Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn²⁺ additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1^−/− neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn²⁺-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn²⁺ induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.     

Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins.

Intercellular adhesion molecule-4 (ICAM-4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins. The location of the ICAM-4 binding sites on CD11a/CD18 and CD11b/CD18 are not known. CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding. ICAM-4 lacks such a residue, which is replaced by an arginine. However, we demonstrate here that ICAM-4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins. The binding was inhibited by anti-I domain and anti-ICAM-4 antibodies and it was dependent on divalent cations. Interestingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced. Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM-4 in a cation dependent manner. Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different. However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized by the ICAM-1 and ICAM-2. Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins.     

A peptide derived from the intercellular adhesion molecule-2 regulates the avidity of the leukocyte integrins CD11b/CD18 and CD11c/CD18

beta 2 integrin (CD11a,b,c/CD18)-mediated cell adhesion is required for many leukocyte functions. Under normal circumstances, the integrins are nonadhesive, and become adhesive for their cell surface ligands, the intercellular adhesion molecules (ICAMs), or soluble ligands such as fibrinogen and iC3b, when leukocytes are activated. Recently, we defined a peptide derived from ICAM-2, which specifically binds to purified CD11a/CD18. Furthermore, this peptide strongly induces T cell aggregation mainly mediated by CD11a/CD18-ICAM-1 interaction, and natural killer cell cytotoxicity. In the present study, we show that the same ICAM-2 peptide also avidly binds to purified CD11b/CD18, but not to CD11c/CD18. This binding can be blocked by the CD11b antibody OKM10. The peptide strongly stimulates CD11b/CD18-ICAM-1-mediated cell aggregations of the monocytic cell lines THP-1 and U937. The aggregations are energy and divalent cation-dependent. The ICAM-2 peptide also induces CD11b/CD18 and CD11c/CD18-mediated binding of THP- 1 cells to fibrinogen and iC3b coated on plastic. These findings indicate that in addition to induction of CD11a/CD18-mediated cell adhesion, the ICAM-2 peptide may also serve as a "trigger" for high avidity ligand binding of other beta 2 integrins.     

Two leukocyte receptors (CD11a/CD18 and CD11b/CD18) mediate transient adhesion to endothelium by binding to different ligands

Upon stimulation with C5a, TNF, or phorbol dibutyrate (PDB), polymorphonuclear leukocytes (PMN) exhibit first an increase then a decrease in adhesion to unstimulated endothelial cells (EC). Essentially all of this adhesion is mediated by the CD18 family of leukocyte integrins on PMN. To determine the individual roles of CD11a/CD18 (LFA-1), CD11b/CD18 (CR3, Mac-1) and CD11c/CD18 (p150,95) in adhesion of PDB-stimulated PMN to unstimulated EC, mAb against the CD11 chains were used. mAb against CD11a or CD11b each blocked adhesion of PMN to EC by approximately 50%, but mAb against CD11c had no effect. Inasmuch as a combination of anti-CD11a and CD11b mAb completely blocked adhesion, it appears that CD11a/CD18 and CD11b/CD18 make approximately equal contributions to binding, and CD11c does not participate. Anti-CD11a or CD11b each blocked adhesion by about 50% throughout the transient time course of PDB-stimulated adhesion, indicating that the capacity of each of these receptors to bind EC is transiently activated by PDB. We next examined the role of ICAM-1 on EC as a ligand for CD18. Two anti-ICAM-1 mAb (LB-2 and 84H10) each inhibited PMN adhesion in a dose-dependent fashion, reaching a maximal inhibition of approximately 50%. Anti-ICAM-1 mAb blocked the CD11a/CD18-dependent portion of adhesion because concomitant use of anti-CD11a and anti-ICAM-1 did not cause additive inhibition. In contrast, anti-CD11b plus anti-ICAM-1 resulted in complete blockade of adhesion. This result suggests that CD11a/CD18 recognizes ICAM-1 on EC, but CD11b/CD18 recognizes a different ligand(s). To determine if CD11b CD18 has the ability to recognize ICAM-1, human macrophages were plated on culture surfaces coated with purified ICAM-1. Interaction of CD11a/CD18 with the surface-bound ICAM-1 resulted in selective down-modulation of CD11a/CD18 from the apical portion of the macrophages. In contrast, ICAM-1-coated surfaces did not down-modulate CD11b/CD18. The data suggest that CD11b/CD18 does not recognize ICAM-1, and that this receptor functions in adhesion of PMN to EC by recognizing novel ligand(s) on EC.     

