Abrogation of Functional -Ligand Expression Reduces Migration of Pathogenic CD8+ T Cells into Heart

This information is current as Yi Hong Cai, Angeles Alvarez, Pilar Alcaide, Paurene of September 24, 2021. Duramad, Yaw-Chin Lim, Petr Jarolim, John B. Lowe, Francis W. Luscinskas and Andrew H. Lichtman J Immunol 2006; 176:6568-6575; ; doi: 10.4049/jimmunol.176.11.6568

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Abrogation of Functional Selectin-Ligand Expression Reduces Migration of Pathogenic CD8؉ T Cells into Heart1

Yi Hong Cai,* Angeles Alvarez,* Pilar Alcaide,* Paurene Duramad,* Yaw-Chin Lim,‡ Petr Jarolim,† John B. Lowe,§ Francis W. Luscinskas,* and Andrew H. Lichtman2*

CD8؉ T cells are involved in autoimmune and infectious myocarditis and cardiac allograft rejection. The role of in cardiac recruitment of CD8؉ T cells is not understood. In this study, the contribution of selectin ligands to effector CD8؉ T cell recruitment into the heart was examined using a model of myocarditis, which depends on transfer of OVA peptide-specific CD8؉ T cells (OT-I) into mice (CMy-mOva) that express OVA in the heart. ␣-(1,3)-Fucosyltransferase (FucT)-VII-deficient OT-I cells displayed over a 95% reduction in their ability to interact with P-selectin under flow conditions in vitro, compared with wild-type OT-I cells. Interaction of FucT-VII-deficient OT-I cells with E-selectin was reduced ϳ50%. FucT-VII-deficient OT-I cells were also less efficiently recruited into a dermal site of Ag and adjuvant injection. Significantly, FucT-VII-deficient OT-I cells Downloaded from were also impaired in their ability to migrate into CMy-mOva hearts, compared with wild-type OT-I cells. Transfer of FucT- VII-deficient T cells caused less severe early myocarditis and myocyte damage than transfer of wild-type T cells. Combined FucT-IV/VII-deficient OT-I cells displayed a more profound reduction in E-selectin interactions in vitro compared with FucT- VII-deficient T cells, and the FucT-IV/VII-deficient T cells also showed less early recruitment and pathogenicity in the CMy-mOva myocarditis model. These results identify a prominent role for selectin ligands in contributing to effector CD8؉ T cell recruitment into the myocardium and indicate that selectin-dependent T cell recruitment is relevant to other tissues besides the skin. The http://www.jimmunol.org/ Journal of Immunology, 2006, 176: 6568–6575.

ffector CD8ϩ T cells contribute significantly to the patho- to E- and P-selectin on endothelial cells (2). The synthesis of these genesis of infectious myocarditis, autoimmune myocardi- ligands in T cells, as well as other leukocytes, is dependent on E tis, and cardiac allograft rejection (1). Nonetheless, the posttranslational modification by ␣-(1,3)-fucosyltransferase mechanisms by which these T cells are recruited to the myocar- (FucT)-IV and FucT-VII, tyrosyl sulfotransferases dium are incompletely understood. Most studies of lymphocyte (TPST-1 and TPST-2), and C2GlcNAcT-I (core 2 ␤-1,6-glu- trafficking into tissues have focused on CD4ϩ T cells, and few of cosaminyltransferase-I). The expression of these enzymes during by guest on September 24, 2021 these studies have addressed trafficking into heart. A better under- effector T cell differentiation is regulated by signals generated by standing of CD8ϩ T cell traffic into the heart is important for cytokine and T cell Ag receptors. In both CD4ϩ and CD8ϩ T cells, design of therapeutic strategies to reduce severity of T cell-medi- FucT-VII is the major fucosyltransferase required for E- and P- ated myocardial disease. selectin ligand synthesis, but FucT-IV may be required for a small The differentiation of effector T cells from naive precursors after fraction of T cell selectin-binding activity (3, 4). Ag recognition results in the acquisition of effector functionality as The analysis of the role of selectin-selectin ligand interactions in well as a change in migratory phenotype. In particular, effector T effector T cell trafficking in vivo is complicated by the fact that cells express chemokine receptors and adhesion molecules not ex- L-selectin, expressed on naive T cells, and L-selectin ligands ex- pressed by naive T cells, and these molecules facilitate recruitment pressed by the of secondary lymphoid into peripheral tissues, especially at sites of inflammation. One organs, are required for trafficking of these cells into lymph nodes important component of the migratory phenotype of effector T where T cell responses are initiated and effector T cells are gen- cells is the expression of functional glycoprotein ligands that bind erated. Therefore, abrogation of selectin ligand synthesis by fuco- syltransferase gene ablation will impair L-selectin ligand synthesis by high endothelial venules, which will impair naive T cell homing *Vascular Research Division and †Laboratory Medicine Division, Department of Pa- to lymph nodes and the generation of effector T cells. Alterna- thology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA tively, T cell trafficking can be studied by adoptive transfer of 02115; ‡Department of Pathology and Physiology and Immunology Program, Faculty of Medicine, National University of Singapore, Singapore; and §Department of Pa- normal effector T cells into mice in which E- and/or P-selectin are thology, Case Western Reserve University School of Medicine, Cleveland, OH 44106 blocked with Abs or into mice with genetic deficiencies in selectin Received for publication December 2, 2005. Accepted for publication March expression. This approach, however, has the limitation that sec- 22, 2006. ondary inflammatory events involving recruitment of endogenous The costs of publication of this article were defrayed in part by the payment of page leukocytes will be inhibited. Secondary inflammation can enhance charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. selectin-independent T cell recruitment. 1 To facilitate the analysis of migration and effector function of a This work was supported by National Institutes of Health Grants AI059610 and ϩ HL072056 (to A.H.L.), HL53993 and HL36028 (to F.W.L.), and 1P01CA71932 (to monospecific population of CD8 T cells that recognize an Ag J.B.L.). A.A. was a recipient of a fellowship from the Spanish Ministerio de Eduacio´n, expressed exclusively in the heart, we have developed a transgenic Cultura, y Deporte. model of CD8ϩ T cell-mediated myocarditis (5). This model uses 2 Address correspondence and reprint requests to Dr. Andrew H. Lichtman, De- partment of Pathology, Brigham and Women’s Hospital, 77 Avenue Louis Pas- teur, New Research Building 752N, Boston, MA 02115. E-mail address: [email protected] 3 Abbreviations used in this paper: FucT, ␣-(1,3)-fucosyltransferase; TnI, troponin I.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 6569

