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Lymphocyte adhesion to CCR5 ligands is reduced by anti-CCR5 gene delivery
Journal of the Neurological Sciences 308 (2011) 25–27
Contents lists available at ScienceDirect
Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Lymphocyte adhesion to CCR5 ligands is reduced by anti-CCR5 gene delivery Elena Marusich, Jean-Pierre Louboutin ⁎, Alena A. Chekmasova, David S. Strayer Department of Pathology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, Room 251, Philadelphia, Pennsylvania 19107, USA
a r t i c l e
i n f o
Article history: Received 28 February 2011 Received in revised form 20 May 2011 Accepted 21 June 2011 Available online 13 July 2011 Keywords: CCR5 PBMC
a b s t r a c t Immune-mediated damage to the central nervous system (CNS) is an important contributor to many CNS diseases, including epilepsy. Chemokines play a role in leukocyte recruitment to, and migration across, the blood–brain barrier (BBB) during many such processes. We previously investigated the role of the chemokine receptor CCR5 in a rat model of epilepsy based on intraperitoneal kainic acid (KA) administration. Before KA injection, rats were given intramarrow inoculations of SV(RNAiR5-RevM10.AU1), which carries an interfering RNA (RNAi) that targets CCR5. Decreased CCR5 expression in blood cells after vector administration reduced expression of CCR5 ligands MIP-1α and RANTES in the microvasculature, and strongly protected from BBB leakage, CNS loss and inflammation and facilitated CNS repair. We show here that rSV40-mediated downregulation of CCR5 in lymphocytes decreased cellular adhesion to surfaces carrying CCR5 ligands. These data suggest that reducing CCR5 in peripheral blood mononuclear cells (PBMCs) might alter their adhesion to the microvasculature and their participation in inflammatory processes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Immune-mediated damage to the central nervous system (CNS) is likely an important contributor to many CNS diseases, including epilepsy, multiple sclerosis and others [1]. Chemokines are involved in leukocyte recruitment to, and migration across, the blood–brain barrier (BBB) during many such processes [2]. In epilepsy, interactions between leukocytes and endothelium may contribute to seizure activity [3]. We have previously studied the role of the chemokine receptor, CCR5, in a rat model of epilepsy based on intraperitoneal kainic acid (KA) administration [4]. Before KA injection, rats were injected into their bone marrow with SV (RNAiR5-RevM10.AU1). This gene delivery vector carries an interfering RNA (RNAi) that targets CCR5, plus a marker epitope (AU1), in an SV40 backbone. Decreased CCR5 expression in blood cells after vector administration reduced expression of CCR5 ligands CCL3 (MIP-1α) and CCL5 (RANTES) in the microvasculature, and strongly protected from BBB leakage, CNS neuronal loss and inflammation, and facilitated CNS repair responses. Once they are immobilized on endothelial surfaces, some chemokines trigger integrin clustering, which arrests circulating lymphocytes at sites of injury and guides them from vascular lumens into the substance of the brain. Among these chemokines, CCL5 (RANTES) increases T cell adhesion to endothelial cells [5]. Here, we tested the extent to which down-
⁎ Corresponding author at: Department of Pathology, Jefferson Medical College, 1020 Locust St., Room 251, Philadelphia, Pennsylvania 19107, United States. Tel.: + 1 215 503 1268; fax: + 1 215 503 1156. E-mail address: [email protected] (J.-P. Louboutin). 0022-510X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2011.06.039
regulation of CCR5 on peripheral blood mononuclear cells (PBMCs) and immortalized T cells altered their adhesion to CCR5 ligands, such as we have found to be expressed on CNS vascular endothelium at sites of injury.
