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The complement system was discovered at the end of the 19th century as a heat-labile plasma component that "complemented" the antibodies in killing microbes, hence the name "complement." Complement is also part of the innate immune system, protecting the host by recognition of pathogen-associated molecular patterns. However, complement is multifunctional far beyond infectious defense. It contributes to organ development, such as sculpting neuron synapses, promoting tissue regeneration and repair, and rapidly engaging and synergizing with a number of processes, including hemostasis leading to thromboinflammation. Complement is a double-edged sword. Although it usually protects the host, it may cause tissue damage when dysregulated or overactivated, such as in the systemic inflammatory reaction seen in trauma and sepsis and severe coronavirus disease 2019 (COVID-19). Damage-associated molecular patterns generated during ischemia-reperfusion injuries (myocardial infarction, stroke, and transplant dysfunction) and in chronic neurologic and rheumatic disease activate complement, thereby increasing damaging inflammation. Despite the long list of diseases with potential for ameliorating complement modulation, only a few rare diseases are approved for clinical treatment targeting complement. Those currently being efficiently treated include paroxysmal nocturnal hemoglobinuria, atypical hemolytic-uremic syndrome, myasthenia gravis, and neuromyelitis optica spectrum disorders. Rare diseases, unfortunately, preclude robust clinical trials. The increasing evidence for complement as a pathogenetic driver in many more common diseases suggests an opportunity for future complement therapy, which, however, requires robust clinical trials; one ongoing example is COVID-19 disease. The current review aims to discuss complement in disease pathogenesis and discuss future pharmacological strategies to treat these diseases with complement-targeted therapies. SIGNIFICANCE STATEMENT: The complement system is the host's defense friend by protecting it from invading pathogens, promoting tissue repair, and maintaining homeostasis. Complement is a double-edged sword, since when dysregulated or overactivated it becomes the host's enemy, leading to tissue damage, organ failure, and, in worst case, death. A number of acute and chronic diseases are candidates for pharmacological treatment to avoid complement-dependent damage, ranging from the well established treatment for rare diseases to possible future treatment of large patient groups like the pandemic coronavirus disease 2019., Competing Interests: Conflict of Interest: T.E.M. is a member of the Scientific Advisory Board of Ra Pharma/UCB. A.J.T. is a consultant for Montis., (Copyright © 2021 by The Author(s).)

Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a β-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins., (© 2015 Authors; published by Portland Press Limited.)

Complement is an important component of the innate immune system that is crucial for defense from microbial infections and for clearance of immune complexes and injured cells. In normal conditions complement is tightly controlled by a number of fluid-phase and cell surface proteins to avoid injury to autologous tissues. When complement is hyperactivated, as occurs in autoimmune diseases or in subjects with dysfunctional regulatory proteins, it drives a severe inflammatory response in numerous organs. The kidney appears to be particularly vulnerable to complement-mediated inflammatory injury. Injury may derive from deposition of circulating active complement fragments in glomeruli, but complement locally produced and activated in the kidney also may have a role. Many kidney disorders have been linked to abnormal complement activation, including immune-complex-mediated glomerulonephritis and rare genetic kidney diseases, but also tubulointerstitial injury associated with progressive proteinuric diseases or ischemia-reperfusion., (© 2013 Elsevier Inc. All rights reserved.)

Neurotransmitter receptors play central roles in basic neurotransmission and synaptic plasticity. Recent studies have revealed that some transmembrane and extracellular proteins bind to neurotransmitter receptors, forming protein complexes that are required for proper synaptic localization or gating of core receptor molecules. Consequently, the components of these complexes contribute to long-term potentiation, a process that is critical for learning and memory. Here, we review factors that regulate neurotransmitter receptors, with a focus on proteins containing CUB (complement C1r/C1s, Uegf, Bmp1) or CCP (complement control protein) domains, which are frequently found in complement system proteins. Proteins that contain these domains are structurally distinct from TARPs (transmembrane AMPA receptor regulatory proteins), and may constitute new protein families that modulate either the localization or function of neurotransmitter receptors. In addition, other CCP domain-containing proteins participate in dendritic patterning and/or synaptic differentiation, although current evidence has not identified any direct activities on neurotransmitter receptors. Some of these proteins are involved in pathologic conditions such as epileptic seizure and mental retardation. Together, these lines of information have shown that CUB and CCP domain-containing proteins contribute to a wide variety of neuronal events that ultimately establish neural circuits., (Copyright © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

The complement system is the main arm of the vertebrate innate immune system against pathogen infection. For the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, subverting the complement system and invading the host cells is crucial to succeed in infection. However, little attention has focused on whether the complement system can effectively control T. cruzi infection. To address this question, we decided to analyse: 1) which complement pathways are activated by T. cruzi using strains isolated from different hosts, 2) the capacity of these strains to resist the complement-mediated killing at nearly physiological conditions, and 3) whether the complement system could limit or control T. cruzi invasion of eukaryotic cells. The complement activating molecules C1q, C3, mannan-binding lectin and ficolins bound to all strains analysed; however, C3b and C4b deposition assays revealed that T. cruzi activates mainly the lectin and alternative complement pathways in non-immune human serum. Strikingly, we detected that metacyclic trypomastigotes of some T. cruzi strains were highly susceptible to complement-mediated killing in non-immune serum, while other strains were resistant. Furthermore, the rate of parasite invasion in eukaryotic cells was decreased by non-immune serum. Altogether, these results establish that the complement system recognizes T. cruzi metacyclic trypomastigotes, resulting in killing of susceptible strains. The complement system, therefore, acts as a physiological barrier which resistant strains have to evade for successful host infection.

