Elastin peptides induced oxidation of LDL by phagocytic cells

The degradation products of one of the major component of vascular wall, elastin, have several important biological activities. Elastin peptides (KE) are mostly generated during vascular aging and the atherosclerotic process. They induce free radical and proteases production from cells, which are the major components of the atherosclerotic process. In the present study, we investigated whether the interaction between elastin peptides and neutrophils as well as monocytes contributes to low density lipoproteins (LDL) oxidation, being one of the most important initiator of the chronic inflammatory process contributing to the development of atherosclerosis. Here, we present data on the link between the elastin degradation products and LDL oxidation by the chemotactically attracted neutrophils and monocytes. The KE as well as the active epitope, the hexapeptide VGVAPG is able, in a differential concentration and time dependence, to induce the oxidation of LDL. KE is able to induce via the production of free radicals by neutrophils the oxidation of LDL very rapidly and in higher concentration compared to monocytes. These effects of KE are occurring through the stimulation of the 67 kDa elastin-laminin receptor (ELR), as demonstrated by the uncoupling effect of lactose. In our present study, the HDL was able to decrease the LDL oxidation by KE. This is a new mechanism by which elastin peptides might participate in the initiation and progression of the atherosclerotic process.     

Trp-Lys-Tyr-Met-Val-D-Met is a chemoattractant for human phagocytic cells

Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a synthetic peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes. The peptide binds to a unique cell surface receptor(s). Recently we had demonstrated that human neutrophils, monocytes, and B lymphocytes express this peptide-specific receptor and that stimulation of human leukocytes with the peptide leads to activation of the oxidative respiratory system and the bactericidal activity of neutrophils or monocytes. In this study we showed that the peptide induces chemotaxis of phagocytic leukocytes and studied the signaling pathway leading to chemotaxis in human monocytes. The peptide-induced monocyte chemotaxis is pertussis toxin (PTX)-sensitive. This fact correlates with the peptide's stimulation of PI hydrolysis and intracellular Ca2+ ([Ca2+]i) release, which is also PTX-sensitive. We demonstrate that the peptide-specific receptor is different from receptor(s) for monocyte chemoattractant protein-1 (MCP-1). We also show that intracellular signaling of WKYMVm leading to monocyte chemotaxis is different from that of MCP-1. The peptide-mediated monocyte chemotaxis is insensitive to protein kinase C (PKC) inhibitor (GF109203X) and butan-1-ol, ruling out PKC and phospholipase D participation in this process. On the other hand, a tyrosine kinase inhibitor (genistein) and RhoA inhibitor (C3 transferase) curtailed the peptide-induced chemotaxis in a concentration-dependent manner, implying the involvement of tyrosine kinase and RhoA, respectively. Treatment of human monocytes with the peptide stimulates tyrosine phosphorylation of several cellular proteins, including p125FAK and Pyk2 and translocation of RhoA from the cytosol to the membrane. We conclude that WKYMVm induces chemotaxis of human phagocytic leukocytes via unique receptors and signaling.     

Generation of chimeric C5a/formyl peptide receptors: towards the identification of the human C5a receptor binding site

We employed the polymerase chain reaction to produce a series of chimeric C5a/formyl peptide receptors. Chinese hamster ovary cells transfected with these constructs were tested for their ability to bind C5a. Substitution of three of the extracellular domains of the C5a receptor with the corresponding domains of the formyl peptide receptor abolished C5a binding, whilst replacement of the first extracellular loop of the C5a receptor with that of the formyl peptide receptor had little effect on the affinity of the receptor for C5a. We therefore conclude that this first outer loop of the C5a receptor does not participate in ligand binding, whilst involvement of the other extracellular domains of the receptor cannot be ruled out.     

Release of histamine from rat mast cells by the complement peptides C3a and C5a.