CMy-mOva mice that express OVA under the control of the car- phate-buffered formalin, embedded in paraffin, and used for preparation of diac myosin H chain promoter, and OT-I cells, which are TCR- H&E-stained sections. The apical portion was fixed in TRIzol reagent (In- transgenic CD8ϩ T cells specific for an OVA peptide bound to the vitrogen Life Technologies) for subsequent RNA extraction. Mice that received footpad immunizations and T cell adoptive transfers class I MHC molecule H-2Kb. Within 4–6 days of adoptive trans- were sacrificed by CO2 inhalation. Sections of footpad were removed by fer into CMy-mOva mice, effector OT-I cells migrate into the heart scalpel and frozen in Tissue-Tek OCT compound for subsequent cryostat and cause a myocarditis. When this model is used with mutant sectioning. OT-1 cells, it affords the opportunity to assess the contributions of Grading myocarditis specific genetic loci to trafficking and posttrafficking functions that contribute to myocarditis. In the present study, we examined the Myocarditis was graded by microscopic examination of H&E stained sec- tions in a blinded fashion by a trained pathologist after examination of the influence of FucT-VII and FucT-IV/VII deficiencies in effector entire area of three sections, using a 0-to-4 scale as follows: 0, no inflam- OT-I cells on their ability to traffic into and cause damage to CMy- mation; 1, one to five distinct inflammatory foci with total involvement of mOva hearts. The results indicate that selectin-ligand dependent 5% or less of the cross-sectional area; 2, more than five distinct inflam- events contribute significantly to CD8ϩ T cell migration into the matory foci, or involvement of Ͼ5% but Ͻ20% of the cross-sectional area; heart. 3, diffuse inflammation involving over 20–50% of the area; and 4, diffuse inflammation involving Ͼ50% of the area (9). Materials and Methods Immunohistochemistry Mice Immunohistochemistry was performed as described previously (10). All mice used in the current study were bred in the pathogen-free facility Briefly, acetone-fixed cryostat sections of heart or footpad were blocked at the Eugene Braunwald Medical Research Center or the Harvard Medical with 1% BSA in PBS. For E-selectin staining, sections were incubated with Downloaded from ␮ School Warren Alpert Building, in accordance with the guidelines of the 10 g/ml unlabeled anti-E-selectin Ig (BD Pharmingen), followed by PBS committee of Animal research at the Harvard Medical School and the Na- wash, and then biotinylated goat anti- Ig, 1/200 dilution (Jackson Im- tional Institutes of Health animal research guidelines. The CMy-mOva munoResearch Laboratories). For Thy1.2 staining, sections were incubated transgenic line that expresses OVA in the heart (5) was carried on with biotinylated anti-CD90.2 Ab (BD Pharmingen). All sections were both C57BL/6-Thy1.2 (CD90.2) and Thy1.1 (CD90.1) backgrounds, and then blocked with 0.3% hydrogen peroxide/PBS at room temperature, and all experimental animals were heterozygous for the OVA transgene. The then incubated with HRP avidin-biotin complex solutions at 1/100 dilution (Vector Laboratories). Specific Ab binding was detected with 3-amino-9-

