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Dynamic Expression of L-Selectin in Cell-to-Cell Interactions between Neutrophils and Endothelial Cellsin Vitro
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
243, 87–93 (1998)
EX984139
Dynamic Expression of L-Selectin in Cell-to-Cell Interactions between Neutrophils and Endothelial Cells in Vitro Q. Liu,1 T. K. Kishimoto,* E. Mainolfi,* R. P. Deleon,* C. Myers,* and R. C. Moretz* Department of Biology, University of Minnesota, Duluth, Minnesota 55812; and *Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368
as migration in a chemotactic gradient has been elucidated [4–7, 28]. Polarized redistribution of cell adhesion molecules in neutrophil response to increasing concentration of chemotactic compound has been suggested [5]. Sullivan et al. [8] further demonstrated neutrophil morphological polarity correlated with polar motion, which may be due to an asymmetric distribution of chemotactic receptors. However, cell adhesion and molecule expression during leukocyte–endothelial cell interactions are still not well defined. The sequential interaction of selectin– carbohydrate, chemoattractant–receptor, and integrin–immunoglobulin family molecules has been proposed in the multiple-step model of neutrophil emigration [1–3, 9 –14]. The L-selectin receptor in part mediates the rolling and initial adherence of neutrophils to endothelial cells [12, 14], while Mac-1 (CD11b/CD18 integrin) strengthens this initial adherence and also facilitates migration of neutrophils through endothelial cells. The role of L-selectin in leukocyte rolling and adhesion has recently been explored both in vitro and in vivo [13, 15, 16]. The spatial distribution of L-selectin molecules on isolated human neutrophils has been characterized [15, 17, 18] and human lymphocytes [19]. Recently, L-selectin distribution in human leukocytes has been quantified using electron microscopy with immunogold labeling [20]. L-selectin is distributed on the tips of microvilli of neutrophils; furthermore L-selectin is rapidly cleaved in response to chemotactic factors. Whether L-selectin is redistributed during neutrophil cell adherence is unknown. In our study, the in situ expression of L-selectin during cell-to-cell interactions between neutrophils and endothelial cells has been examined by confocal microscopic immunofluorescence. Our results demonstrate dynamic L-selectin redistribution at both 2-D and 3-D levels which is associated with neutrophil shape changes during contact with stimulated endothelial cells.
Neutrophil– endothelial cell interactions are regulated by cell adhesion molecules and their cognate ligands. It has been proposed that L-selectin and Mac-1 (CD11b/CD18), two neutrophil adhesion receptors, have sequential roles in neutrophil extravasation during inflammation. In this model, L-selectin mediates rolling and initial adherence of neutrophils to endothelial cells, while Mac-1 strengthens this initial adherence and also facilitates migration of neutrophils through endothelial cells. L-selectin and Mac-1 expression are known to be inversely regulated. Here an in vitro culture system has been developed to investigate in situ expression of L-selectin during cellto-cell interactions between neutrophils and endothelial cell monolayers by confocal immunofluorescence analysis. Neutrophils underwent profound cell shape change from round to polarized cell morphology with pseudopod formation after 5 to 15 min coculture with IL-1-stimulated human endothelial cells. L-selectin was redistributed to the pseudopod of the polarized neutrophils in correlation with such cellular changes. During initial cell attachment, neutrophils bound to IL-1-stimulated endothelial cells expressed a high level of L-selectin in a polarized pattern. L-selectin expression decreased over time during neutrophil– endothelial cell interactions. © 1998 Academic Press
INTRODUCTION
Neutrophil–endothelial interactions during inflammation are a coordinated multistep process involving the function of adhesion receptors and chemokines [1–3]. Neutrophils are the predominant cell type accumulating at sites of acute inflammation. Extravasation during acute inflammation includes adhesive interaction of neutrophils with vascular endothelium and transmigration of the neutrophils into the inflamed tissue. The influence of cell interactions on aspects of neutrophil motility associated with cell polarity and nuclear orientation as well
MATERIAL AND METHODS
1 To whom correspondence and reprint requests should be addressed. Fax: (218) 726-8142. E-mail: [email protected].
Cell preparations. Human blood samples from healthy adults were collected, dextran-sedimented, and centrifuged over Ficoll– 87
0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Phase-contrast images showing adherence of human PMN to HUVEC monolayers with or without IL-1 stimulation at 5 or 15 min. Bar, 15 mm. (A and B) Note that human PMN cells maintained round shapes after 5 (A) or 15 (B) min contact with nonstimulated HUVEC monolayers. (C) PMN cells showed an oval shape (circles mark two examples of the PMN cells) after 5 min contact with stimulated HUVEC monolayers. Some also displayed pseudopod-bearing shapes indicating initial cell migration. (D) Pseudopod-bearing shape (circles mark two examples of the PMN cells) occurred in most PMNs after 15 min contact with stimulated HUVEC monolayers, indicating progression of cell adhesion to migration.
