Neutrophil transepithelial migration: regulation at the apical epithelial surface by Fc-mediated events

Titus A. Reaves1, Sean P. Colgan2, Periasamy Selvaraj1, Mildred M. Pochet1, Shaun Walsh1, Asma Nusrat1, Tony W. Liang1, James L. Madara1, and Charles A. Parkos1

1 Division of Gastrointestinal Pathology, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322; and 2 Department of Anesthesiology, Brigham and Women's Hospital, Boston, Massachusetts 02115


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neutrophil (PMN) transepithelial migration is a major effector of epithelial defense in inflammatory diseases involving mucosal surfaces. However, major receptor-ligand interactions between epithelial cells and PMN remain incompletely characterized. To better define the molecular events involved in PMN interactions with epithelial cells, we produced a monoclonal antibody called g82 that inhibited PMN transepithelial migration in the physiological basolateral-to-apical direction. The g82 antigen localized to the apical surface of human colonic epithelium and was significantly upregulated under inflammatory conditions. Immunoprecipitation revealed two polypeptides of Mr 207 and 32 kDa. F(ab')2 fragments from g82 IgG had no effect on transmigration, suggesting Fc dependence. Further experiments confirmed dependence on the PMN Fc receptor CD32A and that the observed effects were secondary to a failure of PMN to detach from the apical epithelial surface. These Fc-mediated events were epitope specific since binding, isotype-matched antibodies did not affect detachment. These results identify a new mechanism for retention of PMN at the apical epithelial surface following transepithelial migration. This pathway may be important in pathogen clearance and mucosal pathophysiology associated with autoimmunity.

intestine; inflammation; ulcerative colitis; epithelial cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

POLYMORPHONUCLEAR LEUKOCYTE (PMN) migration across columnar epithelia is an important component of mucosal inflammation (22, 31, 46). When PMN reach their target, there is a potent respiratory burst, enzyme release, and phagocytosis that destroys invading pathogens (4, 13, 18) and may lead to tissue injury. Although PMN migration across the vascular endothelium has been extensively characterized on a molecular level (20), much less is known about the sequence of events that regulate PMN-epithelial interactions (34).

Previous studies have shown that PMN transmigration across intestinal (2, 38), lung (43), and urinary tract (1) epithelia is dependent on the leukocyte-specific beta 2-integrin CD11b/CD18. Although this CD11b/CD18-mediated interaction appears to represent an initial adhesive event in PMN-epithelial interactions (36), the epithelial ligand pair for this integrin remains undefined and does not appear to be intracellular cell adhesion molecule-1 (7, 36). At a step following initial beta 2-integrin-dependent adhesion, PMN migration into the intraepithelial space is regulated by the membrane glycoprotein CD47 (9, 37). Although CD47 has been studied in a variety of cell systems (6, 48), the mechanism by which CD47 regulates PMN migration remains obscure.

The apical epithelial surface is a key site for host-pathogen interactions and represents the final destination of migrating leukocytes before they enter the intestinal lumen (27, 49). During active intestinal inflammation, when migrating PMN are retained in close proximity to the apical epithelial surface, crypt abscesses are formed. There is also release of inflammatory mediators that results in alterations in intestinal permeability and epithelial injury (27, 30, 32). Thus aberrant triggering of accumulation of PMN within intestinal crypts could result in further injury and may participate in disease flares characteristic of ulcerative colitis and Crohn's disease (5, 24, 39).

