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Am J Physiol Lung Cell Mol Physiol 290: L809-L817, 2006. First published November 18, 2005; doi:10.1152/ajplung.00311.2005
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TRANSLATIONAL PHYSIOLOGY

Control of intracellular trafficking of ICAM-1-targeted nanocarriers by endothelial Na+/H+ exchanger proteins

Silvia Muro,1,2 Madalina Mateescu,3 Christine Gajewski,1 Mary Robinson,2 Vladimir R. Muzykantov,1,2 and Michael Koval4

1Institute for Environmental Medicine, 2Department of Pharmacology and Targeted Therapeutics Program of the Institute of Translational Medicine and Therapeutics, 3Department of Physiology, University of Pennsylvania Medical School, Philadelphia, Pennsylvania; and 4Division of Pulmonary, Allergy, and Critical Care Medicine, Emory University School of Medicine, Atlanta, Georgia

Submitted 15 July 2005 ; accepted in final form 15 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Targeting nanocarriers (NC) loaded by antioxidant enzymes (e.g., catalase) to endothelial cell adhesion molecules (CAM) alleviates oxidative stress in the pulmonary vasculature. However, antioxidant protection is transient, since CAM-targeted catalase is internalized, delivered to lysosomes, and degraded. To design means to modulate the metabolism and longevity of endothelial cell (EC)-targeted drugs, we identified and manipulated cellular elements controlling the uptake and intracellular trafficking of NC targeted to ICAM-1 (anti-ICAM/NC). BAPTA, thapsigargin, amiloride, and EIPA inhibited anti-ICAM/NC uptake by EC and actin rearrangements induced by anti-ICAM/NC (required for uptake), suggesting that member(s) of Na+/H+ exchanger family proteins (NHE) regulate these processes. Consistent with this hypothesis, an siRNA specific for the plasmalemma NHE1, but not the endosome-associated NHE6, inhibited actin remodeling induced by anti-ICAM/NC and internalization. Anti-ICAM/NC binding to EC stimulated formation of a transient ICAM-1/NHE1 complex. One hour after uptake, ICAM-1 dissociated from NHE1, and anti-ICAM/NC were transported to NHE6-positive vesicles en route to lysosomes. Inhibition of PKC (an activator of intracellular NHE) accelerated nanocarrier lysosomal trafficking. In contrast, monensin, which enhances the endosomal sodium influx and proton efflux maintained by NHE6, inhibited delivery of anti-ICAM/NC to lysosomes by switching their trafficking to a plasma membrane recycling pathway. This markedly prolonged the protective effect of catalase-coated anti-ICAM/NC. Therefore, 1) NHE1 and NHE6 regulate distinct phases of anti-ICAM/NC uptake and trafficking; 2) pharmacological agents affecting these regulatory elements alter the itinerary of anti-ICAM/NC intracellular trafficking; and 3) these agents modulate duration of the therapeutic effects of targeted drugs.

immunoglobulin superfamily cell adhesion molecules; vascular immunotargeting; oxidative stress; endocytosis; sodium proton exchangers

PULMONARY ENDOTHELIUM, which is vulnerable to oxidative stress, represents a specific therapeutic target for protection by antioxidant enzymes, such as catalase and superoxide dismutase (16, 25, 42). For example, vascular immunotargeting of catalase conjugated with monoclonal antibodies to endothelial cell adhesion molecules (CAM) protects against acute pulmonary vascular oxidative stress in animal models (2, 10, 22). In addition to the primary antioxidant effect of targeted antioxidants, a secondary effect of immunoconjugates may be to inhibit leukocyte transmigration mediated by CAM (12, 54). This may provide an additional benefit in attenuating the inflammatory response that accompanies pulmonary oxidative stress in conditions such as acute lung injury/acute respiratory distress syndrome, hyperoxia, and ischemia-reperfusion injury (14).

An immunoglobulin superfamily transmembrane glycoprotein, ICAM-1, is a good candidate for immunotargeting therapeutics to the pathologically altered endothelium, since ICAM-1 is expressed on the luminal surface of endothelial cells (EC) and is upregulated and functionally involved in pathological conditions, including inflammation, thrombosis, and vascular oxidative stress (12, 38). Reporter cargoes and experimental therapeutics, such as catalase, conjugated with anti-ICAM or loaded onto ICAM-targeted nanocarriers (NC), 1) bind to and protect EC against oxidants; 2) accumulate in the pulmonary endothelium after systemic injection; and 3) undergo enhanced targeting to inflamed endothelium (2, 35–37).

The molecular mechanisms regulating cellular uptake, traffic, and metabolism of targeted drugs represent key parameters that determine efficacy, duration, and side effects. For example, we found that lysosomal proteolysis terminates the antioxidant effects of catalase targeted to endothelial CAM within a few hours after binding to cells (36, 52). This is due to the fact that multivalent anti-ICAM/NC and protein conjugates are internalized by EC, which occurs via a novel endocytic pathway called CAM-mediated endocytosis (41). ICAM-1 clustering by multivalent anti-ICAM/NC triggers actin rearrangements and internalization mediated through a multipronged kinase cascade involving PKC, src kinase, and Rho-dependent kinase (ROCK) (41). CAM-mediated endocytosis does not require clathrin or caveolin; however, dynamin is required for internalization (41).

