Activation of the alternative pathway of human complement by autologous cells expressing transmembrane recombinant properdin

Properdin (P) is a serum glycoprotein that stabilizes the labile C3 convertase (C3bBb) of the alternative pathway of the complement system (AP). Thanks to its oligomeric nature, P specifically upregulates AP on surfaces without activating AP in the fluid-phase. We investigated whether human cells, displaying P at their membrane, could activate autologous AP. The cDNAs encoding human P and the transmembrane domain of human platelet derived growth factor receptor were fused together and expressed in human embryo kidney cells (HEK-293). Selected cells displayed P at their surface as shown by FACS. In contact with human serum at 37 degrees C, they triggered AP-mediated C3 deposition. SDS-PAGE analysis showed C3 covalently bound to various membrane proteins, but not to P itself. However, displayed P affinity could bind to serum or purified C3i at 4 degrees C. C3 binding was restricted to the cells displaying P, was inhibited by an anti-P mAb, and did not require serum P. Bound C3 allowed further C5, C7 and C9 deposition as well as cell lysis after blocking CD59 function. In contrast, wild-type cells, cells displaying factor D or truncated P (deleted from its 6th thrombospondin-like repeat) did not activate AP. We hypothesize that displayed P activates AP by stabilizing bystander C3b and/or by capturing serum C3iBb convertase. Finally, we suggest that P could be used for retargeting autologous complement to AP-resistant pathogens and tumor cells.     

Isolation and characterization of the third and fifth components of rabbit complement

Highly purified rabbit C3 and C5 have been obtained from normal rabbit plasma by differential precipitation and column chromatography. Apparent molecular weights of the two proteins as determined by SDS-polyacrylamide gel electrophoresis were 176,500 (C3) and 171,000 (C5), whereas reduction of the samples prior to electrophoresis allowed estimation of the molecular weights of the α and β subunit chains of each protein at 123,000 and 70,100 for C3 and 128,500 and 88,000 for C5. Serum concentrations as determined by rocket immunoelectrophoresis were 1.2 mg/ml for C3 and 0.2 mg/ml for C5, whereas hemolytic activities of C3 and C5 in normal rabbit serum were 2.4 × 10¹⁰ and 3.2 × 10¹⁰ effective molecules per ml, respectively. Amino acid analyses of the C3 and C5 preparations indicated similar but not identical amino acid compositions of the the two rabbit proteins, but there were no unusual amino acids or other features of the composition. Monospecific antisera to each of the two complement components were obtained in goats by immunization with the purified material.     

The alternative complement pathway

The alternative complement pathway comprises three component proteins C3, B, D and three regulatory proteins P, H and I. These plasma proteins represent the major humoral defense mechanism against infection in a non-immune host. The following topics are reviewed: biochemistry of the alternative pathway proteins; molecular mechanisms of activation and regulation of the pathway; involvement of the alternative pathway in human diseases.     

Molecular cloning of the cDNA coding for properdin, a positive regulator of the alternative pathway of human complement

Northern blot analysis indicated that the mRNA for human properdin is approximately 1.5 kb long and that its level in U-937 cells is increased by pretreating the cells with phorbol 12-myristate 13-acetate (PMA). Using a human genomic probe clones coding for human properdin were isolated from a lambda gt10 cDNA library derived from PMA-treated U-937 cells. The sequence of the 1474-bp cDNA insert of the longest clone revealed an open reading fram of 1326 bp coding for the entire 442 amino acids of the mature form of human properdin and 67 bp coding for 22 amino acids of typical, but incomplete leader sequence. Polymerase chain reaction "RACE" experiments identified the start site ATG and revealed the complete, 27-amino acid-long, leader peptide sequence. Within the 81-bp 3' non-translated extension a polyadenylation signal was identified 41 bp downstream from the stop codon, TAA, and 12 bp upstream of a 19 nucleotide long poly(A) tail. The amino acid sequence of human properdin is clearly divided into three distinct regions: a 49 residue-long N-terminal region, a 32 residue-long C-terminal region and a middle region, covering residues 50 to 411, composed of six tandemly repeated thrombospondin repeat (TSR) motifs of the type first described in the adhesive glycoprotein thrombospondin and also known to be present in the C6, C7, C8 alpha, C8 beta and C9 terminal components of complement. Human and mouse properdin sequences show a high (approximately 76%) degree of identity with almost complete conservation of the relatively large number of Cys (44) and Trp (20) residues.     

