Monocytic cell adhesion to intact and plasmin-modified fibrinogen: possible involvement of Mac-1 (CD11b/CD18) and ICAM-1 (CD54)

beta(2)-integrin Mac-1 and immunoglobulin-like ICAM-1 adhesion molecules are expressed by monocytes and both known to bind fibrinogen and its degradation products. Here, we investigated whether fibrinogen cleavage with plasmin modulates the adherence of monocytic cells and what types of adhesion molecules are involved. Using several cell types, characterized by different patterns of Mac-1 and ICAM-1 expression, and monoclonal antibodies against beta(2)-integrins and ICAM-1 we demonstrate, that fibrinogen cleavage evokes gradual decrease in beta(2)-integrin-dependent cell adhesion. Furthermore, generation of the early degradation products, fragments X and Y, by minimum cleavage of fibrinogen stimulates cell adhesion, mediated by ICAM-1.     

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18)

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac- 1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity- purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1- dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.     

A unique recognition site mediates the interaction of fibrinogen with the leukocyte integrin Mac-1 (CD11b/CD18)

Mac-1 (CD11b/CD18), a leukocyte-restricted integrin receptor, mediates neutrophil/monocyte adhesion to vascular endothelium and phagocytosis of complement-opsonized particles. Recent studies have shown that Mac-1 also functions as a receptor for fibrinogen in a reaction linked to fibrin deposition on the monocyte surface. In this study, we have used extended proteolytic digestion of fibrinogen to identify the region of this molecule that interacts with Mac-1. We found that an Mr approximately 30,000 plasmic fragment D of fibrinogen (D30) produced dose-dependent inhibition (IC50 = 1.6 microM) of the interaction of intact 125I-fibrinogen with stimulated neutrophils and monocytes. 125I-D30 bound saturably to these cells with specific association of 136,200 +/- 15,000 molecules/cell in a reaction inhibited by OKM1 and M1/70, monoclonal antibodies specific for the alpha subunit of Mac-1. Direct microsequence analysis and an epitope-mapped monoclonal antibody showed that D30 lacks the COOH-terminal dodecapeptide of the gamma chain as well as the Arg-Gly-Asp sequences in the A alpha chain. We conclude that fibrinogen interacts with the leukocyte integrin Mac-1 through a novel recognition site that is not shared with other known integrins that function as fibrinogen receptors.     

The cell surface glycoprotein Mac-1 (CD11b/CD18) mediates neutrophil adhesion and modulates degranulation independently of its quantitative cell surface expression

It has previously been shown that during degranulation Mac-1 (CD11b/CD18)--a glycoprotein that plays a central role in neutrophil adhesion-is up-regulated on PMN surfaces. It has been assumed that this quantitative change in adhesion Ag expression on the cell surface would in turn lead to increased cellular adhesiveness. In contrast, we found that at an incubation temperature of 16 degrees C, stimulated neutrophil adhesion to plastic tissue culture dishes in the presence of FMLP (2.5 x 10(-6) M), TNF (10 ng/ml), or PAF (1 x 10(-4) M) occurred without cellular degranulation or Mac-1 surface up-regulation as measured cytofluorometrically. As shown by functional inhibition studies employing monoclonal antibodies 60.3 (anti-CD18) and 60.1 (anti-CD11b), adhesion at 16 degrees C, where no CD11b/CD18 up-regulation was seen, is mediated by CD11b/CD18 just as it is at 37 degrees C, where degranulation and CD11b/CD18 up-regulation could be demonstrated. The physiologic importance of these findings was underscored by experiments done on endothelial monolayers, which showed that PMN association with endothelial cells is absolutely independent from the quantitative up-regulation of Mac-1 on PMN surfaces. When neutrophils were stimulated at 37 degrees C by endotoxin, an agent that does not induce aggregation (a form of intercellular adhesion), Mac-1 surface expression increased only after cells had become adherent, whereas cells held in suspension to prevent cell-substrate adhesion neither degranulated nor up-regulated their Mac-1 surface expression. Thus, not only is adherence independent of degranulation and Mac-1 cell surface up-regulation, but both degranulation and Mac-1 surface up-regulation appear to depend on the process of adhesion. Correspondingly, incubation of neutrophils with antibodies 60.1 and 60.3 inhibited not only adhesion of cells stimulated with FMLP at 37 degrees C but degranulation as well. These results indicate that Mac-1 influences degranulation as well as it controls adhesion not by its mere quantity on the cell surface, but rather by an yet undefined molecular modulation.     