Objective: To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia., Design: Prospective cohort study., Setting: Denver complement study (June 2005-June 2008)., Population: A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation., Methods: Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient., Main Outcome Measure: Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined., Results: The mean (+/-SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (+/-0.26) microg/ml and 0.69 (+/-0.2) microg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 +/- 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 +/- 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4-3.1, P < 0.0003) and 0.2 (CI = 0.07-0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors., Conclusions: In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy.

Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host's immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity approximately 200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent.

Despite eradication of smallpox three decades ago, public health concerns remain due to its potential use as a bioterrorist weapon. Smallpox and other orthopoxviruses express virulence factors that inhibit the host's complement system. In this study, our goals were to characterize the ability of the smallpox inhibitor of complement enzymes, SPICE, to regulate human complement on the cell surface. We demonstrate that SPICE binds to a variety of cell types and that the heparan sulfate and chondroitin sulfate glycosaminoglycans serve as attachment sites. A transmembrane-engineered version as well as soluble recombinant SPICE inhibited complement activation at the C3 convertase step with equal or greater efficiency than that of the related host regulators. Moreover, SPICE attached to glycosaminoglycans was more efficient than transmembrane SPICE. We also demonstrate that this virulence activity of SPICE on cells could be blocked by a mAb to SPICE. These results provide insights related to the complement inhibitory activities of poxviral inhibitors of complement and describe a mAb with therapeutic potential.

The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5-30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were approximately 100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.

CD93 is a cell-surface glycoprotein that has been shown to influence defence collagen-enhanced Fc-receptor or CR1-mediated phagocytosis of suboptimally opsonized targets in vitro, and CD93-deficient mice are defective in the clearance of apoptotic cells in vivo. To investigate the mechanism of CD93 modulation of phagocytic activity, GST fusion proteins containing the 47 amino acid intracellular domain (GST-Cyto), or various mutants of the intracellular domain of CD93, were constructed and used to identify intracellular CD93-binding molecules. The intracellular protein moesin, well characterized for its role in linking transmembrane proteins to the cytoskeleton and in cytoskeletal remodelling, bound to GST-Cyto when either cell lysates or recombinant moesin were used as a source of interacting molecules. An association of moesin with CD93 within intact cells was confirmed by co-capping moesin with CD93 in human monocytes. The moesin-binding site on CD93 mapped to the first four positively charged amino acids in the juxtamembrane region of the CD93 cytoplasmic tail. Interestingly, deletion of the last 11 amino acids from the C terminus of CD93 (GST-Cyto-C11) dramatically increased moesin binding to the cytoplasmic tail of CD93 in the cell lysate assay, but not when the binding of purified recombinant moesin was assessed. Furthermore, moesin binding to CD93 was enhanced by the addition of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Taken together, these data suggest that the interaction of moesin with the CD93 cytoplasmic domain is modulated by binding of other intracellular molecules to the C11 region and implies that a PIP(2) signalling pathway is involved in CD93 function.

The innate immune system provides critical protection during initial infections before the generation of an appropriate adaptive (antibody or T cell mediated) immune response. These early defense mechanisms may be particularly critical for neonates in whom the adaptive immune system is not fully operational. Pattern recognition molecules target potential pathogens for destruction by the innate immune system, and likely facilitate the initiation of a pathogen-specific immune response. Defense collagens, such as C1q, MBL and SPA, comprise a family of such proteins that, via specific interactions with phagocytic cells, play a role in this first line of defense. To begin to assess the importance of these innate defense mechanisms in neonates, cord blood plasma and leukocytes were isolated, and responses to these components of the innate defense system were assessed. C1q enhanced the phagocytosis of targets suboptimally opsonized with either IgG or complement components, and this enhancement of phagocytosis was blocked by anti-CD93/C1qRP MAb by 57% to 68%. Flow cytometric analysis demonstrated that neonatal monocytes and neutrophils expressed CD93/C1qRP similarly to adult cells, with several-fold greater expression on monocytes than on neutrophils and essentially no expression on lymphocytes. Superoxide production in response to multivalent C1q by neonatal neutrophils was also comparable to adult cells. We also confirm that C1q and MBL are present in neonate circulation. Thus, the data demonstrate that these recognition and effector mechanisms of the innate system are functional in the newborn and similar to that of adult cells.