Suspensions of rat mast cells were used to study the histamine-releasing actions of anaphylatoxins C3A and C5a in vitro. The peptides, derived from human or porcine complement proteins C3 and C5, were less potent than 48/80 but more potent than bradykinin in stimulating release of histamine from mast cells. The pattern of release resembled that of the anaphylactic release action, e.g. release was limited to less than 30 per cent of the cell histamine, the reaction was calcium-dependent and was potentiated by phosphatidyl serine. When C3a and C5a were added together to mast cell suspensions, the amount of histamine released was additive. Similarly, release by either peptide combined with bradykinin was additive. Histamine-releasing activity (as well as smooth muscle-stimulating activity) was abolished when the peptides were treated with pancreatic carboxy-peptidase B. Active or inactive peptides were bound by mast cells and addition of active C3a in combination with the inactive, des-arginine derivativ, C3ai, resulted in partial inhibition of histamine release.     

Synergistic Enhancement of Chemokine Generation and Lung Injury by C5a or the Membrane Attack Complex of Complement

Complement plays an important role in many acute inflammatory responses. In the current studies it was demonstrated that, in the presence of either C5a or sublytic forms of the complement-derived membrane attack complex (MAC), rat alveolar macrophages costimulated with IgG immune complexes demonstrated synergistic production of C-X-C (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) and C-C (macrophage inflammatory protein-1alpha and monocyte chemoattractant-1) chemokines. In the absence of the costimulus, C5a or MAC did not induce chemokine generation. In in vivo studies, C5a and MAC alone caused limited or no intrapulmonary generation of chemokines, but in the presence of a costimulus (IgG immune complexes) C5a and MAC caused synergistic intrapulmonary generation of C-X-C and C-C chemokines but not of tumor necrosis factor alpha. Under these conditions increased neutrophil accumulation occurred, as did lung injury. These observations suggest that C5a and MAC function synergistically with a costimulus to enhance chemokine generation and the intensity of the lung inflammatory response.     

Upregulation of adhesion molecules induced by broncho-vaxom on phagocytic cells.

Whole blood was incubated with the bacterial extract Broncho-Vaxom (OM85) at various concentrations and for different periods of time. Expression of the beta 2-integrins (LFA-1, CD11a/CD18; MAC-1, CD11b/CD18; p150,95, CD11c/CD18) and ICAM-1 (CD54) by monocytes and granulocytes was studied using flow cytometry. OM85 enhanced the expression of MAC-1 and ICAM-1 on monocytes and granulocytes in a dose-dependent manner. Maximal expression was achieved with 1 mg/ml bacterial extract. The effect on MAC-1 expression was not due to the low concentration of endotoxin contaminating the preparation (less than 1 ng/mg) since polymyxin-B did not substantially affect the adhesion molecule upregulation induced by OM85. In addition, OM85 enhanced the expression of p150,95 on monocytes and granulocytes, and also increased expression of LFA-1 on monocytes, but not on granulocytes. While MAC-1 and p150,95 expression reached peak values between 1 and 6 h, levels of ICAM-1 rose constantly for 10 h. We suggest that the clinical interest of OM85 in the management of recurrent infections could be related to be upregulation of adhesion molecules induced by this bacterial extract.     

Roles of the ribosomal protein S19 dimer and the C5a receptor in pathophysiological functions of phagocytic leukocytes

Monocytes and neutrophils, the major phagocytic leukocytes, migrate to inflammatory sites by sensing chemoattractants such as anaphylatoxin C5a with membrane receptors such as C5a receptor. Upon stimulation, the leukocytes increase cytoplasmic Ca(2+) concentration and generate radical oxygen species. These leukocytes have different functions in inflammation. Neutrophils migrate more rapidly and induce vascular plasma leakage upon infiltration. Monocytes infiltrate tissue more slowly but have superior capacities of phagocytosis and antigen presentation. There must be mechanisms to separately recruit the leukocyte species at an inflammatory site. Ribosomal protein S19 (RP S19) is a component of ribosome. During apoptosis, RP S19 is dimerized and obtains a ligand capacity to C5a receptor. The RP S19 dimer attracts monocytes to phagocytically clear the apoptotic cells that released the dimer molecules. The phagocytic monocytes/macrophages then translocate to regional lymph nodes and present apoptotic cell-derived antigens. Oppositely, the RP S19 dimer inhibits C5a-induced neutrophil migration and promotes apoptosis of neutrophils via the C5a receptor. The RP S19 dimer seems to prevent excessive tissue destruction induced by neutrophils. Skp is a molecular chaperon of Gram-negative bacteria. Skp also attracts monocytes and neutrophils as a ligand of C5a receptor. However, it promotes neither cytoplasmic Ca(2+) enhancement nor radical oxygen generation.     

