The utility of complement assays in clinical immunology: A comprehensive review.

The multi-tasking organ liver, which is the major synthesis site of most serum proteins, supplies humoral components of the innate, - including proteins of the complement system; and, less intensely, also of the acquired immune system. In addition to hepatocyte origins, C1q, factor D, C3, C7 and other protein components of the complement system are produced at various body locations by monocytes/macrophages, lymphocytes, adipocytes, endometrium, enterocytes, keratinocytes and epithelial cells; but the contribution of these alternate sites to the total serum concentrations is slight. The two major exceptions are factor D, which cleaves factor B of the alternative pathway derived largely from adipocytes, and C7, derived largely from polymorphonuclear leukocytes and monocytes/macrophages. Whereas the functional meaning of the extrahepatic synthesis of factor D remains to be elucidated, the local contribution of C7 may up- or downregulate the complement attack. The liver, however, is not classified as part of the immune system but is rather seen as victim of autoimmune diseases, a point that needs apology. Recent histological and cell marker technologies now turn the hands to also conceive the liver as proactive autoimmune disease catalyst. Hosting non-hepatocytic cells, e.g. NK cells, macrophages, dendritic cells as well as T and B lymphocytes, the liver outreaches multiple sites of the immune system. Immunopharmacological follow up of liver transplant recipients teaches us on liver-based presence of ABH-glycan HLA phenotypes and complement mediated ischemia/regeneration processes. In clinical context, the adverse reactions of the complement system can now be curbed by specific drug therapy. This review extends on the involvement of the complement system in liver autoimmune diseases and should allow to direct therapeutic opportunities.

Membrane attack complex-associated molecules from redlip mullet (Liza haematocheila): Molecular characterization and transcriptional evidence of C6, C7, C8ß, and C9 in innate immunity.

The redlip mullet (Liza haematocheila) is one of the most economically important fish in Korea and other East Asian countries; it is susceptible to infections by pathogens such as Lactococcus garvieae, Argulus spp., Trichodina spp., and Vibrio spp. Learning about the mechanisms of the complement system of the innate immunity of redlip mullet is important for efforts towards eradicating pathogens. Here, we report a comprehensive study of the terminal complement complex (TCC) components that form the membrane attack complex (MAC) through in-silico characterization and comparative spatial and temporal expression profiling. Five conserved domains (TSP1, LDLa, MACPF, CCP, and FIMAC) were detected in the TCC components, but the CCP and FIMAC domains were absent in MuC8ß and MuC9. Expression analysis of four TCC genes from healthy redlip mullets showed the highest expression levels in the liver, whereas limited expression was observed in other tissues; immune-induced expression in the head kidney and spleen revealed significant responses against Lactococcus garvieae and poly I:C injection, suggesting their involvement in MAC formation in response to harmful pathogenic infections. Furthermore, the response to poly I:C may suggest the role of TCC components in the breakdown of the membrane of enveloped viruses. These findings may help to elucidate the mechanisms behind the complement system of the teleosts innate immunity.

Characterisation and expression analysis of two terminal complement components: C7 and C9 from large yellow croaker, Larimichthys crocea.

The large yellow croaker Larimichthys crocea, as one of the most economically important marine fish in China and East Asian countries, are facing the fatal attraction of various pathogens in recent years. Elucidation of the organism immunomodulatory mechanism of croaker response to pathogen infection is essential for the disease control. In present study, we reported for the first time the molecular characterization and expression analysis of two terminal complement components (TCCs) of croaker, Lc-C7 and Lc-C9. These two structural conserved TCCs were detected in many tissues in adult healthy fish, with highest levels detected in liver. The transcriptional expression analysis of Lc-C7 and Lc-C9 at different developmental stages showed a continuous increase towards hatch, however the two TCCs mRNA were not detected at the unfertilized stage, hinting the origination of these two TCCs after fertilization. Rapid and drastic responses to Vibrio alginolyticus challenge were observed for Lc-C7 and Lc-C9, suggesting the involvement of component C7 and C9 in innate immune responses to pathogenic invasion in teleost fish. These findings could deepen our understanding about immunomodulatory mechanisms of croaker and shed a new light to the role of component system in teleostean immunomodulation.

