Negative regulation of epithelium-neutrophil interactions via activation of CD44

Mustapha Si-Tahar, Shanthi Sitaraman, Takeshi Shibahara, and James L. Madara

Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Polymorphonuclear neutrophil (PMN) migration across epithelia is a common feature of active inflammation. Given the suggested role of carbohydrates in this process, we examined the receptor CD44. The standard CD44 isoform was expressed at the cell surface of PMN. PMN migration across model polarized intestinal epithelia was reduced (by 60%) if the CD44 receptor was activated by either a specific antibody (clone IM7) or the natural soluble ligand, hyaluronic acid. This inhibitory effect following receptor activation occurred with both basolateral-to-apical- and apical-to-basolateral-directed migration. The anti-CD44 antibody similarly reduced PMN migration through filters in the absence of epithelia, while preincubation of the antibody with the epithelium did not alter subsequent PMN transepithelial migration. These data suggest that PMN, rather than epithelial, CD44 is responsible for these effects. A similar inhibitory effect of anti-CD44 antibody was also observed on migration of intraepithelial lymphocytes. The molecular mechanism involved in such negative signaling following CD44 activation may include modulation of outside-in cell signaling. While neither the anti-CD44 antibody nor CD44 ligand affected PMN mobilization of intracellular Ca2+, both led to increased adenylate cyclase activity, an inhibitory signal for PMN migration. Together, these results suggest that CD44 of PMN may potentially serve as a negative regulator of leukocyte migration across biological surfaces such as columnar epithelia.

adhesion molecules; leukocyte recruitment; inflammation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

POLYMORPHONUCLEAR NEUTROPHILS (PMN) comprise one of the immune system's first lines of defense through phagocytosis and destruction of microorganisms and are important effectors of the acute inflammatory response (35). A key event in these processes is migration of PMN out of the circulation and across a number of tissue barriers in response to chemoattractant stimuli. In many of the major organ systems, the final barrier crossed by PMN is a polarized epithelium, such as that found in the respiratory, urinary, or gastrointestinal tracts (26, 28). A complex series of adhesive and deadhesive events can be envisaged to allow successful migration of PMN through the paracellular spaces of the epithelia, along the basolateral surface of epithelial cells, before reaching the luminal (apical) milieu (26, 28). Although it is known that there are marked differences in the key adhesive molecules involved in PMN interactions with endothelium and epithelia, the current understanding of molecular events in PMN transepithelial migration is far from complete. However, several arguments suggest that this complex cascade of events involves the integrin alpha Mbeta 2 (CD11b/CD18) and the immunoglobulin superfamily member CD47 (2, 10, 26, 28). In addition, while we previously demonstrated that carbohydrate-mediated interactions play a role in transepithelial migration, they appear to be distinct from the well-characterized selectin-based interactions of PMN and endothelial cells (8). Hence, we questioned whether the proteoglycan core protein family member CD44 could contribute to such PMN-epithelial interactions.

CD44 is a glycosylated membrane receptor that is expressed on a large collection of normal and transformed cell types including, among others, epithelial cells and all leukocytes. The CD44 molecule has 10 different splice variants. The hematopoietic form of CD44 represents the basic unit of the CD44 protein and has also been referred to as CD44s, the standard CD44 isoform. It is synthesized as a polypeptide with an apparent molecular weight of 42,000 and is extensively modified with N- and O-linked carbohydrates, reaching a final molecular weight of 80,000-95,000 (17, 21). CD44 has an NH2-terminal link domain involved in binding to its main ligand, hyaluronic acid (4), a high-molecular-weight, highly anionic polysaccharide found both in extracellular matrices and at cell surfaces (4, 17, 21). Several avenues of evidence indicate that recognition of hyaluronic acid is important in various CD44-mediated events, although several other ligands for CD44 exist including the extracellular matrix glycosylated components collagen or fibronectin, the cell surface "adressins" (24), and myosin heavy chain class II invariant chain (25). These later features suggest that non-hyaluronic acid ligands may be capable of signaling through CD44.

The diverse roles proposed for CD44 can be conceptually grouped as manifestations of cellular adhesion in one setting or another. Our interest in CD44 as a glycoprotein that might be important for PMN transepithelial migration was prompted by several observations. It has been recently reported that, during an inflammatory response, lymphocytes are generated that demonstrate an enhanced potential of CD44-hyaluronic acid binding involved in subsequent rolling (11). A role for CD44 in leukocyte trafficking is further suggested by the finding that soluble CD44 can block normal migration of leukocytes to lymphoid organs in mice (16). Soluble hyaluronic acid can downregulate certain PMN responses such as phagocytosis (30). Mikecz et al. (22) also found that anti-CD44 antibody treatment of arthritic mice reduced joint swelling and associated infiltration of inflammatory cells (22). Additional suggestive findings potentially supporting a role of CD44 in the general processes of cell migration include observations identifying this adhesive molecule as a determinant of motility, invasion, and metastasis in selected experimental cancer models (17, 20).

Because of the limited current understanding of events required for PMN migration across epithelial surfaces and in view of the foregoing data, we examined whether CD44 might play a relevant role in this process. We find that CD44s, but not other variant isoforms, is highly expressed on PMN and is involved in the migration properties of this leukocyte through biological surfaces such as polarized human intestinal epithelia.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies and reagents. Anti-human CD44 antibodies used in this study included a monoclonal rat anti-mouse CD44 immunoglobulin (Ig) G2b (clone IM7), which cross-reacts with all human CD44 isoforms, and a mouse anti-human CD44s IgG2b (clone G44-26), both purchased from Pharmingen (San Diego, CA). A polyclonal anti-human CD44var(v3-v10) was obtained from BenderMedSystems (Vienna, Austria). Control isotype-matched antibodies were purchased from Sigma (St. Louis, MO) or Pierce (Rockford, IL). Affinity pure goat Fab fragments for blocking primary rat IgG were obtained from Jackson ImmunoResearch (West Grove, PA). Other antibodies included goat FITC-labeled anti-rat IgG antibody and peroxidase-linked anti-mouse antibody obtained from Pierce and Pharmingen, respectively. Hyaluronic acid was from Worthington (Lakewood, NJ), and collagen-rich solution was prepared in our laboratory by using rat tails, as previously described. N-formyl-Met-Leu-Phe (fMLP) was purchased from Sigma, and indo 1-AM was from Molecular Probes (Eugene, OR). Reagents for SDS-PAGE and nitrocellulose membranes (0.45-µm pores) were from Bio-Rad (Hercules, CA).

