CD99 Is a Key Mediator of the Transendothelial Migration of Neutrophils

Olivia Lou, Pilar Alcaide, Francis W. Luscinskas and William A. Muller
J Immunol January 15, 2007, 178 (2) 1136-1143; DOI: https://doi.org/10.4049/jimmunol.178.2.1136

Abstract

Transendothelial migration of leukocytes is a critical event for inflammation, but the molecular regulation of this event is only beginning to be understood. PECAM (CD31) is a major mediator of monocyte and neutrophil transmigration, and CD99 was recently defined as a second mediator of the transmigration of monocytes. Expression of CD99 on the surface of circulating polymorphonuclear cells (PMN) is low compared with expression of CD99 on monocytes or expression of PECAM on PMN. We demonstrate here that, despite low expression of CD99, Fab of Abs against CD99 blocked over 80% of human neutrophils from transmigrating across HUVEC monolayers in an in vitro model of inflammation. Blocking CD99 on either the neutrophil or endothelial cell side resulted in a quantitatively equivalent block, suggesting a homophilic interaction between CD99 on the neutrophil and CD99 on the endothelial cell. Blocking CD99 and PECAM together resulted in additive effects, suggesting the two molecules work at distinct steps. Confocal microscopy confirmed that CD99-blocked neutrophils lodged in endothelial cell junctions at locations distal to PECAM-blocked neutrophils. The CD99-blocked PMN exhibited dynamic lateral movement within endothelial cell junctions, indicating that only the diapedesis step was blocked by interference with CD99. Anti-CD99 mAb also blocked PMN transmigration in a second in vitro model that incorporated shear stress. Taken together, the evidence demonstrates that PECAM and CD99 regulate distinct, sequential steps in the transendothelial migration of neutrophils during inflammation.

An exuberant neutrophil response is a hallmark of acute inflammation. To enter tissues, neutrophils (polymorphonuclear cells (PMN))3 must traverse the endothelial barrier, usually in a paracellular manner across endothelial cell (EC) borders (1, 2). Exit of neutrophils and other leukocytes from vascular flow is a multistep process that begins with rolling of the leukocyte along the EC wall via interactions between selectins (CD62E, L, and P) and their sialylated receptors (3, 4, 5). The captured leukocytes interact with chemokines or other agents presented on the apical surface of the endothelium, which activate leukocyte β1 and β2 integrins (LFA-1, Mac-1, and VLA-4) to bind tightly to their ligands (ICAM-1, ICAM-2, and VCAM-1) (6, 7, 8, 9). This causes arrest of leukocytes on the endothelial surface. The leukocyte moves from the site of firm adhesion to the nearest junction in a newly identified step termed locomotion, which is mediated by the β2 integrins and their counterreceptors ICAM-1 and ICAM-2 (10). Indeed, LFA-1 and ICAM-1 remain tightly apposed during the subsequent transmigration step (11). The leukocyte is then capable of transendothelial migration (TEM, transmigration, diapedesis), the movement of the leukocyte in amoeboid fashion across the interendothelial cell space and into the extracellular matrix (12, 13). Transmigration is largely an irreversible process for neutrophils and commits these cells to enter the inflammatory site. It is therefore an attractive step for anti-inflammatory therapeutic intervention.

PECAM-1 (PECAM and CD31), a 130-kDa member of the Ig gene superfamily, is a well-characterized molecular mediator of diapedesis (14, 15, 16, 17). Anti-PECAM blocking reagents have been demonstrated to block the transmigration of monocytes (14), neutrophils (14, 18, 19, 20), NK cells (21), and eosinophils (22). A recent study using an in vitro model of inflammation described the involvement of CD99, a heavily glycosylated 32-kDa transmembrane protein, as a second molecule in the transmigration of monocytes across EC borders (23). This study demonstrated that CD99 controlled a process subsequent to PECAM-dependent interaction, suggesting that diapedesis itself can be a multistep process. This was also the first characterization of CD99 as an EC marker.

Considering the dominant role of neutrophils in the acute inflammatory response (24), we set out to determine whether CD99 has a role in neutrophil diapedesis. Whereas PECAM is highly expressed by both neutrophils and monocytes, CD99 is expressed at low density on the surface of neutrophils compared with robust expression on monocytes. Therefore, it was not apparent whether CD99 would play a role in neutrophil transmigration. Using an in vitro model of inflammation that allows simultaneous dissection of molecular interactions during diapedesis (25), we report here that blocking Abs against CD99 inhibit over 80% of neutrophil TEM, demonstrating that CD99 is a critical molecule in neutrophil transmigration across inflamed endothelium.

Materials and Methods

Human subjects

All human subject protocols were approved by the Institutional Review Board.

