Abacavir and didanosine induce the interaction between human leukocytes and endothelial cells through Mac-1 upregulation
De Pablo, Carmena; Orden, Samuela; Apostolova, Nadezdaa; Blanquer, Amandoc; Esplugues, Juan Va,b; Alvarez, Angelesa
aDepartamento de Farmacología and CIBERehd, Facultad de Medicina, Universidad de Valencia, Spain
bFundación Hospital Universitario Dr Peset, Spain
cCentro de Transfusiones de la Comunidad Valenciana, Valencia, Spain.
Received 27 December, 2009
Revised 12 March, 2010
Accepted 23 March, 2010
Correspondence to Juan V. Esplugues, Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia. Avda. Blasco Ibáñez 15-17, 46010 Valencia, Spain Tel: +34 96 3864624; fax: +34 96 3983879; e-mail: Juan.V.Esplugues@uv.es
Objective: Abacavir and didanosine are nucleoside reverse transcriptase inhibitors (NRTI) widely used in therapy for HIV-infection but which have been linked to cardiovascular complications. The objective of this study was to analyze the effects of clinically relevant doses of abacavir and didanosine on human leukocyte–endothelium interactions and to compare them with those of other NRTIs.
Design and methods: The interactions between human leukocytes – specifically peripheral blood polymorphonuclear (PMN) or mononuclear (PBMC) cells – and human umbilical vein endothelial cells were evaluated in a flow chamber system that reproduces conditions in vivo. The expression of adhesion molecules was analyzed by flow cytometry.
Results: Abacavir induced a dose-dependent increase in PMN and PBMC rolling and adhesion. This was reproduced by didanosine but not by lamivudine or zidovudine. Both abacavir and didanosine increased Mac-1 expression in neutrophils and monocytes, but produced no effects on either lymphocytes or the expression of endothelial adhesion molecules. The PMN/PBMC rolling and adhesion induced by abacavir or didanosine did not occur when antibodies against Mac-1 or its ligand ICAM-1 were blocked.
Conclusion: Abacavir induces significant human leukocyte accumulation through the activation of Mac-1, which in turn interacts with its endothelial ligand ICAM-1. The fact that didanosine exhibits similar effects and that lamivudine and zidovudine do not points to a relationship between the chemical structure of NRTIs and the induction of leukocyte/endothelial cell interactions. This mechanism may be especially relevant to the progression of the vascular damage associated with atherosclerosis and myocardial infarction in abacavir and didanosine-treated patients.
Continuous administration of ‘highly active antiretroviral therapy’ (HAART) has made AIDS a chronic illness. However, with the increased longevity of patients there is growing concern about the long-term adverse effects induced by this life-long pharmacological treatment, particularly its role in cardiovascular complications such as atherosclerosis and myocardial infarction . HAART is a combination of at least three drugs: two nucleoside reverse transcriptase inhibitors (NRTI) plus a protease inhibitor and/or a nonnucleoside reverse transcriptase inhibitor (NNRTI) . Although protease inhibitors were originally considered responsible for these deleterious cardiovascular effects, there is recent evidence that also implicates NNRTIs and, particularly, NRTIs .
A potential link between abacavir (ABC), one of the most widely used NRTIs, and myocardial infarction was first highlighted in 2005 , and substantial controversy has surrounded the subject ever since. Recent studies have not only further implicated ABC as agent that raises the risk of myocardial infarction but also highlight a similar risk is, though to a lesser extent, with didanosine (ddI), another NRTI [4–7]. The reasons for these adverse effects are not clear as neither ABC nor ddI seem to exert a negative influence on major predisposing factors such as lipid and/or glucose metabolism. However, the fact that the potential to develop myocardial infarction exists as long as patients are receiving the drugs and decreases when therapy is discontinued points to the existence of a rapid mechanism involving vascular inflammation . There is some evidence that ABC causes endothelial nitric oxide synthase (eNOS) downregulation and superoxide anion production in human endothelial cells , both of which situations can lead to vascular dysfunction and leukocyte accumulation [9,10]. In addition, ABC-treated patients exhibit elevated levels of the inflammatory markers C-reactive protein (CRP) and interleukin-6 .
