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The Journal of Immunology, 2007, 178, 5879 -5887
Copyright © 2007 by The American Association of Immunologists, Inc.
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Neutrophil Transmigration under Shear Flow Conditions In Vitro Is Junctional Adhesion Molecule-C Independent1

Monica Sircar*,{dagger}, Paul F. Bradfield{ddagger}, Michel Aurrand-Lions{ddagger}, Richard J. Fish§, Pilar Alcaide*, Lin Yang*, Gail Newton*, Deanna Lamont*, Seema Sehrawat*, Tanya Mayadas*, Tony W. Liang, Charles A. Parkos||, Beat A. Imhof{ddagger} and Francis W. Luscinskas2,*

* Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; {dagger} Health, Science and Technology Program, Harvard Medical School, Boston, MA 02115; {ddagger} Department of Pathology and Immunology, Centre Médical Universitaire, Geneva, Switzerland; § Service of Angiology and Haemostasis, Department of Internal Medicine, Geneva University Hospital, Geneva, Switzerland; Raven Biotechnologies, South San Francisco, CA 94080; and || Division of Gastrointestinal Pathology, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-{alpha}, IL-1beta, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN beta2 integrins during transendothelial migration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Leukocyterecruitment to sites of inflammation, immune reactions, and injury involves a multistep adhesive and signaling cascade composed of selectin-mediated rolling, subsequent integrin-mediated arrest followed by diapedesis (transmigration) of the vascular endothelium (reviewed in Ref. 1). During arrest and transmigration of polymorphonuclear leukocyte (PMN)3across inflamed or activated endothelium, the beta2-integrins including LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and p150,95 (CD11c/CD18) interact with the endothelial cell receptors ICAM-1 and ICAM-2 (2, 3). In addition, studies both in vitro and in vivo showed that CD31 (also termed PECAM-1), a member of the Ig superfamily expressed on leukocytes and endothelium, is involved in transmigration through homophilic adhesive interactions (4). Other molecules have been identified that participate in leukocyte recruitment including junctional adhesion molecules (JAMs). JAMs are members of the Ig superfamily and serve as cell-cell adhesion receptors. JAMs engage in homophilic interactions among members or through heterophilic interactions with leukocyte beta1 and beta2 integrins (5). JAM-A, JAM-B, and JAM-C partially localize to cell-cell junctions in endothelium and epithelium and recognize leukocyte integrins LFA-1, VLA-4, and Mac-1, respectively (5). JAM-C also is expressed at different levels on endothelial cells of lymphoid organs and peripheral tissues, platelets, T cell subsets, NK cells, and dendritic cells but not on PMN (5). JAM-C is of particular interest because of its reported role in processes including angiogenesis and tumor growth (6), tumor metastasis (7), atherosclerosis (8), and leukocyte recruitment in animal models of inflammation (9, 10) and obstructive nephropathy (11).

Recent studies have characterized JAM-C heterophilic and homophilic adhesive interactions during leukocyte recruitment. Functionally, PMN transendothelial migration (TEM) to MCP-1 in a Transwell assay was blocked by a soluble JAM-C chimera or by mAb to Mac-1 and ICAM-1, and PMN recruitment was reduced by i.v. infusion of soluble murine JAM-C in a murine model of peritonitis (10). These findings suggest that neutrophil recruitment was partially dependent on adhesive interactions mediated by Mac-1 interactions with two different ligands (10). Other in vivo studies (7) have suggested that JAM-C mediates interactions between tumor cells and endothelial cells via homophilic interactions between JAM-C expressed by a lung carcinoma cell line and vascular endothelial (VE) JAM-C. The same group also reported the up-regulation of JAM-C in both endothelial cells and vascular smooth muscle cells in atherosclerotic lesions of ApoE-null mice (8) and that treatment of HUVEC with oxidized low-density lipoprotein induced transmigration of THP-1, a leukocyte cell line, via interactions between Mac-1 and endothelial cell JAM-C and ICAM-1. Although initial studies examining JAM-C null mice detected a sterility defect (12), there are as yet no published results for JAM-C null mice tested in models of inflammation. In normal human intestinal epithelia, JAM-C is abundantly expressed on the basolateral surface and localizes in desmosomes (13). These authors also demonstrated that mAb to JAM-C, which inhibits JAM-C-Mac-1 interactions, or soluble JAM-C/Fc chimeric molecules significantly delayed PMN transepithelial cell migration.

