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Originally published online as doi:10.1189/jlb.1106680 on March 27, 2007

Published online before print March 27, 2007
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(Journal of Leukocyte Biology. 2007;81:1395-1403.)
© 2007 by Society for Leukocyte Biology

CD11c/CD18: novel ligands and a role in delayed-type hypersensitivity

Chanchal Sadhu*,1, Harold J. Ting{dagger}, Brian Lipsky{ddagger}, Kelly Hensley{ddagger}, Leon F. Garcia-Martinez§, Scott I. Simon{dagger} and Donald E. Staunton||

* ICOS Corporation, Bothell, Washington, USA;
{dagger} Department of Biomedical Engineering, University of California, Davis, Davis, California, USA;
{ddagger} Amgen Inc., Seattle, Washington, USA;
§ Alder Biopharmaceutical, Bothell, Washington, USA; and
|| CisThera, Kirkland, Washington, USA

1 Correspondence: ICOS Corporation, 22021 20th Ave., S.E., Bothell, WA 98021, USA. E-mail: casdhu{at}icos.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD11c, a member of the leukointegrin family, is expressed prominently on tissue macrophages and dendritic cells and binds to complement fragment (iC3b), provisional matrix molecules (fibrinogen), and the Ig superfamily cell adhesion molecule, ICAM-1. CD11c has been proposed to function in phagocytosis, cell migration, and cytokine production by monocytes/macrophages as well as induction of T cell proliferation by Langerhans cells. Using assays to quantify CD11c-mediated cell adhesion, we demonstrate that CD11c recognizes ICAM-2 and VCAM-1. The CD11c-binding site on VCAM-1 appears to be different from that used by the integrin {alpha}4. CD11c and {alpha}4ß1 contributed to monocyte capture and transmigration on inflamed human aortic endothelial cells. We discovered that the anti-mouse CD11c mAb N418 blocks CD11c binding to iC3b, ICAM-1, and VCAM-1. Treatment of mice with N418 reduced SRBC-induced delayed-type hypersensitivity significantly. CD11c appeared to contribute predominantly to the sensitization phase and somewhat less to the response to SRBC challenge. This suggests a novel role for CD11c during leukocyte recruitment, antigen uptake, and the survival of APC.

Key Words: integrin • ICAM-1 • VCAM-1 • N418 • DTH


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are a family of heterodimeric cell adhesion receptors, which support cell migration, survival, and proliferation. Integrins bind to cell-surface or matrix-associated ligands, and the binding is tightly regulated [1 ]. Loss of integrin regulation can contribute to hyperproliferative and inflammatory diseases and thus, have been targeted by novel drugs [2 ]. CD11c/CD18 is a member of the CD18 family of integrins, which includes CD11a (LFA-1), CD11b (MAC-1), and CD11d ({alpha}D). Members of this family of integrins share the same ß-chain, namely, ß2 (CD18), but are distinguished by their {alpha}-chains, designated as {alpha}L (CD11a), {alpha}M (CD11b), {alpha}X (CD11c), and {alpha}D (CD11d) [3 4 5 ]. Although their size and amino acid sequences are distinct, the four {alpha}-chains share overall structural organization. CD11a is the largest, with an approximate m.w. of 180 KDa and the most divergent from the rest. CD11c is 150 KDa in size and possesses considerable similarity with CD11b and CD11d in terms of amino acid sequence, expression, and ligand profile. This includes the presence of the inserted or I domain in CD11c, which has been implicated in ligand binding [6 , 7 ].

One of the earliest described properties of CD11c is its ability to mediate phagocytosis of iC3b-opsonized particles [8 ]. Accordingly, CD11c is known as complement receptor 4. CD11c has also been implicated in phagocytosis of latex beads and bacteria in the absence of complement [9 ]. CD11c binds a diverse array of ligands such as cell adhesion molecules (e.g., ICAM-1, ICAM-4) [7 , 10 , 11 ], bacterial cell wall components including LPS [12 ], complement protein (e.g., iC3b) [8 , 13 ], and matrix proteins such as fibrinogen [14 ] and collagen [15 ]. Binding of CD11c to some of the protein ligands is enhanced upon denaturation of the ligand [16 ]. Of the different ligands that bind to CD11c, its binding to fibrinogen has been characterized most extensively [17 ]. Although compared with CD11b, CD11c is expressed at lower levels on neutrophils, TNF-stimulated neutrophils bind to fibrinogen primarily through CD11c [14 ]. Consistent with the report of Davis [16 ], the binding is enhanced upon denaturation or proteolytic degradation of fibrinogen [18 ]. Vorup-Jensen et al. [18 ] demonstrated further that exposure of acidic residues on the denatured protein results in the enhanced binding by CD11c and have proposed that the negatively charged residues serve as a "pattern recognition" signature for the integrin. As a result of the enhanced CD11c-mediated binding of neutrophils to proteolytically degraded fibrinogen and the stable association between CD11c and urokinase-type plasminogen activator, roles for CD11c in innate immunity and immune homeostasis have been predicted [18 ].