Cell culture. The human intestinal epithelial cell line T84 (12) was purchased from the American Type Culture Collection (Rockville, MD). For apical-to-basolateral transmigration experiments, T84 monolayers were grown on permeable, collagen-coated polycarbonate supports (inserts) with a surface area of 0.33 cm2 and a pore size of 5 µm (Costar, Cambridge, MA) and cultured until steady-state resistance was achieved, as previously described (8, 26-28). For physiologically directed basolateral-to-apical transmigration, T84 cells were plated on the underside of permeable filters to produce inverted monolayers (8, 26-28).

PMN isolation. Human PMN were purified from whole blood collected on citrate dextrose and obtained from volunteers by using a gelatin sedimentation technique previously described in detail (27). PMN were resuspended in modified Hanks' balanced salt solution devoid of Ca2+ and Mg2+ [HBSS(-)] at a concentration of 5 × 107 cells/ml. Most of the procedure was performed by using buffers prechilled at 4°C, and isolated PMN were maintained at this temperature until use.

Transmigration and chemotaxis of PMN. PMN transepithelial migration assays were performed in both the physiologically relevant basolateral-to-apical and nonphysiological apical-to-basolateral directions, as previously described (8, 26-28). Briefly, T84 monolayers were washed with Hanks' balanced salt solution containing Ca2+ and Mg2+ [HBSS(+)], followed by apical or basolateral addition of 100 µl of HBSS and 1 × 106 PMN in 25 µl of HBSS(-) pretreated at 4°C (note that to evaluate the influence of incubation temperature, subset experiments were also conducted at room temperature) for 30-45 min with anti-CD44 or control isotype antibody. Other experiments also used PMN or T84 cells pretreated at 4°C and 37°C, respectively, for 30 min with the antibody, followed by extensive washout before the transmigration process was started. Other experiments were also performed on collagen-coated permeable supports lacking epithelial cells to assess the effect of CD44 treatment on chemotaxis properties of PMN across a matrix alone. Migration was quantitated as described previously (8, 26-28). Briefly, transepithelial migration was initiated by the transfer of monolayers to which PMN had just been added to 24-well tissue culture plates containing 1 ml of 1 µM fMLP in HBSS(-) on the side of the monolayer opposite the PMN [except for PMN chemotaxis across filters without epithelial cells, which is efficient at only 10 nM fMLP (27)]. After incubation for 120 min at 37°C, PMN that had migrated across the epithelium into the chemoattractant-containing lower chambers were quantitated by a myeloperoxidase assay (8, 26-28).

Transmigration of lymphocytes. The intraepithelial lymphocyte (IEL) cell line (032891) used in this work was derived from normal human small intestine and has been extensively characterized (33). Lymphocytes were first labeled with the intravital fluorescent dye 5-chloromethylfluorescein diacetate (CMFDA; final concentration 1 µM; Molecular Probes) for 30 min at 37°C. After cells were labeled, they were washed and resuspended in a final volume of 2 × 106 cells/ml before being incubated for 2 h with the control isotype-matched or the anti-CD44 antibody IM7 (40 µg/ml). Fluorescent lymphocytes (100 µl) were then placed onto the upper chamber of 5-µm pore filters (basolateral aspect of T84 monolayers) and allowed to spontaneously migrate into the monolayers at 37°C for 2 h (33). Filters were then washed with HBSS(+), and the fluorescence associated to monolayers [an indicator of migration into the paracellular spaces of the monolayers (33)] was measured with the use of a fluorescence plate reader (CytoFluor 2350; Millipore, Bedford, MA).

Matrix adhesion of PMN or lymphocytes. Both leukocyte populations were separately labeled with CMFDA as described in Transmigration of lymphocytes. The anti-CD44 IM7 or the control antibody was then incubated for 30 min with PMN (at a final concentration of 10 µg/ml) or for 2 h with IEL (at a final concentration of 40 µg/ml). Subsequently, either 1 × 106 PMN or 0.2 × 106 IEL were added to 0.4-µm filters that either had or had not been precoated for 24 h with hyaluronic acid and collagen (100 µl/well; final concentration ~5 mg/ml). PMN were either further stimulated with fMLP (0.5 µM) or not stimulated. All filters were finally incubated at 37°C for 2 h, followed by extensive washing, and quantitation of adherent leukocytes was obtained by measuring fluorescence associated to filters with a fluorescence plate reader.

Flow cytometry. PMN were added to conical bottom 96-well plastic plates (Costar) at 2 × 105 cells/well. Next, plates were centrifuged at 80 g for 5 min, and PMN were subsequently incubated with anti-CD44 antibody IM7 (0.1-20 µg/ml) or anti-CD44var(v3-v10) antibody (0.1-40 µg/ml) or with matched control antibody for 30 min at 4°C; all antibodies were diluted in cold HBSS(-) containing 0.1% (wt/vol) BSA (HBSS/BSA). Cells were further centrifuged at 80 g for 5 min and washed twice with HBSS/BSA before being incubated for 30 min at 4°C with the corresponding second FITC-labeled antibody at an optimal concentration. Finally, PMN were centrifuged as described above, resuspended in HBSS/BSA. Samples were then analyzed with the use of a FACScan flow cytometer (Becton Dickinson Immunocytometry System, Mountain View, CA). Resulting histograms correspond to PMN number (y-axis) vs. fluorescence intensity (x-axis) plotted on a logarithmic scale. Results are also shown as the binding of anti-CD44 antibodies to PMN and are expressed as the fold increase in median fluorescence intensity over control isotype antibody values.

