Phagocytic Integrins: Activation and Signaling.

Phagocytic integrins are endowed with the ability to engulf and dispose of particles of different natures. Evolutionarily conserved from worms to humans, they are involved in pathogen elimination and apoptotic and tumoral cell clearance. Research in the field of integrin-mediated phagocytosis has shed light on the molecular events controlling integrin activation and their effector functions. However, there are still some aspects of the regulation of the phagocytic process that need to be clarified. Here, we have revised the molecular events controlling phagocytic integrin activation and the downstream signaling driving particle engulfment, and we have focused particularly on αMß2/CR3, αXß2/CR4, and a brief mention of αVß5/αVß3integrins.

Integrin αXß2 is a leukocyte receptor for Candida albicans and is essential for protection against fungal infections.

The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin α(X)ß2 in the pathogenesis of fungal infection was assessed. Both purified α(X)ß2 and α(X)ß2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by ß-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of α(M)ß2. Mice deficient in α(X)ß2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in α(X)ß2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of α(X)ß2-deficient mice within 9 d. Organs taken from α(X)ß2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that α(X)ß2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use α(X)ß2 to control fungal invasion.

Complement and antibodies: a dangerous liaison in HIV infection?

Due to ongoing recombination and mutations, HIV permanently escapes from neutralizing antibody (nAb) responses of the host. By the masking of epitopes or shedding of gp120, HIV-1 further impedes an efficient neutralization by Abs. Therefore, nAbs responses of the host are chasing behind a rapidly evolving virus and mainly non-neutralizing antibodies (non-nAbs) are present in the host. At the same time, complement deposition on immune-complexed HIV may counteract the immune response by enhancing the infection. On the other hand, complement-mediated lysis is a putative effector mechanism to control viral replication. Here we review the complex interplay between complement, neutralizing and non-neutralizing Abs during HIV infection and discuss the contribution of Abs and complement in blocking versus enhancing the course of infection.

The apoptotic-cell receptor CR3, but not alphavbeta5, is a regulator of human dendritic-cell immunostimulatory function.

Dendritic cells (DCs) that capture apoptotic cells (ACs) in the steady state mediate peripheral tolerance to self-antigens. ACs are recognized by an array of receptors on DCs, the redundancy of which is not completely defined. We made use of an AC surrogate system to address the individual roles of the alphavbeta5 and complement receptors (CRs) in the phagocytosis and induction of immunity. CR3 and CR4, while substantially less efficient than alphavbeta5 in internalizing ACs, initiate signals that render DCs tolerogenic. Responding T cells show impaired proliferation and IFNgamma production and subsequently die by apoptosis. While tolerogenic DCs are not induced via alphavbeta5, coligation of CR3 and alphavbeta5 maintains the DC's tolerogenic profile. This immunomodulatory role, however, is countered by a significant inflammatory stimulus such as bacterial infection. Overall, our data suggest that under steady-state conditions, signaling via CRs predominates to render DCs tolerogenic.

The physiological relevance of CD4 receptor down-modulation during HIV infection.

Upon binding to the CD4 receptor the HIV envelope protein undergoes conformational changes that culminate in the fusion of the viral and cellular membranes. A few hours later, a sophisticated set of processes is initiated to ensure the down-modulation of the viral receptor. Three viral proteins participate in this process: Nef, Env, and Vpu, suggesting that this function is critical for virus replication. The mechanisms of action of these proteins have been extensively characterized. However, the physiological relevance of the virus-induced CD4 down-modulation remains a focus of controversy, and the impact of this function on the viral life cycle has been underestimated. This review summarizes current hypotheses explaining why HIV needs to reduce expression of its own receptor, and discusses the experimental evidence supporting them. Recent findings indicate that efficient CD4 down-modulation is essential for the production of infectious particles, and highlight the importance of this function in HIV pathogenesis in vivo. Progression to disease correlates with enhanced viral induced CD4 down-modulation, and a subset of long-term nonprogressors carry viruses defective in this function. To date, the HIV-induced CD4 down-modulation has not been targeted for therapeutic intervention. Addressing the reasons why this function is so critical and understanding the interplay between viral and host factors governing surface expression of CD4 may provide clues for the development of new antiviral strategies.