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.     

Mac-1 (CD11b/CD18) mediates adherence-dependent hydrogen peroxide production by human and canine neutrophils

Human neutrophils exposed to protein-coated polystyrene or cultured endothelial monolayers produce large quantities of H2O2 in response to soluble stimuli that elicit little or no secretion of reactive oxygen species from cells in suspension. To characterize the mechanisms involved in this adherence-dependent respiratory burst, we have investigated the possible role of one integrin known to participate in the adhesion of neutrophils to endothelial cells, CD11b/CD18 (Mac-1). H2O2 production was examined with chemotactic factor-stimulated human and canine neutrophils exposed to protein-coated surfaces and cultured human and canine endothelial cells. The two protein-coated surfaces used were type I collagen-coated glass or plastic, a surface to which neither human nor canine neutrophils adhered, and keyhole limpet hemocyanin (KLH)-coated glass or plastic, a surface to which human and canine neutrophils adhered only after chemotactic stimulation. FMLP-stimulated human neutrophils and platelet activating factor-stimulated canine neutrophils failed to produce detectable H2O2 when in contact with type I collagen, but secreted large amounts of H2O2 when adherent to KLH or endothelial cell monolayers. FMLP-stimulated neutrophils from patients with CD18-deficiency failed to adhere to any of these surfaces and failed to produce H2O2 under these conditions. mAb reactive with CD18 and CD11b were equally effective in markedly inhibiting the adhesion of normal human neutrophils to these surfaces and markedly inhibited the production of H2O2. A mAb reactive with CD18 blocked adhesion of stimulated canine neutrophils, and mAb directed against both CD18 and CD11b blocked H2O2 production by canine neutrophils on KLH and endothelium. A nonbinding mAb and a mAb reactive with CD11a did not inhibit H2O2 production of human cells on KLH or endothelial monolayers, and nonbinding and binding control mAb did not inhibit H2O2 production by canine neutrophils. These results indicate that Mac-1 (CD11b/CD18) can mediate adhesion-dependent H2O2 production by human and canine neutrophils exposed to chemotactic factors.     

Modulation of Mac-1 (CD11b/CD18)-mediated adhesion by the leukocyte-specific protein 1 is key to its role in neutrophil polarization and chemotaxis

Leukocyte-specific protein 1 (LSP1) is an intracellular filamentous-actin binding protein which modulates cell motility. The cellular process in which LSP1 functions to regulate motility is not yet identified. In this study, we show that LSP1 negatively regulates fMLP-induced polarization and chemotaxis of neutrophils through its function on adhesion via specific integrins. Using LSP1-deficient (Lsp1(-/-)) mice, we show increased neutrophil migration into mouse knee joints during zymosan-induced acute inflammation, an inflammatory model in which the number of resident synoviocytes are not affected by LSP1-deficiency. In vitro chemotaxis experiments performed by time-lapse videomicroscopy showed that purified Lsp1(-/-) bone-marrow neutrophils exhibit an increased migration rate toward a gradient of fMLP as compared with wild-type neutrophils. This difference was observed when cells migrated on fibrinogen, but not fibronectin, suggesting a role for LSP1 in modulating neutrophil adhesion by specific integrins. LSP1 is also a negative regulator of fMLP-induced adhesion to fibrinogen or ICAM-1, but not to ICAM-2, VCAM-1, or fibronectin. These results suggest that LSP1 regulates the function of Mac-1 (CD11b/CD18), which binds only to fibrinogen and ICAM-1 among the substrates we tested. fMLP-induced filamentous actin polarization is also increased in the absence of LSP1 when cells were layered on fibrinogen, but not on fibronectin. Our findings suggest that the increased neutrophil recruitment in Lsp1(-/-) mice during acute inflammation derives from the negative regulatory role of LSP1 on neutrophil adhesion, polarization, and migration via specific integrins, such as Mac-1, which mediate neutrophil responses to chemotactic stimuli.     

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.     

