Repurposed Drugs That Block the Gonococcus-Complement Receptor 3 Interaction Can Prevent and Cure Gonococcal Infection of Primary Human Cervical Epithelial Cells.

In the absence of a vaccine, multidrug-resistant Neisseria gonorrhoeae has emerged as a major human health threat, and new approaches to treat gonorrhea are urgently needed. N. gonorrhoeae pili are posttranslationally modified by a glycan that terminates in a galactose. The terminal galactose is critical for initial contact with the human cervical mucosa via an interaction with the I-domain of complement receptor 3 (CR3). We have now identified the I-domain galactose-binding epitope and characterized its galactose-specific lectin activity. Using surface plasmon resonance and cellular infection assays, we found that a peptide mimic of this galactose-binding region competitively inhibited the N. gonorrhoeae-CR3 interaction. A compound library was screened for potential drugs that could similarly prohibit the N. gonorrhoeae-CR3 interaction and be repurposed as novel host-targeted therapeutics for multidrug-resistant gonococcal infections in women. Two drugs, methyldopa and carbamazepine, prevented and cured cervical cell infection by multidrug-resistant gonococci by blocking the gonococcal-CR3 I-domain interaction.IMPORTANCE Novel therapies that avert the problem of Neisseria gonorrhoeae with acquired antibiotic resistance are urgently needed. Gonococcal infection of the human cervix is initiated by an interaction between a galactose modification made to its surface appendages, pili, and the I-domain region of (host) complement receptor 3 (CR3). By targeting this crucial gonococcal-I-domain interaction, it may be possible to prevent cervical infection in females. To this end, we identified the I-domain galactose-binding epitope of CR3 and characterized its galactose lectin activity. Moreover, we identified two drugs, carbamazepine and methyldopa, as effective host-targeted therapies for gonorrhea treatment. At doses below those currently used for their respective existing indications, both carbamazepine and methyldopa were more effective than ceftriaxone in curing cervical infection ex vivo This host-targeted approach would not be subject to N. gonorrhoeae drug resistance mechanisms. Thus, our data suggest a long-term solution to the growing problem of multidrug-resistant N. gonorrhoeae infections.

Tumor necrosis-initiated complement activation stimulates proliferation of medulloblastoma cells.

OBJECTIVE AND DESIGN: We sought to determine the effect of necrosis-induced activation of the complement protein C3 in medulloblastoma. MATERIALS/METHODS: Twelve medulloblastoma surgical specimens were evaluated for complement activation using immunohistochemistry, with H&E stains performed on adjacent tissue sections to determine the relationship of complement activation to necrotic tissue. Flow cytometry and Western blot were performed on three established medulloblastoma lines and one surgically-procured cell culture to determine expression of C3a receptor (C3aR) in medulloblastoma. In vitro proliferation of siRNA C3aR knockdown cells was compared to that of control siRNA cells with cell line Daoy. RESULTS: Three surgical specimens were found to have necrosis on H&E sections. In each case, iC3b staining was identified on adjacent sections, limited to the necrotic region. In no case did necrosis occur without iC3b staining on adjacent sections. C3aR protein was demonstrated on both the three established cell lines and on the surgical culture. Proliferation assays of Daoy cells with siRNA knockdown vs. control siRNA revealed significantly reduced proliferation at 72 h (p = 0.001). CONCLUSIONS: Necrosis is associated with complement activation in medulloblastoma. Medulloblastoma cells express C3aR, and siRNA-mediated knockdown of C3aR inhibits proliferation of these cells in vitro.

Targeting gC1qR domains for therapy against infection and inflammation.