ICAM-1 and CD11b/CD18 expression during acute pancreatitis induced by bile-pancreatic duct obstruction: effect of N-acetylcysteine

Different molecules are involved in the recruitment of leukocytes during inflammation. The aim was to investigate (i) the contribution of acinar cells to the overall production of ICAM-1 and (ii) the kinetics of leukocyte CD11b/CD18 expression during acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) to evaluate the contribution of both molecules to leukocyte homing. The role of reactive oxygen species (ROS) as mediators in the expression of ICAM-1 and CD11b/CD18 was examined by using N-acetylcysteine (NAC) as an antioxidant treatment. By mechanisms resistant to NAC treatment, acinar cells were able to produce ICAM-1 at first onset of AP; other cell sources contribute to maintaining increased ICAM-1 plasma levels during AP. By contrast, CD11b/CD18 was overexpressed in leukocytes in the course of AP by oxidant-dependent mechanisms. Since NAC treatment reduced neutrophil infiltration in the pancreas, we conclude that CD11b/CD18 over-expression is required for leukocyte recruitment; however, other adhesion molecules in addition to ICAM-1 seem to contribute to leukocyte homing during BPDO-induced AP.     

Involvement of the CD11b/CD18 integrin, but not of the endothelial cell adhesion molecules ELAM-1 and ICAM-1 in tumor necrosis factor-alpha-induced neutrophil toxicity.

TNF-alpha can incite neutrophil-mediated endothelial cell damage and neutrophil H2O2 release. Both effects require adherent neutrophils. Using specific mAb, we showed in this in vitro study that the CD18 beta 2-chain and the CD11b alpha M-chain of the CD11/CD18 integrin heterodimer have a major role in both TNF-alpha-induced neutrophil-mediated detachment of human umbilical vein endothelial cells and H2O2 release by TNF-alpha-activated human neutrophils. In contrast to anti-CD18 mAb, which consistently prevented neutrophil activation, anti-CD11a mAb and two of three anti-CD11b mAb did not reduce endothelial cell detachment and neutrophil H2O2 release, although they decreased neutrophil adhesion to human umbilical vein endothelial cells. mAb 904, directed against the bacterial LPS binding region of CD11b, reduced endothelial cell detachment for about 40% and neutrophil H2O2 release for more than 50%, demonstrating that CD11b/CD18 is engaged in TNF-induced neutrophil activation. Dependence on CD11b/CD18 could not be overcome by CD18-independent anchoring of neutrophils via PHA. Additionally, neither induction of increased expression of the endothelial cell adhesion molecules ICAM-1 and ELAM-1, nor subsequent addition of specific mAb, influenced endothelial cell injury or H2O2 release by TNF-activated neutrophils. Interaction with ICAM-1 and ELAM-1 therefore appears not to induce additional activation of TNF-stimulated neutrophils. These studies suggest that a specific, CD11b/CD18-mediated signal, instead of adherence only, triggers toxicity of TNF-activated neutrophils.     