CD11c is expressed on the plasma membranes of monocytes, tissue macrophages, NK cells, and most dendritic cells (DCs); a lower level of expression is also observed on neutrophils [19 ]. As a result of its high level of expression on most DCs, CD11c is considered a marker of these dedicated APC [20 , 21 ]. Although involvement of CD11c in diverse functions of DC is relatively unexplored, Meunier et al. [22 ] have demonstrated a complete inhibition of allogeneic T cell proliferation mediated by the cutaneous Langerhans cells in the presence of the CD11c-specific antibody, Leu M5 [23 ]. This observation implies a role of CD11c in antigen presentation by DCs.

In addition to its potential role in antigen presentation by the DCs, CD11c may be involved in inflammation. Antibodies against CD11c inhibit monocyte adhesion to collagen, production of superoxide, and fMLP-induced chemotaxis [9 , 15 ]. Studies with blocking mAb suggest a dominant role of CD11c in monocyte adhesion to melanoma and endothelial cell monolayers, albeit CD11c is expressed on monocytes at a lower level compared with the other ß2 integrins [24 ]. Monocyte CD11/CD18 integrins can interact with multiple ligands on the endothelial cells, which express several cell adhesion molecules, including ICAM-1 and ICAM-2. VCAM-1 is another important Ig-superfamily cell adhesion molecule, which is up-regulated on inflamed endothelium. VCAM-1 plays a central role in the recruitment of monocytes to sites of inflammation [25 26 27 28 ]. Consistent with these observations, VCAM-1 has been implicated in various inflammatory diseases such as atherogenesis [29 ], inflammatory bowel disease [30 ], and multiple sclerosis [31 ]. Thus, the interaction between monocyte integrins, including CD11c and the endothelial Ig-superfamily cell adhesion molecules may play important roles in a variety of inflammatory diseases.

Purified CD11c has been demonstrated to support endothelial cell adhesion, although the ligand(s) of CD11c on the endothelial cells were not identified [32 ]. In an effort to identify the ligands of CD11c on endothelial cells, we tested whether CD11c recognizes purified CAMs. Here, we report that in addition to its known interaction with ICAM-1, CD11c strongly binds ICAM-2 and VCAM-1 during static adhesion, and monocyte CD11c uses VCAM-1 for arrest and transmigration through inflamed aortic endothelial cells. We also report that the antimouse CD11c antibody, N418, which is widely used in identification and characterization of mouse DCs, blocks CD11c binding to its ligands including VCAM-1. Applying N418, we demonstrate a role of CD11c in a mouse model of delayed-type hypersensitivity (DTH) induced by SRBC. Our data suggest that CD11c plays a significant role in the sensitization phase of the SRBC-mediated DTH. Thus, CD11c not only binds to a number of CAMs in vitro, but it also mediates inflammatory responses in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and recombinant proteins
Hybridomas for the anti-human CD11a mAb TS1/22 and the anti-mouse CD11c mAb N418 were from American Type Culture Collection (Manassas, VA, USA). The anti-human {alpha}4-blocking mAb 72A1H was generated at ICOS Corporation (Bothell, WA, USA) and has been described [33 ]. Antibodies were purified from ascites or from hybridoma supernatants using Protein A affinity chromatography. The anti-human CD11c mAb BU15 and 3.9 were from The Binding Site (Birmingham, UK) and BioLegend (San Diego, CA, USA), respectively. The anti-human VCAM-1 mAb 4B9 was a generous gift from Dr. John Harlan (Division of Hematology, University of Washington, Seattle, WA, USA). The hamster anti-{alpha}1 mAb Ha31/8 and control mAb Ha4/8 were purchased from PharMingen (San Diego, CA, USA) [34 ]. The ICAM-1 mAb BBIG-I1 and the VCAM-1 mAb BBIG-V3 were from R&D Systems (Minneapolis, MN, USA). ICAM-1-, ICAM-2-, ICAM-3-, and VCAM-1 (D1–D5)-Fc fusion proteins were generated as described elsewhere [33 ]. The quality of the protein preparations, specifically VCAM-1-Fc, was analyzed by analytical ultracentrifugation, and no aggregate of VCAM-1 was detected.

Generation of cell lines expressing human and mouse CD11c
As some of the characterizations are based on cell adhesion assays, we expressed CD11c in the nonadherent human B lymphoblastoid line JY. Of the four members of ß2 integrins, this cell line only expresses CD11a/CD18 [33 ]. We cloned the mouse- and human-CD11c cDNAs in the vector pNEF38 and electroporated the JY cells with the cDNA expression constructs according to the procedures described elsewhere [35 ]. Transfectants were selected with neomycin in RPMI medium containing glutamine, Na-pyruvate, penicillin, streptomycin, and 10% FCS. The human CD11c-expressing cells were enriched by panning on iC3b-coated plates. The human and mouse CD11c-expressing cells were purified by staining with the mAb 3.9 and N418, respectively, and subsequently sorted on a cell sorter. JY cells expressing human and mouse cDNA were designated as JY[hCD11c] and JY[mCD11c], respectively.