Abstract The receptor for the globular heads of C1q, gC1qR/p33, is a widely expressed cellular protein, which binds to diverse ligands including plasma proteins, cellular proteins, and microbial ligands. In addition to C1q, gC1qR also binds high molecular weight kininogen (HK), which also has two other cell surface sites, namely, cytokeratin 1 and urokinase plasminogen activator receptor (uPAR). On endothelial cells (ECs), the three molecules form two closely associated bimolecular complexes of gC1qR/cytokeratin 1 and uPAR/cytokeratin 1. However, by virtue of its high affinity for HK, gC1qR plays a central role in the assembly of the kallikrein-kinin system, leading to the generation of bradykinin (BK). BK in turn is largely responsible for the vascular leakage and associated inflammation seen in angioedema patients. Therefore, blockade of gC1qR by inhibitory peptides or antibodies may not only prevent the generation of BK but also reduce Clq-induced or microbial-ligand-induced inflammatory responses. Employing synthetic peptides and gClqR deletion mutants, we confirmed previously predicted sites for C1q (residues 75-96) and HK (residues 204-218) and identified additional sites for both C1q and HK (residues 190-202), for C1q (residues 144-162), and for HIV-1 gp41 (residues 174-180). With the exception of residues 75-96, which is located in the alphaA coiled-coil N-terminal segment, most of the identified residues form part of the highly charged loops connecting the various beta-strands in the crystal structure. Taken together, the data support the notion that gC1qR could serve as a novel molecular target for the design of antibody-based and/or peptide-based therapy to attenuate acute and/or chronic inflammation associated with vascular leakage and infection.

Z39Ig is expressed on macrophages and may mediate inflammatory reactions in arthritis and atherosclerosis.

Z39Ig is a transmembrane protein containing two Ig homology domains with unknown functions. Immunohistochemical analyses of human carotid atherosclerotic plaques detected Z39Ig staining in areas rich in foamy macrophages. Z39Ig staining was also observed in macrophages in the lining layers and sublining areas of rheumatoid arthritis synovium. Z39Ig staining in the osteoarthritis synovium was restricted to macrophages in the lining layers. To identify the role(s) of Z39Ig in the function of macrophages, we used human monocytic cell lines TF-1A (Z39Ig-negative) and THP-1 (Z39Ig-positive). The expression of Z39Ig was induced in TF-1A cells ,when they were differentiated into macrophages by treatment with PMA. The stimulation of PMA-treated TF-1A or THP-1 cells with immobilized anti-Z39Ig mAb induced the secretion of IL-8 and matrix metalloproteinase (MMP)-9, which was dependent on NF-kappaB activation. These data indicate that the macrophage Z39Ig is involved in the pathogenesis of inflammatory diseases through chemokine induction, which will promote the migration of inflammatory cells into the lesion area, and MMP-9 induction, which will contribute to cartilage destruction or extracellular matrix degradation.

Microglia/macrophages responses to kainate-induced injury in the rat retina.

The present study was aimed to elucidate how retinal microglia/macrophages would respond to neuronal death after intravitreal kainate injection. An increased expression of the complement receptor type 3 (CR3) and an induction of the major histocompatibility complex (MHC) class II and ED-1 antigens were mainly observed in the inner retina after kainate injection. Prominent cell death revealed by Fluoro Jade B (FJB) staining and ultrastructural examination appeared at the inner border of the inner nuclear layer (INL) at 1 day post-injection. Interestingly, some immunoreactive cells appeared at the outer segment of photoreceptor layer (OSPRL) at different time intervals. Our quantitative analysis further showed that CR3 immunoreactivity was drastically increased peaking at 7 days but subsided thereafter. MHC class II and ED-1 immunoreactivities showed a moderate but steady increase peaking at 3 days and declined thereafter. Double labeling study further revealed that retinal microglia/macrophages expressed concurrently CR3 and ED-1 antigens (OX-42+/ED-1+) or MHC class II molecules (OX-42+/OX-6+) and remained branched in shape at early stage of kainate challenge. By electron microscopy, microglia/macrophages with CR3 immunoreactivity displayed abundant cytoplasm containing a few vesicles and phagosomes. Other cells ultrastructurally similar to Müller cells or astrocytes could also engulf exogenous substances. In conclusion, retinal microglia/macrophages responded vigorously to kainate-induced neuronal cell death that may also trigger the recruitment of macrophages from neighboring tissues and induce the phagocytotic activity of cells other than retinal microglia/macrophages.

Complement component C1q induces endothelial cell adhesion and spreading through a docking/signaling partnership of C1q receptors and integrins.

