Chemokine and chemikine receptor expression by human brain endothelium :an in vitro and in situ study

The infiltration of leukocytes across the BBB is thought to be mediated by chemokines released in the CNS. This study investigated the production of chemokines and expression of chemokine receptors by human brain endothelial cells (HBEC) as well as the consequences of CXCL10 stimulation on brain endothelial function in terms of leukocyte migration and endothelial permeability. Purified primary HBEC as well as a novel human brain endothelial cell line, called hCEMC/D3 and generated in collaboration with the Institute Cochin, were used in the present study. Characterisation of hCMEC/D3 cells was effected by determining the expression and localisation of HBEC markers such as tight junction and adherens junction proteins. Both primary and immortalized HBEC were tested for the production of four chemokines (CCL2, CCL5, CXCL8 and CXCL10) considered to play a role in multiple sclerosis. CXCL8 and CCL2 were constitutively released, whereas CXCL10 and CCL5 could not be detected at basal levels. By contrast, all these chemokines were up-regulated in response to either TNF-a or IFN-y or a combination of both. TNF-a had the most striking effect, up-regulating the production of CCL2, CCL5 and CXCL8, while IFN-y up-regulated CXCL10 exclusively. The chemokine receptors CXCR1 and CXCR3, whose ligands include CXCL8 and CXCL10, were expressed constitutively by HBEC both in vitro and in vivo, and only CXCR3 was up-regulated in response to cytokine stimulation in vitro. Furthermore, CXCR3 on endothelial cells is functional as CXCL10 induces phosphorylation of p42/44 MAP kinase, p38 MAP kinase and JNK/SAPK. However, activation of HBEC by CXCL10 did not induce any permeability change nor did it modulate leukocyte adhesion. These results demonstrate that endothelial cells could play an important role in chemokine production and hence leukocyte infiltration during inflammatory pathologies such as MS. The role of chemokine receptors requires further investigation

Transport Rankings of Non-Steroidal Antiinflammatory Drugs across Blood-Brain Barrier In Vitro Models

The aim of this work was to conduct a comprehensive study about the transport properties of NSAIDs across the blood-brain barrier (BBB) in vitro. Transport studies with celecoxib, diclofenac, ibuprofen, meloxicam, piroxicam and tenoxicam were accomplished across Transwell models based on cell line PBMEC/C1-2, ECV304 or primary rat brain endothelial cells. Single as well as group substance studies were carried out. In group studies substance group compositions, transport medium and serum content were varied, transport inhibitors verapamil and probenecid were added. Resulted permeability coefficients were compared and normalized to internal standards diazepam and carboxyfluorescein. Transport rankings of NSAIDs across each model were obtained. Single substance studies showed similar rankings as corresponding group studies across PBMEC/C1-2 or ECV304 cell layers. Serum content, glioma conditioned medium and inhibitors probenecid and verapamil influenced resulted permeability significantly. Basic differences of transport properties of the investigated NSAIDs were similar comparing all three in vitro BBB models. Different substance combinations in the group studies and addition of probenecid and verapamil suggested that transporter proteins are involved in the transport of every tested NSAID. Results especially underlined the importance of same experimental conditions (transport medium, serum content, species origin, cell line) for proper data comparison

Group transport study of NSAIDs across RBMEC.

*<p>Ratio to Diazepam is calculated by average PE_cell data of the investigated NSAID and the corresponding diazepam value.</p><p>Summary of permeability data of the group study with NSAIDs piroxicam, ibuprofen, meloxicam, tenoxicam and diclofenac across the RBMEC cell layers (n = 3, data are presented as means ± SD).</p

Characterization of the BBB model based on primary rat brain microvascular endothelial cells (RBMEC) and astrocytes.

<p>RBMECs grow in endothelial cell typical spindle-like morphology proven by light and scanning electron microscopy (SEM). Transmission electron microscopic (TEM) images confirmed that RBMEC grow as a monolayer. The enlarged part of the image shows two RBMECs connected to each other directly over a pore of the Transwell insert membrane (A). mRNA expressions of tight junction proteins ZO-1, occludin, claudin-3, claudin-5 and claudin-12, and of adhesion molecules PECAM-1, VCAM, ICAM-1 and CD44. All data were related to endogenous control GAPDH which was set to 1000 (B). Immunofluorescence images of PECAM-1, ZO-1, occludin, claudin-3 and claudin-5 confirmed the protein’s presence and localization in RBMEC layers (C). Transport studies with paracellular marker APTS-dextran ladder confirmed functionality of the barrier. RBMEC layers were able to differentiate between the different dextran fractions in a molecular size-dependent manner. Comparison of the permeability coefficients for APTS-dextran across PBMEC/C1-2, ECV304 and RBMEC layers is presented in the table on the right side (D).</p

Rankings of the group transport studies with NSAIDs across PBMEC/C1-2 layers.