2. Material and methods 2.1. SupT1-CCR5 and PBMCs PBMCs were isolated from buffy coat from peripheral blood of normal human volunteer donors by using centrifugation with Histopaque-1077 (Sigma). The samples were not pooled and the experiments for each individual were done in triplicate. We used only one stock of PBMC from one individual at each time. We previously observed a slightly different (non statistically significant) level of expression of CCR5 in PBMC isolated from buffy coat from peripheral blood of different normal human volunteers donors. Mononuclear cell layer at the plasma-Histopaque-1077 interface was removed, washed, and then incubated 2 days with phytohemagglutinin (PHA, 3 μg/ml) in RPMI-1640 medium, supplemented with 10% of fetal bovine serum (FBS), 2 mM L-glutamine, and containing penicillin (200 U/ml) and streptomycin (100 μg/ml). Then, after washing, PBMCs were stimulated during at least 6 h with 10 ng/μl of IL-2 before adhesion assay. However, PBMCs were starved of IL-2 at least 6 h before adhesion was assayed. SupT1 cells are immortalized human T cells transduced with a retroviral vector to express CCR5 (SupT1-CCR5). They were grown in RPMI-1640 medium, supplemented with 10% newborn calf serum (Hyclone, Logan UT), 2 mM L-glutamine, and containing penicillin (200 U/ml) and streptomycin (100 μg/ml).
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E. Marusich et al. / Journal of the Neurological Sciences 308 (2011) 25–27
2.2. Vectors We then transduced PBMCs or SupT1-CCR5 cells with either of two recombinant SV40-derived vectors designed to target CCR5, either with RNAi, SV(RNAiR5-RevM10.AU1) or with a single chain monoclonal antibody (SFv) against CCR5, SV(2C7) (4). SV(BUGT), which encodes human bilirubin-uridine 5′-diphosphate-glucuronosyl-transferase was used as negative control vector (4). 2.3. Adhesion assays In vitro adhesion assays were performed according to protocols previously reported [3]. Adhesion assays were performed on 8 well glass slides coated with different peptides CCL4, CCL5, CCL2, CCL7 (respectively MIP-1 beta, RANTES, MCP-1, MCP-3) (25 μg/ml) (Santa Cruz, Santa Cruz, CA). These peptides were chosen because CCL4 (MIP-1 beta) and CCL5 (RANTES) are ligands of CCR5, while CCL2 (MCP-1) is ligand of CCR2 and CCL7 (MCP-3) is ligand of CCR1. Cells were treated either with kainic acid (KA) (Tocris, Ellisville, MO) or control saline. In a pilocarpine model of epilepsy, it has been demonstrated that direct pilocarpine treatment does not render T cells hyperadhesive [3]. We previously used a model of KA induced seizures and we tested here the direct effect of KA on SupT1-CCR5 cells and PBMCs to determine if there was an effect of KA on PBMCs and T cells adhesion. We first tested the viability of SupT1-CCR5 cells incubated with different concentrations of KA. Preliminary studies based on Trypan Blue using different concentrations of KA on SupT1-CCR5 cells determined that 10 nm of KA was the optimal concentration. All cells were first clustered then detached for 10 μM and 1 μM KA, suggesting that these cells were all necrotic for these KA concentrations, while the percentages of Trypan Blue-positive cells were almost similar for 100 nM and 10 nM KA (31.6 +/− 4.2% and 31.2 +/− 3.8% respectively; control without KA: 10.5+/− 1.8%; P b 0.01, 10 and 100 nm KA vs. control without KA). For KA treatment, SupT1-CCR5 cells or PBMCs were incubated during 30 min with KA. Cells treated with KA or saline were then incubated at 37 C during 30 min on 8 well slides. Preliminary studies (data not shown) indicated that incubation times ranging from 3 to 30 min all gave comparable results.
