Analysis of cold activation of the contact system in hereditary angioedema with normal C1 inhibitor.

Hereditary angioedema (HAE) attacks are caused by excessive activation of the contact system. Understanding how the contact system is activated in HAE, especially in patients with normal C1 inhibitor (HAEnCI), is essential to effectively treat this disease. Contact system activation involves the cleavage of several proteins including Factor XII (FXII), high molecular weight kininogen (HK), prekallikrein, sgp120 (ITIH4) and C1 inhibitor (C1-INH) before the subsequent generation of bradykinin that mediates HAE. In this study, we evaluated the fragmentation and enzymatic activity of contact system proteins in HAEnCI plasma samples before and after contact system activation induced by incubation in the cold. Our results show that in contrast to normal plasma, cold activation induced contact system activation in the majority of the HAEnCI patient samples we tested, in which each contact system protein exhibited fragmentation, FXII and kallikrein enzymatic activity increased, and C1-INH functional activity decreased. HAEnCI samples with low FXII concentrations or functional activity were not affected by cold activation. One HAEnCI sample with a plasminogen gene mutation activated the fibrinolytic system, as shown by an increase in concentration of plasma D dimers. Our results suggest that cold activation seems to be initiated by the cleavage of prekallikrein, and that it needs FXII in order to occur. Reported to be susceptible to excessive contact system activation after incubation in the cold, we further applied this system of study to the evaluation of plasma from women undergoing estrogen treatment. Similar to plasma from HAEnCI patients, excessive contact system activation was demonstrated.

sgp120 and the contact system in hereditary angioedema: A diagnostic tool in HAE with normal C1 inhibitor.

Mutations in Factor XII, plasminogen gene, angiopoietin-1 gene and kininogen 1 gene have been found in some patients with hereditary angioedema with normal C1 inhibitor (HAE-nl-C1inh), but the underlying disease mechanisms remain unclear. Additionally, there are no accepted biomarkers for this disease. Because the contact system has been implicated in hereditary angioedema with C1 inhibitor deficiency (HAE-C1inh), we studied the fragmentation patterns of serum glycoprotein 120 (sgp120), a protein that is highly susceptible to cleavage by kallikrein, in 31 HAE-C1inh and 13 HAE-nl-C1inh patient plasma samples. Compared to normal controls, the majority of plasma samples from patients with HAE-C1inh contained fragmented sgp120. These samples also showed increased kallikrein amidolytic activity indicating spontaneous contact system activation. In contrast, most samples from HAE-nl-C1inh patients exhibited intact sgp120. However, if these samples were incubated at 4 °C in plastic, significant sgp120 fragmentation and spontaneous contact system activation were observed. Concurrently, there was C1 inhibitor fragmentation that generated the nonfunctional 94 kD fragment and a reduction in C1 inhibitor function. Normal samples did not show sgp120 or C1 inhibitor fragmentation after incubation. We sequenced sgp120 and found it to be identical to inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4). These results suggest that sgp120 or ITIH4 is cleaved when the contact system is activated and that this cleavage could be used as a biomarker in patients with HAE-nl-C1inh.

Complement activation by IgG containing immune complexes regulates the interaction of C1q with its ligands.

Classical pathway activation of the compl ement system is initiated by the binding of the globular head domains of glycoprotein C1q to its corresponding ligand leading to both C1 activation and C3 convertase formation. However, the whereabouts and function of C1q after complement activation have only been marginally investigated. This report presents two mechanisms of action that remove bound C1q from a complement activating IgG immune complex in concentrated serum. The first mechanism details that sequential activation of the classical and alternative pathways releases bound C1q from an immune complex and that the dissociated C1q is subsequently found in complex with complement fragment C3c. The second mechanism is the displacement of C1q from an immune complex by the addition of near physiologic concentrations of purified or serum C1q. This activity can also be demonstrated using serum depleted of C3, normal serum chelated in EDTA, or purified C1. Fresh C1q in C3-depleted serum was found to replace dissociated C1q on the immune complex. C1q dissociated from immune complexes by the mechanism of C1q displacement is able to bind B and T lymphoblastoid cells that express receptors and ligands for both the collagen like region and the globular head domains of C1q. C1q dissociated from immune complexes by the mechanism of C3 activation do not bind these cells. This result suggests that C3 bound to C1q during complement activation and dissociation interferes with the ability of released C1q to access C1q receptors and ligands, particularly receptors for the globular head domains. These underlying mechanisms that regulate the interaction of C1q with its ligands reveal a novel function for complement activation during the immune response.

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21).

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes.

Complement and arthritis: another step in understanding.

In a recent research article in Arthritis Research and Therapy ('Analysis of C204 and the C4 binding protein in the MRL/lpr mouse'), Wenderfer and colleagues report that deficiency in C4 binding protein, a down-regulator of the classic pathway of complement, does not affect the development of autoimmune disease. These data support the earlier finding that the alternative pathway, and not the classic pathway, drives disease progression. However, in a milder variant of the MRL/lpr model, the lpr/lpr mouse, classic pathway deficiency does contribute toward renal pathology and more severe disease. In this editorial we discuss the factors that may cause such a discrepancy.

Role of complement receptors 1 and 2 (CD35 and CD21), C3, C4, and C5 in survival by mice of Staphylococcus aureus bacteremia.

Complement-mediated opsonization and phagocytosis of encapsulated serotype 5 Staphylococcus aureus are essential to host defense. We describe the effects of complement depletion and deficiencies of C4, C5, and complement receptors 1 and 2 on mouse survival after intravenous exposure to S aureus. Depletion of complement proteins in C57BL/6 mice with the use of cobra-venom factor decreased survival compared with that of controls after the induction of bacteremia with mucoid (90% mortality), encapsulated (73%), and unencapsulated (59%) S aureus strains. In this model complement is even more important in the control of infection with encapsulated S aureus (80% of clinical isolates) than in the control of infection by unencapsulated strains. C4-deficient mice demonstrated similar mortality from bacteremia caused by encapsulated S aureus compared with controls, suggesting that in the unimmunized animal the alternative complement pathway contributes more to control of bacteremia caused by encapsulated S aureus than the classical complement pathway or mannan-binding lectin pathway. C5-deficient mice (B10.D2-H2(d) H2-T18(c) Hc(0)/oSnJ) showed similar mortality when subjected to bacteremia caused by encapsulated S aureus compared with C5-sufficient (B10.D2-Hc(1) H2(d) H2-T18(c)/nSnJ) mice, suggesting that in this model the anaphylatoxin C5a and the late complement cascade are not critical to survival of bacteremia induced with the use of these strains. However, C5-deficient mice depleted of C3 with the use of cobra-venom factor had 60% decreased survival compared with untreated C5-deficient mice with bacteremia induced by encapsulated S aureus, suggesting that in this model C3 is more critical than C5 in controlling S aureus bacteremia. Complement receptor 1 (CD35) is the primary receptor for the opsonin C3b. Mice deficient in CD35/CD21 showed a 67% decrease in survival compared with normal mice, suggesting that CD35/CD21 is of major importance in the control of S aureus-induced bacteremia.