OT-I TCR-transgenic mouse strain (6) was provided by W. R. Heath and http://www.jimmunol.org/ F. Carbone (Walter and Eliza Hall Institute of Medical Research, Mel- ethylcarbazole (Vector Laboratories) and counterstained with Gill’s Num- bourne, Australia) and was maintained on a C57BL/6-Thy1.2 (CD90.2) ber 2 hematoxylin solution (Polysciences). ϩ background. The OT-I TCR is expressed on CD8 T cells and is specific Parallel plate flow chamber assays of T cell binding to selectins for the OVA peptide 257–264 (SIINFEKL) bound to the class I MHC molecule H2-Kb (7). Previously derived FucT-VIIϪ/Ϫ and FucT-IVϪ/Ϫ/ T cell adhesion to selectins under flow conditions was analyzed as de- FucT-VIIϪ/Ϫ mice on a C57BL/6 background (8), were cross-bred with scribed previously (11). Briefly, recombinant mouse P-selectin/Fc chimera OT-I mice to generate OT-Iϩ/ϪFucT-VIIϪ/Ϫ and OT-Iϩ/Ϫ FucT-IVϪ/Ϫ/ (20 ␮g/ml) or recombinant mouse E-selectin/Fc chimera (20 ␮g/ml) (R&D Ϫ/Ϫ Ј FucT-VII mice. C57BL/6 mice were purchased from The Jackson Systems) were captured on glass coverslips using goat F(ab )2 anti- Laboratory. Fc Ab as previously detailed (12), and the coverslips were placed in a parallel plate flow chamber. Effector T cells were resuspended in Dulbec- T cell preparations and adoptive transfer experiments co’s PBS containing 0.1% human serum albumin and 20 mM HEPES (pH by guest on September 24, 2021 ϫ 5 T cells were cultured in DMEM (Invitrogen Life Technologies) supple- 7.4), at 37°C (5 10 cells/ml) and were drawn through the flow chamber mented with 10% heat-inactivated FCS (Sigma-Aldrich), 2 mmol/L sodi- at decreasing flow rates for 2.5 min each, i.e., 0.94 ml/min (estimated shear ϭ 2 2 um-pyruvate, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 10 mmol/L stress 1.5 dynes/cm ), 0.78 ml/min (1.0 dynes/cm ), 0.52 ml/min (0.7 dyne/cm2), and 0.26 ml/min (0.4 dyne/cm2). T cell interactions with im- HEPES (Invitrogen Life Technologies). T cell suspensions were prepared ϫ from spleen and lymph nodes of OT-Iϩ/ϪFucT-VIIϪ/Ϫ and OT-Iϩ/Ϫ FucT- mobilized selectins were recorded using a 20 phase contrast objective IVϪ/Ϫ/FucT-VIIϪ/Ϫ mice heterozygous for the OT-I TCR transgene, and and a PC-based videomicroscopy system (Videolab Software; Ed Marcus naive OT-I cells were isolated from the suspensions by CD8 magnetic Laboratories). T cell accumulation was determined after the final minute of beads (Miltenyi Biotec), as described previously (5, 9). The naive T cells each flow rate by counting the number of interacting cells in four different were stimulated in vitro with mitomycin C-treated (Sigma-Aldrich) fields. Specificity of binding was confirmed by adding blocking Abs to the C57BL/6 spleen cell suspensions (APCs) at a T cell to APC ratio of 1:10, perfusate. For this purpose, we used anti-E-selectin Ab (9A9) and anti-P- ␮ selectin Ab (RB40.34), both generously provided by Dr. K. Ley (Univer- and OVA peptide Ag (SIINFEKL) at a final concentration of 1 mol/L. ␮ These cultures were supplemented with 2 ␮g/ml anti-CD28 (clone 37.51; sity of Virginia, Charlottesville, VA), at final concentrations of 10 g/ml. BD Pharmingen), 50 U/ml recombinant mouse IL-2 (R&D Systems), and In vitro analysis of T cell functional phenotype 10 ng/ml recombinant mouse IL-12 (R&D Systems). The cultures were 2 placed in 75-cm flasks and incubated at 37°, 5% CO2. After 3 days of Wild-type or FucT-VII-deficient effector OT-I cells were removed from stimulation, all cultures were diluted 1/1 with fresh medium containing 40 primary activation cultures at day 5, washed and resuspended in RPMI U/ml IL-2 (R&D Systems), and OT-I effectors were harvested for use at 1640 medium, and used for functional assays. Cytotoxic activity of effector day 5. OT-I cells was measured by coculturing the T cells with 51Cr sodium In vitro activated OT-I effector cells were removed from tissue culture, chromate-loaded H-2Kb-expressing EL4 target cells (American Type Cul- washed and resuspended in PBS, and injected i.p. into CMy-mOva mice, as ture Collection) at various E:T cell ratios. Target cells were pulsed with previously described (5, 9), or into C57BL/6 mice immunized 1 h before T SIINFEKL, and released 51Cr in supernatants was detected by gamma cell transfer by footpad injection of 50 ␮g of OVA (Sigma-Aldrich) in 50 counting, as described previously (5). IFN-␥ secretion was measured by ␮l of CFA (Sigma-Aldrich). The number of T cells injected per mouse in culturing 2 ϫ 104 effector T cells with 2 ϫ 105 mitomycin C-treated this study ranged from 0.5 to 2.5 ϫ 106 cells, as indicated. (Sigma-Aldrich) C57BL/6 spleen cells and SIINFEKL (1 ␮g/ml) in 200-␮l microtiter wells. Culture supernatants were harvested at 48 h and IFN-␥ Processing of tissue was detected by ELISA, as described previously (5). At designated times after adoptive transfer, usually 72–96 h, CMy-mOva Serum troponin I (TnI) determination mice received i.p. injection of 2,2,2-tribromoethanol (0.5 mg/kg) to achieve deep plane anesthesia. The chest cavity of each mouse was then opened, Blood was collected from mice at time of sacrifice, and serum fractions the superior vena cava was nicked, and the left ventricle was perfused with were isolated and transferred frozen to the Clinical Chemistry Laboratory cold PBS via a left ventricle apical puncture with an 18° syringe needle. at the Brigham and Women’s Hospital (Boston, MA). Mouse cardiac TnI After exsanguination, the heart was surgically removed, placed in ice-cold was measured using the ADVIA Centaur cTnI assay (Bayer). This auto- RPMI 1640 medium, and cut with a scalpel to yield three contiguous trans- mated sandwich immunoassay uses a primary polyclonal goat anti-TnI Ab verse biventricular sections. The basal section was frozen in Tissue-Tek and a secondary combination of monoclonal mouse anti-Tn Abs. The assay OCT compound (Sakura Finetek) and stored at Ϫ80°C for subsequent im- range is from 0.10 to 50 ␮g/L, sample volume is 100 ␮l, and total impre- munohistochemical staining. The mid-portion was fixed with 10% phos- cision of 3.6% at 43.62 ␮g/L and 6.6% at 0.94 ␮g/L. As indicated below, 6570 SELECTIN-LIGAND-DEPENDENT CTL MIGRATION INTO HEART we found that the ADVIA Centaur human cTnI immunoassay can detect mouse TnI. To verify the utility of this method for a semiquantitative assessment of myocardial damage in mice, we performed serial (up to 100 times) dilutions of mouse specimens with high TnI levels and detected a proportional decrease in signal with decreasing mouse TnI concentrations. analyses All cell preparations were washed twice in staining buffer (Dulbecco’s PBS with 1% BSA). For phenotypic analysis of surface markers, 0.5 ϫ 106 cells were suspended in 100 ␮l of staining buffer containing 1 ␮g each of spe- cific Ab or matched species/isotype control Ig, purchased from BD Pharm- ingen, and incubated on ice for 20 min followed by washing and fixation with 0.5% paraformaldehyde. Stained cell preparations were then analyzed FIGURE 1. Equivalent effector phenotype of FucT-VII-deficient and by flow cytometry using a FACSCalibur instrument and CellQuest soft- wild-type CD8ϩ T cells. A, CTL activity against SIINFEKL (Ag)-pulsed ware (BD Biosciences). Abs used for flow cytometry were as follows: and unpulsed EL4 target cells was measured by 51Cr release. The data are PE-conjugated anti-mouse CD90.1/Thy1.1 (clone OX-7); FITC-conjugated from one experiment of two performed with similar results. B, IFN-␥ se- anti-mouse CD44 (clone IM7); FITC-conjugated anti-mouse CD25 (IL-2R ϩ ␣-chain) (clone 7D4); FITC-conjugated LFA-1 (clone M17/4); PE-conju- cretion by Ag-stimulated FucT-VII-deficient and wild-type CD8 T cells gated L-selectin (MEL14); FITC-conjugated anti-P-selectin glycoprotein was measured by ELISA after 48 h. The data represent mean Ϯ SD of ligand-1/CD162 (clone 2PH1), and FITC-conjugated anti-VLA4 (clone triplicate determinations from one experiment of three performed with sim- 9C10). ilar results.