The Heparan Sulfate Proteoglycan Form of Epithelial CD44v3 Serves as a CD11b/CD18 Counter-receptor during Polymorphonuclear Leukocyte Transepithelial Migration

Leukocyte β₂-integrin CD11b/CD18 mediates the firm adhesion and subsequent transepithelial migration of polymorphonuclear leukocytes, but the identity of its counter-receptor(s) on epithelia remains elusive. Here we identified a monoclonal antibody, clone C3H7, which strongly bound to the basolateral membranes of epithelial cells and inhibited both the adhesion of epithelial cells to immobilized CD11b/CD8 and the transepithelial migration of PMNs in a physiologically relevant basolateral-to-apical direction. C3H7 antigen expression in epithelial monolayers was significantly increased by treatment with proinflammatory cytokine interferon-γ or a combination of interferon-γ and tumor necrosis factor-α. Up-regulation of C3H7 antigen was also observed in the epithelium of inflamed human colon tissues. Microsequencing and Western blotting of the purified antigen showed it to be CD44 variant 3 (CD44v3), a ∼160-kDa membrane glycoprotein. Further studies demonstrated that this epithelial CD44v3 specifically binds to CD11b/CD18 through its heparan sulfate moieties. In summary, our study demonstrates for the first time that the heparan sulfate proteoglycan form of epithelial CD44v3 plays a critical role in facilitating PMN recruitment during inflammatory episodes via directly binding to CD11b/CD18.A major component of many inflammatory diseases is the migration of large numbers of neutrophils (polymorphonuclear leukocytes, PMNs)² across the epithelium and their accumulation within a lumen. Examples include inflammatory bowel disease (IBD), cholangitis, cholecystitis, bronchial pneumonia, bronchitis, pyelonephritis, and cystitis. Under these pathophysiological conditions, epithelial injury and disease symptoms parallel PMN infiltration of the mucosa (1, 2). The current paradigm for migration of PMN across epithelial monolayers envisions a process consisting of sequential molecularly defined events such as CD11b/CD18-mediated firm adhesion of PMN with epithelia (3) followed by CD47-SIRPα interactions at the post-adhesion stage (4). However, although PMN transepithelial migration (TEM) has been widely demonstrated to be CD11b/CD18-dependent, the epithelial counter-receptor(s) for CD11b/CD18 in mediating PMN-epithelia adhesion has not been identified.Function mapping studies using domain-specific antibodies have demonstrated that the inserted domain (I-domain), a stretch of 200 amino acids of the CD11b subunit, is a major binding domain for CD11b/CD18 ligands (5). The I-domain of CD11b is promiscuous in ligand binding and has many known receptors including ICAM-1 (6, 7), fibrinogen (8), collagen (9), Cyr61 (CCN1), and connective tissue growth factor (CCN2) (10), heparin/heparan sulfate (11, 12), elastase (13), iC3b (14), and platelet glycoprotein Ibα (15). However, none of these ligands appear to mediate the firm adhesion of PMNs to the basolateral surfaces of epithelial monolayers at early stages of transmigration. Thus far, no epithelial basolaterally expressed CD11b/CD18 counter-receptor has been identified. ICAM-1, the best characterized cellular ligand for CD11b/CD18, cannot be the intestinal epithelial CD11b/CD18 ligand that mediates PMN firm adhesion because: (a) ICAM-1 is normally not expressed on intestinal epithelia except under inflammatory conditions (16) and (b) when ICAM-1 expression is induced it is up-regulated on the apical rather than basolateral surface of intestine epithelia. In an effort to understand the mechanisms that govern CD11b/CD18-mediated PMN TEM, previous studies by us and others have found that epithelial surface-sulfated proteoglycans (17) and junction adhesion molecule C (JAM-C) play a significant role in regulating PMN transmigration via interaction with leukocyte CD11b/CD18 (18, 19). However, compared with functional inhibitory anti-CD11b antibodies that completely block PMN TEM, soluble carbohydrates or antibodies against JAM-C create only partial inhibition. These results clearly suggest the existence of unknown epithelial adhesion molecule(s) that bind to leukocyte CD11b/CD18 and regulate PMN TEM. Heparin and heparan sulfate have also been shown to block the adhesion and PMN TEM via binding to CD11b/CD18 (11, 12); thus it is reasonable to suggest that a basolateral membrane glycoprotein decorated with heparan sulfate moieties may serve as a counter-receptor for CD11b/CD18. However, the nature of this epithelial heparan sulfate proteoglycan has not been identified.Here we sought to identify novel epithelial adhesive ligand(s) important in PMN transmigration, in particular, a ligand that can bind to CD11b/CD18 on migrating PMNs and mediate the firm adhesion of PMNs to the epithelial basolateral surfaces. To do this, we screened a panel of monoclonal antibodies generated against epithelial plasma membranes. This screening identified one mAb, termed C3H7, that recognized a basolateral membrane protein and inhibited PMN TEM in a physiologically relevant basolateral-to-apical direction. Further study of these results identified the C3H7 antigen as a v3-type human epithelial CD44 variant, a ∼160-kDa glycoprotein that is decorated with heparan sulfate moieties. Subsequent studies revealed that the C3H7 antigen appears to function as a cellular ligand for CD11b/CD18 in regulating the firm adhesion of PMNs to the epithelium during their transmigration process.     