The accumulation of leukocytes in the vessel wall is a hallmark of the early stages of atherosclerosis, acute myocardial infarction and other vascular diseases and is mediated by the interaction between the adhesion molecules expressed on white blood cells and/or endothelial cells. During this process, leukocytes roll along the wall of inflamed vessels before coming to a halt, after which they adhere and transmigrate . In the present study, we demonstrate the capacity of both ABC and ddI to elicit such interactions and explore the molecular mechanisms involved. NRTIs can be classified as purine or pirimidine analogues. Purine analogues can be guanosine (ABC) or adenosine (ddI) derivates. Pirimidine analogues can be thymidine (zidovudine and stavudine) or cytosine (lamivudine, zalcitabine and emtricitabine) derivates [12,13]. Our primary hypothesis was based on the existing clinical evidence obtained with ABC and ddI; however for the sake of comparison, we extended our analysis to include the pirimidine analogues lamivudine and zidovudine.
Human umbilical vein endothelial cell culture
Human umbilical vein endothelial cells (HUVEC) were harvested from freshly obtained umbilical cords by collagenase treatment as previously described . Briefly, umbilical cord veins were rinsed of blood products with warm PBS, after which the vein was filled with collagenase (1 mg/ml) for 17 min at 37°C. The cords were then gently massaged to ensure detachment of endothelial cells from the vessel wall. The digest was collected, centrifuged and pelleted once more. The pellet was resuspended in endothelial cell growth medium (EGM-2) inside T25 culture flasks where cells were cultured until confluence. After reaching confluence, primary cultures were detached with trypsin and transferred into appropriate culture dishes. Passage 1 from these primary cultures was subsequently employed. For adhesion studies, HUVEC were cultured on fibronectin (5 μg/ml)-coated 25-mm plastic coverslips until confluent (∼48 h).
Human peripheral blood mononuclear (PBMC) or polymorphonuclear (PMN) cells were isolated from whole blood anticoagulated with sodium citrate drawn from healthy volunteers . Samples were incubated with dextran (3%) for 45 min. PBMC and PMN in the supernatant were separated by gradient density centrifugation (250 g, 25 min) with Ficoll-PaqueTM Plus. After red blood cell lysis, leukocytes were washed (HBSS without Ca2+ or Mg2+) and resuspended in complete RPMI media.
The medical ethical committee of the Hospital Clínico Universitario de Valencia approved the study and all patients provided written informed consent.
Adhesion assay under flow conditions
The parallel plate flow chamber in-vitro model has been described previously in detail [14,15]. For adhesion assays, coverslips containing confluent HUVEC monolayers were inserted into a circular recess in the bottom plate of the flow chamber (maintained at 37oC), where a portion (5 × 25 mm) of the monolayer was exposed to the flow. The entire chamber was mounted on an inverted microscope (Nikon Eclipse TE 2000-S, Amstleveen, The Netherlands) connected to a video camera (Sony Exware HAD, Koeln, Germany). Experiments were conducted using a 40× objective lens. PMNs or PBMCs were resuspended in flow buffer (DPBS+ containing 20 mmol/l HEPES and 0.1% HSA) at 1 × 106 or 0.5 × 106 cells/ml respectively and drawn across the HUVEC monolayer at a controlled flow rate of 0.36 ml/min (estimated shear stress of 0.7 dyne/cm2). A circular glass window in the top plate of the chamber allowed direct live microscopic examination of the monolayer exposed to the flow. Images were recorded in a single field of view over a 5 min period during which leukocyte parameters were determined. Leukocyte rolling was calculated by counting the number of leukocytes rolling over 100 μm2 of the endothelial monolayer during 1 min period. Velocities of 20 consecutive leukocytes in the field of focus were determined by measuring the time required to travel a distance of 100 μm. Leukocyte adhesion was determined by counting the number of leukocytes that maintained stable contact with the monolayer for 30 s.