Antibodies

Monoclonal mouse-anti-human Abs hec2 (anti-CD99), hec7 (anti-PECAM), and hec1 (anti-VE-cadherin) were produced from hybridomas generated in the lab as described previously (26). The anti-CD99 mAb YG32 was purchased from DiNonA, and 12E7 and O662 were a gift from Dr. A. Bernard (Institut National de la Santé et de la Recherche Médicale Unité 576, Nice, France). Anti-CD11b (OKM1) was purchased from American Type Culture Collection. Polyclonal Ab 177 was raised in rabbits against the extracellular domain of PECAM (Covance) and column purified on protein A-Sepharose (Amersham Biosciences). Anti-junctional adhesion molecule (JAM)-A mAb (clone 1H2A9) was generated as described previously (27). Fab and F(ab′)2 were cut from hec2 IgG1 using immobilized ficin (Pierce) according to the manufacturer’s protocol and purified using protein G column chromatography. Abs for immunofluorescence were coupled to Alexa-488 and Alexa-546 according to the manufacturer’s protocol (Molecular Probes/Invitrogen Life Technologies). Purity of whole Ig or Ab fragments was confirmed by SDS-PAGE and Coomassie staining, and endotoxin levels were confirmed to be at trace levels (Limulus assay; BioWhittaker).

HUVEC culture

HUVEC were isolated by standard methods (26, 28) and cultured on fibronectin-coated tissue culture dishes in medium 199 (M199; Invitrogen Life Technologies) supplemented with 20% heat-inactivated normal human serum (from healthy volunteer donors) and penicillin and streptomycin (Mediatech). HUVEC at passage 2 were cultured on hydrated collagen type I (Vitrogen from Cohesiontech) gels set in 96-well plates with 50 μl of collagen in each well (26). For the neutrophil transmigration assay, HUVEC were stimulated with IL-1β (50 pg/ml) in 5% CO2 at 37°C for the final 4–6 h before transmigration.

Leukocyte isolation from peripheral blood and neutrophil TEM assay

Leukocytes were prepared from heparinized peripheral blood by density sedimentation in a discontinuous gradient of Ficoll (Amersham Biosciences) and Histopaque-1119 (Sigma-Aldrich). Care was taken to minimize physical disruption or temperature changes to the neutrophil preparation to avoid activation by experimental manipulation. All steps were conducted at room temperature until the transmigration step, when the cocultures were placed in a 37°C tissue culture incubator. Low numbers of remaining RBC, which do not interfere with the transmigration assay, were not lysed to avoid osmotic shock to the PMN. The transmigration assay was performed as previously described (25), with slight modifications to study neutrophils. PMN were resuspended in M199 plus 20% autologous serum at a concentration of 5 × 105 cells/ml for transmigration.

EC monolayers activated for 4 h with IL-1β before the experiment were washed in warm M199, then PMN were added and allowed to transmigrate for 20 min at 37°C. In certain experiments, some EC monolayers were left unstimulated or were activated with TNF-α (25 ng/ml) for 4 h as controls. Abs at a concentration of 20 μg/ml were incubated with the PMN at room temperature for 15 min before TEM and allowed to remain for the duration of the assay; in others, the blocking Abs were added to the PMN only or the HUVEC only and washed off before transmigration. In cases where two blocking Abs were used in conjunction, 20 μg/ml of each individual blocking Ab was used. To ensure equal Ab concentration in parallel experimental conditions, the nonblocking hec1 was added to the other sample to bring the final IgG concentration in each well to 40 μg/ml. Nonadherent PMN were removed using several washes with PBS, and the remaining adherent and transmigrated cells were fixed along with the endothelial monolayer by incubation overnight in 2.5% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer (pH 7.4). HUVEC and neutrophil cocultures were differentially stained with Wright-Giemsa and/or silver stain, the collagen gel with the monolayer was removed and placed between a glass slide and coverslip, and adherent and transmigrated cells were counted in multiple fields from six replicates of each condition tested. Total adhesion was calculated as the total number of cells, both adherent and transmigrated, per high-powered field (hpf). For each condition tested, a minimum of 500 PMN were counted from these individual replicate cultures. Transmigration data are expressed as the mean percentage of the total cells that transmigrated below the endothelial layer (%TEM). Statistical significance was determined by one-way ANOVA with Tukey-Kramer and Newman-Keuls Multiple Comparison Test or Student’s t test, where appropriate, using PRISM software (GraphPad).

Flow cytometry

All leukocyte staining and washing steps were conducted in PBS without divalent cations plus 1% heat-inactivated FBS and analyzed with a FACSCalibur (BD Biosciences) using CellQuest software.

