Cutting Edge Cutting Edge: Natalizumab Blocks Adhesion but Not Initial Contact of Human T Cells to the Blood-Brain Barrier In Vivo in an Animal Model of Multiple Sclerosis1 Caroline Coisne,* Wenxian Mao,† and Britta Engelhardt2* The humanized anti-␣4 integrin Ab Natalizumab is an effective treatment for relapsing-remitting multiple sclerosis. Natalizumab is thought to exert its therapeutic efficacy by blocking the ␣4 integrin-mediated binding of circulating immune cells to the blood-brain barrier (BBB). As ␣4 integrins control other immunological processes, natalizumab may, however, execute its beneficial effects elsewhere. By means of intravital microscopy we demonstrate that natalizumab specifically inhibits the firm adhesion but not the rolling or capture of human T cells on the inflamed BBB in mice with acute experimental autoimmune encephalomyelitis (EAE). The efficiency of natalizumab to block T cell adhesion to the inflamed BBB was found to be more effective in EAE than in acute systemic TNF-␣-induced inflammation. Our data demonstrate that ␣4 integrin-mediated adhesion of human T cells to the inflamed BBB during EAE is efficiently blocked by natalizumab and thus provide the first direct in vivo proof of concept of this therapy in multiple sclerosis. The Journal of Immunology, 2009, 182: 5909 –5913. n multiple sclerosis (MS)3 and in its animal model experimental autoimmune encephalomyelitis (EAE), circulating immune cells get access to the CNS where they start the molecular events leading to inflammation, edema formation, and demyelination, all of which set the ground for the development of the disabling clinical picture of the disease. Interaction of circulating immune cells with the endothelial blood-brain barrier (BBB) is thus a critical step in the pathogenesis of EAE and MS. ␣41 integrin was identified as mediating T cell adhesion to inflamed vessels in frozen sections of EAE brains in vitro (1), and Abs blocking ␣4 integrins were shown in a variety of animal models to prevent the development of EAE (summarized in Ref. 2). Based on these findings, the
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*Theodor Kocher Institute, University of Bern, Bern, Switzerland; and †Elan Pharmaceuticals, South San Francisco, CA 94080 Received for publication October 16, 2008. Accepted for publication March 16, 2009.
humanized monoclonal anti-␣4 integrin Ab natalizumab was developed for the treatment of MS with the idea of targeting ␣4 integrin-mediated extravasation of inflammatory cells into the CNS. In clinical trials, natalizumab proved to be highly beneficial in reducing MS disease activity regarding both clinical parameters as well as magnetic resonance intensity measurements of disease activity (3, 4). The rare risk of progressive multifocal leukoencephalopathy (www.nationalmssociety.org/news/news-detail/index.aspx?nid ⫽ 260) and an increased rate of herpes recrudescence in patients receiving natalizumab (natalizumab packaging insert; Biogen Idec) suggest however, that the Ab may have broader immunosuppressive effects. In fact, besides mediating T cell extravasation, ␣4 integrins have been demonstrated in animal models to be involved in multiple immune cell functions including T cell activation and polarization (5), retention of memory T cells in their niches (6), and the localization and maturation of hematopoietic stem cells (7). Thus, better understanding of to what extent the drug natalizumab prevents extravasation of circulating human T cells into the CNS in MS patients would greatly improve our understanding of how natalizumab may exert its immunosuppressive effects. To directly visualize the effect of natalizumab on the interaction of human T cells with the inflamed BBB in vivo, we investigated human T cell interaction with the inflamed spinal cord white matter microcirculation in SJL mice with EAE by intravital fluorescence videomicroscopy. Human T cells were found to initiate contact by rolling or capture and to firmly adhere to the inflamed spinal cord microvasculature in vivo. Whereas natalizumab almost completely blocked the firm adhesion of human T cells to the BBB, it surprisingly left T cell rolling and capture unaffected. Effective blocking of T cell adhesion to the inflamed BBB was seen in mice with EAE as a model for the neuroinflammatory setting of MS. In contrast, natalizumab only partially and transiently reduced T cell interaction with TNF-␣-stimulated spinal cord microvessels in SJL mice. Our study provides the first 2
Address correspondence and reprint requests to Prof. Britta Engelhardt, Theodor Kocher Institute, University of Bern, Freiestrasse 1, CH-3012 Bern, Switzerland. E-mail address:
[email protected]
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1
This work has been supported by the National Multiple Sclerosis Society of the United States, the Swiss Multiple Sclerosis Society, and the Bern University Research Foundation. C.C. has been funded by the Fondation pour la Recherche Me´dicale, the Association pour la Recherche sur la Scle´rose en Plaques, and the National Multiple Sclerosis Society. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803418
Abbreviations used in this paper: MS, multiple sclerosis; BBB, blood-brain barrier; EAE, experimental autoimmune encephalomyelitis; TRITC, tetramethylrhodamine. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
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direct in vivo proof of concept that natalizumab does inhibit T cell extravasation into the CNS by specifically inhibiting T cell adhesion to the inflamed BBB.
