Heterogenous glycosylation of ICAM-3 and lack of interaction with Mac-1 and p150,95

Intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3 have been identified as counter-receptors for the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). The other leukocyte integrins, Mac-1 and p150,95, also interact with ICAM-1. ICAM-1 and ICAM-3 are highly homologous, and an undefined ligand for Mac-1 is presnt on neutrophils where ICAM-3 is well expressed. In addition, glycosylation has been shown to affect the interaction of ICAM-1 with Mac-1. We therefore sought to characterize ICAM-3 heterogeneity and determine whether ICAM-3 was a ligand for either Mac-1 or p150,95. Despite extensive differences in N-linked glycosylation, ICAM-3 purified from lymphoid cells and from neutrophils supports adhesion of LFA-1-bearing cells equally well; however, neither supports adhesion of Mac-1 or p150,95-expressing chinese hamster ovary cell transfectants. Similarly, purified Mac-1 does not support adhesion of ICAM-2 or ICAM-3-expressing L cell transfectants. ICAM-3 on neutrophils does not participate in Mac-1-dependent homotypic aggregation. Thus, ICAM-3 is not a counter-receptor for either Mac-1 or p150,95.     

The β2 integrin Mac-1 but not p150,95 associates with FcγRIIA

In this study we have compared the ligand binding activity of the two closely related β2 integrins, Mac-1 and p150,95, which are expressed separately as receptors permanently transfected into K562 cells. Mac-1 has previously been shown to associate with FcγR, particularly FcγRIII, but K562 cells express only endogenous FcγRIIA. We have, therefore, taken advantage of this situation to examine a possible relationship between FcγRIIA with Mac-1 and p150,95 in the absence of other FcγR. The main finding is that anti-FcγRII mAb have a profound inhibitory effect on cell adhesion mediated by Mac-1, but not on the adhesion mediated by p150,95. Thus, in spite of the fact that Mac-1 and p150,95 bind to the same or at least a very similar selection of ligands, their association with other receptors on the cellular membrane, and therefore their mode of regulation may be different.     

Expression of the H52 epitope on the β2 subunit is dependent on its interaction with the α subunits of the leukocyte integrins LFA-1, Mac-1 and p150,95 and the presence of Ca2+

Integrin-mediated adhesion is a divalent cation-dependent process. Whether divalent cations directly participate in ligand binding or exert their effects indirectly by affecting the overall structure of the integrin heterodimers is not known. In this study we describe the epi tope of the mAb H52 which has been mapped to a predicted disulfide-bonded loop (C386 and C400) in the β2 integrin subunit. In the presence of Ca²⁺ and Mg²⁺ , the H52 epitope is expressed on the monomeric β2 subunit, the LFA-1 and Mac-1 heterodimers but not on p150,95, thus implying that this epitope is masked in p150,95. However, expression of the H52 epitope on Mac-1, but not on LFA-1, or the monomeric β2 subunit, is dependent on the presence of Ca²⁺ , thus suggesting that the chelation of Ca²⁺ causes a conformational change in Mac-1 which results in the loss of the epitope. These results suggest that expression of the H52 epitope on the β2 subunit is dependent on its interaction with the different α sub units. Since the epitope itself is not required for heterodimer formation nor for ligand binding, occupancy of a Ca²⁺ binding site(s) must therefore affect the α β subunit interactions, and thus the overall conformation of Mac-1.     

L1 adhesion molecule on human lymphocytes and monocytes: expression and involvement in binding to αvβ3 integrin

The L1 adhesion molecule is a member of the immunoglobulin (Ig) superfamily initially identified in the nervous system which contains six Ig-like domains. Besides the known L1-L1 homotypic interaction, L1 was recently shown to bind to very late antigen (VLA)-5 in the mouse and αvβ3 in the human. The sixth Ig domain is critical for this function. We now demonstrate that human CD4⁺ peripheral blood T lymphocytes, monocytes and B lymphocytes, but not CD8⁺ T lymphocytes, express L1. When compared to the expression of CD31, another ligand for αvβ3 on T lymphocytes, only a small proportion of cells were CD31⁺L1⁺ double positive. L1 was also detected on the surface of human monocytic and lymphoid tumor lines and was shown to have a molecular mass of ∼220 kDa, similar to the molecule present on neuroblastoma cells. The function of the sixth Ig domain of human L1 as an integrin ligand was also investigated. Using an RGD-containing peptide derived from the sixth Ig domain as well as a fusion protein of the sixth Ig domain of L1 and the Fc portion of human IgG1 (6.L1-Fc), we demonstrated the binding of human MED-B1 (αvβ3^hi, α5β1^lo) tumor cells and this binding was blocked by αv-specific mAb. In contrast, human Nalm-6 cells (αvβ3^lo, α5β1^hi) did not bind to the 6.L1-Fc fusion protein. MED-B1 cells could also be stained with the 6.L1-Fc fusion protein. Our results suggest that human L1 binds predominantly to αvβ3 and that its presence on leukocytes could be important for adhesion and migration.     