Production of stable transfectants expressing huCD99-Fc chimera

The oligonucleotides 5′-CCGGAATTCCGGCCGTGCCCAGCACCTGAAC-3′ and 5′-CGCGGATCCCTATTTACCCGGAGAC-3′ were used to amplify the Fc region of human IgG1 from the DC67 plasmid (gift from Genzyme, Framingham, MA). The oligonucleotides 5′-CCGCTCGAGCGGGCCACCATGGCCCGCG-3′ and 5′-CCGGAATTCCGGGGCGTCGGCCTCTTCCCCT-3′ were used to amplify the extracellular region of human CD99 from a construct encoding human CD99 in pcDNA/neo (23). Human IgG1 Fc and the human CD99 extracellular region were sequentially inserted into the pcDNA3.1(−) vector (Invitrogen Life Technologies), and the resulting construct was transformed into TOP10 chemically competent Escherichia coli (Invitrogen Life Technologies), as per the manufacturer’s protocol. Clones were screened by restriction enzyme digest and verified by DNA sequencing (Genewiz). CD99-Fc-pcDNA3.1 was electroporated into a CHO cell line, ldlD (29), a gift from Dr. M. Krieger (Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA). Clones of stable transfectants were screened by ELISA using an alkaline phosphatase-coupled Ab against the Fc portion of human Ig. CD99-Fc chimera protein was harvested from the supernatant of spent cultures of selected clones, and purified by protein A-Sepharose column chromatography (Amersham Biosciences).

Immunofluorescence staining and confocal microscopy

The transmigration assay was conducted as described in the presence of hec2 or 177 to block transmigration or hec1 as a control. The endothelial monolayers along with adherent and transmigrated PMN were fixed in 2% paraformaldehyde for 15 min. The collagen gels with the fixed EC and PMN were then incubated with hec1-Alexa 488 (anti-VE-cadherin) to visualize EC and anti-CD11b-Alexa 546 to visualize neutrophils. After extensive washing, the collagen gels were examined using a Zeiss LSM510 confocal microscope to acquire a stack of confocal images in representative areas of the monolayer. X-Z orthogonal reconstructions were performed using Metamorph software.

Live imaging of PMN TEM under static conditions

Neutrophils were added to HUVEC cultured to confluency atop collagen gels set into 30-mm tissue culture dishes with glass coverslip bottoms (10). Interactions between neutrophils and EC were visualized by time-lapse photography using a Zeiss Axiovert 200M widefield microscope. Differential interference contrast images were captured at ×40 magnification every 20 s for 20 min by a charge-coupled device camera connected to the microscope and controlled by Metamorph software (Universal Imaging). The images were converted into movie files using Metamorph or Quicktime (Apple Computers).

Live imaging of PMN TEM under fluid shear stress

Neutrophil-endothelial interactions were studied under defined laminar flow in a parallel plate flow chamber as described previously (30). Briefly, confluent HUVEC monolayers grown on 25-mm glass coverslips (Carolina Biological Supply) coated with 5 μg/ml fibronectin (Sigma-Aldrich) were stimulated with 25 ng/ml TNF-α for 4 h before use in transmigration assays. HUVEC monolayers were also incubated with blocking or nonblocking control Abs before being placed in the flow chamber. Neutrophils were suspended to 5 × 105 cells/ml in Dulbecco’s PBS containing 0.2% HSA and incubated at room temperature for 15 min with various Abs before being drawn through the flow chamber at a constant rate of 0.76 dynes/cm2. Abs were present in the flow buffer for the duration of the assay. Images were recorded in real time by videomicroscopy, and adhesion and transmigration were determined from multiple fields using a ×20 objective. Neutrophils moving beneath the EC layer were observed to darken and become “phase-dense” (i.e., in a different plane of focus), and these were scored as transmigrated cells.

Results

Neutrophils freshly isolated from peripheral blood express far less CD99 than monocytes

Homophilic interaction between CD99 on monocytes and CD99 on EC borders is critical for TEM of monocytes (23). However, staining of CD99 by mAb hec2 on freshly isolated, unactivated neutrophils from peripheral blood was low in contrast to its expression on monocytes (Fig. 1⇓). Staining of PMN by other anti-CD99 mAb, 12E7, O62, D44, and YG32 resulted in similar staining profiles (data not shown). It was therefore not immediately suggestive that CD99 would play the same critical role in neutrophil TEM as it does in monocyte transmigration.

FIGURE 1.
FIGURE 1.

Low expression of CD99 on circulating PMN compared with monocytes. PMN (A) or monocytes (Mo) (B) were stained with hec2-FITC or hec7-FITC to assay cell surface expression of CD99 or PECAM, respectively. The histograms represent cells gated by both CD18 expression and forward and side scatter. The unfilled curves represent an isotype-matched (IgG1-FITC) staining control.