Methods Mice and induction of EAE Female SJL mice were obtained from Harlan and used at the age of 12 wk. Animal experiments were performed in accordance with the requirements of the local government. EAE was induced by immunization with proteolipid protein aa 139 –151 exactly as described (8). Experiments were performed with mice suffering from clinical EAE at days 10 –11 postimmunization with clinical scores of 0.5 (limp tail) and 1 (hind leg paraparesis).
Abs and cytokines Human recombinant TNF-␣ was a gift from D. Maennel (University of Regensburg, Regensburg, Germany) and natalizumab was from S. Goelz (Biogen Idec). Human IgG4 Ab was purchased from Sigma-Aldrich.
VCAM-1 binding assay Binding of soluble mouse and human VCAM-1-Fc fusion proteins (R&D Systems) to Jurkat T cells was investigated by FACS analysis exactly as described (9). Briefly, 2 ⫻ 106 Jurkat T cells were incubated for 30 min at room temperature in PBS, 0.1% BSA, 0.2 mM Mn2⫹ with increasing concentrations of soluble recombinant human or mouse VCAM-1/Fc in the presence or absence of the mouse anti-human ␣4 integrin Ab 21/6, the mouse anti-human CD3 (BD Pharmingen), or the mouse anti-human CD18 (Ancell). After washing, FITC-labeled goat anti-human IgG (Vector Laboratories) was applied and fluorescence staining was analyzed on a FACScalibur flow cytometer (BD Biosciences).
Human T cells Jurkat T cells were repeatedly sorted for high ␣4 integrin expression. PBMCs were isolated from buffy coats of healthy donors obtained from a blood bank (Inselspital, Bern, Switzerland) and cultured overnight in RPMI 1640 and 20% FCS. Preparation of PBMCs one day before the assay was necessary due to time constraints that did not allow us to perform all of the experimental procedures within one working day. The protocol used has been approved by the Bernese Cantonal Ethical Review Board (KEK No. 17/05). T cells were purified from PBMCs by negative magnetic selection according to the manufacturer’s protocol (Pan T cell isolation kit II from Miltenyi Biotec), fluorescently labeled with 125 nM calcein-AM (Molecular Probes), and incubated with natalizumab or human IgG4 at 170 g per 4 ⫻ 106 T cells in 300 l of physiological saline (0.9% (v/w) NaCl) 20 min prior to T cell infusion and injection with the cells. Natalizumab binding to human T cells is shown in supplemental Fig. 2.4 The natalizumab dosage used was deduced from the dosage of natalizumab in MS patients. MS patients receive natalizumab once a month as an infusion of 300 mg of injection per patient irrespective of the patient’s body weight. Dosage adjustment for mice was therefore based on blood volume; considering a blood volume of 4 –5 liters for humans and ⬃2. 5 ml for a SJL mouse of 25 g, the corresponding amount of natalizumab is between 120 and 187 g per mouse. Based on the average blood volume of mice used in the study, the injection of 170 g per mouse was chosen.