Using a monoclonal antibody (MAb) approach to study epithelial-leukocyte interactions, we report the identification and characterization of an IgG1 MAb (g82) that, following N-formylpeptide (fMLP)-stimulated PMN migration across polarized monolayers of T84 epithelial cells, inhibits detachment/release of PMN into the lumen. The antigen consists of one or two proteins with relative molecular mass (Mr) of 207 and 32 kDa, is expressed on the apical epithelial surface, and is upregulated under inflammatory conditions. Here we show that the inhibitory effect of g82 IgG on basolateral-to-apical PMN transepithelial migration results from the failure of PMN to detach from the apical epithelial surface. This inhibition is Fc (CD32A)-dependent and unique to the epitope defined by g82, since antibodies reactive with other apically expressed antigens have no effect on PMN transmigration. Our results indicate that transepithelial migration of PMN is not complete after crossing apical intercellular tight junctions but also requires release from the apical domain into the lumen. Control of the latter step might influence critical PMN effector mechanisms, such as clearance of bound pathogens from the apical surface, and mucosal injury in diseases associated with autoimmunity, such as ulcerative colitis (10, 15, 17).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. T84 intestinal epithelial cells (11) (passages 55-80) were grown in a 1:1 mixture of DMEM and Ham's F-12 medium supplemented with 15 mM of HEPES buffer (pH 7.5), 14 mM NaHCO3, 8 µg/ml ampicillin, 90 µg/ml streptomycin, 40 µg/ml penicillin, and 5% newborn calf serum as previously described (37). For apical-to-basolateral and basolateral-to-apical transmigration experiments, T84 cells were grown on collagen-coated, permeable polycarbonate filters (5 µm pore size) with a surface area of 0.33 cm2 (Costar, Cambridge, MA) in both standard and inverted configurations as previously described (2, 37, 38). For ELISA experiments, T84 epithelial cells were cultured to confluence (7 days) in 96-well tissue culture plates. In some experiments, cell monolayers were exposed to recombinant human interferon (IFN)-gamma (106 U/ml; kind gift of Dr. Randall Mrsny, Genentech, San Francisco, CA) for 48 h before use as previously described (7).

Buffers. Hanks' balanced salt solution (HBSS) contains (in g/l) 0.185 CaCl2, 0.098 MgSO4, 0.4 KCl, 0.06 KH2PO4, 8.0 NaCl, 0.048 Na2PO4, 1 glucose, and 10 mM HEPES, pH 7.4. Modified HBSS(-) was prepared as above, except without CaCl2 and MgSO4. Immunoprecipitation wash buffer consisted of 400 mM NaCl, 100 mM NaF, 1 mM EDTA, 1% Triton X-100, and 10 mM Na2HPO4, pH 7.4. For immunoprecipitation experiments, cell lysis buffer contained 100 mM KCl, 30 mM NaCl, 2 mM EDTA, 10 mM HEPES, pH 7.4, and 2% Triton X-100.

PMN isolation. PMN were isolated from whole blood (anticoagulated with citrate/dextrose) obtained from normal human volunteers by using a previously described gelatin sedimentation procedure (16). PMN were suspended in modified HBSS(-) at a concentration of 5 × 107 cells/ml (4°C) and used in transmigration experiments.

Antibodies. T84 cell membranes (21) were used to produce monoclonal antibodies and were screened for inhibition of PMN transepithelial migration as previously described (37). Briefly, female BALB/c mice were immunized by intraperitoneal injection of 1 × 107 cell equivalents emulsified with complete Freud's adjuvant (GIBCO BRL) followed by two subsequent injections of 107 cell equivalents emulsified in incomplete Freud's adjuvant over a 6-wk period. Mice with high titers of antibodies recognizing epithelial surfaces were given a final intravenous immunization by tail vein with 3 × 106 cell equivalents of T84 cell membranes in HBSS. Splenocytes were subsequently harvested (4 days later) and fused with P3U1 myeloma cells. Ten days after fusion, hybridoma supernatants were collected and screened for reactivity with T84 cells compared with a nonepithelial cell type. Hybridoma supernatants demonstrating preferential epithelial reactivity were then screened for inhibition of PMN transepithelial migration. Hybridomas were then subcloned twice by limiting dilution and weaned from selection media, and antibodies were purified by using protein A-Sepharose (Sigma, St. Louis, MO) followed by dialysis against 150 mM NaCl and 10 mM HEPES buffer, pH 7.4. F(ab')2 was commercially prepared (Lampire Biological Laboratories, Coopersburg, PA) by pepsin digestion (100 U/mg, 6 h, 37°C) (33). Purity of antibody digest was confirmed by SDS-PAGE. F(ab') fragments of inhibitory anti-CD32A IgG (26) and anti-CD16B IgG (42) were also commercially prepared by papain digestion in an analogous fashion (Lampire Biological), and binding activity was confirmed by flow cytometry. Control antibodies used in transmigration assays included MAb 44a (inhibitory anti-CD11b), W6/32 (MHC class I, noninhibitory IgG2; antigen is expressed on the apical and basolateral surface of T84 cells), and CLT/479 (IgG1; antigen is expressed on the apical surface of T84 cells) (3, 35, 38, 40, 47).

