An Antibody to the Sixth Ig-like Domain of VCAM-1 Inhibits Leukocyte Transendothelial Migration without Affecting Adhesion

Sukmook Lee, Il-Hee Yoon, Aerin Yoon, Joan M. Cook-Mills, Chung-Gyu Park and Junho Chung
J Immunol November 1, 2012, 189 (9) 4592-4601; DOI: https://doi.org/10.4049/jimmunol.1103803

Abstract

VCAM-1 plays a key role in leukocyte trafficking during inflammatory responses. However, molecular mechanisms underlying this function have not been clearly elucidated. In this study, using phage display technology, we developed a rabbit/human chimeric VCAM-1 Ab, termed VCAM-1 domain 6 (VCAM-1-D6), which specifically recognizes aa 511–599 within the sixth Ig-like domain. We report that the VCAM-1-D6 Ab blocked U937 cell transmigration across activated HUVECs but did not alter adhesion of U937 cells to the HUVECs. We also demonstrate that VCAM-1-D6 does not alter TNF-α–stimulated endothelial cell chemokine or cytokine production. Furthermore, through in vivo efficacy testing using a mouse islet allograft model, we demonstrate that VCAM-1-D6 significantly alleviates allograft rejection by blocking leukocyte infiltration to the grafted islets. Taken together, our results suggest that the VCAM-1-D6 Ab may block VCAM-1–mediated inflammation and could be a useful tool in treating inflammatory diseases.

Introduction

Vascular cell adhesion molecule-1 (also known as CD106) is a cell adhesion molecule induced in activated endothelial cells. During leukocyte recruitment, VCAM-1 binds α4β1 integrin, a leukocyte integrin, in its intermediate-affinity state and, along with selectins and addressins, plays a key role in the rolling step of leukocytes on the endothelium (1–3). Selectin binding on leukocytes or activation of chemokine receptors stimulates “outside-in” signals in leukocytes, which increases the affinity of the integrin family of receptors to bind endothelial cell adhesion molecules, predominantly ICAM-1and VCAM-1 (4–13). This high-affinity interaction results in leukocyte arrest on the endothelium. After the high-affinity adhesion, VCAM-1, together with PECAM-1, plays a critical role in leukocyte transendothelial migration (14).

VCAM-1 is a member of the Ig superfamily. The extracellular region of the full-length form of VCAM-1 contains seven homologous Ig-like domains. Domains 1 and 4, 2 and 5, and 3 and 6 are homologous to each other (15–17). There are two human forms and two mouse forms of VCAM-1. Human VCAM-1 (hVCAM-1) has two splice variants that result in a protein with either seven Ig-like domains or six Ig-like domains (lacking domain 4) (15, 18). Mouse VCAM-1 (mVCAM-1) also has a full-length form with seven Ig-like domains, as well as a truncated form containing only the first three Ig-like domains.

All variants of VCAM-1 have been shown to bind to α4β1 integrin (19–22). In addition to α4β1 integrin, VCAM-1 can bind other integrins, including α4β7, αdβ2, αMβ2, and α9β1 (23–28). It is known that α4β1 integrin binds Ig-like domains 1 and 4 (29–32), and that activated integrins are necessary for firm leukocyte adhesion to the endothelium via Ig-like domains 1 and 4 of VCAM-1 (33).

Leukocyte binding to adhesion molecules, including VCAM-1, activates signals within endothelial cells that allow the opening of narrow vascular passageways between endothelial cells or through an individual endothelial cell for transendothelial migration (34–39). When endothelial cell adhesion molecule signaling is inhibited, leukocytes bind to the endothelium, but transendothelial migration does not occur (40), and leukocytes are often released from the endothelium to continue in the blood flow (41, 42).

In this study, we developed a chimeric rabbit/human VCAM-1 Ab, specific to Ig-like domain 6, using phage display technology. This Ab, VCAM-1 domain 6 (VCAM-1-D6), inhibits the transendothelial migration of leukocytes without hindering the leukocyte adhesion process on endothelial cells in vitro. In a mouse islet allograft model, this Ab successfully inhibits transmigration of leukocytes to the graft site and blocks rejection of grafted pancreatic β cell islets. Our results suggest that the VCAM-1-D6 Ab may block VCAM-1–mediated inflammation.

Materials and Methods

Construction of the Ab library and selection of binders

Four New Zealand white rabbits were immunized and boosted four times with recombinant hVCAM-1 (rhVCAM-1; R&D Systems, Minneapolis, MN) with the approval of the Institutional Animal Care and Use Committee of the Seoul National University Hospital. After the final booster injection, total RNA was prepared from the spleen and the bone marrow. cDNA was synthesized using SuperScript First-Strand cDNA synthesis system (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. A phage-displayed rabbit/human chimeric Fab Ab library was constructed using pComb3X phagemid vector system as described previously (43). After the library construction, Fab clones were selected from the library through six rounds of biopanning as described previously (2). For each round of biopanning, 2.5 μg recombinant mVCAM-1 Fc fusion protein (rmVCAM-1–Fc; R&D Systems)-coated magnetic beads (Dynabeads M-270 epoxy; Invitrogen) were used. After the final round of biopanning, individual phage clones displaying Fab were generated from colonies grown on output plates and tested for reactivity to hVCAM-1–Fc and mVCAM-1–Fc by phage enzyme immunoassay as described previously (4).

Preparation of VCAM-1-D6 Fab and IgG

Top10F′ E. coli cells were transformed with phagemid DNA encoding the VCAM-1-D6 Fab. Bacteria were grown in 1 l Luria\x{2013}Bertani media containing 50 μg/ml carbenicillin at 37°C overnight with constant shaking. The cells were pelleted by centrifugation at 3000 × g for 15 min. Supernatants were collected and concentrated 10 times using the Labscale TFF System (Millipore, Bedford, MA). The VCAM-1-D6 Fab was purified from the concentrated supernatant using hemagglutinin-specific Ab column chromatography as described previously (44, 45). After column chromatography, fractions containing Fab were pooled and analyzed by SDS-PAGE and Coomassie blue staining as described previously (46). The production and purification of VCAM-1-D6 IgG was performed as described previously (47).