Intravital fluorescence videomicroscopy Surgical preparations, intravital microscopy by epi-illumination techniques, and quantitative analysis of the spinal cord microcirculation was performed exactly as described (10 –12) using a custom-made Mikron IVM-500 fluorescence microscope connected to a silicon-intensified target camera (Dage-MTI). Spinal cord microvasculature was visualized with 1% tetramethylrhodamine isothiocyanate (TRITC)-conjugated dextran (0.1 ml of TRITC-dextran, m.w. ⫽ 155,000; Sigma-Aldrich). Systemic injection of three separate 100-l aliquots of fluorescently labeled T cells was achieved by infusion via a catheter placed into the right carotid artery below a ligation, in the direction of the aortic arch. Observations were made using ⫻ 4, ⫻10, and ⫻ 20 long-distance working objectives. Real time microscopic images were recorded using a digital videocassette recorder (DSR-11; Sony) for later off-line analysis, which was performed exactly as described (10, 13). Similarly, hemodynamic parameters were analyzed and calculated based on Hagen-Poiseuille equations precisely as described (10).
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The online version of this article contains supplemental material.
Statistics Mann-Whitney U statistics were used for comparisons between different data sets. Asterisks indicate significant differences (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; and ⴱⴱⴱ, p ⬍ 0.005). For the analysis of the firm adhesion of T cells, quantitative data are given as mean values ⫾ SD calculated from the values in each animal. For analysis of differences between the groups, a Mann-Whitney U test was performed.
Results and Discussion
Inhibition of ␣4 integrin-mediated leukocyte trafficking across the inflamed BBB is thought to be the underlying concept of the therapeutic effect of natalizumab in reducing inflammatory and clinical signs in MS. However, direct in vivo evidence for natalizumab-mediated inhibition of leukocyte trafficking across the BBB is still missing. Imaging techniques allowing study of the interaction of individual circulating white blood cells with the CNS microvascular wall in the brain or spinal cord of humans are not available to date. Therefore, it is desirable to investigate immune cell interaction with the inflamed BBB in an MS-like neuroinflammatory setting in a model organism. In this study we specifically investigated the effect of natalizumab on the interaction of human T cells with the inflamed BBB, as we have recently shown that T cells but not myeloid cells critically rely on 1 integrins to accumulate in the CNS during EAE, suggesting that T cells are the main target of the anti-VLA-4 therapy (12). It is well established that the structural requirements for the binding of human and mouse ␣41 integrin to mouse VCAM-1 are the same involving a cluster of amino acids in domain 1 of mouse VCAM-1, which is identical with the ␣41-binding site in human VCAM-1 (14). Based on these previous observations, we compared the binding of soluble mouse and human recombinant VCAM-1-Fc fusion proteins to human Jurkat T cells expressing high ␣41 integrin levels by FACS analysis. Increasing concentrations of mouse and human soluble VCAM-1 were found to bind with similar affinities to Jurkat T cells in an ␣41 integrin-dependent manner (Fig. 1A). The comparability of human ␣41 integrin-mediated binding to human and mouse VCAM-1 and the observation that circulating human T cells can extravasate across the inflamed BBB into the CNS parenchyma in mice suffering from EAE (Fig. 1, B and C) prompted us to perform live imaging of the interaction of human T cells with the BBB in a mouse model of MS to study the effect of natalizumab. By performing intravital fluorescence videomicroscopy of the spinal cord white matter microvasculature in SJL mice with EAE, we were able to directly visualize the multistep interaction of human T cells with the inflamed BBB under physiological shear forces in vivo in a neuroinflammatory setting modeling MS (10). After visualization of the vascular system by the injection of TRITC-dextran, fluorescently labeled human T cells were infused and could readily be observed passing through the inflamed spinal cord white matter microvessels (supplemental video 1). An average of 22% of the total number of control IgG4-pretreated human T cells observed to pass through a given microvessel over a period of 1 min made initial contact with the inflamed spinal cord microvessels. This initial contact of human T cells with the inflamed BBB was characterized by T cell rolling along the vascular wall with reduced velocity or by transient capture, i.e., the abrupt stop of the T cells on the vessel wall for up to 7 s (Fig. 2a and supplemental video 1A). Surprisingly, natalizumab did not alter the G protein-independent
