Ischemia/reperfusion injury: effect of simultaneous inhibition of plasma cascade systems versus specific complement inhibition.

Ischemia/reperfusion injury (IRI) may occur from ischemia due to thrombotic occlusion, trauma or surgical interventions, including transplantation, with subsequent reestablishment of circulation. Time-dependent molecular and structural changes result from the deprivation of blood and oxygen in the affected tissue during ischemia. Upon restoration of blood flow a multifaceted network of plasma cascades is activated, including the complement-, coagulation-, kinin-, and fibrinolytic system, which plays a major role in the reperfusion-triggered inflammatory process. The plasma cascade systems are therefore promising therapeutic targets for attenuation of IRI. Earlier studies showed beneficial effects through inhibition of the complement system using specific complement inhibitors. However, pivotal roles in IRI are also attributed to other cascades. This raises the question, whether drugs, such as C1 esterase inhibitor, which regulate more than one cascade at a time, have a higher therapeutic potential. The present review discusses different therapeutic approaches ranging from specific complement inhibition to simultaneous inhibition of plasma cascade systems for reduction of IRI, gives an overview of the plasma cascade systems in IRI as well as highlights recent findings in this field.

A Metalloproteinase Mirolysin of Tannerella forsythia Inhibits All Pathways of the Complement System

Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. In this study, we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4, whereas inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium.

Genetic variations in complement factors in patients with congenital thrombotic thrombocytopenic purpura with renal insufficiency.

The congenital form of thrombotic thrombocytopenic purpura (TTP) is caused by genetic mutations in ADAMTS13. Some, but not all, congenital TTP patients manifest renal insufficiency in addition to microangiopathic hemolysis and thrombocytopenia. We included 32 congenital TTP patients in the present study, which was designed to assess whether congenital TTP patients with renal insufficiency have predisposing mutations in complement regulatory genes, as found in many patients with atypical hemolytic uremic syndrome (aHUS). In 13 patients with severe renal insufficiency, six candidate complement or complement regulatory genes were sequenced and 11 missense mutations were identified. One of these missense mutations, C3:p.K155Q mutation, is a rare mutation located in the macroglobulin-like 2 domain of C3, where other mutations predisposing for aHUS cluster. Several of the common missense mutations identified in our study have been reported to increase disease-risk for aHUS, but were not more common in patients with as compared to those without renal insufficiency. Taken together, our results show that the majority of the congenital TTP patients with renal insufficiency studied do not carry rare genetic mutations in complement or complement regulatory genes.

Regulation of T cell function by complement C5A in mice during mycobacterial infection

Tuberculosis is the leading cause of death in the world due to a single infectious agent, making it critical to investigate all aspects of the immune response mounted against the causative agent, Mycobacterium tuberculosis , in order to better treat and prevent disease. Previous observations show a disparity in the ability to control mycobacterial growth between mouse strains sufficient in C5, such as C57BL/6 and B10.D2/nSnJ, and those naturally deficient in C5, such as A/J and B10.D2/nSnJ, with C5 deficient mice being more susceptible. It has been shown that during M. tuberculosis infection, C5 deficient macrophages have a defect in production of interleukin (IL)-12, a cytokine involved in the cyclical activation between infected macrophages and effector T cells. T cells stimulated by IL-12 produce interferon (IFN)-γ, the signature cytokine of T helper type 1 (Th1) cells. It is known that a cell-mediated Th1 response is crucial for control of M. tuberculosis in the lungs of humans and mice. This study demonstrates that murine T cells express detectable levels of CD88, a receptor for C5a (C5aR), following antigen presentation by macrophages infected with mycobacteria. T cells from C5 deficient mice infected with M. tuberculosis were found to secrete less IFN-γ and had a reduced Th1 phenotype associated with fewer cells expressing the transcription factor, T-box expressed in T cells (T-bet). The altered Th1 phenotype in M. tuberculosis infected C5 deficient mice coincided with a rise in IL-4 and IL-10 secretion from Th2 cells and inducible regulatory T cells, respectively. It was found that the ineffective T cell response to mycobacteria in C5 deficient mice was due indirectly to a lack of C5a via poor priming by infected macrophages and possibly by a direct interaction between T cells and C5a peptide. Therefore, these studies show a link between the cells of the innate and adaptive arms of the immune system, macrophages and T cells respectively, that was mediated by C5a using a mouse model of M. tuberculosis infection. ^