SDS-PAGE and Western blot analysis. Resting PMN were solubilized as described previously (34). SDS-PAGE was subsequently performed according to the Laemmli procedure (19). Electrophoresis was performed after each well was loaded on a 6-16% gradient acrylamide gel. Proteins were electrotransferred to nitrocellulose membranes (34) and probed with anti-CD44 antibody (5 µg/ml) or anti-CD44var(v3-v10) antibody (diluted 1:500). Membranes were then probed with the corresponding secondary peroxidase-linked antibody (diluted 1:2,000), washed, and subsequently incubated with enhanced chemiluminescence (ECL) detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ) before being exposed to hyperperformance chemiluminescence films (Amersham). For determination of molecular weight, polyacrylamide gels were calibrated by using standard proteins (Bio-Rad) with molecular weight within the range from 7,700 to 204,000.

Measurement of PMN cytosolic Ca2+ mobilization. PMN were loaded with the fluorescent calcium indicator indo 1 (final concentration 1 µM), and cytoplasmic Ca2+ concentration was measured with the use of a spectrofluorometer (F-4500; Hitachi Scientific Instruments, Mountain View, CA), as previously described (37). PMN (2 × 106) that either had or had not been preincubated with anti-CD44 antibody IM7 (10 µg/ml) or matched control antibody for 30-45 min at 4°C were suspended in 960 µl of HBSS(-) and stimulated under agitation at 37°C with suboptimal (10-9-10-8 M) or optimal concentrations (5 × 10-7 M) of fMLP. A direct Ca2+-mobilization effect of anti-CD44 antibody IM7 was also examined by adding 20 µg/ml of this antibody to resting PMN.

cAMP measurements. Measurements of cAMP were performed on PMN extracts using with the use of a radioimmunoassay kit as described by the supplier (NEN, Boston, MA). Briefly, after PMN were washed with HBSS(-) and had or had not been incubated for 15 and 30 min at 37°C with either anti-CD44 antibody IM7 (10 µg/ml) or a matched control antibody or with hyaluronic acid (500 µg/ml), the reaction was stopped by the addition of a cold extract buffer containing 66% ethanol, 33% HBSS, and 1 mM of the phosphodiesterase inhibitor IBMX. After being centrifuged at 1,500 g for 10 min, an aliquot of each sample (100 µl) was used for the radioimmunoassay.

Data analysis. Unless otherwise indicated, all values are means ± SD of at least three distinct experiments conducted with PMN from different donors. Results were analyzed by using Student's t-test. Differences were considered significant at the P < 0.05 level.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of CD44s, but not CD44var(v3-v10) isoforms, on PMN. CD44 expression on PMN was evaluated by flow cytometry analysis by using IM7, an IgG2b antibody that reacts with all CD44 isoforms and a CD44var(v3-v10) polyclonal antibody specific for these latter isoforms. As shown in Fig. 1A, the large shift of fluorescence intensity between signals associated with IM7 and the irrelevant control isotype-matched antibodies indicates a strong CD44 expression at the surface of PMN (n = 3). By contrast, the level of expression of CD44 var(v3-v10) isoforms on PMN was not significant because a similar labeling was measured for the specific and the control antibodies. The restricted expression of CD44 to the standard form (CD44s) on PMN was further confirmed by SDS-PAGE. As shown in Fig. 1B, immunoblot analysis of PMN-associated CD44 revealed a major band recognized by IM7 but not by the CD44 var(v3-v10) antibody. This band runs at a molecular mass of ~85-95 kDa, which is characteristic of the standard isoform of CD44 (17, 21). By contrast, CD44 is expressed on the human intestinal epithelial cell line T84 (or HT29, not shown) as an ~120-150 kDa protein recognized by the anti-CD44 var(v3-v10).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1.   Expression of CD44 isoforms on polymorphonuclear neutrophils (PMN). A: binding to resting PMN of anti-CD44 antibody either recognizing all isoforms (including the standard isoform, thick line) or only the v3-v10 isoforms (dashed line). Comparison is provided with an isotype-matched control antibody (thin line), and analysis was performed by flow cytometry as described in MATERIALS AND METHODS. B: Western blot of CD44 isoforms in resting PMN and T84 cells of unreduced samples revealed by SDS-PAGE on a 6-16% acrylamide gel with transfer to nitrocellulose membranes and probing with an antibody reacting either with all CD44 isoforms (left) or specifically with v3-v10 isoforms (right). Molecular masses were calculated with respect to calibration standard proteins included in each gel. Fluorescence tracings and Western blots were repeated in 3 separate experiments.