Modern ELISA for determination of functional activity of component C2 and factors B and D, proteinases of a complement system, and component C3, substrate C3-convertases, key complex enzymes of the complement, have been developed. Essential feature of C3-convertases classical (C4bC2a) and alternative (C3bBb) pathways of the complement activation is that their substrate C3 after proteolytic cleavage is converted into C3b, carrying on the surface thioester covalent bond linking C3b with nucleophilic acceptors that results in immobilization of this proteolytic product near the activating enzyme. Cascade character of an activation of complement system allows to create artificial deficit of separate components in the experimental system and to determine (by ELISA) covalently immobilized component C3 during activation, and also to determine functional activity of any of pre-exhausted components. Use of such approach resulted in the development of the ELISA systems suitable for determination of functional activity of component C2 of classical pathway and factors B and D of the alternative pathway by testing quantity of the immobilized C3b at excess C3. The developed methods allow to investigate mechanisms of functioning of complement, inhibition of the cascade activation by endogenic and exogenous inhibitors, and also to find functional deficiency of components in serum and other biological fluids.

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.

Two types of widely coexpressed cell surface C1q-binding proteins (C1q-R): a 60-kDa calreticulin-homolog which binds to the collagen-like "stalk" of C1q and a 33-kDa protein with affinity for the globular "heads" of the molecule, have been described. In this report, we show that the two molecules are also secreted by Raji cells and peripheral blood lymphocytes and can be isolated in soluble form from serum-free culture supernatant by HPLC purification using a Mono-Q column. The two purified soluble proteins had immunochemical and physical characteristics similar to their membrane counterparts in that both bound to intact C1q and to their respective C1q ligands, cC1q and gC1q. In addition, N-terminal amino acid sequence analyses of the soluble cC1q-R and gC1q-R were found to be identical to the reported sequences of the respective membrane-isolated proteins. Ligand blot analyses using biotinylated membrane or soluble cC1q-R and gC1q-R showed that both bind to the denatured and nondenatured A-chain and moderately to the C-chain of C1q. Moreover, like their membrane counterparts, the soluble proteins were found to inhibit serum C1q hemolytic activity. Although cC1q-R was released when both peripheral blood lymphocytes and Raji cells were incubated in phosphate-buffered saline for 1 hr under tissue culture conditions, gC1q-R was releasable only from Raji cells, suggesting that perhaps activation or transformation leading to immortalization is required for gC1q-R release. Subcellular fractionation of Raji cells and analyses by enzyme-linked immunosorbent assay and Western blotting showed that the two molecules are present in the cytosolic fractions as well as on the membrane. The data suggest that soluble forms of both C1q-binding molecules are released from cells and that these molecules may play important roles in vivo as regulators of complement activation.

The complement protein C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) are structurally similar molecules that enhance phagocytic function in vitro. Monoclonal antibodies R3 and R139, which inhibit the enhancement triggered by these three ligands, were used to purify a 126,000 M(r) cell surface protein designated C1qR(P). Amino acid sequence was obtained and the corresponding cDNA was cloned. C1qR(P) is a novel type I membrane protein with the following putative structural elements: a C-type carbohydrate recognition domain, five EGF-like domains, a transmembrane domain, and a short cytoplasmic tail. All peptides identified by amino acid sequencing are encoded by the cDNA. Additionally, an anti-peptide antiserum was generated, which is reactive with C1qR(P). The data indicate that the cloned cDNA encodes the receptor that plays a role in C1q/MBL/SPA-mediated removal or destruction of pathogens and immune complexes by phagocytosis.

The isolation and characterization of two human serum proteins, RHP and N-RHP, are described. N-RHP appears to be the normal counterpart of RHP which is found at elevated levels in sera of patients with rheumatoid arthritis [Rosano et al. (1988b) Inflammation 12, 351 - 360]. Although both proteins crossreact with anti-Factor H and have identical N-terminal amino acid sequences, they differ from Factor H in pI, solubility at low ionic strength, and in glycosylation. RHP differs from Factor H and N-RHP in antigenicity in the rabbit, in effect on the C1q-anti-C1q precipitin reaction, and in ability to disaggregate C1, the first component of the complement system. Removal of RHP, N-RHP and Factor H from binding to C1q is a prerequesite for separation of RHP and N-RHP from Factor H by anion exchange chromatography and isoelectric focusing. The finding of uniquely demonstrable RHP activity (enhancement of C1q-anti-C1q precipitin activity) in unfractionated sera from patients with rheumatoid arthritis, but not in normal sera, suggests that RHP is not an artefact of Factor H produced during isolation.