A hypothesis for the mechanism of superoxide production by phagocytic cells

When arachidonic acid is reacted with Fe2+, the Fe2+ is oxidized to Fe3+, and lipid peroxides are formed as detected by absorption at 232 nm. However, there is a large discrepancy between the amount of arachidonic acid converted to stable oxidized products and the amount of Fe2+ converted to Fe3+. We suggest that under appropriate conditions, electrons from the ferrous iron may reduce oxygen to superoxide in the presence of arachidonic acid. The results are consistent with evidence using bromophenacyl-bromide suggesting that release of fatty acids from cell phospholipids might be required for phagocytes to synthesize superoxide.     

Human C5a and C5a analogs as probes of the neutrophil C5a receptor

Quantitative assessment of the human neutrophil chemotactic response and immunologic evidence demonstrate that human C5a and/or its physiological equivalent, C5a_{des Arg'} serves as the predominant chemotactic factor in complement activated serum. Both C5a and its des Arg74 derivative are capable of promoting directed cellular migration as well as lysosomal enzyme release. When neutrophil chemotaxis is assessed by an ‘under agarose methodology’, in the absence of extraneous human serum proteins. C5a is found to be some 10- to 30-fold more active than C5a_{des Arg} Quantitatively similar results are obtained when each factor is assessed for its ability to promote secretion of azurophilic granular enzymes from cytochalasin B-treated neutrophils. The C5a structural analog C5a-(1–69), lacking five residues of the C-terminal portion of the parent molecule, and a synthetic pentapeptide l-methionyl-l-glutaminyl-l-leucylglycyl-l-arginine, which mimics the C-terminal linear sequence of C5a (Chenoweth et al., 1979a), are found to be devoid of biological activity when examined either-alone or in combination. These findings imply that the C-terminal portions of C5a contribute importantly to modulating ligand-receptor interactions on neutrophils. Based on these observations, a conceptual model of the interaction of human C5a with the neutrophil C5a receptor is proposed. We hypothesize that C5a possesses an internal ‘recognition’ site in addition to the ‘activation’ site contained in the C-terminal region of the molecule.     

Current aspects of chemotaxis of phagocytic cells

Phagocytic cells, the first host defence against microbial attack, are remarkably mobile; they can move very rapidly to the infectious or inflammatory site. They migrate toward a chemotactic factor: C5a, lymphokines, bacterial products, leukotriene B4, etc. The binding of the chemotactic factor to its specific receptor on the cell leads to an activation of the phagocytic cell: the electric potential of the membrane changes, ionic fluxes and free calcium rate increase, arachidonic acid metabolites are produced, cyclic nucleotides are activated. This produces a change in shape, a polarization of the cell and, after cytoskeleton reorganization, migration of the cell towards the chemotactic factor. A constitutional or acquired abnormality of one of these steps induces a defect of chemotaxis for the phagocytic cells and severe infections.     

Expression of the anaphylatoxin C5a receptor in non-myeloid cells

C5, a 74 amino acid peptide cleaved from the complement protein C5, represents the most potent anaphylatoxin and possesses inflammatory as well as immunomodulatory activities. In the past, expression of the receptor for the anaphylatoxin C5a (C5aR) has been thought to be restricted to cells of myeloid origin. However, recent evidence suggests that the C5aR is constitutively expressed in non-myeloid cells including epithelial, endothelial and smooth muscle cells in the human liver and lung. These findings are contrasted by results from our laboratory which demonstrated that in the normal human liver and lung C5aR expression is detectable exclusively in macrophages and macrophage-derived cells (Kupffer cells). Interestingly, we found evidence that C5aR expression may be inducible in epithelial cells as C5aR mRNA was observed in vivo in human keratinocytes of the inflamed but not of the normal skin. Herein we review the work of our laboratory and others on the expression of the C5aR in various human non-myeloid cells types. A better understanding of the expression patterns of this important anaphylatoxin receptor may provide new insights in the pathophysiological role of C5a in vivo.     