Anti-CD44 antibody IM7 inhibits PMN transepithelial migration. To study the role of CD44 molecules in transepithelial migration, we used an in vitro assay that we have extensively characterized (8, 26-28). The effects of IM7 antibody compared with effects of an irrelevant control isotype-matched IgG on PMN migration across T84 monolayers in the physiological basolateral-to-apical direction are depicted in Fig. 2. As shown in Fig. 2A, inset, IM7 antibody binding was detectable at concentrations as low as 0.1 µg/ml and was maximal after 10 µg/ml (n = 2). Strikingly, a marked inhibitory effect of this anti-CD44 antibody on PMN transepithelial migration was observed in the concentration range of IM7 binding. Thus addition of 0.1 or 10 µg/ml IM7 to the basolateral surface of the epithelial monolayers, concomitantly with PMN, resulted in ~35% and ~52% inhibitions, respectively, of PMN transmigration (n = 3, P <=  0.05). Although requiring higher concentrations of antibody (25-75 µg/ml), a similar phenomenon was observed when another anti-CD44 antibody was used (clone G44-26; not shown). The possibility that this latter anti-CD44 antibody influenced transmigration by cross-linking PMN to epithelial cells is unlikely because PMN did not agglutinate under these conditions. More importantly, an inhibitory effect was also observed in experiments in which F(ab')2 fragments (100 µg/ml) were directed against rat IgG added for 30 min to the anti-CD44 antibody-incubated PMN to block all free binding sites of the primary antibody, before washing and the transmigration assay. In addition, Fig. 2A shows that an antibody directed to an unrelated receptor expressed at the surface of PMN, namely, CD87 used at a concentration of 25 µg/ml, does not affect PMN migration (n = 3, P >=  0.05). The role of CD44 in PMN transmigration was further confirmed by the addition (100 µg/ml) to PMN of F(ab') fragments of the anti-CD44 clone NIH44.1 (29). Such treatment also resulted in a significant reduction in transepithelial migration, although less markedly than seen with the intact anti-CD44 antibody IM7 (~25%; n = 3 filters). Also, interestingly, the inhibition of PMN transmigration was not associated with an accumulation of retained PMN in monolayers (monolayer-retained PMN: 6.5 ± 0.6 × 104 or 7.8 ± 0.9 × 104 PMN/ml in the presence or absence of 10 µg/ml anti-CD44 antibody IM7, n = 3; not shown).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibitory effect of anti-CD44 antibody and hyaluronic acid on PMN transepithelial migration or intraepithelial lymphocyte (IEL) migration into epithelial monolayers in the basolateral-to-apical direction. A, left: inhibitory effect of preincubation of PMN for 30-45 min with graded concentrations of the IM7 antibody (solid bars). An isotype-matched control antibody (shaded bar) and an anti-CD87 antibody (open bar) were similarly preincubated at 20 µg/ml with PMN. Inset: concentration-dependent saturation of IM7 binding to PMN, expressed as fold increase in median fluorescence intensity over control isotype antibody (Ab) values. A, right: inhibitory effect of the CD44 natural ligand hyaluronic acid (hatched bars) compared with the absence of ligand addition (shaded bar). PMN transmigration was induced by 5 × 10-7 M N-formyl-Met-Leu-Phe (fMLP) in Hanks' balanced salt solution [HBSS(+)]. After 120 min at 37°C, migrated PMN across monolayers into the chemoattractant-containing lower chambers were quantitated by myeloperoxidase assay. Data represent means ± SD of 3 distinct experiments, each consisting of triplicate determinations. B: inhibitory effect of preincubation for 2 h at 37°C with a saturating concentration of IM7 antibody (solid bars) on IEL migration. An isotype-matched control antibody was incubated with IEL at the same concentration in subsets of control experiments (shaded bar). 5-Chloromethylfluorescein diacetate (CMFDA)-labeled IEL spontaneously migrated at 37°C (2 h) to the interepithelial spaces and were quantitated with a fluorescence plate reader, as previously described (32). Data represent means ± SD of 3 monolayers for each condition and are representative of 2 distinct experiments.

To analyze whether CD44 occupancy by specific natural, soluble ligands could similarly alter PMN transepithelial migration, we replaced the anti-CD44 antibody in the above-mentioned assay system with hyaluronic acid (4, 17, 21). Figure 2A shows that in the presence of 500 µg/ml hyaluronic acid, and to a lesser degree for 200 µg/ml hyaluronic acid, PMN migration through epithelial monolayers was strongly reduced (~59% inhibition upon incubation with 500 µg/ml hyaluronic acid, n = 3, P <=  0.01). It is noteworthy that these concentrations are consistent with those that have been reported to regulate leukocyte (30) or fibroblast biological functions (38).

In another series of experiments, we investigated whether the IM7 inhibitory effect on transepithelial migration of PMN was also seen when other categories of leukocytes were used, such as lymphocytes. Intestinal IEL represent one of the largest lymphoid populations in humans. The mechanisms underlying the final steps in IEL migration from the subepithelial matrix into the intestinal epithelium are largely unknown (33). Figure 2B shows that, as previously reported (33), cultured IEL spontaneously migrated into the intestinal epithelium. This effect was found to be significantly reduced by the preincubation of IEL with the anti-CD44 antibody IM7 (40 µg/ml inhibited IEL migration by ~50% compared with the assay conducted without antibody or in the presence of an irrelevant control antibody, n = 3, P <=  0.05). These data suggest that CD44 is involved in the migration process of IEL, a finding consistent with previous reports showing that a CD44-mediated process contributes to extravasation of peripheral blood lymphocytes at sites of inflammation (11, 21, 25).

Alteration of PMN transepithelial migration by anti-CD44 antibody IM7 depends on an effect restricted to PMN. Experiments were performed to examine whether the effects of anti-CD44 antibodies on the fMLP-induced PMN transepithelial migration were due to antibody effects to PMN or epithelium. Indeed, CD44 is expressed not only on leukocytes but also on various other cell types, including the basolateral surface of the epithelium (11, 17, 20, 21). As indicated in Fig. 1B, CD44 is indeed expressed in the intestinal epithelial cell line T84. However, pretreatment of the epithelium for 45 min with 20-70 µg/ml IM7 antibody did not result in the inhibition of transepithelial migration of PMN. As shown in Fig. 3A, the number of PMN (22.9 ± 7.4 × 104/ml) that migrated through epithelial monolayers preincubated with the anti-CD44 antibody was not different from that seen with preincubation of epithelial monolayers with an irrelevant control antibody under the same conditions (26.9 ± 6.9 × 104 PMN/ml, n = 3, P >=  0.05).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Evidence that the inhibitory effect of anti-CD44 antibody is due to effects on PMN, rather than epithelial cells. A: epithelial cells pretreated with >20 µg/ml IM7 antibody (hatched bar) for 45 min at 37°C before an extensive washout support normal PMN transepithelial migration. An isotype-matched control antibody was also used under the same conditions (shaded bar). B and C: inhibitory effect of preincubation for 30-45 min with 10 µg/ml IM7 antibody (solid bars) on PMN migration through epithelial monolayers in the apical-to-basolateral direction or through collagen-coated or uncoated filters in the absence of epithelium, respectively. An isotype-matched control antibody was used at the same concentration (shaded bars). PMN transmigration through epithelial inserts and filters without epithelium was induced by 5 × 10-7 M fMLP and 10-8 M fMLP, respectively, and further quantified as described in Fig. 2. Data represent means ± SD of 3 distinct experiments, each consisting of triplicate determinations.