INTERACTION OF BACILLUS ANTHRACIS EXOSPORIUM PROTEIN BCLA WITH COMPLEMENT FACTOR H AND SPORE PERSISTENCE IN THE LUNG

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse model. The results suggest that B. anthracis spores have special properties that promote persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence. Moreover, recent discoveries from our laboratory suggest that spores evolved a sophisticated mechanism to interact with the host complement system. The complement system is a crucial part of the host defense mechanism against foreign microorganisms. Knowledge of the specific interactions that occur between the complement system and B. anthracis was limited. Studies performed in our laboratory have suggested that spores of B. anthracis can target specific proteins, such as Factor H (fH) of the complement system. Spores of B. anthracis are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called Bacillus collagen-like protein of anthracis (BclA), which comprises a central collagen-like region and a globular C-terminal domain. BclA is the first point of contact with the innate system of an infected host. In this study, we investigated the molecular details of BclA-fH interaction with respect to the specific binding mechanism and the functional significance of this interaction in a murine model of anthrax infection. We hypothesized that the recruitment of fH to the spore surface by BclA limits the extent of complement activation and promotes pathogen survival and persistence in the infected host. Findings from this study are significant to understanding how to treat post-exposure prophylaxis and improve our knowledge of spores with the host immune system.

Human complement component C3 -binding proteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that uses the host mononuclear phagocyte as a niche for survival and replication during infection. Complement component C3 has previously been shown to enhance the binding of M. tuberculosis to mononuclear phagocytes. Using a C3 ligand affinity blot protocol, we identified a 30 kDa C3-binding protein in M. tuberculosis as heparin-binding hemagglutinin (HbhA). HbhA was found to be a hydrophobic protein that localized to the cell membrane/cell wall fraction of M. tuberculosis, and this protein has previously been shown by others to be located on the surface of M. tuberculosis. The C3-binding activity of HbhA was localized to the C-terminus of the protein, which consists of lysine-alanine repeats. Full-length recombinant HbhA coated onto latex beads was shown to mediate the adherence of the beads to murine macrophage-like cells in both a C3-dependent and a C3-independent manner. An in-frame 576 by deletion in the hbhA gene was created in a virulent strain of M. tuberculosis using a PCR technique known as gene splicing by overlap extension (SOEing). Using the ΔhbhA mutant, HbhA was found not to be necessary for growth of M. tuberculosis in laboratory media or in macrophage-like cells, nor is HbhA required for adherence of M. tuberculosis to macrophage-like cells. HbhA is, however, required for infectivity of M. tuberculosis in mice. Mice infected with the ΔhbhA mutant show decreased growth in the lungs, liver, and spleen compared to mice infected with the wild-type strain. Using the ΔhbhA mutant strain, we were able to purify and identify a second 30-kDa C3-binding protein, HupB. These data demonstrate that HbhA is required for the in vivo but not the in vitro survival of M. tuberculosis and that HbhA is not necessary for the adherence of M. tuberculosis to the macrophage-like cells used in these studies. The expression of two proteins that bind human C3 may aid in the efficient binding of M. tuberculosis to complement receptors for uptake into mononuclear cells, or may influence other aspects of the host-parasite interaction. ^

The function of the murine complement *C3a and *C5a anaphylatoxin receptors in endotoxemia, bacteremia and septic shock

