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The Journal of Immunology, 1998, 160: 5058-5065.
Copyright © 1998 by The American Association of Immunologists

Functional Mapping of CD11b/CD18 Epitopes Important in Neutrophil-Epithelial Interactions: A Central Role of the I Domain1

Leora B. Balsam, Tony W. Liang and Charles A. Parkos2

Divisions of Gastrointestinal Pathology, Departments of Pathology, Brigham and Women’s Hospital, Boston, MA 02115; and Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the intestine, lung, and urinary tract, neutrophil (polymorphonuclear leukocyte, PMN) transepithelial migration is dependent on the leukocyte ß2 integrin CD11b/CD18. While the regions of CD11b involved in recognition of several soluble ligands are known, those that mediate PMN-epithelial interactions have not been investigated. In this study, mAbs reactive with four extracellular regions on CD11b, the NH2-terminal region, I (inserted) domain, cation-binding region, and region proximal to the transmembrane domain (C domain), were analyzed for the ability to block CD11b/CD18-mediated interactions with T84 intestinal epithelial cells. In such a manner, epitope mapping was applied to the complex interactions between CD11b/CD18 and a cell-based ligand system. I domain Abs strongly inhibited both adhesion of PMN to epithelial cells and PMN migration across T84 epithelial monolayers. However, the profile of inhibition was distinct from that of other known ligands of CD11b/CD18. CBRM1/32, an Ab to a discontinuous epitope residing within the NH2- and cation-binding domains, strongly inhibited both adhesion and transmigration responses. C domain Abs had minimal effects on adhesion and transmigration. These findings appear applicable to other epithelia, since similar results were obtained in transmigration experiments with CF15 human airway epithelial cells. Finally, Ab inhibition profiles were confirmed with adhesion assays of isolated epithelial cells to purified CD11b/CD18. These findings demonstrate the central role of the I domain and the participation of a discontinuous region shared by the NH2- and cation-binding domains in mediating PMN-adhesive interactions with epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Active inflammatory diseases of the respiratory system, gastrointestinal tract, and urinary tracts are characterized by neutrophil (polymorphonuclear leukocyte, PMN)3 migration across an epithelium and subsequent collection in the lumenal space (1, 2). In the intestine, such transepithelial migration of PMN results in transient disruption of epithelial barrier function (3, 4) and has functional consequences, including leakage of lumenal contents into the systemic circulation and stimulation of epithelial chloride secretion (5, 6). Epithelial chloride secretion, triggered by the release of 5'-adenosine monophosphate from lumenal PMN (6), represents the basis for secretory diarrhea (7), a common symptom of individuals afflicted with active intestinal inflammation.

Although much research has focused on the process whereby PMN migrate across an endothelium, less is known about the interaction of PMN with epithelia. In the presence of chemotactic peptides such as FMLP, PMN can be driven to migrate across monolayers of epithelial cells grown on permeable membrane supports (8, 9). Because in vivo PMN migration across epithelial monolayers occurs in a polarized manner from the basolateral to apical aspect of the epithelium, assays of PMN transepithelial migration have required modifications to more closely mimic the natural process (8, 9). And although it was shown previously that PMN can migrate across epithelial monolayers in an apical to basolateral or basolateral to apical direction, transmigration of PMN is 5 to 20 times more efficient in the natural basolateral to apical direction. Such transmigration is dependent on the ß2 leukocyte integrin CD11b/CD18, but not on CD11a/CD18 or CD11c/CD18 (8).

The ß2 integrins, a family of four cell surface transmembrane glycoproteins expressed only on leukocytes, include CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1, CR3, or Mo-1), CD11c/CD18 (p150, 95), and CD11d/CD18 (10, 11, 12, 13, 14, 15). They consist of a common ß subunit (CD18) and homologous {alpha} subunits (CD11a-d) that bind noncovalently to form an {alpha}ß heterodimer. Studies with functionally inhibitory mAbs to CD11b/CD18 demonstrate the role of CD11b/CD18 in inflammatory processes, including PMN adhesion to and migration across endothelium (16) and epithelium (8), phagocytosis of opsonized particles (17), and release of PMN hydrolytic enzymes during the respiratory burst (18). Moreover, PMN of patients with leukocyte adhesion deficiency (LAD), characterized by lack of surface expression of ß2 integrins, fail to adhere to and migrate across both endothelium and epithelium (8, 19).

Unlike PMN-endothelial interactions, which have been shown to involve several steps including selectin-mediated rolling, ß2 integrin-dependent adhesion, and CD47-dependent transmigration (20, 21, 22, 23), the interaction between PMN and epithelial cells does not involve selectins or CD11a/CD18 (reviewed in 24 . Initial adhesive events between PMN and epithelial cells appear to be mediated exclusively by CD11b/CD18 (8), followed by CD47-dependent events (22).

