C1-Inhibitor Decreases the Release of Vasculitis-Like Chemotactic Endothelial Microvesicles.

The kinin system is activated during vasculitis and may contribute to chronic inflammation. C1-inhibitor is the main inhibitor of the kinin system. In this study, we investigated the presence of the kinin B1 receptor on endothelial microvesicles and its contribution to the inflammatory process. Compared with controls (n=15), patients with acute vasculitis (n=12) had markedly higher levels of circulating endothelial microvesicles, identified by flow cytometry analysis, and significantly more microvesicles that were positive for the kinin B1 receptor (P<0.001). Compared with microvesicles from wild-type cells, B1 receptor-positive microvesicles derived from transfected human embryonic kidney cells induced a significant neutrophil chemotactic effect, and a B1 receptor antagonist blocked this effect. Likewise, patient plasma induced neutrophil chemotaxis, an effect decreased by reduction of microvesicle levels and by blocking the B1 receptor. We used a perfusion system to study the effect of patient plasma (n=6) and control plasma (n=6) on the release of microvesicles from glomerular endothelial cells. Patient samples induced the release of significantly more B1 receptor-positive endothelial microvesicles than control samples, an effect abrogated by reduction of the microvesicles in the perfused samples. Perfusion of C1-inhibitor-depleted plasma over glomerular endothelial cells promoted excessive release of B1 receptor-positive endothelial microvesicles compared with normal plasma, an effect significantly decreased by addition of C1-inhibitor or B1 receptor-antagonist. Thus, B1 receptor-positive endothelial microvesicles may contribute to chronic inflammation by inducing neutrophil chemotaxis, and the reduction of these microvesicles by C1-inhibitor should be explored as a potential treatment for neutrophil-induced inflammation.

C1-esterase inhibitor for short-term prophylaxis in a patient with hereditary angioedema with normal C1 inhibitor function.

Hereditary angioedema with normal C1-esterase inhibitor (HAE-nC1INH) perioperative is a rare condition which could have potential disastrous ramifications for the anesthesiologist in the perioperative period. However, there is limited evidence and/or guidelines on the optimal way to manage these patients. We present the case of a patient with HAE-nC1INH who was successfully managed in the perioperative period with plasma derived C1-esterase inhibitor (pdC1INH). A 29-year-old woman with a diagnosis of HAE-nC1INH presented to the preoperative consultation in preparation for an upcoming total thyroidectomy. She had a 14-year history of ongoing lip and facial edema sometimes necessitating emergency department visitation. Close consultation with her immunologist, transfusion medicine specialists, and anesthesia care providers allowed for a preoperative plan to provide the patient adequate prophylaxis. Both pdC1INH and tranexamic acid were given preoperatively. The patient underwent surgery with no complications. A multidisciplinary team of clinical immunologists, transfusion medicine specialists, and anesthesiologists facilitated the successful perioperative management of a patient with HAE-nC1INH; pdC1INH may a suitable prophylactic perioperative therapy for this rare patient population.

Distinctive regulation of contact activation by antithrombin and C1-inhibitor on activated platelets and material surfaces.

Activated human plate lets trigger FXII-mediated contact activation, which leads to the generation of FXIIa-antithrombin (AT) and FXIa-AT complexes. This suggests that contact activation takes place at different sites, on activated platelets and material surfaces, during therapeutic procedures involving biomaterials in contact with blood and is differentially regulated. Here we show that activation in platelet-poor plasma, platelet-rich plasma (PRP), and whole blood induced by glass, kaolin, and polyphosphate elicited high levels of FXIIa-C1-inhibitor (C1INH), low levels of FXIa-C1INH and KK-C1INH, and almost no AT complexes. Platelet activation, in both PRP and blood, led to the formation of FXIIa-AT, FXIa-AT, and kallikrein (KK)-AT but almost no C1INH complexes. In severe trauma patients, FXIIa-AT and FXIa-AT were correlated with the release of thrombospondin-1 (TSP-1) from activated platelets. In contrast, FXIIa-C1INH complexes were detected when the FXIIa-AT levels were low. No correlations were found between FXIIa-C1INH and FXIIa-AT or TSP-1. Inhibition of FXIIa on material surfaces was also shown to affect the function of aggregating platelets. In conclusion, formation of FXIIa-AT and FXIIa-C1INH complexes can help to distinguish between contact activation triggered by biomaterial surfaces and by activated platelets. Platelet aggregation studies also demonstrated that platelet function is influenced by material surface-mediated contact activation and that generation of FXIIa-AT complexes may serve as a new biomarker for thrombotic reactions during therapeutic procedures employing biomaterial devices.