PMN transmigration and cell adhesion assays. PMN transepithelial migration experiments were performed in both the apical-to-basolateral and basolateral-to-apical directions with the use of T84 monolayers (34, 38). PMN adhesion to confluent T84 monolayers was also assessed as previously described (35).

Quantitation of PMN adherent to monolayers after basolateral-to-apical transmigration. To quantify PMN associated with epithelial monolayers in basolateral-to-apical transmigration assays, a modified monolayer-washing procedure was developed. After completion of transmigration assays (110 min), T84 monolayers were gently removed from the original reservoirs of 24-well tissue culture plates and transferred to new 24-well tissue culture plates containing 1 ml of HBSS in each well. With the use of a rotor adapted for tissue culture plates (Beckman), the transferred, inverted monolayers were subjected to low-speed centrifugation for 5 min (50 g, 4°C). PMN that detached from the epithelial surface of T84 cell monolayers were then quantified by myeloperoxidase (MPO) assay (38) and reported as adherent PMN. PMN remaining within the filter/monolayer and in the original transmigration chambers were also quantified by MPO assay after nonmigrated PMN were rinsed away and were reported as filter-associated and migrated PMN, respectively.

Immunofluorescence. T84 monolayers were fixed in 3.7% paraformaldehyde in HBSS (10 min, 20°C), washed and incubated in HBSS containing 5% normal goat serum, followed by primary antibody for 2 h (10 µg/ml in 5% normal goat serum) as previously described (36). Labeling was also performed on 4-µm frozen tissue sections of human colonic mucosa, both normal and with active ulcerative colitis, obtained from fresh surgical resection specimens. Tissue sections were mounted on glass coverslips, air dried, and fixed in 3.7% paraformaldehyde. After being washed, monolayers and tissue sections were incubated with FITC-conjugated 2° antibody (Cappel, Durham, NC) for 1 h at 20°C. To visualize nuclei, both monolayers and tissue sections were labeled with propidium iodide (1:1,000 dilution, 20 min, 20°C). Additional control stains for basolateral membrane/tight junction [junction adhesion molecule (JAM); MAb J10.4] (25) and nonspecific murine IgG1 were performed in parallel.

Immunoprecipitation. Immunoprecipitation experiments were performed with the use of biotin surface-labeled T84 cell monolayers as previously described (36, 37). Briefly, monolayers were surface labeled with 1 mM sulfo-NHS-biotin (Pierce Chemical) in HBSS for 20 min (4°C) followed by quenching with 150 mM NH4Cl (36). Monolayers were solubilized in cell lysis buffer containing protease inhibitors followed by centrifugation. The supernatants were then precleared for 2 h with normal mouse IgG-Sepharose [IgG coupled to cyanogen bromide-activated Sepharose at a protein-Sepharose density of 3 mg/ml according to the manufacturer's instructions (Pharmacia, Uppsala, Sweden)]. This was followed by incubation for 2 h (4°C) with 30 µl g82-Sepharose. Immunoprecipitates were then washed with 1% octylglucoside-100 mM sodium phosphate, pH 7.4, denatured by boiling in sample buffer, and subjected to SDS-PAGE followed by Western blot. Nitrocellulose blots were probed with avidin peroxidase and visualized by enhanced chemiluminescence (Amersham, Piscataway, NJ).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibody production and screening. A fusion with splenocytes from mice immunized with T84 cell membranes yielded 915 antibody-producing clones that were screened for binding to T84 epithelial cells and to PMN. By using this approach, 132 clones were identified that preferentially labeled the surface of T84 cells by ELISA. Transepithelial migration experiments were then performed by pooling supernatants in groups of three and adding 50 µl to the upper chamber(s) of the Transwell device as previously described (37). Of the pooled supernatants that initially inhibited migration, one gave consistent inhibition in the screening assays and was subcloned to produce MAb g82 (IgG1). In initial transmigration assays with g82 hybridoma supernatants, antibody was added to the upper chamber (apical surface) in apical-to-basolateral transmigration assays and ~50% inhibition of migration was observed (data not shown). However, the addition of the antibody to the upper chamber (basolateral surface) in basolateral-to-apical transmigration assays resulted in no significant inhibition of migration (data not shown) for reasons that are shown in Fig. 5.