Tumor-driven matrix invasion by infiltrating lymphocytes: involvement of the α1 integrin I-domain

Here we show that tumor cells (TC) from renal cancers regulate the migratory properties of autologous tumor-infiltrating lymphocytes (TIL), enhancing their ability to invade the extracellular matrix. A similar effect is exerted by human recombinant macrophage chemotactic protein 1 (MCP-1) and IL-8, chemokines known to increase T lymphocyte migration both across vascular endothelium and subendothelial matrix. We found that TC freshly derived from renal cell carcinoma surgical specimens constitutively secrete both IL-8 and MCP-1 and that TIL express both specific receptors. TIL matrix invasion elicited by TC is inhibited by the addition of neutralizing antisera specific for IL-8 and MCP-1, demonstrating the direct relationship between chemokine release by TC and TIL invasion. Of note, TIL invasion of the extracellular matrix requires the α1 integrin, which acts through its I-domain that is up-regulated upon culture with MCP-1 and IL-8. Collectively, these findings suggest that TC may actively recruit TIL via the release of chemotactic factors that enhance an α1 integrin-mediated pathway of matrix invasion.     

KIM185, a monoclonal antibody to CD18 which induces a change in the conformation of CD18 and promotes both LFA-1- and CR3-dependent adhesion

Unactivated peripheral blood leukocytes show little tendency to bind to other cells or matrix components, whilst, in the presence of inflammatory mediators, adhesive interactions can rapidly increase. The Leu-CAM (β₂ integrin) family of adhesion molecules have been shown to mediate a variety of these induced adhesion events. Here we describe a monoclonal antibody against CD18, KIM185, which stimulates JY cell homotypic aggregation by a CD11a pathway as well as inducing the adherence of neutrophils to protein-coated plastic by a CD lib-dependent mechanism. The antibody recognizes an epitope distinct from the previously described KIM127 antibody and evidence is presented that the binding of KIM185 can cause a change in the conformation of the CD18 molecule.     

Human T lymphocytes bind to germinal centers of human tonsils via integrin α4/VCAM-1 and LFA-1/ICAM−1 and −2

Binding of T lymphocytes within the different compartments of the secondary lymphoid organs is crucial for the function of the cellular and the humoral immune response. It is still not known which adhesion molecules guide T cells to the distinct areas of the lymphoid microenvironment. In the current study an in situ adhesion assay was used to define the receptors for binding of T cells to human tonsils. The T cell lines Jurkat and MOLT-4 and normal, activated T cells were found to bind exclusively to germinal centers. Jurkat cells used the receptor pair integrin-α4 (VLA-4α)/VCAM-1, whereas activated MOLT-4 cells and normal T cells bound via both adhesion pathways, namely via integrin-α4/VCAM-1 and LFA-1/ICAM-1 and -2. It is suggested that these adhesion mechanisms are involved in the migration of T cells into the germinal centers of secondary lymphoid organs and that they influence the selection of B cells by apoptosis.     

Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin αM290β7 (αEβ7)

The integrin α_M290β7 on the surface of a T cell hybridoma, MTC-1, mediated adhesion of these cells to the mouse epithelial cell line CMT93. This interaction was critically dependent on the presence of divalent cations; Mn²⁺ strongly promoted adhesion, Ca²⁺ was ineffective and Mg²⁺ gave intermediate results. Antibodies to molecules on the surface of CMT93 cells were tested for inhibition of adhesion. One monoclonal antibody (mAb) against E-cadherin, ECCD-2, was found to have significant inhibitory activity. Other mAb to E-cadherin and antibodies to other molecules had no effect. To show that inhibition by ECCD-2 was specific for adhesion mediated by α_M290β7, MTC-1 cells were induced to adhere to CMT93 via the LFA-1/ICAM-1 pathway. For this purpose, the epithelial cells were treated with interferon-γ and tumor necrosis factor-α to induce ICAM-1 expression and, in addition, α_M290β7 on MTC-1 cells was down-regulated by culturing the cells in the absence of transforming growth factor β. Under these circumstances adhesion of MTC-1 cells to CMT93 was inhibited by an antibody to LFA-1 but not by ECCD-2. Transfection of mouse L cells with cDNA for mouse E-cadherin enabled MTC-1 cells to adhere to them through the α_M290β7 integrin; this interaction was inhibited both by ECCD-2 and by blocking antibody against the integrin. These data strongly suggest that E-cadherin is a principal ligand for α_M290β7.     