designed to target CCR5 had to be checked before the adhesion assays were performed in order to exclude a potential effect of CCR5 targeting technique on integrin expression. We tested the expression of α4 integrin on cells transduced with rSV40s targeting CCR5. The slides were washed with PBS and the nuclei of remaining cells were stained using 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Enumeration of bound cells was done using a computerized imaging system (Image-ProPlus software, MediaCybernetics, Bethesda, MD). All cells were counted manually in each well. For example, between 30 × 10 3 and 60 × 10 3 cells transduced with SV (BUGT) were enumerated in one well. Bound cells were expressed as a percentage of the remaining cells transduced by SV(BUGT). 2.5. Statistical analysis Three experiments were performed for this study with 2 replicate wells for each experiment. Comparison of medians between 2 groups was achieved by using the Mann–Whitney test (with a two-tail p value). Comparison of medians between more than 2 groups was done by using the Kruskall–Wallis test. The difference between the groups was considered significant when P b 0.05. On graphs, values are represented as means +/− s.e.m. 3. Results The human T cell line, SupT1-CCR5, and primary human PBMC adhered to plates coated with several chemokines, including CCR5 ligands [CCL4 (MIP-1 beta), CCL5 (RANTES)] as well as ligands for other chemokine receptors [CCL2 (MCP-1), CCL7 (MCP-3)]. In vitro adhesion of SupT1-CCR5 cells to the CCR5 ligands, CCL4 (MIP-1 beta) and CCL5 (RANTES), was significantly reduced (P b 0.01) by anti-CCR5 gene delivery (Fig. 1). Gene delivery to downregulate CCR5 in PBMCs using either RNAi or anti-CCR5 SFv antibody decreased binding to CCR5 ligands but not to ligands for CCR1 [CCL7 (MCP-3)] or CCR2 [CCL2 (MCP-1), CCL7 (MCP-3)]. Addition of KA to SupT1-CCR5 cells had no effect on lymphocyte binding to CCR5 ligands (Fig. 1). Additionally, KA did not alter the cells binding to these ligands of other chemokine receptors (not shown). Similarly, addition of KA to PBMCs had no effect on cell binding to CCR5 ligands. Expression of integrins was not modified by anti-CCR5 gene delivery (not shown).
2.4. Expression of integrins 4. Discussion It has been shown that integrins mediate vascular-leukocyte adhesive interactions in cerebral vessels after status epilepticus [3]. Expression of integrins on cells transduced with rSV40 vectors
There is growing evidence showing that vascular alterations, in particular leukocyte recruitment, might play a key role in
Fig. 1. Gene delivery targeting CCR5 decreases cellular adhesion to CCR5 ligands in vitro. SupT1-CCR5 cells (left panel) or normal human peripheral blood mononuclear cells (PBMCs, central panel), were transduced with SV(RNAiR5-RevM10.AU1), which delivers a RNAi vs. CCR5, SV(2C7), which carries a single chain Fv antibody against CCR5 or SV(BUGT) (control). Adhesion to glass slides coated with chemokines that are (MIP-1 beta, RANTES) or are not (MCP-1, MCP-3) CCR5 ligands was measured by enumerating adherent cells. Results are shown as anti-CCR5-transduced cells binding to plates and expressed as a percentage of SV(BUGT)-transduced cells bound in the same plate. Differences between either PBMCs or SupT1-CCR5 cells transduced with SV(2C7) or SV(RNAiR5-RevM10.AU1) were highly significant for binding to MIP-1 beta and RANTES (Pb0.01). Binding to MCP-1 and MCP-3 was not affected by these treatments (PN0.1). Similarly, treating the gene-modified cells with KA (right panel) did not significantly alter binding (PN0.1).
