Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH), factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001), were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes.This study is the first to show systemic complement activation in AMD patients. This suggests that AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were previously linked to AMD susceptibility.

Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.

Expression levels of the chemokine receptor, CC chemokine receptor 5 (CCR5), at the cell surface determine cell susceptibility to HIV entry and infection. Cellular activation by CCR5 itself, but also by unrelated receptors leads to cross-phosphorylation and crossinternalization of CCR5. This study addresses the underlying molecular mechanisms of homologous and heterologous CCR5 regulation. As shown by bioluminescence resonance energy transfer experiments, CCR5 formed constitutive homo-as well as heterooligomeric complexes together with C5aR but not with the unrelated AT 1a R in living cells. Stimulation with CCL5 of RBL cells, which co-expressed CCR5 together with an N-terminally truncated CCR5-⌬NT mutant, resulted in both protein kinase C (PKC)-and G protein-coupled receptor (GPCR) kinase (GRK)-mediated crossphosphorylation of the mutant unligated receptor, as determined by phosphosite-specific monoclonal antibody. Similarly, both PKC and GRK cross-phosphorylated CCR5 in a heterologous manner after C5a stimulation of RBL-CCR5/C5aR cells, whereas AT 1a R stimulation resulted only in classical PKC-mediated CCR5 phosphorylation. Co-expression of CCR5-⌬NT together with a phosphorylation-deficient CCR5 mutant that neither binds ␤-arrestin nor undergoes internalization partially restored the CCL5-induced association of ␤-arrestin with the homo-oligomeric receptor complex and augmented cellular uptake of 125 I-CCL5. Co-expression of C5aR, but not of AT 1a R, promoted CCR5 co-internalization upon agonist stimulation by a mechanism independent of CCR5 phosphorylation. Co-internalization of phosphorylated CCR5 was also observed in C5a-stimulated macrophages. Finally, co-expression of a constitutively internalized C5aR-US28 CT mutant led to intracellular accumulation of CCR5 in the absence of ligand stimulation. These results show that GRKs and ␤-arrestin are involved in heterologous receptor regulation by cross-phosphorylating and co-internalizing unligated receptors within homo-or hetero-oligomeric protein complexes.Leukocytes express multiple pertussis toxin-sensitive chemoattractant receptors that may be engaged simultaneously or sequentially as these cells are recruited into tissue sites of inflammation. Interaction of such receptors with their cognate ligands results, among other cellular functions, in directional cell movement, integrin activation, and release of granular contents (1). Upon ligand binding, receptors also undergo adaptive changes, which include desensitization and internalization. Two major mechanisms of rapid receptor regulation have been discriminated, namely homologous (agonist-specific) and heterologous (agonist-nonspecific) desensitization, and both mechanisms are believed to be important in fine tuning leukocyte responses (2).Homologous desensitization involves phosphorylation of ligand-occupied receptors by members of the GPCR 3 kinase (GRK) family with, according to the current paradigm, essentially no effect on other receptors expressed in the same cell (3, 4). These kinases often phosphory...

Human CC chemokine receptor 5 (CCR5), a member of the superfamily of G protein-coupled receptors, regulates the activation and directed migration of leukocytes and serves as the main coreceptor for the entry of R5 tropic strains of human immunodeficiency viruses. We have previously shown that RANTES/CCL5 binding to CCR5 induces GPCR kinase (GRK)-and protein kinase C (PKC)-mediated phosphorylation of four distinct C-terminal serine residues. To study these phosphorylation events in vivo, we have generated monoclonal antibodies, which specifically react only with either phosphorylated or nonphosphorylated CCR5. These phosphosite-specific antibodies reveal that following ligand stimulation of the receptor serine 337 is exclusively phosphorylated by a PKC-mediated mechanism, while GRKs phosphorylate serine 349. GRK-mediated receptor phosphorylation proceeds in a regular time-dependent manner (t1 ⁄2 ϳ2 min) with an apparent EC 50 of 5 nM. In contrast, PKC phosphorylates serine 337 at 50-fold lower concentrations and in a very rapid, albeit transient manner. Protein phosphatases that are active at neutral pH and are inhibited by okadaic acid rapidly dephosphorylate phosphoserine 337, but less efficiently phosphoserine 349, in intact cells and in an in vitro assay. Immunofluorescence microscopy demonstrates that phosphorylated receptors accumulate in a perinuclear compartment, which resembles recycling endosomes. This study is the first to analyze in detail the spatial and temporal dynamics of GRK-versus PKCmediated phosphorylation of a G protein-coupled receptor and its subsequent dephosphorylation on the level of individual phosphorylation sites. G protein-coupled receptors (GPCR)1 comprise the largest known family of signal-transducing proteins and respond to a large variety of external stimuli (1, 2). The receptors relay the information encoded by the ligand through the activation of heterotrimeric guanine nucleotide-binding proteins and intracellular effector molecules. Many GPCR undergo a process of rapid desensitization, which involves ligand-induced phosphorylation of serine and threonine residues located in the third intracellular loop or C-terminal domain by two different families of protein kinases. (i) GPCR kinases (GRKs) specifically phosphorylate only the agonist-occupied GPCR and thus mediate agonist-specific or homologous receptor phosphorylation (3, 4). (ii) In contrast, the second messenger-activated kinases, such as cyclic AMP-dependent protein kinase and protein kinase C (PKC), potentially phosphorylate both the ligand-bound GPCR and multiple other receptors in a heterologous manner. Receptor phosphorylation enhances the affinity of the agonistoccupied receptor for interaction with arrestin which interdicts signal transduction between the receptor and G proteins by steric mechanisms. The nonvisual arrestins, ␤-arrestin-1 and ␤-arrestin-2, also promote clathrin-mediated endocytosis of phosphorylated receptors and have been implicated in crosstalk with other signaling pathways. Once internalized, GPCR...