Role of CD11a/CD18-CD54 interactions in suppression of human B cell responsiveness by CD4+ suppressor T cells.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in the suppression of human B cell function by immobilized anti-CD3-activated CD4+ T cells was examined by studying the effects of mAb to these determinants. The suppressive activity was assessed by the effects of CD4+ T cells without mitomycin C treatment activated by immobilized anti-CD3 for 72 hr on the differentiation into Ig-secreting cells of B cells activated for 72 hr with immobilized anti-CD3-stimulated CD4+ T cells that had been treated with mitomycin C (T4 mito). Suppression was not observed when activated CD4+ T cells and B cells were separated by filter membranes, indicating that the suppression requires the direct interactions between anti-CD3-activated CD4+ T cells and activated B cells. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) reversed the suppression of B cell function by suppressor CD4+ T cells significantly. Reversal of suppression of B cell function was most marked when activated B cells were treated with mAb to ICAM-1 and suppressor CD4+ T cells were treated with mAb to LFA-1, but not vice versa. Studies using fluorescence-activated cell sorter revealed marked increase of expression of ICAM-1 on B cells after 72 hr of activation with immobilized anti-CD3-stimulated T4 mito. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the suppressive activity of anti-CD3-activated CD4+ T cells to B cells. Moreover, the data are consistent with a model of T-cell-mediated B cell suppression in which interactions between LFA-1 on suppressor T cells and ICAM-1 on activated B cells play a central role in the suppression of B cell function.     

Occupancy of CD11b/CD18 (Mac-1) divalent ion binding site(s) induces leukocyte adhesion

The group of leukocyte integrins CD11a-c/CD18 coordinate disparate adhesion reactions in the immune system through a regulated process of ligand recognition. The participation of the receptor divalent ion binding site(s) in this mechanism of ligand binding has been investigated. As compared with other divalent cations, Mn2+ ions have the unique property to dramatically stimulate the adhesive functions of the leukocyte integrin CD11b/CD18 (Mac-1), expressed on myelo-monocytic cells. This is reflected in a three- to fivefold increased early monocyte adhesion (less than 20 min) to resting, unperturbed endothelial cells, and increased association of CD11b/CD18 with its soluble ligands fibrinogen and factor X. CD11b/CD18 ligand recognition in the presence of Mn2+ ions is specific, time and concentration dependent, and inhibited by anti-CD11b mAb. At variance with Ca(2+)-containing reactions where CD11b/CD18 functions as an inducible receptor activated by adenine nucleotides or chemoattractants, Mn2+ ions induce per se a constitutive maximal ligand binding capacity of CD11b/CD18, that is not further modulated by cell stimulation. Rather than quantitative changes in surface density, Mn2+ ions increase the affinity of CD11b/CD18 for its complementary ligands up to 10-fold, as judged by Scatchard plot analysis of receptor:ligand interaction under these conditions. Furthermore, monocyte exposure to Mn2+ ions induces the expression of activation-dependent neo-antigenic epitopes on CD11b/CD18, selectively recognized by mAb 7E3. These data suggest that in addition to cell-activating stimuli, favorable engagement of divalent ion binding site(s) can provide an alternative pathway to rapidly regulate the receptor affinity of leukocyte integrins.     