Hypaque gradients according to methods from Smith et al. [4]. Isolated neutrophils were suspended in Dulbecco’s phosphate-buffered saline (PBS). Human umbilical vein endothelial cells (HUVEC) were obtained from Cell Systems Corp. (Kirkland, WA) and cultured in Falcon T-75 flasks containing MCDB131 supplied by Clonetics Corp. and a RPMI 1640-based growth medium (containing 10% fetal calf
serum, antibiotics, heparin, 0.1 mg/ml, and endothelial growth factor, 0.05 mg/ml) for 3– 4 days in a humidified incubator at 37°C and 5% CO2. At confluence, cells were passaged by brief trypsinization. Tissue culture chamber glass slides and coverslips (Nunc, Naperville, IL) were coated with 0.1% gelatin. Trypsinized cells (106 cells/ml) were then seeded into each chamber and cultured for 3 days.
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FIG. 2. Neutrophil cell shape changes (%) in time-course experiments (5 and 15 min) for the cell-adhesion assay in stimulated HUVEC monolayers.
Cell adherence assay. Cultured HUVEC monolayers were stimulated with IL-1 (1 ng/ml) for 4 h. A visual cell adherence assay was performed using the method developed by Smith et al. [24, 25]. Briefly, neutrophils suspended in PBS (106 cells/ml) at 4°C were pipetted onto the stimulated HUVEC monolayers in each culture chamber. After neutrophils settled onto the monolayers for 8 min, the culture chamber and coverslips were then inverted for 8 min and washed with PBS. Only those adherent cells that remained adherent to the monolayers were followed by phase-contrast microscopy. Neutrophils in contact with the monolayer at 5 min were fixed in 1% paraformaldehyde in PBS for analysis of initial adherence. Cells in additional chambers were fixed when a large number of neutrophils became flattened and had undergone major polarized shape changes after 15 min in contact with the stimulated monolayer. Neutrophils in contact with unstimulated HUVEC monolayers for 5 or 15 min were also fixed and used as controls for the cell-adherence assay. Neutrophil cell shape changes were used as morphological markers for initial adherence and its progression to migration. Such cell shape changes were visualized by an inverted microscope and phasecontrast optics [14]. Briefly, cells with spherical shape were scored as round; cells with oval to elongated shape were scored as elongated; neutrophil cells forming a leading edge for migration were identified as polarized. At least three samples of in vitro culture slides and coverslips and 100 cells for each sample were scored for cellular changes after neutrophil contact with stimulated HUVEC monolayers for 5 or 15 min. Morphological changes of neutrophils (round, elongated, and polarized cells) were measured as the percentage of the total cells in each culture slide. Dual in vitro labeling of neutrophils and endothelial cells. In our cell-adherence assay, coordinated in vitro labeling of neutrophils with fluorescein-conjugated anti-L-selectin antibody (Pharmingen, San Diego, CA) and endothelial cells with Cell Tracker orange dye (Molecular Probe, Inc., Eugene, OR) were used. To label endothelial cells in vitro, prewarmed (37°C) 0.1 mM Cell Tracker orange dyecontaining medium was added to each culture chamber after removal of the culture medium according to the manufacturer’s instructions. After incubation of labeled HUVEC under culture conditions for 30 min with the dye-containing medium, fresh prewarmed dye-free medium was added to replace the culture medium, and the cells were cultured under the same conditions for another 30 min. Direct labeling of neutrophils was accomplished by staining isolated PMNs with fluorescein-conjugated anti-L-selectin antibody (DREG-56). The PMNs were incubated at 4°C for at least 15 min with gentle shaking, washed with 2% bovine serum albumin in PBS without azide at least two times, and then added to each HUVEC culture chamber for the cell-adherence assay (1 3 106 PMNs/ml). After 2, 5, 15, 30, and 60 min of cell contact, nonadherent cells were
removed by gentle washings of each chamber with PBS, and the adherent PMNs and HUVEC were fixed with 1% paraformaldehyde in PBS. The nuclei of neutrophils were used as reference markers for the relative cellular location [28] of L-selectin redistribution. After fixation, propidium iodide (PI) was used to stain PMN nuclei without affecting L-selectin redistribution prior to confocal analysis. Fluorescein-conjugated isotype control mouse IgG1 was used as control in the experiment. Confocal microscopy. Immunofluorescent-labeled slides and coverslips were physically separated from the culture chambers and mounted for confocal microscopy analysis. Fluorescein-stained cells were examined on a Phoibos 1000 laser scanning confocal microscope with an argon laser (Molecular Dynamics). The cells were scanned with settings optimized for each fluorophore. Serial horizontal (x and y axes) and vertical (z axis) sections as well as 3-D projections were collected simultaneously to detect in situ distribution of L-selectin during neutrophil adherence to endothelial cells. The 2-D or 3-D projection images were produced by film recorder on Ektachrome 100 film. Final photo images were printed using a computer with Umax PowerLook and photographic quality dye-sublimation printer Textronix Phaser II sdx in the Biology Image Center, University of Minnesota, St. Paul.