E. Marusich et al. / Journal of the Neurological Sciences 308 (2011) 25–27
epileptogenesis. However, the precise mechanisms involved in leukocyte-endothelial interaction/adhesion are not well established. In a model of KA-induced seizures, we previously reported the role of CCR5 and its ligands in the control of vascular inflammation and leukocyte recruitment required for acute excitotoxic seizure induction and neural damage [4]. We show here that rSV40-mediated downregulation of CCR5 decreased cellular adhesion to surfaces carrying CCR5 ligands. These data suggest strongly that reducing CCR5 in PBMCs might alter their adhesion to the microvasculature and their participation in inflammatory processes. In a pilocarpine model of seizures, vascular inflammatory mechanisms and leukocyte-endothelial adhesion contribute to the pathogenesis of seizures and epilepsy. Inhibition of leukocytevascular interactions can have preventive, as well as therapeutic, effects in this model. However, direct pilocarpine treatment did not render T cells hyperadhesive in that model [3]. We previously described a model of KA-induced seizures [4]. We studied here if there was a direct effect of KA on SupT1-CCR5 cells and PBMCs adhesion to CCR5 ligands. Our results show that gene delivery to downregulate cell membrane CCR5 inhibits lymphocyte binding to
27
CCR5 ligands but not to ligands of other chemokine receptors. Further, the epileptogen, KA, does not directly modify PBMC or SupT1-CCR5 cell adhesion to CCR5 ligands. In conclusion, the present results emphasize the role of CCR5 in the adhesion of leukocytes to the ligands of this receptor, which are produced by the endothelium of cerebral microvasculature.
References [1] Kleen JK, Holmes GH. Brain inflammation initiates seizures. Nat Med 2008;14: 1309–10. [2] Man S, Ubogu EE, Ransohoff RM. Inflammatory cell migration into the central nervous system: a few new twists on an old tale. Brain Pathol 2007;17:243–50. [3] Fabene PF, Navarro Mora G, Martinello M, et al. A role for leukocyte-endothelial adhesion mechanisms in epilepsy. Nat Med 2008;14:1377–83. [4] Louboutin JP, Chekmasova AA, Marusich E, Agrawal L, Strayer DS. Role of CCR5 and its ligands in the control of vascular inflammation and leukocyte recruitment required for acute excitotoxic seizure induction and neural damage. FASEB J 2011;25:737–53. [5] Quandt J, Dorovini-Zis K. The beta chemokines CCL4 and CCL5 enhance adhesion of specific CD4+ T cells subsets to human brain endothelial cells. J Neuropath Exp Neurol 2004;63:350–62.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Lymphocyte adhesion to CCR5 ligands is reduced by anti-CCR5 gene delivery Elena Marusich, Jean-Pierre Louboutin ⁎, Alena A. Chekmasova, David S. Strayer Department of Pathology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, Room 251, Philadelphia, Pennsylvania 19107, USA
a r t i c l e
i n f o
Article history: Received 28 February 2011 Received in revised form 20 May 2011 Accepted 21 June 2011 Available online 13 July 2011 Keywords: CCR5 PBMC
a b s t r a c t Immune-mediated damage to the central nervous system (CNS) is an important contributor to many CNS diseases, including epilepsy. Chemokines play a role in leukocyte recruitment to, and migration across, the blood–brain barrier (BBB) during many such processes. We previously investigated the role of the chemokine receptor CCR5 in a rat model of epilepsy based on intraperitoneal kainic acid (KA) administration. Before KA injection, rats were given intramarrow inoculations of SV(RNAiR5-RevM10.AU1), which carries an interfering RNA (RNAi) that targets CCR5. Decreased CCR5 expression in blood cells after vector administration reduced expression of CCR5 ligands MIP-1α and RANTES in the microvasculature, and