 h at 37 °C, the supernatant was collected for ELISA experiments and cells were gently harvested and used for FACS analysis or western blot analysis. Untreated HuMEC-1 served as control. All experiments were performed in triplicates. FACS analysis of ICAM-1, VCAM-1, CD62E and CD62L To characterize the modulation of cell adhesion molecules of HuMEC-1 after MB treatment and followed co-culturing with PBMCs and Jurkat cells, we stained for ICAM-1 [fluorescein isothiocyanate (FITC)-conjugated CD54 antibody (BD, Heidelberg, Germany)], VCAM-1 [phycoerythrin (PE)-conjugated CD106 antibody (BD, Heidelberg, Germany)], CD62L [brilliant violet-421tm conjugated antibody (BioLegend, San Diego, USA)] and CD62E [PeridininChlorophyll (PerCP)-conjugated antibody (abcam, Cambridge, UK)]. Analysis was performed using a FACS Canto flow cytometer (BD, Heidelberg, Germany) and FLOW JO Software. Unstained cells served as controls. Expression levels of adhesion molecules are indicated as fluorescence intensity (FI).
Adhesion molecule expression on microvascular endothelial cells
Enzyme-linked immunoadsorbent assay (ELISA) The soluble ICAM-1, VCAM-1, E-selectin and L-selectin levels in the supernatants after co-culture experiments with MB treated HuMEC-1 were determined using human sICAM-1/CD54 Quantikine ELISA Kit, human sVCAM-1/ CD106 Quantikine ELISA Kit, human sE-selectin/CD62E Quantikine ELISA Kit and human sL-selectin/CD62L ELISA Kit, all from R&D Systems (Minneapolis, USA) in accordance with the manufacturer’s protocol. Western blot analysis To analyze ICAM-1, VCAM-1, E-selectin and L-selectin in HuMEC-1, cell lysates were applied to a 7 % polyacrylamide gel and electrophoresed (90 min, 60 V followed by 100 V). The proteins were then transferred to nitrocellulose membranes (1 h, 100 V). The membranes were blocked with nonfat dry milk for 1 h, and then incubated overnight with monoclonal antibodies directed against human ICAM-1, VCAM-1, E-selectin and L-selectin (all Santa Cruz Biotechnology Inc., Heidelberg, Germany). HRP-conjugated goatanti-mouse IgG (Millipore, Temecula, CA, USA) served as the secondary antibody for ICAM-1, VCAM-1, L-selectin and b-actin. HRP-conjugated goat-anti-rabbit IgG (Millipore, Temecula, CA, USA) served as the secondary antibody for E-selectin. The membranes were briefly incubated with ECL detection reagent (ECLTM, Amersham/GE Healthcare, Mu¨nchen, Germany) to visualize the proteins and then analyzed by the Fusion FX7 system (Peqlab, Erlangen, Germany). b-actin (Sigma, Taufenkirchen, Germany) served as the internal control. The visualized protein bands were analyzed using ImageJ software. The relative amount of protein (RQ) was calculated in relation to b-actin expression. Statistical analysis Data represent mean ± SEM. Statistical analysis was performed with Prism 6 software (Graph Pad) using unpaired Students T test. Differences with p \ 0.05 were considered statistically significant.
Results Surface expression of adhesion molecules on endothelial cells
to control. In case of Jurkat cell co-culture, short time exposure of MB for 30 min lead to a significant drop of ICAM-1 surface expression compared to MB treatment alone (10 lM MB 30 min: 64 ± 2 FI vs. 10lM MB 30 min co-Jurkat: 50.3 ± 2.4 FI; 30 lM MB 30 min: 63.2 ± 0.4 FI vs. 30lM MB 30 min co-Jurkat: 50.85 ± 1.55 FI; 60 lM MB 30 min: 63.85 ± 0.15 FI vs. 60lM MB 30 min co-Jurkat: 53 ± 0.7 FI). This could be observed at 10, 30 and 60 lM MB treatment (Fig. 1a). On the other hand, PBMC co-culture following the same MB treatments did not cause a significant change in ICAM-1 expression compared to MB treatment alone, except at high MB dosage of 60 lM (10 lM MB 30 min: 64 ± 2 FI vs. 10lM MB 30 min co-PBMC: 61.7 ± 