Cross-species function of the pig C1 esterase inhibitor.

BACKGROUND: The use of a bioartificial liver with pig cells for the treatment of fulminant hepatic failure will require research on the plasma complement regulatory proteins of the pig, because the liver produces most of the complement components and plasma complement regulatory proteins. In our previous study, the pig C1 esterase inhibitor (C1-INH), which functions as an inhibitor of the complement reaction in the first step of the classical pathway in the fluid phase, was cloned and some relevant features of the molecule were characterized, especially its cross-species regulation, in comparison with human C1-INH. In a further analysis, the species specificity of C1-INH was examined, using pig endothelial cells (PEC) and several types of sera. MATERIALS AND METHODS: The cDNA of pig C1-INH was used to produce the membrane type pC1-INH, pC1-INH-PI, and inserted into the cloning site of pCXN2 (chicken beta actin promoter). The pCX/pCl-INH-PI plasmid was then transfected into PEC to establish stable PEC with pCl-INH-PI. The expression of the pCl-INH-PI was evaluated by a FACS analysis, and complement-dependent cell lysis with human, dog, rabbit, and mouse sera was then assessed. RESULTS: The transfectant with pig Cl-INH-PI showed a high level of expression on PEC. The PEC transfectants showed an inhibitory effect on complement-dependent PEC lysis. Pig Cl-INH did not show the same suppressive effect for each serum. However, considering the alternative pathway activation of each serum on the pig cell membrane, it can be concluded that pCl-INH has a relatively small species restriction. CONCLUSION: Pig Cl-INH, having a similar structure to human Cl-INH, shows a strong complement regulatory function on other species sera.

A direct role for C1 inhibitor in regulation of leukocyte adhesion.

Plasma C1 inhibitor (C1INH) is a natural inhibitor of complement and contact system proteases. Heterozygosity for C1INH deficiency results in hereditary angioedema, which is mediated by bradykinin. Treatment with plasma C1INH is effective not only in patients with hereditary angioedema, but also in a variety of other disease models, in which such therapy is accompanied by diminished neutrophil infiltration. The underlying mechanism has been explained primarily as a result of the inhibition of the complement and contact systems. We have shown that C1INH expresses the sialyl-Lewis(x) tetrasaccharide on its N-linked glycan, via which it binds to E- and P-selectins and interferes with leukocyte-endothelial adhesion in vitro. Here we show that both native C1INH and reactive center cleaved C1INH significantly inhibit selectin-mediated leukocyte adhesion in several in vitro and in vivo models, whereas N-deglycosylated C1INH loses such activities. The data support the hypothesis that C1INH plays a direct role in leukocyte-endothelial cell adhesion, that the activity is mediated by carbohydrate, and that it is independent of protease inhibitory activity. Direct involvement of C1INH in modulation of selectin-mediated cell adhesion may be an important mechanism in the physiologic suppression of inflammation, and may partially explain its utility in therapy of inflammatory diseases.

C1-inhibitor protects against brain ischemia-reperfusion injury via inhibition of cell recruitment and inflammation.

Previous studies demonstrated that C1-inhibitor (C1-INH), a complement and contact-kinin systems inhibitor, is neuroprotective in cerebral ischemia. To investigate the mechanism of this action, we evaluated the expression of neurodegeneration and inflammation-related factors in mice subjected to 2-h ischemia and 2 or 46 h reperfusion. C1-INH significantly dampened the mRNA expression of the adhesion molecules P-selectin and ICAM-1 induced by the ischemic insult. It significantly decreased the pro-inflammatory cytokine (TNF alpha, IL-18) and increased the protective cytokine (IL-6, IL-10) gene expression. C1-INH treatment prevented the decrease of NFH gene, a marker of cellular integrity and counteracted the increase of pro-caspase 3, an apoptosis index. Furthermore, C1-INH markedly inhibited the activation and/or recruitment of microglia/macrophage, as shown by immunohistochemistry. In conclusion, C1-INH exerts an anti-inflammatory and anti-apoptotic action on ischemia-reperfusion injury. Our present and past data support a major effect of C1-INH on cell recruitment from the vasculature to the ischemic site.

Local and remote ischemia-reperfusion injury is mitigated in mice overexpressing human C1 inhibitor.