Statistical analysis Downloaded from Statistical analyses were performed using the Mann-Whitney U test for data that was not normally distributed, as determined by the Kruskal-Wallis test, and the Students t test for normally distributed data. A value of p Ͻ hesion. The residual E-selectin adhesion could be due to a contri- 0.05 was considered to be significant. bution from FucT-IV because FucT-IV/VII-deficient OT-I cells do not bind E-selectin under shear flow conditions (shown in later Results experiment). FucT-VII deficiency did not influence binding of http://www.jimmunol.org/ Ϫ Ϫ FucT-VII / deficiency does not impair activation or effector OT-I cells to VCAM-1 under identical flow conditions (Fig. 2C). ϩ function of CD8 T cell in vitro These results indicate that the binding of FucT-VII-deficient T The number of CD8ϩ T cells isolated from lymph nodes and cells to E-selectin is significantly reduced, relative to wild-type spleen of FucT-VIIϪ/Ϫ OT-I mice were roughly equivalent to the cells, whereas binding to P-selectin is nearly abolished. As a con- yields from wild-type OT-I mice. This finding is in contrast to a trol, FucT-VII-deficient T cell adhesion to VCAM-1, a VLA-4- decrease in peripheral node CD8ϩ T cells recovered from periph- dependent interaction, was robust and similar to that of wild-type eral nodes of non-TCR-transgenic FucT-VIIϪ/Ϫ mice, as reported OT-I cells. previously (4). This apparent discrepancy can be explained by the ϩ

ϩ FucT-VII deficiency reduces CD8 T cell migration into the by guest on September 24, 2021 fact that in the present study, most of the CD8 T cells are derived skin from the spleen, and there is no evidence that selectin ligands are required for naive T cell migration into the spleen (13). The num- FucT-VII-dependent selectin ligand expression is required for both ϩ ϩ ber of FucT-VIIϪ/Ϫ and wild-type OT-I cells recovered after 5 CD4 and CD8 T cell homing to skin (4, 8, 14). We used a days of Ag stimulation in vitro, was also approximately the same. footpad immunization protocol to ensure that the FucT-VII-defi- Analysis of the expression of CD25, CD44, and L-selectin by cient OT-I cells are also defective in selectin-dependent homing in FACS did not differ significantly between the FucT-VIIϪ/Ϫ and vivo. Significantly more adoptively transferred wild-type OT-I wild-type OT-I cells recovered 5 days after Ag stimulation in vitro cells were present in OVA-immunized footpads compared with (data not shown). These findings indicate FucT-VII-deficient T footpads injected with CFA alone (Fig. 3), indicating that the pres- cells retain a normal capacity to be activated by Ag and to undergo ence of the cognate Ag in the tissues led to enhanced recruitment proliferative responses. FucT-VIIϪ/Ϫ and wild-type OT-I cells ex- and/or enhanced local proliferation of the T cells. However, there pressed equivalent levels of LFA-1, VLA-4, and P-selectin glyco- were significantly fewer FucT-VII-deficient OT-I cells than wild- protein ligand-1 (data not shown). type OT-I cells in sections of footpad 3 days after immunization FucT-VII deficiency did not influence the effector function of with OVA (Fig. 3). These results are consistent with impaired activated OT-I cells. Cytotoxic function of FucT-VIIϪ/Ϫ and wild- migration of the FucT-VII-deficient T cells into the skin. type OT-I cells were equivalent (Fig. 1A). The amount of IFN-␥ Ϫ Ϫ FucT-VII deficiency reduces the ability of adoptively transferred produced by Ag activation of FucT-VII / and wild-type OT-I CD8ϩ T cells to cause myocarditis cells was not significantly different (Fig. 1B).

ϩ We then asked whether T cell interactions with endothelial selec- FucT-VII deficiency reduces CD8 T cell interactions with E- tins contribute to entry of effector CD8ϩ T cells into the heart in and P-selectin the CMy-mOva model of myocarditis, and thereby contribute to The relative abilities of wild-type and FucT-VII-deficient T cells to myocardial histopathology. We examined histological sections of interact with selectins was compared using a flow chamber assay CMy-mOva hearts after adoptive transfer of OT-I effectors, and we and immobilized recombinant P- and E-selectin. FucT-VII-defi- scored the degree of myocarditis. The mean histological myocar- cient OT-I cell binding to P-selectin was negligible at all shear ditis score at 96 h after transfer of 2.5 ϫ 106 FucT-VII-deficient stresses tested, whereas wild-type OT-I cell binding was readily OT-I cells was 56% lower than after transfer of the same number detected (Fig. 2A). FucT-VII deficiency resulted in an ϳ50% re- of wild-type OT-I cells (Fig. 4A). Because the recipient CMy- duction in OT-I cell binding to E-selectin, compared with wild- mOva mice have no ongoing inflammatory processes in the heart type OT-I cells (Fig. 2B). The residual binding of FucT-VII-defi- at the time of T cell transfer, we reasoned that selectin ligand cient OT-I cells was eliminated by a function blocking mAb to expression is not likely to influence the earliest recruitment of T E-selectin (data not shown), demonstrating the specificity of ad- cells. To better discern differences in the migration of selectin The Journal of Immunology 6571

