Michael Mihlan

Factor H related protein 1 (CFHR-1) inhibits complement C5 convertase activity and terminal complex formation

Homozygous deletion of a 84-kb genomic fragment in human chromosome 1 that encompasses the CFHR1 and CFHR3 genes represents a risk factor for hemolytic uremic syndrome (HUS) but has a protective effect in age-related macular degeneration (AMD). Here we identify CFHR1 as a novel inhibitor of the complement pathway that blocks C5 convertase activity and interferes with C5b surface deposition and MAC formation. This activity is distinct from complement factor H, and apparently factor H and CFHR1 control complement activation in a sequential manner. As both proteins bind to the same or similar sites at the cellular surfaces, the gain of CFHR1 activity presumably is at the expense of CFH-mediated function (inhibition of the C3 convertase). In HUS, the absence of CFHR1 may result in reduced inhibition of terminal complex formation and in reduced protection of endothelial cells upon complement attack. These findings provide new insights into complement regulation on the cell surface and biosurfaces and likely define the role of CFHR1 in human diseases.

Monomeric CRP contributes to complement control in fluid phase and on cellular surfaces and increases phagocytosis by recruiting factor H

Complement forms the first defense line of innate immunity and has an important role in the non-inflammatory clearance of apoptotic and necrotic cells. Factor H is one essential complement inhibitor that binds to the acute phase reactant C-reactive protein (CRP). By using recombinant proteins, calcium-independent binding of Factor H to monomeric CRP (mCRP), but not to pentameric CRP (pCRP), was shown. In addition to the two known CRP-binding sites, a novel third site was localized within the C-terminus. This region is frequently mutated in the hemolytic uremic syndrome and the mutant proteins show reduced mCRP binding. In this study, we show that mCRP directs Factor H to the surface of apoptotic and necrotic endothelial cells and identify phosphocholine as one binding moiety for this complex. Factor H–mCRP complexes enhance C3b inactivation both in the fluid phase and on the surface of damaged cells and inhibit the production of pro-inflammatory cytokines. By recruiting the soluble complement inhibitor Factor H to the surface of damaged cells, mCRP blocks the progression of the complement cascade beyond the step of the C3 convertase, prevents the formation of inflammatory activation products, and thus contributes to the safe removal of opsonized damaged cells and particles.

Monomeric C-reactive protein modulates classic complement activation on necrotic cells

The acute‐phase protein C‐reactive protein (CRP) recruits C1q to the surface of damaged cells and thereby initiates complement activation. However, CRP also recruits complement inhibitors, such as C4b‐binding protein (C4bp) and factor H, which both block complement progression at the level of C3 and inhibits inflammation. To define how CRP modulates the classic complement pathway, we studied the interaction of CRP with the classic pathway inhibitor C4bp. Monomeric CRP (mCRP), but not pentameric CRP (pCRP), binds C4bp and enhances degradation of C4b and C3b. Both C1q, the initiator, and C4bp, the inhibitor of the classic pathway, compete for mCRP binding, and this competition adjusts the local balance of activation and inhibition. After attachment of pCRP to the surface of necrotic rat myocytes, generation of mCRP was demonstrated over a period of 18 h. Similarly, a biological role for mCRP, C1q, and C4bp in the disease setting of acute myocardial infarction was revealed. In this inflamed tissue, mCRP, pCRP, C4bp, C1q, and C4d were detected in acetone‐fixed and in unfixed tissue. Protein levels were enhanced 6 h to 5 d after infarction. Thus, mCRP bound to damaged cardiomyocytes recruits C1q to activate and also C4bp to control the classic complement pathway.—Mihlan, M., Blom, A. M., Kupreishvili, K., Lauer, N., Stelzner, K., Bergström, F., Niessen, H. W. M., Zipfel, P. F. Monomeric C‐reactive protein modulates classic complement activation on necrotic cells. FASEB J. 25, 4198–4210 (2011). www.fasebj.org

Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages.

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts.