The possibility that the anti-CD44 antibody IM7 is a general inhibitor of PMN migration across biological barriers per se was tested by two additional series of experiments. The first one demonstrated that PMN transepithelial migration was affected by the anti-CD44 antibody when migration occurred in the nonphysiological apical-to-basolateral direction (6.2 ± 1.5 × 104 vs. 14.0 ± 1.5 × 104 in the presence and absence of the anti-CD44 antibody, respectively; Fig. 3B, n = 3, P <=  0.01). Because epithelial ligands involved in PMN transmigration often demonstrate differential influences on migration based on migration direction (27), such findings were consistent with a primary antibody effect on PMN. Next, PMN migration was measured in a transwell filter system that either had or had not been coated with a complex collagen-rich matrix in the absence of epithelial monolayers. As indicated in Fig. 3C, anti-CD44 antibody IM7 inhibited PMN chemotaxis induced by fMLP by ~60% compared with the value obtained in the presence of control antibody (n = 3, P <=  0.01).

Anti-CD44 antibody IM7 does not globally affect adhesive properties of PMN. The possibility that anti-CD44 IM7 antibody broadly inhibits PMN migration by global effects on adhesion was tested by measuring adhesion of PMN to different supports including hyaluronic acid, collagen, and uncoated cell culture-treated filters. Figure 4A shows that fMLP-activated PMN adhere significantly to the three types of supports. Saturating concentrations of IM7 did not, however, inhibit adhesion to any substrate. For instance, adherence of PMN to the CD44 natural ligand hyaluronic acid was barely altered by the concomitant presence of IM7 antibody, likely because this antibody does not interact with the hyaluronic acid-binding epitope of CD44 (39). It is of note that adhesion properties of IEL to hyaluronic acid, collagen, or uncoated cell culture-treated filters were also unaffected by the anti-CD44 antibody (Fig. 4B). Degranulation responses of PMN were also examined and found to be unaffected by anti-CD44 antibody IM7, as indicated by the same amount of elastase having been released after PMN activation by immune complexes in the presence or absence of IM7 antibody. Additionally, the anti-CD44 antibody did not by itself lead to elastase release (data not shown).


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4.   Lack of regulatory effect of anti-CD44 antibody on leukocyte adhesion. The effect of preincubation of CMFDA-labeled PMN (A) or lymphocytes (B), under conditions described in Fig. 2, with anti-CD44 antibody (solid bar) or a matched control antibody (hatched bars) on leukocyte adhesion to uncoated or hyaluronic acid- and collagen-coated filters is shown. In A, levels of PMN adhesion are compared between resting PMN (open bars) and PMN activated by 5 × 10-7 M fMLP (hatched and solid bars). After extensive washing, quantitation of adherent leukocytes was obtained by measuring fluorescence associated to transwell filters with a fluorescence plate reader. Data represent means ± SD of 3 filters for each condition and are representative of 2 distinct experiments.

Anti-CD44 antibody IM7 activates adenylate cyclase, but not Ca2+ signaling, pathways. To gain further insight as to potential mechanisms by which the anti-CD44 antibody IM7 regulates the PMN transmigration process, we investigated the effect of the antibody on two distinct signaling pathways. Recent studies have suggested that CD44-mediated outside-in signaling is responsible for regulating increases in intracellular Ca2+ and cAMP in some lymphocyte populations (3, 18, 32). Therefore, we first examined whether anti-CD44 antibody IM7 downregulated Ca2+ mobilization in fMLP-activated PMN. As shown in Fig. 5A, preincubation of PMN with IM7 under conditions similar to those used for the transepithelial migration assay did not modify the Ca2+ response induced by the chemotactic agent fMLP. In addition, no direct increase of Ca2+ mobilization could be observed after addition of anti-CD44 antibody IM7 directly into PMN suspensions (not shown). In contrast, Fig. 5B shows that anti-CD44 antibody did stimulate significantly adenylate cyclase activity (cAMP levels detected in PMN incubated for 30 min with the antibody were 2.5 times greater than levels measured in resting PMN, n = 3, P <=  0.05), suggesting that IM7 antibody may mediate the inhibition of PMN transepithelial migration through activation of adenylate cyclase (13-15, 31). Interestingly, the natural ligand of CD44, hyaluronic acid, which also altered PMN migration (cf. Fig. 2), triggered an even higher production of cAMP (up to 10 times the level measured in nontreated PMN after 30-min incubation) (Fig. 5B, n = 3, P <=  0.05).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Stimulation of intracellular Ca2+ and adenylate cyclase activity in PMN incubated with anti-CD44 antibody or hyaluronic acid. A: Ca2+ mobilization in PMN-pretreated for 30-45 min with either the anti-CD44 antibody IM7 (10 µg/ml) or an isotype-matched control antibody under the same conditions, before stimulation by a submaximal concentration of fMLP (10-8 M). The changes in fluorescence, reflecting changes in Ca2+ concentration, were monitored with a spectrofluorometer. Tracings are representative of 3 distinct experiments. B: cAMP production in PMN that were or were not incubated for increasing periods of time with anti-CD44 antibody (10 µg/ml) or with a matched control antibody or hyaluronic acid (500 µg/ml) under the same conditions. cAMP production was measured from PMN lysates with the use of a commercial kit assay. Note that the scale for either graph is different. Data are means ± SD of 3 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Many models of PMN-endothelial cell recognition preceding transendothelial migration of leukocytes have been characterized as a multiple-step process involving sequential, molecularly defined events: 1) a selectin-mediated adhesion that results in leukocyte rolling, 2) an activation of beta 2-integrin-mediated firm adhesion, and 3) a migration through the endothelial cleft. We (26-28) and others (2) have previously provided evidence supporting different paradigms for PMN transmigration across epithelial, as opposed to endothelial, monolayers. Thus transepithelial migration is dependent on CD47 and one beta 2-integrin, namely, CD11b/CD18; the epithelial ligand for this integrin does not appear to be intercellular adhesion molecule-1, further differentiating the two PMN migration systems. Furthermore, compared with PMN-endothelium interactions, in which carbohydrate-based interactions have been extensively studied, far less is known about the contribution of such molecular structures in PMN transepithelial migration (2, 26, 28). We did, however, demonstrate previously (8) that uncharacterized carbohydrates play a critical role in the molecular interactions between PMN and columnar epithelia. The failure of antibodies directed to selectin domains to interfere with epithelium-PMN interactions implies that these interactions substantially differ from those governing carbohydrate-based endothelium-PMN interactions (8). In the present study, we report that antibodies specific for the receptor CD44 strongly inhibit the ability of PMN to cross monolayers of the polarized cryptlike human intestinal epithelial cell line T84. The use of irrelevant control antibodies and F(ab') anti-CD44 fragments, as well as the natural CD44 ligand hyaluronic acid, eliminates the possibility that our results could be confounded with Fc receptor-mediated effects or nonspecific steric consequences. Both the antibody and natural ligand for CD44 stimulate cAMP production in PMN, a known negative regulation signal for PMN movement.

