Differential expression of selectins by mouse brain capillary endothelial cells in vitro in response to distinct inflammatory stimuli

Neuroscience Letters 392 (2006) 216–220

Differential expression of selectins by mouse brain capillary endothelial cells in vitro in response to distinct inflammatory stimuli Caroline Coisne a , Christelle Faveeuw a , Yannick Delplace b , Lucie Dehouck b , Florence Miller a , Rom´eo Cecchelli a , B´en´edicte Dehouck a,∗ a

EA 2465-Universit´e d’Artois, Facult´e des Sciences Jean Perrin, 62307 Lens, France b Cellial Technologies, Facult´ e des Sciences Jean Perrin, 62307 Lens, France

Received 31 May 2005; received in revised form 19 August 2005; accepted 9 September 2005

Abstract Increased lymphocyte trafficking across blood–brain barrier (BBB) is a prominent and early event in inflammatory and immune-mediated CNS diseases. The adhesion molecules that control the entry of leukocytes into the brain have not been fully elucidated. Although the role of ICAM-1 and VCAM-1 has been well documented, the expression and role of selectins is still a matter of controversy. In a mouse syngenic in vitro BBB model, highly relevant for examining immunological events, mouse brain capillary endothelial cells (MBCECs) do not express selectins. Treatment of MBCECs with LPS, induced E- and P-selectin expression, whereas TNF-alpha or IFN-gamma treatments did not. Finally, P-selectin but not E-selectin expression was induced in IL-1beta treated MBCECs. Thus, our study suggests that diverse inflammatory stimuli could differentially regulate selectin expression at the BBB. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: E- and P-selectin expression at the blood–brain barrier; In vitro studies; Cytokines

The presence of blood-borne immune cells in the brain parenchyma of healthy animals and their increased cerebral residence in pathogenic conditions, suggest a recruitment of inflammatory cells into the CNS with the blood–brain barrier (BBB) directly implicated in this phenomenon [17,34]. As in peripheral tissues, leukocyte infiltration through the BBB is thought to proceed via multiple steps, in which different molecules are involved in the selective interaction of blood cells with the cerebral endothelium [8,30]. Studies have shown the importance of CAMs, especially ICAM-1, during the firm adhesion prerequisite to leukocyte extravasation, and VCAM-1 described both in firm adhesion [27] and in leukocyte rolling [11]. This last observation was made in pathological animal models in which a role can not be attributed to selectins as no expression of these molecules has been found on cerebral capillaries [11]. Indeed, the presence and/or role of selectins on cerebral capillaries is still a matter of debate. In peripheral

Abbreviations: BBB, blood–brain barrier; CAM, cell adhesion molecule; MBCEC, mouse brain capillary endothelial cell ∗ Corresponding author. Tel.: +33 321 791 741/758; fax: +33 321 791 736. E-mail address: [email protected] (B. Dehouck). 0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.09.028

tissues, selectins mediate the early transient cell–cell interactions, which occur during tethering and leukocyte rolling along the vessel walls of inflamed tissues. To this end, P-selectin is stored in Weibel-Palade bodies of resting cells and translocated to the membrane surface within 10 min of cytokines or LPS stimulation. New P- and E-selectins are then transcriptively induced to the cell surface [3,14]. However, some ostensible differences have been observed in cerebral endothelial cell responses. In fact, most studies have shown that basal expression of P-selectin is detectable in all organs but the blood vessels of cerebral cortex [2]; authors also agree with the lack of basal E-selectin expression either in peripheral tissues or in CNS [11]. These results raise questions about the expression and role of selectins on cerebral endothelium. In this paper, using a well differentiated in vitro BBB model [9], consisting of a coculture of brain capillary endothelial cells and glial cells, we confirmed the absence of selectin expression on cerebral capillary endothelium and showed their upregulation under specific stimuli, suggesting a selective role for these molecules at the BBB. Primary mouse brain capillary endothelial cell (MBCEC) isolation, primary mouse glial cell preparation and coculture of MBCECs and glial cells, have already been extensively described in Coisne et al. [9]. Endothelial cells and glial