We also found that anti-ICAM/NC uptake and actin stress fiber formation were inhibited by amiloride (41), a pleiotrophic agent that inhibits multiple classes of ion channels, including sodium proton exchangers (Na+/H+ exchanger proteins or NHE) and epithelial sodium channels (ENaC) (20). Amiloride suppresses macropinocytosis in some cell types (23) and inhibits actin stress fiber formation mediated by NHE1 (57), a 91-kDa transmembrane sodium proton exchanger protein, also endowed with actin cross-linking and other ion channel-independent activities, which operates downstream of ROCK-mediated phosphorylation (11, 45). These data, together with the fact that NHE1 is ubiquitously expressed and predominantly localized to the plasma membrane (5), suggest that NHE1 may regulate the initial phases of anti-ICAM/NC endocytosis by EC.

In addition, there are NHE isoforms localized to intracellular compartments that may intervene in anti-ICAM/NC trafficking subsequently to internalization (6, 7, 32, 43). For instance, Nhx1 (an NHE6-related yeast ortholog) is localized to late endosomes and is involved in delivery of endocytosed cargo to lysosomes (6, 7). Whether mammalian NHE isoforms (i.e., intracellular NHE6) play a similar role in regulating vesicular trafficking is not known at present; however, this is plausible given that both Nhx1 and intracellular NHE are required to maintain endosome pH, ion homeostasis, and osmotic balance (5, 43).

In the present study, we used molecular and pharmacological approaches to define potential roles for NHE and endosome Na+/H+ balance in actin rearrangements, endocytosis, and intracellular trafficking of anti-ICAM/NC in EC. By altering endosome homeostasis, we were able to switch the intracellular trafficking of anti-ICAM/NC from a lysosomal degradation pathway to a plasma membrane recycling pathway that preserved anti-ICAM/NC activity. This provides a basis to design a means to prolong the therapeutic effect of ICAM-1-targeted catalase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. MAb to human ICAM-1 were MAb R6.5 (28) and LB-2 (Santa Cruz Biotechnology, Santa Cruz, CA). Polyclonal antibodies to human early endosome antigen (EEA)-1, lysosome-associated membrane protein-1, NHE1, or NHE6 were from Affinity BioReagents (Golden, CO), BD Biosciences/Pharmingen (Franklin Lakes, NJ), or Chemicon International (Temecula, CA). Secondary fluorescent antibodies were from Jackson ImmunoResearch (West Grove, PA) and Molecular Probes (Eugene, OR). Green fluorescent polystyrene (latex) nanospheres, 100 nm in diameter, were from Polysciences (Warrington, PA). Unless otherwise stated, other reagents were from Sigma (St. Louis, MO).

Preparation of anti-ICAM/NC. NC were prepared as described (36) by coating on fluorescently labeled polystyrene nanospheres with either anti-ICAM alone (anti-ICAM/NC) or anti-ICAM and biotinylated catalase at 1:0.5 molar ratio (anti-ICAM/NC/catalase). The final effective diameter of the resulting NC was determined by dynamic light scattering (56). In each case, these protocols yielded preparations with a diameter ranging from 100 to 300 nm.

Cell culture. Internalization of anti-CAM/NC occurs via a common pathway, CAM endocytosis, in a variety of CAM-positive cell types (22, 36, 37, 40, 41). Human umbilical vein EC (HUVEC) and an endothelial-like cell line, EAhy926 (13), were selected to study the uptake and trafficking of anti-ICAM/NC since they provided insights that have subsequently correlated with in vivo models for endothelial targeting of anti-ICAM/NC (37). HUVEC (Clonetics, San Diego, CA) and EAhy926 were cultured at 37°C, 5% CO2, and 95% relative humidity in supplemented medium 199 or DMEM (GIBCO-BRL, Grand Island, NY), respectively (36). Cells were seeded onto 12-mm2 gelatin-coated coverslips in 24-well plates and were treated overnight with TNF-{alpha} before experiments. TNF-{alpha} treatment upregulates ICAM-1 expression; therefore, it enhances anti-ICAM/NC binding to HUVEC, yet it does not affect their internalization kinetics or trafficking (37, 41).

Internalization, trafficking, and stability of anti-ICAM/NC. TNF-{alpha} activated, confluent HUVEC were incubated at 4°C for 30 min with green-labeled anti-ICAM/NC to enable binding to the cell surface. The cells were then washed, warmed to 37°C for various periods of time, cooled to 4°C, washed, and fixed with 2% paraformaldehyde at room temperature for 15 min. Cells were then treated with Texas red goat anti-mouse IgG, which binds anti-ICAM. This protocol preferentially labels anti-ICAM/NC bound to the cell surface vs. internalized NC that can only be labeled upon cell permeabilization with 0.2% Triton X-100 (39). Alternatively, to visualize green fluorescent anti-ICAM/NC in ICAM-1-enriched sites at the plasma membrane, cells were permeabilized with 0.2% Triton X-100 at room temperature, washed, and labeled with red fluorescent LB-2, which recognizes the ICAM-1 cytoplasmic domain (37).