Characterization and evolutionary analysis of duplicated C7 in miiuy croaker.

The complement system, as one of the most sophisticated innate immune system, plays an important role in defense against invading microorganisms. The complement component C7 participates in the cytolytic phase of complement activation through a series of polymerization reactions with other terminal complement components. In this study, we derived two C7 genes from the whole genome of miiuy croaker which were the consequence of the fish-specific genome duplication. Our data showed that miiuy croaker C7-1 and C7-2 genes shared same structure domains. The analysis of gene synteny showed that high degree conserved of synteny was retained between miiuy croaker and other teleosts, and miiuy croaker had a relatively closer relationship with fugu. The expression of C7-1 and C7-2 in miiuy croaker healthy tissues revealed that they were ubiquitously expressed in all ten tested tissues. Besides, the immune response of C7-1 and C7-2 were different in spleen with Vibrio anguillarum, Staphylococcus aureus, poly I:C and LPS at 24 h post-injection, respectively. Furthermore, the expression patterns of C7-1 and C7-2 were different in liver, spleen and kidney after infected with V. anguillarum at different time-point. Evolutionary analysis showed that all the ancestral lineages underwent positive selection except for the ancestral lineages of fish C7-2, indicated that the ancestral lineages of fish C7-1 genes undertook more pressures than C7-2 in defense against the invading microorganisms. Meanwhile, a series of maximum likelihood methods were used to explore the evolutionary patterns on extant vertebrates' C7 genes. Three and one positive selection sites were found in extant mammalian C7 genes and fish C7-2 genes, but no positive selection site was found in extant fishes C7-1 genes. The result showed that extant fish C7-2 genes undertook more pressures compared with C7-1. In conclusion, fish C7-1 and C7-2 gene underwent different evolutionary patterns.

Pseudomonas aeruginosa Psl polysaccharide reduces neutrophil phagocytosis and the oxidative response by limiting complement-mediated opsonization.

Pseudomonas aeruginosa causes chronic lung infections in the airways of cystic fibrosis (CF) patients. Psl is an extracellular polysaccharide expressed by non-mucoid P. aeruginosa strains, which are believed to be initial colonizers. We hypothesized that Psl protects P. aeruginosa from host defences within the CF lung prior to their conversion to the mucoid phenotype. We discovered that serum opsonization significantly increased the production of reactive oxygen species (ROS) by neutrophils exposed to a psl-deficient mutant, compared with wild-type (WT) and Psl overexpressing strains (Psl(++)). Psl-deficient P. aeruginosa were internalized and killed by neutrophils and macrophages more efficiently than WT and Psl(++) variants. Deposition of complement components C3, C5 and C7 was significantly higher on psl-deficient strains compared with WT and Psl(++) bacteria. In an in vivo pulmonary competition assay, there was a 4.5-fold fitness advantage for WT over psl-deficient P. aeruginosa. Together, these data show that Psl inhibits efficient opsonization, resulting in reduced neutrophil ROS production, and decreased killing by phagocytes. This provides a survival advantage in vivo. Since phagocytes are critical in early recognition and control of infection, therapies aimed at Psl could improve the quality of life for patients colonized with P. aeruginosa.

Expression of complement component C7 and involvement in innate immune responses to bacteria in grass carp.

Activation of the complement system, which forms a major part of the innate immune system, results in the formation of the terminal complement complex. The complement component, C7, plays an integral role in the assembly of this complex within target cell membranes. In this study, C7 was isolated and characterized from grass carp, an important cultured fish in China. The predicted amino acid sequence of C7 cDNA (2644 bp) exhibited 55.4 and 48.3% homology with trout C7-1 and zebrafish C7, respectively. The grass carp C7 gene was consisted of 18 exons and 17 introns. C7 gene expression was detected in the trunk kidney, liver, head kidney, skin, spleen, heart and intestine. Significant changes in C7 transcript expression (>20-fold) were detected following Aeromonas hydrophila infection, indicating C7 involvement in innate immune responses to bacteria in teleost fish.