Alpha-tocopherol protects against monocyte Mac-1 (CD11b/CD18) expression and Mac-1-dependent adhesion to endothelial cells induced by oxidized low-density lipoprotein

Alpha-tocopherol supplementation is reported to protect against cardiovascular disease and to influence cells involved in atherogenesis, such as monocytes. Interactions between monocytes and vascular endothelial cells occur early in atherogenesis, and adhesion is mediated by integrins. We evaluated the effects of alpha-tocopherol on expression of Mac-1 (CD11b/CD18) by monocytes after stimulation with oxidized low-density lipoprotein (LDL), which is implicated as a potent chemotactic agent in atherogenesis. Incubation of whole blood with oxidized LDL (100 microg/ml) increased Mac-1 expression on monocytes, and preincubation with alpha-tocopherol reduced this upregulation in a concentration dependent manner. In another experiment, whole blood was obtained from healthy adult volunteers after 10 days of alpha-tocopherol administration (600 mg/day) and was incubated with oxidized LDL (100 microg/ml). There was a decrease in the upregulation of Mac-1 compared with that measured before administration. Adherence of oxidized LDL-stimulated monocytes to human umbilical vein endothelial cells was reduced by pretreatment with alpha-tocopherol, and was also inhibited by an anti-CD18 monoclonal antibody. Experiments with protein kinase C inhibitors suggested that reduction of Mac-1 upregulation by alpha-tocopherol was secondary to a decrease of protein kinase C activity. In conclusion, alpha-tocopherol suppressed the upregulation of Mac-1 expression on monocytes by oxidized LDL.     

A monoclonal antibody reacting with distinct adhesion molecules defines a transition in the functional state of the receptor CD11b/CD18 (Mac-1)

CD11b/CD18 (Mac-1) is a member of the leukocyte integrin family, a group of receptors that have been implicated in various effector functions and cellular collaboration in the immune response. It has been shown previously that CD11b/CD18 on cells of monocyte and myeloid lineage appears to undergo rapid activation and acquire new functional receptor specificities after exposure to selected agonists such as adenosine diphosphate (ADP). We now show that ADP induces a reconformation of the CD11b/CD18 receptor with exposure of new epitopes characteristics of this activated state. By direct binding studies, flow cytometry, and immunoprecipitation experiments, it has been found that the mAb 7E3 reacts with CD11b/CD18 only after ADP-stimulation of the cell suspension. The activated state of CD11b/CD18 induced by ADP and recognized by 7E3 can also be recapitulated by agonists inducing transients in cytosolic Ca2+ such as the chemoattractant FMLP. Moreover, this process of receptor activation does not involve quantitative mobilization of the subcellular storage pool of CD11b/CD18 to the plasma membrane. Because 7E3 also recognizes a qualitative, ADP-mediated activated state of the platelet adhesion receptor GP IIb/IIIa, it is suggested that transients in cytosolic Ca2+ might represent early secondary events for a general pathway of rapid activation of integrin receptors and, as such, represent important signals for cellular interactions in the immune response.     

Sulfite induces adherence of polymorphonuclear neutrophils to immobilized fibrinogen through activation of Mac-1 beta2-integrin (CD11b/CD18)

Sulfite is a major air pollutant which can cause respiratory tract inflammation characterized by an influx of polymorphonuclear neutrophils (PMN). We have previously shown that human PMN can produce sulfite either spontaneously or in response to stimulation with lipopolysaccharide. We now demonstrate that sulfite activates PMN to adhere to immobilized fibrinogen via the beta2-integrin Mac-1 (CD11b/CD18). Mac-1 expression is not altered by treatment with this agent. Although unaffected by pertussis toxin, sulfite-triggered PMN adhesion was abrogated by pretreating cells with the membrane-impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a modifier of thiol groups on the cell surface. These results suggest that sulfite-induced PMN adhesion is dependent on a modification of thiols at the cell surface. Given its potent antioxidant and antimicrobial activities, sulfite may act as an endogenous mediator in host defense and/or inflammation.     

Sulfhydryl reducing agents promote neutrophil adherence without increasing surface expression of CD11b/CD18 (Mac-1, Mo1)

The disulfide reducing agents dithioerythreitol and dithiothreitol, but not oxidized dithiothreitol, induced polymorphonuclear neutrophils to adhere to endothelial cells or to plastic. Adherence was inhibited by monoclonal antibodies 60.1 and 60.3, which are directed to functional epitopes on the CD11b and CD18 polypeptides of the neutrophil membrane adhesion complex (Mac-1, Mo1). The increased adherence induced by the sulfhydryl reducing agents was not accompanied by increased expression of CD11b/CD18. These studies demonstrate that a qualitative alteration in CD11b/CD18 is sufficient to promote neutrophil adherence.     

Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation.

