A novel endocytic pathway induced by clusteri...
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doi: 10.1242/10.1242/jcs.00367
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Journal of Cell Science 116, 1599-1609 (2003)
Copyright © 2003The Company of Biologists Limited
doi: 10.1242/jcs.00367
Research Article
A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1
Silvia Muro2,4, Rainer Wiewrodt3, Anu Thomas2,4, Lauren Koniaris3,4, Steven M. Albelda3, Vladimir R. Muzykantov2,4,* and Michael Koval1,4,*
1 Department of Physiology, University of Pennsylvania School of Medicine, B-400 Richards/6085, 3700 Hamilton Walk, Philadelphia, PA 19104, USA
2 Department of Pharmacology and Medicine, Pulmonary, University of Pennsylvania School of Medicine, B-400 Richards/6085, 3700 Hamilton Walk, Philadelphia, PA 19104, USA
3 Department of Critical Care Division, University of Pennsylvania School of Medicine, B-400 Richards/6085, 3700 Hamilton Walk, Philadelphia, PA 19104, USA
4 Institute for Environmental Medicine, University of Pennsylvania School of Medicine, B-400 Richards/6085, 3700 Hamilton Walk, Philadelphia, PA 19104, USA
* Authors for correspondence (e-mail:mkoval@mail.med.upenn.edu,muzykant@mail.med.upenn.edu)
Accepted 14 February 2003
Antibody conjugates directed against intercellular adhesion molecule (ICAM-1) or platelet-endothelial cell adhesion molecule (PECAM-1) have formed the basis for drug delivery vehicles that are specifically recognized and internalized by endothelial cells. There is increasing evidence that ICAM-1 and PECAM-1 may also play a role in cell scavenger functions and pathogen entry. To define the mechanisms that regulate ICAM-1 and PECAM-1 internalization, we examined the uptake of anti-PECAM-1 and anti-ICAM-1 conjugates by endothelial cells. We found that the conjugates must be multimeric, because monomeric anti-ICAM-1 and anti-PECAM-1 are not internalized. Newly internalized anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with either clathrin or caveolin, and immunoconjugate internalization was not reduced by inhibitors of clathrin-mediated or caveolar endocytosis, suggesting that this is a novel endocytic pathway. Amiloride and protein kinase C (PKC) inhibitors, agents known to inhibit macropinocytosis, reduced the internalization of clustered ICAM-1 and PECAM-1. However, expression of dominant-negative dynamin-2 constructs inhibited uptake of clustered ICAM-1. Binding of anti-ICAM-1 conjugates stimulated the formation of actin stress fibers by human umbilical vein endothelial cells (HUVEC). Latrunculin, radicicol and Y27632 also inhibited internalization of clustered ICAM-1, suggesting that actin rearrangements requiring Src kinase and Rho kinase (ROCK) were required for internalization. Interestingly, these kinases are part of the signal transduction pathways that are activated when circulating leukocytes engage endothelial cell adhesion molecules, suggesting the possibility that CAM-mediated endocytosis is regulated using comparable signaling pathways.
Key words: HUVEC, Vascular endothelium, Cell adhesion, Macropinocytosis, Endocytosis
Endothelial cells internalize natural ligands and artificial macromolecular ligands, that have been designed as carriers for specific drug and gene delivery (Danilov et al., 2001
It is becoming apparent that both PECAM-1 and ICAM-1 may serve as plasma membrane receptors to mediate internalization of natural ligands by different types of cells. For instance, coxsackieviruses and rhinoviruses bind ICAM-1 and are internalized (Shafren et al., 1997a
There are multiple pathways for ligand internalization involving vesicles 100-300 nm in diameter, including clathrin-mediated endocytosis and the clathrin-independent caveolae-mediated pathway (Mukherjee et al., 1997
The regulation of ICAM-1 and PECAM-1 internalization by endothelial cells is not well understood at present. In particular, whether ICAM-1 and PECAM-1 are internalized by similar pathways is not known. In this study, we defined some key elements regulating the internalization of anti-ICAM-1 or anti-PECAM-1 conjugates by endothelial cells. In each case, clustering of the CAM was required for efficient internalization. Given that anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with known endocytic coat proteins and from the analysis of the signaling pathways that regulate the uptake of anti-ICAM-1 and anti-PECAM-1 conjugates, our data suggests that that endothelial cells internalize clustered ICAM-1 and PECAM-1 using a novel endocytic pathway.