The complement alternative pathway and hemostasis

The complement and hemostatic systems are complex systems, and both involve enzymatic cascades, regulators, and cell components—platelets, endothelial cells, and immune cells. The two systems are ancestrally related and are defense mechanisms that limit infection by pathogens and halt bleeding at the site of vascular injury. Recent research has uncovered multiple functional interactions between complement and hemostasis. On one side, there are proteins considered as complement factors that activate hemostasis, and on the other side, there are coagulation proteins that modulate complement. In addition, complement and coagulation and their regulatory proteins strongly interact each other to modulate endothelial, platelet and leukocyte function and phenotype, creating a potentially devastating amplifying system that must be closely regulated to avoid unwanted damage andor disseminated thrombosis. In view of its ability to amplify all complement activity through the C3b-dependent amplification loop, the alternative pathway of complement may play a crucial role in this context. In this review, we will focus on available and emerging evidence on the role of the alternative pathway of complement in regulating hemostasis and vice-versa, and on how dysregulation of either system can lead to severe thromboinflammatory events.     

Assay of classical and alternative pathway activities of murine complement using antibody-sensitized rabbit erythrocytes

Methods for measurement of classical complement pathway activity (CH50) and alternative complement pathway activity (ACH50) in mouse serum using rabbit erythrocytes sensitized with guinea pig anti-rabbit erythrocyte antibody have been established. The assays measured CH50 values in mouse sera that could hardly be determined by the conventional method using antibody-sensitized sheep red blood cells. Mouse serum ACH50 values determined by the method were also 5-7 times higher than those obtained in conventional assays with rabbit erythrocytes. Both the CH50 and ACH50 values varied with the strain among the 25 different strains of mice studied. BALB/c (nu/nu, male), LT/SuJ and Jcl-ICR27 strains exhibited higher CH50 values, and NIH (nu/+), ICR (nu/nu), NOD (male) and AKR strains showed lower values. The ACH50 was higher in C3H/HeN (male), C57BL/6J (male), Jcl-ICR27 and BALB/c (nu/nu, male) mice, and lower in ICR (nu/nu), NOD (female) and AKR mice. Sera from 16 out of the 25 mouse strains showed ACH50 values comparable to or higher than those in man. As for CH50, however, even the highest value seen in BALB/c (nu/nu, male) mice corresponded to about three-fifths of an average value in man. It is concluded that the complement system of mice, especially the alternative pathway of complement activation, functions as actively as that in man. It was also found that male mice have higher CH50 and ACH50 values than female mice. The differences in these parameters between males and females were only slight at the age of 4 weeks and became conspicuous after 6 weeks at which time both the CH50 and ACH50 virtually reached their respective peak levels of activity.     

Determination of alternative pathway of complement activity in mouse serum using rabbit erythrocytes

Rabbit, mouse and sheep erythrocytes expressing different concentrations of membrane sialic acid were used to study possible modes of activation of the alternative complement (C) pathway in mouse, human and guinea pig serum. Mouse erythrocytes activated only human serum, whereas rabbit erythrocytes activated the sera of all three species. Based on the observation that rabbit erythrocytes activate the murine alternative C pathway a method for estimation of alternative C pathway activity (AP50 value) in mouse serum was devised analogous to that used for human AP50 determination. The method is not very sensitive to ageing or to batch variation of the indicator cells. The AP50 value of mouse serum measured by this method is of the same order as for human and guinea pig serum. Mouse serum AP50 activity is partly determined by natural antirabbit erythrocyte antibodies and is sensitive to heating (15′ at 48°C and 4′ at 56°C), and to the actions of cobra venom factor, zymosan and cysteine. Strain and sex differences with respect to AP50 activities of mouse sera were observed.     

Looking back on the alternative complement pathway

The alternative pathway of complement originated from the Properdin pathway originally described by the Pillemer laboratory in the 1950s. This work generated great controversy and it took several decades for a consensus on its components, its reaction sequence and its functions to emerge. This paper reviews this history and attempts to clarify some of the ambiguities that remain.     