Activation of the classical complement pathway is crucially involved in complement-mediated endothelial cell damage in ischemia-reperfusion injury. C1 inhibitor is the only known physiological inhibitor of classical complement pathway activation. Transgenic mice overexpressing human C1 inhibitor were used in a surgical lower torso and a liver ischemia-reperfusion model. Organ-specific endothelial disruption was determined by 125I-tagged albumin extravasation. In the lower torso ischemia-reperfusion model, transgenic mice overexpressing the C1 inhibitor were protected in the muscle and the lungs from endothelial cell damage. In the liver ischemia-reperfusion model, endothelial cell integrity was preserved in transgenic animals in the liver, the gut and the lungs. Our data indicate that inhibiting complement activation by a transgenic approach is effective in protection against ischemia-reperfusion injury.

Mutational analyses of the recombinant globular regions of human C1q A, B, and C chains suggest an essential role for arginine and histidine residues in the C1q-IgG interaction.

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the globular domain (gC1q) of C1q to the Fc regions of aggregated IgG or IgM. Each gC1q domain is a heterotrimer of the C-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Our recent studies have suggested a modular organization of gC1q, consistent with the view that ghA, ghB, and ghC are functionally autonomous modules and have distinct and differential ligand-binding properties. Although C1q binding sites on IgG have been previously identified, the complementary interacting sites on the gC1q domain have not been precisely defined. The availability of the recombinant constructs expressing ghA, ghB, and ghC has allowed us, for the first time, to engineer single-residue substitution mutations and identify residues on the gC1q domain, which are involved in the interaction between C1q and IgG. Because C1q is a charge pattern recognition molecule, we have sequentially targeted arginine and histidine residues in each chain. Consistent with previous chemical modification studies and the recent crystal structure of gC1q, our results support a central role for arginine and histidine residues, especially Arg(114) and Arg(129) of the ghB module, in the C1q-IgG interaction.

Effect of complement fragment 1 esterase inhibition on survival of human decay-accelerating factor pig lungs perfused with human blood.

BACKGROUND: The role of complement in hyperacute lung xenograft rejection has not been fully elucidated. The present study evaluates the effect of complement (C) 1 esterase inhibition on hyperacute rejection of human decay-accelerating factor (hDAF)-positive pig lung by human blood. METHODS: Using a modification of an established ex vivo model, right and left lungs from individual animals were surgically isolated and separately perfused. Pigs homozygous for hDAF were perfused with fresh human blood that was either untreated or treated with complement 1 esterase inhibitor (C1-Inh) at doses of 1 U/ml (n = 5), 5 U/ml (n = 3) or 10 U/ml plasma (n = 5). RESULTS: Only C1-Inh at 10 U/ml prolonged survival time (230 +/- 48.3 minutes) as compared with controls (65.6 +/- 26.5 minutes, p < 0.05) and diminished complement activation (C3a and C5a, p < 0.05). Interestingly, a low concentration of C1-Inh increased the pulmonary vascular resistance (PVR; 1 U/ml: 0.54 +/- 0.3; 10 U/ml: 0.19 +/- 0.08). Sequestration of neutrophils (92 +/- 3%) and platelets (64 +/- 13%) was not prevented by any concentration of C1-Inh. Tissue deposition of C3b and C5b-9 were diminished by hDAF expression, and further blunted by treatment, with 10 U/ml C1-Inh. CONCLUSIONS: Complement plays a critical role in early events of lung hyperacute rejection (HAR). However, even potent inhibition of C1 esterase and C3/C5 convertase, using serum C1-Inh in pig lungs homozygous for hDAF expression, does not prevent rapid lung injury. Our findings implicate innate immune pathways resistant to efficient complement regulation, and suggest a role for neutrophils and platelets in the lung's particular vulnerability.

Complement regulatory protein C1 inhibitor binds to selectins and interferes with endothelial-leukocyte adhesion.

C1 inhibitor (C1INH), a member of the serine proteinase inhibitor (serpin) family, is an inhibitor of proteases in the complement system, the contact system of kinin generation, and the intrinsic coagulation pathway. It is the most heavily glycosylated plasma protein, containing 13 definitively identified glycosylation sites as well as an additional 7 potential glycosylation sites. C1INH consists of two distinct domains: a serpin domain and an amino-terminal domain. The serpin domain retains all the protease-inhibitory function, while the amino-terminal domain bears most of the glycosylation sites. The present studies test the hypothesis that plasma C1INH bears sialyl Lewis(x)-related moieties and therefore binds to selectin adhesion molecules. We demonstrated that plasma C1INH does express sialyl Lewis(x)-related moieties on its N-glycan as detected using mAb HECA-452 and CSLEX1. The data also show that plasma C1INH can bind to P- and E-selectins by FACS and immunoprecipitation experiments. In a tissue culture model of endothelial-leukocyte adhesion, C1INH showed inhibition in a dose-dependent manner. Significant inhibition (>50%) was achieved at a concentration of 250 micro g/ml or higher. This discovery may suggest that C1INH plays a role in the endothelial-leukocyte interaction during inflammation. It may also provide another example of the multifaceted anti-inflammatory effects of C1INH in various animal models and human diseases.