A combination of immunologic, mutagenic, and biochemical approaches has been used to map regions of the CD11b {alpha}-chain involved in ligand recognition. Like other integrin {alpha}-chains, CD11b consists of a short cytoplasmic tail, a single transmembrane domain, and a long extracellular domain (25) (see Fig. 1Go). The extracellular domain consists of seven repeats of ~50 amino acids. Repeats V to VII have sequences similar to the divalent cation-binding "EF-hand" motif. Repeats II and III are separated by an approximately 200-amino-acid I (inserted) domain. This I domain is found only in the {alpha} subunits of ß2 integrins and the {alpha}1 and {alpha}2 subunits of ß1 integrins (10). Recent studies demonstrate that the I domain directly mediates binding a number of ligands including iC3b (26, 27, 28, 29), fibrinogen (26, 29), ICAM-1 (26, 29), ICAM-2 (30), heparin (31), and PMN-inhibitory factor (32, 33), but not to factor X (29). Additionally, the I domain contains a novel Mg2+/Mn2+ binding site that is essential for recognition of certain ligands (27).



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  		FIGURE 1. Schematic representation of the structure of human CD11b showing regions reactive with the Abs used in the present study.

 
In this work, we describe the results of experiments aimed at identifying the regions of CD11b involved in PMN-epithelial interactions. While information exists detailing the interactions of subdomains on CD11b with soluble ligands, little is known about the regions of CD11b that mediate complex functional processes such as transmigration. We have used a panel of mAbs that were previously mapped to four different regions of the CD11b extracellular domain: the NH2 terminus, the I domain, the cation-binding domain, and the COOH terminus (C domain). Performing a combination of transmigration and adhesion experiments with human PMN and epithelial monolayers, as well as adhesion assays with purified CD11b/CD18 and epithelial cell suspensions, we demonstrate that a region of the CD11b I domain mediates adhesion to and transmigration across epithelia and that this recognition site overlaps, but is not identical to previously characterized recognition sites for other known ligands. Moreover, we show participation of a discontinuous region shared by the NH2- and cation-binding domains in such adhesive interactions, suggesting a complex tertiary structure in the epithelial binding domain(s).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

T84 intestinal epithelial cells (34) (passages 60 to 100) were grown in a 1:1 mixture of DMEM and Ham’s F-12 medium supplemented with 15 mM HEPES buffer (pH 7.5), 14 mM NaHCO3, 40 µg/ml penicillin, 8 µg/ml ampicillin, 90 µg/ml streptomycin, and 5% newborn calf serum. Cells were subcultured or harvested every 6 to 8 days with 0.1% trypsin and 1 mM EDTA in Ca2+- and Mg2+-free PBS. For adhesion and transmigration experiments, T84 cells were grown on collagen-coated, permeable polycarbonate filters (inserts) with a surface area of 0.33 cm2 (Costar, Cambridge, MA), as previously described (8, 9). For basolateral to apical transmigration, T84 cells were grown on the underside of permeable filters to produce inverted monolayers (8, 9). For adhesion experiments with purified CD11b/CD18, T84 cells were grown to confluency in 25-cm2 tissue culture flasks and harvested after incubation for 20 min (37°C) in 0.1% trypsin and 0.9 mM EDTA in Ca2+- and Mg2+-free PBS, as previously described (9, 34). A subset of transmigration experiments was performed on CF15 human airway epithelial cells. JME/CF15 cells (35), an SV40 large T Ag-transformed epithelial cell line derived from the nasal airway epithelium of a cystic fibrosis patient, were cultured as previously described (35) with some modifications. CF15 cells were cultured as monolayers to 80 to 100% confluency (~7 days) in 75-cm2 flasks in hormone-defined medium (3 parts DMEM:1 part Ham’s F-12 medium containing 0.18 mM adenine, 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 1.1 µM hydrocortisone, 5 µg/ml insulin, 5 µg/ml transferrin, 2 nM triiodothyronine, 1.64 µM epidermal growth factor (EGF), and 5.5 µM epinephrine), followed by trypsinization as above for passage (1–2 x 106 cells per 75 cm2) or culture on permeable supports. Cells were cultured on permeable supports (5 x 105 cells/cm2) that were pretreated with 50 µl of a 0.5 mg/ml solution of human placental collagen in 0.2% acetic acid (Sigma, St. Louis, MO). For monolayers on permeable supports, on the day after plating, the tissue culture media were removed from the apical (lumenal) surface and maintained as an apical air interface until use 3 days later.