5.7 FI; 30 lM MB 30 min: 63.2 ± 0.4 FI vs. 30lM MB 30 min co-PBMC: 65.45 ± 0.95 FI; 60 lM MB 30 min: 63.85 ± 0.15 FI vs. 60lM MB 30 min co-PBMC: 66.65 ± 0.45 FI). PBMC and Jurkat cell co-culture differed significantly at 30 and 60 lM MB treatment for 30 min (30 lM MB 30 min co-Jurkat: 50.85 ± 1.55 FI vs. 30lM MB 30 min co-PBMC: 65.45 ± 0.95 FI; 60 lM MB 30 min co-Jurkat: 53 ± 0.7 FI vs. 60lM MB 30 min co-PBMC: 66.65 ± 0.45 FI). The long term exposure of MB for 2 h did not significantly change ICAM-1 expression independently of co-culture, whereas similar trend can be seen (Fig. 1a, for more details see supplementary information). Vcam-1/cd106 The evaluation of VCAM-1 (CD106) showed a dose and time dependent increase in surface expression after MB treatment and co-culture experiments (Fig. 1b). MB treatment alone caused a significant up-regulation of VCAM-1 from 10 to 60 lM MB with increasing exposure time (10 lM MB 30 min: 4.27 ± 1.91 FI vs. 60 lM MB 2 h: 22.67 ± 4.94 FI). This increase is also significant at 30 lM MB for 30 min compared to 60 lM MB for 2 h (30 lM MB 30 min: 5.34 ± 2.53 FI vs. 60 lM MB 2 h: 22.67 ± 4.94 FI) (Fig. 1b). Only co-culturing with PBMCs and not Jurkat cells caused significant changes in VCAM-1 surface expression. Short time (30 min) MB treatment for 30 min followed by PBMC co-culture significantly increased VCAM-1 on EC with increasing MB dosages (10 lM MB 30 min co-PBMC: 2.99 ± 0.32 FI vs. 60 lM MB 30 min co-PBMC: 5.81 ± 0.72 FI; 30 lM MB 30 min co-PBMC: 3.49 ± 0.25 FI vs. 60 lM MB 30 min co-PBMC: 5.81 ± 0.72 FI) (Fig. 1b). The contact with Jurkat cells did not cause any significant changes in VCAM-1 expression levels (Fig. 1b, for more details see supplementary information).
Icam-1/cd54 E-selectin/CD62-E In our experiments, treatment of HuMEC-1 with MB caused no significant changes in ICAM-1 (CD54) expression, irrespectively of dose and exposure time (Fig. 1a) in comparison
E-Selectin (CD62-E) was drastically modified by MB and followed co-culture with Jurkat cells and PBMCs (Fig. 1c).
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I. Werner et al. Fig. 1 Changes in adhesion molecule expression on HuMEC-1 after MB treatment, MB treatment followed by Jurkat cell co-culture and MB treatment followed by PBMC co-culture, each for 30 min and 2 h with either 10, 30 or 60 lM MB. a Changes in CD54 expression. b Changes in CD106 expression. c Changes in CD62-E expression. d Changes in CD62-L expression (n = 3, data represent mean ± SEM) (§ p \ 0.05 vs. control, * p \ 0.05)
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Adhesion molecule expression on microvascular endothelial cells
MB treatment alone caused a dose and time dependent increase of E-selectin expression (10 lM MB 30 min: 21.85 ± 4.35 FI vs. 60 lM MB 2 h: 84.5 ± 11.46 FI). The co-culturing of MB treated HuMEC-1 with Jurkat cells lead to a significant MB dose and exposure time dependent increase of E-selectin surface expression (10 lM MB 30 min co-Jurkat: 7.97 ± 2.23 FI vs. 30 lM MB 2 h coJurkat: 33.25 ± 1.55 FI, 60 lM MB 30 min co-Jurkat: 39.25 ± 5.15 FI and 60 lM MB 2 h co-Jurkat: 62.95 ± 11.05 FI; 10 lM MB 2 h co-Jurkat: 14.5 ± 3.1 FI and 30 lM MB 30 min co-Jurkat: 18.2 ± 2.4 FI vs. 30 lM MB 2 h co-Jurkat: 33.25 ± 1.55 FI). Similar significant results were observed after PBMC co-culture with MB treated HuMEC-1 (10 lM MB 30 min co-PBMC: 8.72 ± 1.78 FI and 10 lM MB 2 h co-PBMC: 16.15 ± 4.35 FI vs. 30 lM MB 2 h co-PBMC: 37.95 ± 2.45 FI, 60 lM MB 30 min co-PBMC: 42.55 ± 0.45 FI and 60 lM MB 2 h co-PBMC: 65.1 ± 9 FI; 30 lM MB 30 min co-PBMC: 22.55 ± 3.55 FI vs. 60 lM MB 30 min co-PBMC: 42.55 ± 0.45 FI and 60 lM MB 2 h co-PBMC: 65.1 ± 9 FI) (Fig. 1c). In comparison to ICAM-1 and