RESULTS AND DISCUSSION
PMN cell morphology changed dynamically during the interaction of neutrophils with IL-1-stimulated endothelial cells. Oval to pseudopod-bearing shapes were formed by PMNs after 5 to 15 min of contact with stimulated HUVEC monolayers (Figs. 1C and 1D). In contrast, only a small number of neutrophils were able to adhere to unstimulated HUVEC monolayers, and these human PMNs remained rounded (Figs. 1A and 1B). A large number of neutrophils changed their cell shape from round and elongated to polarized patterns with adherence to stimulated endothelial cells in vitro (Fig. 2). The time of cell contact appeared to affect cell shape change in the cell-adherence assay. Especially, after 15 min, more than 50% of the cells in contact with stimulated HUVEC monolayers had changed to either elongated or polarized cell shapes (Fig. 2). Similar morphological changes of PMN cells were also observed in
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FIG. 3. Serial confocal images showing direct labeling of L-selectin with anti-L-selectin antibody (DREG 56) in in vitro cell-adhesion assays. Bar, 10 mm. (A1–A6) Direct labeling of PMNs after 5 min adhesion with IL-1-stimulated HUVEC monolayers shows that L-selectin was redistributed in a polarized site in cross sections of neutrophils (PI nuclei stain as reference marker of position of neutrophil cells). The six serial sections show changes in L-selectin distribution (arrows), indicating that L-selectin expression decreased from A1 to A3 and disappeared in A4 to A6. (B1–B6) After 15 min of PMN cell contact with IL-1-stimulated HUVEC monolayers, L-selectin was redistributed in a more localized area in PMNs. The serial sections show progressive changes in L-selectin redistribution (arrows), indicating that L-selectin expression decreased from B1 to B3, and disappeared in B4 to B6, comparable to A. FIG. 4. Confocal labeling of L-selectin at different time points of PMN adhesion with IL-1-stimulated HUVEC monolayer. Bar, 5 mm. A
EXPRESSION OF L-SELECTIN IN CELL-TO-CELL INTERACTIONS IN VITRO
the cell-adherence assay (data not shown) using the coverslip method developed by Smith et al. [14]. Dynamic changes of L-selectin expression were documented by serial confocal sections (Figs. 3A and 3B). L-selectin was redistributed (Figs. 3A and 3B) to the leading edge of the PMN cells (also see Figs. 1C and 1D; PI nuclear staining as reference for cell shape change) during their contact with IL-1-stimulated endothelial cells. The pattern of L-selectin redistribution to the leading edge (Figs. 3A and 3B) was associated with the PMN polarized shape changes (Fig. 1) from 5 to 15 min of cell contact with stimulated HUVEC monolayers. Direct labeling of PMNs after 5 min adhesion with IL-1-stimulated HUVEC monolayers showed that L-selectin was redistributed in a polarized site in cross sections of neutrophils (Figs. 3A1–3A6; PI nuclei stain as reference markers of position of neutrophil cells). After 15 min of PMN cell contact with IL-1stimulated HUVEC monolayers, L-selectin was redistributed in a more localized point in PMNs (Figs. 3B1– 3B6). Serial sections (Figs. 3B1–3B6) showed a pattern of progressive changes in L-selectin distribution (arrows indicating changes of L-selectin expression) compared with that in Figs. 3A1–3A6. A similar pattern of L-selectin staining was also observed in the fixed PMNs stained after the cell-adhesion experiment (data not shown). L-selectin redistribution at different time points was also examined by composite 3-D sections and vertical sections (Fig. 4). The composite 3-D image (Fig. 4A) was generated from 10 serial sections (Fig. 3A). This 3-D image (Fig. 4A) shows a polarized pattern of Lselectin redistribution at 5 min of contact with the endothelial monolayer. Furthermore, this pattern of L-selectin redistribution was clearly seen in the vertical sections of the same neutrophil cells (Fig. 4B). A more polarized pattern of L-selectin redistribution was observed after 15 min of PMN cell contact with IL-1stimulated HUVEC monolayers (Fig. 4C). This polarized pattern was also seen in the vertical section (Fig. 4D) of the same cell (Fig. 4C), showing unevenly distributed L-selectin on only one side of cell contact. L-selectin was not detected by 3-D projection on neutrophils after 30 (Fig. 4E) or 60 min (Fig. 4F) of cell contact with IL-1-stimulated HUVEC cells. There was minimal background staining of the cells with mouse MAb fluorescein conjugate controls (data not shown). Our results demonstrated that L-selectin was redis-
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tributed to the leading edge of the neutrophil after contact with stimulated human endothelial cells. This L-selectin redistribution was correlated with profound cell shape changes of neutrophils bound to stimulated human endothelial cells. Initial neutrophil cell polarization occurred after 5 min and a high percentage of neutrophils became polarized after 15 min. These results suggest that both neutrophil cell shape change and L-selectin reorganization occur in response to contact with IL-1-stimulated endothelial cells. Similar results have been reported in other model systems in which lymphocytes formed polarized cell structures bearing pseudopodia during coculture with human endothelial cells. L-selectin reorganized to pseudopods in correspondence with such cell shape changes [21]. Previous studies have also shown that L-selectin was important in the initial attachment of lymphocytes and neutrophils to endothelium [14, 22]. This research extends the investigation on subcellular localization and surface distribution of L-selectin in human neutrophils by both high-resolution field emission SEM and immunoelectron microscopy [15, 18, 20]. L-selectin was localized at different membrane domains from Mac-1 on the surface of inactivated human neutrophils [18]. Borregaard et al. [15] further showed that L-selectin occurred exclusively on the tips of the microvilli on the plasma membrane of unstimulated and stimulated human neutrophils. Our data showed dynamic L-selectin redistribution associated with cell shape changes during neutrophil contact with stimulated endothelial cells. The effects of in vitro cell-labeling dyes on the cellular function of leukocytes have been evaluated [26, 27], demonstrating different degrees of effects on leukocyte function and receptor expression. The effects of membrane-labeling dyes on L-selectin expression and function in the neutrophil cell are largely unknown, thus limiting our experiment to the use of these dyes to colabel the site of L-selectin expression in the cell membrane. However, as an alternative method of labeling, Cell Tracker orange dye was used to label endothelial cells, which greatly improved the image contrast of neutrophil cells in contact with labeled endothelial cells compared with cells in contact with nonlabeled endothelial cells (data not shown). Moreover, developing further cell adhesion experiments under flow conditions will help define the dynamic L-selectin expression in vitro for more physiologically relevant conditions.
