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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.

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.

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.

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-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.

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.

The mechanisms underlying the PF4/heparin immune response are poorly understood. In recent studies, we showed that PF4/heparin complexes, but not PF4 alone or heparin alone, activate complement (C') in a heparin-dependent manner and lead to selective binding of C'-coated antigen (PF4/heparin complexes) to B cells via CD21. In additional studies, we showed that heparinized patients have circulating B cells with C'-coated PF4/heparin complexes (Khandelwal, Blood 2016). To investigate the mechanism by which PF4/heparin complexes activate complement, we performed studies in whole blood using previously described methods assessing binding of C'-fixed PF4/heparin complexes to B cells in whole blood. We incubated blood with inhibitors or conditions known to selectively block specific complement activation pathways, followed by incubation with PF4 and heparin for 1 hour (hr) at 370 C. Binding of PF4/heparin complexes and C' fragments to B-cells was determined by flow cytometry using the murine monoclonal antibody to PF4/heparin complexes (KKO) and antibodies to specific C' activation products (Khandelwal, Blood 2016). To distinguish activation by classical and lectin pathways from alternative pathway of activation, we incubated blood with C1 esterase inhibitor, a protein that inhibits C’ activation by the classical and lectin pathways. As shown in Figure 1, whole blood incubated with PF4/heparin is associated with C’ activation as measured by C3c binding to B cells (Figure 1, column 3), but not if blood is incubated with buffer or PF4 alone (Figure 1, columns 1 & 2). With the addition of C1 esterase inhibitor (10 or 20 U/ml) prior to incubation with PF4/heparin complexes, we noted a dose-dependent reduction in C’ activation (>85% reduction with 10 and 20 U/ml) suggesting that C’ activation by PF4/heparin complexes occurs via the classical or lectin pathways. To further confirm that the alternate pathway is not involved in PF4/heparin mediated C’ activation, we used sensitivity of the alternative pathway to Mg2+ using differential chelation with EDTA and EGTA. EDTA, a chelator of both Ca2+ and Mg2+, inhibits complement activation by all three pathways, whereas EGTA, which preferentially sequesters Ca2+ over Mg2+, permits alternative pathway activation. As shown in Figure 2, incubation of PF4/heparin complexes without chelators allowed for maximal antigen binding to B cells (~100%), while incubation with EDTA (Figure 2, column 3) abolished antigen binding, as did EGTA with and without additional Mg2+ supplementation. We next investigated the role of the classical pathway, a pathway triggered by immune complex mediated-binding and activation of C1. By flow cytometry, we were unable to show C1q deposition although we were able to demonstrate C3c/C4c deposition on antigen-coated B cells from healthy donors (data not shown). We also showed that C’ activation by PF4/heparin complexes was preserved in human serum low in immunoglobulins (IgG and IgM) as well as plasma from mMT mice lacking circulating immunoglobulins (data not shown). To investigate the role of the lectin pathway, we performed competitive inhibition assays by incubating whole blood with PF4/heparin complexes in the presence of mannan. As shown in Figure 3, we show that mannan (500 mg/mL) inhibited binding of antigen to B cells. In other studies, we show that an anti-MBL antibody partially reduced binding of KKO/C’ to B cells. Together, these preliminary studies demonstrate a lack of involvement of the classical and alternative pathways in C’ activation and indicate a contribution by the lectin pathway in C’ activation by PF4/heparin complexes. Further studies are underway to elucidate the role of the lectin pathway in complement activation by PF4/heparin complexes. Insights from these studies will lead to novel interventions that can block the initial steps of immune activation by heparin. Download : Download high-res image (68KB) Download : Download full-size image Download : Download high-res image (62KB) Download : Download full-size image Download : Download high-res image (65KB) Download : Download full-size image Disclosures Arepally: Biokit: Patents & Royalties.

It has been known for over 100 years that immune complexes can cause severe organ pathology. Many investigators have studied mechanisms of immune complexes mediated tissue destruction and how they contribute to the development of autoimmunity, but many critical aspects about immune complex function in causing disease are still unknown. The predisposing factors that govern immune complex tissue deposition and whether or not circulating immune complexes participate in tissue pathology all remain unclear at this time. Nevertheless, a great deal has been learned. It is known that many of the pathological effects of immune complexes are due to their ability to activate complement and bind IgG Fc? receptors. It has been well documented that Fc? receptors may either promote or down-regulate the pathological effects induced by immune complexes. The constituents of immune complexes are also well-defined demonstrating that the isotype and subclass of antibody within the immune complex as well as the nature of the antigen itself all play key roles in pathology. Mediators induced by immune complexes have also been discovered and have been shown to influence pathology. There have been many animal models of disease developed and studied in great detail allowing scientists to understand the mechanisms of clearance of immune complexes from the body. This brief review attempts to clarify the course of events known to take place during immune complex disease and review the basic knowledge gained over the last century.

The online version of the original article can be found under doi:10.1007/s12026-010-8178-6. H. Jiang C. G. Hester M. M. Frank (&) Department of Pediatrics, Duke University Medical Center, Durham, NC, USA e-mail: frank007@mc.duke.edu L. Liao M. M. Frank Department of Medicine, Duke University Medical Center, Durham, NC, USA D. C. Montefiori M. M. Frank Department of Immunology, Duke University Medical Center, Durham, NC, USA D. C. Montefiori Department of Surgery, Duke University Medical Center, Durham, NC, USA

Systemic lupus erythematosus is a complement-mediated autoimmune disease. While genetic deficiencies of classical pathway components lead to an increased risk of developing systemic lupus erythematosus, end organ damage is associated with complement activation and immune complex deposition. The role of classical pathway regulators in systemic lupus erythematosus is unknown. C4 binding protein (C4bp) is a major negative regulator of the classical pathway. In order to study the role of C4bp deficiency in an established murine model of lupus nephritis, mice with a targeted deletion in the gene encoding C4bp were backcrossed into the MRL/lpr genetic background. Compared with control MRL/lpr mice, C4bp knockout MLR/lpr mice had similar mortality and similar degrees of lymphoproliferation. There were no differences in the extent of proteinuria or renal inflammation. Staining for complement proteins and immunoglobulins in the kidneys of diseased mice revealed no significant strain differences. Moreover, there was no difference in autoantibody production or in levels of circulating immune complexes. In comparison with C57BL/6 mice, MRL/lpr mice had depressed C4 levels as early as 3 weeks of age. The absence of C4bp did not impact serum C4 levels or alter classical pathway hemolytic activity. Given that immune complex renal injury in the MRL/lpr mouse is independent of Fc receptors as well as the major negative regulator of the classical pathway, new mechanisms for immune-complex-mediated renal injury need to be considered.