The samples were analyzed with a Nikon Eclipse TE2000-U fluorescence microscope using a x40 PlanApo objective and filters optimized for FITC, Texas red, and Alexa Fluor 350 fluorescence. Images were obtained with a Hamamatsu Orca-1 charge-coupled device camera and analyzed using ImagePro 3.0 software. Merged micrographs were scored automatically by image analysis to obtain the percentage of cell-associated particles that were internalized, as previously described (41, 56).

To examine the effect of inhibitors on uptake and/or trafficking, TNF-{alpha}-activated HUVEC were pretreated at 37°C for 30 min in the presence of either 3 mM amiloride, 20 µM EIPA, 25 µM monensin, 5 µM BAPTA, 1 µM thapsigargin (TG), or 10 µM H7 (a PKC inhibitor). To stain filamentous actin, fixed, permeabilized cells were labeled with phalloidin conjugated to red Alexa Fluor 594.

Given that the fluorophore is embedded in polystyrene particles, green fluorescence emitted by beads used in these protocols is not significantly affected by pH changes. Hence, to identify compartments containing internalized anti-ICAM/NC, TNF-{alpha}-activated HUVEC were incubated with anti-ICAM/NC as described above. After surface labeling of nonpermeabilized cells with blue Alexa Fluor 350 goat anti-mouse IgG, the cells were permeabilized by 15-min incubation with 0.2% Triton X-100 at room temperature, washed, and labeled with polyclonal rabbit anti-NHE1, anti-NHE6, or anti-EEA-1, followed by Texas red goat anti-rabbit IgG. Alternatively, HUVEC lysosomes were labeled with Texas red dextran (10,000 mol wt) internalized by fluid-phase endocytosis (36).

To determine the intracellular stability of anti-ICAM/NC, internalized particles were counterstained with Texas red goat anti-mouse IgG that recognize nondegraded anti-ICAM. Colocalization and particle stability were quantified using the same image analysis utilized to measure anti-ICAM/NC uptake described above.

Coimmunoprecipitation. Cells on 60-mm dishes were washed twice with PBS at 4°C, scraped and resuspended in cold PBS, and centrifuged at 200 g for 5 min. The cells were then resuspended in PBS containing 0.5% Triton X-100, 0.02% SDS, 1 mM PMSF, and 1:100 protease inhibitor cocktail (Sigma) and lysed for 30 min. The cell lysate was precleared by being treated for 1 h with protein A agarose (Invitrogen, Carlsbad, CA) followed by microcentrifugation (16,000 g for 5 min at 4°C). The cleared lysate was incubated with primary antibody at 4°C for 1 h, subsequently incubated with protein A agarose for 1 h, and then microfuged. The pellet was washed three times with PBS, resuspended in SDS-PAGE sample buffer, and analyzed by PAGE and immunoblot.

Small interfering RNA treatment. Predesigned, single-stranded small interfering RNA (siRNA) oligonucleotides to human NHE1 and NHE6 and control oligonucleotides were from Ambion (Austin, TX). Sense and antisense oligonucleotides were resuspended to 100 µM final concentration in annealing buffer and annealed at 37°C for 1 h. For each double-stranded RNA oligo, 2 µg were added to 50 µl of serum-free medium containing 5 µl of GTS Gene Silencer Reagent (Gene Therapy Systems, San Diego, CA), incubated for 5 min at room temperature, and then added to EAhy926 cells plated on 60-mm dishes in 1 ml of medium. After overnight incubation, the medium was changed to medium containing TNF-{alpha}, and the cells were further incubated at 37°C for 16–24 h before further experimental manipulation.

Antioxidant protection by anti-ICAM/NC/catalase. The antioxidant effect of anti-ICAM/NC/catalase was tested at different periods of time after their internalization within control HUVEC or with cells treated with 25 µM monensin. After internalization, the cells were incubated for 15 min at room temperature with 5 mM H2O2 in phenol red-free RPMI. The cells were washed after H2O2 treatment, incubated with 0.1 mM calcein AM and 1 mM ethidium bromide (Live/Dead kit; Molecular Probes) for 15 min at 37°C, and finally scored as percentage of surviving (calcein positive/ethidium negative) cells.

Statistics. Unless otherwise stated, the data were calculated as the means ± SD from a minimum of 70 cells from two independent experiments. Statistical significance was determined by Student's t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NHE1 regulates endocytosis of anti-ICAM/NC. Inhibition of anti-ICAM/NC endocytosis and concomitant actin stress fiber formation by amiloride suggested a role for NHE1 in CAM-mediated endocytosis (41). However, amiloride is a potent inhibitor of other ion channels, such as ENaC (20). Thus we examined the effect of other agents that alter Na+/H+ exchange in cells (EIPA and monensin) on anti-ICAM/NC endocytosis by HUVEC. EIPA, but not monensin, significantly inhibited anti-ICAM/NC uptake to a level comparable with amiloride (Fig. 1). Inhibitors of Ca2+ signaling (BAPTA and TG) also suppressed anti-ICAM/NC internalization by HUVEC. This suggests a possible role for calmodulin/Ca2+ signaling in actin reorganization induced by the anti-ICAM/NC. Calmodulin binds to the NHE1 cytosolic domain upon activation at sites where actin polymerization occurs (45). In agreement with this hypothesis, EIPA inhibited actin filament reorganization induced by binding of anti-ICAM/NC to the cells. To further confirm these results, we used an endothelial-like cell line, EAhy926, transfected with siRNA corresponding to either NHE1 or NHE6, which specifically decreased the expression of these proteins (Fig. 2). Disruption of NHE1, but not NHE6, inhibited anti-ICAM/NC uptake and impaired stress fiber formation.