An in vitro system for testing leucocyte and leukaemic cell line adhesion to synthetic fibres.

Leucocyte adhesion is an important phenomenon in antimicrobial defence, inflammation and immunological mechanisms and has been shown to be dependent upon specialized adhesion molecules. To prevent side-effects related to blood transfusion (e.g. anti-human leucocyte antigen immunization and transmission of infectious agents) leucocyte reduction of blood products is now systematically performed in various countries. The most common system used for leucoreduction is blood filtration. For further understanding of the mechanisms responsible for the interaction between leucocytes and the fibres present in filters we used a flow chamber to study the adhesion of leucocytes and leukaemic cell lines to different types of fibre. Adhesion was quantified using video-microscopy and computer image analysis. Our results demonstrate that adhesion to filter fibres was dependent on the expression of beta2-integrins CD11--CD18 and was inhibited by anti-CD18. The amount of fibres present, their spatial arrangement and the physicochemical characteristics of the fibres were important factors in leucocyte adhesion. Leucocyte adhesion was the highest to polyethylene terephthalate (PET) and polyimide fibres. Lymphocytes or lymphocytic cell lines were poorly adherent to PET fibres. The retaining capacity of leucocyte filters can be improved by taking into account the different parameters for the design of new filters

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.

Structure and flexibility of the multiple domain proteins that regulate complement activation.

In this review we summarise more than 10 years of biophysical exploration into the structural biology of the regulators of complement activation (RCA). The five human proteins responsible for regulation of the early events of complement are homologous and are composed largely from building blocks called "complement control protein (CCP) modules". Unlike most multiple domain proteins they do not contain any of the other widely occurring module types. This apparent simplicity of RCA structure, however, is belied by their sophistication of function. In fact, the structures of the individual CCP modules exhibit wide variations on a common theme while the extent and nature of intermodular connections is diverse. Some neighbouring modules within a protein stabilise each other and some co-operate to form specific binding surfaces. The degree of true "modularity" of CCPs is open to debate. The study of RCA proteins clearly illustrates the value of combining complementary structural biology techniques. The results could have implications for folding, evolution, flexibility and structure-function relationships of other molecules in the large, diverse and little understood category of multiple domain proteins.

Class II-targeted antigen is superior to CD40-targeted antigen at stimulating humoral responses in vivo.

We examined the efficacy of using monoclonal antibodies to target antigen (avidin) to different surface molecules expressed on antigen presenting cells (APC). In particular, we targeted CD40 to test whether the "adjuvant" properties of CD40 signaling combined with targeted antigen would result in enhanced serologic responses. We targeted avidin to class II as a positive control and to CD11c as a negative control. These surface proteins represent an ensemble of surface molecules that signal upon ligation and that are expressed on professional APC, in particular dendritic cells (DC). We observed that targeting class II molecules on APC was superior to targeting CD40, or CD11c. However, CD40 and CD11c could function as targets for antigen bound monoclonal antibodies under certain conditions. Interestingly, inclusion of anti-CD40 mAb with the targeting anti-class II-targeted antigens negatively affects humoral response, suggesting that CD40 signaling under certain conditions may suppress processing and/or presentation of targeted antigen.

Beta 2 (CD11/CD18) integrins can serve as signaling partners for other leukocyte receptors.