Reagents
Murine monoclonal antibodies to human ICAM-1 (R6.5) and PECAM-1 (mAb 62) were provided by Robert Rothlein (Boehringer-Ingelheim, Ridgefield, CN) and M. Nakada (Centocor, Malvern, PA), respectively. Control murine IgG was from Calbiochem (San Diego, CA). Anti-caveolin-1, anti-clathrin, anti-cholera toxin B and anti-transferrin were from Calbiochem (La Jolla, CA). Polyclonal rabbit anti-6-His-Tag was a gift from MBL (Nagoya, Japan). Secondary fluorescent antibodies were from Jackson ImmunoResearch (West Grove, PA) and Molecular Probes (Eugene, OR). Fluorescent transferrin and cholera toxin B were from Molecular Probes. Polystyrene-latex beads 100 nm in diameter, and loaded with a fluorochrome compatible with FITC fluorescence (Fluoresbrite YG microspheres), were purchased from Polysciences (Warrington, PA). Unless otherwise stated, all other reagents were from Sigma (St Louis, MO).
Cell culture
Pooled human umbilical vein endothelial cells (HUVEC) from Clonetics (San Diego, CA) were maintained in M199 medium (GibcoBRL, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS), 2 mM glutamine, 15 µg/ml endothelial cell growth supplement (ECGS), 100 µg /ml heparin, 100 U/ml penicillin and 100 µg/ml streptomycin. EAhy926 cells from an endothelial-like hybrid cell line generated from HUVEC and A549 cells (Edgell et al., 1983
Preparation of immunobeads and immunoconjugates
Fluorescent microspheres were coated with either anti-ICAM-1, anti-PECAM-1 or control murine IgG by incubation at room temperature (RT) for 1 hour as previously described (Wiewrodt et al., 2002
Binding and uptake of anti-ICAM-1 and anti-PECAM-1 immunobeads
Confluent HUVEC or EAhy926 cells were pre-incubated overnight with 250 units of TNF-
To identify compartments containing internalized immunobeads, HUVEC monolayers were incubated with immunobeads for 1 hour at 4°C to allow surface binding, washed, then incubated at 37°C for different time periods to permit endocytosis. The cells were fixed, permeabilized and incubated with rabbit polyclonal anti-human caveolin-1, followed by incubation with goat anti-rabbit IgG conjugated to Alexa Fluor 350. Colocalization with clathrin heavy chain was done in a comparable manner, using TRITC-conjugates anti-clathrin.