In order to study the effects of NRTIs on leukocyte–endothelial cell interactions, we chose one of the analogues of each purine or pirimidine. In this way, leukocytes (PMNs or PBMCs) and HUVEC were treated for 4 h at 37°C with NRTI purine analogues [ABC (0.1–15 μmol/l) or ddI (5 μmol/l)] or NRTI pirimidine analogues [lamivudine (10 μmol/l) or zidovudine (5 μmol/l)] or with a control vehicle. Tumoral necrosis factor (TNF-α, 10 ng/ml, 4 h) and platelet-activating factor (PAF, 1 μmol/l, 1 h) were used as positive controls for HUVEC and leukocytes respectively. Doses were chosen in order to mimic clinical plasma concentrations of the drugs [8,16,17]. To study the effects of blocking antibodies on NRTI-induced leukocyte–endothelial cell interactions, PMN or PBMC were pretreated with anti-lymphocyte function-associated antigen 1 (LFA-1, 10 μg/ml), anti-macrophage 1 antigen (Mac-1, 20 μg/ml), anti-β2 integrins (CD18, 10 μg/ml) or control antibodies (10 μg/ml) for 20 min (4°C, darkness) prior to NRTI administration or HUVEC monolayers were pretreated with anti-intercellular adhesion molecule-1 (ICAM-1, 20 μg/ml) or control antibodies for 30 min at 37°C prior to drug administration. The antibodies were assayed at the previously described doses [18,19].
Analysis of the expression of adhesion molecules in peripheral blood leukocytes and in human umbilical vein endothelial cells
Leukocyte adhesion molecules were analyzed in citrated blood samples from healthy donors (40 μl) as described previously [14,20]. These samples were treated for 4 h at 37°C with the NRTI agents and were then incubated for 20 min on ice in the dark with saturating amounts of the corresponding FITC-conjugated antibody. An automated lysing procedure to remove red blood cells and to fix leukocytes was carried out using the EPICS TQ-PREP system (Coulter Electronics, Hialeah, Florida, USA). Neutrophils, monocytes and lymphocytes were identified in the flow cytometer by their specific size (forward-angle light scatter) and granularity (side-angle light scatter). HUVEC were grown to confluence in six well plates as mentioned earlier. Cells were then stimulated for 4 h at 37°C with NRTI agents. Cells were detached with trypsin and placed in suspension and were then incubated with the corresponding antibody (20 min, ice, darkness), fixed (formaldehyde) and analyzed for protein expression according to forward and side scatter characteristics. For both leukocytes and HUVEC, the median of the specific fluorescence intensity was employed as a marker of the expression of the respective epitope, and all samples were compensated using the appropriate isotype-matched negative control. Ten thousand cells were analyzed in each case. Analysis was performed in an EPICS XL-MCL cytometer (Coulter Electronics).
Dulbecco's PBS with (DPBS+) or without (DPBS-) Ca2+ and Mg2+, EGM-2 culture media and foetal bovine serum were provided by LONZA (Verviers, Belgium). Recombinant TNF-α, human serum albumine (HSA, Albuminate 25%), RPMI1640 supplemented with 20 mM HEPES, HBSS, fibronectin and dextran were purchased from Sigma Chemical Co (St Louis, Missouri, USA). Ficoll-Paque TM Plus was purchased from GE Healthcare (Little Chalfont, Buckinghamshire,UK). Plastic coverslips with a diameter of 25 mm were obtained from Nunc (supplied by Thermo Fisher Scientific). PBS, collagenase, and trypsin were obtained from Gibco Invitrogen. The Immunoprep reagent was acquired from Beckman Coulter. ABC, ddI, lamivudine and zidovudine were from Sequoia Research Products. The following mAb have been reported previously and were used as purified IgG: blocking antibodies against CD11a (clone m38), CD11b (clone ICRF44), β2-integrins and controls were purchased from Calbiochem (San Diego, California, USA). The blocking antibody against ICAM-1 was obtained from BD Bioscience. FITC or PE conjugated control antibodies and antibodies against E-selectin, ICAM-1, vascular cell adhesion molecule (VCAM)-1, CD18, CD11a, CD11b or CD11c were from BD Bioscience.