Cloning of the rat CR3 alphaM (CD11b) subunit, expression and binding assay of recombinant isolated CD11b VA (A-domain) and ICAM-1 Ig modules

The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex. It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration. We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library. The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart. We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E. coli as a soluble recombinant fusion protein with GST. Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E. coli as recombinant soluble (rs) fusion proteins with GST. Recombinant CD11b A-domain was released from the fusion protein by thrombin cut. Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain. These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily.     

A Highlights from MBoC Selection: CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions

CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47^−/− Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47^−/− Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)–activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α–activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 null cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn²⁺ or Mg²⁺/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration.     

Endothelial-leukocyte adhesion molecule-1-dependent and leukocyte (CD11/CD18)-dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine-activated human vascular endothelium

We have examined the contributions of endothelial-leukocyte adhesion molecule-1 (ELAM-1) and the complex of leukocyte surface adhesion molecules designated CD11/CD18 to the adhesion of human polymorphonuclear leukocytes (PMN) to cultured human endothelial cells (HEC), activated by rIL-1 beta for 4 or 24 h. Inhibition of PMN attachment to IL-1-activated HEC was measured in a quantitative in vitro monolayer adhesion assay, after treatment with mAb directed to ELAM-1 (mAb H18/17), and to CD11a (mAb L11), CD11b (mAb 44), CD11c (mAb L29), and CD18 (mAb 10F12), alone or in combination. Pretreatment of activated HEC with mAb H18/7 inhibited PMN adhesion by 47 +/- 8% whereas control mAb had no effect. CD11/CD18-directed mAb significantly blocked PMN adhesion to activated HEC (anti-CD11a, 40 +/- 3%; anti-CD11b, 34 +/- 4%; anti-CD18, 78+/- 6% inhibition). The combination of mAb H18/7 and each of the various anti-CD11/CD18 mAb resulted in greater inhibition of PMN adhesion than any Mab alone. After 24 h of rIL-1 beta treatment, when ELAM-1 was markedly decreased but elevated PMN adhesion was still observed, mAb H18/7 had no effect on PMN adhesion. At this time, CD11/CD18-dependent adhesive mechanisms predominated and a CD11c-dependent mechanism became apparent (anti-CD11a, 67 +/- 4% inhibition; anti-CD11b, 45 +/- 9%; anti-CD11c, 26 +/- 6%; anti-CD18, 97 +/- 1%). In summary, PMN adhesion to IL-1-activated HEC involves both CD11/CD18-dependent mechanisms and an ELAM-1-dependent mechanism, and the relative contribution of these varies at different times of IL-1-induced HEC activation. The additive blocking observed at 4 h with mAb H18/7 in combination with CD11/CD18-directed Mab implies that members of the CD11/CD18 complex do not function as an obligate ligand(s) for ELAM-1.     

ICAM-2 and a peptide from its binding domain are efficient activators of leukocyte adhesion and integrin affinity.

Cell adhesion mediated by the CD11/CD18 integrins and their ligands, the ICAMs, is required for many leukocyte functions. In resting cells the integrins are nonadhesive, but when activated they become adhesive for their ligands. Previous findings have shown that a peptide derived from the first Ig domain of ICAM-2 (P1) binds to LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) and activates leukocyte aggregation. Because its mechanism of action has remained poorly understood, we have now studied the peptide-induced ligand binding in detail. Here we show that P1 was able to induce CD11/CD18-dependent adhesion of human T lymphocytes to immobilized, purified ICAM-1, -2, and -3. The optimal peptide concentration was 150 micrograms/ml, whereas concentrations higher than 400 micrograms/ml did not have any stimulatory effect. The increase in adhesion was detectable within 10 min of treatment with the peptide; it was dependent on energy, divalent cations, temperature, and an intact cytoskeleton but was unaffected by protein kinase C and protein tyrosine kinase inhibitors. Peptide treatment resulted in strong stimulation of the binding of soluble, recombinant ICAMs to T lymphocytes, showing that the integrin affinity toward its ligands was increased. Importantly, soluble ICAM-2Fc was also able to induce T lymphocyte adhesion to purified ICAM-1, -2, and -3, and it was a more potent stimulatory molecule than ICAM-1Fc or ICAM-3Fc.     