C1q, the recognition subunit of the classical complement pathway, has been shown to stimulate host defense mechanisms. It has become increasingly evident that C1q can bind to several different cell types resulting in a variety of functional consequences depending on the cell type. For example, the binding of C1q to monocytes enhances the ability of those cells to ingest pathogens, while interaction with neutrophils, eosinophils, endothelial cells and vascular smooth muscle cells triggers or enhances the generation of toxic oxygen species. While these latter oxygen products play a major role in the destruction of foreign pathogens, in some cases, such as immune-complex-induced vasculitis and myocardial infarction, these toxic oxygen molecules can cause excessive host tissue damage. The differential modulation of C1q-mediated activities by monoclonal antibodies suggest that there is more than one unique type of C1q receptor, which is not necessarily surprising given the diversity of responses reported. This review summarizes what is known about the functional consequences of the interaction of C1q with cells, and describes initial investigations of the biochemical mechanisms/signal transduction pathways involved in these C1q-induced responses. Further investigations of the C1q/C1q receptor system(s) should uncover basic mechanisms and modes of regulation of these defensive responses by the host, and potentially provide information useful for beneficial manipulation of these activities.

In injury and inflammation, complement (C) component C1q, in addition to its central role in initiation of classical pathway of complement activation, modulates diverse cellular functions by binding to specific cell surface receptors. Interaction of substrate-bound C1q with receptors for the collagen-like domain of C1q (C1qRC) of human gingival fibroblasts (HGF) promotes cell attachment. We investigated modulation of the adhesive function and expression of C1qRC by interleukin-1 beta (IL-1 beta) and transforming growth factor-beta (TGF-beta). Confluent fibroblast monolayers were incubated under standard culture conditions with or without cytokines. C1qRC function was measured by attachment assays. IL-1 beta and TGF-beta increased fibroblast adhesion to C1q to 146% and 131% of controls, respectively. Cytokine enhancement of HGF adhesion was concentration-dependent, saturable (20 ng/ml IL-1 beta; 1 ng/ml TGF-beta) and time-dependent (IL-1 beta 12-hr peak; TGF-beta 24-hr peak). Effect of IL-1 beta and TGF-beta on C1qRC expression was assessed by flow cytometry measurements of fluorescence intensity of cells stained with C1q and FITC anti-C1q antibody, and by binding studies with 125I-C1q. Cells treated with cytokines displayed a two- to four-fold increased fluorescence of cell-bound C1q compared to controls. Binding studies indicated the increased fluorescence correlated with increase in number of C1qRC in both IL-1 beta (4.7 x 10(6)/cell) and TGF-beta (3.9 x 10(6)/cell)-treated cells, compared to control (3.0 x 10(6)/cell), but had no effect on binding affinity. Rates of internalization of receptor-bound C1q were similar in cytokine-treated cells and controls. We propose from these data that IL-1 beta and TGF-beta have the ability to upregulate C1qRC expression, and this effect contributes to increased adhesion of HGF to substrate-bound C1q.

Mannan-binding protein is an animal serum lectin (i.e. a molecule with the ability to bind specifically to certain carbohydrate structures). The relevant carbohydrate ligands are found on many pathogenic microorganisms. After binding to suitable carbohydrate ligands, mannan-binding protein is found to be an activator of the classical pathway of complement via an activation of the C1r2C1s2 complex, i.e. antibody and C1q independent. The molecular organization of MBP resembles that of C1q with a distinct division of collagen-like and globular amino acid sequences. This molecular similarity seems to be the basis for the common functional activity of the two proteins. MBP may play an important protective role, especially at early stages of infection prior to the generation of the specific humoral and cellular defence system. The paper explores the structure and the physiological functions of mannan-binding protein.

The human C1q receptor (C1q-R) is a 65-70-kd, highly acidic, hydrophobic glycoprotein that is expressed on a wide variety of cell surfaces. Although the C1q-R itself appears to bind preferentially to C1q, the region of the ligand to which C1q-R binds is the primary binding site for several other molecules, including fibronectin, laminin, and C1q inhibitor (chondroitin 4-sulfate proteoglycan) as well as the complement C1r2C1s2 tetramer. In order to further characterize the C1q-R molecule with regard to its structure and function, highly purified C1q-R was obtained from Raji cells using DEAE-Sephacel and C1q-Sepharose CL-4B chromatography. Studies performed with 125I-labeled C1q-R demonstrated that whereas the C1q-R molecule binds poorly to a variety of human collagens including types II, III, and V, markedly enhanced binding is observed with type IV collagen and moderately enhanced binding with type I collagen. Amino acid composition studies show that the C1q-R molecule contains approximately 44% hydrophobic and 12.6% hydrophilic residues with a ratio of negatively charged to positively charged residues of about 2:1. Treatment of 125I-labeled C1q-R with endoglycosidase F lowers the apparent molecular size from 70 to 58 kd, whereas endoglycosidase H lowered the size to 64 kd. Treatment with neuraminidase, on the other hand, shifted the size of C1q-R to 60 kd. These results suggest the presence of several highly sialylated complex-type or high mannose-type N-linked oligosaccharide side chains. Because purified C1q-R has a blocked amino terminus, amino acid sequences representing internal fragments of the molecule were generated by electroblotting and in situ enzymatic digestion. When these short sequences were searched against the National Biomedical Research Foundation computer data base, a seven-amino-acid sequence, VSWQGQI, showed significant homology (100% and 80% in a five-amino-acid overlap, respectively) with the alpha chains of the human fibronectin (alpha 5 beta 1) and vitronectin (alpha v beta 3) receptors, and to a lesser degree with epidermal growth factor receptor and T cell receptor. A second sequence, ISEDNIR, showed homology with mouse collagen type IV (86% in a six-amino-acid overlap), calmodulin (60% in a seven-amino-acid overlap), and a Leishmania major surface antigen, gp63. These observations seem to predict that C1q-R has pockets of conserved sequences that are similar to those not only present in its ligand(s) but also in other cell surface receptors that may, in part, fulfill similar functions.