Fig. 1 depicts our current thinking about the ways in which Mo1 and p150,95 form cis interactions with other leukocyte receptors. With respect to the associations of Mo1 with Fc gamma RIIIB and uPAR, the inhibitory effect of saccharides such as NADG suggests a lectin-carbohydrate interaction that may involve the recognition of Mo1's beta-glucan site for N-linked carbohydrates4 that are expressed by both Fc gamma RIIIB and uPAR. This hypothesis is supported by the results of Stockl et al., who showed that the binding of C-terminal-specific mAb VIM12 to Mo1, which enhances the phospholipase C-mediated release of Fc gamma RIIIB, was inhibited by NADG. However, unlike the sample lectin-carbohydrate interaction that appears to govern the association between Mo1 and Fc gamma RIIIB, effective Mo1-dependent uPAR signaling also depends on the binding of intact uPA to uPAR (the receptor-binding ATF of uPA proving insufficient to prime neutrophils for an enhanced burst response to FMLP). We speculate that ATF (residues 6-135) binds to uPAR while the carboxyl terminal fragment (residues 136-411), which includes a glycosylation site at residue 144, binds to the lectinlike site of Mo1, thus fostering the linkage between the two receptors. In support of this model is the fact that exposure of neutrophils to ATF reduced the degree of molecular proximity between Mo1 and uPAR (the latter probably occupied by endogenous intact uPA) and increased the molecular association between Mo1 and Fc gamma RIIIB (both as detected by quantitative RET). This hypothesis is analogous to the concept proposed by Nykjaer et al in which plasminogen activator inhibitor-1 initially binds to uPA to form a complex that secondarily binds to the alpha 2 macroglobulin receptor, leading to internalization of the complex. Whereas the contribution of intact uPA to the interaction between Mo1 and uPAR remains speculative (based on the indirect data available), no such ambiguity exists for the role of the LPS/LBP ligand in regulating the association between Mo1 and CD14. In this circumstance, no physical linkage exists between the two receptors without the ligand complex. This observation is consistent with the previously described affinity of the beta 2 integrins for LPS, leading to the notion that the LPS portion of the LPS/LPB complex binds to Mo1, serving to link it with LPS/LBP bound to CD14. The observed reversibility of the interactions between the integrin glycoproteins and uPAR or CD14 illustrates the fact that these associations can be highly dynamic and tied to cellular processes that include directed motility (Mo1-uPAR), adherence to substrates (Mo1-CD14), and energy metabolism (p150,95-uPAR). We speculate that the GPI-anchored receptor proteins serve as rapidly diffusible, expendable "scouts" for the beta 2 integrins, which serve to expand their ligand binding repertoire in a cis-acting fashion.

Phenotypic and functional characterization of CD11c+ dendritic cell population in mouse Peyer's patches.

The antigen-presenting cell system in the gastrointestinal tract, one of three main sites (skin and lung being the others) of primary antigen contact, is poorly understood. Our study focused on dendritic cells (DC) as possible candidates for antigen uptake, processing and presentation in mucosal inductive sites, such as Peyer's patches (PP). To investigate the morphology, immunophenotype and stimulatory activity of intestinal DC, a procedure was developed to obtain a cell population by using collagenase digestion of PP, density centrifugation and cell sorting on the basis of CD11c expression. The resultant low-density cell fraction consisted of a nonadherent cell population expressing different intensities of CD11c that could at least be characterized by typical DC morphology (e.g. abundant cytoplasma with veil-like cytoplasmatic dendrites, irregularly shaped nuclei, multivesicular and multilamellar bodies), constitutive levels of surface MHC class II, the presence of macrophage-specific markers, such as F4/80, Mac-I and Fc receptors, respectively, on subpopulations of CD11c+ sorted cells and expression of adhesion and co-stimulatory receptors like ICAM-1 and CD44. The capability of this low-density CD11c+ fraction to stimulate T cell responses was demonstrated in primary allogeneic mixed-lymphocyte reactions (MLR). Herein, we show that the freshly isolated CD11c+ cells showed weak accessory function, but develop this capacity following short-term culture in vitro in the presence of granulocyte/macrophage colony-stimulating factor. Although the nature and functional capacity of the isolated CD11c+ needs further clarification, these preliminary results describing phenotype and accessory function provide some evidence that these cells isolated from the PP may be immature forms of DC and play a crucial role as antigen-presenting cells with important implications for understanding the complex network regulating intestinal antigen uptake, processing and presentation.

Identification of Sp1-binding sites in the CD11c (p150,95 alpha) and CD11a (LFA-1 alpha) integrin subunit promoters and their involvement in the tissue-specific expression of CD11c.