The Journal of Immunology
FIGURE 1. Human T cells interact with mouse VCAM-1 in vitro and migrate across the inflamed mouse CNS microvasculature in vivo. A, Human and mouse VCAM-1 bind equally well to ␣41 integrin on human T cells in vitro. Binding of human (rh) and mouse (rm) soluble recombinant VCAM-1/Fc fusion proteins to human Jurkat T cells sorted for high ␣4 integrin expression was determined by FACS analysis. Geometrical means of fluorescence intensity obtained after incubation with increasing concentrations of VCAM-1-Fc fusion proteins is shown. Binding of both VCAM-1-Fc fusion proteins to Jurkat T cells was mediated by ␣4 integrins as demonstrated by complete inhibition of binding by the anti-human ␣4 integrin Ab 21/6, but not by Abs directed against CD3 or CD18 (see supplemental Fig. 1). B and C, Human T cells migrate across the inflamed blood-brain barrier into the CNS in mice with EAE. Four hours after T cell infusion mice were perfused with 1% formaldehyde/PBS (pH 7.4) and spinal cords were dissected, embedded in OCT compound (Sakura Finetek), and longitudinal 6-m-thick frozen tissue sections were cut. Slides were screened for the presence of fluorescently labeled T cells and subsequently stained for laminin (blue) to mark the endothelial and parenchymal basement membranes, which define the inner and outer borders of the perivascular space, to determine the presence of human T cells in the mouse spinal cord in relation to blood vessels. In total, 230 tissue sections from three mice were screened for the presence of fluorescently labeled human T cells. Zero to a maximum of three human T cells per section could be detected localized either still inside the vessel lumen, within the perivascular space (B), on its way across the parenchymal basement membrane (C), or very rarely within the CNS parenchyma. T cells shown in A and B were labeled with 2.5 M Cell Tracker Orange before infusion. Bars represent 20 m.
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FIGURE 2. Natalizumab inhibits ␣4 integrin-mediated firm adhesion but not initial contact of human T cells with the BBB during EAE. a, Initial T cell interaction with spinal cord postcapillary venules during EAE. Initial contact of human T cells with the endothelial cells was analyzed by counting all T cells passing through spinal cord microvessels and T cells that visibly initiated contact (either rolling or capture) with the spinal cord microvascular endothelium and thus moved at a slower velocity than the passing T cells in a frame by frame video analysis using the CapImage 8.3 software (Dr. H. Zeintl, Heidelberg, Germany). The rolling and capture fractions represent the percentage of rolling or captured T cells among the total number of T cells passing through a given venule during a 1-min observation period. Each dot represents one venule. Thirty-two venules in eight mice and 28 venules in seven mice for human IgG4 and natalizumab conditions, respectively, are included in this analysis. Quantitative data are given ⫾ SD; the Mann-Whitney U test was performed. b, Adhesion of human T cells to spinal cord microvessels during EAE. Firm adhesion of human T cells to the inflamed spinal cord microvasculature during EAE was evaluated at 10, 30, 60, and 120 min after T cell infusion (4 ⫻ 106 T cells cells/mouse). Eight mice for human IgG4 and natalizumab conditions are included into this analysis. Asterisks indicate significant differences. Quantitative data are given as means ⫾ SD. Mann-Whitney U-Test was performed, asterisks indicate significant (ⴱ, p ⬍ 0.05) and highly significant (ⴱⴱ, p ⬍ 0.01) differences.