The interaction of C1q with endothelial cells elicits a multiplicity of biologic responses. Although these specific responses are thought to be mediated by the interaction of C1q with proteins of the endothelial cell surface, the molecular identity of the participant(s) has not been clearly defined. In this study, we examined the role of two C1q-binding proteins, cC1q-R/CR and gC1q-R/p33, on C1q-mediated adhesion and spreading of human dermal microvascular endothelial cells (HDMVECs). A specific and dose-dependent adhesion and spreading was observed when HDMVECs were cultured in microtiter plate wells coated with concentrations of C1q ranging from 0 to 50 microg/ml. The extent of adhesion and spreading was similar to the adhesion seen on collagen-coated wells. Furthermore, the effect of C1q was mimicked by either polyclonal anti-cC1q-R or mAb 60.11, but not with isotype- and species-matched control IgG. More importantly, however, a 100% inhibition of spreading but not adhesion to C1q-coated wells was observed when HDMVECs were cultured in the presence of 30 mM of the peptide GRRGDSP but not GRRGESP. Furthermore, while anti-beta1 integrin antibody blocked adhesion and spreading, antialpha5 integrin only blocked spreading. Since earlier studies have shown that zinc induces the exposure of hydrophobic sites in the C-terminus of gC1q-R including the putative high-molecular weight kininogen (HK)-binding site corresponding to residues 204-218, we also examined the effect of zinc on antibody binding to cell surface gC1q-R. Flow cytometric data show that the binding of mAb 74.5.2, which recognizes residues 204-218, is greatly enhanced when endothelial cells were incubated in the presence of 50 microM zinc. In summary, our data show that: (a) C1q-mediated endothelial cell adhesion and spreading requires the cooperation of both C1q receptors and 1 integrins, and possibly other membrane-spanning molecules, and (b) zinc can induce the exposure of hydrophobic sites in the C-terminal domain of gC1q-R allowing a more efficient binding of mAb 74.5.2 and HK.

Contractile effect of anaphylatoxin C5a and of a mimetic peptide on the human umbilical artery: further evidence for leukocyte-dependent vasomotion.

Reproducible and concentration-dependent contractile effects were recorded when the human umbilical artery was exposed to recombinant C5a (>or=1 nM). The synthetic mimetic peptide Ac-YSFKPMPLaR was about 100-fold less potent in this respect, and its effect was largely prevented by treatment with the TP prostanoid receptor antagonist SQ 29548. The selective cyclooxygenase-1 inhibitor flurbiprofen, but not by the cyclooxygenase-2 inhibitor L-745337, prevented the contractile effect of both C5a and Ac-YSFKPMPLaR. Cells positive for C5a receptor (CD 88) immunoreactivity were scattered in the umbilical artery structure and were apparently more abundant in the periphery. The macrophage marker CD 68 was also expressed by dispersed cells, with a distribution similar to CD 88; both markers were co-expressed in some cells represented in consecutive sections. Immunoblot for C5a receptors has been applied to tissue or cells extracts: a specific approximately 42-kd major band observed in peripheral blood leukocytes was also expressed by the fresh umbilical artery, but not by cultured smooth muscle cells derived from the umbilical artery. Macrophages dispersed in the connective structure of the vessel wall may, in response to C5a receptor agonists, release prostanoids formed by cyclooxygenase-1 that indirectly contract smooth muscle cells. Leukocyte-dependent vasospasm may be relevant in situations in which complement is activated and may trigger thromboembolic complications via the secretion of thromboxane-like eicosanoids also active on circulatory platelets.

Differential activities of decapeptide agonists of human C5a: the conformational effects of backbone N-methylation.