<p>Permeability coefficient of each substance was normalized to the corresponding permeability coefficient of internal standard diazepam of the same experiment. <b>A):</b> Variant substance compositions - results of the group study with all investigated substances (diazepam, piroxicam, ibuprofen, meloxicam, tenoxicam, diclofenac, celecoxib, carboxyfluorescein = CF) were compared to the study without celecoxib (w/o CC), without celecoxib accomplished in serum-free C6 medium (serum-free), without celecoxib accomplished in PBMEC-Fib medium (GCM  =  glioma conditioned medium) and without celecoxib and carboxyfluorescein (w/o CF). <b>B):</b> Different transport study conditions - results of the group study with all investigated substances (diazepam, piroxicam, ibuprofen, meloxicam, tenoxicam, diclofenac, celecoxib, carboxyfluorescein = CF) were compared to the study without meloxicam (w/o MEL), without meloxicam and with probenecid (with Probenecid) and without meloxicam and with verapamil (with Verapamil). To calculate the statistical significances between the groups, which differed in the substance compositions, a one-way ANOVA was used, to compare the groups with same substance compositions under different experimental transport conditions (in A: w/o CC, serum-free medium, GCM; in B: w/o MEL, with Probenecid, with Verapamil) a two-way ANOVA was accomplished followed by an all pairwise multiple comparison procedure (Holm-Sidak method) with an overall significance level of 0.05. Statistical significance (p<0.05) for each substance is indicated in the figure by * (all vs. w/o CC, all vs. w/o MEL), by # (w/o CC vs. serum-free or GCM; w/o MEL vs. with Probenecid or with Verapamil), by § (serum-free vs. GCM; with Probenecid vs. with Verapamil) or by $ (w/o CC vs. w/o CF). Data are presented as means ± SD (n = 3).</p

Serum binding [%] of NSAIDs in transport media.

*<p>CF  =  carboxyfluorescein.</p><p>Serum binding [%] of NSAIDs, diazepam and carboxyfluorescein. Serum binding was assessed using same substance group compositions as applied for group transport studies with C6 medium containing either 0% or 7.5% serum. Data were presented for serum amounts of 7.5, 50 and 100% (n = 3, means ± SD).</p

Rankings of the group transport studies with NSAIDs across ECV304 layers.

<p>Permeability coefficient of each substance was normalized to the corresponding permeability coefficient of internal standard diazepam of the same experiment. <b>A):</b> Variant substance compositions - results of the group study with all investigated substances (diazepam, piroxicam, ibuprofen, meloxicam, tenoxicam, diclofenac, celecoxib, carboxyfluorescein = CF) were compared to the study without celecoxib (w/o CC), without celecoxib accomplished in serum-free C6 medium (serum-free), without celecoxib accomplished in PBMEC-Fib medium (GCM  =  glioma conditioned medium) and without celecoxib and carboxyfluorescein (w/o CF). <b>B):</b> Different transport study conditions - results of the group study with all investigated substances (diazepam, piroxicam, ibuprofen, meloxicam, tenoxicam, diclofenac, celecoxib, carboxyfluorescein = CF) were compared to the study without meloxicam (w/o MEL), without meloxicam and with probenecid (with Probenecid) and without meloxicam and with verapamil (with Verapamil). To calculate the statistical significances between the groups, which differed in the substance compositions, a one-way ANOVA was used, to compare the groups with same substance compositions under different experimental transport conditions (in A: w/o CC, serum-free medium, GCM; in B: w/o MEL, with Probenecid, with Verapamil) a two-way ANOVA was accomplished followed by an all pairwise multiple comparison procedure (Holm-Sidak method) with an overall significance level of 0.05. Statistical significance (p<0.05) for each substance is indicated in the figure by * (all vs. w/o CC, all vs. w/o MEL), by # (w/o CC vs. serum-free or GCM; w/o MEL vs. with Probenecid or with Verapamil) or by § (serum-free vs. GCM; with Probenecid vs. with Verapamil). Data are presented as means ± SD (n = 3).</p

Group transport studies of NSAIDs across PBMEC/C1-2 and ECV304.

*<p>CF  =  carboxyfluorescein, n.a.  =  not added due to experimental or analytical reasons.</p><p>Summary of permeability data of group transport studies with NSAIDs piroxicam, ibuprofen, meloxicam, tenoxicam, diclofenac and celecoxib across PBMEC/C1-2 as well as ECV304 cell layers. In each transport study the two permeability markers diazepam and carboxyfluorescein were applied at the same time. The permeability of different group compositions (all, without celecoxib, without celecoxib and carboxyfluoresein, without meloxciam), in different transport media (all, serum-free medium, glioma-conditioned medium) and the added transporter inhibitors (without meloxciam with probenecid, without meloxicam with verapamil) was measured (n = 3, data are presented as means ± SD).</p

Time courses of single transport studies of piroxicam across PBMEC/C1-2, ECV304 and RBMEC layers.

<p>Comparison between the cleared volume vs. time graphs of internal standards for the transcellular transport route (diazepam, 100 µM), the paracellular transport route (carboxyfluorescein, 5 µM) and the NSAID piroxicam (100 µM) showed clearly that piroxicam permeated between the two markers across all three BBB (A: PBMEC/C1-2; B: ECV304; C: RBMEC) in vitro models. In addition, it was proved that as tighter the model is a wider frame between the two markers diazepam and carboxyfluorescein could be provided to analyze and compare permeabilities of single drugs. (n = 3 for each time point, data are presented as means ± SD).</p