Live cell imaging experiments have shown that LFA-1 and Mac-1 rapidly redistribute into ring-like structures that colocalize with ICAM-1 during PMN TEM (14, 15). Based on this observation and that Mac-1 binds to JAM-C (13, 16), we examined the hypotheses that JAM-C is required for PMN TEM under shear flow conditions and that JAM-C, like ICAM-1, undergoes transient redistribution during PMN transmigration. Unexpectedly, blocking mAb to JAM-C had no inhibitory effect on PMN transmigration alone and required inhibition of PECAM-1, CD99, and ICAM-1 to detect any contribution by JAM-C mAb. Finally, PMN actively transmigrating induced little or no clustering of JAM-C, whereas ICAM-1 clustering was readily detected as reported previously (14).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

HBSS with Ca2+ and Mg2+ (HBSS+), DPBS (Dulbecco’s PBS) with Ca2+ and Mg2+ (DPBS+) or without (DPBS–), and M199 were obtained from BioWhittaker Bioproducts. Recombinant human TNF-{alpha} was obtained from Genzyme. Human rIL-1beta was a gift from Biogen Idec (Cambridge, MA). Histamine and Gram-negative bacterial LPS was purchased from Sigma-Aldrich (Escherichia coli, serotype 055:B5). Human serum albumin (albuminate 25%) was obtained from Baxter Healthcare. O-Me (8-pCPT-2'O-Me-cAMP) was obtained from BIOLOG Life Sciences Institute. All other chemicals were of the highest grade available from Baker Chemical.

mAbs

The following murine mAbs were used as purified IgG: nonblocking mAb to JAM-A (mAb 1H2A9 (17)), VE-cadherin (Hec-1 (18)); p96 (E1/1 (19)); class I (mAb W6/32, obtained from American Type Culture Collection (ATCC)); blocking anti-ICAM-1 mAb (R6.5, obtained from ATCC (20)); CD99 (hec2) and PECAM-1 (mAb177) (21); mAb to human JAM-C (clone 208206) was obtained from R&D Systems; function-blocking mAb to JAM-C (LUCA14) was described previously (13); and PACA-4 mAb was a gift from Raven Biotechnologies (South San Francisco, CA); three rat mAbs generated against mouse JAM-C that cross-react with human JAM-C (D22, H33, D33) were described previously (22); rabbit anti-ZO-1 and anti-JAM-C polyclonal Abs were obtained from Zymed Laboratories; rabbit anti-von Willebrand factor (vWF) polyclonal Ab was obtained from Sigma-Aldrich. Cy3-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Caltag Laboratories. Alexa 488-conjugated goat anti-mouse IgG was obtained from Invitrogen Life Technologies. The blocking mAb to CD99 (hec2) and the polyclonal Ab to PECAM-1 (177) were gifts from Dr. W. Muller (Weill Medical College, Cornell University, New York, NY).

JAM-C-Mac-1-binding assay

Functionally active human Mac-1 (5 µg/ml (23) was applied to NUNC MaxiSorb plates in HBSS overnight at 4°C, washed once, and incubated in block buffer (HBSS-1% BSA) for 30 min at room temperature (RT) as described (13). During the blocking step, 2 µg/ml JAM-C.Fc (R&D Systems) was preincubated with 5 µg/ml PACA-4 or LUCA14 mAb. JAM-C-Ab mixtures (50 µl) were applied to the appropriate wells and incubated for 1 h at 37°C. Bound JAM-C-Fc was detected by HRP-conjugated goat anti-human Fc (1:1000 in HBSS) followed by colorimetric measurement.

Immunoprecipitation (IP) and blotting of endothelial cell JAM-C

Confluent HUVEC were lysed, subjected to IP using PACA-4 mAb (10 µg), and proteins were separated by SDS-PAGE on a 10% acrylamide gel (Bio-Rad) as described previously (24). Proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell) by wet transfer for 1 h at 4°C at 100 V. The blot was probed with polyclonal Ab to JAM-C (1/300 dilution) and developed using ECL (Pierce Biotechnology).

Construction of enhanced GFP (EGFP)-tagged JAM-C

EGFP was inserted into the hinge region of JAM-C between the membrane proximal C2 domain and the transmembrane region; therefore, this fusion protein left intact the two extracellular Ig domains, the putative phosphorylation sites and the PDZ-binding domain in the C terminus, as described previously (9). This construct when expressed in CHO cells is functional because it binds to JAM-B and these heterophilic interactions are prevented by anti-JAM-C mAb. The JAM-C GFP cDNA was transferred to a lentivirus expression vector, and high-titer virus stocks were produced. These stocks were titrated by a previously described protocol for expression in HUVEC (24). A dose of virus was chosen that routinely gave expression of JAM-C GFP in 70–80% of endothelial cells after 5 days of infection, which resulted in an increase in JAM-C expression of 4.4 ± 1.3-fold (n = 4 separate experiments) as compared with that in uninfected HUVEC. These infected monolayers were morphologically identical to medium- or GFP lentivirus-infected cells as assessed by phase contrast microscopy and permeability measurements (see Fig. 4E). Lentivirus infection did not activate endothelial cells, as evidenced by a lack of induction of E-selectin or VCAM-1 (data not shown). The JAM-C fusion protein was anticipated to migrate at ~72 kDa with the added mass of the EGFP tag, as confirmed by SDS-PAGE gel electrophoresis (see Fig. 4C).