composite 3-D image (A) was generated from 10 serial sections (Fig. 3A, left arrow). A polarized pattern of L-selectin redistribution related to the endothelial monolayer was seen in A and in the vertical sections of the same neutrophil cells (B). The 3-D projection (C) of the same cell as Fig. 3B (right arrow) shows polarized L-selectin distribution associated with polarized PMN cell changes (Fig. 1D). Vertical sections (D) through the same neutrophil cell as in C (arrow) show unevenly distributed L-selectin on only one particular side of the cell relative to nuclear staining. Composite 3-D images of L-selectin labeling after 30 (E) or 60 min (F) of PMN adhesion with IL-1-stimulated HUVEC monolayer. L-selectin distribution was not visible on PMN cells in these 3-D projections.
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The cytoplasmic domain of L-selectin regulates the initial cell adhesion of leukocytes to endothelial cells [16]. Cytoskeletal interactions with the cytoplasmic domain of L-selectin appear to play an important role in this process. It is critical to understand whether cytoskeletal proteins are involved in mobilizing L-selectin molecules to form clusters on the polarized cell tip or if other selective molecular expression mechanisms are involved. Pavalko et al. [23] have reported that the cytoplasmic domain of L-selectin interacted with cytoskeletal proteins through a-actinin, but such interactions were not required in receptor positioning. Future exploration of the precise role of cytoskeletal interactions with the cytoplasmic domain of L-selectin should assist in better understanding the mechanism of regulation of cell adhesion during inflammation. In conclusion, this confocal microscopic immunofluorescence analysis provides evidence for dynamic changes of in situ expression of L-selectin at the 3-D cellular level during neutrophil adhesion. The regulated redistribution of L-selectin associated with neutrophil cell shape changes in the time-course experiment appeared to be an endothelial cell contact-dependent mechanism. Selective shedding and dynamic redistribution of L-selectin may be dependent on the intercellular interactions occurring during the early stages of neutrophil extravasation. We are grateful for research support provided by Dr. James Stevenson, help from the image analysis lab in the Analytical Department of Boehringer Ingelheim Pharmaceuticals, Inc., and technical suggestions from Dr. Dave Hazal from Molecular Dynamics, Inc. The insightful comments on this paper from Dr. Merry Jo Oursler are appreciated. We also express our thanks for help in printing from the Cell Image Center, New York University Medical School, and from the Biology Image Center, University of Minnesota.
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Kishimoto, T. K., Jutila, M. A., Berg, E. L., and Butcher, E. C. (1989). Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. Science 245, 1238 – 1241.
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Smith, C. W., Kishimoto, T. K., Abbassi, O., Hughes, B., Rothlein, R., McIntire, L. V., Butcher, E., and Anderson, D. C. (1991). Chemotactic factors regulate lectin adhesion molecule 1 (LECAM-1)-dependent neutrophil adhesion to cytokine-stimulated endothelial cells in vitro. J. Clin. Invest. 87, 609 – 618.
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Borregaard, N., Kjeldsen, L., Sengelov, H., Diamond, M. S., Springer, T. A., Anderson, D. C., Kishimoto, T. K., and Bainton, D. F. (1994). Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators. J. Leukocyte Biol. 56, 80 – 87.
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Kansas, G. S., Ley, K., Munro, M., and Tedder, T. F. (1993). Regulation of leukocyte rolling and adhesion to high endothelial venules through the cytoplasmic domain of L-selectin. J. Exp. Med. 177, 833– 838.
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Hasslen, S. R., Vonandrian, U. H., Butcher, E. C., Nelson, R. D., and Erlandsen, S. L. (1995). Spatial distribution of L-selectin (CD62L) human lymphocytes and transfected murine L1-2 cells. Histochem. J. 27, 547–554.