strongly protected from BBB leakage, CNS loss and inflammation and facilitated CNS repair. We show here that rSV40-mediated downregulation of CCR5 in lymphocytes decreased cellular adhesion to surfaces carrying CCR5 ligands. These data suggest that reducing CCR5 in peripheral blood mononuclear cells (PBMCs) might alter their adhesion to the microvasculature and their participation in inflammatory processes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Immune-mediated damage to the central nervous system (CNS) is likely an important contributor to many CNS diseases, including epilepsy, multiple sclerosis and others [1]. Chemokines are involved in leukocyte recruitment to, and migration across, the blood–brain barrier (BBB) during many such processes [2]. In epilepsy, interactions between leukocytes and endothelium may contribute to seizure activity [3]. We have previously studied the role of the chemokine receptor, CCR5, in a rat model of epilepsy based on intraperitoneal kainic acid (KA) administration [4]. Before KA injection, rats were injected into their bone marrow with SV (RNAiR5-RevM10.AU1). This gene delivery vector carries an interfering RNA (RNAi) that targets CCR5, plus a marker epitope (AU1), in an SV40 backbone. Decreased CCR5 expression in blood cells after vector administration reduced expression of CCR5 ligands CCL3 (MIP-1α) and CCL5 (RANTES) in the microvasculature, and strongly protected from BBB leakage, CNS neuronal loss and inflammation, and facilitated CNS repair responses. Once they are immobilized on endothelial surfaces, some chemokines trigger integrin clustering, which arrests circulating lymphocytes at sites of injury and guides them from vascular lumens into the substance of the brain. Among these chemokines, CCL5 (RANTES) increases T cell adhesion to endothelial cells [5]. Here, we tested the extent to which down-
⁎ Corresponding author at: Department of Pathology, Jefferson Medical College, 1020 Locust St., Room 251, Philadelphia, Pennsylvania 19107, United States. Tel.: + 1 215 503 1268; fax: + 1 215 503 1156. E-mail address: [email protected] (J.-P. Louboutin). 0022-510X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2011.06.039
regulation of CCR5 on peripheral blood mononuclear cells (PBMCs) and immortalized T cells altered their adhesion to CCR5 ligands, such as we have found to be expressed on CNS vascular endothelium at sites of injury.
2. Material and methods 2.1. SupT1-CCR5 and PBMCs PBMCs were isolated from buffy coat from peripheral blood of normal human volunteer donors by using centrifugation with Histopaque-1077 (Sigma). The samples were not pooled and the experiments for each individual were done in triplicate. We used only one stock of PBMC from one individual at each time. We previously observed a slightly different (non statistically significant) level of expression of CCR5 in PBMC isolated from buffy coat from peripheral blood of different normal human volunteers donors. Mononuclear cell layer at the plasma-Histopaque-1077 interface was removed, washed, and then incubated 2 days with phytohemagglutinin (PHA, 3 μg/ml) in RPMI-1640 medium, supplemented with 10% of fetal bovine serum (FBS), 2 mM L-glutamine, and containing penicillin (200 U/ml) and streptomycin (100 μg/ml). Then, after washing, PBMCs were stimulated during at least 6 h with 10 ng/μl of IL-2 before adhesion assay. However, PBMCs were starved of IL-2 at least 6 h before adhesion was assayed. SupT1 cells are immortalized human T cells transduced with a retroviral vector to express CCR5 (SupT1-CCR5). They were grown in RPMI-1640 medium, supplemented with 10% newborn calf serum (Hyclone, Logan UT), 2 mM L-glutamine, and containing penicillin (200 U/ml) and streptomycin (100 μg/ml).