Human aortic endothelial cell (HAEC) culture
HAECs were purchased at Passage 4 (Cascade Biologics, UK) after ~13 cell divisions from initial isolation, expanded in T75 flasks to 80–90% confluence, and then seeded onto six-well, tissue-culture plates (Becton Dickinson, San Jose, CA, USA) between Passages 5 and 6 (16–19 total cell divisions). HAECs were conditioned with freshly isolated very low-density lipoprotein (vLDL) by incubation for 2 h/day for up to 3 days (at 2.5 mg/mL) as described previously [28 ]. Acute inflammation was stimulated by incubating conditioned HAECs with TNF-{alpha} at 0.3 ng/mL for 4 h and then washed twice before use in the monocyte adhesion assay.

Monocyte isolation
Blood was drawn from healthy donors into heparin-coated syringes according to Protocol #200311635-6 of the University of California, Davis, Institutional Review Board (Davis, CA, USA). Mononuclear cells were isolated by sedimentation over Lymphosep density separation media from MP (Aurora, OH, USA) as described [28 ]. The cells thus obtained were purified further from platelets by resuspending in HEPES buffer + 2% human serum albumin (HSA) and then centrifuging at 250 g at 15°C for 10 min. This process was repeated three times. Monocytes were isolated from other mononuclear cells using a negative isolation kit from Dynal (Brown Deer, WI, USA), resuspended in HEPES buffer + 0.2% HSA + 1.5 mM CaCl2 at a concentration of 107 monocytes/mL, and kept at room temperature until use.

Immunostaining and flow cytometry
Cells (1x106) were stained with the appropriate mAb at a concentration of 10 µg/mL, followed by incubation with FITC-labeled sheep anti-mouse secondary antibody. All of the test antibodies were of IgG1 subtype. The IgG1 mAb, 1B7, was included in each experiment to establish nonspecific binding. For sorting, cells expressing human or mouse CD11c were stained with labeled 3.9 or N418, respectively, and the top 1% of the stained cells were sorted.

Cell adhesion assays
Static adhesion assays of the JY transfectants were carried out as described elsewhere [33 ] with the following modifications. Briefly, 96-well, flat-bottom Immulon 4 plates were coated overnight at 4°C with the CAMs or the specified mAb at 10 µg/mL unless mentioned otherwise. The plates were blocked with 1% HSA for 1 h at room temperature and washed with PBS. Cells were diluted the day before assay to 2.5 x 105 cells/mL and grown overnight. For the adhesion assays, cells were harvested, washed, and resuspended at 9 x 105 cells/mL. After appropriate treatments, 0.9 x 105 cells were added to each well (final volume, 300 µL) in triplicate and allowed to adhere to the protein-coated wells for 40 min at 37°C in a humidified incubator maintained at 5% CO2. PMA was used at 20 ng/mL, and the blocking mAbs were used at 10 µg/mL. The adhered cells were fixed with glutaraldehyde, and unbound cells were removed by washing. Bound cells were quantified using crystal violet staining. The percentage of attached cells was calculated as (absorbance of sample wells/absorbance of mAb capture wells) x 100.

For the measurement of adhesion under flow, a custom-designed silicone rubber-based parallel plate flow chamber was constructed using soft photolithography technology as described [36 ]. Briefly, silicone disks of 25 mm diameter were cast on top of a replica master to form four independent sets of continuous channels (200 µm widex100 µm highx2 mm long), which were vacuum-sealed above a confluent monolayer of HAECs in a single well of a six-well, tissue-culture plate. A syringe pump (Harvard Apparatus, Holliston, MA, USA) in withdrawal mode was connected to the channel outlet via Teflon tubing to achieve a defined flow rate and constant shear stress of 2 dyne/cm2. These flow chambers were denoted vascular mimetic devices, as they are scaled to the geometry of blood vessels and allow the delivery of media, cytokines, and lipoproteins under precise shear conditions to a monolayer of endothelium and provide a means to visualize monocyte recruitment. Monocytes or antibodies were introduced in a 10-µL bolus into an open reservoir at the channel inlet. Adhesion molecule inhibition of monocytes (anti-integrins) or HAECs (anti-ICAM-1 and anti-VCAM-1) was accomplished by pretreatment with antibodies for 15 min at 37°C prior to introduction to the flow channel. Digital image sequences were acquired, and monocyte rolling, arrest (i.e., for a period of 30 s), and transmigration underneath the HAEC monolayer were quantified as described previously [37 ].