Given the significance of CD40-CD40 ligand interactions in chronic inflammatory diseases including atherosclerosis, the transcriptional regulation of CD40 expression as a potential therapeutic target was investigated in human umbilical vein cultured endothelial cells. Exposure to interferon-␥ (IFN-␥) plus tumor necrosis factor-␣ resulted in a marked synergistic de novo expression of CD40, which, according to electrophoretic mobility shift analysis, was attributable to activation of the transcription factors nuclear factor-B (NF- B IntroductionCD40 (or TNFR5) is a cell-surface receptor belonging to the tumor necrosis factor (TNF) receptor superfamily that is principally expressed by B cells, but also by other antigen-presenting cells and, in addition, by a variety of nonimmune cells such as smooth muscle cells, fibroblasts, and endothelial cells. 1,2 The corresponding ligand (CD40L or CD154) has been cloned and identified as a CD4 ϩ T-cell activation antigen. 3 CD40-CD154 interactions play a critical role in the regulation of both humoral and cellular immunity. 4 In endothelial cells, CD40 stimulation causes a TNF-␣-like increase in expression of adhesion molecules and chemokines that promote the homing and extravasation of leukocytes at sites of inflammation. 5,6 Moreover, in addition to monocytes, endothelial cells produce bioactive interleukin-12 (IL-12) in response to CD40 stimulation. 7 IL-12 is a potent differentiation factor for naive T-helper cells, promoting their clonal expansion into Th1 cells. 8 These aforementioned events also seem to be important for the development of atherosclerosis, and all of the principal cells present in human atherosclerotic lesions, such as endothelial cells, macrophages, smooth muscle cells, and T-helper cells, express CD40, CD154, or both. 9,10 Moreover, anti-CD154 antibodies are capable of reducing the size of atherosclerotic lesions in hyperlipidemic mice 11 and limiting heart-transplant atherosclerosis in the same species. 12 However, because of adverse side effects, the use of such antibodies may be limited in patients with chronic inflammatory diseases. 13 Moreover, as yet no low-molecularweight antagonist for CD40 has been developed, and anti-CD40 antibodies stimulate rather than inhibit CD40 signaling in cells expressing the receptor. 14 Suppression of CD40 expression in CD154 target cells may thus provide a feasible therapeutic alternative.Cytokine-inducible expression of CD40 in rat vascular smooth muscle cells is mediated by the transcription factors nuclear factor-B (NF-B) and signal transducer and activator of transcription-1 (STAT-1). 15 In the mouse macrophage cell line RAW 264.7, STAT-1 and 2 Ets family members (PU.1 and Spi-B) are involved in interferon-␥ (IFN-␥) induction of CD40 gene expression. 16 Although sequenced in part, the promoter of the human CD40 gene has not been functionally characterized, 17 so the transcription factors governing CD40 expression in human cells are not yet known. Therefore, we investigated the transcriptional regulation of cyt...

Upon agonist binding, the C5a anaphylatoxin receptor (C5aR) is rapidly phosphorylated on phosphorylation sites that are located within the C-terminal domain of the receptor. Previous studies suggested that C5aR phosphorylation proceeds in a hierarchical manner with serine 334 presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. To better understand the dynamics of Ser-334 phosphorylation, we generated site-specific monoclonal antibodies that specifically react with phosphoserine 334. In differentiated U937 cells, which endogenously express C5aR, stimulation with low C5a concentrations resulted in a very rapid (t 1 ⁄ 2 ϳ 20 s), albeit transient, receptor phosphorylation. Whole cell phosphorylation assays with specific inhibitors as well as in vitro phosphorylation assays with recombinant enzymes and peptide substrates revealed that phosphorylation of Ser-334 is regulated by protein kinase C-␤ and a calyculin A-sensitive protein phosphatase. Surprisingly, at high concentrations (>10 nM) of C5a, the protein kinase C-mediated phosphorylation of Ser-334 was essentially blocked. This could be attributed to the even faster (t 1 ⁄ 2 < 5 s) binding of ␤-arrestin to the receptor. Analysis of C5aR Ser/Ala mutants that possess a single intact serine residue either at position 334 or at neighboring positions 327, 332, or 338 revealed functional redundancy of C-terminal phosphorylation sites since all 4 serine residues could individually support C5aR internalization and desensitization. This study is among the first to analyze in a detailed manner, using a non-mutational approach, modifications of a defined phosphorylation site in a G protein-coupled receptor and to correlate these findings with functional parameters of receptor deactivation.