Analogues of the potent, conformationally biased, decapeptide agonist of human C5a anaphylatoxin, C5a(65-74)Y65,F67,P69,P71,D-Ala73 (YSFKPMPLaR, peptide 54), were synthesized with methyl groups occupying specific amide nitrogen atoms along the peptide backbone. This N-methylation induced crucial extended backbone conformations in a manner similar to the two Pro residues, but without eliminating the contributions made by the side-chain of the residue for which Pro was substituted. The presence of backbone N-methyl groups on peptide 54 analogues had pronounced detrimental effects on the ability to bind and activate C5aRs expressed on human PMNs, but not on the ability to contract smooth muscle of human umbilical artery. Several N-methylated analogues of peptide 54 (peptides 56, 67, 124, 125, and 137) were significantly more selective for smooth muscle contraction, which is mediated by tissue resident macrophages, than for enzyme release from PMNs. Indeed, peptide 67, YSFKDMP(MeL)aR was almost 3000-fold more selective for smooth muscle contraction than for PMN enzyme release. Consistent with these differential activities was the observation that peptide 67 expressed a significantly greater binding affinity to C5aRs expressed on rat macrophages than on rat PMNs. This differential activity was also observed in vivo in the rat where peptide 67 induced a hypotensive response similar to peptide 54 and rhuC5a, but without accompanying neutropenia.

Targeting complement in therapy.

With increasing evidence that complement activation significantly contributes to the pathogenesis of a large number of inflammatory diseases, strategies that interfere with its deleterious action have become a major focus in pharmacological research. Endogenous soluble complement inhibitors (C1 inhibitor, recombinant soluble complement receptor 1, antibodies) blocking key proteins of the cascade reaction, neutralizing the action of the complement-derived anaphylatoxin C5a, or interfering with complement receptor 3 (CR3, CD18/11b)-mediated adhesion of inflammatory cells to the vascular endothelium have successfully been tested in various animal models over the past years. Promising results consequently led to clinical trials. Furthermore, incorporation of membrane-bound complement regulators (decay-accelerating factor (CD55), membrane co-factor protein (CD46), CD59) in transgenic animals has provided a major step forward in protecting xenografts from hyperacute rejection. At the same time, the poor contribution of complement to the antitumor response, which is caused by multiple resistance mechanisms that hamper the efficacy of antibody-based tumor therapy, is increasingly recognized and requires pharmacologic intervention. First attempts have now been made to interfere with the resistance mechanisms, thereby improving complement-mediated tumor cell destruction.

Therapeutic inhibition of the complement system.

The use of powerful methodologies in molecular biology, biochemistry, and physiology in the last 2 decades had led to impressive progress in our understanding of the mechanisms of complement activation and its role as either a protective or a pathogenic factor in human disease. With respect to disease pathogenesis, the complexity of the complement cascade provides opportunities for several different therapeutic targets within the complement pathways. More than a century after complement was first described, we are about to witness in the near future the availability of a variety of complement inhibitors for specific therapies. Progress in the area of xenotransplantation has been substantial, but formidable obstacles remain to selective inhibition of the factors that block successful clinical xenotransplantation. Bispecific antibodies, designed to enhance rather than inhibit existing complement pathways, hold strong promise for the clearance of viral and bacterial pathogens from the circulation.

Chemoattractant receptors for interleukin-8 and C5a: expression on peripheral blood leukocytes and differential regulation on HL-60 and AML-193 cells by vitamin D3 and all-trans retinoic acid.

Two homologous high-affinity receptors for the chemoattractant interleukin-8, IL-8RA and IL-8RB, and one for the chemoattractant C5a (C5aR) have been cloned. These membrane proteins are members of the rhodopsin superfamily of G-protein coupled seven-transmembrane segment receptors. New monoclonal antibodies (mAb) directed against the deduced N-terminal sequences of the IL-8RA (mAb SE2) and IL-8RB (mAb HC2) were generated to determine the IL-8R expression on human blood leukocytes and two human myeloid cell lines. The C5aR expression was detected using the mAb W17/1. Approximately 107,000 C5aR, 55,000 IL-8RA, and 25,000 IL-8RB molecules per cell could be detected on human granulocytes by flow cytometric analysis. On peripheral blood monocytes, 42,000 C5aR molecules/cell and 3000 IL-8RB molecules/cell were expressed. However, we were unable to quantitate IL-8RA expression, which was detectable but below 2500 molecules per cell and thus outside the standard range for the quantitation of receptor molecules by flow cytometry. On AML-193 cells, only the IL-8RB was constitutively expressed, whereas on HL-60 cells, we could not detect expression of any of the three receptors. Vitamin D3 (250 ng/ml, 7 days), which has been shown to induce differentiation of AML-193 and HL-60 cells into the monocytic phenotype, led to an up-regulation of IL-8RB and C5aR in both cell lines in the absence of any expression of IL-8RA. In contrast, all-trans retinoic acid (0.1 microM, 7 days), which induces differentiation into the granulocytic phenotype, led to an up-regulation of IL-8RB in AML-193 cells and to an expression of IL-8RB and C5aR in HL-60 cells. Again, neither cell line expressed IL-8RA. These findings suggest that regulation of IL-8RA expression differs from that of its IL-8RB homolog and may be a late event in leukocyte maturation.