Buffers

HBSS (HBSS(+)) contained (in g/L): 0.185 CaCl2, 0.098 MgSO4, 0.4 KCl, 0.06 KH2PO4, 8 NaCl, 0.048 Na2P04, 1 glucose, and HEPES buffer added to 10 mM (pH 7.4). Modified HBSS (HBSS(-)) was prepared as HBSS(+), except without CaCl2 or MgSO4.

PMN isolation

PMN were isolated from whole blood of normal human volunteers using a gelatin sedimentation technique previously described (36). PMN were resuspended at 5 x 107 cells/ml in HBSS(-) and stored at 4°C for a maximum of 1 to 4 h before use in experiments.

Antibodies

A panel of murine mAbs that had previously been shown to bind to defined regions of the CD11b extracellular domain and to have effects on the binding of known ligands to CD11b/CD18 (37) was obtained as ascites fluids. The extracellular regions of CD11b reactive with the Abs are illustrated in Figure 1Go. The panel included: CBRM1/9 (C domain, IgG2a), CBRM1/20 (cation-binding domain, IgG1), CBRM1/23 (C domain, IgG2a), CBRM1/24 (I domain, IgG1), CBRM1/26 (C domain, IgG2b), CBRM1/27 (I domain, IgG1), CBRM1/29 (I domain, IgG1), CBRM1/31 (I domain, IgG2a), CBRM1/32 (NH2- and cation-binding domain, IgG1), and CBRM1/34 (I domain, IgG1). CBRM1/9 and CBRM1/32 were also used after precipitation of ascites fluids in saturated ammonium sulfate, followed by resuspension and dialysis against saline HEPES (150 mM NaCl and 10 mM HEPES, pH 7.4). CBRM1/23 used in transmigration and PMN adhesion assays was purified from ascitic fluid using protein A-Sepharose (Sigma) and dialyzed against normal saline containing 10 mM HEPES, pH 7.4. Other CD11b mAbs used included OKM1 (C domain, purified, IgG2b) (17), LM2/1 (I domain, purified, IgG1) (37), and 44a (I domain, purified, IgG2a) (27, 38). As controls, functionally inhibitory mAbs to CD11a (TS1/22, subclass IgG1) (39), CD11c (SHCL3, subclass IgG2a) (40), and CD18 (TS1/18, subclass IgG1) (39) were also used as ascites.

Saturating concentrations of purified mAbs, ascitic fluids, and ammonium sulfate-precipitated ascites were determined by ELISA on adherent PMN. A quantity amounting to 1 x 105 PMN was added to each well of a 96-well microtiter plate in 0.15 ml HBSS(+), followed by incubation for 20 min (37°C) to allow for settling and spreading. Adherent PMN were then stimulated with PMA (0.1 µg/ml) for 10 min (37°C) to up-regulate CD11b/CD18. After blocking nonspecific protein binding with 10% goat serum in HBSS(+), PMN-coated wells were incubated with varying concentrations of the anti-CD11b mAbs in blocking buffer, followed by incubation with alkaline phosphatase-conjugated secondary Ab in blocking buffer. Color development was performed using a standard alkaline phosphatase substrate assay, and the OD at 405 nm was determined using a microtiter plate reader.

Saturating concentrations of mAbs for assays with purified CD11b/CD18 were determined using a similar protocol. However, instead of using PMN, microtiter plates were coated for 2 h (20°C) with 50 µl of a solution of CD11b/CD18, purified as previously described (26), which was diluted in 150 mM NaCl, 2 mM MgCl2, and 25 mM Tris, pH 7.3, for a final integrin concentration of approximately 5 to 10 µg/ml. Subsequent steps in the assay were the same as above, except for the use of a modified blocking buffer containing 2 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 0.5% heat-aggregated BSA in Dulbecco’s PBS, pH 7.4. Saturating Ab concentrations were estimated as above assuming 2 x 105 copies of CD11b/CD18 per PMN. The maximum OD values obtained for the various Abs ranged from 0.6 to 1.5 in a typical experiment with purified integrin, and from 0.2 to 0.5 on intact PMN, indicating good binding of the Abs. From these experiments, the following final saturating Ab concentrations were determined for both transmigration and adhesion assays: 1/200 dilution of ascitic fluid in HBSS(+), 1/50 dilution of ammonium sulfate-precipitated CBRM1/9 and CBRM1/32, 5 µg/ml OKM1, 20 µg/ml LM2/1, 20 µg/ml CBRM1/23, and 10 µg/ml 44a.

