Beatrix Pollok-Kopp

Genetic control of the alternative pathway of complement in humans and age-related macular degeneration

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.

G Protein-coupled Receptor Kinases Promote Phosphorylation and β-Arrestin-mediated Internalization of CCR5 Homo- and Hetero-oligomers

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 cross-internalization 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 AT1aR in living cells. Stimulation with CCL5 of RBL cells, which co-expressed CCR5 together with an N-terminally truncated CCR5-ΔNT mutant, resulted in both protein kinase C (PKC)- and G protein-coupled receptor (GPCR) kinase (GRK)-mediated cross-phosphorylation 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 AT1aR stimulation resulted only in classical PKC-mediated CCR5 phosphorylation. Co-expression of CCR5-ΔNT together with a phosphorylation-deficient CCR5 mutant that neither binds β-arrestin nor undergoes internalization partially restored the CCL5-induced association of β-arrestin with the homo-oligomeric receptor complex and augmented cellular uptake of 125I-CCL5. Co-expression of C5aR, but not of AT1aR, 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-US28CT mutant led to intracellular accumulation of CCR5 in the absence of ligand stimulation. These results show that GRKs and β-arrestin are involved in heterologous receptor regulation by cross-phosphorylating and co-internalizing unligated receptors within homo- or hetero-oligomeric protein complexes.

Polymorphism of CC chemokine receptors CCR2 and CCR5 in Crohn's disease

Crohns disease (CD) is a chronic inflammatory disease of the intestine that is characterized by mononuclear cell infiltration and a predominant Th1 lymphocyte response. We tested the hypothesis that CC chemokine receptors CCR2 and CCR5 might be important in the regulation of the intestinal immune response in this disease, and we speculated that carriers of a defective 32 base pair deletion mutant of CCR5, CCR5Delta32, which results in a non-functional receptor, might be protected from CD. Using polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) gene frequencies of CCR5Delta32 and of CCR2-641 (replacement of valine-64 by isoleucine in the CCR2 gene) in healthy controls (n=346) and in CD patients (n=235) were determined. In CD patients, subgroup phenotypic analyses were performed according to the Vienna classification. The overall gene frequency of CCR5Delta32 (9.8%) and CCR2-641 (7.6%) in CD patients did not deviate significantly from healthy controls (9.2 and 8.2%, respectively), nor did we observe a significant deviation from the predicted Hardy-Weinberg distribution. No significant differences in the CD phenotype classification for the different CCR5 and CCR2 alleles were observed, except for a trend to disease sparing of the upper gastrointestinal tract (carrier frequency 0 versus 19.6%, Delta=1 9.6%, P=0.079) as well as a more stricturing disease behaviour (23.5 versus 16.2%, Delta=7.3%, P=0.136) in carriers of the mutant CCR5Delta32 allele. These results indicate that the different CCR5 but not CCR2 alleles may influence disease behaviour and thereby contribute to the observed heterogeneity of CD. However, the associations observed are limited and await replication in other datasets. CCR2 and CCR5 polymorphisms are unlikely to be important determinants of overall disease susceptibility.

Rat complement factor H: molecular cloning, sequencing and quantification with a newly established ELISA.