Complement Regulation at Necrotic Cell Lesions Is Impaired by the Age-Related Macular Degeneration-Associated Factor-H His402 Risk Variant

Age-related macular degeneration is a leading form of blindness in Western countries and is associated with a common SNP (rs 1061170/Y402H) in the Factor H gene, which encodes the two complement inhibitors Factor H and FHL1. However, the functional consequences of this Tyr402 His exchange in domain 7 are not precisely defined. In this study, we show that the Tyr402 His sequence variation affects Factor H surface recruitment by monomeric C-reactive protein (mCRP) to specific patches on the surface of necrotic retinal pigment epithelial cells. Enhanced attachment of the protective Tyr402 variants of both Factor H and FHL1 by mCRP results in more efficient complement control and further provides an anti-inflammatory environment. In addition, we demonstrate that mCRP is generated on the surface of necrotic retinal pigment epithelial cells and that this newly formed mCRP colocalizes with the cell damage marker annexin V. Bound to the cell surface, Factor H–mCRP complexes allow complement inactivation and reduce the release of the proinflammatory cytokine TNF-α. This mCRP-mediated complement inhibitory and anti-inflammatory activity at necrotic membrane lesions is affected by residue 402 of Factor H and defines a new role for mCRP, for Factor H, and also for the mCRP–Factor H complex. The increased protective capacity of the Tyr402 Factor H variant allows better and more efficient clearance and removal of cellular debris and reduces inflammation and pathology.

Changes and regulation of the C5a receptor on neutrophils during septic shock in humans.

During experimental sepsis, excessive generation of the anaphylatoxin C5a results in reduction of the C5a receptor (C5aR) on neutrophils. These events have been shown to result in impaired innate immunity. However, the regulation and fate of C5aR on neutrophils during sepsis are largely unknown. In contrast to 30 healthy volunteers, 60 patients in septic shock presented evidence of complement activation with significantly increased serum levels of C3a, C5a, and C5b-9. In the septic shock group, the corresponding decrease in complement hemolytic activity distinguished survivors from nonsurvivors. Neutrophils from patients in septic shock exhibited decreased C5aR expression, which inversely correlated with serum concentrations of C-reactive protein (CRP) and clinical outcome. In vitro exposure of normal neutrophils to native pentameric CRP led to a dose- and time-dependent loss of C5aR expression on neutrophils, whereas the monomeric form of CRP, as well as various other inflammatory mediators, failed to significantly alter C5aR levels on neutrophils. A circulating form of C5aR (cC5aR) was detected in serum by immunoblotting and a flow-based capture assay, suggestive of an intact C5aR molecule. Levels of cC5aR were significantly enhanced during septic shock, with serum levels directly correlating with lethality. The data suggest that septic shock in humans is associated with extensive complement activation, CRP-dependent loss of C5aR on neutrophils, and appearance of cC5aR in serum, which correlated with a poor outcome. Therefore, cC5aR may represent a new sepsis marker to be considered in tailoring individualized immune-modulating therapy.

Insulin secretory granules control autophagy in pancreatic β cells

Too hungry to eat, too hungry not to eat Pancreatic beta cells, the source of insulin in response to food, employ an unusual mechanism to adapt to nutrient depletion. Goginashvili et al. found that starvation of beta cells induced selective degradation of newly formed insulin granules through their fusion with lysosomes, the cells garbage disposal units (see the Perspective by Rutter). The nutrient sensor mTOR is recruited to these lysosomes, leading to its local activation and the suppression of autophagy—a process by which cells “eat” their own constituents. Protein kinase D, a major regulator of insulin granule biogenesis, controls this granule degradation in response to nutrient availability. Thus, unlike most other cells, autophagy is not the strategy of choice in beta cells to adapt to starvation. Science, this issue p. 878; see also p. 826 Newly formed insulin granules are degraded by lysosomes to prevent insulin release upon fasting. [Also see Perspective by Rutter] Pancreatic β cells lower insulin release in response to nutrient depletion. The question of whether starved β cells induce macroautophagy, a predominant mechanism maintaining energy homeostasis, remains poorly explored. We found that, in contrast to many mammalian cells, macroautophagy in pancreatic β cells was suppressed upon starvation. Instead, starved β cells induced lysosomal degradation of nascent secretory insulin granules, which was controlled by protein kinase D (PKD), a key player in secretory granule biogenesis. Starvation-induced nascent granule degradation triggered lysosomal recruitment and activation of mechanistic target of rapamycin that suppressed macroautophagy. Switching from macroautophagy to insulin granule degradation was important to keep insulin secretion low upon fasting. Thus, β cells use a PKD-dependent mechanism to adapt to nutrient availability and couple autophagy flux to secretory function.