Transmigration and adhesion experiments

PMN transmigration experiments were performed in the presence of various mAbs using both standard (apical to basolateral migration) and inverted (basolateral to apical migration) T84 epithelial cell monolayers exactly as described previously (8). Before use, confluency of epithelial monolayers was assessed by measurement of transepithelial resistance. T84 monolayers with transepithelial resistances of 250 to 1000 ohm · cm2 were then washed extensively with HBSS(+) to remove residual medium, and a solution of Ab and PMN was added to the upper chamber of the transwell device to a final volume of 200 µl. Transmigration was initiated by transfer of monolayers containing the mAb/PMN solution to 24-well tissue culture plates containing 1 ml of the chemoattractant FMLP (0.1 µM) in HBSS(+). After a 110-min incubation (37°C), PMN transmigration was quantitated by myeloperoxidase (MPO) assay (8). Recovery of PMN determined by this MPO assay averaged 94 ± 9% of the total number of applied cells. In apical to basolateral transmigration assays, migrated PMN were defined as the sum of cells that migrated into the monolayers plus cells appearing in the chemoattractant-containing lower reservoirs. In these experiments, after application of 2 x 106 PMN, migrated cells comprised approximately 5 to 30% of the total. In addition, as has been observed previously, more than 50% of the migrated PMN appeared in the lower reservoirs (8). For basolateral to apical transmigration assays, migrated PMN were defined as cells appearing in the chemoattractant-containing lower reservoirs. In these assays, typically 20 to 60% of the applied PMN migrated, with greater than 75% appearing in the lower reservoirs, in accordance with previous findings (8). A subset of basolateral to apical transmigration experiments was performed exactly as above with monolayers of CF15 airway epithelial cells instead of T84 cells.

The percentage of control transmigration was determined by the equation: 100 x (number transmigrated PMN in sample + experimental mAb)/(number transmigrated PMN in sample + control mAb (TS1/22 or SHCL3)). The percentage of inhibition by mAb was determined according to the equation: 100 - (percent control transmigration).

Adhesion assays between PMN and T84 monolayers were performed as previously described (22, 41, 42). T84 monolayers were washed free of media with HBSS(-), incubated with 2 mM EDTA in HBSS(-) for 12 min (37°C), and rinsed with HBSS(+). A solution (150 µl) containing saturating concentrations of mAb, FMLP (100 nM final concentration), and 2 x 106 PMN was layered on the apical surface. Monolayers were subjected to low speed centrifugation (250 x g, 4 min, 22°C) to ensure uniform PMN-epithelial cell contact, incubated for 15 min (37°C), and washed with HBSS(+) (22°C). Adherent PMN were quantified by MPO assay (8). The percentage of control adherence was determined by the equation: 100 x (number of adherent cells in sample + experimental mAb)/(number of adherent cells in sample + control mAb (TS1/22)). The percentage of inhibition by mAb was determined according to the equation: 100 - (percent control adherence).

Adhesion of T84 cells to purified CD11b/CD18

Functionally active CD11b/CD18 was purified by LM2/1 immunoaffinity chromatography, as described by Diamond et al. (26). SDS-PAGE of the purified integrin, followed by Coomassie blue staining, revealed two prominent protein bands with Mr values of ~150 and 95 kDa, characteristic of CD11b and CD18, respectively (not shown). As detailed previously (22, 42) and above, microtiter plates were coated with purified CD11b/CD18 (50 µl of ~1–2 µg/ml CD11b/CD18 per well) and nonspecific binding blocked by incubation with a blocking buffer containing 2 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 0.5% heat-aggregated BSA in Dulbecco’s PBS, pH 7.4. Saturating concentrations of mAbs diluted in blocking buffer were added in a total volume of 50 µl and incubated with CD11b/CD18-coated wells for 1 h (4°C). Isolates of T84 cells were then fluorescently labeled by incubation with 5 µg/ml BCECF-AM (2', 7'-bis(2-carboxyethyl)-5 (and 6)-carboxyfluorescein acetoxymethyl ester; Molecular Probes, Eugene, OR) for 10 min (37°C), followed by addition (50 µl, ~2.5 x 105 cells/well) to the integrin-coated wells. After 1 h of incubation on a stationary surface (37°C), the microtiter wells were gently washed, and adhesion was quantified by measuring the excitation/emission wavelength of 485/535 nm using a fluorescence microtiter plate reader (Millipore, Milford, MA).

The percentage of adherence was calculated as: 100 x (postwash fluorescence)/(prewash fluorescence). The percentage of control adhesion was determined by the equation: 100 x (percent adherence of sample + experimental mAb)/(percent adherence of sample + control mAb (SHCL3)). The percentage of inhibition by mAb was determined according to the equation: 100 - (percent control adhesion).