Complement factor C7 contributes to lung immunopathology caused by Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) remains a significant global health burden despite the availability of antimicrobial chemotherapy. Increasing evidence indicates a critical role of the complement system in the development of host protection against the bacillus, but few studies have specifically explored the function of the terminal complement factors. Mice deficient in complement C7 and wild-type C57BL/6 mice were aerosol challenged with MTB Erdman and assessed for bacterial burden, histopathology, and lung cytokine responses at days 30 and 60 post-infection. Macrophages isolated from C7 -/- and wild-type mice were evaluated for MTB proliferation and cytokine production. C7 -/- mice had significantly less liver colony forming units (CFUs) at day 30; no differences were noted in lung CFUs. The C7 deficient mice had markedly reduced lung occlusion with significantly increased total lymphocytes, decreased macrophages, and increased numbers of CD4+ cells 60 days post-infection. Expression of lung IFN-γ and TNF-α was increased at day 60 compared to wild-type mice. There were no differences in MTB-proliferation in macrophages isolated from wild-type and knock-out mice. These results indicate a role for complement C7 in the development of MTB induced immunopathology which warrants further investigation.

C7 is expressed on endothelial cells as a trap for the assembling terminal complement complex and may exert anti-inflammatory function.

We describe a novel localization of C7 as a membrane-bound molecule on endothelial cells (ECs). Data obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, Northern blot analysis, and mass spectrometry revealed that membrane-associated C7 (mC7) was indistinguishable from soluble C7 and was associated with vimentin on the cell surface. mC7 interacted with the other late complement components to form membrane-bound TCC (mTCC). Unlike the soluble SC5b-9, mTCC failed to stimulate ECs to express adhesion molecules, to secrete IL-8, and to induce albumin leakage through a monolayer of ECs, and more importantly protected ECs from the proinflammatory effect of SC5b-9. Our data disclose the possibility of a novel role of mC7 that acts as a trap for the late complement components to control excessive inflammation induced by SC5b-9.

Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes. Studies on C9 binding and incorporation into high molecular weight complexes.

As part of a broader analysis of the mechanism(s) by which the most sensitive (type III) paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes are excessively sensitive to reactive lysis by isolated C5b6, C7, C8, and C9, we have compared type III PNH (PNH-III) and normal human E in respect to both total specific binding of 125I-C9 and the proportion of cell-bound C9 appearing in high molecular weight (HMW) complexes. In a previous report, we found that after exposure to purified C5b6 and 125I-C7, specific C7 binding and, by implication, EC5b-7 formation were equal for PNH-III E and normal E. In the present study, C8-dependent binding of 125I-C9 to PNH-III EC5b-7 and normal EC5b-7 was also similar, although lysis of the PNH-III E was up to five times greater; that is, PNH-III E required fewer bound C9 molecules to produce an effective lytic site than did normal E. To quantify radioactivity in monomeric and HMW forms of membrane-bound C9, lysed and unlysed E were subjected to low ionic strength buffers to convert all E to ghosts. These ghosts were solubilized in 0.1 or 2% SDS (without reduction) and electrophoresed on 2.4-11% polyacrylamide gradient gels followed by autoradiography and densitometric scanning. With 0.1% SDS, broad, heterodisperse zones of HMW C9 were recovered from both PNH and normal ghosts; the amounts of C9 incorporated into the HMW complexes were similar for PNH-III E and normal E. In selected experiments, 125I-C7 could be shown in these same HMW bands. When membranes were solubilized in 2% SDS, the overall proportion of HMW C9 complexes compared with dimer and monomer C9 was reduced on both PNH and normal membranes. In many, but not all experiments, more of the highest mol wt C9 complexes were detected from PNH-III E membranes solubilized in 2% SDS than from normal or PNH-II E membranes similarly treated. When antibody-sensitized E were lysed by purified C1-C9, PNH-III EA bound far more C9 than did normal EA, and both lysis and C9 incorporation into HMW complexes were markedly and proportionately increased over normal; however, lytic efficiency of 125I-C9 bound to PNH EA was equal to or less than that bound to normal EA.(ABSTRACT TRUNCATED AT 250 WORDS)

The pore-forming protein (perforin) of cytolytic T lymphocytes is immunologically related to the components of membrane attack complex of complement through cysteine-rich domains.