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cells were prepared from 4 to 5 weeks old and newborn OF1 mouse cerebral cortex, respectively (Charles River, L’Arbresle, France). Reagents: LPS (Escherichia coli, serotype 055:B5) and mouse recombinant IFN-gamma were obtained from Sigma Chemicals; Human recombinant TNF-alpha from R&D systems; mouse recombinant IL-1beta from biosource. Exposure: Confluent cocultured MBCECs were incubated, during 4 and 17 h at 37 ◦ C, with either 100 ng/mL of LPS, 100 ng/mL of cytokines or control medium. Then, immunocytochemistry or flow cytometry analyses were performed. ICAM-1 (YN1) Ab was a generous gift from Dr J. Boucraut (Marseille, F). VCAM-1 (9DB3), ICAM-2 (3C4), E-Selectin (UZ4), P-selectin (RB40. 34) and 9B5 as an isotype-matched control, were kindly provided by Pr B. Engelhardt (Bern, CH). AlexaFluor 488-conjugated goat anti-rat IgG and Rat anti-mouse CD31 mAb conjugated to PE were obtained from Serotec Limited. Immunofluorescence experiments were performed either on live cells (surface staining) or on fixed and permeabilized cells (intracellular staining). For immunofluorescence, MBCECs were fixed for 10 min in 1% paraformaldehyde (PFA), followed by primary and secondary Ab reactions. For intracellular staining, MBCECs were incubated for 2 min with cold methanol/acetone (v/v) before primary and secondary Ab reactions. In both cases, the primary and secondary (AlexaFluor 488) Abs were incubated for 30 min at room temperature. After LPS or cytokine treatment, MBCECs were trypsinized to carry out FACS analyses. A total of 1 × 105 to 2 × 105 cells were pelleted and resuspended in 50 ␮L of medium (PBS supplemented with 3 mM sodium azide and 2% FBS) or culture supernatant containing mAbs specific for each cell surface molecule.

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After 30 min of incubation on ice, cells were washed twice. AlexaFluor 488-conjugated goat anti-rat IgG was added as the secondary Ab for 20 min. After two washes, the binding sites of the secondary Ab were blocked by addition of PBS + 10% rat serum before adding a rat anti-mouse CD31 mAb directly conjugated to PE. The cells were washed twice, resuspended in PBS containing 2% PAF and analyzed on a FACScalibur flow cytometer using the CellQuest software (Becton Dickinson). Cells were double gated according to (1) the forward and side scatter parameters and (2) the CD31 staining. The number of cells analyzed varied from 3000 to 10,000. We have previously shown on MBCEC monolayers (1) a basal expression of ICAM-1, VCAM-1 and ICAM-2; (2) an LPS-dependent upregulation of ICAM-1 and VCAM-1 expressions; (3) no change in ICAM-2 expression after LPS treatment [9]. To extend these observations, CAM expression was tested following IL-1beta, TNF-alpha and IFN-gamma, treatments known to modulate adhesion molecule expression and transmigration of inflammatory cells [4,12,23,29,38]. FACS analyses revealed an upregulation of ICAM-1 and VCAM-1 expression, with a less pronounced effect of IFN-gamma on ICAM-1 increase. Constitutive ICAM-2 expression stayed unchanged (Fig. 1). These results are in agreement with previous findings [13,18,29] and attest to the MBCEC responsiveness to IL-1beta, TNF-alpha and IFN-gamma. Then, constitutive selectin expression on cerebral endothelium was studied and compared with selectin levels after LPS, IL-1beta, TNF-alpha and IFN-gamma treatments. FACS analyses performed on live endothelial cells did not show any constitutive expression of E- and P-selectins (Fig. 2). However, following 4- and 17-h LPS or IL-1beta-treatment, Pselectin appeared on the MBCEC surface. In contrast, only 4-h

Fig. 1. CAM expression on confluent MBCECs cocultured with glial cells. ICAM-1 (a–d), VCAM-1 (e–h), ICAM-2 (i–l) basal expression (open histogram) was compared to LPS (a, e and i), IL-1beta (b, f and j), TNF-alpha (c, g and k), and IFN-gamma (d, h and l) induced expression (dark grey solid histogram) and to isotype control (light grey solid histogram). Isotype control profiles are similar with or without inflammatory stimulus activation (data not shown). Cells were double gated within cell scatter and on positive fluorescence for CD31.

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Fig. 2. Selectin expression on confluent MBCECs cocultured with glial cells. P-Selectin (a–d) and E-selectin (e–h) basal expression (light grey solid histogram) was compared to a 4-h treatment (open histogram) and a 17-h treatment (dark grey solid histogram) with LPS (a and e), IL-1beta (b and f), TNF-alpha (c and g) and IFN-gamma (d and h). Isotype control profiles (with or without inflammatory stimulus activation) are similar to the basal expression outlined for each condition (data not shown). Cells were double gated within cell scatter and on positive fluorescence for CD31.