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Spertini, O., Luscinskas, F. W., Kansas, G. S., Munro, J. M., Griffin, J. D., Gimbrone, M. A., Jr., and Tedder, T. F. (1991). Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts
EXPRESSION OF L-SELECTIN IN CELL-TO-CELL INTERACTIONS IN VITRO with an inducible endothelial cell ligand to support leukocyte adhesion. J. Immunol. 147, 2565–2573. 23. Pavalko, F. M., Walker, D. M., Goheen, L. G. M., Doerschuk, C. M., and Kansas, G. S. (1995). The cytoplasmic domain of L-selectin interacts with cytoskeletal protein via a-actinin: Receptor positioning in microvilli does not require interaction with a-actinin. J. Cell Biol. 129, 1155–1164. 24. Smith, C. W., Marlin, S. D., Rothlein, R., Toman, C., and Anderson, D. C. (1989). Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J. Clin. Invest. 83, 2008 –2017. 25. Smith, C. W., Rothlein, R., Hughes, B. J., Mariscalco, M. M., Schmalstieg, F. C., and Anderson, D. C. (1988). Recognition of an endothelial determinant for CD 18-dependent neutrophils Received August 13, 1997
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adherence and transendothelial migration. J. Clin. Invest. 82, 1746 –1756. 26. Clerck, L. S. D., Bridts, C. H., Mertens, A. M., Moens, M. M., and Stevens, W. J. (1994). Use of fluorescent dyes in the determination of adherence of human leukocytes to endothelial cells and the effect of fluorochromes on cellular function. J. Immunol. Methods 172, 115–124. 27. Samlowski, W. E., Robertson, B. A., Draper, B. K., Prystas, E., and McGregor, J. R. (1991). Effect of supravital fluorochromes used to analyze the in vivo homing of murine lymphocytes on cellular function. J. Immunol. Methods 144, 101–115. 28. Walter, R. J., Berlin, R. D., and Oliver, J. M. (1980). Asymmetric Fc receptor distribution on human PMN oriented in a chemotactic gradient. Nature 286, 724 –725.
243, 87–93 (1998)
EX984139
Dynamic Expression of L-Selectin in Cell-to-Cell Interactions between Neutrophils and Endothelial Cells in Vitro Q. Liu,1 T. K. Kishimoto,* E. Mainolfi,* R. P. Deleon,* C. Myers,* and R. C. Moretz* Department of Biology, University of Minnesota, Duluth, Minnesota 55812; and *Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368
as migration in a chemotactic gradient has been elucidated [4–7, 28]. Polarized redistribution of cell adhesion molecules in neutrophil response to increasing concentration of chemotactic compound has been suggested [5]. Sullivan et al. [8] further demonstrated neutrophil morphological polarity correlated with polar motion, which may be due to an asymmetric distribution of chemotactic receptors. However, cell adhesion and molecule expression during leukocyte–endothelial cell interactions are still not well defined. The sequential interaction of selectin– carbohydrate, chemoattractant–receptor, and integrin–immunoglobulin family molecules has been proposed in the multiple-step model of neutrophil emigration [1–3, 9 –14]. The L-selectin receptor in part mediates the rolling and initial adherence of neutrophils to endothelial cells [12, 14], while Mac-1 (CD11b/CD18 integrin) strengthens this initial adherence and also facilitates migration of neutrophils through endothelial cells. The role of L-selectin in leukocyte rolling and adhesion has recently been explored both in vitro and in vivo [13, 15, 16]. The spatial distribution of L-selectin molecules on isolated human neutrophils has been characterized [15, 17, 18] and human lymphocytes [19]. Recently, L-selectin distribution in human leukocytes has been quantified using electron microscopy with immunogold labeling [20]. L-selectin is distributed on the tips of microvilli of neutrophils; furthermore L-selectin is rapidly cleaved in response to chemotactic factors. Whether L-selectin is redistributed during neutrophil cell adherence is unknown. In our study, the in situ expression of L-selectin during cell-to-cell interactions between neutrophils and endothelial cells has been examined by confocal microscopic immunofluorescence. Our results demonstrate dynamic L-selectin redistribution at both 2-D and 3-D levels which is associated with neutrophil shape changes during contact with stimulated endothelial cells.
Neutrophil– endothelial cell interactions are regulated by cell adhesion molecules and their cognate ligands. It has been proposed that L-selectin and Mac-1 (CD11b/CD18), two neutrophil adhesion receptors, have sequential roles in neutrophil extravasation during inflammation. In this model, L-selectin mediates rolling and initial adherence of neutrophils to endothelial cells, while Mac-1 strengthens this initial adherence and also facilitates migration of neutrophils through endothelial cells. L-selectin and Mac-1 expression are known to be inversely regulated. Here an in vitro culture system has been developed to investigate in situ expression of L-selectin during cellto-cell interactions between neutrophils and endothelial cell monolayers by confocal immunofluorescence analysis. Neutrophils underwent profound cell shape change from round to polarized cell morphology with pseudopod formation after 5 to 15 min coculture with IL-1-stimulated human endothelial cells. L-selectin was redistributed to the pseudopod of the polarized neutrophils in correlation with such cellular changes. During initial cell attachment, neutrophils bound to IL-1-stimulated endothelial cells expressed a high level of L-selectin in a polarized pattern. L-selectin expression decreased over time during neutrophil– endothelial cell interactions. © 1998 Academic Press
INTRODUCTION
Neutrophil–endothelial interactions during inflammation are a coordinated multistep process involving the function of adhesion receptors and chemokines [1–3]. Neutrophils are the predominant cell type accumulating at sites of acute inflammation. Extravasation during acute inflammation includes adhesive interaction of neutrophils with vascular endothelium and transmigration of the neutrophils into the inflamed tissue. The influence of cell interactions on aspects of neutrophil motility associated with cell polarity and nuclear orientation as well
MATERIAL AND METHODS
1 To whom correspondence and reprint requests should be addressed. Fax: (218) 726-8142. E-mail: [email protected].
Cell preparations. Human blood samples from healthy adults were collected, dextran-sedimented, and centrifuged over Ficoll– 87
0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
88
LIU ET AL.
