Adenosine receptors: G protein-mediated signalling and the role of accessory proteins.

Ever since the discovery of the effects of adenosine in the circulation, adenosine receptors continue to represent a promising drug target. Firstly, this is due to the fact that the receptors are expressed in a large variety of cells; in particular, the actions of adenosine (or, respectively, of the antagonistic methylxanthines) in the central nervous system, in the circulation, on immune cells and on other tissues can be beneficial in certain disorders. Secondly, there exists a large number of ligands, which have been generated by introducing several modifications in the structure of the lead compounds (adenosine and methylxanthine), some of them highly specific. Four adenosine receptor subtypes have been identified by molecular cloning; they belong to the family of G protein-coupled receptors, which transfer signals by activating heterotrimeric G proteins. It has been appreciated recently that accessory proteins impinge on the receptor/G protein interaction and thus modulate the signalling reaction. These accessory components may be thought as adaptors that redirect the signalling pathway to elicit a cell-specific response. Here, we review the recent literature on adenosine receptors and place a focus on the role of accessory proteins in the organisation of adenosine receptor signalling. These components have been involved in receptor sorting, in the control of signal amplification and in the temporal regulation of receptor activity, while the existence of others is postulated on the basis of atypical cellular reactions elicited by receptor activation.     

Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis

Besides its traditional role in hemostasis, factor XIII subunit A (FXIII-A) is supposed to function as a cellular transglutaminase and to be involved in certain intracellular processes, including cytoskeletal remodeling. To investigate its intracellular role, the aim of the present study was to follow changes in FXIII-A production in combination with the receptor-mediated phagocytic activities of monocytes/macrophages and to examine the phagocytic functions of monocytes in patients with FXIII-A deficiency. Human blood monocytes were isolated from the buffy coats of healthy volunteers and cultured for 4 days. The FcgammaR-mediated phagocytosis of sensitized erythrocytes (EA) and the complement receptor (CR)-mediated phagocytosis of complement-coated yeast particles were studied during monocyte/macrophage differentiation. Changes in the gene expression of FXIII-A were detected by real-time quantitative RT-PCR. FXIII-A protein production was investigated with fluorescent image analysis at single cell level and Western immunoblot analysis. Both the FcgammaR and CR-mediated phagocytosis increased during culturing, which peaked on day 3. The phagocytic activity of the cells could be markedly inhibited with monodansylcadaverine, an inhibitor of the transglutaminase-induced crosslinking of proteins. The phagocytosis of EA, complement-coated and uncoated yeast particles was found to be strongly diminished in monocytes of FXIII-A deficient patients. The phagocytic functions of cultured cells showed a change in parallel with the alterations in FXIII-A mRNA expression, as well as with that in FXIII-A in protein synthesis detected by image and Western immunoblot analyses in concert. Our results suggest that FXIII-A plays a role in the Fcgamma and complement receptor-mediated phagocytic activities of monocytes/macrophages.     

Masugi's nephritis: preparation of an active nephrotoxic serum

An active nephrotoxic serum (NTS) was prepared by immunization of rabbits with isolated renal glomeruli of rats and Freund's complete adjuvant. The glomeruli were isolated from the renal cortex by the sieving procedure. Intravenous injection of the NTS to rats induced the development of marked proteinuria and the nephrotic syndrome. Histological and electron microscopic studies of renal tissue performed by days 5 and 15 since the first injection of the NTS revealed extracapillary proliferative glomerulonephritis. IgG depositions on the basal membranes of the glomerular capillaries were linear on day 5 and granular on day 15 since the first injection of the NTS.     

Interaction of West Nile virus with primary murine macrophages: role of cell activation and receptors for antibody and complement

We have measured growth of West Nile virus in mouse primary peritoneal macrophages (resident, thioglycolate elicited, and Mycobacterium bovis BCG activated) and in macrophagelike (P338D1) and nonmacrophage (L929, PS clone D) cell lines infected in the absence or presence of specific antibodies (immunoglobulin G ([IgG], IgM), and complement. Monoclonal antibodies directed against Fc receptors (IgG1/2b, 2.4G2) and type 3 complement receptors (Mac-1) were used to define the role of each receptor. Virus yield depended on a balance between enhancement and neutralization and was influenced by the physiologic state of the macrophage, the receptor pathway of viral entry, the mouse strain and age of donor. BCG-activated macrophages displayed a greater ability to restrict West Nile virus than nonactivated cells only in the presence of antiviral IgM, with or without complement; the Fc receptors for various classes of IgG mediated striking enhancement. These studies identify some of the complex innate and acquired factors that determine the interaction between West Nile virus and primary macrophages in vitro.     