Data analysis and statistics
Data are mean ± SEM of n at least four experiments. Statistical significance was considered to be less than 0.05 by one-way ANOVA analysis of variance with Newman–Keuls post-test correction to compare multiple variances.
Effects of nucleoside reverse transcriptase inhibitors on leukocyte–endothelium interactions
ABC induced a significant and dose-dependent increase in the rolling flux (Fig. 1a and 1c) and adhesion (Fig. 1b and 1d) of PMN and PBMC. Concomitantly, ABC induced a decrease in the rolling velocity of PMN (veh: 732 ± 104, ABC 5 μmol/l: 542 ± 58 μm/s, P < 0.05, n = 5) and PBMC (veh: 751 ± 69, ABC 5 μmol/l: 469 ± 120 μm/s, P < 0.01, n = 6). PBMC were more sensitive than PMN to the effects of ABC, and this difference reached significance with doses 10 times lower (1 μmol/l). Leukocyte–endothelium interactions were significantly increased by ddI but not by zidovudine or lamivudine (Fig. 2).
Endothelial cells were not activated by ABC or ddI
In HUVECs, the highest concentrations of ABC (15 μmol/l, n = 4) or ddI (10 μmol/l, n = 4) had no effect on the expression (as % of control) of E-selectin (ABC: 103 ± 5%, ddI: 99 ± 2%), ICAM-1 (ABC: 99 ± 15, ddI: 93 ± 7%) or VCAM-1 (ABC: 98 ± 7%, ddI: 109 ± 18%).
Role of Mac-1 in the activation of leukocytes
Monocyte and neutrophil adhesion to the endothelium is generally mediated by the interaction of β2-integrins LFA-1 and/or Mac-1 with their endothelial ligand ICAM-1. However, Mac-1 has other nonendothelial matrix ligands. β2-integrins share a common β subunit (CD18) and have a specific α subunit (CD11a for LFA-1 and CD11b for Mac-1) . Treatment of neutrophils and monocytes with ABC or ddI increased the expression of CD18 and CD11b (Fig. 3 and Supplemental Figure 1, http://links.lww.com/QAD/A33) but had no effect on the expression of CD11a, CD49d and L-selectin. Once again, monocytes were more sensitive than neutrophils to the effects of ABC. Neither ABC nor ddI had any effect on the adhesion molecules of lymphocytes (data not shown). The interactions induced by ABC or ddI were prevented by antibodies against CD11b, CD18 or ICAM-1, but not by antibodies against CD11a (Fig. 4 and Supplemental Figure 2, http://links.lww.com/QAD/A34), thus suggesting that a CD11b-CD18 (Mac-1) mechanism is responsible for the accumulation of leukocytes.
This study demonstrates for the first time that both ABC and ddI induce the interaction between human leukocytes and endothelial cells by activating Mac-1 in neutrophils and monocytes, but not in lymphocytes, which in turn interacts with the ICAM-1 that is present on endothelial cells.
We used an in-vitro model in which human leukocytes flow over a monolayer of human endothelial cells with a shear stress similar to that observed in vivo . This reproduces the processes that precede the formation of an inflammatory focus in vivo (rolling and adhesion) and which are critical for hemostasis and vascular cell integrity. However, an exacerbation of these interactions contributes to the vascular dysfunction and injury associated with many vascular diseases (e.g., atherosclerosis, diabetic vasculopathy, hypercholesterolemia, hypertension, ischemia–reperfusion,…) . Our dynamic experimental system has been widely used to visualize and analyze the multistep recruitment of leukocytes in these diseases; moreover, it allows the mechanisms of action implicated in this recruitment to be assessed [22,23].