of LPS assay was able to induce E-selectin expression. Indeed, neither 4- or 17-h IL-1beta treatment induced the appearance of this selectin. These results show the induction of selectins and suggest a different time course of E- and P-selectin expression after endotoxin treatment. These observations were confirmed by immunocytochemistry performed on live endothelial cell monolayers (Fig. 3). Under basal conditions, no selectin cell surface expression was observed. Intracellular expression of P-selectin, was also investigated. Although MBCECs expressed von Willebrand factor related antigen, known to be present in Weibel-Palade bodies [9], no intracellular P-selectin staining was detected on endothelial cells (data not shown). MBCECs were also negative for E-selectin (data not shown). Following LPS treatment, P-selectins appeared as distinct dots at the cell surface by immunostaining. Dot density differed from cell to cell; going from low to high density. Similar observations have been reported after IL-1beta treatment (data not shown). These immunostaining data correlate with the FACS analyses which clearly showed a broad spectrum in mean fluorescence density after LPS (Fig. 2a) and IL-1beta (Fig. 2b) treatments. A switch to high fluorescence intensity was observed for 35% of cells after LPS challenge and for 50% and 67% of cells after a 4- and 17-h IL-1beta treatment, respectively.

Although some in vitro data has shown a constitutive expression of selectins on brain-derived endothelial cells [37], most authors agree with the lack of basal expression of selectins at the BBB [2,11,15] and an increase in expression following LPS treatment [16]. Gotsch et al. [15] and Piccio et al. [26] have shown LPS-dependent P-selectin expression on cerebral vessels in vivo. However, these investigations were performed on large brain vessels and the question of selectin induction on brain capillaries still remains. With regards to IL-1beta, Bernades-Silva et al. [5] have shown an upregulation of both selectins after an in vivo IL-1beta challenge in contradiction to our results. However, IL-1beta was injected intracerebrally which would indicate exposure of the abluminal side of cerebral endothelium to cytokine as well as the involvement of parenchyma cells in this phenomenon. In our experiments, IL-1beta challenge occurred from the luminal side of BBB, which may explain the discrepancy in E-selectin induction. These authors also reported P-selectin dependent recruitment of neutrophils to the brain parenchyma. Indeed, while intravenous injection of P-selectin neutralizing monoclonal Abs inhibited brain neutrophil invasion, E-selectin blockade had no effect on this phenomenon, suggesting a predominant role of P-selectin in IL-1beta-induced neutrophil recruitment into the brain.

Fig. 3. P-Selectin immuno-localization on confluent MBCECs cocultured with glial cells. Cell surface expression after a 4- and 17-h LPS activation (a and b, respectively) was compared to basal condition (c) (bar = 25 ␮m).

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Our LPS/IL-1beta results showed that, although the BBB does not constitutively express selectins, these molecules can be induced after specific stimuli. This may help to explain in vivo data looking at the influence of selectin deficiency on acute brain pathological disorders, which suggest a direct role of these molecules in leukocyte infiltration into brain parenchyma. Indeed, Tang et al. [32] have shown that the increase in BBB permeability associated with meningitis in adult mice is clearly inhibited in double P- and E-selectins deficient animals. Furthermore, Combes et al. [10] reported that mice deficient in endothelial P-selectin did not show any sign of cerebral malaria. TNF-alpha or IFN-gamma stimulation did not induce any selectin expression either on the cell surface (Fig. 2) or within the cell (data not shown). In contrast, ICAM-1 and VCAM-1 upregulation has been shown after incubation with these cytokines (Fig. 1). Therefore, the lack of E- and P-selectins does not correspond to the lack of cellular responsiveness to these stimuli. In contrast with our data, TNF-alpha dependent expression of E-selectin by cerebral endothelial cells has been described in different studies. However, it is worth mentioning that these studies were in vitro studies using human [16] or rat [35] brain endothelial cells isolated from their in vivo environment. Engelhardt et al. [11], showed that endothelial cell/glial cell interactions are important in BBB selectin expression regulation. The in vitro model used for our investigation consisted of a coculture of brain endothelial cells and glial cells [9]. Furthermore, Engelhardt et al. [11] showed that neither expression of E- nor P-selectin is induced in BBB-forming endothelium after initiation of experimental autoimmune encephalomyelitis (EAE), in spite of the well known presence and involvement of TNF-alpha in this pathology [6]. As suggested by our results, TNF-alpha may not be involved in selectin dependent process. Indeed, TNF-alpha is a common cytokine found in both acute and chronic inflammation and its presence is usually associated with other cytokines depending on the inflammatory context. TNF-alpha and IL-1beta co-expression is usually found in acute inflammation caused by infectious agents (sepsis [19]; meningitis [28]; malaria [7,33] and ischemia [36]). In this study, IL-1beta was able to induce selectin expression without requiring TNF-alpha involvement. With respect to IFN-gamma, the potential effect of this cytokine on selectin expression on cerebral endothelium is not well documented. In vitro studies, performed on non-cerebral endothelial cells, showed that IFN-gamma inhibits activationinduced expression of E- and P-selectins [21,20,31]. These results can be related to the anti-inflammatory effect of IFNgamma described in vivo [18], although these data are in disagreement with others [24,25]. The absence of IFN-gamma dependent induction of selectins may be explained by the fact that this cytokine is almost exclusively produced by activated T cells. These cells are involved in CNS chronic inflammation characterized by T cell infiltration [22]. In this case, selectinindependent rolling is thought to be mediated by alpha4 integrins, which interact with VCAM-1 [1,11]. To conclude, our results raise new insights into the controversial expression of selectins at the BBB. They show that, in contrast to ICAM-1 and VCAM-1 for which upregulation after