C3a and C5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway.

Mast cells are known to accumulate at sites of inflammation, however, the chemotaxins involved are undefined. Since most natural leukocyte secretagogues also induce cell migration, and since the anaphylatoxins C3a and C5a are mast cell secretagogues, we hypothesized that both C3a and C5a are also mast cell chemotaxins. Here we report that C3a and C5a are, in fact, potent chemotaxins for the human mast cell line HMC-1. The optimal concentrations, half-maximal effective concentrations (a measure of agonist potency) and the efficacy (response at the optimal concentration) compared with medium control were, for C3a: 10 nM, 0.5 nM, and 256%, respectively; for C5a: 1 nM, 10 pM and 145%. Chemotaxis of HMC-1 cells to both C3a and C5a was blocked by pertussis toxin, suggesting that Gi-coupled receptors are involved in signal transduction. C3a and C5a also induced transient pertussis toxin-inhibitable increases in [Ca2+]i (ED50 = 1 nM for both) that could be homologously but not heterologously desensitized, suggesting that the receptors for C3a and C5a are distinct. These results make C3a the most effective mast cell chemotaxin identified to date. The chemotactic potency described here for C3a is also 100- to 1000-fold greater than for all of its previously described cellular actions. Direct chemoattraction of mast cells by C3a and C5a may help explain the rapid accumulation of mast cells at sites of inflammation.

Granulocyte functions in children with cancer are differentially sensitive to the toxic effect of chemotherapy.

To analyze the toxicity associated to chemotherapy upon granulocytes, different functional assays were performed, within days of drug exposure and at time of bone marrow recovery, on polymorphonuclear neutrophils (PMN) from children with cancer. There were no significant postchemotherapy changes in the expression of the different receptors studied nor in the phagocytosis of Staphylococcus aureus 42D. By contrast, a significant decrease was observed in H2O2 production in PMN recently exposed to chemotherapy with both cytofluorometric and chemiluminescence assays. There was also a decrease in the production of O2- and in chemotaxis; finally, the intracellular killing of S. aureus 42D and Escherichia coli was reduced. In patients having recovered from drug-induced bone marrow aplasia, PMN functions were found to be normal except for bactericidal activity which was still defective. The observations indicate that, in patients exposed to chemotherapy, some PMN functions are transiently altered, whereas microorganism cell killing is continuously impaired.

TNF-alpha suppresses IL-1 alpha and IL-6 upregulation of C4b-binding protein in HepG-2 hepatoma cells.

C4b-binding protein (C4BP) is a regulatory protein involved in the regulation of the classical pathway of complement and the natural anticoagulant pathway. C4BP is synthesized by the liver, a target organ of the IL-6 proinflammatory cytokine. C4BP is an acute-phase protein and its basal levels may increase by as much as 4-fold during an inflammatory response. IL-6 which plays a major role in the modulation of the acute-phase proteins, including C4BP, also has been shown by our group to significantly increase hepatocyte production of the anticoagulant protein, protein S. In this study, we have examined the role of cytokine combinations on the production of the C4BP regulatory protein in the HepG-2 hepatoma cell line and report that IL-1 alpha and IL-6 in combination as well as IL-1 alpha and Oncostatin M (OSM) were approximately additive in the upregulation of C4BP while IL-6 and OSM were not and that TNF-alpha blocked both IL-1 alpha and IL-6 but not OSM upregulation of C4BP.