Regulation of the mannan-binding lectin pathway of complement on Neisseria gonorrhoeae by C1-inhibitor and alpha 2-macroglobulin.

We examined complement activation by Neisseria gonorrhoeae via the mannan-binding lectin (MBL) pathway in normal human serum. Maximal binding of MBL complexed with MBL-associated serine proteases (MASPs) to N. gonorrhoeae was achieved at a concentration of 0.3 microg/ml. Preopsonization with MBL-MASP at concentrations as low as 0.03 microg/ml resulted in approximately 60% killing of otherwise fully serum-resistant gonococci. However, MBL-depleted serum (MBLdS) reconstituted with MBL-MASP before incubation with organisms (postopsonization) failed to kill at a 100-fold higher concentration. Preopsonized organisms showed a 1.5-fold increase in C4, a 2.5-fold increase in C3b, and an approximately 25-fold increase in factor Bb binding; enhanced C3b and factor Bb binding was classical pathway dependent. Preopsonization of bacteria with a mixture of pure C1-inhibitor and/or alpha(2)-macroglobulin added together with MBL-MASP, all at physiologic concentrations before adding MBLdS, totally reversed killing in 10% reconstituted serum. Reconstitution of MBLdS with supraphysiologic (24 microg/ml) concentrations of MBL-MASP partially overcame the effects of inhibitors (57% killing in 10% reconstituted serum). We also examined the effect of sialylation of gonococcal lipooligosaccharide (LOS) on MBL function. Partial sialylation of LOS did not decrease MBL or C4 binding but did decrease C3b binding by 50% and resulted in 80% survival in 10% serum (lacking bacteria-specific Abs) even when sialylated organisms were preopsonized with MBL. Full sialylation of LOS abolished MBL, C4, and C3b binding, resulting in 100% survival. Our studies indicate that MBL does not participate in complement activation on N. gonorrhoeae in the presence of "complete" serum that contains C1-inhibitor and alpha(2)-macroglobulin.

Increased vascular permeability in C1 inhibitor-deficient mice mediated by the bradykinin type 2 receptor.

Heterozygosity for C1 inhibitor (C1INH) deficiency results in hereditary angioedema. Disruption of the C1INH gene by gene trapping enabled the generation of homozygous- and heterozygous-deficient mice. Mating of heterozygous-deficient mice resulted in the expected 1:2:1 ratio of wild-type, heterozygous, and homozygous-deficient offspring. C1INH-deficient mice showed no obvious phenotypic abnormality. However, following injection with Evans blue dye, both homozygous and heterozygous C1INH-deficient mice revealed increased vascular permeability in comparison with wild-type littermates. This increased vascular permeability was reversed by treatment with intravenous human C1INH, with a Kunitz domain plasma kallikrein inhibitor (DX88), and with a bradykinin type 2 receptor (Bk2R) antagonist (Hoe140). In addition, treatment of the C1INH-deficient mice with an angiotensin-converting enzyme inhibitor (captopril) increased the vascular permeability. Mice with deficiency of both C1INH and Bk2R demonstrated diminished vascular permeability in comparison with C1INH-deficient, Bk2R-sufficient mice. These data support the hypothesis that angioedema is mediated by bradykinin via Bk2R.

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.

Clinical, biochemical, and genetic characterization of a novel estrogen-dependent inherited form of angioedema.