Small molecule agonists of integrin CD11b/CD18 do not induce global conformational changes and are significantly better than activating antibodies in reducing vascular injury

Background

CD11b/CD18 is a key adhesion receptor that mediates leukocyte adhesion, migration and immune functions. We recently identified novel compounds, leukadherins, that allosterically enhance CD11b/CD18-dependent cell adhesion and reduce inflammation in vivo, suggesting integrin activation to be a novel mechanism of action for the development of anti-inflammatory therapeutics. Since a number of well-characterized anti-CD11b/CD18 activating antibodies are currently available, we wondered if such biological agonists could also become therapeutic leads following this mechanism of action.

Methods

We compared the two types of agonists using in vitro cell adhesion and wound-healing assays and using animal model systems. We also studied effects of the two types of agonists on outside-in signaling in treated cells.

Results

Both types of agonists similarly enhanced integrin-mediated cell adhesion and decreased cell migration. However, unlike leukadherins, the activating antibodies produced significant CD11b/CD18 macro clustering and induced phosphorylation of key proteins involved in outside-in signaling. Studies using conformation reporter antibodies showed that leukadherins did not induce global conformational changes in CD11b/CD18 explaining the reason behind their lack of ligand-mimetic outside-in signaling. In vivo, leukadherins reduced vascular injury in a dose-dependent fashion, but, surprisingly, the anti-CD11b activating antibody ED7 was ineffective.

Conclusions

Our results suggest that small molecule allosteric agonists of CD11b/CD18 have clear advantages over the biologic activating antibodies and provide a mechanistic basis for the difference.

General significance

CD11b/CD18 activation represents a novel strategy for reducing inflammatory injury. Our study establishes small molecule leukadherins as preferred agonists over activating antibodies for future development as novel anti-inflammatory therapeutics.     

Integrin cross-talk modulates stiffness-independent motility of CD4+ T lymphocytes

To carry out their physiological responsibilities, CD4+ T lymphocytes interact with various tissues of different mechanical properties. Recent studies suggest that T cells migrate upstream on surfaces expressing intracellular adhesion molecule-1 (ICAM-1) through interaction with leukocyte function-associated antigen-1 (α_Lβ₂) (LFA-1) integrins. LFA-1 likely behaves as a mechanosensor, and thus we hypothesized that substrate mechanics might affect the ability of LFA-1 to support upstream migration of T cells under flow. Here we measured motility of CD4+ T lymphocytes on polyacrylamide gels with predetermined stiffnesses containing ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), or a 1:1 mixture of VCAM-1/ICAM-1. Under static conditions, we found that CD4+ T cells exhibit an increase in motility on ICAM-1, but not on VCAM-1 or VCAM-1/ICAM-1 mixed, surfaces as a function of matrix stiffness. The mechanosensitivity of T-cell motility on ICAM-1 is overcome when VLA-4 (very late antigen-4 [α₄β₁]) is ligated with soluble VCAM-1. Last, we observed that CD4+ T cells migrate upstream under flow on ICAM–1-functionalized hydrogels, independent of substrate stiffness. In summary, we show that CD4+ T cells under no flow respond to matrix stiffness through LFA-1, and that the cross-talk of VLA-4 and LFA-1 can compensate for deformable substrates. Interestingly, CD4+ T lymphocytes migrated upstream on ICAM-1 regardless of the substrate stiffness, suggesting that flow can compensate for substrate stiffness.     

Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by beta-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcgammaRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) beta(2) integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by beta-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by beta-glucan. A single CD11b lectin site for beta-glucan and uPAR was suggested because the binding of either beta-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of (125)I-labeled beta-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of (125)I-labeled beta-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.     