ELISA

rhVCAM-1–Fc (R&D Systems) and rmVCAM-1–Fc were dissolved in 50 μl PBS and added to the wells of a microtiter plate. The amount of VCAM-1–Fc added to each well was 5, 10, 20, or 50 ng. After incubation overnight at 4°C and washing three times with PBST (PBS containing 0.05% [v/v] Tween 20), the microtiter plate was incubated for 2 h at 37°C with 3% (w/v) BSA in PBS. After washing with PBST, the plate was incubated with 2 nM VCAM-1-D6 Fab or 2 nM of unrelated Fab for 1 h at 37°C, and washed twice with PBST. The plate was incubated with anti-human Fab Ab conjugated to HRP (Sigma, St. Louis, MO) diluted 1000-fold in 3% (w/v) BSA in PBS. After washing with PBST, 50 μl 3,3,5,5-tetramethylbenzidine substrate solution (GenDEPOT, Barker, TX) was added to each well. OD was measured at 450 nm using a microtiter plate reader (Labsystems, Barcelona, Spain).

Flow cytometry

HUVECs (Lonza, Baltimore, MD) and human aortic endothelial cells (HAECs; Lonza, Switzerland) were maintained in endothelial cell growth medium-2 in a humidified incubator at 37°C with 5% CO2 according to the manufacturer’s instructions. Mouse vascular endothelial cells (MVECs) were a kind gift of Dr. Saito (Tsurumi University, Tsurumi, Japan) and were maintained in Medium 199 supplemented with 5% (v/v) FBS, 10 μg/ml insulin, 2.4 μg/ml hydrocortisone, and 1% (v/v) penicillin/streptomycin. The endothelial cells were stimulated with 20 ng/ml human (hTNF-α) or mouse TNF-α (mTNF-α; Millipore) for 24 h. Cells were incubated with 10 μg/ml VCAM-1-D6 Fab, an anti-integrin αIIbβ3 Fab that was used as a negative control (48), or the mouse anti–hVCAM-1 Ab 51-10C9 (BD Bioscience, San Diego, CA) (49), diluted in flow cytometry buffer (1% [w/v] BSA in PBS containing 0.05% [w/v] sodium azide) at 37°C for 1 h. After centrifugation at 1000 × g for 10 min, cells were resuspended in the flow cytometry buffer. This washing step was then repeated two more times. The cells were then incubated at 37°C for 1 h with FITC-labeled anti-human Fab (Jackson Immunoresearch Laboratory, West Grove, PA) or Alexa Fluor 488 labeled goat anti-mouse IgG Ab (Invitrogen) diluted 100-fold in flow cytometry buffer. The samples were analyzed by flow cytometry. For all flow cytometry measurements, a FACS Canto II instrument (BD Biosciences, San Jose, CA) equipped with a 488-nm laser was used. Ten thousand cells were detected per measurement with no gating, and the results were analyzed using the FlowJo software program (Tree Star, Ashland, OR).

Immunocytochemistry

Immunocytochemistry was performed as described previously (50). In brief, coverslips were incubated with 1 μg/ml poly-l-lysine for 1 h; then HUVECs, HAECs, and MVECs were grown on coverslips in the absence or presence of hTNF-α or mTNF-α at the concentration of 20 ng/ml. After rinsing with 1X PBS two times, the cells were fixed with 3.7% (w/v) paraformaldehyde for 30 min at 37°C. After washing with 1X PBS and blocking with the 1X PBS containing 5% (w/v) BSA and 0.1% Triton X-100 for 4 h at 4°C, the cells were incubated overnight at 4°C with 10 μg/ml VCAM-1-D6 Fab, control Fab, or the mouse anti–hVCAM-1 Ab 51-10C9. The cells were washed five times with 1X PBS containing 0.05% Triton X-100 and then incubated in the same buffer for 1 h with FITC-labeled goat anti-human Fab or Alexa Fluor 488-labeled goat anti-mouse IgG Ab (1:100), and 2 μg/ml Hoechst (Invitrogen) for 1 h to visualize VCAM-1 and the nucleus, respectively. Slides were then examined under a fluorescence microscope (Olympus, Melville, NY).

Real-time interaction analysis

The kinetic parameters of the interactions between either the VCAM-1-D6 Fab and VCAM-1, or VCAM-1-D6 IgG and VCAM-1, were determined using the BIAcore system X-100 (Biacore AB, Uppsala, Sweden). In brief, hVCAM-1–Fc or mVCAM-1–Fc was immobilized on a CM5 dextran sensor chip (Biacore AB) in 10 mM sodium acetate buffer (pH 4.0) at a flow rate of 5 μl/min using the Amine Coupling Kit (Biacore AB). VCAM-1-D6 Fab or IgG in HEPES-buffered saline containing 0.005% surfactant P20, 3 mM EDTA, and 0.15 M NaCl were injected over 150 s at the flow rate of 30 μl/min at 37°C. After each analysis, the surface was regenerated with 1 M NaCl/50 mM NaOH as described previously (37). Biacore X-100 evaluation software version 1.1 (Biacore AB) was used to calculate Kon and Koff constants.