DTH
SRBC-induced DTH studies in mice were conducted as described [38 ]. Balb/c mice (6- to 8-week-old, female) were purchased from Taconic Farms (Germantown, NY, USA). Antibodies were injected i.p. at the indicated dose 1 h prior to sensitization (Day 0). Sensitization was carried out by injection of 2 x 107 SRBC, which were resuspended in endotoxin-free PBS at 2 x 105/µL. Each mouse was injected s.c. with 100 µl SRBC suspension under isoflurane anesthesia. Mice were anesthetized on Day 5, and baseline measurements of hind footpad thickness were obtained with a spring-loaded caliper, injected with appropriate antibodies. One hour after antibody injection, animals were challenged with 25 µL SRBC in the right hind footpad and 25 µL sterile saline in the left hind footpad. Footpad thickness measurements were obtained 20 h postchallenge under anesthesia. Percent increase in footpad thickness was calculated using the following formula: 100 x (thickness at the end of experiment–baseline thickness)/baseline thickness.

Statistical analysis
Analysis of data was performed using GraphPad Prism v4.0 software (GraphPad Software, Inc., San Diego, CA, USA.). All data are reported as mean ± SD or SE, as indicated. Nonparametric grouping of data was analyzed by ANOVA and secondary analysis for significance with Tukey post-tests. Gaussian-distributed mean values were analyzed by Student’s t-test unless otherwise indicated. Group comparisons were deemed significant for two-tailed P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of a stable cell line expressing functionally active human CD11c
To study the function and regulation of cellular CD11c, we expressed the integrin in the human B lymphoblastoid cell line JY. This cell line expresses one other CD18 integrin, CD11a/CD18. Integrins expressed by JY show a basal level of binding to ligand, which can be stimulated further through activation of protein kinase C by PMA [33 ]. Figure 1 demonstrates integrin expression by the JY (upper panels) and the JY transfectants JY[hCD11c] (lower panels), as determined by mAb binding and FACS analysis. The two CD11c mAbs, BU15 (anti-CD11c-1) and 3.9 (anti-CD11c-2), bound JY[hCD11c] cells at moderate levels, and binding to untransfected cells was similar to that of the isotype-matched, control antibody. Also shown in Figure 1 are expression patterns of CD11a and the integrin {alpha}4. Expression of CD11a was lower in the CD11c-transfected cells compared with the untransfected cells, although no such difference was observed in the expression of {alpha}4.


Figure 1
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  		Figure 1. Expression of integrins in JY (upper panels) and JY[hCD11c] (lower panels). For comparison, staining pattern with the isotype-matched, control antibody is shown (gray lines) with that of the test mAb (dark lines). Antibodies used were BU15 (anti-CD11c-1), 3.9 (anti-CD11c-2), TS1/22 (anti-CD11a), and 72A1H (anti-{alpha}4).

 
Several laboratories have reported ICAM-1 as a ligand of CD11c [7 , 10 , 39 ]. To test the functionality of CD11c in JY[hCD11c], we measured CD11c-dependent binding of the cells to immobilized ICAMs, including ICAM-1. JY cells express CD11a, which uses ICAMs as ligands. We used the CD11a-blocking mAb, TS1/22, to suppress CD11a binding to the ICAMs. Under our assay conditions, JY cells expressing only CD11a bound to ICAM1, and the binding was abolished by TS1/22 (Fig. 2A ). As shown in Figure 2B , similar to the JY cells, the JY[hCD11c] cells also bound to ICAM-1, and the binding was stimulated by PMA. However, in the presence of TS1/22, at a concentration that completely abolished JY cell binding to ICAM-1 (Fig. 2A) , nearly 50% of the maximal JY[hCD11c] cell binding was retained. Inclusion of a CD11c-blocking mAb in addition to the CD11a-blocking mAb in the assay mixture strongly inhibited cell adhesion. These data indicate that the CD11c molecule is well expressed and functional on the JY[hCD11c] and binds cooperatively with CD11a to an ICAM-1 substrate.


Figure 2
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  		Figure 2. JY and JY[hCD11c] adhesion to ICAM-1. Immulon 4 (96-well) plates were coated with ICAM-1 or glycophorin (Glyco.; as negative control) at 10 µg/mL, and cell adhesion was performed essentially as described in Materials and Methods. Raw adhesion values (absorbance at 570 nm) are shown along the y-axis. Cell adhesion to ICAM-1 was performed in the absence or in the presence of PMA (20 ng/mL). TS1/22 (anti-CD11a) and BU15 (anti-CD11c) were used at 10 µg/mL. Adhesion of JY and JY[hCD11c] is shown in A and B, respectively. One representative each of at least three experiments is shown.

 
Using the JY[hCD11c] cell adhesion assay in the presence of the CD11a-blocking mAb, we determined whether CD11c can bind other ICAMs, such as ICAM-2 and ICAM-3. As shown in Figure 3 , compared with the glycophorin control, the JY[hCD11c] cells showed significant binding to ICAM-1 and ICAM-2 in the absence of PMA; insignificant adhesion to ICAM-3 was observed under these conditions. Furthermore, like the ICAM-1 binding, the JY[hCD11c] binding to ICAM-2 was stimulatable by PMA. In the presence of PMA, the cells bound significantly to ICAM-3, but the binding was weaker compared with that of ICAM-1 and ICAM-2.