FIGURE 2. Impaired interaction of FucT-VII-deficient CD8ϩ T cells with E- and P-selectin under flow. The data represent the accumulation of wild-type (WT) and FucT-VII-deficient (FucT-VII KO) T cells on glass coverslips coated with recombinant P-selectin Ig (A), E-selectin Ig (B) or VCAM-1 (C) under the indicated shear stresses. The data represent the mean Ϯ SD values from three separate experiments, with triplicate determinations in each experiment. Downloaded from ligand-deficient and wild-type OT-I cells into the heart, we pre- FucT-VII deficiency impairs the ability of adoptively transferred treated mice with LPS (75 ng/gm) i.p. 4 h before transfer of T cells. CD8ϩ T cells to enter the myocardium The mean histological myocarditis score 96 h after transfer of Although the histological analysis of myocarditis is consistent ϫ 6 2.5 10 wild-type OT-I cells into LPS-treated CMy-mOva mice with reduced migration of FucT-VII OT-I cells into the heart was 184% higher than the mean histological score after transfer of compared with wild-type T cells, the histological score reflects http://www.jimmunol.org/ the same number of cells into untreated CMy-mOva mice (Fig. 4), the degree of inflammatory infiltrate, irrespective of cell type, indicating that LPS treatment did enhance the OT-I-induced in- including endogenous T cells and neutrophils. The degree of flammatory response in the heart. The histological score after secondary inflammation that may occur in response to OT-I transfer of FucT-VII-deficient OT-I cells into LPS-pretreated infiltration may not be linearly related to the number of T cells CMy-mOva mice was 46% lower than after transfer of the same that enter the heart, and therefore differences in myocarditis number of wild-type OT-I cells into LPS-treated animals (Fig. 4B). score may not be a sensitive indicator of differences in T cell Thus the FucT-VII dependency of the OT-I-mediated inflamma- migration. To more specifically assess whether FucT-VII defi- tory response was moderately reduced in the presence of LPS pre- ciency results in less migration of OT-I cells into the heart, we treatment. The reduced pathogenicity of FucT-VII-deficient T cells adoptively transferred Thy1.2 (CD90.2) expressing wild-type or by guest on September 24, 2021 suggests that selectin ligands are expressed on heart endothelial FucT-VII-deficient OT-I cells into Thy1.1 (CD90.1) homozy- cells during OT-I-mediated myocarditis. We confirmed this to be gous CMy-mOva mice, and used immunohistochemistry to the case by immunohistochemical detection of E-selectin in heart quantify the number of Thy1.2 OT-I cells in myocardial sec- sections from mice with ongoing OT-I-mediated myocarditis (Fig. tions at 96 h after transfer. This time point was chosen because 5). We could not detect E- and/or P-selectin expression by immu- nohistochemistry in hearts of CMy-mOva mice that did not receive OT-I transfers (data not shown).

FIGURE 4. FucT-VII deficiency reduces ability of CD8ϩ T cells to FIGURE 3. Reduced migration of FucT-VII-deficient CD8ϩ T cells cause myocarditis. Mice were sacrificed and hearts processed for histologic into dermal site of injection of Ag and adjuvant. Thy1.2ϩ/ϩ C57BL/6 mice analysis 96 h after transfer of T cells. A, A total of 2.5 ϫ 106 wild-type were injected in one foot pad with CFA with our without OVA (50 ␮g/ (WT) or FucT-VII-deficient (FucT-VII KO) OT-I cells were transferred mouse), followed 5 h later by i.p. injection of 5 ϫ 106 wild-type (WT) or into untreated CMy-mOva hosts. B, A total of 2.5 ϫ 106 wild-type or FucT-VII-deficient (FucT-VII KO) OT-I Thy1.1ϩ/ϩ T cells. Three sections FucT-VII-deficient OT-I cells transferred into mice pretreated with LPS were examined per mouse, and three mice were included in each group. (75 ng/mouse, i.p.), 4 h before T cell transfer. Each data point represents Each data point represents the mean number of Thy1.1ϩ T cells per footpad the mean histological score of triplicate sections from a single mouse, and section in each mouse. The horizontal lines represent the medians of each the horizontal bars represent the median scores for each group. The dif- group. The differences in medians of OVA-treated wild-type and FucT- ferences between medians of wild-type and FucT-VII-deficient groups in VII-deficient mice were significant (p Ͻ 0.05). both experiments were significant (p Ͻ 0.05). 6572 SELECTIN-LIGAND-DEPENDENT CTL MIGRATION INTO HEART Downloaded from