For microscopy, samples mounted onto glass slides were observed using an Olympus IX70 inverted fluorescence microscope, 40x or 60x PlanApo objectives and filters optimized for fluorescent immunobeads (excitation BP460-490 nm, dichroic DM505 nm, emission BA515-550 nm), TxR fluorescence (excitation BP530-550 nm, dichroic DM570 nm, emission BA590-800+ nm) and Alexa Fluor 450 (excitation BP360-370 nm, dichroicDM400 nm, emission BA420-460 nm) (Chroma Technology, Brattleboro, VT). Separate images for each fluorescence channel were acquired using a Hamamatsu Orca-1 CCD camera. The images were then merged and analyzed with ImagePro 3.0 imaging software (Media Cybernetics, Silver Spring, MD) as previously described (Wiewrodt et al., 2002
Mechanisms of ICAM-1- and PECAM-1-mediated uptake
Mammalian pcDNA3 expression vectors encoding for 6-His-tagged versions of human dynamin-2 [wild-type and dominant-negative forms (K44A), (PH*)] were gifts from Drs S. Schmid (Scripps Research Institute, La Jolla, CA) (Altschuler et al., 1998
For studies using pharmacological inhibitors, TNF-
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Fig. 10. Model for uptake mediated by cell adhesion molecules. The model shown is for signaling pathways stimulated when monomeric ICAM-1 or anti-PECAM-1 are clustered by binding to immunoconjugates. Pharmacological inhibitors and activators are indicated by italics. Cell adhesion molecules have the capacity to bind proteins that mediate direct interactions with the actin cytoskeleton, such as
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Fig. 4. Anti-ICAM-1 conjugates are not internalized by clathrin or caveolae-mediated endocytosis. TNF-
Monomeric antibodies to PECAM-1 are poorly internalized by endothelial cells (Muzykantov et al., 1999
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Fig. 1. Small ICAM-1 immunoconjugates are internalized by HUVEC. HUVEC were treated with 250 U TNF-
On average,
Because dynamin-2 is frequently involved in vesicle-mediated internalization and phagocytosis (Altschuler et al., 1998
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Fig. 2. Anti-ICAM-1 immunobead uptake is inhibited by dominant-negative dynamin constructs. EAhy926 cells were transfected with 1.5 µg of DNA encoding either wild-type (A-C) or dominant-negative (K44A; e, PH*; f) dynamin-2. Nontransfected cells are shown in (D). Twelve hours post-transfection, cells were stimulated with TNF-
We also found that the dominant-negative dynamin-2 constructs inhibited the uptake of Alexa Fluor 594-conjugated cholera toxin (FL-cholera toxin, S.M., R.W. and A.T. et al., unpublished) that is internalized by caveolae-mediated endocytosis (Schnitzer et al., 1994
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Fig. 3. Anti-ICAM-1 immunobeads do not colocalize with caveolin or clathrin. TNF-
We therefore used a series of pharmacological inhibitors to further characterize internalization of anti-ICAM-1 and anti-PECAM-1 conjugates by HUVEC. The specificity of different inhibitors was confirmed using fluorescent transferrin and cholera toxin as controls for clathrin-mediated and caveolar endocytosis, respectively (Fig. 4). As shown inFig. 5, inhibitors of clathrin-mediated transferrin endocytosis (MDC, potassium depletion) did not inhibit the uptake of anti-ICAM-1 or anti-PECAM-1 immunobeads. Furthermore, inhibitors of caveolae-dependent cholera toxin uptake (filipin, genestein) were not effective at inhibiting anti-ICAM-1 or anti-PECAM-1 immunobead internalization. Because uptake of anti-ICAM-1 and anti-PECAM-1 immunobeads appeared to be through a unique internalization pathway, we examined the effect of other inhibitors on immunobead endocytosis. Previous work has indicated that amiloride, an inhibitor of the sodium/proton pump, can inhibit macropinocytosis by dendritic cells (West et al., 1989
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Fig. 5. Effect of endocytosis inhibitors on anti-ICAM-1 and anti-PECAM-1 uptake. Uptake of anti-ICAM-1 and anti-PECAM-1 immunobeads was quantified as mean±s.d. by fluorescence microscopy using control cells, potassium-depleted cells or cells pretreated for 30 minutes at 37°C before incubation with immunobeads with either 50 µM MDC, 50 µM genistein, 1 µg/ml filipin, 3 mM amiloride or 25 µM monensin. Cells incubated with anti-ICAM-1 or anti-PECAM-1 immunobeads at 4°C are controls for no internalization. *P<0.05.
Because protein kinase C (PKC) has been reported to play a pivotal role in macropinocytosis and phagocytosis by macrophages (Araki et al., 1996
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Fig. 6. Uptake of anti-ICAM-1 or anti-PECAM-1 immunobeads is PKC-mediated. TNF-
In fact, the formation of F actin stress fibers is frequently associated with ICAM-1 crosslinking (Thompson et al., 2002
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Fig. 7. Internalized anti-ICAM-1 immunobeads associate with the actin cytoskeleton. (A,b) HUVEC incubated in either the absence (A) or presence (B) of anti-ICAM-1 immunobeads for 15 minutes were fixed and then treated with rhodamine phalloidin to label filamentous actin. Note the stimulation of actin stress fibers by anti-ICAM-1 immunobeads. Bar, 10 µm. (C,D) HUVEC were incubated with anti-ICAM-1 immunobeads for 15 (C) or 30 (D) minutes, fixed, then immunostained to double-label surface-bound material (blue, arrowheads). Arrows denote internalized immunobeads in vesicles associated with stress fibers. Bar, 10 µm.