Binding of the antibody to the surface of T84 cell monolayers could be readily detected by ELISA (Fig. 1), and such binding was increased approximately twofold after treatment of T84 cells with IFN-gamma for 48 h. Surface reactivity of g82 IgG with T84 cells was confirmed by flow cytometry (data not shown). In addition, such flow cytometric analyses failed to demonstrate binding of g82 IgG to PMN (data not shown).


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Fig. 1.   The antigen for g82 is expressed on the surface of T84 epithelial cells and upregulated after stimulation with interferon (IFN)-gamma . Surface binding of g82 IgG (20 µg/ml) to control and IFN-gamma -stimulated T84 monolayers was assessed by ELISA after incubation with IFN-gamma for various times as detailed in MATERIALS AND METHODS. Values represent the optical density (OD) for binding of g82 IgG with values obtained for control, nonspecific murine IgG1 subtracted out. For reference, OD values for control IgG on both nonstimulated and IFN-gamma -stimulated monolayers were 0.05 ± 0.01. Values are means ± SE; n = 10-12 monolayers in each condition (P < 0.025 by ANOVA for both).

g82 IgG immunoprecipitates two proteins from surface-labeled T84 cells. Labeling and immunoprecipitation experiments were performed using g82 IgG. T84 cell monolayers were surface labeled with biotin, detergent solubilized, and immunoprecipitated with g82 IgG as detailed in MATERIALS AND METHODS. Western blots were then analyzed by gel scanning. Compared with protein standard controls, two proteins of ~207 and ~32 kDa Mr (Fig. 2) were immunoprecipitated by g82 IgG under both reducing and nonreducing conditions. Immunoprecipitation experiments with IFN-gamma -stimulated T84 cells produced two proteins with identical Mr values to those obtained from nonstimulated epithelial cells (data not shown). Western blotting of T84 cell extracts or immunoprecipitates with g82 IgG were unsuccessful. Thus it is not clear which of the two proteins, or both, represents the antigen.


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Fig. 2.   Immunoprecipitation of the antigen for g82 IgG. As detailed in MATERIALS AND METHODS, T84 cells were biotinylated, detergent solubilized, and immunoprecipitated with g82 IgG. Samples were subjected to SDS-PAGE under reducing conditions on a 6-16% gradient gel followed by Western blot and probed with streptavidin peroxidase. CTL, immunoprecipitation with control mouse IgG; g82, immunoprecipitation with g82 IgG demonstrating 2 labeled protein bands of 207 and 32 kDa.

The antigen for g82 is expressed on the apical surface of T84 cells and human colonic epithelium. To localize the epitope defined g82 IgG, immunofluorescence labeling was performed using confluent monolayers of T84 cells cultured on permeable supports. Fig. 3, A and B, represent computer-reconstructed confocal micrographs of g82 IgG staining in the x-z plane. Nuclear staining is shown in red. An intense linear staining pattern consistent with apical surface expression of the g82 antigen is observed, which is significantly increased after IFN-gamma treatment (Fig. 3B). For comparison, staining for the tight junction/basolateral membrane protein JAM (25) is shown in Fig. 3C. Background fluorescence from nonspecific isotype-matched IgG was not significant (not shown).