Statistics

Data are presented as the mean ± SD and compared by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of apical to basolateral transmigration by mAbs

We have demonstrated previously that PMN migration across cultured T84 epithelial cell monolayers is dependent on a CD11b/CD18-mediated adhesive event (8). However, the structural epitopes involved in this interaction have not been defined. Using a panel of previously characterized mAbs that recognize different epitopes on the CD11b extracellular domain (37), we attempted to map the regions that mediate recognition of epithelial cell counter-receptors. In the presence of a gradient of the chemotactic peptide FMLP, PMN layered on the apical surface of T84 monolayers were driven to transmigrate across the epithelium in an apical to basolateral direction. Transmigration was quantified by assaying for MPO activity. Consistent with previous reports (8), when no mAb was added to the apical compartment, after 110 min of incubation, 27.4 ± 2.4% PMN were found between the monolayer and filter, and 72.5 ± 2.4% PMN were found in the chemoattractant-containing lower reservoir. Since transepithelial migration is defined as PMN migration across tight junctions (3, 43), we report the sum of monolayer-associated PMN and PMN within the lower reservoir as transmigrated PMN. The results of the apical to basolateral transmigration experiments are shown in Figure 2Go. In the presence of the binding Ab control (TS1/22; negative control), 1.3 ± 0.33 x 105 PMN transmigrated, similar to the number of PMN transmigrating in the absence of mAb (1.8 ± 0.24 x 105). Compared with negative controls, with the exception of one Ab (LM2/1), mAbs recognizing epitopes on the I domain inhibited transmigration with a mean of 86 ± 10% (p < 0.001), whereas mAbs recognizing the C domain only inhibited transmigration 9.9 ± 28.1% (NS). Six of seven mAbs that map to the I domain inhibited transmigration strongly (>75%), whereas no mAb directed to the C domain inhibited transmigration >30% (Table IGo). LM2/1 was the only I domain mAb that consistently had no inhibitory effect on transmigration in the apical to basolateral direction. CBRM1/32, which maps to a discontinuous epitope consisting of both the NH2 domain and divalent cation-binding region (37), inhibited transmigration 94.6 ± 2.2% compared with controls (p < 0.01). The mAb that mapped to the divalent cation-binding region, CBRM1/20, had no inhibitory effect.



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  		FIGURE 2. Effect of anti-CD11b mAbs on PMN migration across T84 cell monolayers in the apical to basolateral (A to B) direction. PMN (2 x 106 cells/monolayer) were incubated with saturating concentrations of the indicated mAbs and induced to migrate across T84 monolayers by a gradient of the chemotactic peptide FMLP for 110 min, followed by quantitation, as detailed in Materials and Methods. As controls, migration is shown in the presence of no Ab or mAb TS1/22, anti-CD11a. With the exception of LM2/1, mAbs directed against the I domain strongly inhibit transmigration. In addition, CBRM1/32 also strongly inhibits transmigration. mAbs directed against the C domain do not inhibit transmigration. Each condition represents the mean of three to five individual monolayers ± SD (60 monolayers total). One of three representative experiments.

 

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  		Table I. Summary of mAb effects on CD11b-mediated interactions with epithelial cells1

 
Inhibition of basolateral to apical transmigration by mAbs

PMN migration across T84 monolayers in the direction that occurs naturally (basolateral to apical) was investigated using a modified transwell apparatus (8, 9). Inverted monolayers of T84 cells were grown such that the apical surface was in direct contact with the chemoattractant-containing lower reservoir. In the presence of our panel of defined anti-CD11b mAbs, PMN were layered on the filter surface and were driven to migrate across the filter and epithelial monolayer by an FMLP gradient. PMN that had crossed epithelial tight junctions and entered the reservoir below were quantified and defined as having transmigrated. PMN migration was similar in the presence of negative control Ab TS1/22 as in the absence of any mAb (2.6 ± 0.55 x 105 vs 1.8 ± 0.5 x 105) (Fig. 3Go). As can be seen in the figure and in Table IGo, when compared with controls, I domain mAbs inhibited transmigration with a mean of 94 ± 6.5% (p < 0.01), and C domain mAbs inhibited transmigration with a mean of 30 ± 15% (NS). Five of six I domain mAbs inhibited transmigration strongly (>80%) (Table IGo). Such inhibition was not due to Fc-mediated interactions since the effects of I domain Abs were not influenced by the presence of functionally blocking anti-CD16 F(ab')2 (44) and/or anti-CD32 IgG (45) (data not shown). The results of basolateral to apical transmigration experiments in the presence of mAb LM2/1 were inconsistent. In some experiments, there was no inhibition, and in others there was pronounced inhibition (data not shown). CBRM1/32 inhibited transmigration strongly (89.7 ± 8.5%, p < 0.01), whereas addition of CBRM1/20 resulted in variable and partial inhibition (47.7 ± 24.8% inhibition; NS).