VCAM-1, in this scenario no significant difference between MB treatment alone and the two coculture experiments could be observed. L-selectin/CD62-L In case of L-selectin (CD62-L) it seems that MB in contact to circulating blood cells does not influence the surface expression of this selectin. Even though a kind of modification can be seen in Fig. 1d but neither significant changes nor any trend could be observed. Protein expression of adhesion molecules in endothelial cells Western blot analysis did not reveal significant changes in CD54 expression comparing MB treatment and MB treatment followed by co-culture experiments. MB treatment alone significantly reduced CD54 expression compared to control after long-term treatment and higher dosages (ctr 1.51 ± 0.21 RQ vs. 30 lM MB 2 h 0.17 ± 0.01 RQ and 60 lM MB 2 h 0.16 ± 0.01 RQ). CD106 analysis has shown no appreciable significant differences comparing all experimental groups, except a dose-dependent increase from 30 lM MB 30 min coPBMCs to 60 lM MB 30 min co-PBMCs (30 lM MB 30 min co-PBMC 1.12 ± 0.05 RQ vs. 60 lM MB 30 min coPBMC 1.45 ± 0.01 RQ) and higher CD106 levels in 60 lM MB 30 min co-PBMCs compared to 60 lM MB treatment alone (60 lM MB 30 min co-PBMC 1.45 ± 0.01 RQ vs. 60 lM MB 30 min 0.38 ± 0.07 RQ). CD62-E was dosedependent reduced after short term MB treatment and dose dependent elevated after long term MB treatment (30 lM MB
30 min 1.11 ± 0.07 RQ vs. 60 lM MB 30 min 0.76 ± 0.00 RQ; 10 lM MB 2 h 0.85 ± 0.01 RQ vs. 60 lM MB 2 h 1.06 ± 0.01 RQ; p \ 0.05). High dose MB treatment caused a time-dependent up-regulation of CD62-E expression (60 lM MB 30 min 0.76 ± 0.00 RQ vs. 60 lM MB 2 h 1.06 ± 0.01 RQ; p \ 0.05). PBMC co-culture was able to significantly reduce CD62-E levels after 30 lM MB 30 min treatment (30 lM MB 30 min 1.11 ± 0.07 RQ vs. 30 lM MB 30 min co-PBMC 0.61 ± 0.04 RQ). CD62-L expression levels were only significantly modulated by Jurkat cell co-culture. Lowdose MB treatment caused less CD62-L expression compared to Jurkat cell co-culture (10 lM MB 30 min 1.33 ± 0.00 RQ vs. 10 lM MB 30 min co-Jurkat 1.73 ± 0.09 RQ). Furthermore, a dose and time dependent modification of CD62-L was observed after MB treatment followed by Jurkat cell co-culture (30 lM MB 30 min co-Jurkat 1.70 ± 0.00 RQ vs. 60 lM MB 30 min co-Jurkat 1.30 ± 0.0.06 RQ and 30 lM MB 30 min co-Jurkat 1.70 ± 0.00 RQ vs. 30 lM MB 2 h co-Jurkat 1.09 ± 0.13 RQ). Soluble forms of adhesion molecules sICAM-1, sVCAM-1, sE-selectin and sL-selectin released after MB treatment and co-culture sICAM-1 in the supernatant did not differ significantly in any group compared to control. The sICAM-1 levels after MB treatment compared to MB treatment followed by co-culture with Jurkat cells and PBMCs was significantly higher in the 10 lM MB 30 min group, 30 lM MB 2 h group and 60 lM MB 2 h group (Fig. 2a). MB treatment only has shown a dose dependent increase in the long-term treatment group (Fig. 2a). Soluble CD106 was not detectable in every experimental group. Low-dose MB treatment caused higher sCD106 release after PBMC co-culture than after Jurkat cell co-culture by trend. After high dose MB treatment the trend is reversed. Jurkat cell co-culture led to a time dependent elevation of sCD106 comparing 30 min vs. 2 h treatment (Fig. 2b) Soluble CD62-E was detected only in very low concentrations (Fig. 2c). PBMC co-culture showed higher levels of sCD62-E after low-dose MB compared to Jurkat cell co-culture and MB treatment only, by trend. Soluble CD62-L was only measurable in the PBMC co-culture groups and after short time and low-dose MB treatment (Fig. 2d). Longterm MB treatment combined with PBMC co-culture caused a significant dose dependent drop in sCD62-L levels.