Preparation of VCAM-1 constructs as Fc fusion proteins

RNA was prepared from HUVECs treated with hTNF-α using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized using the SuperScript First-Strand cDNA synthesis system (Invitrogen) following the manufacturer’s instructions. The extracellular domain of hVCAM-1 (hVCAM-1–WT; aa 1–698) was amplified using the following primers: 5′-GGCCCAGGCGGCCATGCCTGGGAAGATGGTCG-3′ and 5′-GGCCCAGGCGGCCATGCCTGGGAAGATGGTCG-3′. The primers were designed to add SfiI restriction digestion sites at both the 5′ and 3′ ends. The hVCAM-1 construct hVCAM-C1 (aa 1–599) was created using the following primers: 5′-GGCCCAGGCGGCCATGCCTGGGAAGATGGTCG-3′ and 5′-GGCCCCACCGGCCCCAGTAACTTGGATAATTAATTCCAC-3′. The hVCAM-C2 (1–510) construct was generated using the following primers: 5′-GGCCCAGGCGGCCATGCCTGGGAAGATGGTCG-3′ and 5′-GGCCCCACCGGCCCC GGCAACATTGACATAAAGTG-3′. The hVCAM-C3 (1–407) construct was generated using the following primers: 5′-GGCCCAGGCGGCCATGCCTGGGAAGATGGTCG-3′ and 5′-GGCCCCACCGGCCCCTGGATCTCTAGGGAATGAGT-3′. The hVCAM-C4 (1–311) construct was generated using the following primers: 5′-GGCCCAGGCGGCCATGCCTGGGAAGATGGTCG-3′ and 5′-GGCCCCACCGGCCCC TTTCTCTTGAACAATTAATT-3′. The hVCAM-C5 (1–222) construct was generated using the following primers: 5′-GGCCCAGGCGGCCATGCCTGGGAAGATGGTCG-3′ and 5′-GGCCCCACCGGCCCCTGATATGTAGACTTGCAATT-3′. The hVCAM-D1 (511–599) construct was generated using the following primers: 5′-GGCCCAGGCGGCCCCCAGAGATACAACCGTCTT-3′ and 5′-GGCCGGCCTGGCC GTAACTTGGATAATTAATT-3′. The PCR fragments were digested with SfiI and cloned into modified pcDNA 3.1 vectors (Invitrogen) encoding the hinge region and CH2-CH3 domain of human IgG1 at the 3′ region of the cloning site. Ligated products were transformed into calcium chloride-competent E. coli DH5α cells, and plasmid DNA was prepared as described previously (38). HEK293F cells (4.0 × 107 cells; Invitrogen) were transfected with 50 μg of each DNA using 50 μg polyethylenimine (Polysciences, Warrington, PA) as described previously (51). The transfected cells were maintained in Freestyle 293 expression media (Invitrogen) supplemented with 0.5% (v/v) penicillin/streptomycin in a humidified incubator at 37°C with 8% CO2. After 7 d, the culture media was collected and the fusion proteins were purified using protein A affinity chromatography as described previously (44, 45).

Immunoblot analysis

SDS-PAGE and immunoblot analysis were performed as described previously (5). The purified proteins were dissolved in a Laemmli sample buffer and loaded into each well of 4–12% Tris-Glycine Gel (Novex NuPAGE; Invitrogen). After electrophoresis, the proteins were transferred to nitrocellulose membranes using the wet transfer system (GE Healthcare Life Sciences, Pittsburgh, PA). The membrane was incubated with TTBS (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.05% [v/v] Tween 20) containing 5% (w/v) skim milk at room temperature for 1 h, followed by incubation with 10 μg/ml VCAM-1-D6 Fab in TTBS containing 5% (w/v) skim milk at room temperature for 2 h. The membrane was washed with TTBS and incubated with anti-human Fab-HRP diluted 1000-fold in TTBS containing 5% (w/v) skim milk at room temperature for 2 h. In a parallel experiment, the membranes were incubated with HRP-conjugated rabbit anti-human Fc (Pierce, Rockford, IL) diluted 5000-fold in TTBS containing 5% (w/v) skim milk at room temperature for 30 min. The membranes were washed with TTBS three times, and protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to the manufacturer’s instructions.

Human monocyte isolation

Heparin-treated blood was collected from healthy volunteers after an informed consent was signed, and in regulation accordance with the ethical guidelines of Seoul National University hospital. PBMCs were isolated by Ficoll density gradient centrifugation (Ficoll-Paque; Amersham, Uppsala, Sweden) and resuspended in DMEM supplemented with 10% FBS, 2-ME, l-glutamine, gentamicin, essential and nonessential amino acids, and HEPES (Life Technologies, Grand Island, NY). CD14+ monocytes were then isolated by negative selection using the monocyte isolation kit II (Miltenyi Biotec, Auburn, CA) and an AutoMACS instrument (Miltenyi Biotec), according to the manufacturer’s protocol. The purity of the monocytes was evaluated by fluorescent staining with FITC-labeled mouse anti-CD14 Ab (Ancell, Bayport, MN) and flow cytometry analysis. This procedure yielded a monocyte fraction containing 83% CD14+ cells.

Transendothelial cell migration assay

HUVECs (5.0 × 104 cells/well) were added to the upper chambers of a 24-transwell plate with polycarbonate membranes of 8.0-μm diameter pores (Corning, Corning, NY) and incubated overnight in a humidified incubator at 37°C with 5% CO2. Cells were treated with hTNF-α diluted in endothelial cell growth medium-2 (Lonza, Baltimore, MD) to a final concentration of 20 ng/ml for 24 h. VCAM-1-D6 Fab, VCAM-1-D6 IgG, or rhVCAM-1 domain 6 (aa 511–599)-Fc were added to the upper chamber at various concentrations. Then U937 human monocytic cells (2.0 × 105 cells/well) were added to the upper chamber. An anti-αIIβ3 Fab (48) and an anti-respiratory syncytial virus Ab (palivizumab; MedImmune, Baltimore, MD) were used as the control Fab and control IgG, respectively. The mouse anti–hVCAM-1 Ab 51-10C9 was used as a positive control. Transmigration assays were also performed using isolated with human monocytes, following the procedures as described earlier. The final concentrations of VCAM-1-D6 Fab, VCAM-1-D6 IgG, mouse anti–hVCAM-1 Ab 51-10C9, and control Fab were adjusted to 10 μg/ml. The final concentrations of control IgG and the rhVCAM-1 domain 6-Fc were 20 μg/ml. RPMI 1640 media containing 50 ng/ml human stromal cell-derived factor-1α (SDF-1α; R&D Systems) was placed in the lower chamber. After 8 h, cells that migrated to the lower chamber were collected and counted under a light microscope.