Previous studies identified specific receptors for C1q on human blood platelets in purified systems using monomeric C1q. To assess the physiologic potential of platelet C1q receptors, C1q binding was evaluated in whole blood and in the presence of immune complexes or aggregated IgG. Blood was obtained from healthy volunteers and collected directly into EDTA (1 vol 100mM EDTA:9 vol whole blood) and purified, 125I-labeled C1q or 125I-C1q associated with albumin-anti-albumin immune complexes. Samples were incubated at 22 or 37 degrees C for 60 min, and total cell bound C1q and platelet associated C1q were quantified. Platelet-bound, monomeric C1q or immune complex-associated C1q represented 40-50% of total peripheral blood cell-associated C1q. C1q binding was unaffected by the incubation temperature, but the preincubation of 125I-C1q with immune complexes enhanced binding two- to threefold. This binding was partially inhibited by preincubating platelets with either the collagen-like amino-terminal fragments of C1q (c-C1q) or a monoclonal antibody Fab fragment recognizing platelet Fc receptors. A more complete inhibition was achieved if platelets were preincubated with both agents. Similar observations were made using washed platelets and 125I-C1q associated with aggregated IgG. The role of C1q and platelet C1q receptors in enhancing aggregated-IgG binding to platelets was further supported by experiments demonstrating increased 125I-aggregated IgG binding to platelets not only after preincubation of 125I-aggregated IgG with C1q but also following platelet preincubation with C1q. These data suggest that C1q receptors may participate in the localization and presentation of C1q-associated immune complexes on the platelet surface and demonstrate that platelets contribute significantly to the C1q binding activity of peripheral blood.

In injury and inflammation, interactions of complement C1q with C1q receptors may provide attachment sites for cell localization and tissue regeneration. Cultured smooth muscle cells (58%), epithelial cells (26%), and endothelial cells (25%) attach to C1q-coated surfaces, while only 6% of cultured B cells (Raji) attach. Endothelial and Raji cells express C1q receptors, but C1q receptors (C1qR) on smooth muscle cells and epithelial cells have not previously been demonstrated. Evidence is provided that smooth muscle cells express an average of 1.5 x 10(6) C1qR/cell (K alpha = 10(8) M-1) and that epithelial cells express an average of 0.7 x 10(6) C1qR/cell (K alpha = 1.4 x 10(8) M-1). Binding properties of C1qR, and immunoreactivity to anti-C1qR antibodies, are characterized. The antibodies specifically recognize a 67-kDa component of smooth muscle cell lysates and inhibit cell attachment to C1q substrates. We conclude that distribution of C1qR may be ubiquitous; binding properties, size, and antigenicity of various C1qR may be related, but adhesive function may be tissue specific.

The serum half-lives of three recombinant mouse monoclonal antibodies, differing radically in their ability to bind to Clq or FcRI but only minimally in structure, were determined in the BALB/c mouse following intravenous administration. The wild-type antibody, a chimaeric antibody comprising variable domains binding 3-iodo-4-hydroxy-5-nitrophenylacetate and constant domains of the mouse IgG2b isotype, was eliminated from the bloodstream with biphasic kinetics: alpha-phase, 0.5 days; beta-phase, 7.0 days. The alpha- and beta-phase half-lives of mutant recombinant antibodies with single amino acid substitutions, either Glu 235-Leu allowing binding to the mouse FcRI, or Lys 322-Ala reducing Clq binding 30-fold, were indistinguishable from those of the wild-type antibody demonstrating that the biological half-life of intact mouse IgG is independent of the ability to bind Clq or FcRI. The major implication of the present study is that IgG molecules which have been genetically engineered to eliminate interaction with other components of the immune system should retain the long half-life typical of natural antibodies.

A receptor binding to the C1q subcomponent of complement has been reported by many workers. In this paper we report for the first time that C1q receptor binds not only to C1q, but also to three other structurally similar ligands, namely mannan binding protein (MBP), conglutinin, and lung surfactant protein (SP-A). All these ligands have been reported to enhance removal of species bound to their globular domain from blood (MBP, conglutinin, C1q) or lung (SP-A) through phagocytosis. One of the possible roles for ligand-receptor binding may be initiation of phagocytosis.

