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doi: 10.1242/10.1242/jcs.00367

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A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1

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

View larger version (37K): [in a new window]   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 -actinin (-act) and ERM proteins. On the basis of our inhibitor data and results from the literature, we propose that clustering of ICAM-1 or PECAM-1 can also stimulate PKC, Src kinase and ROCK signal transduction pathways. This could help regulate the recruitment of other cofactors, such as dynamin-2 (dyn2), to the plasma membrane. Also, ERM proteins and NHE transporters are downstream targets for phosphorylation by the Rho/ROCK pathway, which might further serve to recruit actin to sites of immunoconjugate uptake in response to ICAM-1 or PECAM-1 clustering.

View larger version (90K): [in a new window]   Fig. 4. Anti-ICAM-1 conjugates are not internalized by clathrin or caveolae-mediated endocytosis. TNF--activated HUVEC were untreated (A-C), potassium-depleted (D-F), or treated for 30 minutes at 37oC with 1 µg/ml filipin (G-I), or 3 mM amiloride (J-L). The cells were incubated in the presence or absence of inhibitors for 1 hour at 37°C with fluorescent transferrin (Tf: A,D,G,J), fluorescent cholera toxin (CT: B,E,H,K) or anti-ICAM-1 immunobeads (C,F,I,L), then fixed and counterstained to double-label surface-bound material (yellow, arrowheads). As shown, potassium depletion specifically inhibited transferrin uptake by clathrin-mediated endocytosis (D), filipin specifically inhibited caveolar uptake of cholera toxin (H) and amiloride specifically inhibited uptake of anti-ICAM-1 immunobeads (L).

View larger version (60K): [in a new window]   Fig. 1. Small ICAM-1 immunoconjugates are internalized by HUVEC. HUVEC were treated with 250 U TNF- for 24 hours. Confluent monolayers were incubated at either 4°C (c) or 37°C (A,C-F) in the presence of either large (A; >1000 nm diameter), small (b; <500 nm diameter) biotin-anti-ICAM-1/streptavidin conjugates, anti-ICAM-1 immunobeads (C,D) or beads previously coated with control murine IgG (E,F). The cells were subsequently washed, fixed and counterstained with fluorescent goat anti-mouse IgG. Merged images corresponding to representative samples were pseudocolored to show single-labeled, internalized immunoconjugates/immunobeads as green (arrows) and double-labeled immunoconjugates/immunobeads on the cell surface as yellow (arrowheads). The phase-contrast image shown in e corresponds to the fluorescence image shown in F. Bar, 10 µm. (G) Uptake of anti-ICAM-1 (l) and anti-PECAM-1 (°) immunobeads by TNF--activated HUVEC was determined for different incubation times as the mean percentage of internalized (single labeled) immunobeads per cell. Error bars corresponding to s.d. were smaller than the size of the symbols used for the graph.

View larger version (44K): [in a new window]   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- for 36 hours and then incubated for 2 hours at 37°C with anti-ICAM-1 immunobeads. The cells were then washed, fixed and surface-bound particles were counterstained with TxR goat anti-mouse IgG. The cells were then permeabilized and stained with rabbit anti-6-His antibody followed by Alexa Fluor 350 goat anti-rabbit IgG to identify transfected cells expressing dynamin (A). The corresponding phase-contrast image is shown in (B). Merged images corresponding to representative samples of transfected (C,E,F) or control (D) cells are shown, where single-labeled, internalized immunobeads are green (arrows) and double-labeled immunobeads on the cell surface are yellow (arrowheads). Blue fluorescence of transfected cells is omitted in panels (C,E,F) to enable better visualization of red and green fluorescence. (G) The percentage of immunobead uptake was calculated as described as mean±s.d. *P<0.05.

View larger version (67K): [in a new window]   Fig. 3. Anti-ICAM-1 immunobeads do not colocalize with caveolin or clathrin. TNF--stimulated HUVEC were incubated with control anti-ICAM-1 immunobeads (A,C) or Alexa 594-conjugated cholera toxin B subunit (B) for 15 minutes at 37°C. The cells were then washed and fixed, and surface-bound material was counterstained with TxR goat anti-mouse IgG (A,C) or goat anti-cholera toxin followed by fluorescein rabbit anti-goat IgG (B). After permeabilization, the cells were then labeled with rabbit anti-human caveolin-followed by Alexa 350-conjugated goat anti-rabbit IgG (A,B) or TRITC-conjugated anti-clathrin heavy chain (C). Insets show images magnified twofold. The image color channels were selected to facilitate the comparison between panels in the figure, and they are: green, internalized immunobeads or cholera toxin; blue, surface-bound immunobeads or cholera toxin; red, caveolin-1 (arrowheads) or clathrin (arrows). There was little, if any, colocalization of anti-ICAM-1 immunobeads with caveolin-1 or clathrin, as evidenced by the lack of yellow labeling in A and C and areas showing internalized immunobeads with little caveolin-1 or clathrin nearby (see insets).

View larger version (26K): [in a new window]   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.

View larger version (63K): [in a new window]   Fig. 6. Uptake of anti-ICAM-1 or anti-PECAM-1 immunobeads is PKC-mediated. TNF--activated HUVEC were treated for 30 minutes with vehicle alone (A), 0.1 µM PMA (B), 0.1 µM BIM-1 (C) or 10 µM H-7 (D), then incubated with anti-ICAM-1 or anti-PECAM-1 immunobeads for 1 hour at 37°C, then fixed and immunostained to double-label surface-bound material (yellow, arrowheads). Arrows denote internalized immunobeads. Uptake of anti-ICAM-1 and anti-PECAM-1 immunobeads was quantified as mean±s.d. by fluorescence microscopy for these treatments, expressed as a percentage of immunobead uptake for the PKC inhibitors (E). For PMA, this is expressed as the total number of internalized particles per cell (F), as the percent internalization was equivalent for control and PMA stimulated cells. *P<0.05.

View larger version (153K): [in a new window]   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.

View larger version (55K): [in a new window]   Fig. 8. Uptake of anti-ICAM-1 immunobeads requires actin regulatory proteins. TNF- activated HUVEC were treated for 30 minutes with vehicle alone (A), 10 µM latrunculin A, 20 µM nocodazole, 10 µM radicicol (B), 10 µM Y-27632 (C) or 0.5 µM wortmannin (D), then incubated with anti-ICAM-1 immunobeads for 1 hour at 37°C, then fixed and immunostained to double-label surface-bound material (yellow, arrowheads). Arrows denote internalized immunobeads. Uptake of anti-ICAM-1 immunobeads was quantified as mean±s.d. by fluorescence microscopy for these treatments, expressed as a percentage of immunobead uptake. Uptake required both Src kinase activity and ROCK activity, since it was inhibited by radicicol and Y27632, but did not appear to require PI-3 kinase activity, as wortmannin had no measurable effect on uptake. *P<0.05.

View larger version (164K): [in a new window]   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.

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