Promoter region of the human gene coding for beta-chain of C4b binding protein. Hepatocyte nuclear factor-3 and nuclear factor-I/CTF transcription factors are required for efficient expression of C4BPB in HepG2 cells.

Differential expression of the human genes coding for the alpha and beta polypeptides of the human C component C4b binding protein (C4BP) modulates the levels of C4BP molecules containing C4BP beta polypeptides, providing a mechanism to avoid the potential harmful effects of elevated concentrations of C4BP beta in plasma. To understand how the expression of the C4BPB gene is controlled, we have examined, in the major promoter of the human C4BP B gene, potential regulatory elements. A region from nucleotide -126 to +25 was able to drive high expression of a reporter gene in the human hepatoma cell line HepG2. A small subfragment of this region (from -126 to -90) is responsible for more than 90% of the promoter activity. Electrophoretic mobility shift assays revealed that transcription factors of the hepatocyte nuclear factor-3 (HNF-3) and nuclear factor-I (NFI/CTF) families were able to bind to this region in a sequence-specific manner. We have characterized binding sites for these transcription factors and determined their relative contribution to the activity of the C4BPB promoter. The results suggest that cooperative interaction between HNF-3 and NF-I/CTF is required to obtain a full C4BPB promoter activity. Comparison of the structures of the C4BPA and C4BPB promoters reveals significant differences that could explain the differential transcription of the C4BP alpha and C4BP beta polypeptides during the acute phase response.

IFN-gamma up-regulates the human C5a receptor (CD88) in myeloblastic U937 cells and related cell lines.

On human mature monocytes the immunomodulator IFN-gamma has been shown to down-regulate the receptor for the anaphylatoxic peptide C5a (CD88, C5aR). In this study, we show that in immature myelo-/monoblastic U937, HL60, and MonoMac6 cells, IFN-gamma induces C5aR-ligand binding activity. In U937 cells, this induction cannot be blocked by the protein kinase C inhibitor staurosporine. An increase in free cytosolic Ca2+ upon ligand binding indicates functional coupling of this receptor in U937 and HL-60 cells. G-Proteins involved in this C5a responsiveness after IFN-gamma induction are completely pertussis toxin sensitive. Our data suggest that an additional pertussis toxin-resistant pathway exists in U937 cells after induction by dibutyryl cAMP. However, this is not due to changes in the mRNA level of the pertussis toxin-insensitive G-protein subunit G alpha 16. Induction by dibutyryl cAMP, but not that by IFN-gamma, resulted in C5a-dependent release of N-acetyl-beta-D-glucosaminidase, further highlighting functional differences in the effects of the inducers. Our data show an IFN-gamma-dependent increase in C5aR expression and suggest a maturation-related change in signaling of the C5aR, presumably at the level of receptor coupling.

Influence of suramin on the expression of Fc receptors and other markers on human monocytes and U937 cells, and on their phagocytic properties.

Suramin, a polyanionic and polycyclic compound, was initially used for the treatment of trypanosomiasis and onchocerciasis. In the last decade, it has been used in therapy of cancer and acquired immune deficiency syndrome (AIDS). The influence of suramin on the expression of various markers by human mononuclear phagocytes is not known and was, therefore, presently investigated. Suramin inhibited the proliferation of U937 cells and mitogen-induced T-cell proliferation in a dose-dependent manner. The constitutive and cytokine-driven expression of Fc receptors for IgG (Fc gamma RI and Fc gamma RII), IgE (Fc epsilon RII) and IgA (Fc alpha R) on blood monocytes and U937 cells was suppressed by suramin. The basal level, as well as cytokine-induced major histocompatibility complex (MHC) class II antigens, was markedly diminished on suramin-treated monocytes. Furthermore, suramin dramatically reduced expression of CD14 and partially reduced complement receptor type 3 (CR3) and CR4 expression on monocytes. In contrast, suramin slightly induced MHC class I antigens on monocytes and CD71 on U937 cells. The capacity of monocytes to phagocytose IgG-sensitized ox erythrocytes, opsonized Escherichia coli, or fluorescein isothiocyanate (FITC)-conjugated latex beads was significantly inhibited. Northern blot analysis showed that the amount of Fc epsilon RII-specific mRNA was only partially reduced, suggesting that other mechanisms may be involved in the regulation of Fc epsilon RII expression. Our data demonstrate that suramin suppresses the expression of various cell-surface structures on human mononuclear phagocytes and impairs their phagocytic capacity.