Anti-CD99 Abs block neutrophil transmigration

The presence of anti-CD99 mAb did not affect adhesion of PMN to the EC monolayers (Fig. 2⇓A). However, anti-CD99 mAb had a major effect on PMN transmigration across IL-1β-treated HUVEC, arresting neutrophils on the apical surface of the EC monolayer (Fig. 2⇓C). This was despite the low expression of CD99 on unactivated PMN. Blocked PMN were observed to lodge within EC junctions, particularly at tricellular corners. To confirm that the PMN were arrested at EC junctions, we stained the PMN-EC cocultures after transmigration with silver nitrate, a procedure that defines EC junctions by the deposition of silver precipitate in EC junctions (Fig. 2⇓B).

FIGURE 2.
FIGURE 2.

Reagents against CD99 block TEM of neutrophils. PMN were allowed to transmigrate for 20 min in the presence of 20 μg/ml of the indicated agents. A depicts the average number of adherent PMN per hpf; 20–30 hpf were examined for each condition. B illustrates the arrest of CD99-blocked PMN on IL-1β-stimulated HUVEC stained with silver nitrate before fixation. Note that some of the CD99-blocked PMN are arrested at tricellular junctions (white arrows). C shows the average percentage transmigration. The block by anti-CD99 Fab or F(ab′)2 is equivalent to that by anti-CD99 whole IgG, indicating that this block is independent of FcγR engagement. All blocks are significantly different from the “no Ab” and nonblocking VE-cadherin (anti-VE-cad) conditions (p < 0.001). The data are shown as mean adhesion or transmigration ± SEM from six replicates of each condition, and the data are representative of a total of 5 (YG32, CD99 Fab, F(ab′)2, and CD99-Fc chimera) to 20 (VE-cadherin, PECAM, and CD99) independent experiments with different blood donors.

The anti-CD99 mAb hec2 generally blocked >80% of PMN from transmigrating across IL-1β-activated HUVEC in our in vitro system (Fig. 2⇑C). An equivalent blockade was achieved with YG32, another anti-CD99 mAb (Fig. 2⇑C). Quantitatively, the block obtained with anti-CD99 mAb was generally greater than that obtained with 177, an anti-PECAM polyclonal rabbit Ab (Figs. 2–4⇑⇓⇓). Treating PMN with Fab or F(ab′)2 of hec2 was comparable to using whole Ig as a blocking agent, indicating that the blockade of PMN transmigration by anti-CD99 reagents was independent of PMN FcR engagement (Fig. 2⇑). To demonstrate that blockade of TEM is due to specific interference with CD99 and not merely steric hindrance by junctional Ab, we used nonblocking anti-VE-cadherin mAb (hec1), which stains endothelial junctions similarly to hec2 and 177, as a control in the transmigration assay. hec1 at the same or higher concentration did not block PMN adhesion or transmigration (Fig. 2⇑, compare anti-VE-cadherin to no Ab), demonstrating that the block in transmigration was not due to the physical presence of Abs “jamming up” the junction, or forming immune complexes for PMN to ingest.

FIGURE 3.
FIGURE 3.

Blocking CD99 on EC, PMN, or both EC and PMN inhibits transmigration equivalently. Anti-CD99 or anti-PECAM was added for 20 min to PMN (▪) or EC (□), then unbound Ab was washed off before transmigration. For PMN+EC samples (▧), blocking Abs were added to both EC and PMN and allowed to remain during the transmigration. After transmigration, monolayers were washed, fixed, and stained with Wright-Giemsa before PMN were counted under Nomarski optics. The data are shown as mean transmigration ± SEM (as for Fig. 2⇑C), and the data are representative of five independent experiments. All blocks with anti-CD99 or anti-PECAM differed significantly (p < 0.001) from the nonblocking VE-cadherin condition. Percentage of transmigration did not differ significantly (p > 0.05) when the Ag was blocked on the neutrophil side, the endothelial side, or both, for any of the Ab conditions. Block in transmigration by anti-CD99 differed significantly (p < 0.05) than the block by anti-PECAM under all conditions.

FIGURE 4.
FIGURE 4.

Blocking both PECAM and CD99 has an additive effect in blocking PMN transmigration. The transmigration assay was conducted under standard conditions. Before transmigration, the EC monolayer was treated for 30 min at 37°C with 20 μg/ml of each of the Abs indicated. Blocking PECAM, CD99, or both PECAM and CD99 resulted in transmigration percentages that differed significantly (p < 0.001) from no Ab or the nonblocking VE-cadherin (VE-cad) control. The CD99 block (∗) was significantly better than the PECAM block (p < 0.001). Blocking both PECAM and CD99 (∗∗) resulted in a block of transmigration significantly better (p < 0.01) than blocking either PECAM or CD99 alone. These data are representative of five independent experiments from different blood donors.