Follicular dendritic cells and the persistence of HIV infectivity: the role of antibodies and Fcgamma receptors

Large quantities of HIV are found trapped on the surface of follicular dendritic cells (FDCs), and virus persists on these cells until they ultimately die. We recently found that FDCs maintain HIV infectivity for long periods in vivo and in vitro. Because FDCs trap Ags (and virus) in the form of immune complexes and are rich in FcgammaRs, we reasoned that Ab and FcgammaRs may be required for FDC-mediated maintenance of HIV infectivity. To investigate this hypothesis, HIV immune complexes were formed in vitro and incubated for increasing times with or without FDCs, after which the remaining infectious virus was determined by HIV-p24 production in rescue cultures. FDCs maintained HIV infectivity in vitro in a dose-dependent manner but required the presence of specific Ab for this activity regardless of whether laboratory-adapted or primary X4 and R5 isolates were tested. In addition, Abs against either virally or host-encoded proteins on the virion permitted FDC-mediated maintenance of HIV infectivity. We found that the addition of FDCs to HIV immune complexes at the onset of culture gave optimal maintenance of infectivity. Moreover, blocking FDC-FcgammaRs or killing the FDCs dramatically reduced their ability to preserve virus infectivity. Finally, FDCs appeared to decrease the spontaneous release of HIV-1 gp120, suggesting that FDC-virus interactions stabilize the virus particle, thus contributing to the maintenance of infectivity. Therefore, optimal maintenance of HIV infectivity requires both Ab against particle-associated determinants and FDC-FcgammaRs.     

Age and the course of nephrotoxic nephritis in rats]

In experiments on two groups of mongrel rats (4 weeks old and 4 months old) with induced nephrotoxic nephritis it was revealed that in comparison with adult rats the course of nephritis in ratlings was characterized by lesser proteinuria, selective in nature, by lesser reducticn of endogenous creatinine clearance and diuresis. The acido- and ammo-niogenesis decreased in ratlings and adult rats to the same extent. Morphological changes in the kidneys of ratlings were less pronounced than in adult animals, and were mostly localized in the convoluted tubules. The level of DNA-synthetic activity of the epithelial nuclei of the glomeruli prevailed over this index of the convoluted tubules epithelium. The weight index of the kidneys increased less in ratlings with nephritis than in adult rats. beta-lipoproteinemia in ratlings increased 8 times. Normalization of the urine and blood indices occurred more rapidly in ratlings than in adult rats.     

Binding of human C-reactive protein to mouse macrophages is mediated by distinct receptors

Human C-reactive protein (CRP) is known to activate mouse macrophages (M phi) to a tumoricidal state and to serve as an opsonin for M phi. Therefore, cell surface receptors for CRP on mouse M phi were characterized and their relationship to the IgG FcR determined. The specific binding of 125I-CRP to resident or elicited mouse M phi was saturable, reversible, and involved both a high and a low affinity receptor population. Binding of CRP to the mouse M phi cell lines PU5 1.8 and J774 was nearly identical to that observed with peritoneal M phi. The high affinity receptor population had a calculated K of 10 nM and a receptor density of approximately 10(5) sites per cell. Mouse Ig of the IgG2a, IgG2b, or IgG1 isotypes inhibited binding of 125I-CRP to PU5 1.8 cells at concentrations five-fold greater than that of the homologous ligand. In the converse experiment, unlabeled CRP failed to inhibit specific binding of 125I-labeled IgG2a, IgG2b or IgG1. Isolation of CRP binding proteins from surface iodinated PU5 1.8 cells by ligand-affinity chromatography or chemical cross-linking yielded a major protein band of 57 to 60 kDa which appeared to be distinct from the IgG1/IgG2b FcR (FcR-II) membrane proteins. Removal of radiolabeled IgG2b/IgG1 binding membrane proteins by affinity chromatography did not remove CRP-binding proteins. The rat mAb 2.4G2 which inhibits binding of radiolabeled mouse IgG2b, did not inhibit the binding of CRP. A rat polyclonal antiserum to CRP-binding membrane proteins of PU5 1.8 cells inhibited 125I-CRP binding, but not 125IgG2b binding. The rat polyclonal antibody reacted with two 57 to 60 kDa membrane proteins from PU5 1.8 cells that appear to be of a similar size on Western blots. The 125I-CRP was internalized via endosomes and intact CRP subunits could be detected intracellularly. The findings suggest that binding of CRP occurs through a receptor that is distinct from the IgG FcRs, but that CRP-R activity may be influenced by an association with an IgG FcR.     