C5a stimulus-secretion coupling in rat basophilic leukaemia (RBL-2H3) cells transfected with the human C5a receptor is mediated by pertussis and cholera toxin-sensitive G proteins.

Rat basophilic leukaemia cells (RBL-2H3) were transfected with either the wild type human C5a receptor or a truncated form lacking the last 23 C-terminal residues. Transfected cells bound human C5a specifically, with affinities in the range 3-20nM, and 12-166,000 receptors per cell, similar values to those obtained on human neutrophils and monocytic cells. The stimulation of secretion by human C5a was completely inhibited by pertussis toxin and partially sensitive to cholera toxin, indicating that both wild-type and mutated receptors are coupled to G proteins. Cells transfected with the mutated receptor were equally sensitive to hC5a, suggesting that this portion of the C terminus is not an absolute requirement for signal transduction.

Regulation of eosinophil migration by adult T cell leukemia-derived factor.

Adult T cell leukemia-derived factor (ADF), originally defined as an IL-2 receptor alpha-chain (IL-2R alpha)/p55 (Tac) inducer, is a human thioredoxin homologue and has many cytokine-like activities. In this study, we examined the regulatory effect of ADF on eosinophil migration using human eosinophils and an eosinophilic subline of HL-60 human promyelocytic leukemia cells, YY-1. rADF induced migration of eosinophils from patients with hypereosinophilia, although rADF exhibited little activity on eosinophils from healthy donors. When human eosinophils were incubated with rADF (0.1-10 micrograms/ml) at 37 degrees C for 24 h, both chemotactic and chemokinetic activity of the complement anaphylatoxin peptide C5a on eosinophil migration was markedly enhanced in a dose-dependent manner. Similarly, this enhancing effect of rADF was observed in the migration assay using YY-1 cells. In contrast, rADF showed no modulation of migratory behavior of human eosinophils and YY-1 cells by IL-3, IL-5, nor granulocyte-macrophage colony-stimulating factor. Scatchard analysis of C5a receptors on YY-1 cells using 125I-C5a showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. Furthermore, mutant ADF (mADF), which had no reducing activity, had no enhancing effect on C5a-induced eosinophil migration. These results indicate a possible involvement of ADF in the recruitment of eosinophils through redox regulation by a dithiol reductase activity.

Lipopolysaccharide-induced suppression of erythrocyte binding and phagocytosis via Fc gamma RI, Fc gamma RII, Fc gamma RIII, and CR3 receptors in murine macrophages.

In this study we have investigated the ability of lipopolysaccharide (LPS) to suppress binding and phagocytosis of erythrocytes via various receptors on mouse macrophages. Thioglycollate-elicited peritoneal macrophages were treated in vitro with LPS and the ability to bind and phagocytose radiolabeled sheep red blood cells was determined. We show that LPS can directly suppress phagocytosis of immunoglobulin G-opsonized and nonopsonized sheep red blood cells (SRBCs) by inflammatory macrophages. Suppression was dose dependent and was observed after 4 h of exposure. This effect lasted for at least 24 h following the removal of LPS. LPS suppressed the binding, rate, and absolute level of phagocytosis via Fc receptors. Phagocytosis via all Fc receptors (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) was suppressed by LPS. Furthermore, suppression was not limited to Fc receptor-mediated phagocytosis because binding and uptake of C3bi-opsonized SRBCs to CR3 receptors was also decreased following LPS treatment. LPS did not exert its effects via the production of interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha, or interferon alpha/beta, because antibodies to these cytokines did not abrogate the effect. The ability of LPS to suppress binding and phagocytosis of microorganisms may contribute to the toxic effects of LPS during gram-negative sepsis by preventing or delaying elimination of bacteria by host macrophages.