FIGURE 6. Reduced migration of FucT-VII-deficient CD8ϩ T cells into heart. Thy1.1ϩ/ϩ CMy-mOva mice received 2.5 ϫ 106 or 1.0 ϫ 106 wild-type (WT) or FucT-VII-deficient (FucT-VII KO) OT-I Thy1.2ϩ/ϩ T

cells, hearts were removed from euthanized animals 96 h later, and frozen http://www.jimmunol.org/ sections were stained for Thy1.2-positive cells. Representative stained sec- tions of hearts from CMy-mOva mice receiving 2.5 ϫ 106 wild-type (WT) OT-I (A) or FucT-VII-deficient (FucT-VII KO) T cells (B) are shown. Four FIGURE 5. E-selectin expression in inflamed CMy-mOVa hearts. Low fields were counted per section and three sections were counted per heart, (A) and high (B) power photomicrographs of frozen sections stained for to derive a mean value per field for each heart, represented by each data E-selectin are shown. Tissue was harvested 96 h after transfer of 2.5 ϫ 106 point (C). The horizontal lines in C represents the median values for each OT-I effector cells. group. The differences between the median of wild-type and FucT-VII- deficient groups were significant (p Ͻ 0.05). by guest on September 24, 2021 previous work with our model indicates that transferred wild- type OT-I effectors are first detectable in a mediastinal draining data, and indicate that functional selectin ligands play an important lymph node, but they have left this node and are readily role in the migration of the effector CD8ϩ T cells into detectable in the heart by 96 h (5). The analysis revealed myocardium. that FucT-VII deficiency caused a 98% reduction in T cell mi- gration into the heart when 1.0 ϫ 106 cells were transferred, and Reduced pathogenicity of FucT-IV/VII-deficient T cells an 88% reduction when 2.5 ϫ 106 T cells were transferred The residual capacity of FucT-VII-deficient OT-I cells to enter (Fig. 6). the heart and cause myocarditis may reflect selectin-indepen- dent migration and/or residual selectin ligand synthesis depen- Impaired migration of FucT-VII-deficient T cells into the heart dent on FucT-IV. Although previous studies have reported that results in reduced myocyte damage only a small percentage of the normal amount selectin binding The data discussed so far indicate there is still a significant histo- activity is retained in T cells from FucT-VII null mice, our in logically identifiable inflammatory response in the CMy-mOva vitro flow studies with FucT-VII-deficient OT-I cells (see Fig. hearts after selectin ligand-deficient CD8ϩ T cell transfer, even 2) indicate significant residual capacity to bind E-selectin. To though the actual number of these T cells that enter the heart is address this issue, we performed experiments with combined only a small percent of the number of normal CD8ϩ T cells that FucT-IV/VIIϪ/Ϫ OT-I mice. These mice are more difficult to enter the heart under identical conditions. OT-I effector T cells breed, limiting the number of experiments that could be per- have direct cytotoxic activity against OVA expressing cells, such formed. In vitro flow chamber experiments indicated that al- as the cardiac myocytes in CMy-mOva mice. We decided to mea- most all P- and E-selectin binding activity seen in normal OT-I sure myocyte damage as another comparative indicator of the abil- effector cells was lost in FucT-IV/VIIϪ/Ϫ OT-I effector cells ity of selectin ligand-deficient and normal T cells to leave the (Fig. 8). The almost complete loss of E-selectin binding by circulation, enter myocardium and thereby promote myocardial pa- FucT-IV/VIIϪ/Ϫ OT-I cells is in contrast to the significant re- thology. Previously we have shown that serum levels of cardiac sidual binding by FucT-VIIϪ/Ϫ OT-I effector cells, shown in TnT are a sensitive and specific indicator of myocyte damage in Fig. 2. However, the pathologic consequences of adoptive the CMy-mOva model (5). In this study, we adapted a clinical transfer of FucT-IV/VIIϪ/Ϫ OT-I cell into CMy-mOva mice laboratory assay for cardiac TnI to follow mouse heart damage. were similar to those described above after transfer of FucT- Significantly elevated TnI levels were detectable 96 h after T cell VIIϪ/Ϫ OT-I. In particular, the mean myocardial inflammation transfer of normal OT-I cells into CMy-mOva mice, and the levels score after FucT-IV/VIIϪ/Ϫ OT-I transfer was ϳ50% less than were 88% reduced in mice receiving FucT-VII-deficient T cells after wild-type OT-I transfer (Fig. 9A). Serum TnI levels after (Fig. 7). These data are consistent with the immunohistochemical FucT-IV/VIIϪ/Ϫ OT-I transfer were 84% less than after transfer The Journal of Immunology 6573

FIGURE 9. Reduced myocarditis and myocyte damage induced by FucT-IV/VII-deficient T cells. CMy-mOva mice were sacrificed 96 h post- transfer of 2.5 ϫ 106 wild-type (WT) or FucT-IV/VII-deficient (FucT-IV/ VII KO) OT-I cells. Histological grade of myocarditis (A), and serum TnI FIGURE 7. Reduced myocyte damage induced by FucT-VII-deficient levels (B) were determined. Each data point represents the mean value from ϩ CD8 T cells. Blood samples were taken from CMy-mOva mice 96 h after a single mouse, as described in previous experiments. The horizontal line transfer of 2.5 ϫ 106 wild-type (WT) or FucT-VII-deficient (FucT-VII KO) represents median values for each group. The difference between median Downloaded from OT-I cells, and serum TnI was determined. The data represent the mean Ϯ values in A, but not B, is significant (p Ͻ 0.05). SEM TnI concentration from 10 animals per group. The mean values were significantly different (p Ͻ 0.01). genesis of cardiac allograft rejection and both viral and auto- immune myocarditis. Although E-selectin likely plays an im- of wild-type OT-I cells (although the difference was not statis- portant role in the acute inflammatory response associated with tically significant). These results confirm the important role that ischemia reperfusion injury in the heart (15), there is little pub- http://www.jimmunol.org/ fucosyltransferase-dependent selectin ligand synthesis plays in lished information about endothelial selectins in T cell-medi- ϩ migration of CD8 migration into the heart, but they do not ated cardiac inflammation. E-selectin is reported to be up-reg- support the hypothesis that FucT-IV expression accounts for ulated in murine T cell mediated myocarditis (16). FucT-VII residual cardiac migratory capacity seen in the FucT-VII-defi- deficiency in murine recipients of cardiac allograft prolonged ϩ cient CD8 T cells. A caveat in the interpretation of these re- survival of the grafts (17), and a selectin ligand mimic has been sults is the limited number of mice studied, leaving the possi- shown to reduce the inflammatory response in rat cardiac allo- bility that the double knockout OT-I cells may be less graft (18). These studies did not distinguish the importance of pathogenic than the single knockout cells. T cell selectin ligands from the ligands on other leukocytes. by guest on September 24, 2021 We are not aware of any previous studies specifically Discussion addressing selectin-dependent migration of CD8ϩ T cells into In this study, we have observed that functional selectin ligands the heart. contribute significantly to the migration of cardiac Ag specific Previous work has clearly demonstrated that endothelial se- CD8ϩ effector T cells into the heart. In particular, abrogation of lectins and/or their leukocyte ligands are required for the mi- fucosyltransferase-dependent E- and P-selectin binding activity gration of effector CD4ϩ and CD8ϩ T cells of the Th1/Tc1 markedly reduced the capacity of CD8ϩ effector T cells to enter phenotype into dermal inflammatory sites (19–25) and experi- the heart and cause myocyte damage. Our model of CD8ϩ T mentally induced peritonitis (26). However, the importance of cell-mediated myocarditis was developed to study migration endothelial selectins and their ligands for CD4ϩ and CD8ϩ T and effector functions of T cells specific for an Ag expressed cell migration into tissues other than skin has been questioned. exclusively in the heart, and is therefore relevant to the patho- For example, CD4ϩ T cell-dependent inflammatory response in