Figure 4
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  		FIGURE 4. Characterization of JAM-C GFP expression and localization in HUVECs. A, HUVECs were infected with lentivirus JAM-C GFP or lentivirus GFP and cultured for 4 days. JAM-C GFP expression was quantified by indirect immunofluorescence and flow cytometry using mAb clone 208206. Solid gray shows nonbinding mAb K16/16; dashed line is JAM-C surface expression level on resting HUVECs; and solid plot is GFP signal from HUVECs infected with JAM-C GFP. B, HUVEC-expressing JAM-C GFP were fixed with 10% buffered formalin, permeabilized, and stained with JAM-C mAb clone 208206 followed by staining with a Cy-3-tagged anti-murine IgG secondary mAb. Stained tissues were examined by confocal microscopy as detailed in Materials and Methods. In the single z-slice shown, JAM-C GFP is localized at both cell-cell borders and the apical surface. Two noninfected cells show staining at junctions but not in the cytosol. C, IP of JAM-C and JAM-C GFP from unstimulated or 4-h TNF-{alpha}-stimulated HUVECs. HUVECs were biotinylated using a Pierce chemiluminescence kit according to manufacturer’s directions, lysed, and JAM-C was immunoprecipitated with Luca14 mAb. Proteins were resolved by SDS-PAGE and biotinylated JAM-C Ag was detected by chemiluminescence (25 ). The JAM-C and JAM-C GFP had the predicted masses of ~40 kDa and ~70 kDa, respectively. D, Immunofluorescence microscopy indicates that the cellular location of adherens junction markers VE-cadherin and tight-junction marker ZO-1 were not altered by expression of GFP or JAM-C GFP. Images were obtained with a x40 objective and epifluorescence microscopy. E, TER of HUVECs is not affected by overexpression of JAM-C or GFP. HUVECs were plated for analysis of TER (Materials and Methods) and infected with medium (control), lentivirus GFP (GFP), or lentivirus JAM-C GFP (JAM-C). TER was monitored in replicate wells for 130 h. TER was then monitored following the addition of a 3',5'-cAMP analog (O-Me, 500 µM) to raise barrier function, or histamine (10 µM) to lower barrier function. There was no significant difference in TER among the medium, GFP, or JAM-C GFP-treated monolayers.

 
Human cell culture and leukocyte isolation

Pooled HUVECs were described previously (24). HUVEC monolayers (subculture 2) were grown to confluence and were used 48 h later. Human PMN were isolated from anticoagulated whole blood obtained from healthy volunteer donors as described previously (25). The Human Use Committee Board of Brigham and Women’s Hospital approved all protocols involving human subjects.

Leukocyte adhesion and transmigration under flow

The parallel plate flow chamber and computer-controlled, digital microscopy system used for leukocyte transmigration assays has been described previously (26). Confluent HUVEC monolayers were treated with 25 ng/ml TNF-{alpha} for 4–6 h and treated with test or control mAb for 30 min before adhesion assays. A bolus of PMN (1 x 106 in 100 µl) was drawn across the HUVEC monolayer at 0.35 ml/min (a calculated shear stress of 0.7 dyne/cm2), and the flow rate was then increased to 0.5 ml/min (1.0 dynes/cm2). This protocol minimizes leukocyte string formation (i.e., secondary adhesion) that otherwise hampers detailed analysis (27). Transmigrated PMN were distinguished from those interacting with the apical surface by their phase-dark morphology (24). The total number of accumulated PMN was determined by counting the number of adherent and transmigrated cells in five to seven random fields. The percentage of TEM was calculated as the total transmigrated PMN ÷ [total adhered + transmigrated PMN] x100.

For detachment assays using immobilized proteins, glass coverslips were coated with 10 µg/ml indicated proteins as described previously (28). PMN were drawn across immobilized proteins at 0.35 ml/min (0.7 dynes/cm2) and paused for 60 s. Flow was re-established and fluid shear stress was increased stepwise every 20 s. PMN remaining adherent after each step in flow was determined, and the percentage of PMN remaining adherent was calculated for each time point.

Live cell fluorescence image acquisition and analysis of JAM-C GFP

Live cell imaging was performed with MetaMorph 4.6 software (Molecular Devices) to control a digital fluorescence imaging system coupled to a Nikon model TE2000-inverted microscope equipped with an Excite fluorescence lamp system (14). For time-lapse experiments, sequential differential interference contrast (DIC) and fluorescent images were collected every 15 s from the same field. Images were analyzed and processed with MetaMorph software, version 5.0, and ImageJ, as described previously (14).