Blocking CD99 on either neutrophil or EC reduces transmigration, suggesting a homophilic interaction

Previous studies showed that CD99 on monocytes and CD99 on EC interacted by a homophilic mechanism (23). We sought to determine whether CD99 on PMN would also interact homophilically with CD99 on EC and whether the CD99 expressed on PMN was as important during TEM as EC CD99. Blocking CD99 on either the PMN or the EC reduced TEM to the same extent (Fig. 3⇑), which was equivalent to blocking both sides of the PMN-EC interaction simultaneously. This is consistent with a requirement for a homophilic interaction between neutrophil CD99 and endothelial CD99 during TEM. More direct evidence was provided by the fact that we could block TEM using a soluble CD99-Fc decoy protein (Fig. 2⇑), which interacts with CD99 in a homophilic manner.

CD99 blocks downstream of PECAM during neutrophil diapedesis

When optimal blocking concentrations of anti-CD99 and anti-PECAM Abs were added together during the in vitro assay, the percentage of PMN blocked from transmigrating was significantly greater than with either Ab alone (Fig. 4⇑). On average, over 90% of PMN were halted from transmigrating over the 20-min assay when treated with both blocking agents. This suggested they acted at different steps in TEM. To characterize the spatial relationship of CD99-blocked PMN to EC, we used confocal microscopy (Fig. 5⇓). PMN were incubated with HUVEC monolayers in the presence of mAb as for the quantitative transmigration assays (Figs. 2–4⇑⇑⇑). The monolayers were then fixed and stained for VE-cadherin (to define EC junctions) and CD11b (to stain PMN) to characterize the location of PMN blockade in the junction by confocal microscopy. X-Z orthogonal sections revealed that, in the presence of anti-PECAM Ab, PMN are arrested on top of the apical surface of the EC monolayer as described previously (14). In contrast, CD99-blocked PMN appeared lodged within the EC junctions (Fig. 5⇓), similar to the appearance of monocytes blocked from transmigrating by anti-CD99 mAb (23). We conclude that CD99 inhibition of neutrophil transmigration occurs at a point later in the transmigration process than the PECAM block.

Effects of CD99 blockade are independent of the presence and type of cytokine stimulation

While all of the data presented thus far show PMN interactions with IL-1β-activated HUVEC monolayers, the effects of blocking CD99 and PECAM were seen whether HUVEC monolayers were cytokine activated or not, and whether they were activated with IL-1β or TNF-α (Fig. 6⇓). Due to low levels of basal chemokine secretion (see Discussion), PMN adhere at low levels to unstimulated HUVEC monolayers. Adhesion increased when HUVEC were treated with either cytokine. As previously shown for monocytes (14, 23), anti-CD99 and anti-PECAM did not block adhesion of PMN to EC monolayers, while anti-CD18 (used as a positive control) did block adhesion (Fig. 6⇓A). Very few cells adhere in the presence of anti-CD18 (Fig. 6⇓A), but of those that do adhere, virtually 100% transmigrate (Fig. 6⇓B). The low numbers of PMN that adhere in the absence of cytokine stimulation transmigrate efficiently, and this TEM is blocked effectively with anti-CD99, anti-PECAM, or both. Anti-CD99 mAb was equally effective at blocking the relatively low numbers of PMN binding to unactivated HUVEC as it was at blocking PMN TEM across IL-1β- or TNF-α-activated HUVEC (Fig. 6⇓B).

FIGURE 6.
FIGURE 6.

Adhesion and percentage of transmigration of PMN across unstimulated HUVEC and HUVEC stimulated with IL-1β or TNF-α. Before transmigration, HUVEC were left untreated (▩) or treated with 50 pg/ml IL-1β (▪) or 25 ng/ml TNF-α (□). The transmigration assay was conducted under standard conditions, with blocking Abs added to both PMN and EC. A shows an average of the total number of PMN adhering per hpf examined. B shows the corresponding average percentage of transmigration under the same conditions. Values shown are mean ± SEM for six replicates for each condition. Value of p < 0.001 for %TEM of all blocked conditions when compared with the “no Ab” or VE-cadherin conditions. For all conditions, there was no significant difference (p > 0.05) in the %TEM when the HUVEC were unstimulated or stimulated with IL-1β or TNF-α. Values of p < 0.001 for all CD99 blocks in comparison with all PECAM blocks, and p < 0.01 for CD99+PECAM blocks in comparison with CD99 blocks for HUVEC stimulated with IL-1β or TNF-α.