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Fig. 3.   g82 IgG labels the apical surface of T84 monolayers. Polarized monolayers of T84 cells were stimulated with IFN-gamma or media alone followed by localization of the g82 antigen by indirect immunofluorescence and confocal laser scanning microscopy. All images are shown in the vertical or x-z plane of section (computer reconstructed, Zeiss LSM 5.10). Nuclei are stained with propidium iodide and shown in red. A: linear apical membrane staining for g82 IgG (arrows, green). B: significantly increased apical and subapical membrane staining with g82 IgG (arrows, green) following treatment of T84 monolayers with IFN-gamma . For comparison, C depicts the typical staining pattern for a lateral membrane protein [junction adhesion molecule (JAM), monoclonal antibody (MAb) J10.4; arrowheads].

We next assessed whether the epitope recognized by g82 IgG was similarly expressed by human intestinal epithelia and if the apical polarity of expression as predicted by the in vitro model was also present. Immunofluorescence photomicrographs of g82-labeled frozen sections of human colonic mucosa are shown in Fig. 4, A and C. Figure 4, A-C, are from sections of normal colonic mucosa, whereas D-F are from sections of colonic mucosa with active ulcerative colitis. Cell nuclei are stained with propidium iodide and shown in red. Figure 4A represents g82 IgG staining of normal colonic epithelium and demonstrates an apical staining pattern on surface epithelial cells similar to that observed with T84 cells in Fig. 3, which is substantially increased in active ulcerative colitis (Fig. 4D). For comparison, Fig. 4, B and E, represent tight junction and basolateral staining for JAM (25), which is also increased in active ulcerative colitis (Fig. 4E). Figure 4, C and F, represent nonspecific staining with IgG1.


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Fig. 4.   Immunolocalization of the antigen for MAb g82 in human colonic mucosa. Cryosections of normal colonic mucosa (A-C) and of colonic mucosa from patients with active ulcerative colitis (D-F) were examined for localization of the g82 antigen by indirect immunofluorescence and confocal laser scanning microscopy as detailed in MATERIALS AND METHODS. A: apical membrane staining pattern for MAb g82 (arrows) is observed in normal colonic crypt epithelial cells. D: staining with MAb g82 is markedly increased in the apical pole of crypt epithelia from a patient with active ulcerative colitis. B and E: tight junction (arrow) and lateral membrane staining (arrowheads) for JAM is shown for normal colonic epithelium (B) and ulcerative colitis (E). C and F: staining with nonspecific murine IgG1 for normal colonic epithelium (C) and ulcerative colitis (F). As can be seen, there is occasional staining of immune cell (plasma cells) in the lamina propria.

Because the epitope recognized by g82 IgG was expressed in a polarized (apical) fashion on cultured and natural epithelia, and because in our screening assays g82 IgG inhibited apical-to-basolateral PMN transmigration, we then assessed whether g82 IgG inhibited transepithelial migration in a physiologically distinct manner. For these experiments, basolateral-to-apical PMN transmigration assays were performed by placing antibody in the lower chamber (apical epithelial membrane) of the Transwell device. As depicted in Fig. 5A, incubation of g82 IgG in the lower chamber of inverted Transwells would result in antibody access to antigens expressed on the apical surface. Migration assays were then performed as previously described, and the results are depicted in Fig. 5B. As shown, there was ~88% inhibition of transepithelial migration in the basolateral-to-apical direction by inclusion of g82 IgG in the apical compartment of the Transwell compared with migration in the presence of a control binding antibody W6/32 (P < 0.001).


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Fig. 5.   g82 IgG inhibits detachment of transmigrated neutrophils (PMN) from the apical epithelial surface. A: g82 IgG was added to the lower reservoir of inverted T84 monolayers to facilitate antibody binding to apically expressed antigen (Y). Basolateral-to-apical PMN transepithelial migration assays were then performed as previously described. B: after 110 min of transmigration, both migrated and filter-associated PMN were quantified by myeloperoxidase (MPO) assay. C: PMN associated with the apical surface (underside of monolayer) after transmigration assays were dislodged by low-speed centrifugation of monolayers followed by MPO assay of dissociated PMN. Data are means ± SE (n = 4) for g82 IgG and a binding control, W6/32, both used at 20 µg/ml.