Immunofluorescence microscopy and laser scanning confocal microscopy

Confluent HUVEC monolayers on 12-mm glass coverslips were stimulated for 4 h with medium alone (control) or with medium containing 25 ng/ml TNF-{alpha}, 10 U/ml IL-1beta, and 1 µg/ml LPS, washed three times with cold DPBS+, then fixed for 8 min at RT with 10% neutral-buffered formalin. The fixed cells were permeabilized (DPBS+ with 0.5% Triton X-100 for 15 min at RT) and then incubated for 1 h at 4°C with blocking buffer (DPBS+ with 1% horse serum, 1% goat serum, and 0.1 mg/ml salmon testes DNA). Monolayers were incubated with primary Ab (mAbs, 10 µg/ml in blocking buffer; polyclonal Ab, 2 µg/ml in blocking buffer) overnight at 4°C, washed, then incubated with Cy-3-conjugated goat anti-mouse F(ab')2 IgG or goat anti-rabbit F(ab')2 for 1 h at 4°C. The coverslips were washed three times with DPBS+ and mounted. Immunofluorescence microscopy was performed using MetaMorph 5.0 software (Molecular Devices) to control a digital fluorescence microscope described above. Double staining of HUVEC for vWF and JAM-C was performed as described above, using Cy3-conjugated goat anti-rabbit IgG and Alexa 488-conjugated goat anti-mouse IgG secondary Ab at dilutions of 1/300 and 1/500, respectively. A single z-slice is shown for HUVECs transduced with lentivirus JAM-C GFP (see Fig. 4B). Confocal images were acquired with a Radiance 2100 laser scanning confocal head (Bio-Rad) attached to an inverted microscope (Nikon TE2000) through a x60 oil immersion objective (Nikon PlanApo, 1.4 NA). The confocal iris was set to provide a 0.5-µm slice thickness and samples were scanned along the z-axis from basolateral to apical aspects. Images were analyzed in Lasersharp2000 version 4.3 (Bio-Rad) and ImageJ version 1.31, and figures were generated in Photoshop version 7.0. Bars on confocal images are 20 µm.

Measurement of transendothelial electrical resistance (TER)

HUVECs (subculture 2) were plated at 50% confluence (6,000 cells per 0.8 cm2) on gold microelectrodes and then infected immediately with lentivirus GFP or lentivirus JAM-C GFP. Monolayers were used 4 days after plating when the TER had stabilized. TER was measured with an electrical cell-substrate impedance sensing system (ECIS; Applied BioPhysics) as detailed previously (29). Cell adhesion and spreading of endothelial cells results in an increase in TER, whereas cell retraction, rounding, or loss of cell-cell lateral junction adhesion leads to a decrease (negative) in TER. These measurements provide a sensitive assay that reflects changes in paracellular permeability. TER values from each electrode were pooled and plotted vs time. Data are representative of five separate experiments performed in duplicate.

Data analysis

Data are expressed as mean ± SD. Statistical significance was assumed to be p < 0.05 using one-way ANOVA, or by Student’s t test, where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PACA mAb recognizes human JAM-C and inhibits soluble JAM-C-Mac-1 binding

Anti-JAM-C mAb PACA-4 detects a ~40-kDa band in unstimulated HUVEC lysates as determined by IP and Western blotting. This result is consistent with a previous report (10). We next examined whether PACA-4 mAb blocked JAM-C Fc binding to immobilized human Mac-1. PACA-4 mAb clearly inhibited binding of JAM-C Fc chimera to immobilized active Mac-1. The data in Fig. 1 show that PACA-4 recognizes JAM-C and inhibits JAM-C binding to Mac-1.


Figure 1
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  		FIGURE 1. PACA-4 recognizes human endothelial cell JAM-C and inhibits JAM-C binding to immobilized Mac-1 integrin. A, HUVEC lysates were subjected to IP using PACA-4 mAb, isolated proteins were separated by 10% SDS-PAGE, and JAM-C was detected by Western blot analysis with polyclonal anti-JAM-C Ab as detailed in Materials and Methods. B, Direct binding of JAM-C Fc chimera to immobilized Mac-1 was performed. LUCA14 and PACA-4 mAb inhibit this interaction. Data are mean ± SE of two independent experiments performed in triplicate.

 
JAM-C is expressed on the surface and predominantly in intracellular compartments in HUVEC monolayers

Previous studies have reported that JAM-C is expressed in vascular endothelium and partially localizes to cell-cell borders (10, 30). Our initial goal was to confirm these data in HUVECs using JAM-C mAb. FACS analysis demonstrated that the level of JAM-C surface expression on resting HUVECs is low (Fig. 2A) and does not increase following 4 h (solid lines) or 24 h (dashed lines) of treatment with inflammatory stimuli known to induce an increase in the expression of endothelial cell adhesion molecules such as ICAM-1. We next evaluated the pattern of JAM-C staining in resting and cytokine- or LPS-activated HUVECs by immunofluorescence microscopy. In resting HUVECs, JAM-C staining was low but uniform on the apical surface with moderate junctional staining as compared with VE-cadherin, which localizes to endothelial cell-cell junctions (Fig. 2B). Interestingly, examination of detergent-permeabilized monolayers revealed a significant intracellular pool of JAM-C, which has not been previously appreciated. The staining pattern of JAM-C was not significantly altered by 4- (Fig. 2C) or 24-h (data not shown) treatment with IL-1beta, TNF-{alpha}, or LPS. A similar staining pattern was obtained with anti-JAM-C mAb LUCA14 (data not shown). As a control, staining with VE-cadherin was performed, and its pattern was not altered significantly (Fig. 2B and data not shown).