Nomenclature of the alternative activating pathway of complement

This terminology note outlines for the first time a standard nomenclature for the alternative activating pathway of complement. It was drafted by a group of experts working under the auspices of the International Union of Immunological Societies (IUIS), and has been approved by the Nomenclature Committee of the IUIS.     

Nomenclature of the alternative activating pathway of complement

This terminology note outlines for the first time a standard nomenclature for the alternative activating pathway of complement. It was drafted by a group of experts working under the auspices of the International Union of Immunological Societies (IUIS), and has been approved by the Nomenclature Committee of the IUIS.     

Low-molecular weight inhibitors of the alternative complement pathway

Dysregulation of the alternative complement pathway predisposes individuals to a number of diseases. It can either be evoked by genetic alterations in or by stabilizing antibodies to important pathway components and typically leads to severe diseases such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, C3 glomerulopathy, and age-related macular degeneration. In addition, the alternative pathway may also be involved in many other diseases where its amplifying function for all complement pathways might play a role. To identify specific alternative pathway inhibitors that qualify as therapeutics for these diseases, drug discovery efforts have focused on the two central proteases of the pathway, factor B and factor D. Although drug discovery has been challenging for a number of reasons, potent and selective low-molecular weight (LMW) oral inhibitors have now been discovered for both proteases and several molecules are in clinical development for multiple complement-mediated diseases. While the clinical development of these inhibitors initially focuses on diseases with systemic and/or peripheral tissue complement activation, the availability of LMW inhibitors may also open up the prospect of inhibiting complement in the central nervous system where its activation may also play an important role in several neurodegenerative diseases.     

Mechanisms of lipopolysaccharide-initiated rabbit platelet responses: alternative complement pathway dependence of the lytic response.

Experiments were performed to examine the relationship of endotoxin-initiated complement activation and rabbit platelet lysis. The results of these experiments supported the concept that activation of the alternative pathway is required for endotoxin-initiated complement-dependent rabbit platelet lysis. Our data demonstrated that preparations of endotoxin or isolated lipid A, which activate selectively the classical pathway, are incapable of initiating platelet lysis. Essentially equivalent results were obtained in citrated or heparinized plasma, although the latter anticoagulated plasma appeared to be more efficient in supporting lysis. Additional data support the concept that natural antibody to either the polysaccharide or the lipid A region of the lipopolysaccharide, which might be present in rabbit plasma, probably did not play a prominent role in the complement-mediated lytic response.     

New synthetic inhibitor to the alternative complement pathway

The inhibitory effects of 6-amidino-2-naphthyl 4-guanidinobenzoate (FUT-175) on the activities of factor B, factor D and cobra venom factor (CVF) X Bb were examined. FUT-175 bound specifically to the Bb fragment of factor B or CVF X Bb. FUT-175 was a non-competitive inhibitor of the esterolysis of L-leucyl-L-alanyl-L-arginine naphthylester by factor B and CVF X Bb. FUT-175 also inhibited the haemolytic activity of factor B, the C3 convertase activity of CVF X Bb and the factor B-cleaving activity of factor D. The concentration of FUT-175 causing 50% inhibition of these activities was 10(-5) - 10(-4)M.     

Activation of the properdin pathway of complement in patients with gram-negative of bacteremia.

To determine the pathway used for activation of complement component C3, serum levels of components C1, C4, C2, C3, C5, C6, and C9 and two properdin factors, properdin and factor B, were measured in 42 patients with gram-negative bacteremia, in 19 of whom shock subsequently developed. Mean levels of the classical components C1, C4, and C2 in bacteremic patients in whom shock subsequently developed did not differ significantly (p greater than 0.05) from those of patients with uncomplicated bacteremia. Levels of properdin, factor B and C3, C5, C6, and C9 were significantly (p less than 0.05) decreased in patients with shock in comparison with those with uncomplicated bacteremia. Taken together, these findings are consistent with activation of C3 and the terminal complement sequence, C5-C9, occurring primarily by the properdin pathway, in patients with gram-negative bacteremia eventuating in shock. Biologically active products released during activation of C3-C9 may contribute to the development of shock.