FIGURE 8. Impaired interaction of FucT-IV/VII-deficient CD8ϩ T cells with E- and P-selectin under flow. The data represent the accumulation of wild-type (WT) and FucT-IV/II-deficient (FucT-IV/VII KO) T cells on glass coverslips coated with recombinant P-selectin Ig (A), E-selectin Ig (B)or VCAM-1 (C) under the indicated shear stresses. The data represent the mean Ϯ SD accumulation on three separate coverslips per group. 6574 SELECTIN-LIGAND-DEPENDENT CTL MIGRATION INTO HEART the brain in experimental autoimmune encephalitis appears in- cell-mediated cardiac inflammatory processes, such as allograft tact in the absence of endothelial selectins (27). Furthermore, rejection. CD8ϩ T cell-dependent clearance of lymphocytic choriomen- ingitis virus from several tissues, including liver, lung, and Acknowledgments spleen is not impaired in E-/P-selectin null mice (28). Similarly, We thank Helena Skalsky-Stossel (Brigham and Women’s Hospital Clin- CD8ϩ-dependent clearance of lymphocytic choriomeningitis ical Laboratories), for performing serum troponin assays on mouse serum virus or vesicular stomatitis virus from several organs, includ- samples and Hong Pang for excellent technical assistance. ing brain, ovary, liver, lung, and spleen, was equivalent in Disclosures FucT-VII-deficient and wild-type mice (14). Our results do not The authors have no financial conflict of interest. contradict these previous studies because they did not address cardiac inflammation. We believe that regulation of inflamma- References tory responses in the heart likely differs from other tissues, 1. Binah, O. 2002. Cytotoxic lymphocytes and cardiac electrophysiology. J. Mol. especially those tissues in which exposure to environmental Cell. Cardiol. 34: 1147–1161. 2. Ley, K., and G. S. Kansas. 2004. Selectins in T-cell recruitment to non-lymphoid pathogens is a normal and frequent occurrence (29). Further- tissues and sites of inflammation. Nat. Rev. Immunol. 4: 325–335. more our disease model, in which effector T cells are transferred 3. Knibbs, R. N., R. A. Craig, P. Maly´, P. L. Smith, F. M. Wolber, N. E. Faulkner, into quiescent CMy-mOva mice, differs significantly from the J. B. Lowe, and L. M. Stoolman. 1998. ␣(1,3)-Fucosyltransferase VII-dependent synthesis of P- and E-selectin ligands on cultured T lymphoblasts. J. Immunol. situation of a systemic viral infection, in which strong innate 161: 6305–6315. immune responses with accompanying inflammation are likely 4. Smithson, G., C. E. Rogers, P. L. Smith, E. P. Scheidegger, B. Petryniak, J. T. Myers, D. S. L. Kim, J. W. Homeister, and J. B. Lowe. 2001. Fuc-TVII is Downloaded from to generate multiple, redundant molecular signals for T cell required for T helper 1 and T cytotoxic 1 lymphocyte selectin ligand expression recruitment. and recruitment in inflammation, and together with Fuc-TIV regulates naive T Although our data demonstrate selectin-dependent migration of cell trafficking to lymph nodes. J. Exp. Med. 194: 601–614. 5. Grabie, N., M. W. Delfs, J. R. Westrich, V. A. Love, G. Stavrakis, F. Ahmad, T cells into the heart, it is unclear whether the earliest T cell mi- C. E. Seidman, J. G. Seidman, and A. H. Lichtman. 2003. IL-12 is required for gration into the myocardium is selectin-dependent in our model. differentiation of pathogenic CD8ϩ T cell effectors that cause myocarditis. J. Clin. Invest. 111: 671–680. Morphologic and analyses indicate that in the ab- 6. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, and http://www.jimmunol.org/ sence of OT-I transfer, CMy-mOva hearts are not inflamed (5, 9), F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. and endothelial selectin expression is not detectable by immuno- Cell 76: 17–27. 7. Carbone, F. R., and M. J. Bevan. 1989. Induction of ovalbumin-specific cytotoxic histochemistry before OT-I transfer. Nonetheless, early T cell im- T cells by in vivo peptide immunization. J. Exp. Med. 169: 603–612. migrants rapidly trigger an inflammatory response. Induced endo- 8. Homeister, J. W., A. D. Thall, B. Petryniak, P. Maly´, C. E. Rogers, P. L. Smith, R. J. Kelly, K. M. Gersten, S. W. Askari, G. Cheng, et al. 2001. The ␣(1,3)fucosyl- thelial selectin expression clearly enhances the recruitment of OT-I transferases FucT-IV and FucT-VII exert collaborative control over selectin-depen- cells, as they are likely to enhance recruitment of endogenous leu- dent leukocyte recruitment and lymphocyte homing. Immunity 15: 115–126. kocytes, including neutrophils. It remains possible that the unin- 9. Grabie, N., D. T. Hsieh, C. Buono, J. R. Westrich, J. A. Allen, H. Pang, G. Stavrakis, and A. H. Lichtman. 2003. Neutrophils sustain pathogenic CD8ϩ T flamed myocardial microvasculature may express low basal levels cell responses in the heart. Am. J. Pathol. 163: 2413–2420.

