Binding of LFA-1 (CD11a) to Intercellular Adhesion Molecule 3 (ICAM-3; CD50) and ICAM-2 (CD102) Triggers Transmigration of Human Immunodeficiency Virus Type 1-Infected Monocytes through Mucosal Epithelial Cells

Transmigration of human immunodeficiency virus (HIV)-infected mononuclear cells through the genital mucosa is one of the possible mechanisms of sexual transmission of HIV. Here, we investigated the transmigration of cell-associated R5-tropic HIV type 1 (HIV-1) through a tight monolayer of human epithelial cells in vitro. We show that this process is dependent on an initial interaction between alphaLbeta2 integrin CD11a/CD18 on infected monocytic cells and intercellular adhesion molecule 2 (ICAM-2; CD102) and ICAM-3 (CD50) on the apical membrane of epithelial cells. The CD50 and CD102 ligands were overexpressed on epithelial cells when the cells were activated by proinflammatory cytokines in the cellular microenvironment. An accumulation of proviral DNA was found in the transmigrated cells, clearly reflecting the preferential transepithelial migration of HIV-1-infected cells under proinflammatory conditions. Our observations provide new insights supporting the hypothesis that HIV-infected mononuclear cells contained in genital secretions from infected individuals may cross the epithelial genital mucosa of an exposed receptive sexual partner, particularly under inflammatory conditions of damaged genital tissue. Understanding the fundamental aspects of the initial HIV entry process during sexual transmission remains a critical step for preventing human infection and developing further vaccinal strategies and virucidal agents.     

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.     

T-cell-mediated immunity to lymphocytic choriomeningitis virus in beta2-integrin (CD18)- and ICAM-1 (CD54)-deficient mice.

The T-cell response to lymphocytic choriomeningitis virus was studied in mice with deficient expression of beta2-integrins or ICAM-1. In such mice, the generation of virus-specific cytotoxic T lymphocytes was only slightly impaired and bystander activation was as extensive as that observed in wild-type mice. T-cell-mediated inflammation, assessed as primary footpad swelling and susceptibility to intracerebral infection, was slightly compromised only in beta2-integrin-deficient mice. However, adoptive immunization of mutant mice soon after local infection did reveal a reduced capacity to support the inflammatory reaction, indicating that under conditions of more limited immune activation both molecules do play a role in formation of the inflammatory exudate. Finally, virus control was found to be somewhat impaired in both mutant strains. In conclusion, our results indicate that although LFA-1-ICAM-1 interaction is important for certain aspects of the T-cell-mediated response to viruses, T-cell activation is surprisingly intact in these mutant mice, indicating extensive functional redundancy within cell interaction molecules.     

Fibrinogen interactions with ICAM-1 (CD54) regulate endothelial cell survival.

We tested hypothesis that the interaction of fibrinogen (Fg) with intercellular adhesion molecule 1 (ICAM-1) mediates cellular adhesion and cell proliferation. Our results demonstrate that Fg : ICAM-1 ligation mediates endothelial cell survival and has an anti-apoptotic effect via activation of the MAP kinase pathway. Fg : ICAM-1 ligation in endothelial cells treated with tumor necrosis factor (TNF)alpha resulted in the hyperphosphorylation of extracellular signal-regulated kinase (ERK)-1/2 (eightfold to 10-fold) at 5-30 min. The specificity of ERK-1/2 phosphorylation was verified using the recognition peptides Fg-gamma-(117-133) and ICAM-1(8-22). ERK-1/2 hyperphosphorylation was dependent on intact cytoskeleton, as treatment with cytochalasin B and nocodazole blocked this activity. The attachment of TNFalpha-treated endothelial cells to fibrinogen or Fg-gamma-(117-133) resulted in cell survival, as assessed by an annexin V binding assay. ICAM-1(8-22) blocked the survival process. The MEK-1 inhibitor PD 98059 blocked ERK-1/2 phosphorylation, and treatment of endothelial cells with PD 98059 resulted in apoptosis even upon Fg : ICAM-1 ligation. Cells transfected with dominant-negative ERK-1/2 underwent apoptosis upon Fg : ICAM-1 ligation. Cell survival factor A1 was specifically upregulated upon adhesion of TNFalpha-stimulated endothelial cells to Fg. A1 expression was blocked by ICAM-1(8-22) and PD 98059. The Fg : ICAM-1 endothelial cell survival pathway appears to be mediated via the activation and upregulation of ERK-1/2 and A1.     