Figure 3
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  		Figure 3. Selectivity of CD11c-dependent binding of JY[hCD11c] to ICAMs. Cell adhesion was performed essentially as described in Materials and Methods. Glycophorin was used as a negative control. All of the proteins were used at 10 µg/mL to coat the 96-well plate. BU15 (anti-CD11c-1) and 3.9 (anti-CD11c-2) were used at 10 µg/mL each. Cell adhesion was normalized using values from a set of wells in which BU15 was coated to determine the maximum number of cells that can adhere in a CD11c-dependent manner. In these experiments, TS1/22 was used at a concentration of 10 µg/mL to suppress CD11a binding to the ICAMs. *, P < 0.05, compared with adhesion to glycophorin.

 
Characterization of VCAM-1 as a novel ligand of CD11c
JY[hCD11c] demonstrated high levels of binding to VCAM-1-coated wells, which were stimulated further by PMA (Fig. 4A ). JY cells express the integrin {alpha}4ß7 (Fig. 1) but not {alpha}4ß1 [33 ]. Therefore, to demonstrate and compare CD11c-dependent binding to VCAM-1, we used the {alpha}4-blocking mAb 72A1H to suppress activity of {alpha}4ß7 on the JY[hCD11c] cells. Pretreatment of the JY[CD11c] cells individually with 72A1H or either of the two CD11c mAb did not reduce cell adhesion significantly. Similar treatment of JY cells with the anti-{alpha}4 mAb blocked adhesion to VCAM-1 completely (see Fig. 4D ). In contrast, JY[hCD11c] adhesion to VCAM-1 was inhibited when {alpha}4ß7 and CD11c were blocked simultaneously with the respective antibodies. For example, 72A1H (anti-{alpha}4) and 3.9 (anti-CD11c-2) together reduced cell adhesion by ~80%. The combination of 72A1H, 3.9, and BU15 (anti-CD11c-1) reduced binding to a background level (Fig. 4A) . This indicates that CD11c expression confers {alpha}4-independent binding of JY[CD11c] to VCAM-1.


Figure 4
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  		Figure 4. JY[hCD11c] binds to VCAM-1 in a CD11c-dependent manner. Cell adhesion was performed according to the procedure described in Materials and Methods and normalized using values from a set of wells in which the mAb 72A1H was coated to determine the maximum number of cells that can adhere. (A) Relative contribution of CD11c and {alpha}4 for binding to VCAM-1. BU15 (anti-CD11c-1), 3.9 (anti-CD11c-2), and 72A1H (anti-{alpha}4) were used at 10 µg/mL each. *, P < 0.05, compared with cell adhesion to VCAM-1 in the presence of PMA and in the absence of any blocking mAb. (B) Concentration-dependent binding of JY[hCD11c] to VCAM-1 and ICAM-1. To suppress CD11a- and {alpha}4ß7-mediated cell adhesion, TS1/22 and 72A1H were used at a concentration of 10 µg/mL, respectively. #, P < 0.05, compared with cell adhesion to VCAM-1 at 10 µg/mL (Student-Newman-Keuls method). (C) Comparison of the requirement of divalent cations for CD11c and {alpha}4ß7. Untransfected JY cells were used for {alpha}4ß7-mediated adhesion studies. For CD11c binding to VCAM-1, JY[hCD11c] cells were used in the presence of mAb 72A1H to suppress {alpha}4ß7-mediated binding to VCAM-1. (D) CD11c binding site on VCAM-1 is different from that of {alpha}4. (Upper) JY cell binding to VCAM-1; (lower) JY[hCD11c] binding to VCAM-1.

 
We further examined the concentration-dependent adhesion of JY[hCD11c] to VCAM-1 as compared with ICAM-1. Beyond the coating concentration of 5 µg/mL, cell adhesion to ICAM-1 increased marginally, whereas adhesion to VCAM-1 increased significantly. Compared with that of ICAM-1, cell adhesion to VCAM-1 was moderately higher at 10 µg/mL (Fig. 4B) . These data suggest that VCAM-1 can function as an Ig-superfamily ligand for CD11c-depedent cell adhesion.

Divalent cations are required for integrins to shift conformation and bind ligands. We therefore tested whether CD11c-mediated adhesion of JY[hCD11c] cells to VCAM-1 is dependent on divalent cations. As shown in Figure 4C , chelating Ca2+ and Mg2+ with increasing concentration of EDTA in the media reduced CD11c-mediated JY[hCD11c] cell adhesion progressively to VCAM-1, such that only 50% binding was observed at 0.5 mM EDTA. A similar inhibition of {alpha}4ß7-dependent JY cell adhesion to VCAM-1 was also observed, although in this case, 50% reduction was observed at ~1.5 mM EDTA. Thus, like other integrin-ligand interactions, the CD11c-VCAM-1 interaction is also strictly dependent on divalent cations.