Analysis of the above results revealed that the number of PMN recovered from assays using g82 IgG was reduced compared with recoveries from controls. We surmised that for inhibition to occur at the level of the apical epithelial surface there should be an increase in PMN associated with the epithelium/filter. Moreover, this observation could result from a failure of PMN to detach from the apical surface following migration through the filter, paracellular epithelial spaces, and across subapical epithelial intercellular junctions. However, as shown in Fig. 5B, we first noted recovery of relatively small numbers of PMN from the filter/epithelium in the presence of g82 IgG and suspected that we might be losing apically attached PMN in the washing steps that preceded MPO analysis of filter-associated PMN.

Thus we modified our monolayer-washing procedure to quantify loosely adherent PMN that failed to detach from the apical epithelial surface following basolateral-to-apical transmigration assays. As detailed in MATERIALS AND METHODS, immediately after basolateral-to-apical transmigration assays, monolayers were gently transferred to 24-well tissue culture plates containing HBSS and subjected to low-speed centrifugation at ~50 g for 5 min. PMN that were dislodged from the epithelial surface were then quantified in the tissue culture wells and referred to as apically adherent PMN. As shown in Fig. 5C, by using this technique we found a marked increase in adherent PMN in the g82-treated condition compared with controls. In addition, the number of recovered PMN after treatment with g82 IgG was essentially the same as those obtained for controls (42.5 × 104 total recovered PMN vs. 37.0 × 104 and 43.8 × 104 total recovered PMN for g82 vs. W6/32 and no antibody controls, respectively; P = not significant).

Inhibition of detachment of PMN from the apical surface of epithelia is specific for the g82 antigen and mediated by Fc receptors. We surmised that the results in Fig. 5 could be explained by PMN Fc receptors interacting with g82 IgG bound to the apical surface. To test this hypothesis, the Fc portion of g82 IgG was removed to form F(ab')2 fragments, which were then used in transmigration assays. F(ab')2 fragments produced from g82 IgG showed similar binding characteristics to T84 cells as intact IgG, thus indicating preservation of specific binding affinity (data not shown). As indicated in Fig. 6A, the data are normalized with respect to migration in the absence of antibody. Although apical surface detachment was inhibited 93.8% by intact g82 IgG, there was no significant inhibition of surface detachment by the g82 F(ab')2.


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Fig. 6.   Fc interactions mediate g82 IgG effects on detachment of migrated PMN from the apical surface of the epithelium. A: effects of g82 F(ab')2 and apical binding control IgGs W6/32 and CLT/479 were tested for effects on basolateral-to-apical PMN transepithelial migration as detailed in MATERIALS AND METHODS. Inset: results of a T84 cell surface binding ELISA comparing OD values obtained for g82 IgG and CLT/479 IgG are shown and demonstrate comparable labeling for 2 concentrations of applied MAb. Transmigration assays were also performed in the presence of apically applied g82 IgG with PMN exposed to 5 µg/ml of functionally inhibitory F(ab') of anti-FcR MAbs (CD32A) or (CD16B). Black bars represent migration into the lower reservoir (does not include apically adherent PMN) and are expressed as %control migration in the absence of antibody. Data are means ± SE (n = 4). B: effect of g82 IgG on PMN adhesion to T84 monolayers was examined. g82 IgG was added to the apical surface of T84 monolayers followed by the addition of PMN and stimulated with fMLP to enhance adhesion. As in A, g82 F(ab')2 failed to enhance adhesion. Furthermore, F(ab') of anti-CD32A but not CD16B blocked the g82-mediated increase in adhesion. Data are means ± SE (n = 4) and are representative of three separate experiments.

To examine whether the Fc-mediated inhibitory effects of g82 IgG were antigen/epitope specific or represented a more generalized effect that could be induced with any apically bound antibody, we obtained an isotype-matched antibody with surface labeling characteristics similar to those observed with g82 IgG. Figure 6A, inset, shows the optical density values of a T84 cell surface binding ELISA demonstrating comparable levels of binding with MAb CLT/479 IgG compared with g82 IgG. Furthermore, immunofluorescence staining of T84 monolayers with CLT/479 IgG showed an apical staining pattern similar to those observed with g82 IgG (data not shown). However, as can be seen in Fig. 6A, when intact CLT/479 IgG or W6/32 IgG were applied to the apical surface of T84 monolayers, there was no significant inhibition of PMN detachment from the apical surface following transepithelial migration. These findings suggest that the Fc-mediated effects on PMN attachment/detachment to apical surfaces vary with respect to the nature of the epitope recognized.