Combinatorial model of chemokine involvement in glomerular monocyte recruitment: role of CXC chemokine receptor 2 in infiltration during nephrotoxic nephritis

A sequential model involving chemokines has been proposed for leukocyte extravasation into areas of inflammation; however, site-specific aspects remain to be elucidated. Hence, we studied the role of chemokines produced by mesangial (MC) or glomerular endothelial cells (GEC) and their receptors in glomerular recruitment of monocytes. Stimulation of MC with TNF-alpha up-regulated mRNA and protein of CC and CXC chemokines but not constitutive expression of the CX(3)C chemokine fractalkine. While growth-related activity (GRO)-alpha was immobilized to MC proteoglycans, monocyte chemotactic protein (MCP)-1 was secreted into the soluble phase. Firm adhesion and sequestration of monocytes on activated MC was supported by the GRO-alpha receptor CXCR2 and to a lesser extent by CX(3)CR, whereas the MCP-1 receptor CCR2 contributed to their transendothelial chemotaxis toward activated MC. In contrast, fractalkine mRNA and protein was induced by TNF-alpha in transformed rat GEC, and both CXCR2 and CX(3)CR mediated monocyte arrest on GEC in shear flow. The relevance of these mechanisms was confirmed in a rat nephrotoxic nephritis model where acute glomerular macrophage recruitment was profoundly inhibited by blocking CXCR2 or CCR2. In conclusion, our results epitomize a combinatorial model in which chemokines play specialized roles in driving glomerular monocyte recruitment and emphasize an important role for CXCR2 in macrophage infiltration during early phases of nephrotoxic nephritis.     

Structural basis of the interaction between IgG and Fcgamma receptors

The binding of multivalent antigen-antibody complexes to receptors for the Fc portion of IgG (FcgammaR) induces the clustering of the FcgammaR and triggers cell activation leading to defence reactions against pathogens. The Fc portion of IgG consists of two identical polypeptide chains which are related to each other by a 2-fold axis and are folded in two structural domains, the C(H)2 domain, near the flexible hinge region of the IgG molecule, and the C(H)3 domain. We studied the interaction in solution between the Fc fragment of mouse IgG2b and the extracellular region of mouse FcgammaRII. We find that one Fc molecule binds one FcgammaRII molecule only. Using NMR spectroscopy, we show that FcgammaRII binds to a negatively charged area of the C(H)2 domain, corresponding to the lower hinge region, and that the binding of FcgammaRII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. We therefore propose a model that explains why IgG molecules are unable to trigger FcgammaR-mediated cellular responses spontaneously in the absence of crosslinking by multivalent antigens.     

Class A scavenger receptors, macrophages, and atherosclerosis

The scope of this review is to discuss the new advances in our understanding of the role of scavenger receptor class A in the initiation and modulation of the atherosclerotic process. Through the approaches of gene manipulation in the mouse model, a substantial body of literature has accumulated that depicts scavenger receptor class A as a central player in atherogenesis. In studies of scavenger receptor class A overexpression in macrophages through bone marrow transplantation using transgenic donor material, recipient mice with hyperlipidemia caused either by apolipoprotein E or LDL receptor deficiency did not show convincing changes in the degree of atherosclerosis development compared with controls. Conversely, the deletion of the scavenger receptor class A gene in the mouse has shown, in a consistent and significant fashion, that this receptor serves a pro-atherogenic function under hyperlipidemic conditions, as both apolipoprotein E and LDL receptor-deficient mice had reduced atherosclerosis in the absence of scavenger receptor class A. In addition, we have recently shown that C57BL/6 mice are protected from diet-induced atherosclerosis when they lack scavenger receptor class A, and that the macrophage is the cell type responsible for the effect of scavenger receptor class A deficiency in reducing lesion formation in C57BL/6 and LDL receptor null mice. Together, these results demonstrate that macrophage scavenger receptor class A contributes significantly to atherosclerotic lesion formation, and suggest that the uptake of oxidized or modified lipoproteins by vessel wall macrophages is a central process in atherogenesis.     

Fcgamma receptors in the initiation and progression of systemic lupus erythematosus

Systemic lupus erythematosus, a systemic autoimmune disorder, is characterized by the production of autoantibodies to nuclear constituents and inflammatory lesions in multiple organ systems. Although the pathogenesis of the disease is largely unknown, recent studies have suggested that disturbances in apoptosis and/or clearance of apoptotic cells may play an important role in the induction and perpetuation of autoantibody production. When autoantibodies subsequently complex to autoantigens present on apoptotic cells, ligation of Fcgamma receptor will result in inflammation and disease development. Indeed, mice deficient in activating Fcgamma receptors were protected against inflammation in models of immune complex-mediated autoimmune disease, whereas deletion of the inhibitory Fcgamma receptors increased autoantibody production and susceptibility to immune complex-induced inflammation. Additionally, functional polymorphisms in Fcgamma receptors were shown to be associated with development of human systemic lupus erythematosus. This review focuses on the role of Fcgamma receptors in the initiation of autoantibody production, inflammatory handling of immune complexes, and disease development in systemic lupus erythematosus.     

Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase

The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells.     

Release of arachidonic acid and formation of oxygenated derivatives after complement attack on macrophages: role of channel formation

Treatment of [3H]arachidonic acid ([3H]C20:4)-labeled, antibody-sensitized mouse resident peritoneal macrophages with rabbit serum complement, or C6-deficient rabbit serum + C6, caused hydrolytic release of incorporated [3H]C20:4 from phospholipids, followed by conversion to oxygenated derivatives. The C6 dose-response curve for release of C20:4 plus its metabolites was monotonic, which indicates dependence on channel formation, whereas the dose-response curve for lysis displayed multi-hit behavior. High-performance liquid chromatography demonstrated that the major radiolabeled products in the aqueous phase co-eluted with C20:4, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin E2. Kinetic studies of the release of 6-keto-PGF1 alpha, the major metabolite, displayed biphasic characteristics; a moderate amount of this prostaglandin was released before the onset of cell lysis. Experimental evidence obtained by freeze-thaw or by incubation of these cells with melittin or A23187 indicated that cell lysis does not necessarily result in the production of inflammatory mediators. Furthermore, when macrophages were treated with serum complement, it was apparent that the major part of the release was due to C5b-9 and not to the action of C5a. We conclude that release of C20:4 and its derivatives from complement-treated macrophages does not depend on cytolysis, but is a consequence of insertion and channel formation.     

Protection from Streptococcus pneumoniae infection by C-reactive protein and natural antibody requires complement but not Fc gamma receptors

Streptococcus pneumoniae is an important human pathogen and the most common cause of community-acquired pneumonia. Both adaptive and innate immune mechanisms provide protection from infection. Innate immunity to S. pneumoniae in mice is mediated by naturally occurring anti-phosphocholine (PC) Abs and complement. The human acute-phase reactant C-reactive protein (CRP) also protects mice from lethal S. pneumoniae infection. CRP and anti-PC Ab share the ability to bind to PC on the cell wall C-polysaccharide of S. pneumoniae and to activate complement. CRP and IgG anti-PC also bind to Fc gamma R. In this study, Fc gamma R- and complement-deficient mice were used to compare the mechanisms of protection conferred by CRP and anti-PC Ab. Injection of CRP protected wild-type, FcR gamma-chain-, Fc gamma RIIb-, and Fc gamma RIII-deficient mice from infection. Complement was required for the protective effect of CRP as cobra venom factor treatment eliminated the effect of CRP in both gamma-chain-deficient and wild-type mice, and CRP failed to protect C3- or C4-deficient mice from infection. Unexpectedly, gamma-chain-deficient mice were extremely sensitive to pneumococcal infection. This sensitivity was associated with low levels of natural anti-PC Ab. Gamma-chain-deficient mice immunized with nonencapsulated S. pneumoniae produced both IgM- and IgG PC-specific Abs, were protected from infection, and were able to clear the bacteria from the bloodstream. The protection provided by immunization was eliminated by complement depletion. The results show that in this model of systemic infection with highly virulent S. pneumoniae, protection from lethality by CRP and anti-PC Abs requires complement, but not Fc gamma R.     

Concurrent and independent binding of Fcgamma receptors IIa and IIIb to surface-bound IgG

Fc receptor-antibody interactions are key mechanisms through which antibody effector functions are mediated. Neutrophils coexpress two low-affinity Fcgamma receptors, CD16b (FcgammaRIIIb) and CD32a (FcgammaRIIa), possessing overlapping ligand binding specificities but distinct membrane anchor and signaling capacities. Using K562 cell transfectants as a model, the kinetics of both separate and concurrent binding of CD16b and CD32a to surface-bound IgG ligands were studied. CD16b bound human IgG with 2-3 times higher affinity than did CD32a (A(c)K(a) = 4.1 and 1.6 x 10(-7) microm(4), respectively) and both FcgammaRs had similar reverse kinetic rates (k(r) = 0.5 and 0.4 s(-1), respectively). Because CD16b is expressed on neutrophils at a 4-5 times higher density than CD32a, our results suggest that CD16b plays the dominant role in binding of neutrophils to immobilized IgG. The question of possible cross-regulation of binding affinity between CD16b and CD32a was investigated using our multispecies concurrent binding model (Zhu and Williams, Biophys. J. 79:1850-1857, 2000). Because the model assumes independent binding (no cooperation among different species), the excellent agreement between the model predictions and the experimental data suggests that, when coexpressed on K562 cells, these two FcgammaRs do not interact in a manner that alters the kinetic rates of either molecule.