The complement system functions as a major effector for both the innate and adaptive immune response. Activation of the complement cascade by either the classical, alternative, or lectin pathway promotes the proteolysis of C3 and C5 thereby generating C3a and C5a. Referred to as anaphylatoxins, the C3a and C5a peptides mediate biological effects upon binding to their respective receptors; C3a binds to the C3a receptor (C3aR) while C5a binds to the C5a receptor (C5aR, CD88). Both C3a and C5a are known for their broad proinflammatory effects. Elevated levels of both peptides have been isolated from patients with a variety of inflammatory diseases such as COPD, asthma, RA, SLE, and sepsis. Recent studies suggest that C5a is a critical component in the acquired neutrophil dysfunction, coagulopathy, and progressive multi-organ dysfunction characteristic of sepsis. The primary hypothesis of this dissertation was that preventing C3a-C3aR and C5a-C5aR mediated pro-inflammatory effects would improve survival in endotoxic, bacteremic and septic shock. To test this hypothesis, the murine C3aR and C5aR genes were disrupted. Following disruption of both the C3aR and C5aR genes, no abnormalities were identified other than the absence of their respective mRNA and protein. In models of both endotoxic and bacteremic shock, C3aR deficient mice suffered increased mortality when compared to their wild type littermates. C3aR deficient mice also had elevated circulating IL-1β levels. Using a model of sepsis, C3aR deficient mice had a higher circulating concentration of IL-6 and decreased peritoneal inflammatory infiltration. While these results were unexpected, they support an emerging role for C3a in immunomodulation. In contrast, following endotoxic or bacteremic shock, C5aR deficient mice experienced increased survival, less hemoconcentration and less thrombocytopenia. It was later determined that C5a mediated histamine release significantly contributes to host morbidity and mortality in bacteremic shock. These studies provide evidence that C5a functions primarily as a proinflammatory molecule in models of endotoxic and bacteremic shock. In the same models, C3a-C3aR interactions suppress the inflammatory response and protect the host. Collectively, these results present in vivo evidence that C3a and C5a have divergent biological functions. ^

Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

Humoral response to herpes simplex virus is complement-dependent

The complement system represents a cascade of serum proteins, which provide a major effector function in innate immunity. Recent studies have revealed that complement links innate and adaptive immunity via complement receptors CD21/CD35 in that it enhances the B cell memory response to noninfectious protein antigens introduced i.v. To examine the importance of complement for immune responses to virus infection in a peripheral tissue, we compared the B cell memory response of mice deficient in complement C3, C4, or CD21/CD35 with wild-type controls. We found that the deficient mice failed to generate a normal memory response, which is characterized by a reduction in IgG antibody and germinal centers. Thus, complement is important not only in the effector function of innate immunity but also in the stimulation of memory B cell responses to viral-infected cell antigens in both blood and peripheral tissues.

Abrogation of the alternative complement pathway by targeted deletion of murine factor B

To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P = 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

Molecular basis for a link between complement and the vascular complications of diabetes

Activated terminal complement proteins C5b to C9 form the membrane attack complex (MAC) pore. Insertion of the MAC into endothelial cell membranes causes the release of growth factors that stimulate tissue growth and proliferation. The complement regulatory membrane protein CD59 restricts MAC formation. Because increased cell proliferation characterizes the major chronic vascular complications of human diabetes and because increased glucose levels in diabetes cause protein glycation and impairment of protein function, we investigated whether glycation could inhibit CD59. Glycation-inactivation of CD59 would cause increased MAC deposition and MAC-stimulated cell proliferation. Here, we report that (i) human CD59 is glycated in vivo, (ii) glycated human CD59 loses its MAC-inhibitory function, and (iii) inactivation of CD59 increases MAC-induced growth factor release from endothelial cells. We demonstrate by site-directed mutagenesis that residues K41 and H44 form a preferential glycation motif in human CD59. The presence of this glycation motif in human CD59, but not in CD59 of other species, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.

Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.