of endothelial E- and P-selectin, which are below the detection 10. Buono, C., C. E. Come, G. Stavrakis, G. F. Maguire, P. W. Connelly, and by guest on September 24, 2021 ␥ limit of immunohistochemistry, but nonetheless contribute to early A. H. Lichtman. 2003. Influence of interferon- on the extent and phenotype of diet-induced atherosclerosis in the LDLR-deficient mouse. Arterioscler. Thromb. T cell recruitment. Basal expression of E-selectin has been de- Vasc. Biol. 23: 454–460. tected in dermal microvessels (30), and this reflects the impor- 11. Lim, Y. C., H. Xie, C. E. Come, S. I. Alexander, M. J. Grusby, A. H. Lichtman, and F. W. Luscinskas. 2001. IL-12, STAT4-dependent up-regulation of CD4ϩ T tance of the skin in immune surveillance. In contrast, immune cell core 2 ␤-1,6-N-acetylglucosaminyltransferase, an enzyme essential for bio- surveillance is not likely to be an adaptive function that has synthesis of P-selectin ligands. J. Immunol. 167: 4476–4484. evolved in the heart (29), and therefore constitutive expression 12. Lim, Y. C., L. Henault, A. J. Wagers, G. S. Kansas, F. W. Luscinskas, and A. H. Lichtman. 1999. Expression of functional selectin ligands on Th cells is of selectins on myocardial microvascular endothelial cells differentially regulated by IL-12 and IL-4. J. Immunol. 162: 3193–3201. would not be expected. 13. Nolte, M. A., A. Hamann, G. Kraal, and R. E. Mebius. 2002. The strict regulation of lymphocyte migration to splenic white pulp does not involve common homing Previous studies suggest that L-selectin ligand on endothelial receptors. Immunology 106: 299–307. cells mediates recruitment of T cells into human and rat cardiac 14. Erdmann, I., E. P. Scheidegger, F. K. Koch, L. Heinzerling, B. Odermatt, G. Burg, J. B. Lowe, and T. M. Ku¨ndig. 2002. Fucosyltransferase VII-deficient allografts (31, 32). In our adoptive transfer model, FucT-VII ϩ mice with defective E-, P-, and L-selectin ligands show impaired CD4 and deficiency is limited to the transferred T cells, and L-selectin CD8ϩ T cell migration into the skin, but normal extravasation into visceral or- ligand expression by endothelial cells is therefore not altered. gans. J. Immunol. 168: 2139–2146. 15. Lefer, A. M. 1995. Role of selectins in myocardial ischemia-reperfusion injury. Because we see marked reduction of FucT-VII-deficient OT-I Ann. Thorac. Surg. 60: 773–777. cells into skin and heart, it appears that these T cells rely more 16. Wang, Y., M. Afanasyeva, S. L. Hill, and N. R. Rose. 1999. Characterization of on endothelial selectins for recruitment rather than on endothe- murine autoimmune myocarditis induced by self and foreign cardiac myosin. Autoimmunity 31: 151–162. lial L-selectin ligand binding to T cell L-selectin. In fact, there 17. La´cha, J., A. Bushell, K. Smetana, P. Rossmann, P. Pøibylova´, K. Wood, and is little L-selectin expression on the effector OT-I cells that we P. Maly´. 2002. Intercellular cell adhesion molecule-1 and selectin ligands in acute cardiac allograft rejection: a study on gene-deficient mouse models. J. Leukocyte transfer. It is possible that the kinetics of clearance of the adop- Biol. 71: 311–318. tively transferred T cells from the circulation, relative to the 18. Renkonen, R., M. N. Fukuda, L. Petrov, T. Paavonen, J. Renkonen, P. Hayry, and timing of endothelial L-selectin expression, in our model does M. Fukuda. 2002. A peptide mimic of selectin ligands abolishes in vivo inflam- mation but has no effect on the rat heart allograft survival1. Transplantation not recapitulate what happens during a T cell response that is 74: 2–6. endogenously initiated. 19. Austrup, F., D. Vestweber, E. Borges, M. Lo¨hning, R. Bra¨uer, U. Herz, H. Renz, ϩ R. Hallmann, A. Scheffold, A. Radbruch, and A. Hamann. 1997. P- and E-selectin In summary, our data demonstrate that effector CD8 T cells mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues. specific for a cardiac Ag use selectin ligand interactions with car- Nature 385: 81–83. diac endothelial selectins as part of the process of migration into 20. Bullard, D., E. Kunkel, H. Kubo, M. Hicks, I. Lorenzo, N. Doyle, C. Doerschuk, K. Ley, and A. Beaudet. 1996. Infectious susceptibility and severe deficiency of myocardium. The requirement of selectin-selectin ligand interac- leukocyte rolling and recruitment in E-selectin and P-selectin double mutant tion is likely to limit potentially dangerous entry of activated T mice. J. Exp. Med. 183: 2329–2336. 21. Frenette, P. S., T. N. Mayadas, H. Rayburn, R. O. Hynes, and D. D. Wagner. cells into uninflamed myocardium. Based on these results, it is 1996. Susceptibility to infection and altered hematopoiesis in mice deficient in reasonable to consider selectin blockade for the prevention of T both P- and E-selectins. Cell 84: 563–574. The Journal of Immunology 6575

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