We used the VCAM-1 mAb 4B9 to test whether CD11c and {alpha}4 recognize overlapping region(s) of VCAM-1 [25 ]. This antibody blocks both {alpha}4-binding sites in VCAM-1 Domains 1 and 4 [40 , 41 ]. As shown in Figure 4D (upper panel), addition of 4B9 or 72A1H totally abolished adhesion of JY to VCAM-1. This is consistent with the fact that the JY cells only express one receptor, {alpha}4ß7, which can bind to VCAM-1. In contrast to JY adhesion, 4B9 reduced adhesion of JY[hCD11c] to VCAM-1 by ~25% only (Fig. 4D , lower panel). Taken together, these data suggest that a major recognition site of CD11c in binding to VCAM-1 is distinct from Domains 1 and 4, which are recognized by {alpha}4ß7. Consistent with this hypothesis is the observation that no further inhibition of CD11c-dependent binding to VCAM-1 was observed when {alpha}4 mAb was included along with the VCAM-1 mAb. However, binding was abolished totally when in addition to 4B9, the CD11c-blocking mAb (BU15 and 3.9) were included in the assay mixture.

The antimouse CD11c mAb N418 blocks CD11c-mediated cell adhesion
To determine the function of CD11c in mice, we antagonized binding of mouse CD11c to various ligands with blocking mAb. The JY[mCD11c] cells were used to test mouse CD11c binding to the established CD11c ligands, such as iC3b and ICAM-1. Similar to the JY[hCD11c]/ICAM-1 adhesion assays, the JY[mCD11c]/ICAM-1 assays were conducted in the presence of TS1/22 to block CD11a-mediated cell adhesion to ICAM-1. As shown in Figure 5 , in the absence of PMA, the JY[mCD11c] cells bound poorly to iC3b and ICAM-1. However, nearly 100% of the PMA-stimulated JY[mCD11c] cells bound to iC3b, whereas 40% of the input cells bound to ICAM-1. Similarly, upon PMA stimulation, the JY[mCD11c] cells bound strongly to VCAM-1, essentially reflecting the pattern of binding as seen with iC3b. Furthermore, JY[mCD11c] binding to all three ligands was abolished completely by a mouse CD11c-specific mAb, N418 [20 ].


Figure 5
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  		Figure 5. N418 (antimouse CD11c) blocks binding of mouse CD11c to its ligands. Immulon 4 (96-well) plates were coated with the ligands or N418 at 10 µg/mL each, and adhesion of JY[mCD11c] to iC3b, ICAM-1, and VCAM-1 was studied as described in Materials and Methods. Adhesion was normalized using the values obtained from the N418-coated wells as 100%. For N418-mediated blocking, the mAb was used at 10 µg/mL.

 
Monocyte adhesion to inflamed endothelium expressing ICAM-1, ICAM-2, and VCAM-1 is supported by {alpha}4ß1 and ß2 integrins
An early event in inflammation and atherogenesis is monocyte recruitment from the circulation to sites of arterial inflammation. We examined monocyte recruitment to vLDL-primed and TNF-{alpha}-inflamed endothelium using a custom microfluidic shear flow channel under a stress of 2 dynes/cm2 (Fig. 6 ). This model of inflammatory atherogenesis provides an ideal system to examine monocyte recruitment supported by CD11c and {alpha}4ß1, as costimulation with vLDL and TNF-{alpha} up-regulates VCAM-1 by up to threefold that expressed on TNF-{alpha}-inflamed endothelium [28 ]. Recruitment of monocytes onto HAECs was imaged by phase-contrast microscopy and categorized as the number of cells rolling to arrest and transmigrated across the endothelial monolayer. Using blocking mAb, we examined the adhesion molecules supporting monocyte recruitment in the vascular mimetic flow channels. Monocyte arrest and transmigration were supported by ICAM-1 and VCAM-1, as pretreatment with antibody to each virtually abrogated firm adhesion, but not rolling, which was supported by E-selectin, as determined by antibody inhibition [28 ]. {alpha}4ß1 and CD11c accounted for 80% of monocyte arrest on inflamed HAECs, as blocking each with antibody added up to the control level of ~12 monocytes per field. A distinct contribution of CD11c binding to VCAM-1 in monocyte arrest and transmigration was detected by simultaneously blocking ICAM-1, CD11a, CD11b, and {alpha}4ß1. Under these conditions, recruitment was not significantly less than blocking {alpha}4ß1 alone, indicating that VCAM-1 is the predominant ligand bound by CD11c on inflamed HAECs. By comparison, monocyte adhesion supported by CD11a and CD11b binding to ICAM-1 (i.e., under conditions with anti-{alpha}4ß1 and anti-CD11c) accounted for ~50% that of CD11c binding to VCAM-1 (i.e., anti-{alpha}4ß1, anti-CD11a, anti-CD11b, and anti-ICAM-1). Taken together, these data show clearly that CD11c and {alpha}4ß1 are the predominant integrins supporting monocyte arrest and transmigration on vLDL-primed and TNF-{alpha}-inflamed HAECs.