2E8 Binds to the High Affinity I-domain in a Metal Ion-dependent Manner: A SECOND GENERATION MONOCLONAL ANTIBODY SELECTIVELY TARGETING ACTIVATED LFA-1*

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α_L subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K_D) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn²⁺ was replaced sequentially by Mg²⁺ and Ca²⁺. Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4⁺ and CD8⁺ T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.     

Expression and polarization of intercellular adhesion molecule-1 on human intestinal epithelia: consequences for CD11b/CD18-mediated interactions with neutrophils.

BACKGROUND: Epithelial dysfunction and patient symptoms in inflammatory intestinal diseases such as ulcerative colitis and Crohn's disease correlate with migration of neutrophils (PMN) across the intestinal epithelium. In vitro modeling of PMN transepithelial migration has revealed distinct differences from transendothelial migration. By using polarized monolayers of human intestinal epithelia (T84), PMN transepithelial migration has been shown to be dependent on the leukocyte integrin CD11b/CD18 (Mac-1), but not on CD11a/CD18 (LFA-1). Since intercellular adhesion molecule-I (ICAM-1) is an important endothelial counterreceptor for these integrins, its expression in intestinal epithelia and role in PMN-intestinal epithelial interactions was investigated. MATERIALS AND METHODS: A panel of antibodies against different domains of ICAM-1, polarized monolayers of human intestinal epithelia (T84), and natural human colonic epithelia were used to examine the polarity of epithelial ICAM-1 surface expression and the functional role of ICAM-1 in neutrophil-intestinal epithelial adhesive interactions. RESULTS: While no surface expression of ICAM-1 was detected on unstimulated T84 cells, interferon-gamma (IFN gamma) elicited a marked expression of ICAM-1 that selectively polarized to the apical epithelial membrane. Similarly, apically restricted surface expression of ICAM-1 was detected in natural human colonic epithelium only in association with active inflammation. With or without IFN gamma pre-exposure, physiologically directed (basolateral-to-apical) transepithelial migration of PMN was unaffected by blocking monoclonal antibodies (mAbs) to ICAM-1. In contrast, PMN migration across IFN gamma-stimulated monolayers in the reverse (apical-to-basolateral) direction was inhibited by anti-ICAM-1 antibodies. Adhesion studies revealed that T84 cells adhered selectively to purified CD11b/CD18 and such adherence, with or without IFN gamma pre-exposure, was unaffected by ICAM-1 mAb. Similarly, freshly isolated epithelial cells from inflamed human intestine bound to CD11b/CD18 in an ICAM-1-independent fashion. CONCLUSIONS: These data indicate that ICAM-1 is strictly polarized in intestinal epithelia and does not represent a counterreceptor for neutrophil CD11b/CD18 during physiologically directed transmigration, but may facilitate apical membrane-PMN interactions after the arrival of PMN in the intestinal lumen.     

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

Forcing Switch from Short- to Intermediate- and Long-lived States of the αA Domain Generates LFA-1/ICAM-1 Catch Bonds

Binding of lymphocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion under force. Using a biomembrane force probe capable of measuring single bond interactions, we showed ICAM-1 binding to LFA-1 at different conformations, including the bent conformation with the lowest affinity. We quantify how force and conformations of LFA-1 regulate its kinetics with ICAM-1. At zero-force, on-rates were substantially changed by conditions that differentially favor a bent or extended LFA-1 with a closed or open headpiece; but off-rates were identical. With increasing force, LFA-1/ICAM-1 bond lifetimes (reciprocal off-rates) first increased (catch bonds) and then decreased (slip bonds). Three states with distinct off-rates were identified from lifetime distributions. Force shifted the associated fractions from the short- to intermediate- and long-lived states, producing catch bonds at low forces, but increased their off-rates exponentially, converting catch to slip bonds at high forces. An internal ligand antagonist that blocks pulling of the α₇-helix suppressed the intermediate-/long-lived states and eliminated catch bonds, revealing an internal catch bond between the αA and βA domains. These results elucidate an allosteric mechanism for the mechanochemistry of LFA-1/ICAM-1 binding.