Multiple isoforms of CD44 exist as a result of alternative splicing of a series of exons and the differential use of glycosaminoglycan attachment sites on the extracellular domain (17, 21). In addition, although CD44 is an adhesion receptor that is broadly distributed, including sites on both leukocytes and epithelial tissues (such as the model epithelium used herein), we have provided evidence that only one isoform of CD44 (CD44s) exists on peripheral blood PMN and influences the process of transepithelial migration. CD44 has long been known to mediate cell interactions in numerous systems, with described roles ranging from extracellular matrix binding, lymphocyte homing, and activation to lymphopoiesis and metastasis (7, 17, 20, 21). Various isoforms of CD44 have been selectively associated with regulation of migration and invasion of subsets of cell types. The regulation of movement observed can be negative, as shown here, or positive, depending on the cell type. For example, CD44s promotes melanoma cell migration (17, 20, 21). In addition to our current data analyzing IEL migration into epithelial monolayers (cf. Fig. 2B), several lines of evidence point to an important in vivo role for the interaction of CD44 expressed on lymphocytes and either hyaluronic acid or other matrix carbohydrated-based ligands. First, CD44 has been shown to be required for acute extravasation and movement of activated T cells to inflamed peritoneum induced by local administration of superantigen (11). In addition, antibodies to CD44 and alpha 4-integrin prevent model inflammation of the central nervous system by targeting the primary influx of T cells and thus presumably affecting the secondary influx of other leukocytes (6). Strikingly, direct evidence identifying CD44 as a regulator of PMN mobility per se has not been described prior to our study, although our findings are consistent with earlier work showing that the key ligand of CD44, i.e., hyaluronic acid, regulates PMN functions including phagocytosis, locomotion, and metabolism (30).

As indicated above, CD44 has long been shown to be an adhesion receptor for matrix components. However, whether CD44 mediates cell migration in general, and PMN transmigration in particular, by a direct interaction with such matrix molecules or through other unknown counterreceptors exposed at the surface of adjacent cells (e.g., the epithelial cells) remains to be defined. Also, the facts that anti-CD44 antibody-treated PMN are not retained in the epithelial monolayer and that anti-CD44 antibody does not affect leukocyte adhesion but inhibits PMN migration through simple uncoated filters suggest that the inhibitory effect of anti-CD44 antibody likely occurs at a step preceding PMN contact with the epithelium. One potential mechanism is that the anti-CD44 antibody with the PMN surface may eventually modulate outside-in cell signaling events specifically critical for PMN mobility properties. Similar effects have been observed in PMN-endothelium interactions with the use of an antibody directed against the CD47 adhesion molecule (10, 27). Furthermore, recent findings support the notion that the function of CD44 as a signaling molecule is as important as its function as a cell adhesion receptor (3, 18, 32). For instance, the functional importance of the cytoplasmic domain of CD44 has been demonstrated because it is involved in the indirect interaction with cytoskeletal components and adenylate cyclase or in the mobilization of intracellular Ca2+ (5, 18, 23, 32). However, confocal analysis of PMN labeled with phalloidin-rhodamine to evaluate the polymerization state of the actin cytoskeleton in PMN incubated for different time periods (1, 5 and 30 min) with anti-CD44 antibody did not reveal any cytoskeletal reordering (not shown). These data suggest that the inhibitory effect of IM7 antibody is not related to an alteration of PMN actin cytoskeleton, a structure known to facilitate leukocyte migration across the colonic epithelial barrier (17a).

It has been demonstrated that the ability of an anti-CD44 antibody, different from IM7 IgG, to inhibit activation of T lymphocytes is based on its induction of an elevation of intracellular cAMP (32). In the present study, to begin to address the issue of whether anti-CD44 antibodies regulate PMN mobility by triggering an inhibitory cell signal, we measured Ca2+ levels in PMN, which either had or had not been preincubated with IM7 antibody, in response to a chemotactic mediator. In addition, we evaluated cAMP concentrations in PMN treated with the anti-CD44 antibody or with the natural, soluble ligand of CD44, hyaluronic acid. It is established that cAMP is a potent inhibitory signal of several leukocyte proinflammatory activities such as NADPH oxidase or granule exocytosis in neutrophils. Also, several reports (13-15, 31) have previously shown that activation of adenylate cyclase-dependent signaling pathways in PMN downregulates those that govern leukocyte locomotion. Interestingly, we found that while the Ca2+ response was not significantly affected, the anti-CD44 antibody and hyaluronic acid induced a substantial cAMP response. These observations suggest that the blocking antibody used is an activator of outside-in signaling, comparable to the natural soluble ligand. This feature is of particular interest because previous reports have shown that the increase of intracellular cAMP concentration results in the decrease of PMN motility in vitro and in vivo (1, 9, 13-15, 31) without significantly altering the peak of Ca2+ mobilization (1, 36). The underlying mechanism is not entirely elucidated but may include a decreased exposure by cAMP of specific adhesive receptors involved in the transmigration process. For instance, it has been demonstrated that elevation of intracellular levels of cAMP blocks chemoattractant stimulation of beta 2-integrin in PMN. This effect is mediated by protein kinase A (PKA), and this PKA-dependent inhibition occurs downstream of the small GTPase RhoA, a critical mediator of chemoattractant to integrin signaling (20a). A similar process is likely to occur in our CD44-regulated migration system, although our results suggest that the anti-CD44 antibody IM7 specifically affects postadhesive signals before PMN transepithelial migration. The identity of such signals and their putative regulation by adenylate cyclase-dependent pathways remains to be elucidated.