Transplant recipients who have had sensitizing events such as pregnancies, blood transfusions and previous transplants often develop antibodies directed against human leukocyte antigen (HLA)-molecules of the donor tissue. These pre-formed donor-specific antibodies (DSA) represent a high risk of organ failure as a consequence of antibody-mediated hyper-acute or acute allograft rejection. As a first assay to detect DSA, the complement-dependent lymphocytotoxicity assay (CDC) was established more than 40 years ago. However, this assay is characterized by several drawbacks such as a low sensitivity and a high susceptibility to various artificial factors generally not leading to valid and reliable outcomes under several circumstances that are reviewed in this article. Furthermore, only those antibodies that exert complement-fixing activity are detected. As a consequence, novel procedures that act independently of the complement system and that do not represent functional assays were generated in the format of solid phase assays (SPAs) (bead- or ELISA-based). In this article, we review the pros and cons of these sensitive SPA in comparison with the detection of DSA through the use of the traditional methods such as CDC and flow cytometric analyses. Potential drawbacks of the alternative methodological approaches comprising high background reactivity, susceptibility to environmental factors and the possible influence of subjective operators' errors concerning the interpretation of the results are summarized and critically discussed for each method. We provide a forecast on the future role of SPAs reliably excluding highly deleterious DSA, thus leading to an improved graft survival.

SUMMARY:The 155-kd soluble complement regulator factor H (FH), which consists of 20 short consensus repeats, increases the affinity of complement factor I (FI) for C3b by about 15 times. In addition to its cofactor activity, it prevents factor B from binding to C3b and promotes the dissociation of the C3bBb complex. The primary site of synthesis of FH, as well as of FI, is the liver, but the cell types responsible for the hepatic synthesis of both factors have not yet been clearly identified. In contrast to FI-mRNA, which was detectable only in hepatocytes (HC), FH-specific mRNA was identified in both HC and Kupffer cells (KC). As calculated for equal amounts of mRNA isolated from both cell types, FH-specific mRNA was found to be nearly 10-fold higher in KC than in HC, leading to the conclusion that KC are an abundant source of FH. Of the investigated proinflammatory cytokines IL-6, TNF-␣, IL-1␤, and IFN-␥, only IFN-␥ up-regulated FH-specific mRNA up to 6-fold in both primary HC and KC. This was also demonstrable on the protein level. However, FH-specific mRNA was not inducible in the rat hepatoma cell line H4IIE, which did not express FH-specific mRNA and could not be up-regulated in FAO cells that constitutively expressed FH-specific mRNA. This demonstrates that transformed cell lines do not reflect FH regulation in isolated primary HC. In addition to IFN-␥, the endotoxin lipopolysaccharide (LPS) up-regulated FH-specific mRNA nearly 10-fold in KC after stimulation at concentrations of 10 or 1 ng/ml. In contrast, concentrations of up to 2 g LPS/ml did not show any effect on HC. Our data suggest that LPS does not regulate the expression of FH in HC. (Lab Invest 2002, 82:183-192).T he regulation of complement activation at the level of C3 and C4 is mediated by several receptors and soluble regulatory proteins. These include complement receptor 1 (CD35), complement receptor 2 (CD21), decay-accelerating factor (CD55), membrane cofactor protein (CD46), factor I (FI), and factor H (FH). These proteins, with the exception of FI, form the "regulators of complement activation" family (Hourcade et al, 1989). Their genes are closely linked on chromosome 1 in man and mouse. This illustrates that this region on the long arm of chromosome 1 in humans is analogous to that region in mice (Klickstein et al, 1985). The serine protease FI could be located to chromosome 4 in man (Goldberger et al, 1987) and to chromosome 3 in mouse (Minta et al, 1996).The potentially deleterious complement system is strictly regulated to prevent damage in the absence of pathologic situations Mulligan et al, 1992;Piddlesden et al, 1994;Smith et al, 1993). On some host cell surfaces, membrane-bound complement regulatory factors are expressed only at low levels. Thus, reduced protection against complement attack may be compensated by the soluble regulator FH, which has a critical role in complement inactivation in the fluid phase (Meri and Pangburn, 1990; Pangburn and Müller-Eberhardt, 1978) and acts in concert with FI. FH is a single-chain protein...