C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure.

The kinetics of inhibition of the complement serine protease, C1s, by its only known inhibitor, C1 inhibitor, have been measured by a variety of methods. One method continuously monitors the loss of esterolytic activity with a synthetic substrate coupled to a chromogen while another monitors the formation of a stable (covalent) complex by high-pressure size-exclusion chromatography under dissociating conditions. Additional methods employ fluorescence probes to follow the formation of bimolecular complexes but are not expected to distinguish between covalent product and noncovalent (reversible) intermediates. There was good agreement between rate constants obtained by the various methods over a broad range of inhibitor concentrations, suggesting that noncovalent intermediates do not accumulate to a significant extent. The reaction appears to be pure second order with a bimolecular rate constant of 6.0 X 10(4) M-1 s-1 at 30 degrees C, independent of Ca2+, and an activation energy of 11.0 kcal/mol. The rate increases up to 35-fold in the presence of heparin which was shown to bind to all three components (enzyme, inhibitor, and complex) with similar affinity (Kd = 2.0-3.3 microM). The fluorescent probe 1,1'-bis(anilino)-4-,4'-bi(naphthalene)-8,8'-disulfonate [bis(ANS)] bound to the complex with Kd = 0.26 microM under conditions where the individual components had little affinity for the dye, consistent with the generation of one or more hydrophobic binding sites on the protein surface during complex formation.

Biologic and immunogenic activities of semisynthetic human monocomponent insulins were examined in insulin-dependent diabetic patients (group 1). Patients treated with porcine monocomponent (group 2) and conventional (group 3) insulins were studied for control purposes. The patients were examined before the beginning of insulin treatment and for a 6-mo follow-up period. The data collected during the study show that insulin antibody levels were significantly lower in group 1 than in groups 2 and 3. Furthermore, the prevalence of immune complexes assays with the C1q solid phase technique failed to reveal any differences between the three groups. When the conglutinin binding test was used, the prevalence of immune complexes showed a slight but not significant reduction in group 1 and a significant increase in group 3. The metabolic control was similar in the three groups during follow-up and the insulin requirement was lower, but not significantly, in group 1 than in groups 2 and 3. These data suggest that with human monocomponent insulins equivalent glycemic control may be achieved at similar doses than those required with porcine monocomponent insulins. Furthermore, human insulin is the least immunogenic of the present available insulins.

The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4.

Circulating immune complexes (CICs) have been detected in the sera of patients with non-Hodgkin's lymphoma (NHL), Hodgkin's disease, chronic myeloid leukemia, and acute lymphoblastic leukemia by using C1q-binding and L1210-binding assays. Both assays gave broadly similar patterns of reactivity in terms of frequency and magnitude, though there are some differences. Significantly elevated CIC levels were observed in all pathologic groups. However, sera from NHL patients with an unfavorable prognosis consistently exhibited the highest frequency of positive values and mean CIC levels in both these assays. The two tests showed concordance in 66.6% of the NHL patients' sera and were significantly correlated. Of the sera from NHL patients 12.7% were positive in the C1q-binding assay only and 15.9% in the L1210-binding assay only. Both the assays gave positive results in some patients, and a degree of overlap indicates the presence of different types of CIC in cancer patients' sera. The combined use of two methods for detecting CICs may be useful for evaluation of the activity, the extent, and the prognosis of the malignant disease.

C1q, a subcomponent of the first complement component, of a 60-year-old female patient with hypocomplementemic vasculitis-urticaria syndrome (HVUS) was characterized. The C1q-precipitin activity (C1q-p) could not be detected by a routine method with 0.6% agarose in 10 mM Na-phosphate buffer containing 10 mM EDTA (pH 7.2). Hemolytic activity of her serum complement (CH50) and levels of C1 and C4 were significantly reduced at the exacerbation stage, but levels of other complement components were almost within the normal range throughout her clinical course. The specific activity of C1q at her exacerbation stage was significantly low, and its elution position on Sephacryl S-300 column was spread toward the low molecular weight in comparison with that of normal plasma. Molecular weights of the delayed fraction of C1q were estimated to be approximately 300,000 on the Sephacryl and 440,000 by the polyacrylamide gel electrophoresis (PAGE) containing sodium dodecyl sulfate (SDS) followed by immunoblotting, respectively. On reduction of her plasma, two bands with molecular weights equivalent to those of B and C chains of the normal C1q in an approximate molar ratio of 2:1 were immunostained. Plasma at her exacerbation stage showed only one precipitation line against anti-human C1q-antiserum which was completely fused with that formed between purified normal human C1q and the same antiserum. The probable structural change of the hypofunctional C1q in the case of this HVUS is discussed.