FIG. 1. Phase-contrast images showing adherence of human PMN to HUVEC monolayers with or without IL-1 stimulation at 5 or 15 min. Bar, 15 mm. (A and B) Note that human PMN cells maintained round shapes after 5 (A) or 15 (B) min contact with nonstimulated HUVEC monolayers. (C) PMN cells showed an oval shape (circles mark two examples of the PMN cells) after 5 min contact with stimulated HUVEC monolayers. Some also displayed pseudopod-bearing shapes indicating initial cell migration. (D) Pseudopod-bearing shape (circles mark two examples of the PMN cells) occurred in most PMNs after 15 min contact with stimulated HUVEC monolayers, indicating progression of cell adhesion to migration.
Hypaque gradients according to methods from Smith et al. [4]. Isolated neutrophils were suspended in Dulbecco’s phosphate-buffered saline (PBS). Human umbilical vein endothelial cells (HUVEC) were obtained from Cell Systems Corp. (Kirkland, WA) and cultured in Falcon T-75 flasks containing MCDB131 supplied by Clonetics Corp. and a RPMI 1640-based growth medium (containing 10% fetal calf
serum, antibiotics, heparin, 0.1 mg/ml, and endothelial growth factor, 0.05 mg/ml) for 3– 4 days in a humidified incubator at 37°C and 5% CO2. At confluence, cells were passaged by brief trypsinization. Tissue culture chamber glass slides and coverslips (Nunc, Naperville, IL) were coated with 0.1% gelatin. Trypsinized cells (106 cells/ml) were then seeded into each chamber and cultured for 3 days.
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FIG. 2. Neutrophil cell shape changes (%) in time-course experiments (5 and 15 min) for the cell-adhesion assay in stimulated HUVEC monolayers.
Cell adherence assay. Cultured HUVEC monolayers were stimulated with IL-1 (1 ng/ml) for 4 h. A visual cell adherence assay was performed using the method developed by Smith et al. [24, 25]. Briefly, neutrophils suspended in PBS (106 cells/ml) at 4°C were pipetted onto the stimulated HUVEC monolayers in each culture chamber. After neutrophils settled onto the monolayers for 8 min, the culture chamber and coverslips were then inverted for 8 min and washed with PBS. Only those adherent cells that remained adherent to the monolayers were followed by phase-contrast microscopy. Neutrophils in contact with the monolayer at 5 min were fixed in 1% paraformaldehyde in PBS for analysis of initial adherence. Cells in additional chambers were fixed when a large number of neutrophils became flattened and had undergone major polarized shape changes after 15 min in contact with the stimulated monolayer. Neutrophils in contact with unstimulated HUVEC monolayers for 5 or 15 min were also fixed and used as controls for the cell-adherence assay. Neutrophil cell shape changes were used as morphological markers for initial adherence and its progression to migration. Such cell shape changes were visualized by an inverted microscope and phasecontrast optics [14]. Briefly, cells with spherical shape were scored as round; cells with oval to elongated shape were scored as elongated; neutrophil cells forming a leading edge for migration were identified as polarized. At least three samples of in vitro culture slides and coverslips and 100 cells for each sample were scored for cellular changes after neutrophil contact with stimulated HUVEC monolayers for 5 or 15 min. Morphological changes of neutrophils (round, elongated, and polarized cells) were measured as the percentage of the total cells in each culture slide. Dual in vitro labeling of neutrophils and endothelial cells. In our cell-adherence assay, coordinated in vitro labeling of neutrophils with fluorescein-conjugated anti-L-selectin antibody (Pharmingen, San Diego, CA) and endothelial cells with Cell Tracker orange dye (Molecular Probe, Inc., Eugene, OR) were used. To label endothelial cells in vitro, prewarmed (37°C) 0.1 mM Cell Tracker orange dyecontaining medium was added to each culture chamber after removal of the culture medium according to the manufacturer’s instructions. After incubation of labeled HUVEC under culture conditions for 30 min with the dye-containing medium, fresh prewarmed dye-free medium was added to replace the culture medium, and the cells were cultured under the same conditions for another 30 min. Direct labeling of neutrophils was accomplished by staining isolated PMNs with fluorescein-conjugated anti-L-selectin antibody (DREG-56). The PMNs were incubated at 4°C for at least 15 min with gentle shaking, washed with 2% bovine serum albumin in PBS without azide at least two times, and then added to each HUVEC culture chamber for the cell-adherence assay (1 3 106 PMNs/ml). After 2, 5, 15, 30, and 60 min of cell contact, nonadherent cells were
removed by gentle washings of each chamber with PBS, and the adherent PMNs and HUVEC were fixed with 1% paraformaldehyde in PBS. The nuclei of neutrophils were used as reference markers for the relative cellular location [28] of L-selectin redistribution. After fixation, propidium iodide (PI) was used to stain PMN nuclei without affecting L-selectin redistribution prior to confocal analysis. Fluorescein-conjugated isotype control mouse IgG1 was used as control in the experiment. Confocal microscopy. Immunofluorescent-labeled slides and coverslips were physically separated from the culture chambers and mounted for confocal microscopy analysis. Fluorescein-stained cells were examined on a Phoibos 1000 laser scanning confocal microscope with an argon laser (Molecular Dynamics). The cells were scanned with settings optimized for each fluorophore. Serial horizontal (x and y axes) and vertical (z axis) sections as well as 3-D projections were collected simultaneously to detect in situ distribution of L-selectin during neutrophil adherence to endothelial cells. The 2-D or 3-D projection images were produced by film recorder on Ektachrome 100 film. Final photo images were printed using a computer with Umax PowerLook and photographic quality dye-sublimation printer Textronix Phaser II sdx in the Biology Image Center, University of Minnesota, St. Paul.