rolling or capture of human T cells to the inflamed BBB during EAE as compared with control T cells pretreated with human IgG4, indicating that ␣4 integrins are not dominantly involved in mediating the initial contact of T cells with the inflamed BBB in MS in vivo (Fig. 2a and supplemental video 2A). This observation is in apparent contrast to our previous observation made under noninflammatory conditions, where the ␣4 integrin was observed to mediate the G protein-independent
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capture of freshly activated encephalitogenic T cell blasts to low levels of constitutively expressed VCAM-1 in the noninflamed spinal cord microvasculature in healthy mice (10). Thus, upregulation of endothelial adhesion molecules other than VCAM-1 on the inflamed BBB in EAE seems to allow for ␣4 integrin-independent initial contact of human T cells with the inflamed microvascular wall. In accordance with this, we recently demonstrated that 1 integrin-deficient T cells are able to capture and roll on inflamed CNS microvessels during EAE (12). Because P-selectin is found to be up-regulated in inflamed CNS microvessels during EAE (8) and can mediate T cell rolling via P-selectin glycoprotein ligand-1 (15, 16), the inhibition of T cell rolling in inflamed CNS microvessels during EAE might require the simultaneous blockage of several adhesion mechanisms. Natalizumab was, however, found to strongly reduce the firm adhesion of human T cells to the BBB in EAE by 70% as determined 10 min after cell infusion compared with controls (supplemental videos 1B and 2B). This potent natalizumab-mediated reduction of human T cell adhesion to the inflamed spinal cord microvascular wall was sustained 30, 60, and 120 min after T cell infusion (Fig. 2b), providing evidence that in the presence of ␣4 integrin-blocking natalizumab, T cells are unable to maintain G protein-dependent, integrin-mediated firm contact with the inflamed BBB in vivo. Our observations therefore provide the first live visual proof that human T cells use ␣4 integrins to bind to the inflamed BBB in vivo and that natalizumab blocks T cell entry into the CNS by specifically inhibiting the firm adhesion of human T cells to the BBB in a neuroinflammatory setting modeling MS. To define whether natalizumab generally inhibits the adhesion of human T cells to the inflamed BBB in vivo, we next modeled an acute systemic inflammation by i.v. application of TNF-␣ in SJL mice 4 h before an investigation of human T cell interaction with the acutely stimulated spinal cord white matter microvascular wall. Blood vessels were again visualized by TRITC-dextran. Upon infusion, fluorescently labeled human T cells were found to initiate contact with the inflamed spinal cord microvascular wall by rolling and capture, similarly as observed in EAE (Fig. 3a and supplemental video 3A). Interestingly, under acute TNF-␣-stimulated conditions, natalizumab significantly reduced the percentage of T cells rolling along the activated spinal cord white matter microvascular wall but did not inhibit T cell capture (supplemental video 4A). Taken together, natalizumab therefore again failed to significantly inhibit the initial contact of human T cells to the TNF-␣ stimulated BBB, confirming that under inflammatory conditions ␣4 integrins are not required to initiate the interaction of human T cells with the BBB in vivo. In contrast to the observations made in EAE, natalizumab only partially and transiently (at 10 min but no longer at 30 or 60 min after infusion) reduced the firm adhesion of human T cells to the TNF-␣-stimulated BBB in vivo, suggesting that the drug is less efficient in inhibiting T cell interaction with the acutely inflamed BBB (Fig. 3b and supplemental videos 3B and 4B). The different inhibitory capacity of natalizumab in EAE vs TNF-␣ induced inflammatory conditions may be due to different ␣4 integrin ligand densities expressed on the BBB upon stimulation with TNF-␣ vs during the complex neuroinflammatory situation in EAE. Additionally, differences in hemodynamic flow parameters in the spinal cord microvasculature in SJL mice with EAE vs TNF-␣ injected SJL mice may have an impact on natalizumab-mediated inhi-
