Georgia Sfyroera

A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma

Complement component 5 (C5) has been described as either promoting or protecting against airway hyperresponsiveness (AHR) in experimental allergic asthma, suggesting pleomorphic effects of C5. Here we report that local pharmacological targeting of the C5a receptor (C5aR) prior to initial allergen sensitization in murine models of inhalation tolerance or allergic asthma resulted in either induction or marked enhancement of Th2-polarized immune responses, airway inflammation, and AHR. Importantly, C5aR-deficient mice exhibited a similar, increased allergic phenotype. Pulmonary allergen exposure in C5aR-targeted mice resulted in increased sensitization and accumulation of CD4+ CD69+ T cells associated with a marked increase in pulmonary myeloid, but not plasmacytoid, DC numbers. Pulmonary DCs from C5aR-targeted mice produced large amounts of CC chemokine ligand 17 (CCL17) and CCL22 ex vivo, suggesting a negative impact of C5aR signaling on pulmonary homing of Th2 cells. In contrast, C5aR targeting in sensitized mice led to suppressed airway inflammation and AHR but was still associated with enhanced production of Th2 effector cytokines. These data suggest a dual role for C5a in allergic asthma, i.e., protection from the development of maladaptive type 2 immune responses during allergen sensitization at the DC/T cell interface but enhancement of airway inflammation and AHR in an established inflammatory environment.

Complement inhibition decreases the procoagulant response and confers organ protection in a baboon model of Escherichia coli sepsis.

Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late after Escherichia coli challenge in a baboon model of sepsis-induced multiple organ failure. Compstatin infusion inhibited sepsis-induced blood and tissue biomarkers of complement activation, reduced leucopenia and thrombocytopenia, and lowered the accumulation of macrophages and platelets in organs. Compstatin decreased the coagulopathic response by down-regulating tissue factor and PAI-1, diminished global blood coagulation markers (fibrinogen, fibrin-degradation products, APTT), and preserved the endothelial anticoagulant properties. Compstatin treatment also improved cardiac function and the biochemical markers of kidney and liver damage. Histologic analysis of vital organs collected from animals euthanized after 24 hours showed decreased microvascular thrombosis, improved vascular barrier function, and less leukocyte infiltration and cell death, all consistent with attenuated organ injury. We conclude that complement-coagulation interplay contributes to the progression of severe sepsis and blocking the harmful effects of complement activation products, especially during the organ failure stage of severe sepsis is a potentially important therapeutic strategy.

Cloning and structure of three rainbow trout C3 molecules: a plausible explanation for their functional diversity ☆

We have previously identified and characterized three distinct trout C3 proteins (C3-1, C3-3 and C3-4) that differ in their electrophoretic mobility, glycosylation patterns, reactivity with monospecific C3 antibodies, partial amino acid sequence and binding to various complement activators. To study the structural elements that determine the observed functional differences, we have cloned and sequenced the three C3 isoforms. Comparison of the deduced amino acid sequences showed that the sequence identity/similarity of C3-3 to C3-4 is 76/81%, whereas those of C3-3 and C3-4 to C3-1 are 55/67% and 54/67%, respectively. It is interesting that the beta-chain of C3-4 contains two insertions of 65 (residues 504-569) and 23 amino acids (residues 123-146), while the beta-chain of C3-1 contains a 14-amino acid insertion (residues 143-157). The C3 convertase cleavage site (Arg-Ser) is conserved in the three trout isoforms; however, the factor I cleavage sites are Arg-Ala (for C3-1 and C3-4) and Arg-Thr (C3-3) instead of Arg-Ser at position 1281 of human C3, and Arg-Thr (C3-1, C3-3) instead of Arg-Ser for C3-4 at position 1298 of human C3. Of special interest is the absence of the His(1126) and Glu(1128) (human C3 numbering) from C3-4 and of Glu(1128) from C3-3. These residues are thought to play an important role in determining the binding specificity of the thioester-containing proteins. Accordingly, we postulate that the distinct binding reactions of the trout C3 isoforms with various complement activators could be due at least in part to the observed changes in the His and Glu residues.

Protection of Nonself Surfaces from Complement Attack by Factor H-Binding Peptides: Implications for Therapeutic Medicine

Exposure of nonself surfaces such as those of biomaterials or transplanted cells and organs to host blood frequently triggers innate immune responses, thereby affecting both their functionality and tolerability. Activation of the alternative pathway of complement plays a decisive role in this unfavorable reaction. Whereas previous studies demonstrated that immobilization of physiological regulators of complement activation (RCA) can attenuate this foreign body-induced activation, simple and efficient approaches for coating artificial surfaces with intact RCA are still missing. The conjugation of small molecular entities that capture RCA with high affinity is an intriguing alternative, as this creates a surface with autoregulatory activity upon exposure to blood. We therefore screened two variable cysteine-constrained phage-displayed peptide libraries for factor H-binding peptides. We discovered three peptide classes that differed with respect to their main target binding areas. Peptides binding to the broad middle region of factor H (domains 5–18) were of particular interest, as they do not interfere with either regulatory or binding activities. One peptide in this group (5C6) was further characterized and showed high factor H-capturing activity while retaining its functional integrity. Most importantly, when 5C6 was coated to a model polystyrene surface and exposed to human lepirudin-anticoagulated plasma, the bound peptide captured factor H and substantially inhibited complement activation by the alternative pathway. Our study therefore provides a promising and novel approach to produce therapeutic materials with enhanced biocompatibility.