RESULTS AND DISCUSSION
PMN cell morphology changed dynamically during the interaction of neutrophils with IL-1-stimulated endothelial cells. Oval to pseudopod-bearing shapes were formed by PMNs after 5 to 15 min of contact with stimulated HUVEC monolayers (Figs. 1C and 1D). In contrast, only a small number of neutrophils were able to adhere to unstimulated HUVEC monolayers, and these human PMNs remained rounded (Figs. 1A and 1B). A large number of neutrophils changed their cell shape from round and elongated to polarized patterns with adherence to stimulated endothelial cells in vitro (Fig. 2). The time of cell contact appeared to affect cell shape change in the cell-adherence assay. Especially, after 15 min, more than 50% of the cells in contact with stimulated HUVEC monolayers had changed to either elongated or polarized cell shapes (Fig. 2). Similar morphological changes of PMN cells were also observed in
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FIG. 3. Serial confocal images showing direct labeling of L-selectin with anti-L-selectin antibody (DREG 56) in in vitro cell-adhesion assays. Bar, 10 mm. (A1–A6) Direct labeling of PMNs after 5 min adhesion with IL-1-stimulated HUVEC monolayers shows that L-selectin was redistributed in a polarized site in cross sections of neutrophils (PI nuclei stain as reference marker of position of neutrophil cells). The six serial sections show changes in L-selectin distribution (arrows), indicating that L-selectin expression decreased from A1 to A3 and disappeared in A4 to A6. (B1–B6) After 15 min of PMN cell contact with IL-1-stimulated HUVEC monolayers, L-selectin was redistributed in a more localized area in PMNs. The serial sections show progressive changes in L-selectin redistribution (arrows), indicating that L-selectin expression decreased from B1 to B3, and disappeared in B4 to B6, comparable to A. FIG. 4. Confocal labeling of L-selectin at different time points of PMN adhesion with IL-1-stimulated HUVEC monolayer. Bar, 5 mm. A
EXPRESSION OF L-SELECTIN IN CELL-TO-CELL INTERACTIONS IN VITRO
the cell-adherence assay (data not shown) using the coverslip method developed by Smith et al. [14]. Dynamic changes of L-selectin expression were documented by serial confocal sections (Figs. 3A and 3B). L-selectin was redistributed (Figs. 3A and 3B) to the leading edge of the PMN cells (also see Figs. 1C and 1D; PI nuclear staining as reference for cell shape change) during their contact with IL-1-stimulated endothelial cells. The pattern of L-selectin redistribution to the leading edge (Figs. 3A and 3B) was associated with the PMN polarized shape changes (Fig. 1) from 5 to 15 min of cell contact with stimulated HUVEC monolayers. Direct labeling of PMNs after 5 min adhesion with IL-1-stimulated HUVEC monolayers showed that L-selectin was redistributed in a polarized site in cross sections of neutrophils (Figs. 3A1–3A6; PI nuclei stain as reference markers of position of neutrophil cells). After 15 min of PMN cell contact with IL-1stimulated HUVEC monolayers, L-selectin was redistributed in a more localized point in PMNs (Figs. 3B1– 3B6). Serial sections (Figs. 3B1–3B6) showed a pattern of progressive changes in L-selectin distribution (arrows indicating changes of L-selectin expression) compared with that in Figs. 3A1–3A6. A similar pattern of L-selectin staining was also observed in the fixed PMNs stained after the cell-adhesion experiment (data not shown). L-selectin redistribution at different time points was also examined by composite 3-D sections and vertical sections (Fig. 4). The composite 3-D image (Fig. 4A) was generated from 10 serial sections (Fig. 3A). This 3-D image (Fig. 4A) shows a polarized pattern of Lselectin redistribution at 5 min of contact with the endothelial monolayer. Furthermore, this pattern of L-selectin redistribution was clearly seen in the vertical sections of the same neutrophil cells (Fig. 4B). A more polarized pattern of L-selectin redistribution was observed after 15 min of PMN cell contact with IL-1stimulated HUVEC monolayers (Fig. 4C). This polarized pattern was also seen in the vertical section (Fig. 4D) of the same cell (Fig. 4C), showing unevenly distributed L-selectin on only one side of cell contact. L-selectin was not detected by 3-D projection on neutrophils after 30 (Fig. 4E) or 60 min (Fig. 4F) of cell contact with IL-1-stimulated HUVEC cells. There was minimal background staining of the cells with mouse MAb fluorescein conjugate controls (data not shown). Our results demonstrated that L-selectin was redis-