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E. Marusich et al. / Journal of the Neurological Sciences 308 (2011) 25–27
2.2. Vectors We then transduced PBMCs or SupT1-CCR5 cells with either of two recombinant SV40-derived vectors designed to target CCR5, either with RNAi, SV(RNAiR5-RevM10.AU1) or with a single chain monoclonal antibody (SFv) against CCR5, SV(2C7) (4). SV(BUGT), which encodes human bilirubin-uridine 5′-diphosphate-glucuronosyl-transferase was used as negative control vector (4). 2.3. Adhesion assays In vitro adhesion assays were performed according to protocols previously reported [3]. Adhesion assays were performed on 8 well glass slides coated with different peptides CCL4, CCL5, CCL2, CCL7 (respectively MIP-1 beta, RANTES, MCP-1, MCP-3) (25 μg/ml) (Santa Cruz, Santa Cruz, CA). These peptides were chosen because CCL4 (MIP-1 beta) and CCL5 (RANTES) are ligands of CCR5, while CCL2 (MCP-1) is ligand of CCR2 and CCL7 (MCP-3) is ligand of CCR1. Cells were treated either with kainic acid (KA) (Tocris, Ellisville, MO) or control saline. In a pilocarpine model of epilepsy, it has been demonstrated that direct pilocarpine treatment does not render T cells hyperadhesive [3]. We previously used a model of KA induced seizures and we tested here the direct effect of KA on SupT1-CCR5 cells and PBMCs to determine if there was an effect of KA on PBMCs and T cells adhesion. We first tested the viability of SupT1-CCR5 cells incubated with different concentrations of KA. Preliminary studies based on Trypan Blue using different concentrations of KA on SupT1-CCR5 cells determined that 10 nm of KA was the optimal concentration. All cells were first clustered then detached for 10 μM and 1 μM KA, suggesting that these cells were all necrotic for these KA concentrations, while the percentages of Trypan Blue-positive cells were almost similar for 100 nM and 10 nM KA (31.6 +/− 4.2% and 31.2 +/− 3.8% respectively; control without KA: 10.5+/− 1.8%; P b 0.01, 10 and 100 nm KA vs. control without KA). For KA treatment, SupT1-CCR5 cells or PBMCs were incubated during 30 min with KA. Cells treated with KA or saline were then incubated at 37 C during 30 min on 8 well slides. Preliminary studies (data not shown) indicated that incubation times ranging from 3 to 30 min all gave comparable results.
designed to target CCR5 had to be checked before the adhesion assays were performed in order to exclude a potential effect of CCR5 targeting technique on integrin expression. We tested the expression of α4 integrin on cells transduced with rSV40s targeting CCR5. The slides were washed with PBS and the nuclei of remaining cells were stained using 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Enumeration of bound cells was done using a computerized imaging system (Image-ProPlus software, MediaCybernetics, Bethesda, MD). All cells were counted manually in each well. For example, between 30 × 10 3 and 60 × 10 3 cells transduced with SV (BUGT) were enumerated in one well. Bound cells were expressed as a percentage of the remaining cells transduced by SV(BUGT). 2.5. Statistical analysis Three experiments were performed for this study with 2 replicate wells for each experiment. Comparison of medians between 2 groups was achieved by using the Mann–Whitney test (with a two-tail p value). Comparison of medians between more than 2 groups was done by using the Kruskall–Wallis test. The difference between the groups was considered significant when P b 0.05. On graphs, values are represented as means +/− s.e.m. 3. Results The human T cell line, SupT1-CCR5, and primary human PBMC adhered to plates coated with several chemokines, including CCR5 ligands [CCL4 (MIP-1 beta), CCL5 (RANTES)] as well as ligands for other chemokine receptors [CCL2 (MCP-1), CCL7 (MCP-3)]. In vitro adhesion of SupT1-CCR5 cells to the CCR5 ligands, CCL4 (MIP-1 beta) and CCL5 (RANTES), was significantly reduced (P b 0.01) by anti-CCR5 gene delivery (Fig. 1). Gene delivery to downregulate CCR5 in PBMCs using either RNAi or anti-CCR5 SFv antibody decreased binding to CCR5 ligands but not to ligands for CCR1 [CCL7 (MCP-3)] or CCR2 [CCL2 (MCP-1), CCL7 (MCP-3)]. Addition of KA to SupT1-CCR5 cells had no effect on lymphocyte binding to CCR5 ligands (Fig. 1). Additionally, KA did not alter the cells binding to these ligands of other chemokine receptors (not shown). Similarly, addition of KA to PBMCs had no effect on cell binding to CCR5 ligands. Expression of integrins was not modified by anti-CCR5 gene delivery (not shown).