β1 and β4 integrin expression in methacarn and formalin-fixed material from in situ ductal carcinoma of the breast

The integrins are αβ heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Previous distribution studies on integrin expression have been limited by the requirement for cryostat sectioned tissues, and consequent poor resolution. We have examined 40 examples of ductal carcinoma in situ (DCIS) for the expression of both β1 and β4 integrin chains. These showed strong polarized membrane staining of residual myoepithelial cells (correlating with expression of smooth muscle specific actin) and of the basement membrane region with β1 and β4 antibodies respectively. In 12 out of 40 cases, the DCIS was negative for the β1 chain and a variable pattern of reactivity was seen in the remaining cases. The β4 chain was detected focally and weakly in the tumour cells of 7/40 DCIS and strongly in one; all of these cases were also positive for the β1 chain. Of the 22 cases where co-existent invasion was present, the infiltrating component showed either a similar degree or a diminution of the extent of immunostaining when compared with the in situ component; only one showed enhanced staining (β1 only). This study demonstrates that two of the main β chains, β1 and β4, can be effectively demonstrated on methacarn and cold (4°C) formalin-fixed tissues by avidin-biotin indirect immunoperoxidase staining and that the results are similar to those achieved using frozen tissue.     

Role of the lectin domain of Mac-1/CR3 (CD11b/CD18) in regulating intercellular adhesion

Leukocytediapedesis requires that Mac-1/CR3-dependent adhesion be regulated so that cells can move from one attachment site to another. The high affinity adhesion state of Mac-1/CR3 is generated when it forms alectin-dependent complex with the receptor for urokinase plasminogen activator (uPAR; CD87). The extensively glycosylated uPAR binds to the same C-terminal lectin domain of CD11b that had previously been shown to prime Mac-1/CR3 for cytotoxic degranulation in response to β-glucan uPAR and β-glucan compete for a lectin site that is near to the CBRM1/23 epitope (residues 943–1047) at the C-terminus of CD11b, and thus the lectin domain is critical to both the adhesion and cytotoxic functions of Mac-1/CR3. Adhesion is reversed when the uPA enzyme is captured by its receptor (uPAR), causing uPAR to bind to CD11b at a second site (residues 424–440) that is in between the N-terminal I-domain and the divalent cation binding region.     

Recombinant integrin CD11b A-domain blocks polymorphonuclear cells recruitment and protects against skeletal muscle inflammatory injury in the rat

The beta2 integrin CD11b/CD18 (CR3) is a major adhesion receptor of neutrophils, normally utilized to fend off infections. This receptor contributes, however, to multiple forms of non-infectious inflammatory injury when dysregulated as shown in gene knock-outs and through the use of blocking monoclonal antibodies. The major ligand recognition site of CR3 has been mapped to the A-domain in the CD11b subunit (CD11bA). The recombinant form of this domain exhibits a ligand binding profile similar to that of the holoreceptor. To assess the potential anti-inflammatory activity of CD11bA as a competitive antagonist of CR3 in vivo, we assessed its effects on a developed animal model of traumatic skeletal muscle injury in the rat. Recombinant soluble rat CD11bA-domain fused to glutathione-S-transferase (GST) was administered intravenously in a single dose at 1 mg/kg to nine groups of Wistar rats, five in each group, 30 min before inducing traumatic skeletal muscle injury. Control animals received either a function-blocking anti-CD11b/CD18 monoclonal antibody (1 mg/kg), non-functional mutant forms of the CD11bA (D140GS/AGA, T209/A, D242/A), recombinant GST or buffer alone. In control animals, the wounded muscle showed oedema, erythrocyte extravasation and myonecrosis both within and outside the immediate wounded area (5-10 mm zone) and influx of neutrophils was detected 30 min post-wound, followed by a second wave 3 hr later. Wild-type CD11bA- or anti-CD11b monoclonal antibody (mAb)-treated rats showed a comparable and significant decrease in the number of infiltrating PMN (78 + 4%, n = 70 and 86 +/- 2%, n = 50, respectively) and preservation of the muscular fibres outside the immediate zone of necrosis (75 + 4%, n = 70, 84 +/- 1%, n = 50, respectively), compared to controls. These data demonstrate that CD11bA can be an effective tissue-preserving agent in acute inflammatory muscular injury.     

Role of polymorphonuclear neutrophil leukocytes and their integrin CD11a (LFA-1) in the pathogenesis of severe murine malaria.