Factor H (FH) is the predominant soluble regulatory protein of the complement system. With a concentration of 300–600 µg/ml in human plasma it acts as a cofactor for the FI‐mediated cleavage of the component C3b to iC3b. Furthermore, it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex (i.e. it has decay accelerating activity). FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of nearly 60 amino acid residues. For the screening of a rat liver cDNA library, we used two hybridization probes which had been produced by polymerase chain reaction (PCR). The probes were generated using degenerated primers which corresponded to conserved parts of the human and the murine factor H nucleotide sequences. The entire rat sequence spanned 4240 nucleotides with an open reading frame of 3708 nucleotides. These were preceded by 23 nucleotides of the 5′ untranslated region, followed by a stop codon and a 3′ untranslated region of 478 nucleotides including the polyadenylation‐signal up to the beginning of the poly A tail. Comparison of the rat cDNA‐derived coding sequence revealed identities of 74% to the human and 87% to the mouse FH nucleotide sequence. The translation product of rat FH mRNA was 1236 aa in length (leader sequence included) with an identity of 63% to the human and 81.5% to the murine protein. The degree of glycosylation of rat FH‐Mr is about 9.5%. To quantitate FH in rat serum and supernatants of primary cultures of rat hepatocytes (HC), a reliable and sensitive sandwich‐enzyme‐linked immunosorbent assay (ELISA) was established. The concentration of FH in rat serum was calculated to be 238 µg ± 21 µg/ml (mean ± SD). Its concentration in the culture supernatants of HC was upregulated about three‐fold by interferon (IFN)‐γ (100 U/ml).

Novel solid phase-based ELISA assays contribute to an improved detection of anti-HLA antibodies and to an increased reliability of pre- and post-transplant crossmatching

Antibodies directed against HLA antigens of a given organ donor represent the dominating reason for hyper-acute or acute allograft rejections. In order to select recipients without donor-specific antibodies, a standard crossmatch (CM) procedure, the complement-dependent cytotoxicity assay (CDC), was developed. This functional assay strongly depends on the availability of isolated vital lymphocytes of a given donor. However, the requirements of the donor’s material may often not be fulfilled, so that the detection of the antibodies directed against HLA molecules is either impaired or becomes completely impossible. To circumvent the disadvantages of the CDC procedure, enzyme-linked immunosorbent assay (ELISA)-based and other solid phase-based ELISA-related techniques have been designed to reliably detect anti-HLA antibodies in recipients. Due to the obvious advantages of these novel technologies, when compared with the classical CDC assay, there is an urgent need to implement them as complementary methods or even as a substitution for the conventional CDC crossmatch that is currently being applied by all tissue typing laboratories.

Constitutive Expression and Regulation of Rat Complement Factor H in Primary Cultures of Hepatocytes, Kupffer Cells, and Two Hepatoma Cell Lines

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-α, IL-1β, and IFN-γ, only IFN-γ 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-γ, 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 μg LPS/ml did not show any effect on HC. Our data suggest that LPS does not regulate the expression of FH in HC.

Dynamics of Protein Kinase C-mediated Phosphorylation of the Complement C5a Receptor on Serine 334

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 (t½ ∼ 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-β 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 (t½ < 5 s) binding of β-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.

Artificially Positive Crossmatches Not Leading to the Refusal of Kidney Donations due to the Usage of Adequate Diagnostic Tools

Allografting patients with human leukocyte antigens (HLA) which are recognized by preformed antibodies constitutes the main cause for hyper-acute or acute rejections. In order to select recipients without these donor-specific antibodies, the complement-dependent cytotoxicity crossmatch (CDC-CM) assay was developed as a standard procedure about forty years ago. The negative outcome of pretransplant crossmatching represents the most important requirement for a successful kidney graft survival. The artificially positive outcomes of CDC-based crossmatches due to the underlying disease Systemic Lupus Erythematosus (SLE), however, may lead to the unjustified refusal of adequate kidney grafts. Two prospective female recipients destined for a living as well as for a cadaver kidney donation, respectively, exhibited positive CDC-based crossmatch outcomes although for both patients no historical immunizing events were known. Furthermore, solid phase-based screening or antibody differentiation analyses never led to positive results. Immediate reruns of the CDC-based crossmatch assays using the alternative antibody monitoring system (AMS-)crossmatch ELISA resulted in unequivocally negative outcomes. Consequently both transplantations were performed without any immunological complications for the hitherto follow-up time of 25 and 28 months, respectively. We here show two case reports demonstrating an alternative methodical approach to circumvent CDC-based artefacts and point to the urgent need to substitute the CDC-based crossmatch procedure at least for special groups of patients.