Cell adhesion and neutralization assay

The full-length cDNA of hVCAM-1 was ligated into the KpnI or XhoI site of the pcDNA3.1 (+) vector (Invitrogen). After transforming calcium chloride-competent E. coli DH5α cells, plasmid DNA was prepared as described previously (38). For negative control (mock), pcDNA3.1 (+) vector was also prepared for transfection. HEK293 cells (5.0 × 104 cells) were transfected with 4 μg of each plasmid DNA using 6 μl TurboFect (Fermentas International, Burlington, Ontario, Canada) following the manufacturer’s recommendations. The transfected cells were cultured in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin, and 400 μg/ml G418 as a selection marker. Clones stably expressing full-length hVCAM-1 or mock-transfected clones were selected and harvested, respectively, and the expression of hVCAM-1 was determined using flow cytometry.

Leukocyte adhesion assays were performed as described previously, with minor modifications (50). In brief, 3.0 × 105 hVCAM-1 or mock-transfected HEK293 cells, HUVECs, or HAECs were plated on six-well dishes. Human endothelial cells were then stimulated with 20 ng/ml hTNF-α for 24 h. The VCAM-1-D6 Fab, control Fab (48), or mouse anti–hVCAM-1 IgG 51-10C9 was individually added to a final concentration of 10 μg/ml and incubated for 1 h at 37°C inside a humidified incubator with 5% CO2. U937 cells were labeled with CFSE (Molecular Probes, Eugene, OR), and 6.0 × 105 cells were added to each well. The plate was incubated for 1 h at 37°C inside a humidified incubator with 5% CO2. Wells were washed five times with PBS containing 0.2 mM CaCl2 and 0.1 mM MgCl2 to remove unbound cells. Bound cells were detached by treating with trypsin/EDTA (Life Technologies, Toronto, ON, Canada) for 5 min and then washed two times with DMEM containing 10% FBS. In flow cytometry, cells showing fluorescence were counted as CFSE-labeled U937 cells attached to mock- or hVCAM-1–transfected HEK293 cells, HUVECs, or HAECs. The assay was performed using a FACS CantoII instrument (BD Biosciences) with excitation at 488 nm and the emission filter set to a 530/30 bandpass. In each measurement, 10,000 cells were counted with no gating. Results are expressed as the percentage of CFSE+ cell counts over the total counted events.

Measurement of HUVEC-secreted cytokines and chemokines

HUVECs (3.0 × 105 cells) were added to each well of a 6-well plate and treated with 20 ng/ml hTNF-α for 24 h at 37°C inside a humidified incubator with 5% CO2. The VCAM-1-D6 IgG or the mouse anti–hVCAM-1 Ab 51-10C9 was added to each well to a final concentration of 10 μg/ml. The plate was incubated for 24 h at 37°C inside a humidified incubator with 5% CO2. The media were harvested and subjected to the Bio-Plex Multiplex Chemokine and Cytokine assay (Bio-Rad Laboratories, Hercules, CA) or an IL-8 ELISA (Invitrogen) with a dynamic range between 15.6 pg/ml and 1 ng/ml, following the manufacturers’ recommendations.

Mice

Female C57BL/6 and BALB/c inbred mice, aged 8–10 wk, were purchased from Charles River Laboratories (Wilmington, MA) and maintained in the Seoul National University specific pathogen-free animal facility. These mice were bred at the Biological Services Unit of the Seoul National University according to the Institutional Animal Care and Use Committee guidelines.

Isolation of mouse pancreatic islets

Murine islets were isolated as described previously (7). In brief, the pancreas from BALB/c mice was exposed and injected with HBSS (Mediatech, Herndon, VA) containing 0.55 mg/ml collagenase P (Roche, Indianapolis, IN) via the common bile duct until adequate distension was achieved. The distended pancreas was digested at 37°C for 20 min with gentle shaking. The digestion was terminated by putting the pancreas on ice. The islets were filtered through a metal mesh and were purified on Euro-Ficoll gradients (Sigma, St. Louis, MO) by centrifugation at 700 × g at 8°C for 15 min. This protocol routinely yielded preparations of >90% purity. Islets were stabilized by culturing overnight in RPMI 1640 media supplemented with 11 mM glucose, 2 mM l-glutamine, 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin before transplantation.

Induction of diabetes mellitus and islet transplantation

Diabetes mellitus was induced in mice (8–10 wk old) by i.p. administration of 250 mg/kg streptozotocin (STZ; Sigma) freshly dissolved in citrate buffer (pH 4.5). Blood glucose in nonfasting conditions was measured from the snipped tail by a portable glucometer (Lifescan, Milpitas, CA). Two consecutive nonfasting blood glucose readings of >250 mg/dl were obtained from whole blood. Seven days after STZ administration, mice were anesthetized with isoflurane. For islet transplantation, the left kidney was exposed and 500 islet equivalents were delivered beneath the kidney capsule using PE-50 polyethylene tube (Becton Dickinson, Parsippany, NJ). Mice were treated with either VCAM-1-D6 IgG or control IgG (0.1 mg, i.p. injection on days −1, 0, 1, 2, 3, 4, 5, 6, and 7. Day 0 is defined as the time of islet transplantation.

Immunohistochemistry

The kidney with the islet graft was removed and embedded in OCT compound (Tissue-Tek, Sakura, Torrance, CA). Frozen sections were obtained on a cryostat (CM1850; Leica, Bannockburn, IL) at 5-μm thickness and fixed in acetone for 10 min at 4°C. Sections were washed three times with PBS, followed by incubation in an endogenous peroxide-blocking solution for 5 min at room temperature. Nonspecific staining was prevented by treating the sections with 1% (v/v) FBS in PBS for 30 min at room temperature. The primary Abs used were guinea pig anti-insulin (1:20 dilution; 0.7 mg/ml; Dako, Carpinteria, CA), rat anti-CD4 (1:50 dilution; 0.62 μg/ml, RM4-5; BD Biosciences, San Jose, CA), and rat anti-macrophage marker F4/80 Ab (1:50 dilution; 10 μg/ml; eBiosciences, San Diego, CA). Primary Abs were applied for 60 min at room temperature. HRP-conjugated anti-guinea pig or anti-rat secondary Abs were applied for 40 min at room temperature. Color development was performed using a VECTASTAIN Elite ABC kit (PK6101; Vector Laboratories, Burlingame, CA). The sections were observed and visualized using a light microscope (Axiocam; Carl Zeiss, Oberkochen, Germany).