Role of ICAM-1 (CD54) in the development of murine cerebral malaria

In susceptible mouse strains, infection of mice with Plasmodium berghei ANKA (PbA) results in a lethal complication, cerebral malaria. Cerebral malaria is due to the immune response induced by the parasite, which results in an increased production of TNF, known to increase the expression of adhesion molecules on the endothelia. To investigate the role of the adhesion molecule ICAM-1 (CD54), we infected wild-type (+/+) and ICAM-1-deficient (-/-) mice with PbA. While +/+ mice died 6-8 days after infection, -/- mice survived > 15 days. Parasitaemia was similar in +/+ and -/- mice. Serum TNF concentration was increased by the infection and was significantly higher in infected +/+ than in -/- mice. However, TNF mRNA levels in spleen, lungs, and brain were elevated in both infected +/+ and -/- mice. For IFN-gamma, serum levels were similar in both groups. A breakdown of the blood-brain barrier was evident in infected +/+ mice only. Interestingly, thrombocytopenia was profound in infected +/+, but practically absent in -/- mice. Moreover, macrophage sequestration was evident in brain venules and lung capillaries of +/+ mice and was significantly less important in the alveolar capillaries of infected -/- mice. In contrast, neutrophil sequestration in the lung was similar in both +/+ and -/- mice. Sequestration of parasitized red blood cells was significantly greater in the alveolar capillaries from +/+ than -/- mice. These results indicate that while the immune response is similar in both +/+ and ICAM-1(-/-) mice, the absence of mortality in ICAM(-/-) mice correlates with a decrease of macrophage and parasitized RBC trapping and a less severe thrombocytopenia.     

ICAM-3 (CD50) is expressed by human mast cells: induction of homotypic mast cell aggregation via ICAM-3

Intercellular adhesion molecule-3 (ICAM-3, CD50), an adhesion receptor of the immunoglobulin superfamily, is suggested to play a key role in adhesive cellular interactions during the initial phase of an immune response. We here provide evidence that ICAM-3 is abundantly expressed by cells of the human mast cell line HMC-1 and, to a lower degree, by purified skin mast cells, as demonstrated by flow-cytometry, ELISA and RT-PCR. ICAM-3 immunoprecipitated from surface biotinylated HMC-1 cells migrates as a broad band of Mr 124,000 by Western blot analysis. We also demonstrate that monoclonal antibodies directed against ICAM-3 are capable of inducing rapid HMC-1 cell aggregation, the extent of which strongly depends on the epitope recognized by the mAb applied. Interestingly, although inhibitable by two of six mAbs against LFA-1, HMC-1 aggregation induced via ICAM-3 appears to be mediated by an adhesive pathway independent of LFA-1. Dermal mast cells are also aggregated with anti-ICAM-3 mAbs, a phenomenon which has not been described before for isolated tissue mast cells. However, this process displays slower kinetics, as compared to HMC-1 cells. That anti-ICAM-3 mAbs are able to mediate biological effects is further illustrated by their capability to increase stimulation-dependent release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 from HMC-1 cells. Taken together, these results indicate that ICAM-3 is not only expressed by immature and mature human mast cells, but also possesses functional relevance and may therefore play a significant role in mast cell associated processes.     

Intercellular adhesion molecule-2 (ICAM-2) and Pseudomonas aeruginosa ocular infection

In a previous study, ICAM-1-deficient knockout (KO) mice were able to recruit inflammatory cells into Pseudomonas aeruginosa-infected eyes and resolve the infection as well as wild-type (WT) mice. Based on this observation, it was hypothesized that ICAM-2 could serve as a surrogate receptor for leukocyte recruitment in lieu of ICAM-1. To test this hypothesis, ICAM-2 expression was first examined in both uninfected and P. aeruginosa-infected eyes (6 h postinfection) by immunohistochemistry and RT-PCR. Similar to ICAM-1, ICAM-2 was constitutively expressed on the vascular endothelium of the iris, ciliary body, and conjunctiva of uninfected eyes. Unlike ICAM-1, ICAM-2 was not expressed in the cornea nor upregulated following P. aeruginosa infection. The role of ICAM-2 in P. aeruginosa ocular infection was then addressed through a monoclonal antibody (MAb) blockade of ICAM-2 in infected ICAM-1 KO and WT mice. MAb blockade of ICAM-2 resulted in fewer infiltrating inflammatory cells (as ascertained by histopathology) in the anterior chamber of eyes of ICAM-1-KO and WT mice 24 h postinfection. However, a myeloperoxidase assay of infected corneas showed no statistical difference (P > 0.11) between the two groups in infiltrating PMN. Collectively, these data suggest that constitutively expressed ICAM-2 does play a role in recruiting inflammatory cells into the anterior chamber of the eye during P. aeruginosa infection. Furthermore, inflammatory cell recruitment into the P. aeruginosa-infected cornea appears to be mediated by an ICAM-independent pathway.     

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.     

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.     