Structural, functional and immunological similarities between the ninth component of complement (C9) and the lymphocyte pore-forming protein (PFP, perforin) have recently been described (8-10). PFP is shown here to be immunologically related to all other components of the membrane attack complex (MAC) of human complement, namely, C5b-6, C7, C8, and C9. Polyclonal antibodies raised against purified human C5b-6, C7, C8, or C9 react with other components of the MAC and with mouse lymphocyte PFP. The antigenic epitopes shared by human complement proteins and mouse lymphocyte PFP are limited to cysteine-rich domains. Only complement proteins that have been reduced and alkylated elicit the production of crossreactive antibodies when used as immunogens. The nonreduced forms of complement components or lymphocyte PFP neither react with these antibodies nor give rise to crossreactive antibodies. The homologous domains of complement proteins and lymphocyte PFP may play related functions in their attachment to lipid membranes and assembly of membrane lesions.

Cooperative interaction of factor B and other complement components with mononuclear cells in the antibody-independent lysis of xenogeneic erythrocytes.

Synergistic cytotoxicity is a term used to describe a cytotoxic system in which xenogeneic erythrocyte target cells are lysed in the presence of nonimmune human mononuclear effector cells and antibody-depleted normal human serum. Neither the mononuclear cells nor the serum alone are cytolytic to the target erythrocytes. Previous studies have shown that the serum activity is not immunoglobulin and is heat-labile, suggesting a similarity to serum complement. In this report, sera deficient in various complement components as well as highly purified single complement components were tested with whole mononuclear cell populations and purified monocytes and lymphocytes to further characterize this cytotoxicity system. Whole mononuclear cell populations failed to mediate target cell lysis in sera deficient in C5 or factor B. However, C3-deficient serum, even in the presence of anti-C3 antibody, supported synergistic cytotoxicity normally. Purified lymphocytes were also normally cytotoxic in C3-deficient serum but failed to lyse targets in sera deficient in C5, C7, C8, or depleted of factor B. Purified monocytes failed to lyse the target cells only in factor B-depleted serum and could lyse the target cells in serum-free medium when purified factor B alone was added. Monocyte-mediated cytotoxicity induced by factor B was inhibited 73-100% by adding lymphocytes back to the purified monocytes. Thus, both lymphocytes and monocytes can serve as effector cells in this form of cytotoxicity but require cooperative interaction with different sets of complement components. In addition, lymphocytes can modulate the monocyte-mediated form of target cell lysis associated with factor B.

Amastigotes of Trypanosoma cruzi escape destruction by the terminal complement components.

We studied the effect of complement on two life cycle stages of the protozoan parasite Trypanosoma cruzi: epimastigotes, found in the insect vector, and amastigotes, found in the mammalian host. We found that while both stages activate vigorously the alternative pathway, only epimastigotes are destroyed. The amounts of C3 and C5b-7 deposited on the amastigotes were similar to those bound to the much larger epimastigotes. Binding of C9 to amastigotes was four to six times less than binding to epimastigotes, resulting in a lower C9/C5b-7 ratio. Although a fairly large amount of C9 bound stably to amastigotes, no functional channels were formed as measured by release of incorporated 86Rb. The bound C9 had the characteristic properties of poly-C9, that is, it expressed a neo-antigen unique to poly-C9, and migrated in SDS-PAGE with an apparent Mr greater than 10(5). The poly-C9 was removed from the surface of amastigotes by treatment with trypsin, indicating that it was not inserted in the lipid bilayer. Modification of amastigote surface by pronase treatment rendered the parasites susceptible to complement attack. These results suggest that amastigotes have a surface protein that binds to the C5b-9 complex and inhibits membrane insertion, thus protecting the parasites from complement-mediated lysis.