To determine which PMN Fc receptor was mediating these results, we tested functionally inhibitory anti-Fc receptor F(ab') fragments for the ability to block g82 IgG effects. As shown in Fig. 6A, incubation of PMN with anti-CD32A F(ab') followed by basolateral-to-apical migration in Transwells containing g82 IgG resulted in loss of the inhibitory effects observed with g82 IgG. Interestingly, functionally inhibitory F(ab') fragments of anti-CD16B IgG were ineffective in blocking the effect of intact g82 IgG. These results suggest that the functional effect of g82 IgG is mediated by a specific interaction with PMN low-affinity Fc receptor CD32A.

Effects of g82 IgG on PMN adhesion to T84 monolayers. The results of the above migration experiments suggested that Fc-mediated PMN adhesion to the apical surface of T84 monolayers could have significant effects on terminal events in the PMN transepithelial migration cascade. Therefore, we examined the ability of g82 IgG to promote adhesion of PMN when directly applied to the apical surface of confluent T84 monolayers. As shown in Fig. 6B, PMN adhesion to monolayers pretreated with binding-control antibodies was not significantly increased compared with binding in the absence of applied antibody. In contrast, PMN adhesion to T84 monolayers pretreated with g82 IgG was increased nearly threefold. Furthermore, when T84 monolayers were stimulated with IFN-gamma , there was an additional twofold increase in adhesion observed with g82 IgG compared with controls (data not shown). These findings parallel previous observations of generally increased PMN adhesion to T84 monolayers after stimulation with IFN-gamma (8). Lastly, as can be seen in Fig. 6B, the g82 IgG-induced increase in adhesion was reduced to control levels by pretreatment of PMN with anti-CD32A F(ab') but not by anti-CD16B F(ab').


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we demonstrate that Fc-mediated adhesive interactions can modulate the PMN transepithelial migration response to the chemotactic peptide fMLP. In particular, we show that g82 IgG specifically inhibits PMN detachment following basolateral-to-apical transepithelial migration and is dependent on CD32A, one of the low-affinity Fc receptors on PMN (13, 18). Since this Fc-mediated effect appears to be epitope specific, it suggests that geographical constraints (antigen clustering, compartmentalization, etc.) and/or signaling events at the level of the apical membrane might influence PMN detachment from the epithelial surface after transmigration. In addition, signaling events from such contact could result in altered epithelial functions such as permeability that, in turn, may modulate subsequent PMN migration.

Although we were not able to demonstrate direct adhesive interactions between the g82 antigen and PMN, it has been suggested that induction of other apical ligands for PMN such as intracellular cell adhesion molecule-1 might function to promote clearance of apically attached bacteria (19, 36). If this is true, then one could envision similar Fc-mediated events to those described here. In particular, since the apical epithelial surface is the initial site of pathogen adherence (14, 28, 29, 49), opsonization followed by antibody-mediated adhesion/phagocytosis would facilitate clearance of offending microorganisms. Indeed, a characteristic histological feature of infectious colitis is the presence of abundant crypt abscesses, collections of PMN within colonic crypts (22, 23), in close proximity to the apical epithelial surface.

IgG-mediated PMN-epithelial adhesive interactions are also likely to play a role in the pathophysiology of mucosal diseases associated with autoantibodies such as ulcerative colitis. In the active phase of this disease, when there are abundant crypt abscesses, patient symptoms are most pronounced. Similarly, studies have shown that ulcerative colitis is associated with IgG autoantibodies directed against intestinal epithelial cells in a majority of patients (10, 15, 17). In one of these studies, abundant IgG1 binding to the apical intestinal epithelial surface was found in 7 of 11 patients with active ulcerative colitis (15). Thus, although we do not have evidence for a direct association between antibodies against the antigen for g82 and human disease, our findings provide a mechanism for PMN retention in colonic crypts under the above conditions.