Both the integrins LFA-1 and Mac-1 bind to ICAM-1, an immunoglobulin superfamily member. Previously, we localized the binding sites of LFA-1 and the major group of human rhinoviruses to the first NH2-terminal immunoglobulin-like domain of ICAM-1. Here, we show that the binding site on ICAM-1 for Mac-1 is unexpectedly distinct from that for LFA-1 and maps to the third NH2-terminal immunoglobulin-like domain. These findings provide a function for the tandem duplication of immunoglobulin-like domains in ICAM-1 and have implications for other immunoglobulin superfamily members. Mutations at two sites in the third domain that destroy consensus sequences for N-linked glycosylation enhance binding to purified Mac-1. Agents that interfere with carbohydrate processing provide evidence that the size of the N-linked oligosaccharide side chains on ICAM-1 affects binding to Mac-1 but not to LFA-1. Thus, we suggest that the extent of glycosylation on ICAM-1 may regulate adhesion to LFA-1 or Mac-1 in vivo.     

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.     

Leishmania promastigotes require opsonic complement to bind to the human leukocyte integrin Mac-1 (CD11b/CD18)

Previous reports have suggested that Leishmania spp. interact with macrophages by binding to Mac-1 (CD1 1b/CD18), a member of the leukocyte integrin family. To better define this interaction, we tested the ability of leishmania promastigotes to bind to purified leukocyte integrins and to cloned integrins expressed in COS cells. We show that leishmania promastigotes bind to cellular or purified Mac-1 but not lymphocyte function-associated antigen-1 in a specific, dose-dependent manner that requires the presence of serum. Binding is inhibited with specific monoclonal antibodies to Mac-1. In the absence of complement opsonization, three different species of leishmania tested fail to bind directly to any of the three leukocyte integrins. We show that binding to Mac-1 requires the third component of complement (C3). Organisms incubated in heat-inactivated serum or serum that has been immunologically depleted of C3 fail to bind to Mac-1. Because the addition of purified C3 to C3-depleted serum restores leishmania binding to Mac-1, we suggest that parasites gain entry into macrophages by fixing complement and subverting a well-characterized adhesive interaction in the immune system between Mac-1 and iC3b.     

Participation of beta2-integrins CD11b/CD18 and CD11c/CD18 in adhesion and migration of cells on fibrinogen

The role of beta2-integrins CD11b/CD18 and CD 11c/CD 18 in adhesion and migration of leukocytes on fibrinogen was studied. The monoclonal antibodies against CD11b inhibited the spontaneous adhesion of monocytic THP-1 cells on fibrinogen, whereas antibodies to CD11c more effectively inhibited the adhesion stimulated by chemokine MCP-1. By the RNA-interference method the clones of THP-1 with reduced expression of CD11b and general beta2-subunit CD18 were obtained. MCP-I stimulated the adhesion to fibrinogen of THP-1 cells of wild-type and mutant cells with reduced expression of CD11b (THP-1-CD11b-low), but not of cells with low expression of CD18 (THP-1-CD18-low). THP-1-CD18-low cells were also characterized by the impaired chemotaxis in presence of MCP-1. The data obtained suggest that spontaneous cell adhesion to fibrinogen is mediated to a greater extent by CD11b/CD18 integrins, while chemokine-stimulated adhesion and migration is mostly dependent on CD11c/CD18 molecules.     

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.     

Mac-1 (CD11b/CD18) as accessory molecule for Fc alpha R (CD89) binding of IgA

IgA, the principal ligand for FcalphaRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcalphaRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the beta(2) integrin Mac-1 (Mac-1(-/-)) were unable to bind SIgA, despite expression of FcalphaRI. Consistent with this, serum IgA stimulated Mac-1(-/-) PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1(-/-) PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcalphaRI interactions.     

Effect of integrin beta 2 subunit truncations on LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) assembly, surface expression, and function

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the beta2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized beta2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated beta2 variants, we showed that the subregion Q23-D300 of the beta2 subunit is sufficient to combine with the alphaL and alphaM subunits intracellularly. However, only the beta2 variants terminating after Q444 promote cell surface expression of LFA-1 and Mac-1. Thus, the major cysteine-rich region and the three highly conserved cysteine residues at positions 445, 447, and 449 of the beta2 subunit are not required for LFA-1 and Mac-1 surface expression. The surface-expressed LFA-1 variants are constitutively active with respect to ICAM-1 adhesion and these variants express the activation reporter epitope of the mAb 24. In contrast, surface-expressed Mac-1, both the wild type and variants, require 0. 5 mM MnCl2 for adhesion to denatured BSA. These results suggest that the role of the beta2 subunit in LFA-1- and Mac-1-mediated adhesion may be different.     

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.