A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen

We report that a subpopulation (10%) of the Mac-1 (CD1 1b/CD18) molecules on activated neutrophils mediates adhesion to ICAM-1 and fibrinogen. We describe a novel mAb (CBRM1/5) that binds to an activation-specific neoepitope on a subset of Mac-1 molecules on neutrophils and monocytes after stimulation with chemoattractants or phorobol esters but does not recognize Mac-1 on resting myeloid cells. CBRM1/5 immunoprecipitates a subpopulation of Mac-1 molecules from detergent lysates of neutrophils, binds to immunoaffinity-purified Mac- 1, and localizes to the I domain on the alpha chain of Mac-1. Because CBRM1/5 recognizes a fraction of Mac-1 on activated neutrophils, but still blocks Mac-1-dependent adhesion to fibrinogen and ICAM-1, we suggest that only a small subset of Mac-1 molecules is competent to mediate adhesion.     

Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking

The integrin LFA-1 interacts with a variety of ligands termed ICAMs. ICAM-1 and ICAM-2 are both expressed on endothelium and serve as counterreceptors during lymphocyte trafficking. In this study, we analyzed their relative contribution to lymphocyte recirculation through lymph nodes and to recruitment into lung and inflamed skin by blocking mAbs against ICAM-1 and ICAM-2 and mice deficient for ICAM-1. The entry of lymphocytes into peripheral and mesenteric lymph nodes was found to be unaffected by the functional deletion of either ICAM-1 or ICAM-2. However, when both pathways were blocked, recirculation through lymph nodes was strongly reduced. Trapping of lymphocytes in the lung after i.v. injection is partly mediated by LFA-1/ICAM interactions; the data presented in this study show an exclusive role of ICAM-1 in LFA-1-dependent lung trapping. Similarly, ICAM-1, but not ICAM-2, was required for the migration of T effector cells into the inflamed skin. These results indicate that ICAM-1 and ICAM-2 have redundant functions in lymphocyte recirculation through lymph nodes, but ICAM-1 is unique in supporting migration into inflamed sites and trapping within the lung.     

CD2-associated protein (CD2AP) expression in podocytes rescues lethality of CD2AP deficiency

Mice born without CD2-associated protein (CD2AP) develop renal failure and nephrotic syndrome about 4 weeks after birth and die around 6 weeks of age. Although CD2AP is widely expressed, the severity of the renal failure precludes a clear determination of the role of CD2AP in other tissues. Here we generated transgenic mice expressing CD2AP using a podocyte-specific promoter. Podocyte-specific expression of CD2AP prevented the development of proteinuria, demonstrating that the renal failure is solely due to loss of CD2AP in podocytes and not in other renal or in immune cells. CD2AP-deficient mice are long-lived and appear phenotypically normal. Histological analysis demonstrated testicular abnormalities that were age-related. CIN85, a paralog of CD2AP, is poorly expressed in both the podocyte and the basal seminiferous tubule, suggesting that the loss of CD2AP in specific tissues may be compensated for by CIN85.     

ICAM-2 peptides mediate lymphocyte adhesion by binding to CD11a/CD18 and CD49d/CD29 integrins.

Three fifteen-amino-acid polypeptides designated peptides 1, 2 and 3 were synthesised as likely candidates for mimicking the role of ICAM-2 as a ligand. The ability of each peptide to bind lymphoid cells was tested. Peptide 2 largely mediated cell attachment of unstimulated cells and this binding was only marginally increased by stimulating the cells with phorbol dibutyrate (P(Bu)2). Peptide 3 mediated minimal spontaneous cell attachment, but this binding was significantly enhanced following P(Bu)2 stimulation. Peptide 1 had no effect on cell attachment with or without stimulation. The cell attachment to peptide 2 was both temperature- and cation-dependent. Studies using specific monoclonal antibodies showed that with unstimulated cells, anti-VLA-4 alpha(CD49d) or beta chain (CD29) antibodies (KD4-13 and 4B4) and anti-CD18 (1B4) each partially inhibited the cell binding. Monoclonal antibodies against CD54 (ICAM-1; 84H10 or LB2), MHC class 1 (W6/32) and control mouse IgG had no effect. When anti-CD29 and anti-CD18 monoclonal antibodies were used concurrently, there was almost complete inhibition of the cell attachment. These observations indicated that cell adhesion via ICAM-2 is mediated: (i) predominantly by peptide 2 in unstimulated and P(Bu)2-stimulated cells, and also, to some extent, by peptide 3 in P(Bu)2-stimulated cells and (ii) by binding to both CD11/CD18 and CD49d/CD29 integrins.     