Figure 6
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  		Figure 6. Monocyte recruitment to HAECs under shear stress is supported by CD11c and {alpha}4ß1. HAEC monolayers were pretreated with vLDL particles (250 mg/dL) and then stimulated with TNF-{alpha} (30 U/mL) for 4 h. Monocytes (106/mL) were isolated via negative selection from whole blood and sheared at 2 dyne/cm2 in a vascular mimetic flow channel. The indicated antibodies were added at 10 µg/mL. n > 4 for all experiments. *, Statistical analysis indicates that the number of arrested cells blocked by anti-CD11c and {alpha}4ß1 was different at P < .05 from blocking these molecules singly.

 
CD11c functions in a SRBC-mediated DTH response in the mouse
We used the mouse CD11c-blocking mAb N418 in an in vivo model of DTH reaction to determine whether blockade of CD11c affects the response. The DTH response was initiated by sensitizing mice with SRBC. Five days postsensitization, the animals were challenged in the footpad with SRBC, and swelling of the footpads was measured after 20 h. To study the effect of CD11c blocking, mice were dosed i.p. with the antimouse CD11c mAb N418 or an isotype-matched, irrelevant antibody at 500 µg per animal. To investigate the stage at which blocking of CD11c is maximally effective in the inhibition of the DTH response, we treated mice with N418 in the following three different protocols: before sensitization as well as before challenge with SRBC; before sensitization only; and before challenge only. In the isotype-matched, antibody-treated group, no reduction of footpad swelling was observed compared with the untreated mice. However, treatment with N418 strongly inhibited the DTH response; in this group of mice, 70–75% reduction of the footpad swelling was observed compared with the control mice (Fig. 7 ). Furthermore, N418 was most efficacious when dosed according to Protocol 1 or 2. Although a statistically significant reduction in footpad swelling was observed, the efficacy of the mAb was diminished substantially when dosed according to Protocol 3. Conversely, N418 inhibited DTH similarly when administered according to Protocol 1 or 2.


Figure 7
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  		Figure 7. CD11c plays a role in SRBC-medicated DTH in mice. Groups of mice (five per group) were sensitized (Sens) with SRBC after appropriate mAb treatment (Day 0) as indicated. As a positive control, a group of mice was treated with the hamster anti-VLA-1 mAb Ha31/8. As a negative control, the hamster anti-keyhole limpet hemocyanin mAb Ha4/8 was used. On Day 5, baseline measurements of footpad thickness were obtained, and the mice were again treated with mAb as indicated and subsequently challenged (Chal) with SRBC. Footpad thickness was measured 20 h later. One representative of three independent experiments is shown. Data are presented as percent increase in footpad thickness over the baseline values (a, P<0.001; b, P<.0014; c, P<0.0003; d, P<0.0507; all P values were calculated compared with the negative control mAb treatment data).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report several novel findings related to the leukocyte integrin CD11c/CD18: ICAM-2 and VCAM-1 can serve as its ligands, the anti-mouse CD11c mAb N418 blocks CD11c function, and in vivo, CD11c plays a role in inflammatory response. We expressed human and mouse CD11c in the lymphoblastoid cell line JY and studied the adhesive capacity of CD11c using cell adhesion assays. Our data suggest that in the absence of stimulation by phorbol ester, CD11c can bind to ICAM-1 and ICAM-2 but not ICAM-3. Although CD11c binding to ICAM-1 has been reported earlier, to our knowledge, this is the first demonstration of CD11c binding to ICAM-2. In the presence of PMA, CD11c binds weakly to ICAM-3, demonstrating selectivity of CD11c binding among the different ICAM ligands. Considering the weak binding of CD11c to ICAM-3, it is not surprising that an earlier report failed to observe CD11c binding to ICAM-3 [42 ]. Thus, these data suggest that CD11c is expressed in JY cells in a functional and regulatable manner, and ICAM-2 is a novel ligand of CD11c.

We also demonstrate that CD11c expression in lymphoid cells confers {alpha}4-independent binding to VCAM-1, which is inducible with phorbol ester and is cation-dependent. CD11c-dependent adhesion to VCAM-1 was equivalent to that supported by {alpha}4ß7 and was inhibited specifically by the CD11c mAbs, BU15 and 3.9. The mAb 3.9 binds to the I domain of CD11c (C. Sadhu, Lee Hendrickson, Ken Dick, Tamara Potter, S. Staunton, manuscript submitted, and ref. [43 ]), which suggests that the I domain of CD11c mediates binding to VCAM-1. Thus, CD11c binding to VCAM-1 displays typical features of an integrin-ligand interaction.