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  		FIGURE 3. Effect of anti-CD11b mAbs on PMN migration across epithelial cell monolayers in the basolateral to apical (natural; B to A) direction. In A, inverted T84 monolayers were grown on the underside of permeable membrane filters, as described in Materials and Methods, and transmigration experiments were performed as described above for Figure 2Go, except that one-half of the number of PMN (1 x 106) were used per transwell. As controls, migration is shown in the presence of no Ab or mAb TS1/22, anti-CD11a. As in apical to basolateral transmigration, I domain mAbs and CBRM1/32 strongly inhibit transmigration. C domain mAbs do not affect transmigration. mAb LM2/1 (data not shown) gave inconsistent results. mAb 44a, another I domain-specific Ab, was not used, but has been shown previously to block basolateral to apical PMN transmigration by greater than 70% (8). B shows the effects of CD11b Abs on basolateral to apical PMN transmigration across monolayers of CF15 nasal airway epithelial cells, performed as described above and in Materials and Methods. As can be seen, compared with control migration, the Ab inhibition profile with CF15 monolayers is indistinguishable from that observed in A with T84 monolayers. Each condition represents the mean of three individual monolayers ± SD. In A, each condition represents the mean of six individual monolayers ± SD (78 monolayers total) from two separate experiments.

 
To confirm the relevance of the findings with other epithelia, the Ab panel was tested for effects on basolateral to apical PMN migration across a model respiratory epithelium, CF15. This cell line, derived from the nasal airway of a patient with cystic fibrosis, forms monolayers with good barrier function and has a defect in the cystic fibrosis transmembrane conductance regulator (35). Indeed, monolayers of CF15 epithelial cells achieved transepithelial resistances of 600 to 840 ohm · cm2 after 5 to 7 days in culture, indicating good barrier function (data not shown). The effects of the panel of anti-CD11b Abs on PMN migration across CF15 cells are shown in Figure 3GoB. As demonstrated, the profile of inhibition is indistinguishable from that observed with T84 cells. The five I domain Abs tested inhibited transmigration 88.3 ± 10.4% when compared with noninhibitory controls (TS1/22 and SHCL3). The three C domain Abs tested had minimal effects, resulting in only 7.8 ± 16.2% inhibition. As with T84 cells, CBRM1/32 inhibited as strongly as the I domain mAbs, and CBRM1/20 had only minor effects. Such studies help to confirm the importance of the I domain in PMN migration across epithelia other than the intestinal type.

Effect of mAbs on PMN adhesion to T84 cell monolayers

We previously described an assay for investigating PMN-adhesive interactions with T84 cell monolayers (41). Following this protocol, PMN and mAb were layered on epithelial monolayers and exposed to FMLP to enhance ß2 integrin-mediated adhesive interactions. Typically, 1 to 7 x 104 PMN adhered to T84 monolayers under such conditions (data not shown). Similar numbers of adherent PMN were found on monolayers exposed to the negative control mAb TS1/22 as on monolayers to which no mAb was added (92.8 ± 21.4% of adhesion in the absence of any added Ab) (Fig. 4Go). With the exception of LM2/1, when compared with controls, I domain mAbs inhibited adhesion with a mean of 79 ± 5.2% (p < 0.001), and C domain mAbs inhibited adhesion 12.9 ± 22.8% (NS). While six of seven I domain mAbs consistently and strongly inhibited adhesion (Table IGo), LM2/1 had no effect (6.8 ± 17.7% inhibition compared with controls). C domain mAbs consistently had minimal effects on adhesion. CBRM1/32 inhibited adhesion as strongly as that observed with the I domain mAbs (74.2 ± 13.4% inhibition compared with controls; p < 0.001). CBRM1/20 was similar to C domain mAbs, having minimal effects on adhesion (-1.7 ± 40.2% inhibition compared with controls; NS).



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  		FIGURE 4. Effect of anti-CD11b mAbs on PMN adhesion to T84 monolayers. PMN were layered on T84 monolayers in the presence of saturating concentrations of anti-CD11b mAbs, and adhesion was assessed, as detailed in Materials and Methods. For each condition, adherence is normalized with respect to PMN adhesion in the presence of the noninhibitory binding control mAb TS1/22 (anti-CD11a). As in Figures 2Go and 3Go, I domain mAbs, with the exception of LM2/1, inhibited adhesion strongly, as did mAb CBRM1/32. C domain mAbs had minimal effects. Each condition represents the mean of six to nine individual monolayers ± SD from three separate experiments (108 monolayers total).