In our experiments, concentrations of ABC (0.1–15 μmol/l) and ddI (5 μmol/l) that mimic those present in patients (1–8 and 3–10 μmol/l respectively) [8,24,25] induced leukocyte–endothelial cell interactions (rolling and adhesion). The magnitude of the increases obtained with both drugs was smaller than that observed after direct stimulation with proinflammatory agents such as TNF-α, interleukin-4 or RANTES [26,27]; however, it was substantially greater than that exhibited in unstimulated PMNs or PBMCs from patients with different vascular conditions [28–30]. Leukocyte accumulation induced by ABC or ddI occured simultaneously with the selective upregulation of Mac-1 on the surface of human neutrophils and monocytes. Nevertheless, the expression of other adhesion molecules in these leukocytes [CD11a/CD18 or very late antigen (VLA)-4] or in endothelial cells (E-selectin, ICAM-1 or VCAM-1) was not affected. Thus, our results suggest that ABC and ddI promote the recruitment of leukocytes before activating the endothelium, and thus before dysfunction appears which needs a much longer period of exposure (24 h) to develop .
HIV-infection itself is associated with a more pronounced adhesion of leukocytes to endothelial cells  and with an elevated cardiovascular risk . Both situations could be related to the high levels of the endothelial dysfunction markers (ICAM-1, VCAM-1 and E-selectin) that are present in these patients . Since ABC has been specifically associated with an impaired endothelial function in HIV-infected patients  and given the difference between the activation profile of adhesion molecules observed in our experiments and that of HIV patients, the effects of antiretrovirals and those of the virus could feasibly be accumulative. In other words, the virus may cause endothelial activation  and antiretrovirals could activate leukocytes. This is an interesting hypothesis, but further clinical and experimental studies would be necessary before any solid conclusion can be drawn.
Mac-1 is mobilized from intracellular secretory vesicles to the cell surface within minutes of stimulating neutrophils and monocytes. Although it may interact with ICAM-2, iC3B, factor X or fibrinogen, its main ligand, ICAM-1 is constitutively expressed on the surface of the vascular endothelium . Thus, the fact that blocking either Mac-1 or ICAM-1 significantly reduced the effects of ABC and ddI point to a role for both these molecules in the rolling and adhesion induced by these two antiretrovirals. Although their involvement in adhesion is to be expected, rolling is considered to be mediated by selectins and/or VLA-4/VCAM-1 . However, there is growing evidence of the implication of Mac-1/ICAM-1 in rolling in both the activated and resting endothelium [35,36].
ABC and ddI specifically affect PMN and PBMC. This is of relevance given that there is a substantial increase in the levels of neutrophils and monocytes during acute myocardial infarction and in the expression of Mac-1 among the two cell populations . In addition, it has been reported that Mac-1 induces the binding of neutrophils to activated cardiac myocytes , and recent evidence points to the mediation of the leukocyte engagement of platelets as the link between cellular adhesion and thrombosis by Mac-1 [36,39].
Finally, the fact that similar effects to those of ABC were observed with ddI but not with lamivudine or zidovudine suggests a relationship between the chemical structure of NRTIs and the induction of leukocyte/endothelial cell interactions. It is tempting to speculate that purine analogues such as ABC or ddI have the potential to interfere with purine-signaling pathways and to provoke cardiovascular complications with inflammatory components (such as atherosclerosis and myocardial infarction) by reducing the levels of adenosine and increasing those of proinflammatory ATP. However, it is necessary to evaluate the actions, on the one hand, of other NRTIs such as tenofovir and emtricitabine, both of which are potential alternatives to ABC for patients with an elevated risk of cardiovascular disease, and on the other hand, of NNRTIs and protease inhibitors in this and other experimental settings before a sound clinical conclusion can be established.
C.D.P. performed the research; S.O., N.A. and A.B. helped perform the research and J.V.E. and A.A. designed the research and wrote the paper. J.V.E. and A.A. contributed equally to this study.
This study has been supported by grants SAF2007-60021 from Ministerio de Educación y Cultura; GV/2007/074, GVACOMP2009-266, ACOMP2009-194 and CS2009-AP-030 from Generalitat Valenciana and CD06/04/0071 (CIBERehd) and PI081325 from Ministerio de Sanidad y Consumo. C.D.P and S.O. have been supported by grants from Ministerio de Educación y Cultura and from Fundación Juan Esplugues respectively.
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