Figure 1
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  		Fig. 1. Na+/H+ exchanger protein (NHE) inhibitors and Ca2+ blockers suppress cell adhesion molecule (CAM)-mediated endocytosis and actin remodeling. A–F: TNF-{alpha}-activated human umbilical vein endothelial cells (HUVEC) were incubated with fluorescent anti-ICAM/nanocarriers (NC) for 1 h at 37°C in either control medium (A) or medium containing amiloride (B), EIPA (C), monensin (D), BAPTA (E), or thapsigargin (TG; F), fixed, and counterstained with Texas red-labeled goat anti-mouse IgG. Double-labeled yellow particles represent anti-ICAM/NC bound to the cell surface (arrows), and single-labeled green particles are anti-ICAM/NC internalized within the cells (arrowheads). Bar = 10 µm. G: quantification of CAM-mediated endocytosis, calculated as means ± SD (n ≥ 70 cells from 2 independent experiments) (*P < 0.05). H–O: cells were incubated with anti-ICAM/NC in either the absence (H–K) or presence (L–O) of EIPA for 0 min (H and L), 5 min (I and M), 1 h (J and N), or 3 h (K and O) and fixed, and F-actin was labeled using Alexa Fluor 594-conjugated phalloidin. Control cells showed formation of stress fibers (arrows) induced by anti-ICAM/NC as opposed to EIPA-treated cells. Bar = 10 µm.

 

Figure 2
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  		Fig. 2. NHE1-specific small interfering RNA (siRNA) inhibits internalization and actin reorganization induced by anti-ICAM/NC. A and B: EAhy926 cells were transfected with siRNA targeted to either a nonrepresented sequence (control), NHE1, or NHE6, and then incubated for 2 days and harvested, and the amount of NHE1 or NHE6 protein was determined by immunoblot (IB). By densitometry (B), siRNA significantly reduced protein expression to ~40% of control values (P < 0.05, n = 3). C: cells treated with control, NHE1, or NHE6 siRNA were incubated for 24 h and then assayed for the amount of anti-ICAM/NC internalized during a 1-h incubation at 37°C. Data are means ± SE (n ≥ 70 cells from 2 independent experiments) normalized to the level of internalization for control transfected cells (*P < 0.01). D–F: cells treated with control (D), NHE1 (E), or NHE6 (F) siRNA were incubated for 24 h and then assayed for actin stress fibers (arrows) induced by anti-ICAM/NC using by Alexa Fluor 594-conjugated phalloidin. Bar = 10 µm. Con, control.

 
Consistent with a role for NHE1 in regulating uptake by CAM-mediated endocytosis, HUVEC incubated for 15 min at 37°C in the presence of anti-ICAM/NC showed a redistribution of NHE1, which colocalized with both membrane-bound and internalized NC, as determined by immunofluorescence microscopy (Fig. 3). In contrast, there was significantly less colocalization of anti-ICAM/NC with NHE6 at this time point. The change in NHE1 distribution and colocalization with anti-ICAM/NC suggested that ICAM-1 clustering induced the formation of an NHE1/ICAM-1 complex, which was confirmed by coimmunoprecipitation. Cells incubated with anti-ICAM/NC showed an increased amount of NHE1 that coimmunoprecipitated with ICAM-1. Interestingly, pretreatment of cells with EIPA did not inhibit NHE1/ICAM-1 complex formation, despite the inhibitory effect of EIPA on uptake and cytoskeletal rearrangement, suggesting that NHE1/ICAM-1 interaction was a distinct event upstream from these processes.


Figure 3
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  		Fig. 3. Clustering by anti-ICAM/NC induces complex formation between ICAM-1 and NHE1. A and B: TNF-{alpha}-activated human umbilical vein endothelial cells (HUVEC) incubated in the absence (A) or presence (B) of anti-ICAM/NC, fixed, and immunostained for NHE1. Anti-ICAM/NC induced clustering of NHE1 (arrowheads). Bar = 10 µm. C: cells were incubated with anti-ICAM/NC for 15 min at 37°C, fixed, and then triple labeled to show surface-localized anti-ICAM/NC (Alexa Fluor 350, blue), NC (green), and NHE1 or NHE6 (Texas red). White particles represent sites of NHE1 clustering by anti-ICAM/NC at the cell surface (arrow in high-magnification insets), whereas yellow color represents colocalization within intracellular vesicles (arrowhead in insets). Bar = 10 µm; inset bar = 1 µm. D: quantification of cells labeled as in C, showing the percentage colocalization of markers (NHE1 or NHE6) with anti-ICAM/NC. E: control cells or cells treated with anti-ICAM/NC for 30 min at 4°C were harvested, solubilized, and then immunoprecipitated (IP) with antibodies against the cytosolic domain of ICAM-1. By immunoblot, cells incubated with anti-ICAM/NC formed coimmunoprecipitable NHE1/ICAM-1 complexes, as quantified in F, where data are means ± SE (n = 3). Pretreatment of cells with EIPA had little effect on NHE1/ICAM-1 complex formation. G: at steady state, in the absence of anti-ICAM/NC, few ICAM-1 molecules form a complex with NHE1. NHE1/ICAM-1 complex formation is induced by binding of anti-ICAM/NC, which clusters ICAM-1 in the plasma membrane. In this model, the signal cascade initiated by anti-ICAM/NC binding to ICAM-1 triggers the formation of actin fibers through interactions with the cytosolic domain of NHE1. Uptake and stress fiber formation were inhibited by EIPA; however, NHE1/ICAM-1 complex formation was not. ERM, ezrin, radixin, moesin protein family; {alpha}-Act, {alpha}-actinin.