C7 deficiency and meningococcal infection susceptibility in two spanish families.

In this work, we report the genetic basis of C7 deficiency in two different Spanish families. In family 1, by using exon-specific polymerase chain reaction and sequencing, a recently described mutation was found in homozygosity in the patient; a single base change in exon 15 (C2107T) leading to a stop codon that causes truncation of the C-terminal portion of C7 (Q681X). Patient's father, mother and sister were heterozygous for this mutation. Interestingly, patient's parents were not related. In family 2, a new single base mutation in exon 2 (G90A), leading to a stop codon that causes the premature truncation of C7 (W8X), was found in the patient, mother and sister 1. Additionally, patient 2, her father and sisters, displayed a missense mutation in exon 9 (G1135C) resulting in a change of aminoacid (G357R). Although sister 1 bore the same mutations in the C7 gene that patient 2, she remains asymptomatic. Because both mutations were found in the patient and her sister, we analyse other defence mechanisms such as FcgammaR polymorphisms as well as mannose-binding lectin alleles (MBL2 gene) and MBL levels. Results showed that both siblings bore identical combinations of FcgammaR allotypes and different MBL2 alleles, exhibiting patient 2 a MBL-insufficient genotype. Normal MBL levels were found in patient 1 and in two previously studied C7-deficient siblings, suggesting the involvement of other mechanisms of immunity distinct of FcgammaR variants and the MBL pathway, for the absence of meningococcal recurrent infections in certain C7-deficient individuals.

Evaluation of Serum and Gene Expression of Galectin-4, Interleukin-27, and Complement-7 in Hepatitis C Virus-Infected Egyptian Patients.

BACKGROUND: Hepatitis C virus (HCV) is considered a major global public health problem. Recently, there are great advances in HCV therapy, but there are some limitations that are creating an urgent need for assessment of some cytokines that have a potent antiviral effect in the immune system and anti-inflammatory effects to provide a potential novel immunotherapeutic target in HCV infection. OBJECTIVE: This study was directed to assess the serum levels and gene expression levels of Galectin-4 (LEG4), Interleukin-27 (IL-27), and Complement-7 (C-7) and their correlation with the viral load in HCV infection. Subjects and Methods. This work was conducted on 80 subjects, Group 1 (n = 40) early detected HCV patients and Group 2 (n = 40) healthy controls. LEG4, IL-27, and C-7 were assessed at the protein levels by ELISA, and their gene expression was assessed by RT-qPCR. The viral load was measured by PCR. RESULTS: There were significant elevations in the mean levels of gene expression and serum levels of all studied parameters LEG4, IL-27, and C-7 in the HCV group compared to the control group. Significant negative correlations between the viral load and each of the serum proteins and gene expressions of both LEG4 and IL-27 in HCV patients were found. The gene expression levels of LEG4, IL-27, and C-7 were positively correlated with their corresponding serum proteins in HCV patients. CONCLUSION: LEG4 and IL-27 showed significant negative correlations with the viral load, which could be an immune response to the control of the extent of hepatic inflammation, thus creating a potential novel immunotherapeutic approach in HCV infection for further studies or therapeutic clinical trials.

Complement protein C3 binding to Bacillus anthracis spores enhances phagocytosis by human macrophages.

Alveolar macrophages are thought to play a central role in the pathogenesis of inhalational anthrax. Receptors present on macrophages that mediate phagocytosis of Bacillus anthracis spores have yet to be completely defined. To begin to determine if soluble factors that are present in the lung such as immunoglobulin and complement are involved, we characterized the binding of human IgG and C3 to the surface of B. anthracis spores at different concentrations of nonimmune human serum. Furthermore we investigated the uptake of B. anthracis spores by human monocyte-derived macrophages in the presence of nonimmune human serum. Here we show that C3b is bound to B. anthracis spores and is activated through the classical pathway by IgG bound to the spore surface. Furthermore, we show that C3 serves as an opsonin for B. anthracis spores resulting in enhanced phagocytosis by human macrophages. These studies provide evidence that nonimmune serum contains IgG which binds to B. anthracis spores but is not sufficient to initiate phagocytosis. However, surface-bound IgG does initiate the classical pathway of complement activation, which is active in the lung, resulting in deposition of the opsonin C3b on the spore surface.