 
Effect of mAbs on binding of T84 cells to purified CD11b/CD18

To more directly test the role of the CD11b extracellular subdomains in PMN-epithelial adhesive interactions, we examined the ability of T84 cells to bind to purified CD11b/CD18 in the presence of the panel of mAbs (Fig. 5Go). Microtiter plates coated with CD11b/CD18 were incubated with mAbs, and then a suspension of T84 cells was added to each well. Typically, 20 to 30% of applied T84 cells adhered to the CD11b/CD18-coated microtiter wells. This adhesion was unaffected by the addition of control, nonbinding mAb. As shown in Figure 5Go, compared with adhesion in the presence of control Ab, I domain mAbs (with the exception of LM2/1) strongly inhibited adhesion with a mean of 82.2 ± 13.7% (p < 0.001). As summarized in Table IGo, four of seven I domain mAbs inhibited adhesion of T84 cells to CD11b/CD18 by >80%. In general agreement with our other functional assays, the only I domain Ab that did not consistently block adhesion was LM2/1. C domain mAbs had somewhat variable but minor effects, with a mean inhibition of 8.5 ± 32.1% (NS). OKM1 and CBRM1/26 were the two C domain Abs that appeared to have some inhibitory effect on adhesion (43.4 ± 14.2 and 38.3 ± 21% inhibition; p < 0.01, 0.02, respectively), but such effects were significantly less than I domain mAbs CBRM1/24, CBRM1/29, CBRM1/34, and 44a. Also consistent with the other functional studies, CBRM1/32 strongly inhibited adhesion (89.4 ± 6.3%; p < 0.001), whereas CBRM1/20 lacked inhibitory effect (-30.7 ± 16.6% inhibition).



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  		FIGURE 5. Effect of CD11b mAbs on T84 cell adhesion to purified CD11b/CD18. As described in Materials and Methods, labeled T84 cells were assayed for adhesion to functionally active CD11b/CD18 in the presence of the panel of CD11b mAbs. For each condition, adherence is normalized with respect to PMN adhesion in the presence of control mAb TS1/22, as detailed in Materials and Methods. As in Figures 2 to 4GoGoGo, with the exception of LM2/1, I domain mAbs and CBRM1/32 strongly inhibit adhesion. As can be seen, C domain mAbs had only minor inhibitory effects on adhesion. Each condition represents the mean of nine determinations ± SD from three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
While there are other reports of the effects of epitope-mapped Abs on binding of CD11b/CD18 to various ligands, this study is the first to examine the regions of CD11b important in complex functional assays of PMN transmigration. To identify the domains of CD11b/CD18 important in PMN migration, we used an epithelial cell-based assay system since initial adhesive events between PMN and epithelial cells appear to be mediated exclusively by CD11b/CD18 (22, 41), thereby simplifying the interpretation of results. In particular, a panel of mAbs directed against various epitopes of the CD11b extracellular domain was used to identify regions of the CD11b molecule that mediate adhesion to and migration across model epithelial cell monolayers. We find that regions of the I domain are important recognition sites for epithelial cell counter-receptors. In addition, we find that the discontinuous NH2/cation-domain epitope recognized by CBRM1/32 (separated in the linear {alpha}-chain sequence by the I domain) is essential for ligand recognition.

All but one (LM2/1) of the I domain mAbs examined were strongly inhibitory in transmigration assays regardless of the direction of PMN migration. Similarly, all I domain mAbs except LM2/1 inhibited PMN adhesion to the cell monolayers. The findings in these functional assays were confirmed in assays utilizing purified CD11b/CD18 and T84 epithelial cells. In this study, the I domain-specific mAbs 44a, CBRM1/29, CBRM1/31, and CBRM1/34 strongly inhibited T84 cell binding; CBRM1/24 and CBRM1/27 yielded moderate inhibition, whereas LM2/1 was minimally inhibitory. The Abs to the C domain and one limited to the cation-binding region had somewhat variable but minor effects on adhesion and transmigration. However, the cation-binding domain must be in close proximity to important functional site(s), as one Ab that recognizes a discontinuous epitope shared both by the cation-binding domain and NH2 terminus was strongly inhibitory.

These data are consistent with the conclusion that the recognition site on the CD11b molecule for epithelial cells includes portions of the I domain and a discontinuous epitope formed by the NH2-terminal end and the cation-binding domain. While the I domain of CD11b has been shown to directly mediate binding to a variety of ligands, including iC3b (27, 28, 29, 37), fibrinogen (29, 37), ICAM-1 (29, 37), and heparin (31), the profile of inhibition by mAbs to various I domain epitopes is different from that which we have demonstrated in our epithelial cell adhesion and transmigration experiments. Clearly though, certain regions of the CD11b I domain important in the recognition of iC3b, fibrinogen, and ICAM-1 are also important in binding to epithelial cells. Such findings are consistent with the conclusion that these ligands share overlapping but not identical binding sites in the CD11b I domain.