Generation of Immune Responses against Hepatitis C Virus by Dendritic Cells Containing NS5 Protein-Coated Microparticles

Dendritic cells (DCs) internalize and process antigens as well as activate cellular immune responses. The aim of this study was to determine the capacity of DCs that contain antigen-coated magnetic beads to induce immunity against the nonstructural hepatitis C virus (HCV) antigen 5 (NS5). Splenocytes derived from Fms-like tyrosine kinase receptor 3 (Flt3) ligand-pretreated BALB/c mice were incubated with magnetic beads coated with HCV NS5, lipopolysaccharide (LPS), and/or anti-CD40; purified; and used for immunization. Cellular immunity was measured using cytotoxic T-lymphocyte (CTL) and T-cell proliferation assays, intracellular cytokine staining, and a syngeneic tumor challenge using NS5-expressing SP2/0 myeloma cells in vivo. Splenocytes isolated from animals vaccinated with DCs containing beads coated with NS5, LPS, and anti-CD40 secreted elevated levels of interleukin-2 (IL-2) and gamma interferon in the presence of NS5. The numbers of CD4⁺, IL-2-producing cells were increased >5-fold in the group immunized with DCs containing beads coated with NS5, LPS, and anti-CD40, paralleled by an enhanced splenocyte proliferative response. Immunization promoted antigen-specific CTL activity threefold compared to the level for control mice and significantly reduced the growth of NS5-expressing tumor cells in vivo. Thus, strategies that employ NS5-coated beads induce cellular immune responses in mice, which correlate well with the natural immune responses that occur in individuals who resolve HCV.An estimated 170 million individuals (3% of the world''s population) are infected with hepatitis C virus (HCV), leading to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. The current standard-of-care therapy for chronically infected HCV patients is the combined administration of pegylated alpha interferon (IFN-α) and ribavirin (28). A sustained response is seen in approximately 50 to 60% of individuals (33). Treatment is long term (6 to 12 months), costly, and associated with substantial toxicity (42). Clearly, more-effective regimens are needed (10).Clinical studies of the adaptive host immune response to acute HCV infection suggest a rationale for immunization approaches. Clearance of HCV is associated with early, multispecific, strong CD8⁺ T-cell immunity that is matched by vigorous and sustained CD4⁺ T-cell proliferation in response to multiple recombinant structural and nonstructural viral proteins (13, 18, 47). Activated T cells secrete proinflammatory cytokines (TH1 type), such as IFN-γ, coinciding with large reductions in viral load during acute infection (47). HCV infections that are successfully controlled result in durable memory populations (44). This observation is supported by a substantially lower rate of HCV persistence in reexposed humans with a history of acute resolving HCV (29). Rechallenge experiments with chimpanzees showed that antibody-mediated depletion of CD4⁺ T cells resulted in HCV persistence, which is in contrast to a marked reduction in duration and peak of viremia in nontreated animals (4, 20). The importance of CD4⁺ T cells is further emphasized by the loss of immune protection against reexposure to HCV and correlated with low CD4⁺ T-cell counts in intravenous drug users who had recovered from HCV but subsequently acquired human immunodeficiency virus infection (29). Whether recovery from acute HCV coincides ultimately with virus eradication is still a matter of debate (34). The disappearance of HCV-specific antibodies in some individuals 10 to 20 years after viral clearance indicates that a subgroup of patients achieves complete virus elimination (44).Persistent HCV infection can be attributed to a number of viral evasion strategies. First, the rapid spread of HCV in a host outpaces the immune response by several weeks (39, 46). Second, HCV is a strong inducer of type I IFN but appears to render hepatocytes resistant to antiviral activity (45, 46). Third, NK cell function can be inhibited by cross-linking tetraspanin CD81 on the cell surface with the major envelope protein of HCV (HCV E2), thus blocking NK cell activation, proliferation, and cytokine production (12). Finally, persistent HCV infections correlate with the permanent loss of HCV-specific T-cell proliferation during acute HCV infection, the functional exhaustion of an initially vigorous response, and the inability of effector T cells to migrate into the infected liver (10, 13, 18, 36, 39, 46). Insufficient CD4⁺ T-cell activity appears to be a key event leading or contributing to chronic HCV. Failure to sustain the CD4⁺ helper response renders virus-specific CD8⁺ T cells inadequate (e.g., loss of cytotoxicity and IFN-γ production), thus contributing to persistent viremia (39).Recapitulating successful immune responses and addressing HCV-specific defects in immunity are mandatory in efforts to improve current treatment options. The purported involvement of dendritic cells (DCs) in the impaired immune responses observed in patients with persistent HCV infection makes DCs a principal target for immunomodulatory approaches. Access to a sufficient quantity of mature DCs is a critical issue since only mature DCs are capable of targeting the antigen and inducing cellular immunity rather than tolerance. Previously, we outlined a novel method of generating large numbers of DCs in vivo (17). These, in turn, are enriched in vitro by phagocytosis of magnetic beads and separation in a magnetic field. Here we further demonstrate that, by immunizing mice with DCs that contain beads coated with the nonstructural HCV antigen NS5 and compounds known to induce DC maturation, significant levels of cellular immunity are elicited. The key elements of this approach reside in the combined enrichment, maturation, and antigen targeting of DCs in a single step and the generation of immune responses of the type known to promote viral clearance in humans.     