FIGURE 3. Natalizumab only partially and transiently reduces ␣4 integrinmediated T cell adhesion to TNF-␣-activated spinal cord microvessels. a, T cells passing through the spinal cord microvessels and T cells that visibly initiated contact with the activated spinal cord microvascular endothelium and thus moved at a slower velocity than the main blood stream were counted in 42 postcapillary venules in nine mice for the IgG4 condition and in 53 postcapillary venules in 14 mice for the natalizumab condition in a frame by frame analysis of the videos. Shown is the percentage of rolling or captured T cells among the total number of T cells passing through a given venule during a 1-min observation period. Each dot represents one venule. Natalizumab significantly reduces the percentage of T cells rolling along but not captured at the vascular wall. b, Evaluation of human T cell adhesion to the spinal cord vessel wall. T cells permanently adhering within the inflamed spinal cord white matter microvasculature counted at 10 min after cell infusion (4 ⫻ 106 T cells cells/ mouse) are shown. Ten and 15 mice for human IgG4 and natalizumab conditions, respectively, were included in the analysis. Quantitative data are given as means ⫾ SD; asterisks indicate significant (ⴱ, p ⬍ 0.05) and highly significant (ⴱⴱ, p ⬍ 0.01) differences.
bition of T cell interaction with the BBB. Although neither inflammatory condition by itself produced significantly different hemodynamic flow parameters compared with those determined by us in the spinal cord white matter microcirculation of healthy SJL mice previously (10), direct comparison of the hemodynamic flow parameters between the spinal cord microvasculature under EAE or TNF-␣-stimulated conditions revealed significant differences (supplemental Fig. 3). Specifically, the mean velocity of circulating T cells, wall shear rates, and wall shear stress were found to be significantly lower in spinal cord
The Journal of Immunology
microvessels during EAE when directly compared with those in mice injected with TNF-␣ (supplemental Fig. 3). These findings suggest that lower shear forces may favor ␣4 integrin-mediated T cell adhesion to the inflamed CNS microvasculature, and thus natalizumab can efficiently block this step of the multistep T cell migration cascade across the inflamed BBB during EAE. Taken together, our data provide the first direct in vivo proof of concept of natalizumab-mediated inhibition of stable adhesion of human T cells to the inflamed BBB in a mouse model of MS. Surprisingly, natalizumab-mediated inhibition of T cell adhesion to the inflamed BBB was found to be efficient in EAE modeling the neuroinflammation in MS, but not in acute systemic inflammation induced by TNF-␣. Thus, our data suggest that the clinical efficacy of anti-␣4 integrin therapy in MS is at least in part due to the inhibition of ␣4 integrin-mediated T cell adhesion (and surprisingly not T cell rolling or capture) to the BBB, thus preventing T cell entry into the CNS.
Acknowledgments We owe special thanks to Heidi Tardent for excellent microsurgical preparations and to Jens Stein for patient advice about performing and analyzing the intravital fluorescence videomicroscopy experiments. We thank Biogen Idec for the gift of natalizumab and Fre´de´rique Bard and Ted Yednock (Elan Pharmaceuticals) for very fruitful discussions.
Disclosures The authors have no financial conflict of interest.
References 1. Yednock, T. A., C. Cannon, L. C. Fritz, F. Sanchez-Madrid, L. Steinman, and N. Karin. 1992. Prevention of experimental autoimmune encephalomyelitis by antibodies against ␣41 integrin. Nature 356: 63– 66. 2. Engelhardt, B., and R. M. Ransohoff. 2005. The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends Immunol. 26: 485– 495.