2.4. Expression of integrins 4. Discussion It has been shown that integrins mediate vascular-leukocyte adhesive interactions in cerebral vessels after status epilepticus [3]. Expression of integrins on cells transduced with rSV40 vectors
There is growing evidence showing that vascular alterations, in particular leukocyte recruitment, might play a key role in
Fig. 1. Gene delivery targeting CCR5 decreases cellular adhesion to CCR5 ligands in vitro. SupT1-CCR5 cells (left panel) or normal human peripheral blood mononuclear cells (PBMCs, central panel), were transduced with SV(RNAiR5-RevM10.AU1), which delivers a RNAi vs. CCR5, SV(2C7), which carries a single chain Fv antibody against CCR5 or SV(BUGT) (control). Adhesion to glass slides coated with chemokines that are (MIP-1 beta, RANTES) or are not (MCP-1, MCP-3) CCR5 ligands was measured by enumerating adherent cells. Results are shown as anti-CCR5-transduced cells binding to plates and expressed as a percentage of SV(BUGT)-transduced cells bound in the same plate. Differences between either PBMCs or SupT1-CCR5 cells transduced with SV(2C7) or SV(RNAiR5-RevM10.AU1) were highly significant for binding to MIP-1 beta and RANTES (Pb0.01). Binding to MCP-1 and MCP-3 was not affected by these treatments (PN0.1). Similarly, treating the gene-modified cells with KA (right panel) did not significantly alter binding (PN0.1).
E. Marusich et al. / Journal of the Neurological Sciences 308 (2011) 25–27
epileptogenesis. However, the precise mechanisms involved in leukocyte-endothelial interaction/adhesion are not well established. In a model of KA-induced seizures, we previously reported the role of CCR5 and its ligands in the control of vascular inflammation and leukocyte recruitment required for acute excitotoxic seizure induction and neural damage [4]. We show here that rSV40-mediated downregulation of CCR5 decreased cellular adhesion to surfaces carrying CCR5 ligands. These data suggest strongly that reducing CCR5 in PBMCs might alter their adhesion to the microvasculature and their participation in inflammatory processes. In a pilocarpine model of seizures, vascular inflammatory mechanisms and leukocyte-endothelial adhesion contribute to the pathogenesis of seizures and epilepsy. Inhibition of leukocytevascular interactions can have preventive, as well as therapeutic, effects in this model. However, direct pilocarpine treatment did not render T cells hyperadhesive in that model [3]. We previously described a model of KA-induced seizures [4]. We studied here if there was a direct effect of KA on SupT1-CCR5 cells and PBMCs adhesion to CCR5 ligands. Our results show that gene delivery to downregulate cell membrane CCR5 inhibits lymphocyte binding to
27
CCR5 ligands but not to ligands of other chemokine receptors. Further, the epileptogen, KA, does not directly modify PBMC or SupT1-CCR5 cell adhesion to CCR5 ligands. In conclusion, the present results emphasize the role of CCR5 in the adhesion of leukocytes to the ligands of this receptor, which are produced by the endothelium of cerebral microvasculature.
References [1] Kleen JK, Holmes GH. Brain inflammation initiates seizures. Nat Med 2008;14: 1309–10. [2] Man S, Ubogu EE, Ransohoff RM. Inflammatory cell migration into the central nervous system: a few new twists on an old tale. Brain Pathol 2007;17:243–50. [3] Fabene PF, Navarro Mora G, Martinello M, et al. A role for leukocyte-endothelial adhesion mechanisms in epilepsy. Nat Med 2008;14:1377–83. [4] Louboutin JP, Chekmasova AA, Marusich E, Agrawal L, Strayer DS. Role of CCR5 and its ligands in the control of vascular inflammation and leukocyte recruitment required for acute excitotoxic seizure induction and neural damage. FASEB J 2011;25:737–53. [5] Quandt J, Dorovini-Zis K. The beta chemokines CCL4 and CCL5 enhance adhesion of specific CD4+ T cells subsets to human brain endothelial cells. J Neuropath Exp Neurol 2004;63:350–62.