Receptors for IgA on phagocytic cells

IgA receptors have been detected on monocytes, polymorphonuclear neutrophils (PMNs) and eosinophils, and on phagocytic cells at mucosal sites. These receptors bind both secretory and serum forms of immunoglobulin A (IgA) and require the Ca2 region of the IgA molecule for ligand recognition. Monocytes and PMNs modulate their expression of the IgA receptor upon treatment with cytokines, such as granulocyto-macrophage colony-stimulating factor, and lipopolysaccharide. Purified IgA receptors appear as heavily glycosylated molecules with an average molecular weight of 60 kD, dropping to 32 and 36 kD upon treatment with N-glycanase. The cDNA sequence encoding the IgA receptor has been determined by expression cloning, and predicts that the receptor consists of two Ig-like extracellular domaines, a transmembrane region and a cytoplasmic tail of 41 residues. Ligation of IgA receptors on phagocytic cells by multivalent IgA complexes induces a variety of responses, including superoxide generation, release of inflammatory mediators, phagocytosis, and killing of various pathogenic microorganisms. Thus the apparent role of these receptors is to amplify the protective effects of the IgA antibody, a function of potential importance to mucosal defense.     

Plasma clearance of the human C5a anaphylatoxin by binding to leucocyte C5a receptors.

The C5a anaphylatoxin is a potent complement-derived mediator of inflammation with chemotactic activity. In this study the possible role of specific high-affinity binding sites for C5a on peripheral blood leucocytes for the removal of C5a from human blood plasma was investigated. The addition of purified granulocytes or mononuclear cells to complement-activated plasma resulted in the rapid and dose-dependent removal of up to 80% of plasma C5a, as determined by ELISA. The specific role of leucocyte C5a receptors (C5aR) in the plasma clearance of C5a was demonstrated by the inhibition of C5a uptake by the preincubation of cells with the C5aR-specific monoclonal antibody S5/1. Furthermore, U937 cells which had been induced by db-cAMP to express C5aR, but not undifferentiated U937 cells, were capable of removing C5a from plasma. The inhibition of C5aR internalization by monensin did not affect C5a uptake by leucocytes. The co-incubation with leucocytes had no effect on the plasma clearance of complement activation products C3a or terminal complement complex (TCC), as determined by this in vitro assay. The binding of the C5a anaphylatoxin to cellular receptors represents an effective control mechanism that protects the organism from systemic effects of this potent phlogistic mediator.