Monoclonal antibodies against LFA-1 or its ligand ICAM-1 accelerate CD2 (T11.1 + T11.2)-mediated T cell proliferation

Activation of human-purified T cells can be mediated by pairwise combinations of monoclonal antibodies directed against T11.1 and T11.2 epitopes on the CD2 molecule. Monoclonal antibodies (mAbs) reactive with either the alpha and beta chains of the lymphocyte-function-associated antigen-1 (LFA-1) molecule or one of its ligands, intercellular adhesion molecule-1 (ICAM-1), were found to accelerate anti-CD2-induced proliferation. This effect was seen on thymocytes and resting or preactivated T cells (phytohemagglutinin blasts and alloproliferative T cell clones) and could be observed, following the introduction of anti-LFA-1 or -ICAM-1 mAbs, up to 50 hr after the CD2 stimulatory signal. This effect was equally abrogated by 55 kDa anti-interleukin-2 (IL-2) receptor mAb, but neither the expression of IL-2 receptor nor the production of IL-2 was modified. The effects of anti-LFA-1 or anti-ICAM-1 on T cell activation through the CD2 pathway were therefore opposite to those observed in the CD3 pathway, where both mAbs strongly delayed T cell proliferation.     

The human leucocyte antigen CD48 (MEM-102) is closely related to the activation marker Blast-1

The glycosyl phosphatidylinositol (GPI)-linked antigen recognized by monoclonal antibody (mAb) MEM-102 is expressed on all peripheral blood lymphocytes, both resting and activated. Its properties are very similar to a previously described activation antigen, Blast-1. The amino acid sequence deduced from the structure of cloned cDNA is identical to that of the Blast-1 antigen except for a single amino acid residue. There are several other minor differences in the nucleotide sequence of the Blast-1 and MEM-102 cDNAs that do not affect the predicted structure of the polypeptide product. The amino acid sequence of the first 15 N-terminal residues of the antigen purified from Raji cells is found in the deduced sequence close to the presumed boundary between the leader peptide and mature polypeptide. Properties of the recombinant product expressed in COS cells are similar to the antigen isolated from peripheral blood mononuclear cells (PBMNCs) or B-and T-cells lines. The antigen purified on immobilized mAb MEM-102 is recognized by all six known CD48 mAbs under western blotting conditions. COS cells transfected with MEM-102 cDNA react with all the CD48 mAbs. It is concluded that mAb MEM-102 is directed against the as yet poorly characterized antigen CD48, which is therefore structurally closely related to Blast-1. Several possibilities are discussed that might account for the apparent discrepancy between the broad pan-leucocyte expression of the MEM-102/CD48 antigen and much more restricted expression of the epitope recognized by the previously described mAb defining the Blast-1 antigen.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 37766.     

In vivo effects of monoclonal antibody to ICAM-1 (CD54) in nonhuman primates with renal allografts

These studies test whether allograft rejection can be blocked by interference with leukocyte adhesion, using a murine IgG2a mAb (R6.5) reactive with monkey ICAM-1 (CD54). In 16 Cynomolgus renal allograft recipients, R6.5 was administered prophylactically as the sole immunosuppressive agent for 12 days (0.01 to 2 mg/kg/day). Survival in 14 recipients with technically successful grafts was significantly prolonged (24.2 +/- 2.4 vs 9.2 +/- 0.6 days for controls; p less than 0.001). Intercellular adhesion molecule-1 (CD54) (ICAM-1) was expressed on vascular endothelium in the kidney and other organs in the monkey in a pattern similar to that in humans. During cellular rejection in controls, ICAM-1 expression increased on endothelial cells, infiltrating mononuclear leukocytes and tubular cells. Biopsies during R6.5 administration showed decreased T cell infiltration (CD2, CD8, CD4) compared with controls and decreased arterial endothelial inflammation. No changes occurred in circulating T cells, aside from variable coating with mIgG. In six of eight other recipients R6.5 administration (0.5 to 2 mg/kg/day for 10 days) reversed preexisting rejection that resulted from taper of Cyclosporine to subtherapeutic levels. Responding grafts showed decreased edema and hemorrhage but no consistent change in the infiltrate. At 1 h after the first dose, mouse IgG deposited primarily on the graft vascular endothelium without any change in the inflammatory infiltrate. Mouse IgG also deposited on the endothelium of normal organs without eliciting an inflammatory response and was cleared from the endothelium within 4 days. Inasmuch as the principal site of binding was the vascular endothelium, we hypothesize that the antibody blocks adhesion to graft ICAM-1 molecules on the vessels. Anti-ICAM-1 also binds to recipient cells and may interfere with Ag presentation and/or T cell interactions. Whatever the mechanism(s), these studies indicate that an anti-ICAM-1 antibody inhibits T cell mediated injury in vivo, and that ICAM-1 is a critical molecule in the pathogenesis of allograft rejection.