Results

An Ab isolated from a rabbit/human chimeric Fab library reacts with hVCAM-1 and mVCAM-1

Rabbits were immunized and boosted four times with purified rhVCAM-1. Total RNA was prepared from spleen and bone marrow, and subjected to cDNA synthesis. Using this cDNA, we generated a rabbit/human chimeric Fab library containing rabbit variable regions and human constant regions with a complexity of 5.7 × 109. After six rounds of biopanning on immobilized mVCAM-1–Fc, clones were randomly selected, rescued by infection of helper phage, and tested for their reactivity to hVCAM-1 and mVCAM-1 in phage enzyme immunoassay. Ab clones reactive to both hVCAM-1 and mVCAM-1 were selected for further study.

After expression in E. coli and subsequent purification, the Fab purity (>90%) was confirmed by SDS-PAGE and Coomassie blue staining (data not shown). Using an ELISA, we determined that 0.02 nM VCAM-1-D6 Fab bound to both hVCAM-1– and mVCAM-1–coated ELISA plates (Fig. 1A). In a Western blot, the VCAM-1-D6 Fab detected 5–50 ng rhVCAM-1 and rmVCAM-1 (Fig. 1B).

FIGURE 1.
FIGURE 1.

VCAM-1-D6 Ab has cross-species reactivity to hVCAM-1 and mVCAM-1. (A) After rhVCAM-1–Fc and rmVCAM-1–Fc were coated onto 96-well plates, ELISA was performed as described with purified control Fab (□) or VCAM-1-D6 Fab (▪). These results represent the mean ± SD obtained from triplicate wells. (B) The indicated amounts of hVCAM-1 and mVCAM-1 recombinant protein were examined by immunoblot analysis with VCAM-1-D6 Fab and anti-human Fab-HRP. (C) HAECs, HUVECs, and MVECs were cultured in the absence (dotted line) or presence (solid line) of hTNF-α or mTNF-α for 24 h, respectively. The cells were then analyzed using flow cytometry with the VCAM-1-D6 Fab Ab, a control Fab, and the mouse anti–hVCAM-1 Ab 51-10C9, followed by FITC-labeled goat anti-human Fab or Alexa Fluor 488-labeled goat anti-mouse IgG, respectively. These results are representative of three independent experiments. (D) HAECs, HUVECs, and MVECs were cultured in the absence or presence of hTNF-α or mTNF-α, and incubated with the VCAM-1-D6 Fab, control Fab, or the mouse anti–hVCAM-1 Ab 51-10C9. Then cells were treated with FITC-labeled goat anti-human Fab Ab or Alexa Fluor 488-labeled goat anti-mouse IgG Ab, respectively (original magnification ×400).

In real-time interaction analysis using VCAM-1–coated chips (Table I), the KD constant for VCAM-1-D6 Fab interaction with hVCAM-1 was 1.35 ± 0.02 × 10−8. For the interaction with mVCAM-1, the KD constant was 4.78 ± 0.06 × 10−10. When using the IgG form of the VCAM-1-D6 Ab, the KD constant for interaction with hVCAM-1 and mVCAM-1 was 3.06 ± 0.04 × 10−10 and 7.31 ± 0.42 × 10−11, respectively (Table I). These data indicate a strong affinity for VCAM-1-D6 Fab and IgG with either hVCAM-1 or mVCAM-1. The reactivity of VCAM-1-D6 Fab to endogenous VCAM-1 expressed on human and mouse endothelial cells, and its localization pattern were investigated using flow cytometry and immunocytochemistry (Fig. 1C, 1D). VCAM-1-D6 Fab reacted to VCAM-1 on HAECs, HUVECs, and MVECs stimulated by hTNF-α or mTNF-α, but not to unstimulated cells. In the immunocytochemical study, the localization pattern of VCAM-1 obtained using VCAM-1-D6 Fab was similar to that obtained with the mouse anti–hVCAM-1 Ab 51-10C9 that was used as a positive control.

View this table:
Table I. Affinity constants (Ka, Kd) evaluated by BIAcore for VCAM-1-D6 Fab and IgG/hVCAM-1 and mVCAM-1 interactions

The VCAM-1-D6 Ab specifically recognizes the sixth Ig-like domain of VCAM-1

To identify the epitope region for the VCAM-1-D6 Fab, we constructed expression vectors for six truncated forms of the extracellular domain of hVCAM-1 as Fc fusion proteins (Fig. 2A). These constructs were transfected into HEK293F cells, and the Fc fusion proteins were purified by protein A column chromatography. An equal amount of each fusion protein was subjected to immunoblot analysis. The VCAM-1-D6 Fab detected hVCAM-1-WT (1–698)–Fc and hVCAM-1-C1 (1–599)–Fc, whereas it did not bind to hVCAM-1-C2 (1–510)–Fc, suggesting hVCAM-1 aa 511–599 as an epitope for VCAM-1-D6. The HRP-conjugated human Fc Ab reacted to all of the Fc fusion proteins (Fig. 2B).

FIGURE 2.
FIGURE 2.

The VCAM-1-D6 Ab specifically recognizes the sixth Ig-like domain of VCAM-1. (A) The indicated wild-type and C-terminal domain serial deletion mutants were constructed and prepared as Fc-fusion proteins. In addition, a fragment containing aa 511–599 of the hVCAM-1 extracellular domain was fused to the Fc domain. (B) The same amount of purified hVCAM-1 wild-type and C-terminal domain serial deletion mutant Fc fusion proteins (0.1 μg) was subjected to immunoblot analysis using the VCAM-1-D6 Fab, followed by anti-human Fab-HRP (upper panel) or anti-human Fc-HRP (lower panel). (C) Purified wild-type hVCAM-1 and the aa 511–599 fragment Fc fusion proteins were analyzed by immunoblot using VCAM-1-D6 Fab, followed by anti-human Fab-HRP (lower panel) or anti-human Fc-HRP (upper panel). In the control lane, recombinant human Fc was loaded as a negative control. These results are representative of three independent experiments.