Given the dramatic association of anti-ICAM-1 immunobeads with actin in HUVEC, we also examined the cytoskeletal requirements for the uptake of anti-ICAM-1 and anti-PECAM-1 immunobeads. In contrast to macropinocytosis by macrophages (Racoosin and Swanson, 1992
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Fig. 8. Uptake of anti-ICAM-1 immunobeads requires actin regulatory proteins. TNF-
We also examined inhibitors that affect kinases known to play a role in regulating actin organization. In contrast to macropinocytosis by phagocytes (Araki et al., 1996
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Fig. 9. Agents that inhibit anti-ICAM-1 immunobead uptake disrupt actin rearrangements induced by immunobeads. HUVEC were pretreated with 10 µM latrunculin A (A), 10 µM radicicol (B), 10 µM Y-27632 (C) or 3 mM amiloride (D), incubated with anti-ICAM-1 immunobeads for 15 minutes then fixed and stained for filamentous actin using rhodamine phalloidin. Each of these agents that inhibit uptake of anti-ICAM-1 immunobeads also inhibited actin stress-fiber formation. Bar, 10 µm.
In this study, we found that endothelial cells internalize clustered ICAM-1 and clustered PECAM-1 by a novel endocytic pathway (Table 1). CAM-mediated endocytosis was distinct from caveolae-mediated uptake (McIntosh et al., 2002
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Table 1. Comparison of CAM-mediated endocytosis with phagocytosis and macropinocytosis
On the basis of amiloride sensitivity and PKC dependence, internalization of clustered anti-ICAM-1 and anti-PECAM-1 seemed to be related to macropinocytosis (Lamaze and Schmid, 1995
The uptake of anti-ICAM-1 and anti-PECAM-1 conjugates required clustering of cell adhesion molecules (Muzykantov et al., 1999
Dynamin, via interactions with endophilin and profilin, helps recruit actin to sites of endocytic activity (Farsad et al., 2001
Another major distinction from macropinocytosis and phagocytosis is that uptake of anti-ICAM-1 and anti-PECAM-1 conjugates larger than 500 nm in diameter was poor (Fig. 1) (Wiewrodt et al., 2002
Consistent with a size threshold for ICAM-1-mediated internalization, an ICAM-1 enriched structure is formed at the contact site between lymphocytes and HUVEC, where the endothelial cell appears to partially engulf the lymphocyte (Barreiro et al., 2002
The size threshold for internalization of clustered ICAM-1 and PECAM-1 might also enable endothelial cells to distinguish small apoptotic fragments from intact cells bound to the endothelial cell surface, such as other endothelial or blood cells (Barreiro et al., 2002
Understanding the mechanisms that regulate uptake of anti-ICAM-1 and anti-PECAM-1 conjugates will probably help to extend the utility of these agents as the basis for endothelium-specific drug-targeting vehicles (Li et al., 2000
We thank B. Daugherty for a critical reading of the manuscript. S.M. is supported by a fellowship from Fundación Ramón Areces (Spain). R.W. is a postdoctoral fellow of the Mildred Scheel Stiftung fur Krebsforschung der Deutschen Krebshilfe e.V. (D/98/02288). Supported by an American Heart Association grant-in-aid 9950389N (M.K.) National Institutes of Health SCOR in Acute Lung Injury, HL60290, Project 4, (V.R.M., S.M.A.); grants HL/GM 71175-01 (V.R.M.), GM61012 (M.K.) and P01 HL019737-26, Project 3 (M.K.) and Department of Defense Grant PR 012262 (V.R.M.).
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