Figure 2
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  		FIGURE 2. JAM-C expression and localization in HUVECs. A, Confluent HUVEC monolayers were incubated in complete culture medium (sham) or medium containing TNF-{alpha} (20 ng/ml), IL-1beta (10 U/ml), and LPS (1 µg/ml) for 4 (solid line) or 24 h (dashed line). The expression of JAM-C on HUVECs was assessed with two different mAbs by indirect immunofluorescence and FACS analysis (see Materials and Methods). ICAM-1 expression is significantly elevated after 4 (solid line) or 24 h (dashed line) of cytokine treatment (47 ) and serves as a positive control. Data are representative of four or more separate studies. B, JAM-C is detected in resting HUVECs in the intracellular compartment by immunofluorescence microscopy. JAM-C was detected using PACA-4 mAb and secondary Cy-3-tagged F(ab')2 goat anti-mouse mAb. VE-cadherin staining with mAb Hec-1 serves as a marker of junctions. C, HUVEC monolayers were incubated with cytokines or medium for 4 h as described in A above, and the JAM-C expression using PACA-4 mAb and localization was determined with or without detergent permeabilization of cells followed by immunofluorescence microscopy. D, Confocal microscopic analysis of HUVECs stained for JAM-C and vWF Ag. Permeabilized monolayers were stained with JAM-C (PACA-4 mAb, left panel, green) or vWF Ag (red, middle panel). The red and green images were overlaid (merge). Bars, 20 µm.

 
vWF and P-selectin, a well-characterized leukocyte adhesion molecule, are sequestered in endothelial intracellular storage granules termed Weibel-Palade bodies (WPB) (31). We therefore evaluated the staining pattern of JAM-C and vWF in resting, permeabilized HUVEC monolayers. By confocal microscopy, the JAM-C staining was brightest at the basolateral surface in most cells (Fig. 2D; basolateral). Anti-JAM-C mAb LUCA14 gave similar results. In contrast, the staining pattern of vWF Ag was primarily localized to rod-shaped granules throughout the cytoplasm and showed little overlap with JAM-C. Z-sections taken 0.5 µm higher (apical) gave a similar result. Some overlap of signal in the merged image can be seen occasionally adjacent to the nucleus and is likely due to colocalization in the protein biosynthetic pathway of the Golgi apparatus (31). These initial findings suggest that the majority of JAM-C is intracellular and exhibits some colocalization with vWF in WPB storage granules.

Function-blocking mAb to JAM-C do not reduce PMN adhesion or transmigration under shear flow

As a starting point for our analysis, we used a fluid shear detachment assay to measure the ability of purified JAM-C to bind PMN. JAM-C did not support PMN adhesion above the background level seen for control protein A (Fig. 3A), whereas both E-selectin and ICAM-1 retained the majority (>80%) of PMN initially bound across a broad range of laminar shear stresses used to detach cells. PMN adhesion to JAM-C has been reported to be weak as compared with Mac-1 recognition of ICAM-1 and fibrinogen (32), and this effect may explain why the PMN failed to bind in the more stringent detachment assay.


Figure 3
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  		FIGURE 3. Immobilized JAM-C does not support PMN adhesion under shear flow, and function-blocking Abs to JAM-C do not reduce PMN transmigration under shear flow. A, PMN were perfused across the immobilized proteins as described in Materials and Methods. Soluble JAM-C was prepared as previously described (13 ) and does not support PMN adhesion under shear flow conditions, whereas E-selectin and ICAM-1 exhibit robust actions, as reported previously (48 49 ). B and C, The JAM-C mAb LUCA14, PACA-4, D22, H33, and D33 alone do not reduce PMN transmigration; however, PACA-4 and LUCA14 exhibit synergistic inhibition of TEM when combined with mAb to ICAM-1, PECAM-1, and CD99 (combo blk). For comparison, anti-ICAM-1 mAb (R6.5) significantly reduced TEM under shear flow, whereas control nonblocking mAb JAM-A or a combination of mAb to VE-cadherin, p96, and HLA class I had no inhibitory effect (combo cont). PMN transmigration was determined under shear flow conditions as detailed in Materials and Methods. Data are pooled from n = 3–7 separate experiments performed in duplicate.

 
Previous studies reported that PMN chemotactic migration to chemokines in a Transwell assay was partially blocked by soluble JAM-C and by a Mac-1 mAb or PECAM-1 (10). To investigate the role of JAM-C in PMN adhesion and transmigration of cytokine-activated endothelial monolayers under shear flow, we used a panel of function-blocking mAb to JAM-C. Interestingly, mAb LUCA14 and PACA-4, which block Mac-1 binding to immobilized JAM-C (Ref. 13 and Fig. 1), as well as other mAb had no inhibitory effect on PMN transmigration in this system (Fig. 3, B and C). Similarly, mAb D22, H33, D33 to JAM-C, which inhibit interactions between JAM-C and JAM-B in vitro and reduce leukocyte recruitment in murine experimental models of inflammation (9, 33), did not reduce transmigration (Fig. 3B). As a control, function-blocking mAb to ICAM-1 significantly reduced PMN transmigration. We next examined whether JAM-C mAb would synergize with a combination of blocking mAb to ICAM-1, PECAM-1, and CD99 (combo). Interestingly, either JAM-C mAb together with the combo blocking showed greater inhibition than the combo alone, suggesting a small role for JAM-C in PMN transmigration in this system.