To further confirm that hVCAM-1 aa sequence 511–599 includes the epitope of the VCAM-1-D6 Fab, we prepared a Fc fusion protein containing these residues (hVCAM-1-D6 [511–599]–Fc; Fig. 2A). This purified Fc fusion protein was subjected to immunoblot analysis with VCAM-1-D6 Fab. VCAM-1-D6 Fab successfully recognized hVCAM-1-D6 (511–599)–Fc (Fig. 2C). The IgG form of the VCAM-1-D6 Ab also reacted to hVCAM-1-D6 (511–599)–Fc (data not shown). From these data, we concluded that the VCAM-1-D6 Fab specifically recognizes a C-terminal region (aa 511–599) of hVCAM-1, which represents the sixth Ig-like domain of hVCAM-1 (aa 511–595).

The VCAM-1-D6 Ab significantly inhibits U937 cell and human monocyte transmigration across activated endothelial cells without inhibiting U937 cell or human monocyte adhesion

To investigate whether the VCAM-1-D6 Ab regulates leukocyte transmigration across activated endothelial cells, we performed transendothelial cell migration assays with HUVECs and U937 human monocytic cells. HUVECs were added to the upper chamber of transwells, allowed to grow to confluence, and then treated with hTNF-α for 24 h to induce VCAM-1 expression. After adding U937 cells to the upper chambers, the chemoattractant human SDF-1α was added to the lower chambers. After 24 h, the U937 cells that migrated through the HUVEC monolayer to the lower chamber were collected and counted. When VCAM-1-D6 Fab or VCAM-1-D6 IgG was added to the upper chamber, the number of migrating U937 cells decreased significantly in a concentration-dependent manner. We also found that both the VCAM-1-D6 Fab and IgG almost completely inhibited the migration at 10 μg/ml (Fig. 3A, 3B). To check whether rhVCAM-1 domain 6 is directly involved in leukocyte transendothelial migration, we performed a transmigration assay with rhVCAM-1 domain 6 (aa 511–599)-Fc and control IgG. rhVCAM-1 domain 6-Fc significantly inhibited the transmigration of U937 cells across activated HUVECs in a concentration-dependent manner (Fig. 3C). We also performed the transmigration assay with monocytes isolated from human peripheral blood. VCAM-1-D6 Fab or VCAM-1-D6 IgG and rhVCAM-1-domain 6-Fc inhibited the transmigration of human monocytes across activated HUVECs (Fig. 3D).

FIGURE 3.
FIGURE 3.

The VCAM-1-D6 Ab specifically inhibits U937 and human monocyte transendothelial migration across TNF-α–activated HUVECs. HUVECs (2.0 × 105 cells) were plated on the upper surface of transwell plates and cultured in the absence or presence of TNF-α for 24 h. After a 1-h preincubation with the indicated concentrations of VCAM-1-D6 Fab (A), control Fab (A), VCAM-1-D6 IgG (B), mouse anti–hVCAM-1 IgG (B), control IgG (B), rhVCAM-1 domain 6-Fc (C), or control IgG (C), U937 cells were added to the upper part of the transwell plate. Recombinant SDF-1α (50 ng/ml) was added to the lower part of the transwell. After 24 h, cells that migrated were counted under a light microscopy. (D) The transendothelial migration assays with human monocytes were performed by preincubating HUVECs with 10 μg/ml of the Fabs or 20 μg/ml rhVCAM-1 domain 6-Fc or control IgG in the same experimental settings described earlier. Results represent the mean ± SD obtained from experiments performed in triplicate. *p > 0.05.

The effect of the VCAM-1 D6 Fab on leukocyte adhesion to VCAM-1 was examined using hVCAM-1– or mock-transfected HEK293 cells, as well as HUVECs and HAECs induced to express VCAM-1 by treatment with hTNF-α. Cell surface expression in hVCAM-1-transfected HEK293 cells was confirmed by flow cytometric analysis (Fig. 4A). For adhesion assays, the cells were grown to confluence on six-well dishes. U937 cells were labeled with CFSE and then added to each well in the absence or presence of 50 μg/ml VCAM-1-D6 Fab, control Fab, or the mouse anti–hVCAM-1 Ab 51-10C9. After 1 h, U937 cells attached to hVCAM-1 overexpressing HEK-293 cells, HUVECs, or HAECs were detached by trypsinization and counted using flow cytometry. The adhesion blocking Ab for domain 1 of VCAM-1, mouse anti–hVCAM-1 Ab 51-10C9, significantly inhibited U937 adhesion to hVCAM-1–overexpressing HEK 293 cells, HUVECs, or HAECs, whereas VCAM-1-D6 Fab and rhVCAM-1 domain 6-Fc did not affect U937 cell attachment to the VCAM-1–overexpressing cells (Fig. 4B–D).

FIGURE 4.
FIGURE 4.

The VCAM-1-D6 Ab does not alter VCAM-1–mediated binding of U937 cells to TNF-α–activated HUVECs. (A) HEK293 cells overexpressing VCAM-1 or control vector were analyzed by flow cytometry in the absence (dotted line) or presence (solid line) of VCAM-1-D6 Fab. HAECs (B), HUVECs (C), or VCAM-1–transfected HEK293 cells (D) were incubated in the absence or presence of VCAM-1-D6 Fab, control Fab, or the mouse anti–hVCAM-1 IgG 51-10C9 for 1 h. CFSE-labeled U937 cells were then added for 1 h. Bound cells were examined using flow cytometry as described. Results shown represent the mean ± SD obtained from three separate experiments performed in duplicate. *p > 0.05.