Discussion These data show that MB may have an impact on adhesion molecule expression of endothelial cells. E- and L-selectin are both critical for leukocyte capture and rolling in the circulation [18]. In our study, expression of E-selectin
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I. Werner et al. Fig. 2 Changes in soluble adhesion molecules in the supernatant after MB treatment, MB treatment followed by Jurkat cell co-culture and MB treatment followed by PBMC co-culture, each for 30 min and 2 h with either 10 lM, 30 lM or 60 lM MB. a Changes in sCD54 levels. b Changes in sCD106 levels. c Changes in sCD62-E levels. d Changes in sCD62-L levels (n = 3, data represent mean ± SEM) (§ p \ 0.05 vs. control, * p \ 0.05)
significantly increased in a dose dependent manner following MB. ICAM-1 and VCAM-1 were also modulated by MB. In this respect, MB may potentially affect the firm
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adhesion and transmigration of immune cells into inflammed tissue. The fact, that E-selectin is highly expressed on activated inflamed endothelial cells [19] indicates that MB
Adhesion molecule expression on microvascular endothelial cells
potentially induces endothelial cell activation at high doses or prolonged intervals of exposure. Given that ICAM-1 and VCAM-1, known to be highly expressed on activated endothelial cells [20], are in part also higher expressed due to MB and thus would support this theory. As the interaction between activated endothelial cells and circulating blood cells is essential in inflammatory processes, we co-incubated HuMEC-1 with human circulating blood cells after MB treatment. To get a deeper insight, if different circulating blood cell types may affect the adhesion molecule expression on endothelial cells with contact to MB, two different cell types were used for coculturing experiments, human PBMCs and human T-lymphocytes. Comparing the effects of PBMC and Jurkat cell co-culture we found significant differences in ICAM-1 surface expression. It seems that in case of short term MB exposure the change of ICAM-1 on endothelial cells is predominantly T-lymphocyte dependent. The change in adhesion molecule expression observed in the co-culture experiments seems not to be a result of the removal of MB but the interaction of circulating blood cells and endothelial cells after MB exposure. Would the modification of adhesion molecules just be a result of the displacement of MB from the endothelial cells, no significant differences between PBMC and Jurkat cell co-cultures should be observed. Furthermore, the increase of soluble ICAM-1 after isolated MB treatment may be related to an increased apoptosis of endothelial cells after MB, as previously described [21]. Co-incubation with PBMCs or T-lymphocytes would have a protective effect in our hypothesis. Interestingly, this fact could not be demonstrated for VCAM-1. We showed increased expression of VCAM-1 in co-cultures with both PBMCs and T-lymphocytes. The effect on these types of cells seems to be time and dose dependent, confirming our flow-cytometric results. However, we observed an increased expression of VCAM-1 in the supernatants of co-cultures with T-lymphocytes after high dosed MB, probably related to an increased cellular damage. The changes of L- and E-Selectin were not significant, probably related to the surface endothelial nature of these adhesion molecules. The deviating results of our western blot analysis may be related to total cell protein isolation, thus reflecting the amount of intra and extracellular protein levels. A recent study showed that MB treatment in high concentrations of 100 lM inhibited cytokine and chemokine secretion from microglia in an in vitro model of inflammation [22]. Other studies suggested that MB administered to tumor-bearing mice was able to decrease TNF production. [23]. Our results confirm the immunomodulatory effect of MB, although in our experiments, the expression of adhesion molecules on endothelial cells was mainly increased after administration. As some adhesion
molecules are lesser expressed in our experimental set up, it could be hypothesized that MB has a slight anti-inflammatory effect on endothelial cells in contact with circulating blood cells. However, the overall time/dose dependent increased expression of adhesion molecules confirm some clinical reports, related to increased extravasation and toxic effects of MB [17, 24]. Even though MB is more often used in daily clinics, our research is not focused on reproducing the clinic setup of MB application, but on the elementary effect of MB on adhesion molecules and the potential accompanied influence on inflammatory processes. Thus, our study may have some limitations regarding the translation to the clinical use of MB. In our experimental model we applied a constant volume and concentration of MB to the endothelial cells. In real life, MB is distributed in the body via blood flow when injected intravenously (IV). We applied MB to the endothelial cells once in a static setup and thus cannot mimic the distribution. Animal studies have demonstrated that depending on administering MB IV or oral, substantially higher concentrations of MB can be found in some organs than in blood [25]. MB is eliminated in the bile, feces and urine as leucomethylene blue, the metabolized form of MB [16]. Consequently, the MB concentration is usually not stable throughout the body. Our in vitro study does not mimic this scenario and therefore our data should be food for thought when further investigating MB in vitro as well as in vivo. In daily clinical routine, MB is often used as a rescue therapeutical option in patients with refractory vasoplegic shock. Patients suffering from this condition show an increased inflammatory response. From our data, MB may modulate adhesion molecule expression on endothelial cells and thus may lead to increased inflammatory processes, including infiltration and capillary leakage. Therefore, more clinical data are needed to confirm the possible pro-inflammatory effects of MB in critically-ill patients. Acknowledgments We thank Maryam Tabib and Falko Seyffarth for excellent technical support and Dr. V. Mirakaj, University Tu¨bingen, Department of Anesthesiology and Intensive Care Medicine for providing the HuMEC-1 cell line.
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