JAM-C GFP localizes to endothelial cell-cell junctions but does not enhance endothelial cell transelectrical resistance or leukocyte adhesion and transmigration

Because JAM-C does not localize focally to endothelial cell-cell junctions (Fig. 2) or support adhesion under shear flow conditions (Fig. 1A), and because function-blocking mAb to endothelial JAM-C alone do not reduce PMN transmigration (Fig. 3, B and C), we hypothesized that a more robust junctional expression of JAM-C is necessary for its participation in transmigration. This line of reasoning is in keeping with two recent reports indicating that exposure of endothelium to certain stimuli, such as in vascular beds of tumors where significant angiogenesis occurs or in atherosclerotic lesions in ApoE-null mice, does not alter JAM-C protein levels but instead trigger redistribution of JAM-C to cell-cell junctions (6, 8). In the study using an atherosclerotic model, a role for JAM-C in leukocyte transmigration was more apparent under atherogenic conditions. To achieve elevated expression of JAM-C at cell-cell borders, we transduced HUVEC monolayers with lentivirus containing JAM-C GFP. This approach achieved a 4.4-fold increase over the baseline level of surface JAM-C and dramatically increased JAM-C expression at cell-cell junctions (Fig. 4, A and B).

We next characterized expression of JAM-C in HUVECs transduced with JAM-C GFP. SDS-PAGE analysis showed that native JAM-C and JAM-C GFP fusion expressed in HUVECs migrated at the predicted molecular mass (Fig. 4C) of 40 and 70 kDa in resting or 4-h TNF-{alpha}-activated HUVECs. HUVECs transduced with JAM-C-GFP showed normal morphology by phase-contrast microscopy (data not shown) and no alterations in the localization of VE-cadherin, a marker of endothelial adherens junctions, or of ZO-1, a marker of tight junctions (Fig. 4D). JAM-C overexpression had no gross effect on overall barrier integrity because TER was similar to resting HUVECs over a 4-day period. As a control, HUVECs infected with JAM-C GFP lentivirus, GFP lentivirus, or medium alone also responded similarly to the barrier-enhancing effects of a cAMP analog O-Me (34) and exhibited a reduction in TER in response to the barrier-disrupting agonist histamine (Fig. 4E).

JAM-C GFP does not enhance leukocyte adhesion and transmigration under shear flow in vitro

We next turned to test whether monolayers expressing abundant junctional JAM-C exhibited elevations in PMN adhesion or transmigration. As shown in Fig. 5, PMN did not show enhanced adhesion or transmigration of JAM-C GFP-transduced monolayers as compared with GFP transduced or sham-transduced HUVEC monolayers. The ability of function-blocking JAM-C mAb LUCA14 was then assessed (Fig. 6A). Under these conditions, no reduction in PMN adhesion or transmigration was observed after preincubation with a saturating dose of LUCA14 mAb. In contrast, a blockade of ICAM-1 alone did reduce transmigration. Additional studies examined whether the combination of mAb with both ICAM-1 and JAM-C is necessary to detect a role for JAM-C in transmigration. Interestingly, the combination of mAb did not block adhesion or TEM beyond that seen with ICAM-1 mAb alone. A combination of mAb to VE-cadherin and JAM-A had no inhibitory effect on adhesion or TEM.


Figure 5
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  		FIGURE 5. JAM-C overexpression in HUVECs does not enhance PMN adhesion or transmigration under shear flow. HUVECs were infected with JAM-C GFP virus (4.4 ± 1.4-fold increase, over basal expression (n = 5)) and PMN adhesion (top panel), and percentage of transmigration (bottom panel) was determined as described in Materials and Methods (n = 3 separate experiments).

 

Figure 6
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  		FIGURE 6. Dynamics of endothelial cell JAM-C during PMN transmigration. A, The role of JAM-C in PMN transmigration of HUVEC-overexpressing JAM-C GFP was probed with function-blocking JAM-C mAb LUCA14 in an in vitro TEM assay as detailed in Materials and Methods. B, Paired fluorescence and DIC images of neutrophil-transmigrating HUVECs transduced with JAM-C GFP or immunolabeled with a nonblocking ICAM-1 mAb were captured by time-lapse fluorescence imaging microscopy as detailed previously (14 35 36 ). Note that ICAM-1 clusters around adherent and transmigrating PMN, whereas JAM-C forming a gap in fluorescence signal (arrows) does not.