Alternatively, the antibody IM7 has been shown to modify the adhesive properties of leukocyte CD44 by inducing a shedding of the molecule, leading to reduced expression of CD44 on the surface of leukocytes within 12-20 h of incubation of antibody exposure (22). However, we performed flow cytometry experiments (not shown) demonstrating that CD44 did not shed after PMN incubation with IM7 antibody under our conditions, likely because the time of treatment (<1 h) is insufficient to trigger such process.

Additional investigations are needed to further dissect signaling mechanisms by which the CD44 adhesion molecule tightly regulates leukocyte motility. Our findings indicate that CD44 ligation by an antibody or the natural ligand is a negative regulator of PMN migration across biological surfaces including complex matrices and polarized epithelia. These findings have substantial clinical relevance in the identification of regulatory mechanisms of active inflammatory responses.


    ACKNOWLEDGEMENTS

We are indebted to Dr. Charles A. Parkos for helpful comments and suggestions during the preparation of the manuscript. We also thank Dr. David Segal [National Institutes of Health, Bethesda, MD] for generously providing F(ab') fragments of the anti-CD44 clone NIH44.1.


    FOOTNOTES

These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-35932 and DK-47622 (to J. L. Madara).

Present address of M. Si-Tahar: Unite de Pharmacologie Cellulaire, Unite Associet IP/INSERM 285, Institut Pasteur, Paris, France (E-mail: msitahar{at}pasteur.fr).

Address for reprint requests and other correspondence: J. L. Madara, Epithelial Pathobiology Unit, Emory Univ. Hospital, Dept. of Pathology and Laboratory Medicine, Rm. H184, 1364 Clifton Road, Atlanta, GA 30322 (E-mail: james_madara{at}emory.org).

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 22 February 2000; accepted in final form 14 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahmed, MU, Hazeki K, Hazeki O, Katada T, and Ui M. Cyclic AMP-increasing agents interfere with chemoattractant-induced respiratory burst in neutrophils as a result of the inhibition of phosphatidylinositol 3-kinase rather than receptor-operated Ca2+ influx. J Biol Chem 270: 23816-23822, 1995[Abstract/Free Full Text].

2.   Albelda, SM. Endothelial and epithelial cell adhesion molecules. Am J Respir Cell Mol Biol 4: 195-203, 1991[ISI][Medline].

3.   Aruffo, A. CD44: one ligand, two functions. J Clin Invest 98: 2191-2192, 1996[Free Full Text].

4.   Aruffo, A, Stamenkovic I, Melnick M, Underhill CB, and Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell 61: 1303-1313, 1990[ISI][Medline].

5.   Bourguignon, LYW, Lokeshwar VB, Chen X, and Kerrick WGL Hyaluronic acid-induced lymphocyte signal transduction and HA receptor (GP85/CD44)-cytoskeletal interaction. J Immunol 151: 6634-6644, 1993[Abstract/Free Full Text].

6.   Brocke, S, Piercy C, Steinman L, Weissman IL, and Veromaa T. Antibodies to CD44 and integrin alpha 4, but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment. Proc Natl Acad Sci USA 96: 6896-6901, 1999[Abstract/Free Full Text].

7.   Camp, RL, Scheynius A, Johansson C, and Pure E. CD44 is required for optimal contact allergic responses but is not required for normal leukocyte extravasation. J Exp Med 178: 497-507, 1993[Abstract].

8.   Colgan, SP, Parkos CA, McGuirk D, Brady HR, Papayianni AA, Frendl G, and Madara JL. Receptors involved in carbohydrate binding modulate intestinal epithelial-neutrophil interactions. J Biol Chem 270: 10531-10539, 1995[Abstract/Free Full Text].

9.   Condino-Neto, A, Vilela MM, Cambiucci EC, Ribeiro JD, Guglielmi AA, Magna LA, and De Nucci G. Theophylline therapy inhibits neutrophil and mononuclear cell chemotaxis from chronic asthmatic children. Br J Clin Pharmacol 32: 557-561, 1991[ISI][Medline].

10.   Cooper, D, Lindberg FP, Gamble JR, Brown EJ, and Vadas MA. Transendothelial migration of neutrophils involves integrin-associated protein (CD47). Proc Natl Acad Sci USA 92: 3978-3982, 1995[Abstract/Free Full Text].

11.   DeGrendele, HC, Estess P, and Siegelman MH. Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science 278: 672-675, 1997[Abstract/Free Full Text].

12.   Dharmsathaphorn, K, and Madara JL. Established intestinal cell lines as model systems for electrolyte transport studies. Methods Enzymol 192: 354-389, 1990[Medline].

13.   Elferink, JGR, and de Koster BM. Inhibition of interleukin-8-activated human neutrophil chemotaxis by thapsigargin in a calcium- and cyclic AMP-dependent way. Biochem Pharmacol 59: 369-375, 2000[ISI][Medline].

14.   Elferink, JGR, Huizinga TW, and de Koster BM. The effect of pentoxifylline on human neutrophil migration: a possible role for cyclic nucleotides. Biochem Pharmacol 54: 475-480, 1997[ISI][Medline].

15.   Elferink, JGR, and VanUffelen BE. The role of cyclic nucleotides in neutrophil migration. Gen Pharmacol 27: 387-393, 1996[Medline].

16.   Guo, YJ, Wong JH, Lin SC, Aruffo A, Stamenkovic I, and Sy MS. Disruption of T lymphocytes reappearance in anti-Thy-1-treated animals in vivo with soluble CD44 and L-selectin molecules. Cell Immunol 154: 202-208, 1994[ISI][Medline].

17.   Herrera-Gayol, A, and Jothy S. Adhesion proteins in the biology of breast cancer: contribution of CD44. Exp Mol Pathol 66: 149-156, 1999[ISI][Medline].