Molecular and functional characterization of a CD59 analogue from large yellow croaker Pseudosciana crocea.

CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. Here, the cDNA of a CD59 analogue was cloned from large yellow croaker (Pseudosciana crocea), a marine fish (LycCD59), by expressed sequence tags (EST) and RACE techniques. The open reading frame (ORF) of 351 nucleotides (nt) of LycCD59 encodes a polypeptide of 117 amino acids (aa), which includes a putative 20-aa NH(2)-signal peptide and a 97-aa coding region with a putative GPI-anchoring site at Asn(71). The deduced LycCD59 protein shared the structural feature of mammalian CD59, including a conserved cysteine skeleton responsible for the formation of disulfide bonds, and a similar pattern of hydrophobic termini. RT-PCR analysis showed that LycCD59 mRNA was broadly expressed in various tissues examined, except for intestine. And Northern blot analysis revealed a single LycCD59 transcript of approximately 1.0kb. LycCD59 expression in blood, spleen, and kidney was significantly up-regulated during 24h of induction with poly(I:C) or inactivated trivalent bacterial vaccine as determined by a relative quantitative real-time PCR analysis, and a coordinated up-regulation of LycCD59 and complement C3 and C7 mRNA was also found in these three tissues post-induction although their up-regulation pattern and extent were somewhat different in various tissues with poly(I:C) or bacterial vaccine. The recombinant protein of LycCD59 produced in E. coli was shown to significantly inhibit the erythrocyte lysis of tilapia (Oreochromis niloticus) in an in vitro hemolytic system, which was mediated by serum from large yellow croaker and tilapia, respectively, but not from mouse and chicken, suggesting that LycCD59 has a species-selective inhibition of complement activation. These results represent the first functional identification of a CD59 analogue in teleost fish, strongly suggesting the presence of regulatory mechanism for terminal complement pathway in teleost fish.

Complement lysis of human erythrocytes. Differeing susceptibility of two types of paroxysmal nocturnal hemoglobinuria cells to C5b-9.

Although enhanced sensitivity of erythrocytes to complement-mediated lysis is a hallmark of paroxysmal nocturnal hemoglobinuria (PNH), subpopulations of erythrocytes in such patients vary significantly in this respect. One PNH erythrocyte subpopulation (termed type III) comprises exquisitely sensitive cells, whereas type II PNH erythrocytes are intermediate in complement sensitivity between PNH type III and normal human erythrocytes. Differences in the action of the terminal complement components that would account for the differing lytic behavior of types II and III PNH erythrocytes have been proposed but not directly demonstrated. The present studies, making use of carefully selected cases with pure populations of type II or type III erythrocytes, confirm a prior observation that antibody-coated PNH erythrocytes of both types II and III display comparably supranormal C3 binding in whole human serum. However, when lysis was induced by the isolated C5b-9 membrane attack mechanism, bypassing the requirement for C3 binding, only type III PNH cells exhibited greater than normal lysis. This finding suggests that type III PNH erythrocytes have an additional membrane abnormality not present in type II cells. Thus, the differing lytic behavior of these two cell types in whole serum may reflect the additive effects on type III cells of both exaggerated C3 binding and enhanced sensitivity to C5b-9, whereas the more moderate lysis of type II PNH cells may be determined mainly or entirely by the earlier-acting mechanism producing augmented C3 binding. The failure of guinea pig C8 and C9, as opposed to human C8 and C9, to reveal the true lytic sensitivity of PNH-III E in our earlier study is illustrated, and its implications briefly discussed.

Characterization of the complement sensitivity of paroxysmal nocturnal hemoglobinuria erythrocytes.