 
Sorting of internalized anti-ICAM/NC to NHE6-positive endosomes. Anti-ICAM/NC remained in NHE1-positive compartments during ~30 min after internalization, but both coimmunoprecipitation of NHE1 and ICAM-1 and colocalization of NHE1 and anti-ICAM/NC were markedly decreased 1 h after internalization (Fig. 4). The dissociation of NHE1, ICAM-1, and anti-ICAM/NC coincided with an increase in colocalization of anti-ICAM/NC with NHE6: 1 h after internalization, ~60% of anti-ICAM/NC were localized in an NHE6-positive compartment. At this time point, >80% of internalized anti-ICAM/NC were in EEA-1-positive endosomes (see Fig. 6), suggesting partial remodeling of the NHE1 and NHE6 composition of endocytic vesicles containing anti-ICAM/NC. Although NC localized to NHE6-positive vesicles at 1 h after internalization, we found that ICAM-1 did not form complexes retrievable with anti-NHE6 (Muro, Muzykantov, and Koval, unpublished observations). Consistent with the lack of ICAM-1 binding to NHE6, we previously found that ICAM-1 had recycled to the plasma membrane 1 h after internalization (37).


Figure 4
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  		Fig. 4. Transport of anti-ICAM/NC from NHE1- to NHE6-positive endosomes. A–D: HUVEC were incubated with anti-ICAM/NC at 37°C for either 30 min (A and C) or 60 min (B and D) and then fixed and immunostained for either NHE1 (A and B) or NHE6 (C and D). Internalized anti-ICAM/NC colocalizing with either NHE1 or NHE6 are denoted by arrowheads in high-magnification insets. Bar = 10 µm; inset bar = 1 µm. E: quantification of anti-ICAM/NC colocalized with either NHE1 or NHE6 at 30 or 60 min after internalization (means + SD, n ≥ 70 cells from 2 independent experiments). F and G: cells treated with anti-ICAM/NC at 37°C for 15 or 60 min, harvested, solubilized, and immunoprecipitated with anti-ICAM or anti-NHE1 antibodies. By immunoblot, the amount of coimmunoprecipitable NHE1/ICAM-1 complexes decreased with increasing time at 37°C, as quantified in G, where data are means ± SE (n = 3) and the dashed line corresponds to the amount of NHE1/ICAM complex formed at the plasma membrane (see Fig. 3, E and F).

 

Figure 6
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  		Fig. 6. Monensin enhances anti-ICAM/NC recycling to the plasma membrane. HUVEC were incubated with anti-ICAM/NC (green) for 1 (A and D), 2 (B and E), or 3 h (C and F) in control (A–C) or monensin-containing medium (D–F), fixed, immunolabeled for surface-bound anti-ICAM/NC (blue), permeabilized, and finally immunostained for an early endosome marker, early endosome antigen (EEA)-1 (red). Arrowheads denote anti-ICAM/NC localized to EEA-1-positive endosomes. Arrows denote anti-ICAM/NC localized to the plasma membrane. Bar = 10 µm. G: transport of anti-ICAM/NC through early endosomes was comparable for control cells (black bars) and monensin-treated cells (gray bars). H: cells treated with monensin show the majority of anti-ICAM/NC localized to the plasma membrane 3 h after internalization. In contrast, control cells showed few anti-ICAM/NC localized to the plasma membrane; instead, anti-ICAM/NC internalized by these cells were delivered to lysosomes (see Fig. 5). Means ± SD of n ≥ 70 cells from 2 independent experiments.

 
NHE6 mediates Na+/H+ exchange in early and recycling endosomes, which has the potential to regulate acidification and osmolarity in these compartments and, by analogy to the yeast ortholog Nhx1, lysosomal biogenesis (6, 7). Although monensin did not inhibit anti-ICAM/NC internalization (Fig. 1), it enhances Na+ influx and H+ efflux from endosomes and other intracellular compartments, which mimics the activity of a constitutively activated intracellular NHE. Given this, we examined the effect of monensin on the intracellular trafficking of anti-ICAM/NC. Monensin markedly altered anti-ICAM/NC trafficking. At 3 h after internalization, <15% of internalized anti-ICAM/NC were transported to lysosomes in monensin-treated cells compared with ~75% of internalized anti-ICAM/NC transported to lysosomes in untreated control cells (Fig. 5). In contrast to the monensin effect, EIPA did not inhibit lysosomal trafficking of anti-ICAM/NC (Fig. 5), which is consistent with a reported low sensitivity of intracellular NHE to this drug as opposed to NHE1 (45, 47). In addition, pharmacological inhibition of PKC by H7 resulted in accelerated anti-ICAM/NC trafficking to lysosomes (Fig. 5). Because PKC is required for NHE activation (45), inhibiting PKC should inhibit NHE6 activity. The contrasting effects of monensin and H7 on lysosomal delivery of anti-ICAM/NC are consistent with a potential role for NHE6 in regulating this process.