 
Characterization of JAM-C localization during PMN adhesion and transmigration by live cell fluorescence imaging under shear flow in vitro

Endothelial cell ICAM-1 binds leukocyte integrins Mac-1 and LFA-1, and ICAM-1 has been shown to redistribute and cluster around adherent and transmigrating PMN (15, 35, 36). Although our findings described so far do not support a role for JAM-C in transmigration of PMN, we nonetheless wanted to evaluate the spatial behavior of endothelial cell JAM-C GFP during PMN adhesion and transmigration by live cell fluorescence imaging and suspected that direct visualization of this molecule during TEM may clarify our above results. Fig. 6B shows the spatial pattern of JAM-C at sites of PMN transmigration using time-lapse fluorescence and DIC digital microscopy. JAM-C GFP had a strikingly different behavior during TEM as compared with ICAM-1. JAM-C did not cluster or accumulate around stably adherent or transmigrating PMN as we and others have shown previously for ICAM-1 (Fig. 6B; ICAM-1 panels). In contrast, JAM-C formed a small gap at sites where PMN transmigrated similar to the behavior described previously for endothelial cell VE-cadherin (24, 37). This behavior is best illustrated in Fig. 6B, from time (t) = 0.75 to t = 1.25 min.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We examined the hypotheses that endothelial cell JAM-C is required for PMN transendothelial cell migration under shear flow conditions and that JAM-C undergoes redistribution during PMN transmigration. We performed experiments in a well-characterized in vitro flow adhesion model that mimics physiological laminar shear flow conditions (14, 38) and had several unanticipated results regarding endothelial cell JAM-C expression and its role in PMN transmigration.

JAM-C is localized in intracellular pools and minimally expressed at cell-cell borders

Although we did reproduce previous reports that JAM-C is constitutively expressed in HUVECs and partially localizes to cell-cell junctions (6, 10), surprisingly, we found that a significant level of JAM-C Ag is intracellular (Fig. 2). Interestingly, surface expression of JAM-C is not dramatically elevated by treatment with prototypic proinflammatory stimuli, including LPS, IL-1beta, or TNF-{alpha}, and distribution of JAM-C also is not altered by these stimuli. Numerous studies have demonstrated that endothelial cells contain storage granules of bioactive molecules such as vWF and P-selectin sequestered in WPB as well as recently described chemokine-containing storage granules (39, 40). Confocal analysis showed that the majority of JAM-C Ag was intracellular and did not demonstrate significant colocalization with the vWF Ag. There was occasionally observed colocalization of signal adjacent to the nucleus, which could be just coincidental because of colocalization at sites of protein biosynthesis (endoplasmic reticulum and Golgi apparatus (31)). Further studies are necessary to understand the biosynthetic secretory pathway of JAM-C, to identify potential storage pools, if any, that contain JAM-C and to assess the endocytic trafficking route(s) of JAM-C in endothelium.

Recent studies with HUVECs cultured on Matrigel have reported that treatment with VEGF induced rapid redistribution of JAM-C toward cell-cell borders (6). In contrast, treatment of HUVECs with oxidized low-density lipoprotein promoted a 2-fold increase in JAM-C surface expression and its redistribution away from cell-cell borders (8). Because the vascular endothelium exhibits phenotypic heterogeneity in vivo (reviewed in Ref. 41), the current data raise the intriguing possibility that intracellular and extracellular localization of JAM-C varies depending on the vascular bed and pathophysiological stimuli present. Hence, ultrastructural characterization of JAM-C expression and localization in endothelium of peripheral tissues and organs as well as endothelium cultured in vitro from different anatomical sites will be necessary to understand how heterophilic binding to JAM-B modulates its adhesive capacity for leukocyte Mac-1 in various animal models of disease.

Function-blocking mAbs do not reduce PMN transmigration in vitro

In this study, we show that JAM-C mAb that block interactions with Mac-1 (LUCA-14 and PACA-4) or JAM-B binding sites (H33 and D22) do not prevent PMN migration across cytokine-activated HUVECs under shear flow conditions (Fig. 3, B and C). This lack of effect is likely explained by the low expression of JAM-C at cell-cell borders, which is the location where most (>95%) PMN transmigrate in our system (14, 35). However, a small inhibitor effect on PMN transmigration could be demonstrated whether the JAM-C mAb PACA-4 or Luca14 was combined with blocking mAb to CD99, PECAM-1, and ICAM-1 (Fig. 3C). The physiological relevance of this observation is not clear because the combo block of mAb itself inhibits >75% of TEM.