Living Schistosoma mansoni of various developmental stages were studied with respect to their ability to activate the complement system in sera of humans, mice and rats. Immunofluorescence assays demonstrated that binding of human C3 occurred on fresh schistosomula as well as on schistosomula prepared from mouse lymph-nodes or lungs and on adult schistosomes. However, rodent C3 was deposited only on fresh schistosomula. Deposition of human C3 on the worms' surface required activation of the complement system. The alternative pathway was shown to be involved in deposition of human C3 on schistosomes of all ages, whereas activation of the classical pathway was demonstrable only with fresh schistosomula. Immunoelectrophoretic studies demonstrated a dose-dependent cleavage of human C3 and conversion of factor B by living adult schistosomes. The results demonstrate that the ability of living schistosomes to activate complement in vitro is dependent not only on their development stage but also on the species of the serum.

Sera from 41 patients with interstitial cystitis were tested for immunoglobulins (IgA, G, M), complement components (C1q, C3, C4) and for C1-esterase inhibitor. There was a statistically highly significant depletion of the serum levels of complement component C4 (p less than 0.001) and immunoglobulin G was markedly elevated (p less than 0.001). Our results suggest the classical pathway activation of the complement system and support the possibility that a chronic local immunological process is involved in the pathogenesis of the disease.

We examined the effect of endogenous C1q on the sensitivity of the fluid-phase C1q binding assay (C1qBA) in detecting an immune complex (IC) model, heat-aggregated IgG (HAIgG), at concentrations of 10-10,000 micrograms/ml sample. Results in normal human serum (NHS) or plasma (NHP) were compared with those in heat-inactivated NHS (NHS/56) in which most endogenous C1q was depleted by heat denaturation. Higher HAIgG concentrations were required in NHP and NHS to produce the same 125I-C1q precipitation seen in NHS/56. This decreased sensitivity varied from 70% at low HAIgG concentrations to 0% at high concentrations, as predicted for a large pool of endogenous C1q, in equilibrium with 125I-C1q, but in excess of that which could bind to all but the highest concentrations of IC model. In serum depleted of functional C1q on an immunoadsorbant of HAIgG, the precipitation of radiolabeled HAIgG under C1qBA conditions was concentration dependent and generated a saturation curve, showing that only a fraction of IC are usually precipitated in this assay. HAIgG precipitation was enhanced 1.4-fold in NHS/56 (8 micrograms C1q/ml) and three-fold in NHS (67 micrograms C1q/ml) suggesting that IC size is increased by endogenous C1q. In dual label experiments using 131I-HAIgG, the precipitation of 125I-C1q in NHS/56 was directly proportional to IC model precipitation, but markedly discordant in NHP, showing the measurement of IC in heat-inactivated sera superior to that in native serum. A comparison of the C1q:HAIgG ratio in PEG precipitates with that in samples, indicated that equilibrium was established between C1q and IC model. Thus the precipitation of 125I-C1q in the C1qBA represents (1) the fraction of total C1q bound to IC, and (2) the fraction of IC precipitated by PEG.

The functional properties of the secreted form of C4 (C4s), which has a Mr approximately 5000 greater than the predominant C4 molecule found in plasma (C4p), two incompletely processed two-chain C4 molecules (beta - alpha + gamma and beta + alpha - gamma), and the extracellular C4 precursor (designated pro-C4(E)] were evaluated. All four molecules are secreted in parallel by a human hepatoma-derived cell line (Hep G2). Secretion of hemolytically active C4 is linear up to approximately 12 hr, peaks at 24 hr, and then progressively decreases over the next 48 hr. This loss of C4s functional activity parallels the proteolytic conversion of C4s to C4bs. To compare the hemolytic efficiencies of C4s and C4p, a solid-phase competitive radioimmunoassay was developed to permit measurement of the small quantities of C4 antigen in these cultures. The hemolytic efficiencies of C4s and C4p were similar. These results indicate that extracellular processing of C4s to C4p does not modulate the hemolytic activity of the molecule. Consistent with their ability to bind methylamine, both the alpha s-chain and the alpha - gamma subunit undergo denaturation-induced autolysis. The extracellular and intracellular pro-C4 molecules are also sensitive to autolytic cleavage. Interestingly, the beta - alpha subunit is resistant to autolysis. In experiments in which C4s and C4p were cleaved by C1-s to C4bs, C4(beta - alpha + gamma), C4(beta + alpha - gamma), and pro-C4(E) were resistant to C1-s cleavage and thus hemolytically inactive relative to C4s. These data indicate that processing of C4 to a three-chain structure is required to provide the proper conformation for efficient activation by C1.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.