5913 3. Polman, C. H., P. W. O’Connor, E. Havrdova, M. Hutchinson, L. Kappos, D. H. Miller, J. T. Phillips, F. D. Lublin, G. Giovannoni, A. Wajgt, et al. 2006. A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N. Engl. J. Med. 354: 899 –910. 4. Rudick, R. A., W. H. Stuart, P. A. Calabresi, C. Confavreux, S. L. Galetta, E. W. Radue, F. D. Lublin, B. Weinstock-Guttman, D. R. Wynn, F. Lynn, et al. 2006. Natalizumab plus interferon 1a for relapsing multiple sclerosis. N. Engl. J. Med. 354: 911–923. 5. Mittelbrunn, M., A. Molina, M. M. Escribese, M. Yanez-Mo, E. Escudero, A. Ursa, R. Tejedor, F. Mampaso, and F. Sanchez-Madrid. 2004. VLA-4 integrin concentrates at the peripheral supramolecular activation complex of the immune synapse and drives T helper 1 responses. Proc. Natl. Acad. Sci. USA 101: 11058 –11063. 6. Sixt, M., M. Bauer, T. Lammermann, and R. Fassler. 2006. 1 integrins: zip codes and signaling relay for blood cells. Curr. Opin. Cell Biol. 18: 482– 490. 7. Bungartz, G., S. Stiller, M. Bauer, W. Muller, A. Schippers, N. Wagner, R. Fassler, and C. Brakebusch. 2006. Adult murine hematopoiesis can proceed without 1 and 7 integrins. Blood 108: 1857–1864. 8. Do¨ring, A., M. Wild, D. Vestweber, U. Deutsch, and B. Engelhardt. 2007. E- and P-selectin are not required for the development of experimental autoimmune encephalomyelitis in C57BL/6 and SJL Mice. J. Immunol. 179: 8470 – 8479. 9. Yednock, T. A., C. Cannon, C. Vandevert, E. G. Goldbach, G. Shaw, D. K. Ellis, C. Liaw, L. C. Fritz, and L. I. Tanner. 1995. ␣41 integrin-dependent cell adhesion is regulated by a low affinity receptor pool that is conformationally responsive to ligand. J. Biol. Chem. 270: 28740 –28750. 10. Vajkoczy, P., M. Laschinger, and B. Engelhardt. 2001. ␣4-integrin-VCAM-1 binding mediates G protein-independent capture of encephalitogenic T cell blasts to CNS white matter microvessels. J. Clin. Invest. 108: 557–565. 11. Engelhardt, B., P. Vajkoczy, and M. Laschinger. 2003. Detection of endothelial/lymphocyte interaction in spinal cord microvasculature by intravital videomicroscopy. Methods Mol. Med. 89: 83–93. 12. Bauer, M., C. Brakebusch, C. Coisne, M. Sixt, H. Wekerle, B. Engelhardt, and R. Fassler. 2009. 1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity. Proc. Natl. Acad. Sci. USA 106: 1920 –1925. 13. Stein, J. V., G. Cheng, B. M. Stockton, B. P. Fors, E. C. Butcher, and U. H. von Andrian. 1999. L-selectin-mediated leukocyte adhesion in vivo: microvillous distribution determines tethering efficiency, but not rolling velocity. J. Exp. Med. 189: 37–50. 14. Renz, M. E., H. H. Chiu, S. Jones, J. Fox, K. J. Kim, L. G. Presta, and S. Fong. 1994. Structural requirements for adhesion of soluble recombinant murine vascular cell adhesion molecule-1 to ␣41. J. Cell Biol. 125: 1395–1406. 15. Kerfoot, S. M., M. U. Norman, B. M. Lapointe, C. S. Bonder, L. Zbytnuik, and P. Kubes. 2006. Reevaluation of P-selectin and ␣4 integrin as targets for the treatment of experimental autoimmune encephalomyelitis. J. Immunol. 176: 6225– 6234. 16. Battistini, L., L. Piccio, B. Rossi, S. Bach, S. Galgani, C. Gasperini, L. Ottoboni, D. Ciabini, M. D. Caramia, G. Bernardi, et al. 2003. CD8⫹ T cells from patients with acute multiple sclerosis display selective increase of adhesiveness in brain venules: a critical role for P-selectin glycoprotein ligand-1. Blood 101: 4775– 4782.