Infection of CBA mice with Plasmodium berghei ANKA results in severe malaria, which is characterized by mortality 6 to 10 days after infection and is associated with alterations of the brain microcirculation. These alterations consist of (i) intravascular sequestration of monocytes, (ii) an increase in vascular permeability as documented by Evans blue diffusion, and (iii) microhemorrhages. This syndrome may be due to an increase of production of tumor necrosis factor alpha which upregulates the endothelial expression of ICAM-1 and thus leads to adhesion of CD11a/CD18 (LFA-1)-bearing cells. During severe malaria, we found an important sequestration of the CD11a-bearing polymorphonuclear neutrophil leukocytes (PMN) in the lung but not in the brain. Treatment with a monoclonal antibody (MAb) against PMN, which induces profound neutropenia, prevented mortality and Evans blue diffusion in the brain and the lung, while it unexpectedly increased the occurrence of microhemorrhages. The anti-PMN MAb abolished PMN sequestration in the lung and also partially decreased monocyte sequestration in the brain and the lung. Treatment with an anti-CD11a MAb also prevented mortality, Evans blue diffusion, and PMN and monocyte sequestration. This study shows that PMN contribute to the mortality and the microvascular lesions resulting from severe malaria. This may be due to their CD11a-dependent sequestration in the lung and also to their indirect influence on vascular permeability and the sequestration of monocytes.     

Characterization of four CD18 mutants in leucocyte adhesion deficient (LAD) patients with differential capacities to support expression and function of the CD11/CD18 integrins LFA-1, Mac-1 and p150,95.

Leucocyte adhesion deficiency (LAD) is a hereditary disorder caused by mutations in the CD18 (beta2 integrin) gene. Four missense mutations have been identified in three patients. CD18(A270V) supports, at a diminished level, CD11b/CD18 (Mac-1, alphaMbeta2 integrin) and CD11c/CD18 (p150,95, alphaXbeta2 integrin) expression and function but not CD11a/CD18 (LFA-1, alphaLbeta2 integrin) expression. Conversely, CD18(A341P) supports a limited level of expression and function of CD11a/CD18, but not of the other two CD11/CD18 antigens. CD18(C590R) and CD18(R593C) show a decreasing capacity to associate with the CD11a, CD11c and CD11b subunits. Transfectants expressing the CD11a/CD18 with the C590R and R593C mutations are more adhesive than transfectants expressing wild-type LFA-1, and express the reporter epitope of the monoclonal antibody 24 constitutively. Thus, the four mutations affect CD18 differently in its capacities to support CD11/CD18 expression and adhesion. These results not only provide a biochemical account for the clinical diversity of patients with leucocyte adhesion deficiency, but also offer novel insights into the structural basis of interaction between the alpha and beta subunits, which is an integral component in our understanding of integrin-mediated adhesion and its regulation.     

Distinct structural and functional epitopes of the αEβ7 integrin

Intestinal intraepithelial lymphocytes (iIEL) are predominantly CD3⁺, CD8⁺ T lymphocytes located above or adjacent to the mucosal basement membrane. Although they are positioned to interact with intercellular luminal antigen or with enterocytes, the function of iIEL remains unknown. Most (> 85%) of the iIEL express the α^Eβ₇ integrin which appears to be involved in the adhesion of lymphocytes to epithelial cells. We report the characterization of three monoclonal antibodies (mAb) termed αE7-1, αE7-2, and αE7-3, that react with the α^Eβ₇ integrin recognized by the previously described mAb HML-1 as demonstrated by identical sodium dodecyl sulfate – polyacrylamide gel electrophoresis mobility and charge. Flow cytometric analysis of antibody cross-blocking indicated that these mAb recognize distinct epitopes of α^Eβ₇. While all of the mAb were capable of blocking the adhesion of cultured iIEL to a breast epithelial cell line, only HML-1 and αE7-1 (which recognize an identical or closely related epitope) were co-stimulatory with suboptimal concentrations of anti-CD3 mAb in inducing proliferation of cultured iIEL. Thus, these mAb appear to recognize functionally distinct epitopes of α^Eβ₇ and will be useful to study relationships between the structure and function of this integrin.     