17a.   Hofman, P, d'Andrea L, Guzman E, Selva E, Le Negrate G, Far DF, Lemichez E, Boquet P, and Rossi B. Neutrophil F-actin and myosin but not microtubules functionally regulate transepithelial migration induced by interleukin 8 across a cultured intestinal epithelial monolayer. Eur Cytokine Netw 10: 227-236, 1999[ISI][Medline].

18.   Isacke, CM. The role of the cytoplasmic domain in regulating CD44 function. J Cell Sci 107: 2353-2359, 1994[Free Full Text].

19.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

19a.   Laudanna, C, Campbell JJ, and Butcher EC. Elevation of intracellular cAMP inhibits RhoA activation and integrin-dependent leukocyte adhesion induced by chemoattractants. J Biol Chem 272: 24141-24144, 1997[Abstract/Free Full Text].

20.   Lesley, J, Hyman R, English N, Catterall JB, and Graham T. CD44 in inflammation and metastasis. Glycoconj J 14: 611-622, 1997[ISI][Medline].

21.   Lesley, JR, Hyman R, and Kincade PW. CD44 and its interaction with extracellular matrix. Adv Immunol 54: 271-335, 1993[ISI][Medline].

22.   Mikecz, K, Brennan FR, Kim JH, and Glant TT. Anti-CD44 treatment abrogates tissue oedema and leukocyte infiltration in murine arthritis. Nat Med 6: 558-563, 1995.

23.   Moffat, FL, Jr, Han T, Li ZM, Peck MD, Falk RE, Spalding PB, Jy W, Ahn YS, Chu AJ, and Bourguignon LY. Involvement of CD44 and the cytoskeletal linker protein ankyrin in human neutrophil bacterial phagocytosis. J Cell Physiol 168: 638-647, 1996[ISI][Medline].

24.   Nakache, M, Berg EL, Streeter PR, and Butcher EC. The mucosal vascular addressin is a tissue-specific endothelial cell adhesion molecule for circulating lymphocytes. Nature 337: 179-182, 1989[ISI][Medline].

25.   Naujokas, MF, Morin M, Anderson MS, Peterson M, and Miller J. The chondroitin sulfate form of invariant chain can enhance stimulation of T cell responses through interaction with CD44. Cell 74: 257-268, 1993[ISI][Medline].

26.   Parkos, CA. Molecular events in neutrophil transepithelial migration. Bioessays 19: 865-731, 1997[ISI][Medline].

27.   Parkos, CA, Colgan SP, Liang TW, Nusrat A, Bacarra AE, Carnes DK, and Madara JL. CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia. J Cell Biol 132: 437-450, 1996[Abstract].

28.   Parkos, CA, Colgan SP, and Madara JL. Interactions of neutrophils with epithelial cells: lessons from the intestine. J Am Soc Nephrol 5: 138-152, 1994[Abstract].

29.   Pericle, F, Sconocchia G, Titus JA, and Segal DM. CD44 is a cytotoxic triggering molecule on human polymorphonuclear cells. J Immunol 157: 4657-4663, 1996[Abstract].

30.   Pisko, EJ, Turner RA, Soderstrom LP, Panetti M, Foster SL, and Treadway WJ. Inhibition of neutrophil phagocytosis and enzyme release by hyaluronic acid. Clin Exp Rheumatol 1: 41-44, 1983[ISI][Medline].

31.   Roch-Arveiller, M, Boquet P, Bradshaw D, and Giroud JP. Inhibition by cholera toxin of rat polymorphonuclear leukocyte chemotaxis demonstrated in vitro and in vivo. Infect Immun 25: 187-190, 1979[ISI][Medline].

32.   Rothman, BL, Kennure N, Kelley KA, Katz M, and Aune TM. Elevation of intracellular cAMP in human T lymphocytes by an anti-CD44 mAb. J Immunol 151: 6036-6042, 1993[Abstract/Free Full Text].

33.   Shaw, SK, Hermanowski-Vosakta A, Shibahara T, McCormick BA, Parkos CA, Carlson SL, Ebert EC, Brenner MB, and Madara JL. Migration of intestinal intraepithelial lymphocytes into a polarized epithelial monolayer. Am J Physiol Gastrointest Liver Physiol 275: G584-G591, 1998[Abstract/Free Full Text].

34.   Si-Tahar, M, Pidard D, Balloy V, Moniatte M, Kieffer N., Van Dorsselaer A, and Chignard M. Human neutrophil elastase proteolytically activates the platelet integrin alpha IIbbeta 3 through cleavage of the carboxyl terminus of the alpha IIb subunit heavy chain. Involvement in the potentiation of platelet aggregation. J Biol Chem 272: 11636-11647, 1997[Abstract/Free Full Text].

35.   Smith, JA. Neutrophils, host defense, and inflammation: a double-edged sword. J Leukoc Biol 56: 672-686, 1994[Abstract].

36.   Villagrasa, V, Navarrete C, C, Sanz Berto L, Perpina M, Cortijo J, and Morcillo EJ. Inhibition of phosphodiesterase IV and intracellular calcium levels in human polymorphonuclear leukocytes. Methods Find Exp Clin Pharmacol 18: 239-245, 1996[ISI][Medline].

37.   Wilson, HA, Greenblatt D, Taylor CW, Putney JW, Tsien RY, Finkelman FD, and Chused TM. The B lymphocyte calcium response to anti-Ig is diminished by membrane immunoglobulin cross-linkage to the Fc gamma receptor. J Immunol 138: 1712-1718, 1987[Abstract/Free Full Text].

38.   Yoneda, M, Yamagata M, Suzuki S, and Kimata K. Hyaluronic acid modulates proliferation of mouse dermal fibroblasts in culture. J Cell Sci 90: 265-273, 1988[Abstract].

39.   Zheng, Z, Katoh S, He Q, Oritani K, Miyake K, Lesley J, Hyman R, Hamik A, Parkhouse RM, and Farr AG. Monoclonal antibodies to CD44 and their influence on hyaluronan recognition. J Cell Biol 130: 485-495, 1995[Abstract].


Am J Physiol Cell Physiol 280(3):C423-C432
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society