Imaging of live neutrophil transmigration

We directly observed neutrophil interactions with EC during transmigration by time-lapse videomicroscopy to study the effect of CD99 blocking agents. As a control, neutrophils in the absence of blocking Ab rapidly migrated across the EC monolayer as evidenced by a visual change in phase density. In contrast, neutrophils in the presence of anti-CD99 mAb did not transmigrate, but instead were highly dynamic, moving laterally along EC junctions (Fig. 7⇓ and data not shown). Often, the CD99-blocked neutrophils could be seen deforming the junctional membrane and pushing aside the cytoplasm of the EC, further evidence that the neutrophil was in fact stuck in the junction and had not transmigrated.

CD99 plays a role in neutrophil diapedesis in an in vitro model of fluid shear stress

In the vasculature, EC are subjected to biomechanical forces generated by blood flow. EC can respond to these forces, modifying their physical structure as well as up-regulating or down-regulating particular sets of genes (31). Hence, we investigated whether the variable of laminar fluid shear stress would affect the ability of blocking Abs against CD99 to inhibit neutrophil transmigration. The parallel plate flow chamber used for these experiments has been described previously (30). HUVEC grown to confluence on thin glass plates were prestimulated with TNF-α and set into the flow chamber. Neutrophils were perfused in the presence of CD99-blocking Abs at a steady shear rate of 0.76 dynes/cm2, parallel to the monolayer to provide laminar shear stress. The perfusion of neutrophils was followed by perfusion of buffer containing blocking Ab to ensure exposure to the Abs as the neutrophils were transmigrating.

Under these experimental conditions, half the PMN were prevented from transmigrating by the presence of the anti-CD99 mAb, hec2 (Fig. 8⇓ and data not shown). In contrast, mAb against JAM-A had no significant effect, as reported previously (11). The live imaging of PMN transmigration under shear stress closely resembled the studies under static conditions (Fig. 7⇓ and data not shown). CD99-blocked neutrophils demonstrated a wide range of morphologies. Whether under static or fluid shear conditions, some CD99-blocked neutrophils appeared flattened and spread laterally along a junction whereas others would appear to be diving perpendicular to the EC monolayer, but with a pseudopod or a significant proportion of the cytoplasmic body lodged in the EC junction.

Discussion

We demonstrate here that CD99 is a key molecule mediating the diapedesis of neutrophils in an in vitro model of inflammation. CD99 expression on circulating neutrophils varies according to donor but is consistently low in comparison to its expression on monocytes (Fig. 1⇑). Nevertheless, we find that blocking agents against CD99 inhibit over 80% of adherent neutrophils from migrating across HUVEC treated with the proinflammatory cytokine, IL-1β (Fig. 2⇑), or TNF-α (Fig. 6⇑). Fab and F(ab′)2 of blocking mAb, as well as CD99-Fc chimera selectively inhibit TEM as efficiently as intact IgG. Engaging leukocyte PECAM with Abs often enhances their adhesion to endothelial monolayers via inside-out activation of β2 integrins (Fig. 2⇑A and Ref. 21); they are nonetheless prevented from transmigrating by the same Ab (Fig. 2⇑C and Ref. 14). No effect on adhesion is observed with anti-CD99 reagents.

Preliminary experiments (data not shown) indicate that there is a large pool of CD99 within neutrophils that becomes exteriorized when the PMN are activated. Thus, a PMN attached to the surface of the EC has significantly higher CD99 surface expression than the unactivated PMN in circulation. The exact conditions and signaling mechanisms that regulate this process are complex and are currently under investigation.

We find that binding of mAb to either neutrophil CD99 or endothelial CD99 results an equivalent quantitative block of transmigration, suggesting a homophilic interaction for CD99 (Fig. 3⇑). The fact that a CD99-Fc soluble chimera blocks PMN transmigration (Fig. 2⇑C) lends additional support for a homophilic interaction between the chimera and CD99 on the EC or PMN. CD99 homophilic adhesion was previously demonstrated by increased cell aggregation among L cells transfected with CD99; this increased aggregation could be specifically blocked with the anti-CD99 blocking mAb, hec2 (23). Thus it appears that homophilic PECAM-PECAM and CD99-CD99 interactions are involved in the diapedesis of monocytes (23), and as demonstrated here, neutrophils.

In agreement with the earlier study in monocyte transmigration (23), we demonstrate here that CD99 likewise comprises a “later step” in PMN transmigration. We show that CD99 homophilic interactions between EC and PMN occur downstream of PECAM-PECAM interactions (Fig. 5⇓). Analysis by Nomarski optics (Fig. 2⇑) and confocal microscopic imaging (Fig. 5⇓) show that while PECAM-blocked PMN arrest on the EC surface overlying the junctions, CD99-blocked neutrophils are lodged within the intraendothelial junctional space. The mechanism by which CD99-blocked PMN become lodged downstream of those blocked by PECAM remains to be elucidated.