Figure 5
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  		Fig. 5. Monensin inhibits delivery of anti-ICAM/NC to lysosomes. HUVEC were preincubated with Texas red dextran to label lysosomes and then further incubated with fluorescent anti-ICAM/NC (green) for 1 (A, D, G, J), 2 (B, E, H, K), or 3 h (C, F, I, L) in control medium (A–C) or medium containing EIPA (D–F), H7 (G–I), or monensin (J–L), fixed, and imaged by fluorescence microscopy. Control cells transported anti-ICAM/NC to Texas red-labeled lysosomes (arrows), which was accelerated by H7. In contrast, anti-ICAM/NC internalized by monensin-treated cells showed little colocalization with lysosomes (arrowheads). Bar = 10 µm. M: quantification of anti-ICAM/NC delivery to Texas red-labeled lysosomes. Shown are the means ± SD of n ≥ 70 cells from 2 independent experiments. By 3 h, the majority of internalized anti-ICAM/NC was transported to lysosomes by control cells (black bars) and EIPA-treated cells (lined bars) with comparable kinetics. In contrast, cells treated with H7 (white bars) had more rapid transport of NC to lysosomes, and cells treated with monensin showed little, if any, NC delivery to lysosomes (gray bars).

 
In contrast to the effect of monensin on lysosomal trafficking of internalized anti-ICAM/NC, there was little effect of monensin on anti-ICAM/NC sorting to EEA-1-positive endosomes (Fig. 6). Because we recently found that internalized ICAM-1 is recycled to the plasma membrane after dissociating from anti-ICAM/NC (37), we tested whether monensin stimulated recycling of anti-ICAM/NC to the plasma membrane. Consistent with this, cells treated with monensin recycled >70% of the internalized anti-ICAM/NC to the plasma membrane compared with control cells, which showed very little anti-ICAM/NC recycling (Fig. 6). Moreover, the majority of recycled NC resided in ICAM-1-enriched sites at the plasma membrane (80 ± 9% of recycled NC), suggesting that monensin inhibits anti-ICAM/NC dissociation from ICAM-1. Therefore, monensin induced the recycling of ICAM-1 complexed to anti-ICAM/NC, which ordinarily does not occur unless ICAM-1 dissociates from the immunoconjugate (37).

Given that lysosomal degradation is the major mechanism that inactivates anti-ICAM/NC based therapeutic agents (36), the results described above suggested that a "monensin switch" from lysosome delivery to plasma membrane recycling would decelerate degradation of drugs delivered by anti-ICAM/NC. As shown in Fig. 7, this was the case by two criteria. First, using an immunofluorescence assay for degradation of antibodies coating anti-ICAM/NC, we found that internalized anti-ICAM was preserved in monensin-treated cells (Fig. 7). Second, the ability of anti-ICAM/NC coated with catalase (anti-ICAM/NC/catalase) to protect HUVEC from H2O2-induced injury was markedly prolonged by monensin, where monensin-treated cells showed more than a threefold prolongation of the duration of antioxidant protection by anti-ICAM/NC/catalase compared with control cells. Thus, monensin treatment, by diverting anti-ICAM/NC/catalase from lysosomes to a recycling pathway, decreased their degradation and prolonged the duration of their antioxidant effect.


Figure 7
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  		Fig. 7. Monensin delays anti-ICAM/NC degradation and prolongs the antioxidant effect of anti-ICAM/NC/catalase. A–F: control or monensin-treated HUVEC were incubated with fluorescent anti-ICAM/NC for 1 (A and D), 2 (B and E), or 3 h (C and F), fixed, permeabilized, and immunostained for intact anti-ICAM using Texas red-labeled anti-mouse IgG. Intact yellow anti-ICAM/NC are denoted by arrows, and NC with degraded anti-ICAM are denoted by arrowheads. Bar = 10 µm. G: quantification of anti-ICAM/NC degradation. Relative stability was determined as the percent of yellow anti-ICAM/NC, determined as means ± SD of n ≥ 70 cells from 2 independent experiments. Nearly all of the anti-ICAM coating was degraded by control cells (black bars) after 3 h at 37°C. In contrast, most of the anti-ICAM/NC were intact at this time point in the case of monensin-treated cells (gray bars). H: control HUVEC (black bars) or HUVEC treated with monensin (gray bars) were incubated with anti-ICAM/NC/catalase for various periods of time and then challenged with H2O2 to induce oxidative injury. Percent of cell survival was quantified by fluorescence microscopy using the Live/Dead assay from at least 500 cells/condition and represent means ± SD. Monensin treatment significantly prolonged antioxidant protection of anti-ICAM/NC/catalase.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Targeting therapeutic cargoes to endothelial CAM provides a platform for the targeted delivery of drugs to the vascular endothelium (2, 10, 22, 35, 38, 42). There are several approaches to the production of therapeutic agents targeted to CAM, including pharmacologically active NC in the 100- to 300-nm diameter size range conjugated to anti-CAM antibodies (anti-CAM/NC). NC offer several advantages for drug delivery, including size, permitting intravascular administration, multifunctionality, modularity, and the ability to be targeted to specific cells (24). The efficacy of this drug delivery system has been demonstrated by its capacity to protect endothelial cells from oxidant-induced toxicity both in vitro and in vivo (2, 22, 36, 37, 56). Results of this study indicate that the endothelial NHE, NHE1 and NHE6, regulate the uptake, intracellular trafficking, and subsequent metabolism of anti-ICAM/NC (Fig. 8). Our data further support a differential role for plasma membrane NHE1 vs. intracellular NHE6 regulating early vs. late steps of CAM-mediated endocytosis, i.e., internalization vs. vesicular trafficking.