Polyanion has previously been shown to inhibit generation of amplification pathway convertase of complement and to inhibit H-mediated decay of this complex. Polycations, protamine sulfate, and poly-l-lysine (PLL) were examined in this study for direct effects on alternative amplification pathway mechanisms and for ability to regulate polyanion-induced inhibiton; they were found to inhibit generation of EAC4b,3b,Bb, and EAC4b,3b,Bb,P in a dose-related manner. The generation of EAC4b,3b,B was also inhibited by higher doses but was augmented by lower doses of polycation. Polycation interfered with the effective consumption of B in the fluid phase, bound minimally to EAC4b,3b, and did not cause accelerated decay of a preformed convertase. Polycation diminished the ability of polyanion (heparin) to inhibit generation of both cell-bound and fluid-phase amplification pathway convertase. In contrast, polycation enhanced the ability of polyanion to cause decay of preformed convertase even though polycation had no effect itself. These studies demonstrate that both polyionic substances have the capacity to regulate amplification pathway mechanisms directly.

In order to study the relationship between the presence of circulating immune complexes (CIC) and the severity and duration of acute dysentery, we studied 11 adults and 45 children in Bangladesh with bacillary and amebic dysentery, using the Raji cell and solid-phase C1q (C1q-SPA) assays. CIC were found in 70% of patients with shigellosis and in all eight cases of amebic dysentery. Mild shigellosis was associated with positive samples in the first week of clinical illness, whereas severe cases, including those with the hemolytic-uremic syndrome, had negative admission assays but positive convalescent assays. Samples positive in the first two weeks of illness were more likely positive by the Raji cell assay alone whereas samples in the third and fourth weeks of illness were positive more often by the C1q-SPA assay. Only one shigellosis sample was positive by both assays. In amebiasis 11 of 13 samples were positive by the Raji assay alone. In dysenteric disease circulating immune complexes probably represents the failure of the inflamed mucosa to exclude microbial and dietary antigens, and suggests that the presence of CIC in any intestinal disease must be interpreted with caution.

A cultured fibroblast subtype with growth and synthetic properties expected of cells residing in healing wounds and in inflammatory lesions binds the Clq component of complement with a functional affinity much higher than that of the remaining cell population. In this study we examined the optimal conditions that favor the interaction between purified 125I-labeled Clq and this cell subtype, following its isolation from the parent culture using a cell sorter, and assessed the biologic consequences of binding. Binding of 125I-Clq to the cell surface is specific, saturable and reversible. It is maximal between pH 5.5 and 8.5 at an ionic strength of mu = 0.10 and decreases as a function of increasing salt concn, with half saturation near physiologic ionic strength. Scatchard analysis of binding data indicates a single class of sites with an average association constant in the order of 1.5 x 10(9)/M and an average number of 2.5 x 10(6) binding sites per cell. Unlabeled globular fragments of Clq inhibit intact 125I-Clq binding by 64%, while unlabeled collagen-like fragments have no effect. Thus it appears that binding of Clq to this high-affinity site is mediated by a region of the globular domain of the molecule. Only the fibroblast subtype with binding sites for the globular domain of Clq appear to have the capacity to induce non-immune activation of the classical complement cascade, as assessed by the generation of C4a and C4d fragments in normal AB serum following exposure to the cells. This activation may generate products that account for a previously reported complement mitogenicity for fibroblasts.

Indicators of immune-mediated disease were studied in calves with severe natural bovine respiratory syncytial virus infection. Although antigen and antibody were detected concurrently in most calves, immune complexes were not detected by use of immunofluorescence, ELISA, and binding of the 1q component of complement. Complement component C3, however, was observed by immunofluorescence in the cranioventral, virus-infected portion of the lungs of 19 of 25 calves. Reductions in the amount of histamine and in the numbers of mast cells and mast cell granules in the virus-positive cranioventral and virus-negative caudodorsal portions of the lungs, indicated activation of mast cells and liberation of their granule contents. On the basis of these and previous findings, a model for the pathogenesis of bovine respiratory syncytial virus-induced disease was proposed.

After a polyethylene glycol extraction which removes monomeric immunoglobulin G (IgG), circulating immune complexes were assayed by the FIAX fluorescence assay (Whittaker M. A. Bioproducts, Walkersville, Md.). The extraction portion of the procedure can be completed within a few hours after an overnight precipitation step, and the fluorescence assay takes about 0.5 h. The fluorescence assay is similar to the routine FIAX method for measuring IgG in cerebrospinal fluid except that a 10-microliter sample from sera is substituted for a 50-microliter sample. Good parallelism between endogenous immune complexes, monomeric IgG, and aggregated human globulin, along with good between-run precision (coefficients of variation, less than 10%), indicates that monomeric IgG calibrators from the kit could be used to standardize the assay. This standardization eliminates the need for aggregated human globulin, which is unstable and difficult to prepare. A reference range of 9 to 63 mg/liter followed a gaussian distribution. Correlation data indicate that the test provides information similar to the C1q-binding test (rho = 0.87; n = 30) but has better diagnostic sensitivity and better discrimination in the high range. Because of its simplicity, good reproducibility, and accessibility to equipment available in many laboratories, the method described here may be a preferred technique for measuring circulating immune complexes.