We next used a lentivirus construct to enhance JAM-C expression at cell borders and evaluated whether enhanced junctional expression affected PMN adhesion and under flow. Despite expression at cell-cell borders, JAM-C overexpression did not enhance PMN adhesion or extent of PMN transmigration (Figs. 5 and 6) and had minimal effects on overall barrier integrity as assessed by TER. Furthermore, JAM-C did not cluster around adherent and transmigrating PMN, whereas ICAM-1 certainly did (Fig. 6B). It was not possible to obtain adequate live cell images of JAM-C with fluorescent-tagged nonblocking mAb because of its low level of expression at HUVEC cell-cell borders. Previous studies demonstrate that this fusion protein expressed in cell lines can recognize JAM-B and support Mac-1 binding of a leukocyte cell line. The expressed JAM-C GFP fusion protein is of correct mass and mimics wild-type distribution of JAM-C (Fig. 4). Importantly, previous studies have shown that JAM-C GFP is functional because when it is expressed in Chinese hamster ovary or Madin-Darby canine kidney cells, the fusion protein recognizes JAM-B (42). This JAM-C-JAM-B heterophilic interaction is reported to occur through interactions on both Ig domains of JAM-C: the membrane-distal V-type Ig domain (RIE66) and the C2 domain, and the interaction is blocked by JAM-C mAb (42). These data support the conclusion that JAM-C GFP is functionally active and capable of interacting with ligands in our model.

Our findings, taken together, do not support a role for JAM-C in PMN transmigration in this system; however, several caveats merit discussion of these findings compared with previously published reports implicating JAM-C in transmigration. One explanation for the lack of effect by JAM-C mAb is that, in the HUVEC system, ICAM-1 is the preferred endothelial cell ligand for both LFA-1 and Mac-1, bypassing a need for JAM-C. Consistent with this idea, we have shown in previous studies that, depending on the specific mAb to ICAM-1 used, >90% of PMN transmigration can be inhibited (35) (unfortunately, this mAb 282 used in our previous study (35) is no longer available). Another likely explanation is that other adhesion pathways including PECAM-1 and CD99 (43, 44) are operative, and perhaps dominant, during leukocyte transmigration at endothelial junctions, which is the location of >95% of TEM events in the current model (14, 35). This result is supported by our data that a role for JAM-C is detected only if these other adhesion pathways are also blocked (Fig. 3B). We speculate that the participation of JAM-C in leukocyte TEM depends on the vascular bed and the inflammatory stimulus. For example, the IL-1beta, TNF-{alpha}, or LPS-activated HUVEC monolayers, whereas characterized extensively and used by many investigators to study leukocyte-endothelial cell interactions, may not be optimal to evaluate JAM-C in TEM and may require a different cytokine stimulus or a combination of cytokines and inflammatory mediators. It is of interest to note that previous studies reported that soluble human JAM-C partially reduced PMN transendothelial cell migration to MCP-1 in a static Transwell assay and that infusion of soluble murine JAM-C partially blocked PMN recruitment in a model of acute peritonitis (10). Although PMN-directed migration to MCP-1 is unexpected, nonetheless the inhibition by soluble JAM-C may work through ligation of Mac-1 on blood leukocytes and subsequent down-regulation of this adhesion receptor or by preventing platelet-leukocyte interactions, which are known to participate in leukocyte recruitment in both in vitro and in vivo models (45, 46). One other possibility is that soluble JAM-C binds endothelial surface-expressed JAM-C and negatively signals the endothelium during transmigration, although this possibility has not yet been explored.

In summary, we conclude that JAM-C has a minimal, if any, role in neutrophil transendothelial cell transmigration in this model and that ICAM-1 is the preferred ligand for neutrophil beta2 integrins. Once leukocytes have exited the vasculature, epithelial cell JAM-C is likely to be involved in leukocyte migration along the lateral epithelial membrane toward the apical surface (13). In light of the in vitro and in vivo studies showing a role for JAM-C in leukocyte recruitment, we suggest that the relationship between the endothelial cell ICAM-1 and JAM-C pathways may be more complex in vivo and that the role played by JAM-C is strongly influenced by its level of expression and location in endothelium as well as the epithelium.


   Acknowledgments
 
We thank Kay Case and Vanessa Davis, (Center of Excellence in Vascular Biology, Brigham and Women’s Hospital, Boston, MA) for providing well-characterized HUVECs, and Nancy Voynow at the Brigham and Women’s Hospital Editorial Services for proofreading our manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by National Institutes of Health Grants DK72564, HL72124, and DK61379 (to C.A.P.), HL36028 (to T.M.), and HL36028, HL53993, and HL56985 (to F.W.L.); Swiss National Science Foundation Grants 310000-112551/I (to M.A.-L.) and 3100A0-100697 (to B.A.I.); and grants from Oncosuisse (to B.A.I.) and Thorn Foundation (to M.A.-L.). Back

2 Address correspondence and reprint requests to Dr. Francis W. Luscinskas, Brigham and Women’s Hospital, Harvard University, Vascular Research Division, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: fluscinskas{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; JAM, junctional adhesion molecule; TEM, transendothelial migration; VE, vascular endothelial; DPBS, Dulbecco’s PBS; vWF, von Willebrand factor; RT, room temperature; IP, immunoprecipitation; EGFP, enhanced GFP; DIC, differential interference contrast; TER, transendothelial electrical resistance; WPB, Weibel-Palade bodies. Back

Received for publication October 2, 2006. Accepted for publication February 20, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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