Figure 8
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  		Fig. 8. Model for endocytosis and sorting of anti-ICAM/NC. In control cells, NHE6 exchanges Na+ for H+ to help regulate endosome acidification by vacuolar H+/ATPase (vATPase). Acidification in the early endosome favors dissociation of anti-ICAM/NC from ICAM-1 and NHE1, thus permitting delivery of anti-ICAM/NC to lysosomes and recycling of ICAM-1 to the plasma membrane. Monensin enhances Na+/H+ exchange independently of NHE6 and thus inhibits endosome acidification and increases endosome Na+ content, resulting in a net influx of H2O into the endosome lumen. This inhibits anti-ICAM/NC dissociation from ICAM-1, prevents lysosomal maturation, and favors recycling of internalized anti-ICAM/NC to the plasma membrane.

 
Clustering of ICAM-1 by multivalent anti-ICAM/NC stimulated the formation of an ICAM-1/NHE1 complex, which was a key event required for remodeling of the actin cytoskeleton and CAM-mediated endocytosis, and thus responsible for intracellular delivery of anti-ICAM/NC. Coclustering of ICAM-1 and NHE1 was upstream of CAM-mediated endocytosis and stress fiber formation since ICAM-1/NHE1 complexes formed in the presence of EIPA, which inhibited the latter two processes. Previous studies have demonstrated interaction of ICAM-1 with {alpha}-actinin and ezrin, radixin, moesin protein family (ERM), as well as stimulation of actin filament formation by ICAM-1 clustering (1, 3, 9). In light of our results, it seems likely that NHE1 may function as a docking element mediating cytoskeletal rearrangements via recruiting ERM scaffold proteins (11, 57). The RhoA-ROCK pathway [which can be activated by ICAM-1 clustering (41)] is known to promote NHE1 activation via serine phosphorylation (45). This prompts conformational changes that can allow Ca2+-dependent binding of calmodulin to NHE1 at sites of actin reorganization (45). Given that both ROCK (41) and Ca2+ inhibitors attenuate anti-ICAM/NC uptake (Fig. 1), it is plausible that additional signal cascades initiated by ICAM-1 clustering activate NHE1. The pathway by which ICAM-1 clustering induced by anti-ICAM/NC activates NHE1 remains to be studied. One possibility is that this is comparable to the plasma membrane and actin reorganization observed in EC upon ICAM-1 engagement by leukocyte lymphocyte function antigen-1, which regulates inflammation and endothelial permeability (3, 9, 17).

Although roles for intracellular NHE in regulating endocytosis and membrane trafficking are just beginning to be elucidated, some insights can be gained from studies of the yeast ortholog Nhx1 (6, 7). Yeast deficient in Nhx1 expression, so-called E type vacuolar protein sorting mutants, are incapable of sorting endocytosed material to the vacuole (6). Nhx1 and mammalian NHE isoforms (such as NHE6 in EC) play a role in maintaining lumen pH balance in endosomes by mediating the exchange of Na+ and K+ for H+ (6, 7, 43). Whereas changes in luminal acidity are known to regulate some elements of intracellular trafficking, such as ligand-receptor dissociation and lysosome enzyme function (29, 31, 34), regulation of vesicle formation, budding, and targeting by changes in luminal pH are not well understood.

By analogy to its yeast ortholog, Na+/H+ exchange by NHE6 (which has been localized to early and recycling endosomes positive for EEA-1 and rab11) is believed to maintain moderate low pH within the endosomal lumen, and to regulate ion balance and osmolarity, required for ligand/receptor dissociation and vesicular trafficking (7, 43). This is likely to be the case for intracellular trafficking after CAM-mediated endocytosis. For instance, the kinetics of transport of anti-ICAM/NC to an NHE6-enriched compartment, which was relatively slow (~1 h), is consistent with our previous observation that internalized anti-ICAM/NC resided in EEA-1-positive endosomes for 1–2 h before delivery to lysosomes and degradation (36). Therefore, disruption of ICAM/NHE1 and anti-ICAM/NC complexes after internalization may be mediated by NHE6 in endosomal compartments, likely followed by rapid recycling of ICAM-1, and maybe NHE1, to the plasma membrane (Fig. 8). It has also been postulated that further inactivation of NHE6 may prompt acidification of the vesicular lumen and lysosomal biogenesis (15), which is consistent with lysosomal delivery of anti-ICAM/NC (36).