Allosteric inhibition of complement function by a staphylococcal immune evasion protein

The complement system is a major target of immune evasion by Staphylococcus aureus. Although many evasion proteins have been described, little is known about their molecular mechanisms of action. Here we demonstrate that the extracellular fibrinogen-binding protein (Efb) from S. aureus acts as an allosteric inhibitor by inducing conformational changes in complement fragment C3b that propagate across several domains and influence functional regions far distant from the Efb binding site. Most notably, the inhibitor impaired the interaction of C3b with complement factor B and, consequently, formation of the active C3 convertase. As this enzyme complex is critical for both activation and amplification of the complement response, its allosteric inhibition likely represents a fundamental contribution to the overall immune evasion strategy of S. aureus.

Electrostatic modeling predicts the activities of orthopoxvirus complement control proteins.

Regulation of complement activation by pathogens and the host are critical for survival. Using two highly related orthopoxvirus proteins, the vaccinia and variola (smallpox) virus complement control proteins, which differ by only 11 aa, but differ 1000-fold in their ability to regulate complement activation, we investigated the role of electrostatic potential in predicting functional activity. Electrostatic modeling of the two proteins predicted that altering the vaccinia virus protein to contain the amino acids present in the second short consensus repeat domain of the smallpox protein would result in a vaccinia virus protein with increased complement regulatory activity. Mutagenesis of the vaccinia virus protein confirmed that changing the electrostatic potential of specific regions of the molecule influences its activity and identifies critical residues that result in enhanced function as measured by binding to C3b, inhibition of the alternative pathway of complement activation, and cofactor activity. In addition, we also demonstrate that despite the enhanced activity of the variola virus protein, its cofactor activity in the factor I-mediated degradation of C3b does not result in the cleavage of the α′ chain of C3b between residues 954–955. Our data have important implications in our understanding of how regulators of complement activation interact with complement, the regulation of the innate immune system, and the rational design of potent complement inhibitors that might be used as therapeutic agents.

A Molecular Insight into Complement Evasion by the Staphylococcal Complement Inhibitor Protein Family

Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its β-chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies.

Molecular cloning of the β subunit of complement component eight of rainbow trout

Complement-mediated killing of pathogens through the lytic pathway is an important effector mechanism of the innate immune response. C8 is one of the components of the lytic pathway and is composed of an alpha, beta, and gamma subunit. In the present study we report the cloning and characterization of the primary structure of the C8beta subunit in the rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequence of trout C8beta shows 72 and 47% identity with that of Japanese flounder and human, respectively. It also contains many of the same structural motifs as those found in mammalian lytic components. The C8beta gene appears to exists as a single copy in the trout genome and is expressed primarily in the liver. The protein encoded by the gene was identified by Western blotting using an anti-peptide antibody and was approximately 65kDa.

Restoration of complement-enhanced neutralization of vaccinia virus virions by novel monoclonal antibodies raised against the vaccinia virus complement control protein

ABSTRACT The vaccinia virus complement control protein (VCP) is secreted by infected cells and has been shown to inhibit complement activation through interactions with C3b/C4b. It contains four short consensus repeat (SCR) domains. It has been suggested that all four SCRs are required for VCPs activity. To elucidate which SCR domains are involved in abolishing complement-enhanced neutralization of vaccinia virus virions, we generated and characterized a panel of mouse monoclonal antibodies (MAbs) raised against VCP. Ten MAbs were isolated and all recognized VCP on Western blots under reducing conditions as well as native-bound VCP in a sandwich enzyme-linked immunosorbent assay. Three of the 10 MAbs (2E5, 3D1, and 3F11) inhibited VCPs abolition of complement-enhanced neutralization of vaccinia virus virions. These MAbs blocked the interaction of VCP with C3b/C4b. The seven remaining MAbs did not alter VCP function in the complement neutralization assay and recognized VCP bound to C3b/C4b. To understand MAb specificity and mode of interaction with VCP, we mapped the MAb binding regions on VCP. The seven nonblocking MAbs all bound to the first SCR of VCP. One of the blocking MAbs recognized SCR 2 while the other two recognized either SCR 4 or the junction between SCRs 3 and 4, indicating that structural elements involved in the interaction of VCP with C3b/C4b are located within SCR domains 2 and 3 and 4. These anti-VCP MAbs may have clinical significance as therapeutic inhibitors of VCPs complement control activity and may also offer a novel approach to managing vaccinia virus vaccine complications that occur from smallpox vaccination.

C5a Receptor-Dependent Cell Activation by Physiological Concentrations of Desarginated C5a: Insights from a Novel Label-Free Cellular Assay

The complement anaphylatoxins C3a, C5a, and desarginated C5a (C5adesArg) play critical roles in the induction of inflammation and the modulation of innate and acquired immune responses after binding to their G protein-coupled receptors, C3a receptor and C5a receptor (C5aR). The role of C5adesArg in inducing cell activation has been often neglected, because the affinity of C5adesArg for C5aR has been reported to be much lower than that of C5a. We have used a novel label-free cellular assay to reassess the potential of C5adesArg to induce activation of transfected and primary immune cells. Our results indicate that physiological levels of C5adesArg induce significant levels of cell activation that are even higher than those achieved by stimulating cells with analogous concentrations of C5a. Such activation was strictly dependent on C5aR, because it was completely abrogated by PMX-53, a C5aR antagonist. Pharmacological inhibition of specific G proteins located downstream of C5aR indicated differential involvement of Gα proteins upon C5aR engagement by C5a or C5adesArg. Further, mass spectrometric characterization of plasma-derived C5a and C5adesArg provided important insight into the posttranslational modification pattern of these anaphylatoxins, which includes glycosylation at Asn64 and partial cysteinylation at Cys27. Although the context-specific physiological contribution of C5adesArg has to be further explored, our data suggest that C5adesArg acts as a key molecule in the triggering of local inflammation as well as the maintenance of blood surveillance and homeostatic status.