The tissue/epithelial distribution studies in this report provide additional evidence supporting the physiological relevance of our findings. First, the antigen for g82 is similarly expressed at the apical surface in both T84 cells and natural human colonic epithelium. Secondly, such expression is significantly upregulated in T84 monolayers after stimulation with the inflammatory cytokine IFN-gamma . In parallel, the antigen is similarly upregulated in colonic epithelium from patients with active ulcerative colitis. Thus, under inflammatory conditions associated with enhanced PMN-epithelial interactions and crypt abscess formation, there is appropriate upregulation of a crypt surface antigen that supports Fc-mediated PMN adhesion.

Although we have illustrated the potential importance of IgG antibodies against specific antigens in the gastrointestinal tract during disease, how would IgG gain access to such luminal antigens? Although IgA is the predominant secreted immunoglobulin, recent studies by Dickinson et al. (12) have demonstrated that IgG antibodies are actively transported across epithelial barriers in the basolateral-to-apical direction. IgG movement into the lumen would be further enhanced under conditions associated with disruption of epithelial barrier function. This is indeed the case for ulcerative colitis, which is characterized by widespread mucosal ulceration leading to abundant seepage of serum components into the intestinal lumen (22, 23, 41). These observations provide a mechanism by which IgG can gain access to apically restricted antigens that is enhanced under specific inflammatory conditions.

As depicted in Fig. 7, the results from the current study can be incorporated into a multistep model of PMN transepithelial migration and crypt abscess formation. In this model, extravasated PMN migrate to the basolateral aspect of the crypt epithelium, where initial contact is dependent on the PMN beta 2-integrin CD11b/CD18 (2, 38). Subsequently, PMN migrate across the epithelium by CD47-dependent processes (37) to reach the apical aspect (lumen) of the epithelium. Here, in the presence of apically bound IgG, PMN are retained in close proximity to the epithelium, contributing to crypt abscess formation. Close apposition of PMN to apical epithelial membranes would facilitate the action of paracrine factors released from PMN on epithelial function such as cAMP-mediated chloride secretion (27, 44, 45). The above scenario thus provides a mechanism for secretory diarrhea that is commonly observed in individuals with abundant crypt abscesses.


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Fig. 7.   Model of crypt abscess formation incorporating Fc-mediated PMN adhesion to crypt epithelial surfaces. As shown, PMN transepithelial migration is a multistep process that is dependent on initial adhesion events involving the beta 2-integrin CD11b/CD18 followed by CD47-dependent transmigration. On entry into colonic crypts, transmigrated PMN may interact with epithelial bound antibodies (e.g., g82) and remain adherent to the apical epithelial surface rather than detach and enter the luminal space. By this mechanism, PMN associated with the epithelium would be retained in the crypt rather than being flushed from the lumen by fluid flow. Conditions with abundant crypt abscesses where this process might occur include infectious colitis from epithelial adherent microorganisms and diseases associated with autoantibodies and mucosal damage (e.g., ulcerative colitis).

Results of our immunoprecipitation experiments indicate that g82 IgG recognizes at least one of two proteins with Mr values of 207 and 32 kDa. However, since the antibody does not Western blot, it remains unclear which protein harbors the recognized epitope. Further characterization of the epitope awaits purification/sequencing and/or molecular cloning of the proteins recognized.


    ACKNOWLEDGEMENTS

We thank Denice Esterly for expert tissue culture and technical assistance.


    FOOTNOTES

This research was supported by Grants HL-60540, HL-54229, HL-60569, DK-50189, DK-47662, DK-35932, and DK-57827 from the National Institutes of Health and a Biomedical Sciences Grant from the Arthritis Foundation.

Address for reprint requests and other correspondence: T. A. Reaves, Dept. of Pathology and Laboratory Medicine, Emory Univ., 1639 Pierce Drive, Atlanta, GA 30322 (E-mail: treaves{at}emory.edu).

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

Received 26 May 2000; accepted in final form 14 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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