Signaling through the LFA-1 leucocyte integrin actively regulates intercellular adhesion and tumor necrosis factor-α production in natural killer cells

The LFA-1 leucocyte integrin is known to participate in natural killer (NK) cytolytic activity, mediating effector target interactions. The possibility that LFA-1 may also play an active regulatory role in NK cells has been explored. To this end, we have employed a monoclonal antibody (HP1N) raised against recombinant interleukin-2 (rIL-2)-activated NK cells, which recognizes the α chain of the LFA-1 heterodimer (CD11a). In contrast to other anti-CD11a mAb the HP1N and its F(ab)₂ fragment did not affect NK cell-mediated cytotoxicity and triggered a strong homotypic adhesion of NK cells and other LFA-1⁺ cells. Cellular aggregation was inhibited by anti-CD18 mAb, anti-ICAM-1 mAb, and other anti-CD11a mAb. Remarkably, the HP1N mAb was also shown to induce tumor necrosis factor-α (TNF-α) production from NK cells upon costimulation with anti-CD16 mAb. Such an effect appeared to be independent from homotypic adhesion since it took place in Mg²⁺-free medium, where NK cell aggregation was inhibited. Moreover, incubation with the HP1N mAb triggered a Ca²⁺ influx into the cytosol; this effect was clearly observed upon cross-linking of cell bound HP1N and was also substantiated with other anti LFA-1 (CD11a and CD18) mAb. Taken together these results indicate that the LFA-1 molecule is capable of transducing signals in NK cells, which regulate the intercellular interaction with its ligand, and enhance the activation via Fey receptor type III.     

A mixed population of immature and mature leucocytes in umbilical cord blood results in a reduced expression and function of CR3 (CD11b/CD18)

Neonatal neutrophils express less membrane and cytoplasmic CR3 (iC3b-receptor, Mac-1, α_Mβ₂-integrin) than do adult neutrophils, and it has been suggested that this renders neonatal neutrophils deficient in diapedesis and bactericidal activity. The reason(s) for this deficiency are unknown. In this study, CR3 expression and the CR3-dependent respiratory burst activity of individual neonatal neutrophils are quantified in comparison with adult leucocytes using flow cytometry. Monocytes and neutrophils are defined as CD14^highCD15^low and CD14^lowCD15^high, respectively. Although neonatal neutrophils bore less CR3 on average than did adult neutrophils, neonatal neutrophils were more heterogeneous and many neonatal neutrophils expressed adult levels of CR3. Because of higher neutrophil concentrations in cord versus adult blood, the calculated number of neutrophils in cord blood expressing high amounts of CR3 was equivalent to that of adult blood. Similar findings were made with monocytes. The size of the CR3-dependent respiratory burst stimulated by particulate β-glucan correlated directly with the expression of CR3 by individual neutrophils. With neonatal and adult neutrophils having comparable CR3 densities, the respiratory burst activities were equivalent. Wright–Giemsa differential staining of the subset of neonatal neutrophils with low CR3 levels isolated by fluorescence-activated cell sorting showed a higher proportion of immature cells than the sorted population expressing high CR3 levels. Therefore, higher proportions of immature cells in cord blood probably explain previous reports of deficient CR3 expression and function. The typical neutrophilia of cord blood may compensate for this apparent deficiency by providing adult concentrations of mature neutrophils.     

Contribution of CD11a/CD18, CD11b/CD18, ICAM-1 (CD54) and −2 (CD102) to human monocyte migration through endothelium and connective tissue fibroblast barriers

Recently we reported that monocyte migration through a barrier of human synovial fibroblasts (HSF) is mediated by the CD11/CD18 (β₂) integrins, and the β₁ integrins VLA-4 and VLA-5 on monocytes. Here we investigated in parallel the role of β₂ integrin family members, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) on monocytes, and the immunoglobulin supergene family members, ICAM-1 and ICAM-2 on HSF and on human umbilical vein endothelial cells (HUVEC), in monocyte migration through HSF and HUVEC monolayers. Using function blocking monoclonal antibodies (mAb), when both VLA-4 and VLA-5 on monocytes were blocked, treatment of monocytes with mAb to both LFA-1 and to Mac-1 completely inhibited monocyte migration across HSF barriers, although blocking either of these β₂ integrins alone had no effect on migration, even when VLA-4 and VLA-5 were blocked. This indicates that optimal β₂ integrin-dependent monocyte migration in synovial connective tissue may be mediated by either LFA-1 or Mac-1. Both ICAM-1 and ICAM-2 were constitutively expressed on HSF and on HUVEC, although ICAM-2 was only minimally expressed on HSF. Based on results of mAb blockade, ICAM-1 appeared to be the major ligand for LFA-1-dependent migration through the HSF. In contrast, both ICAM-1 and ICAM-2 mediated LFA-1-dependent monocyte migration through HUVEC. However, neither ICAM-1 nor ICAM-2 was required for Mac-1-dependent monocyte migration through either cell barrier, indicating that Mac-1 can utilize ligands distinct from ICAM-1 and ICAM-2 on HSF and on HUVEC during monocyte transmigration.