VCAM-1-D6 IgG does not activate endothelial cells

We used a fluorescence multiplex immunoassay to investigate whether VCAM-1-D6 IgG activates cells via VCAM-1 cross-linking. HUVECs were treated with 10 μg/ml VCAM-1-D6 IgG or the mouse anti–hVCAM-1 Ab 51-10C9 (as a positive control) (23, 52) for 24 h. In parallel experiments, HUVECs were pretreated with 20 ng/ml hTNF-α for 24 h before incubation with the VCAM-1 Abs. The endothelial cell culture supernatant was examined for two chemokines (GM-CSF and IL-8), as well as nine cytokines (IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IFN-γ, and TNF-α). The agonistic mouse anti–hVCAM-1 Ab 51-10C9 significantly enhanced TNF-α–stimulated endothelial cell IL-8 production (Fig. 5) but did not affect production of the other cytokines (data not shown). In contrast, the VCAM-1-D6 Ab did not enhance IL-8 production (Fig. 5), nor did it enhance any of the other cytokines or chemokines tested (data not shown).

FIGURE 5.
FIGURE 5.

The VCAM-1-D6 Ab does not induce IL-8 secretion of TNF-α–activated HUVECs. HUVECs and TNF-α–activated HUVECs (3.0 × 105 cells) were plated in 6-well plates and cultured in the absence or presence of 10 μg/ml of the agonistic VCAM-1 Ab (mouse anti–hVCAM-1 Ab 51-10C9) or VCAM-1-D6 IgG for 24 h. The culture supernatants were centrifuged to remove cell debris, and IL-8 secretion was analyzed by ELISA. Results shown represent the mean ± SD obtained from three separate experiments performed in duplicate.

VCAM-1-D6 IgG protects against grafted islet rejection by blocking leukocyte infiltration

To investigate the effect of the VCAM-1-D6 Ab on inflammation in vivo, we used an MHC-mismatched mouse islet allograft model. Diabetes was induced by STZ and the experimental animals received VCAM-1-D6 IgG, whereas control animals received an irrelevant IgG. Abs were injected into mice a total of nine times (0.1 mg/injection) before islet transplantation on day 7 (Fig. 6A). Blood glucose levels were monitored twice per week. Median graft survival in the control IgG group was 28.2 ± 8.6 d. In contrast, animals treated with the VCAM-1-D6 Ab showed no evidence of graft rejection at the time of their sacrifice, 110 d posttransplantation (Fig. 6B, 6C). In addition, the islet grafts from control and VCAM-1-D6 IgG-treated animals were histologically examined. The islet tissue from the control animals demonstrated clear evidence of immune rejection with the loss of insulin-positive cells (Fig. 7A), as well as significant mononuclear leukocytic infiltrate into the graft (Fig. 7B, 7C). In contrast, the islet grafts from the VCAM-1-D6 Ab-treated mice were intact with many insulin-positive β cells (Fig. 7D). Interestingly, the long-term functioning grafts (>110 d) from the VCAM-1-D6 IgG-treated mice showed persistent peri-islet mononuclear cellular infiltration of both CD4+ T cells (Fig. 7E) and macrophages (Fig. 7F). However, these mononuclear cells were rarely observed within the islets.

FIGURE 6.
FIGURE 6.

The VCAM-1-D6 Ab maintains long-term graft survival in allogeneic islet transplantation assay. (A) Schematic depiction of Ab treatment protocol. (B) Islets harvested from BALB/c mice were transplanted into the subcapsular space of kidney. Blood glucose levels were monitored twice per week from control IgG (○) or VCAM-1-D6 IgG (□)-treated groups. (C) Treatment with the VCAM-1-D6 Ab induces prolonged graft survival in C57BL/6 recipients (mean survival day > 110 d, n = 5). Control IgG treatment group was hyperglycemic after a brief normoglycemia period (mean survival day = 28.2 d, n = 5). Graft survival is presented as Kaplan–Meier survival curves.

FIGURE 7.
FIGURE 7.

The VCAM-1-D6 Ab significantly promotes islet cell survival by blocking leukocyte migration inside the grafted islets. The kidneys of control animals were isolated at the time of rejection and kidneys fromVCAM-1-D6 Ab-treated mice were isolated 110 d after transplantation. Histological comparison of islet grafts from the two groups was performed using immunohistochemical staining. (A and D) Islet cell staining using an insulin Ab. (B and E) CD4 T cell staining using a CD4 Ab. (C and F) Macrophage staining using the F4/80 Ab. Images are representative of three independent experiments. Arrows indicate the stained cells. Original magnification ×100 (A–D), ×200 (E, F).

Discussion

In this study, we demonstrate that VCAM-1-D6 binds to the sixth Ig-like domain of VCAM-1, a domain that does not mediate leukocyte binding. However, VCAM-1-D6 binding to this VCAM-1 domain blocks leukocyte transendothelial migration. Furthermore, VCAM-1-D6 IgG does not alter TNF-α–stimulated endothelial cell chemokine or cytokine production. In addition, VCAM-1-D6 IgG inhibited the recruitment of leukocytes to transplanted islets and blocked rejection of islet transplants.

Leukocyte α4β1 integrin binding to domains 1 and 4 of VCAM-1 on the endothelium plays a critical role in recruiting leukocytes during the initiation and progression of inflammation. Abs that block this interaction have anti-inflammatory properties. Natalizumab, a humanized α4β1 integrin Ab that inhibits the interaction between the integrin and domains 1 and 4 of VCAM1, was approved for treatment of multiple sclerosis (39). In animal experiments, the rat anti-mVCAM-1 Abs MK1.9 and MK2.7 were shown to successfully inhibit the progression of inflammatory processes. MK1.9 blocks Ramos cell binding to murine endothelioma lines (40) and also increased the graft survival rates of C3H/H3J mice that received tail skin grafts from B10.BR mice (53). In an experimental autoimmune encephalomyelitis model, MK1.9 reduced disease severity (54). Another VCAM-1 Ab, MK2.7, allowed long-term islet allograft survival of >100 d with a survival rate of 75% of the islet grafts (55). In a mouse model of Crohn’s disease, MK2.7 treatment yielded a 70% resolution of the acute inflammation (56). Furthermore, MK2.7 also reduced the development of arthritis in a collagen-induced arthritis model (57) and inhibited inflammatory cell infiltration into the skin in a keratin-14 IL-4 transgenic mouse atopic dermatitis model (58). The mouse anti–hVCAM-1 Abs 4B9, BBIG-V1, 1G11, and P3H12 block binding of lymphoma cell lines to VCAM-1–expressing endothelial cells (52, 59, 60). However, these Abs are species specific. A desirable property for a VCAM-1 Ab for therapeutic development is its reactivity to both hVCAM-1 and animal VCAM-1. The VCAM-1-D6 Ab described in this study has broad reactivity to hVCAM-1, mVCAM-1, and pig VCAM-1 (Fig. 1A and data not shown). This broad species reactivity allows evaluation of this Ab in various animal disease models, as well as in human disease interventions.