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various inflammatory stimuli is commonly observed, selectin expression on cerebral endothelium is only induced by selective mediators, suggesting a non-systematic involvement of these molecules in inflammatory events which may lead to the selective entrance of different leukocyte subsets into the brain. Acknowledgements This work was supported by grants from the Ministry of Research of France (fellowship to C. Coisne) and by the “Conseil R´egional du Nord-Pas-de-Calais” (fellowship to F. Miller). We are very grateful to Pr B. Engelhardt (Theodor Kocher Institute, University of Bern, Switzerland) and Dr. J. Boucraut (Laboratoire NICN UMR 6184, Facult´e de M´edecine Timone, Marseille) to their generous gifts of Abs. The authors thank Dr. Val´erie Bu´ee for the critical reading of the manuscript. References [1] R. Alon, P.D. Kassner, M.W. Carr, E.B. Finger, M.E. Hemler, T.A. Springer, The integrin VLA-4 supports tethering and rolling in flow on VCAM-1, J. Cell. Biol. 128 (1995) 1243–1253. [2] F.J. Barkalow, M.J. Goodman, M.E. Gerritsen, T.N. Mayadas, Brain endothelium lack one of two pathways of P-selectin-mediated neutrophil adhesion, Blood 88 (1996) 4585–4593. [3] F.J. Barkalow, M.J. Goodman, T.N. Mayadas, Cultured murine cerebral microvascular endothelial cells contain von Willebrand factor-positive Weibel-Palade bodies and support rapid cytokine-induced neutrophil adhesion, Microcirculation 3 (1996) 19–28. [4] D.M. Barten, N.H. Ruddle, Vascular cell adhesion molecule-1 modulation by tumor necrosis factor in experimental allergic encephalomyelitis, J. Neuroimmunol. 51 (1994) 123–133. [5] M. Bernardes Silva, D.C. Anthony, A.C. Issekutz, V.H. Perry, Recruitment of neutrophils across the blood–brain barrier: the role of E- and P-selectins, J. Cereb. Blood. Flow. Metab. 21 (2001) 1115–1124. [6] L.A. Boos, A.J. Szalai, S.R. Barnum, Murine complement C4 is not required for experimental autoimmune encephalomyelitis, Glia 49 (2005) 158–160. [7] H. Brown, G. Turner, S. Rogerson, M. Tembo, J. Mwenechanya, M. Molyneux, T. Taylor, Cytokine expression in the brain in human cerebral malaria, J. Infect. Dis. 180 (1999) 1742–1746. [8] E.C. Butcher, Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity, Cell 67 (1991) 1033–1036. [9] C. Coisne, L. Dehouck, C. Faveeuw, Y. Delplace, F. Miller, C. Landry, C. Morissette, L. Fenart, R. Cecchelli, P. Tremblay, B. Dehouck, Mouse syngenic in vitro blood–brain barrier model: a new tool to examine inflammatory events in cerebral endothelium, Lab. Invest. 85 (2005) 734–746. [10] V. Combes, A.R. Rosenkranz, M. Redard, G. Pizzolato, H. Lepidi, D. Vestweber, T.N. Mayadas, G.E. Grau, Pathogenic role of P-selectin in experimental cerebral malaria: importance of the endothelial compartment, Am. J. Pathol. 164 (2004) 781–786. [11] B. Engelhardt, D. Vestweber, R. Hallmann, M. Schulz, E- and Pselectin are not involved in the recruitment of inflammatory cells across the blood–brain barrier in experimental autoimmune encephalomyelitis, Blood 90 (1997) 4459–4472. [12] Z. Fabry, D.J. Topham, D. Fee, J. Herlein, J.A. Carlino, M.N. Hart, S. Sriram, TGF-beta 2 decreases migration of lymphocytes in vitro and homing of cells into the central nervous system in vivo, J. Immunol. 155 (1995) 325–332. [13] Z. Fabry, M.M. Waldschmidt, D. Hendrickson, J. Keiner, L. Love Homan, F. Takei, M.N. Hart, Adhesion molecules on murine brain microvascular endothelial cells: expression and regulation of ICAM-1 and Lgp 55, J. Neuroimmunol. 36 (1992) 1–11.

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