FIGURE 5.
FIGURE 5.

Anti-CD99-treated PMN are blocked at a location downstream of anti-PECAM-blocked PMN. PMN and EC were pretreated with anti-VE-cadherin (negative control), anti-PECAM, or anti-CD99 before transmigration. Collagen gels were fixed along with the PMN-EC cocultures with 2% paraformaldehyde and stained with anti-VE-cadherin-Alexa 488 (green) to visualize EC junctions and anti-CD11b-Alexa 546 (red) to identify PMN. The images shown are along the X-Z plane (orthogonal to monolayer). The green channel was intentionally amplified to mark the location of the endothelial monolayer. VE-cadherin staining appears intermittent, since it is only present where the plane of imaging crosses a cell junction. In the VE-cadherin sample, the PMN is distinctly under the monolayer. White arrows in the CD99 samples pinpoint the contact between PMN and EC, emphasizing that the PMN is partway through the junction. These images are representative of >250 cells examined for each condition in multiple experiments.

Still images of CD99-blocked PMN reveal a diverse morphology, with some blocked neutrophils stretched out and lodged laterally in the junction whereas others have the bulk of their cell bodies in the luminal or abluminal side, but with a portion of their cytoplasm in the junctional space (data not shown). Live imaging (Fig. 7⇓ and data not shown) reveals that CD99-blocked PMN are in fact not firmly arrested, but moving dynamically within EC junctions, sometimes back and forth along the same junctional space as if looking for a place to transmigrate. This is reminiscent of the appearance of monocytes blocked on TEM by anti-PECAM, which “walked” along the tops of the EC borders (10). The live imaging illustrates the capacity of the neutrophil to deform the membrane of the EC to accommodate its movement (data not shown). In our studies, transmigration was observed to occur paracellularly (between EC) as opposed to transcellularly (through the body of the cell) (32, 33). It is not known whether CD99 plays a role in transcellular migration, which is thought to comprise a small percentage (10% at most), of transmigration events.

FIGURE 7.
FIGURE 7.

CD99-blocked PMN interact dynamically with EC membranes but are prevented from transmigrating across junctions. The montage shown represents the movement of a single representative PMN treated with 20 μg/ml anti-CD99 before transmigration. The montage represents a total of 420 s from the time the PMN landed onto the EC monolayer, with each frame representing a 20-s lapse. The polar ends of the PMN, the leading edge and the uropod, are indicated by arrowheads. Three arrowheads represent a landing or a bifurcation. Differential contrast images were captured at ×40 magnification every 20 s for a total transmigration time of 20 min. To aid the reader, the EC junctions have been identified using white dotted lines in every fourth panel of the montage (time points 0, 80, 160, etc.) The images are selected from a video representation (data not shown).

We primarily studied neutrophil transmigration in an assay devoid of shear stress. Our static assay, which employs HUVEC plated atop collagen gels set in 96-well plates (25), allows the EC to form a physiologic basal lamina and physiological matrix into which they can migrate. Findings from this assay have all been verified in vivo (34, 35). The diapedesis step, unlike earlier steps of rolling and adhesion, occurs largely independently of shear (36, 37). However, there are reports that TEM efficiency is enhanced in the presence of shear stress in vitro (38, 39, 40). Therefore, we sought to verify our findings in a different in vitro model under flow conditions and found that under fluid shear stress as well, blocking CD99 inhibits neutrophils from transmigrating (Fig. 8⇓). Our mAb hec2 was quantitatively less effective at blocking TEM under these conditions. This is not likely to be due to the fact that these EC were activated by TNF-α, since in the static assay, hec2 blocks equivalently under all conditions tested (Fig. 6⇑). The differences could be due to the assay conditions (static vs shear), and/or the differences in preparation of the PMN. In the static assay all PMN are subjected to transmigration at the same time-as soon as they are isolated. In the shear assay, since only one condition can be tested at a time, the isolated PMN are held in suspension for longer periods. We have found that PMN tend to become activated upon standing and this may affect their sensitivity to Ab blockade.

FIGURE 8.
FIGURE 8.