The epitopes of VCAM-1 neutralizing Abs have been reported to be either domain 1 or 4, the domains involved in α4β1 integrin binding (23, 24). The MK2.7 Ab is reactive to VCAM-1 domains 1 and 4 (61). The epitope for the mouse anti–hVCAM-1 Abs, 51-10C9 and 4B9, is localized to domain 1 (23, 24).

In this study, we showed that an Ab reactive to VCAM-1 domain 6 inhibited the transmigration. Because rhVCAM-1 domain 6 (aa 51–599) Fc fusion also inhibited the transmigration of U937 cells and human monocytes across HUVECs, it is more likely that the domain 6 directly interacts with another VCAM-1 molecule or with tetraspanins CD9, CD81, CD151 in tetraspanin-enriched microdomains to initiate downstream signaling (62, 63). This may regulate transendothelial migration because VCAM-1 cross-linking activates downstream signaling molecules in endothelial cells that are required for leukocyte transendothelial migration (64). For example, treatment of human and mouse endothelial cells with anti–mVCAM-1–coated beads (clone MV-CAM.A) increased the intracellular level of reactive oxygen species, tyrosine phosphatase activity, and protein kinase Cα activity, all of which are required for leukocyte migration (49). VCAM-1 cross-linking with the VCAM-1 Ab (1G11) induced the activation of Rac1, a small GTP binding protein, reactive oxygen species production, and p38 MAPK activation (65). VCAM-1 activation by beads coated with the mouse anti–hVCAM-1 Ab 51-10C9 stimulates NADPH oxidase, metalloproteinase-2, and metalloproteinase-9 activity (42). In this study, the agonistic mouse anti–hVCAM-1 Ab 51-10C9 enhanced TNF-α–stimulated endothelial cell production of IL-8. However, an anti–VCAM-1 domain 6 Ab did not alter TNF-α–stimulated production of chemokines or cytokines. Nevertheless, the VCAM-1-D6 Ab has the unique function of blocking leukocyte transendothelial migration without affecting leukocyte adhesion. In vivo intravital microscopy studies demonstrate bound leukocytes that do not migrate are often released and continue with the blood flow (41).

In this study, the in vivo efficacy of the VCAM-1-D6 Ab was demonstrated in a mouse islet allotransplantation model. VCAM-1 plays a critical role in recruitment of leukocytes to allografts (66–68). VCAM-1 is overexpressed in grafted islets through inflammatory cytokines such as IFN-γ, TNF-α, and IL-1 (14, 68). The mVCAM-1–specific Ab MK2.7 blocks adhesion to domain 1 and protects against mouse islet allograft rejection by blocking leukocyte adhesion to endothelium. In contrast, we found that VCAM-1-D6 IgG did not block adhesion but did block leukocyte transendothelial migration. In addition, it successfully inhibited islet graft transplant rejection for at least 110 d after transplantation. Immunohistochemical studies also indicated an inhibition of CD4+ T cell and macrophage infiltration toward the grafted islets. This infiltration of CD4 T cells and macrophages in the peri-islet space without invasion into the graft site was previously observed when immunological tolerance was achieved (69). Thus, we have identified a unique function for VCAM-1-D6 Abs in blocking islet graft transplant rejection and blocking leukocyte recruitment.

The unique property of the VCAM-1-D6 Ab to not block leukocyte adhesion may provide some advantages. It is reported that in lymphoid tissues, the interaction of lymphocyte VLA-4 with VCAM-1 on stromal cells activates IL-7 production, a pre-B cell growth factor (70). The MK1.9 and MK2.7 Abs inhibited B lymphocyte development in bone marrow culture in vitro (40). VCAM-1 is expressed at low levels on interstitial dendritic cells and could have a role in Ag presentation for lymphocyte activation (68). Abs that block lymphocyte adhesion may inhibit T lymphocyte activation by interfering with the interaction between interstitial dendritic cells and lymphocytes.

In summary, VCAM-1-D6 Ab, which is reactive to VCAM-1 domain 6, inhibits transmigration but not adhesion, suggesting that the sixth IgG-like domain may participate in transmigration (36, 50).

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • ↵3 C.-G.P. and J.C. codirected this work.

  • This work was supported by Grant 2011K000737 from the Korea Biotechnology Research and Development Group of Next-Generation Growth Engine Project of the Ministry of Education, Science and Technology, Republic of Korea, and by a grant from Korea Healthcare Technology Research and Development Project, Ministry of Health and Welfare, Republic of Korea (Project A040004 to C.-G.P.).

  • Abbreviations used in this article:

    HAEC
    human aortic endothelial cell
    hTNF-α
    human TNF-α
    hVCAM-1
    human VCAM-1
    mTNF-α
    mouse TNF-α
    mVCAM-1
    mouse VCAM-1
    MVEC
    mouse vascular endothelial cell
    rhVCAM-1
    recombinant human VCAM-1
    rhVCAM-1–Fc
    recombinant human VCAM-1 Fc fusion protein
    rmVCAM-1
    recombinant mouse VCAM-1
    rmVCAM-1–Fc
    recombinant mouse VCAM-1 Fc fusion protein
    SDF-1α
    stromal cell-derived factor-1α
    STZ
    streptozotocin
    TTBS
    10 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.05% (v/v) Tween 20
    VCAM-1-D6
    VCAM-1 domain 6.

  • Received January 5, 2012.
  • Accepted August 29, 2012.

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