Human complement factor H-related protein 4 binds and recruits native pentameric C-reactive protein to necrotic cells

Human complement factor H-related protein 4 (CFHR4) is a plasma glycoprotein which appears in two isoforms. CFHR4 is a member of the factor H protein family, and shares structural similarity and sequence homology with the other CFHR proteins and with the complement regulator factor H. Given the structural and sequence similarity, we hypothesized that similar to factor H, CFHR4 binds to C-reactive protein (CRP). We have recombinantly expressed the two CFHR4 isoforms and analyzed their binding to both native and denatured, monomeric CRP. Here, we show that both CFHR4 isoforms bind in the presence of calcium to native pentameric CRP, but not to modified CRP. This is in contrast to factor H, which binds to modified CRP independent of calcium. Comparison of the two CFHR4 isoforms and a recombinant CFHR4 fragment for CRP binding indicates that the first domain of CFHR4 is relevant for this interaction. Interaction of the native proteins was demonstrated by co-precipitation of CFHR4 and CRP from serum of sepsis patients with elevated CRP levels. CFHR4 bound to necrotic cells and was localized in necrotic tumor tissue as demonstrated by immunohistological analyses. In addition, CFHR4 facilitated binding of native CRP to the surface of necrotic cells. Altogether these data identify CFHR4 as a novel ligand for native CRP, and suggest a role for CFHR4 in opsonization of necrotic cells.

Molecular basis of C-reactive protein binding and modulation of complement activation by factor H-related protein 4☆

C-reactive protein (CRP) is a pattern recognition molecule that binds several microbial and host ligands. Ligand-bound CRP activates the complement system via the classical pathway. Previously, we identified human complement factor H-related protein 4 (CFHR4), a member of the factor H protein family, as a CRP binding protein. Here, we investigated the molecular basis and the functional relevance of the interaction of CFHR4 with native CRP. Using recombinantly expressed CFHR4 fragments, the CRP binding site was localized to the first short consensus repeat (SCR) domain of CFHR4. Peptide arrays identified residues 35-41 of CFHR4 to be involved in CRP binding. Substitutions of the positively charged amino acids of this motif resulted in strongly reduced CRP binding. Sequence comparisons revealed that such a motif is not present in the related SCR6 domain of factor H, or in the homologous domains of the four other CFHR proteins. Homology modelling based on SCR6 of factor H showed that the CRP binding site is surface exposed on SCR1 of CFHR4. CFHR4-bound CRP was able to activate complement, determined by C3 fragment deposition. Recombinant CFHR4 proteins with mutations in the identified binding site showed reduced CRP binding, which in turn resulted in reduced complement activation. In summary, these data reveal the molecular basis of the specific interaction of CFHR4 with native CRP and suggest a role for CFHR4 in enhancing opsonization via CRP binding.

Human complement factor H is a novel diagnostic marker for lung adenocarcinoma

Human complement factor H (CFH), a central complement control protein, is a member of the regulators of complement activation family. Recent studies suggested that CFH may play a key role in the resistance of complement-mediated lysis in various cancer cells. In this study, we investigated the role of CFH in human lung cancer. Expression of CFH was analyzed in lung cancer cell lines by RT-PCR, Western blotting and immunofluorescence. In primary lung tumors, the protein expression of CFH was evaluated by immunohistochemistry (IHC) on tissue microarray (TMA). Binding of CFH to lung cancer cells was detected by flow cytometry. mRNA expression of CFH was detected in 6 out of 10 non-small cell lung cancer (NSCLC) cell lines, but in none of the small cell lung cancer (SCLC) cell lines. In line with Western blotting, immunofluorescence analysis demonstrated CFH protein expression in 3 NSCLC cell lines, and the immunoreaction was mainly associated with cell cytoplasm and membrane. In primary lung tumors, 54 out of 101 samples exhibited high expression of CFH and high expression was significantly correlated with lung adenocarcinoma (p=0.009). Also, in adenocarcinoma of the lung, Kaplan-Meier survival analysis showed a tendency that CFH-positive tumors had worse prognosis in comparison to CFH-negative tumors (p=0.082). Additionally, shorter survival time of patients with adenocarcinoma (<20 months) was associated with higher staining of CFH (p=0.033). Our data showed that non-small cell lung cancer cells expressed and secreted CFH. CFH might be a novel diagnostic marker for human lung adenocarcinoma.