Anti-CD99 mAb block PMN TEM in an in vitro model incorporating shear stress. Blocking with anti-CD99 results in a transmigration percentage significantly different from the other TEM bars (p < 0.01). HUVEC monolayers were stimulated with 25 ng/ml TNF-α for 4 h before transmigration. Neutrophils (5 × 105 cells/ml in Dulbecco’s PBS) were drawn through the flow chamber at a constant rate of 0.76 dynes/cm2. PMN and EC were incubated with 20 μg/ml of the indicated Abs before transmigration, and Abs were present in the flow buffer for the duration of the assay. Images were taken by videomicroscopy every 15 s for a total of 10 min of transmigration. Fifteen to 20 cells were analyzed for each condition. The percentage of PMN transmigrating in each condition was determined from multiple fields using a ×20 objective. Neutrophils moving beneath the EC layer (i.e., in a different plane of focus) were scored as transmigrated cells. Data represent the mean ± SEM of three separate experiments. ∗, Significantly different from no Ab or JAM-A (p < 0.01).

Blocking CD99 alone in our model prevents >80% of PMN from transmigrating; blocking PECAM generally prevents 70–75% of PMN from transmigrating. We do not know why some PMN manage to transmigrate despite the presence of blocking Abs. Perhaps PMN in different stages of their lifespan or different states of activation have a different sensitivity to CD99 or PECAM. Blocking both PECAM and CD99 does have an additive effect, blocking >90% of PMN (Figs. 4⇑ and 6⇑). The residual PMN that transmigrate in the presence of the combined PECAM plus CD99 blockade do not represent PMN that would have transmigrated in the absence of cytokine stimulation. The low numbers of PMN that do adhere to HUVEC monolayers in the absence of cytokine stimulation (due to low levels of chemoattractants made by EC and in serum that accumulate in the collagen gels (25)) transmigrate efficiently, and their transmigration is effectively blocked by Abs against PECAM and CD99 (Fig. 6⇑). It is possible that some functional redundancy exists between PECAM and CD99; however, our data are more suggestive of a sequential relationship with CD99 functioning downstream of PECAM. The temporal and spatial relationship between these molecules and their signaling will be an important area for research.

Other molecules have recently been implicated in diapedesis. The existence of PECAM- and CD99-independent pathways is supported by our data, because even with our blocking Abs at saturating conditions, a small proportion of neutrophils or monocytes are capable of transmigrating. Several members of the JAM family have been implicated in transmigration. Several reports suggest JAM-A mediates TEM of leukocytes (41, 42), although the transmigration of human neutrophils (11) and monocytes (10) in vitro is unaffected by anti-JAM-A mAb. JAM-B and -C have been implicated in leukocyte extravasation in vitro and in vivo (43, 44, 45). Poliovirus receptor is a member of a related family of junctional proteins on EC and binds to CD226 on leukocytes. Blocking either poliovirus receptor or CD226 reduced monocyte TEM in an in vitro assay (46). Thus, it appears that diapedesis is regulated by a number of molecular interactions, some homophilic and some heterophilic. It will be important to determine whether in vivo these molecular gatekeepers act in series or in concert, and whether they overlap in function or whether each one is required to move a leukocyte across EC junctions.

Inflammation is a critical part of the innate immune response against infection; at the same time, a degree of inflammation is present in most disease processes. For CD99 and PECAM to be considered as anti-inflammatory drug targets, it is important to understand the relative importance of each molecular interaction in the diapedesis of different types of leukocytes. Neutrophils dominate in acute inflammatory responses (such as those in response to infection or following ischemia), whereas monocytes/macrophages take over in chronic inflammation. We demonstrate here that CD99 plays a profound role in the transmigration of neutrophils. Similar to findings in monocyte TEM, we find that CD99 appears to act at a step downstream of PECAM during diapedesis. Blocking both molecules has an additive effect in blocking almost all neutrophils and monocytes from transmigrating. It will be important to extend these studies into in vivo models. A recent study demonstrated that polyclonal Ab against mouse CD99 inhibited homing of T cell clones into inflamed skin (47). Generating reagents to block CD99 function in vivo and the generation of CD99-deficient mice should shed further insight into CD99 function in the inflammatory response.

Acknowledgments

We thank Ron Liebman for excellent technical assistance, and Drs. Alan Schenkel and Oliver Florey for comments on the manuscript.

Disclosures

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 HL064774 and HL046849 (to W.A.M.) and HL53993 and HL36028 (to F.W.L.), a Fulbright-Spanish Ministry of Education and Science (to P.A.), and a predoctoral fellowship from the Cancer Research Institute (to O.L.).

  • ↵2 Address correspondence and reprint requests to Dr. William A. Muller, Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, 1300 York Avenue C-312, New York, NY 10021. E-mail address: wamuller{at}med.cornell.edu

  • ↵3 Abbreviations used in this paper: PMN, polymorphonuclear cell; EC, endothelial cell; hpf, high-powered field; JAM, junctional adhesion molecule; M199, medium 199; TEM, transendothelial migration.

  • Received July 11, 2006.
  • Accepted October 24, 2006.

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