The I domain-specific mAb LM2/1 has been shown previously to inhibit CD11b/CD18 interactions with both fibrinogen (37) and factor X (46). In this study, LM2/1 was noninhibitory in assays of adhesion and in apical to basolateral transmigration assays. The data for basolateral to apical transmigration assays were inconsistent, with strong inhibition observed in some experiments and minimal effects in others. While these results are inconclusive, it is also possible that the region of the I domain on CD11b/CD18 reactive with LM2/1 plays some role in polarized migration of PMN across epithelial cells and the interaction with more than one ligand, as discussed below. CD11b/CD18 binding to several of the above ligands (31, 37), as well as epithelial cells (this study), was also abrogated by CBRM1/32, suggesting that the discontinuous epitope recognized by this Ab is involved in their recognition. Thus, it is possible this epitope may be directly involved in ligand binding, forming part of the binding pocket. Alternatively, it may be involved indirectly, maintaining the functional conformation of the holoreceptor. In this case, binding of CBRM1/32 to its epitope would result in the actual ligand binding site being sterically inaccessible. This hypothesis has been proposed for other anti-CD11b/CD18 Abs. Specifically, others have argued that the inhibitory effects of several anti-CD11b/CD18 Abs, including LM2/1 and OKM1, may be the result of Ab-induced conformational changes in the holoreceptor (29). Indeed, experimental evidence to support this hypothesis comes from studies with the CD18-reactive mAbs KIM 127 and KIM 185. However, the binding of these Abs to CD11b/CD18 has been shown to induce or enhance ligand-binding capacity (47, 48).

From the data in this study, it is unclear whether the C domain plays any direct role in CD11b/CD18 binding to epithelial cells. Perhaps the variable, partial inhibition observed with some of the C domain Abs in this study might be explained by participation of the C domain in the binding site tertiary structure. Additional mapping studies might clarify this issue.

While multiple ligands for CD11b/CD18 have been identified, the epithelial counter-receptor(s) for transmigrating PMN remains to be defined. One such candidate receptor is ICAM-1. While this glycoprotein is not expressed on normal intestinal epithelium, its expression is up-regulated on the apical epithelial membrane during states of inflammation, after stimulation with inflammatory cytokines (42) or after colonization with bacterial pathogens (49). Under such conditions, PMN can adhere to apically expressed ICAM-1 in a CD11b/CD18-dependent manner. However, the physiologic relevance of this CD11b/CD18-ICAM-1 interaction is unclear since transmigrating PMN would not have access to apically expressed ICAM-1 until after crossing the epithelium (42). This observation and others, including the lack of involvement of other known ligands for CD11b/CD18 (serum factors and heparin) in PMN-epithelial interactions (8) (Parkos, unpublished observations), and the fact that unactivated epithelia support PMN adhesion/transmigration (8, 41), clearly demonstrate the existence of other novel epithelial ligand(s) for CD11b/CD18. Difficulties in identifying such epithelial counter-receptors for CD11b/CD18 are most likely related to the multiple and diverse ligand specificities of CD11b/CD18.

Since CD11b/CD18-mediated adhesion is an initial event in PMN-epithelial interactions, a detailed understanding of the functional regions of CD11b relevant to these cell-based interactions may provide insights into new therapeutic modalities that inhibit PMN transepithelial migration. Inflammatory diseases that might be treated by such therapies are many and include ulcerative colitis, Crohn’s disease, bronchitis, bronchiectasis, pneumonia, arthritis cystitis, and pyelonephritis. Toward this end, one group (50) recently solved the crystal structure of a recombinant form of the CD11b I domain. The recombinant protein has been reported to mimic some, but not all, of the ligand-binding properties of CD11b/CD18 (27, 29). Further work with these reagents in cell-based ligand assay systems may prove useful in future studies aimed at attenuating the deleterious sequelae of PMN interactions with epithelial tissues.


   Acknowledgments
 
We thank Mike Diamond and Tim Springer for generously providing the CBRM panel of Abs and Periasamy Selvaraj for Fc mAbs. In addition, we thank Denice Carnes and Rachel Kerner for expert tissue culture support. Finally, we thank Jim Madara for useful comments and suggestions.


   Footnotes
 
1 This work was supported in part by grants from National Institutes of Health HL54229, DK47662, and HL60540, and a research grant from Crohn’s and Colitis Foundation of America. Back

2 Address correspondence and reprint requests to Dr. Charles A. Parkos, Emory University Department of Pathology and Laboratory Medicine, WMRB Room 2309, Atlanta, GA 30322. Back

3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; I domain, inserted domain; MPO, myeloperoxidase. Back

Received for publication September 22, 1997. Accepted for publication January 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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