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tributed to the leading edge of the neutrophil after contact with stimulated human endothelial cells. This L-selectin redistribution was correlated with profound cell shape changes of neutrophils bound to stimulated human endothelial cells. Initial neutrophil cell polarization occurred after 5 min and a high percentage of neutrophils became polarized after 15 min. These results suggest that both neutrophil cell shape change and L-selectin reorganization occur in response to contact with IL-1-stimulated endothelial cells. Similar results have been reported in other model systems in which lymphocytes formed polarized cell structures bearing pseudopodia during coculture with human endothelial cells. L-selectin reorganized to pseudopods in correspondence with such cell shape changes [21]. Previous studies have also shown that L-selectin was important in the initial attachment of lymphocytes and neutrophils to endothelium [14, 22]. This research extends the investigation on subcellular localization and surface distribution of L-selectin in human neutrophils by both high-resolution field emission SEM and immunoelectron microscopy [15, 18, 20]. L-selectin was localized at different membrane domains from Mac-1 on the surface of inactivated human neutrophils [18]. Borregaard et al. [15] further showed that L-selectin occurred exclusively on the tips of the microvilli on the plasma membrane of unstimulated and stimulated human neutrophils. Our data showed dynamic L-selectin redistribution associated with cell shape changes during neutrophil contact with stimulated endothelial cells. The effects of in vitro cell-labeling dyes on the cellular function of leukocytes have been evaluated [26, 27], demonstrating different degrees of effects on leukocyte function and receptor expression. The effects of membrane-labeling dyes on L-selectin expression and function in the neutrophil cell are largely unknown, thus limiting our experiment to the use of these dyes to colabel the site of L-selectin expression in the cell membrane. However, as an alternative method of labeling, Cell Tracker orange dye was used to label endothelial cells, which greatly improved the image contrast of neutrophil cells in contact with labeled endothelial cells compared with cells in contact with nonlabeled endothelial cells (data not shown). Moreover, developing further cell adhesion experiments under flow conditions will help define the dynamic L-selectin expression in vitro for more physiologically relevant conditions.
composite 3-D image (A) was generated from 10 serial sections (Fig. 3A, left arrow). A polarized pattern of L-selectin redistribution related to the endothelial monolayer was seen in A and in the vertical sections of the same neutrophil cells (B). The 3-D projection (C) of the same cell as Fig. 3B (right arrow) shows polarized L-selectin distribution associated with polarized PMN cell changes (Fig. 1D). Vertical sections (D) through the same neutrophil cell as in C (arrow) show unevenly distributed L-selectin on only one particular side of the cell relative to nuclear staining. Composite 3-D images of L-selectin labeling after 30 (E) or 60 min (F) of PMN adhesion with IL-1-stimulated HUVEC monolayer. L-selectin distribution was not visible on PMN cells in these 3-D projections.
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The cytoplasmic domain of L-selectin regulates the initial cell adhesion of leukocytes to endothelial cells [16]. Cytoskeletal interactions with the cytoplasmic domain of L-selectin appear to play an important role in this process. It is critical to understand whether cytoskeletal proteins are involved in mobilizing L-selectin molecules to form clusters on the polarized cell tip or if other selective molecular expression mechanisms are involved. Pavalko et al. [23] have reported that the cytoplasmic domain of L-selectin interacted with cytoskeletal proteins through a-actinin, but such interactions were not required in receptor positioning. Future exploration of the precise role of cytoskeletal interactions with the cytoplasmic domain of L-selectin should assist in better understanding the mechanism of regulation of cell adhesion during inflammation. In conclusion, this confocal microscopic immunofluorescence analysis provides evidence for dynamic changes of in situ expression of L-selectin at the 3-D cellular level during neutrophil adhesion. The regulated redistribution of L-selectin associated with neutrophil cell shape changes in the time-course experiment appeared to be an endothelial cell contact-dependent mechanism. Selective shedding and dynamic redistribution of L-selectin may be dependent on the intercellular interactions occurring during the early stages of neutrophil extravasation. We are grateful for research support provided by Dr. James Stevenson, help from the image analysis lab in the Analytical Department of Boehringer Ingelheim Pharmaceuticals, Inc., and technical suggestions from Dr. Dave Hazal from Molecular Dynamics, Inc. The insightful comments on this paper from Dr. Merry Jo Oursler are appreciated. We also express our thanks for help in printing from the Cell Image Center, New York University Medical School, and from the Biology Image Center, University of Minnesota.
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