The affected erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH II and PNH III cells) are abnormally sensitive to complement-mediated lysis. Normal human erythrocytes chemically modified by treatment with 2-amino-ethylisothiouronium bromide (AET) have been used as models for PNH cells inasmuch as they also exhibit an enhanced susceptibility to complement. To investigate the bases for the greater sensitivity of these abnormal cells to complement-mediated lysis, we compared binding of C3 and constituents of the membrane attack complex to normal, PNH II, PNH III, and AET-treated cells after classical pathway activation by antibody and fluid-phase activation by cobra venom factor complexes. When whole serum complement was activated by antibody, there was increased binding of C3 and C9 to PNH II, PNH III, and AET-treated cells, although the binding of these complement components to PNH II and PNH III cells was considerably greater than their binding to the AET-treated cells. In addition, all of the abnormal cell types showed a greater degree of lysis per C9 bound than did the normal erythrocytes. PNH III and AET-treated cells were readily lysed by fluid-phase activation of complement, whereas normal and PNH II erythrocytes were not susceptible to bystander lysis. The greater hemolysis of PNH III and AET-treated cells in this reactive lysis system was due to a quantitative increase in binding of constituents of the membrane attack complex. This more efficient binding of the terminal components after fluid-phase activation of whole serum complement was not mediated by cell-bound C3 fragments. These investigations demonstrate that the molecular events that characterize the enhanced susceptibility of PNH II, PNH III, and AET-treated erythrocytes to complement-mediated lysis are heterogeneous.

The Syk Tyrosine Kinase Is Required for Skin Inflammation in an In Vivo Mouse Model of Epidermolysis Bullosa Acquisita.

The inflammatory form of epidermolysis bullosa acquisita is caused by autoantibodies against type VII collagen (C7), a component of the dermal-epidermal junction. We have previously shown that myeloid Src family kinases mediate skin inflammation triggered by anti-C7 antibodies. Here we identify the Syk tyrosine kinase as a critical component of autoantibody-induced skin inflammation downstream of Src family kinases. Immobilized C7-anti-C7 immune complexes triggered neutrophil activation and Syk phosphorylation in a Src family kinase-dependent manner. Bone marrow chimeric mice lacking Syk in their hematopoietic compartment were completely protected from skin inflammation triggered by anti-C7 antibodies despite normal circulating anti-C7 levels. Syk deficiency abrogated the accumulation of CXCL2, IL-1ß, and leukotriene B4 at the site of inflammation and resulted in defective in vivo neutrophil recruitment. Syk-/- neutrophils had a normal intrinsic migratory capacity but failed to release CXCL2 or leukotriene B4 upon activation by immobilized C7-anti-C7 immune complexes, indicating a role for Syk in the amplification of the inflammation process. These results identify Syk as a critical component of skin inflammation in a mouse model of epidermolysis bullosa acquisita and as a potential therapeutic target in epidermolysis bullosa acquisita and other mechanistically related inflammatory skin diseases such as bullous pemphigoid.

Magnetic bead based assays for complement component C5.

Two novel magnetic agarose bead based assays have been developed to measure complement component C5 interaction with C3b and the Factor I Modules (FIMs) of C7. One innovation was to couple C3b onto the magnetic agarose bead using the alternative pathway C3 convertase, which resulted in a linkage of the ligand by a covalent ester bond. A second innovation was to employ nickel ion charged N,N,N'-tris(carboxymethyl)ethylene-diamine-magnetic agarose to capture recombinantly prepared C7 FIMs that were expressed with an oligo-histidine linker followed by an acidic domain that provided a spacer enabling the C7 modules exposure to C5. Detection was brought about by peroxidase coupled to C5. Both assays exhibited adequate statistics suitable for screening. As examples of the utility of these new methods, we chose to examine influence of natural products on C5 interaction. Fucoidan and ß-glucans were observed to inhibit C3b-C5 interaction, and dextran sulfate was similarly active; however, rosmarinic acid had no measurable effect. In contrast only ß-glucans from two species of macrofungi were able to interfere with interaction of C5 with the FIMs of C7.