The leukocyte integrins LFA-1 (CD11a/CD18) and p150,95 (CD11c/CD18) mediate cell-cell and cell-extracellular matrix interactions during inflammatory responses and signal transduction into the cytoplasm. While the CD11a integrin subunit is expressed on all leukocytes, CD11c is almost exclusively expressed on cells of the myeloid lineage and on activated B lymphocytes. Its expression is regulated during cell activation and differentiation by transcriptional mechanisms. We have previously demonstrated that the proximal region of the CD11c promoter directs tissue-restricted and developmentally-regulated expression of reporter genes. Structural studies by electrophoretic mobility shift assays have demonstrated the presence of two Sp1-binding sites at -70 (Sp1-70) and -120 (Sp1-120) which mediate the Sp1 transactivation of the CD11c promoter in Sp1-defective SL2 cells, and which are involved in cell lineage-specific DNA-protein interactions, as demonstrated by footprinting in vivo. More importantly, mutation of either Sp1 site inhibited the activity of the CD11c promoter both in myeloid U937 cells and the CD11c-expressing B lymphoblastoid JY cell line, while the opposite effect was observed in the CD11c-negative epithelial HeLa cell line, demonstrating the involvement of both Sp1-binding sites in the basal and the tissue-restricted expression of the CD11c integrin subunit gene. Interestingly, the analysis of the CD11a proximal promoter also revealed the existence of an Sp1-binding site at -70, indicating a common role for these cis-acting elements in the transcription of the leukocyte integrin alpha subunit genes. The binding of Sp1 to the regulatory regions of the leukocyte integrin genes raises the possibility that the retinoblastoma susceptibility gene product is implicated in integrin expression through its functional interaction with Sp1, thus establishing a link between integrin-dependent leukocyte adhesiveness and the state of cellular differentiation/proliferation.

CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide.

CD11c/CD18 is a member of the leukocyte integrin family, heterodimeric adhesion molecules that interact with a diverse repertoire of ligands, including bacterial lipopolysaccharide (LPS). Their role as signal transducing receptors remains uncertain. We used a heterologous expression system to determine if CD11c/CD18 was capable of initiating signal transduction in response to LPS-binding, as assessed by the induced translocation of nuclear factor-kappa B. We have previously reported that Chinese hamster ovary (CHO)-K1 fibroblasts, normally unresponsive to LPS, acquire serum-dependent macrophage-like responses to LPS when transfected with CD14 (Golenbock, D.T., Y. Liu, F. Millham, M. Freeman, and R. Zoeller. 1993. J. Biol. Chem. 268:22055-22059), a known LPS receptor. In contrast, CHO cells acquired serum-independent responses to Gram-negative bacteria and LPS when transfected with CD11c/CD18 (CHO/CD11c). In comparison to CHO cells transfected with CD14 (CHO/CD14), responses in CHO/CD11c cells were slower, required higher endotoxin concentrations for maximal response, and were not inhibited by the presence of antibodies to CD14. CD11c/CD18 is, thus, the second phagocyte receptor, in addition to CD14, which has been shown to have the capacity to activate cells after binding to LPS. The function of this receptor in normal phagocytes may be limited to the recognition of LPS in infected tissues, where LPS-CD14 interactions are not favored because of the absence of serum proteins.

[Arachidonic acid metabolism affected by cellular interactions regulating leukocyte adherence].

Increased leukocyte adherence to endothelium is recognized to be the first step following the development of unstable angina and cerebrovascular disease. In this paper, the methods to determine leukocyte adherence and the conditions which influence the result of the assay are discussed. We administered aspirin, TxA2 synthetase inhibitor or TxA2 receptor antagonist to healthy volunteers. Leukocyte adherence was inhibited following the administration of these antiplatelet agents. The effect on leukocytes may prevent thrombosis formation. Although the role of the leukocyte adherence in the pathogenesis of thrombosis has not been fully elucidated. Several forms of stimuli may cause expression of adhesive glycoproteins, which have a RGD sequence, on the surface of leukocyte and thus lead to thrombotic formation.