To compare the VCAM-1-binding site for CD11c relative to {alpha}4, we used mAb 4B9. VCAM-1 can possess six or seven Ig-like domains in its ectodomain via alternative splicing. The predominant form of VCAM-1 possesses seven Ig-like domains and an {alpha}4ß1-binding site in Domains 1 and 4 each [41 ]. The antibody 4B9 binds VCAM-1 Domain 1 yet blocks {alpha}4ß1 binding to VCAM-1, possessing Domains 1 and 4. We observed only ~25% inhibition of CD11c-mediated binding to VCAM-1 in the presence of 4B9. The low level of 4B9 inhibition of CD11c/CD18 binding suggests a predominant VCAM-1-binding site distinct from that of {alpha}4ß1. This is similar to the distinct sites recognized by CD11a, CD11b, and CD11c on ICAM-1. For example, CD11a and CD11b bind to Domains 1 and 3 of ICAM-1, respectively [44 , 45 ], whereas CD11c binds to Domain 4 [39 ]. Previously, we demonstrated that {alpha}Dß2, which possesses 77% amino acid homology to CD11c, also binds VCAM-1 [46 ]. {alpha}Dß2 binding to VCAM-1 was partially dependent on the {alpha}4ß1 VCAM-1-binding site. Thus, {alpha}Dß2 may bind two VCAM-1 sites with one being distinct from that recognized by {alpha}4ß1. It is not clear that the second {alpha}Dß2-binding site on VCAM-1 is the same or differs from that of CD11c.

Atherosclerosis exhibits many common elements of chronic inflammatory diseases including up-regulation of E-selectin, ICAM-1, and VCAM-1 in arterial lesions. Recruitment of monocytes to inflamed endothelium is an early and critical event in atherogenesis. A novel vascular mimetic device was applied to image the multistep process of monocyte recruitment to inflamed HAECs. Monocyte rolling transitioned to arrest and transmigration within a few cell diameters of capture, as also observed by intravital microscopy of mouse models of atherosclerosis. To study if CD11c might function in the recruitment of monocytes to atherogenic endothelium, we determined if it could support cell adhesion to vLDL-primed and TNF-{alpha}-inflamed HAECs under conditions of shear flow. HAECs were pretreated with vLDL for 2 h per day over 3 days to model postprandial exposure to native, triglyceride-rich lipoproteins. We have reported recently that vLDL and TNF-{alpha} up-regulate membrane expression of VCAM-1 up to threefold compared with acute stimulation with TNF-{alpha} alone [28 ]. The data are consistent with the conclusion that CD11c and VLA-4 are equally important in mediating arrest and transmigration of human monocytes on atherogenic HAECs under shear stress. A smaller contribution to adhesion and migration (i.e., ~10%) was detected for CD11a/CD11b binding to ICAM-1. A previous report has demonstrated that CD11a and CD11b interacting with ICAM-1 and ICAM-2 were essential for monocyte transmigration across cytokine-inflamed, venous endothelium in a static adhesion assay [47 ]. This underscores the prominence of {alpha}4ß1, CD11c, and VCAM-1 and ICAM-1 interactions under conditions of shear stress, as blocking integrin or VCAM-1 also decreases the efficiency of initial tethering on vLDL-primed and cytokine-inflamed HAECs [28 ].

The hamster anti-mouse CD11c mAb N418 has been used widely in studies involving isolation and characterization of mouse DCs. As described in the original report by Metlay et al. [20 ], N418 binds selectively to DCs, relative to macrophages. However, no inhibition of DC:T cell binding was observed in the presence of N418. Therefore, N418 has been considered a nonblocking mAb. In the JY[hCD11c] adhesion assays described in this report, CD11c-blocking mAb had no effect on JY[hCD11c] adhesion to ICAM-1 or ICAM-2, unless CD11a is blocked (data not shown). Therefore, in the studies reported by Metlay et al. [20 ], other integrin/ligand pathways may have masked the function of N418.

As CD11c is able to bind multiple ligands, it can function at several stages of the DTH reaction. Spleen plays an essential role in the induction of DTH response by SRBC. Splenectomy abolished DTH response when performed as late as 6 days after immunization with SRBC [48 ]. In vivo, iC3b is likely to opsonize and facilitate immune, cell mediated uptake of the SRBC. CD11c is a major iC3b receptor on the DCs, which are present in spleen. Therefore, it seems possible that in this model of DTH, CD11c on the splenic DCs may be active in antigen uptake. CD11c on the DCs and macrophages may also function at the antigen-presentation step via binding to ICAM-1 on T cells. Such a function of CD11c has been inferred from the observed inhibition of Langerhans cell-mediated, allogenic T cell proliferation by the CD11c mAb, Leu M5 [22 ]. The CD11c binding to ICAM-1 can furnish additional binding strength between the APC and T cells, particularly when CD11a expression is relatively low. Engagement of CD11c may also lead to cytokine production by the APC. In fact, proinflammatory cytokine production upon engagement of monocyte CD11c has been documented [49 ]. However, our data show that CD11c blocking with N418 only at the SRBC challenge step reduces its efficacy of the mAb. Therefore, compared with the antigen-uptake step, a role of CD11c in APC:T cell interaction seems secondary in this model.


   ACKNOWLEDGEMENTS
 
We thank Dr. John Harlan for his generous gift of the anti-VCAM-1 antibody 4B9. We also thank Lee Adams and Padma Ravikumar for excellent assistance in the preparation of the CD11c expression vectors and generation of the CD11c-expressing cells, Vince Florio for critical reading of the manuscript, and Tony DeMaggio for help with Figure 1 .

Received November 17, 2006; revised January 23, 2007; accepted February 21, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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