It has long been appreciated that monensin, an ionophore that effectively enhances Na+/H+ exchange in endosomes and mimics constitutively activated NHE6, has the capacity to interfere with vesicle sorting (33). In particular, monensin inhibits trafficking from the cis to medial aspects of the Golgi apparatus (27, 55) and has also been shown to inhibit recycling of many plasma membrane receptors, such as transferrin receptors, LDL receptors, and beta2-adrenergic receptors (4, 26, 49, 53). However, this effect is not universal, since recycling of chemokine coreceptor-5 chemokine receptors and beta1-adrenergic receptors is not inhibited by monensin (26, 48). In most cases, the effect of monensin on receptor recycling is due to the component of receptor traffic that is transported through the Golgi, as opposed to the rab11-dependent recycling pool that bypasses the Golgi apparatus (21, 49, 53), although it remains to be determined whether there are also monensin-sensitive plasma membrane recycling pathways that bypass this compartment.

We found that monensin stimulated the recycling of anti-ICAM/NC to the plasma membrane (Fig. 8). To our knowledge, this is the first demonstration that monensin can stimulate a plasma membrane recycling pathway, which may occur as a reminiscence of ICAM-1 recycling pathway (37). Although one effect of monensin is to alkalinize the endosome lumen, enhanced anti-ICAM/NC recycling was not simply due to increased endosomal pH, since we previously found that chloroquine is also effective at increasing endosome pH; however, endocytosed anti-ICAM/NC are still transported to lysosomes in chloroquine-treated cells (36). Because monensin also enhances ion transport into endosomes (18, 43), one possibility is that endosome ion content may help regulate sorting of membrane trafficking. For instance, unrepressed Na+/H+ exchange by monensin may secondarily lead to Cl– influx to the endosomal lumen to maintain ion balance, which if accompanied by H2O influx, may cause aberrant engorgement of endosomes (Fig. 8), favoring exocytosis (15). However, the precise role for endosome ion content in regulating transport remains obscure at present.

Monensin treatment acted as a "switch" that both inhibited delivery to lysosomes and enhanced plasma membrane recycling of internalized anti-ICAM/NC. This in turn prevented degradation and prolonged the duration of anti-ICAM/catalase/NC to protect against oxidant-mediated toxicity. This offers another method to modulate the duration and, perhaps, efficacy of NC-based therapeutic agents that complements other approaches including: 1) modifying NC geometry to influence internalization rate and traffic, 2) prolonging NC retention time in endosomes by inhibiting delivery to lysosomes using nocodazole, 3) inhibiting NC degradation within lysosomes by increasing lysosome pH using weak bases, and 4) the use of sustained treatment by multiple doses of NC administration (36, 37, 56). Monensin treatment differs from these methods in that it provides a means to enable anti-ICAM/NC to be simultaneously and stably localized to the plasma membrane and recycling endosomal compartments. In the case of anti-ICAM/NC containing catalase, this strategy may help maximize interception of both extracellular and intracellular oxidants. Although toxicity should be taken into consideration as a limiting factor in terms of in vivo therapies (8, 44), pharmacological agents with the ability to inhibit trafficking of endocytosed anti-ICAM/NC to lysosomes and to enhance plasma membrane recycling are likely to provide a useful adjunct to enhance the duration and efficacy of NC-based drug delivery systems. Our findings suggest that understanding roles for NHE in regulating vesicle targeting is a key to the successful implementation of auxiliary pharmacological agents that optimize the application of targeted NC as a drug delivery platform.

From a more general perspective, results of this study are relevant to endothelial biology and transport. For example, NHE1-mediated ICAM-1-dependent cytoskeletal reorganization, similar to that observed in this work, may be involved in regulation of vascular permeability during pathological events. Indeed, actin filament formation controls endothelial barrier properties (46). Furthermore, microvascular endothelial cell NHE have been implicated in ischemia-reperfusion injury by enhancing ICAM-1 surface density via Ca2+ signaling (17), a mediator known to increase microvessel permeability during lung injury (30, 50, 51). Whether NHE also regulate protein transport across the lung epithelial barrier, as in the case of transcytosis involving caveoli or clathrin-coated pits (19), remains to be determined.

In conclusion, NHE1 and NHE6 regulate anti-ICAM/NC-induced actin reorganization and subsequent internalization and vesicular trafficking by EC, which may play physiological and pathophysiological roles in a variety of processes, including endothelial drug delivery. Pharmacological manipulations of these endothelial NHE may help modulate the subcellular delivery, longevity, and effects of therapeutics targeted to endothelium by anti-ICAM/NC.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants R01-HL-71175, P01-HL-079063 (Project 3), and Department of Defense PR-012262 (V. R. Muzykantov), R01-GM-61012 and P01-HL-019737 (to M. Koval), and American Heart Association SDG 0435481N (to S. Muro).


   ACKNOWLEDGMENTS
 
The authors thank John Leferovich (Institute for Environmental Medicine, Univ. of Pennsylvania Medical School, Philadelphia, PA) for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. R. Muzykantov (drug delivery and vascular immunotargeting), Univ. of Pennsylvania School of Medicine, Institute for Environmental Medicine, 1 John Morgan/6068, 3620 Hamilton Walk, Philadelphia, PA 19104 (e-mail: muzykant{at}mail.med.upenn.edu); or M. Koval (cell biology and endocytosis), Emory Univ., Pulmonary Medicine, Ste. 205, 615 Michael St., Atlanta, GA 30322 (e-mail: mhkoval{at}emory.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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