BACKGROUND: Two genetic forms of hereditary angioedema (HAE) are currently recognized. Both are transmitted in an autosomal dominant manner and are characterized by recurrent episodes of localized angioedema. Involvement of the gut leads to episodes of severe abdominal pain, and laryngeal involvement can lead to airway obstruction and even death. One type results from heterozygosity for a nonexpressed C1 inhibitor allele, and the other results from heterozygosity for a nonfunctional C1 inhibitor allele. OBJECTIVE: This report identifies a third type of HAE, with a unique estrogen-dependent phenotype. METHODS: Detailed medical histories were obtained from family members, and a pedigree was constructed to ascertain the mode of inheritance. Determination of serum complement factors, C1 inhibitor protein, C1 inhibitor function, coagulation factor XII, plasma prekallikrein, high molecular weight kininogen, and selected DNA sequences were performed in affected members by using standard assays. RESULTS: Episodes of angioedema were clinically indistinguishable from those associated with previously described forms of HAE; however, these occurred only during pregnancy or the use of exogenous estrogens. Patients were otherwise asymptomatic, except for one patient who had acetyl salicylic acid/nonsteroidal anti-inflammatory drug-related angioedema later in life. History was available for members spanning 4 generations, and affected individuals were identified in 3 generations. Of 46 family members, phenotype could be determined in 13 members. Seven were affected, and 6 were not. One male of undetermined phenotype was an obligate carrier. The unique estrogen-dependent nature of the phenotype means that the status of several members in the third and fourth generation remains unknown. The disorder appears to be transmitted in an autosomal dominant fashion, although other modes of inheritance cannot be excluded entirely. C1 inhibitor protein, C1 inhibitor function, C2, C4, C1q, coagulation factor XII, prekallikrein, and high molecular kininogen were normal in 3 affected family members during asymptomatic periods. DNA sequencing revealed no abnormality in 3 patients in the coding region of the gene encoding C1 inhibitor or in the 5' flanking regions of the genes encoding C1 inhibitor and factor XII. CONCLUSIONS: This family appears to have a novel form of inherited angioedema that does not result from C1 inhibitor deficiency or dysfunction. The phenotype is uniquely estrogen dependent. Implications for diagnosis and treatment are discussed. Further studies are required to define the exact nature of the genetic abnormality involved.

Hereditary angioedema with normal C1-inhibitor activity in women.

BACKGROUND: Hereditary angioedema (HAE) is a well defined autosomal dominant disease (Mendelian Inheritance in Man #106100) that results from an inherited deficiency of C1 (the activated first component of complement) inhibitor function. We report an unusual variant of HAE with normal biochemical C1-inhibitor function, occurring only in women. METHODS: We screened 574 patients with recurrent angioedema of the skin for presence of HAE. 283 patients were selected, in whom angioedema was associated with abdominal pain attacks or recurrent life-threatening episodes of upper-airway obstruction, or both, rather than with urticaria. We measured C1-inhibitor concentration and functional activity as well as complement C4 concentration and took pedigrees to characterise patients. FINDINGS: 94 HAE cases with C1-inhibitor deficiency, positive family history, or both were identified. Biochemical testing showed that 84 patients from 49 families had a functional C1-inhibitor deficiency. 11 of these patients had no affected family members (probably representing de-novo mutations). Ten women with HAE, from ten families, had normal C1-inhibitor protein concentrations and function, and normal C4 concentration. A more detailed study of these families identified another 26 affected members, who were also all women. Of those women, 14 could be studied and also had normal C1-inhibitor concentration and function. The disease was seen in successive generations, and in offspring of affected mothers, the sex ratio (M/F) was shifted to 1/1.5. INTERPRETATION: HAE with normal C1-inhibitor concentration and function represents a unique genetic disease arising only in women. The formal genetics of this entity are suggestive of an X-linked dominant mode of inheritance. For this disorder we propose the term hereditary angioedema type 3 (HAE III).

C1-Esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema.

C1-esterase inhibitor (C1-Inh) therapy was introduced in clinical medicine about 25 years ago as a replacement therapy for patients with hereditary angioedema caused by a deficiency of C1-Inh. There is now accumulating evidence, obtained from studies in animals and observations in patients, that administration of C1-Inh may have a beneficial effect as well in other clinical conditions such as sepsis, cytokine-induced vascular leak syndrome, acute myocardial infarction, or other diseases. Activation of the complement system, the contact activation system, and the coagulation system has been observed in these diseases. A typical feature of the contact and complement system is that on activation they give rise to vasoactive peptides such as bradykinin or the anaphylatoxins, which in part explains the proinflammatory effects of either system. C1-Inh, belonging to the superfamily of serine proteinase inhibitors (serpins), is a major inhibitor of the classical complement pathway, the contact activation system, and the intrinsic pathway of coagulation, respectively. It is, therefore, endowed with anti-inflammatory properties. However, inactivation of C1-Inh occurs locally in inflamed tissues by proteolytic enzymes (e.g., elastase) released from activated neutrophils or bacteria thereby leading to increased local activation of the various host defense systems. Here we will give an overview on the biochemistry and biology of C1-Inh. We will discuss studies addressing therapeutic administration of C1-Inh in experimental and clinical conditions. Finally, we will provide an explanation for the therapeutic benefit of C1-Inh in so many different diseases.