Novel Assays to Distinguish Between Properdin-Dependent and Properdin-Independent C3 Nephritic Factors Provide Insight Into Properdin-Inhibiting Therapy.

C3 glomerulopathy (C3G) is an umbrella classification for severe renal diseases characterized by predominant staining for complement component C3 in the glomeruli. The disease is caused by a dysregulation of the alternative pathway (AP) of the complement system. In more than half of C3G patients C3 nephritic factors (C3NeFs) are found. These autoantibodies bind to the AP C3 convertase, prolonging its activity. C3NeFs can be dependent or independent of the complement regulator properdin for their convertase-stabilizing function. However, studies to determine the properdin-dependency of C3NeFs are rare and not part of routine patient workup. Until recently, only supportive treatments for C3G were available. Complement-directed therapies are now being investigated. We hypothesized that patients with properdin-dependent C3NeFs may benefit from properdin-inhibiting therapy to normalize convertase activity. Therefore, in this study we validated two methods to distinguish between properdin-dependent and properdin-independent C3NeFs. These methods are hemolytic assays for measuring convertase activity and stability in absence of properdin. The first assay assesses convertase stabilization by patient immunoglobulins in properdin-depleted serum. The second assay measures convertase stabilization directly in patient serum supplemented with the properdin-blocking agent Salp20. Blood samples from 13 C3NeF-positive C3G patients were tested. Three patients were found to have properdin-dependent C3NeFs, whereas the C3NeF activity of the other ten patients was independent of properdin. The convertase-stabilizing activity in the samples of the patients with properdin-dependent C3NeFs disappeared in absence of properdin. These data indicate that inhibition of properdin in patients with properdin-dependent C3NeFs can normalize convertase activity and could represent a novel therapy for normalizing AP hyperactivity. Our assays provide a tool for identifying C3G patients who may benefit from properdin-inhibiting therapy and can be incorporated into standard C3G laboratory investigations.

The Challenges and Promise of Complement Therapeutics for Ocular Diseases.

Ocular inflammation is a defining feature of sight threating diseases and its dysregulation can catalyze and or propagate ocular neurodegenerative maladies such as age-related macular degeneration (AMD). The complement system, an intrinsic component of the innate immunity, has an integral role in maintaining immune-surveillance and homeostasis in the ocular microenvironment; however, overstimulation can drive ocular inflammatory diseases. The mechanism for complement disease propagation in AMD is not fully understood, although there is accumulating evidence showing that targeted modulation of complement-specific proteins has the potential to become a viable therapeutic approach. To date, a major focus of complement therapeutics has been on targeting the alternative complement system in AMD. Recent studies have outlined potential complement cascade inhibitors that might mitigate AMD disease progression. First-in-class complement inhibitors target the modulation of complement proteins C3, C5, factor B, factor D, and properdin. Herein, we will summarize ocular inflammation in the context of AMD disease progression, current clinical outcomes and complications of complement-mediated therapeutics. Given the need for additional therapeutic approaches for ocular inflammatory diseases, targeted complement modulation has emerged as a leading candidate for eliminating inflammation-driven ocular maladies.

The role of properdin in complement-mediated renal diseases: a new player in complement-inhibiting therapy?

Properdin is known as the only positive regulator of the complement system. Properdin promotes the activity of this defense system by stabilizing its key enzymatic complexes: the complement alternative pathway (AP) convertases. Besides, some studies have indicated a role for properdin as an initiator of complement activity. Though the AP is a powerful activation route of the complement system, it is also involved in a wide variety of autoimmune and inflammatory diseases, many of which affect the kidneys. The role of properdin in regulating complement in health and disease has not received as much appraisal as the many negative AP regulators, such as factor H. Historically, properdin deficiency has been strongly associated with an increased risk for meningococcal disease. Yet only recently had studies begun to link properdin to other complement-related diseases, including renal diseases. In the light of the upcoming complement-inhibiting therapies, it is interesting whether properdin can be a therapeutic target to attenuate AP-mediated injury. A full understanding of the basic concepts of properdin biology is therefore needed. Here, we first provide an overview of the function of properdin in health and disease. Then, we explore its potential as a therapeutic target for the AP-associated renal diseases C3 glomerulopathy, atypical hemolytic uremic syndrome, and proteinuria-induced tubulointerstitial injury. Considering current knowledge, properdin-inhibiting therapy seems promising in certain cases. However, knowing the complexity of properdin's role in renal pathologies in vivo, further research is required to clarify the exact potential of properdin-targeted therapy in complement-mediated renal diseases.

Anti-mouse properdin TSR 5/6 monoclonal antibodies block complement alternative pathway-dependent pathogenesis.

The complement alternative pathway (AP) is a major contributor to a broad and growing spectrum of diseases that includes age-related macular degeneration, atypical hemolytic uremic syndrome, and preeclampsia. As a result, there is much interest in the therapeutic disruption of AP activity. Properdin, the only positive regulator of the AP, is a particularly promising AP target. Several issues need to be clarified before the potential for properdin-directed therapy can be realized. In this report we use a portion of the mouse properdin protein, expressed in a bacterial system, to raise rabbit polyclonal and hamster monoclonal antibodies that block properdin-dependent pathogenesis. These antibodies, when employed with AP-dependent mouse disease models, can help evaluate the feasibility of properdin-directed therapy.

A novel antibody against human properdin inhibits the alternative complement system and specifically detects properdin from blood samples.

The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1-10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80-125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13-30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.

Blocking properdin, the alternative pathway, and anaphylatoxin receptors ameliorates renal ischemia-reperfusion injury in decay-accelerating factor and CD59 double-knockout mice.

Complement is implicated in the pathogenesis of ischemia-reperfusion injury (IRI). The activation pathway(s) and effector(s) of complement in IRI may be organ specific and remain to be fully characterized. We previously developed a renal IRI model in decay-accelerating factor (DAF) and CD59 double-knockout (DAF(-/-)CD59(-/-)) mice. In this study, we used this model to dissect the pathway(s) by which complement is activated in renal IRI and to evaluate whether C3aR- or C5aR-mediated inflammation or the membrane attack complex was pathogenic. We crossed DAF(-/-)CD59(-/-) mice with mice deficient in various complement components or receptors including C3, C4, factor B (fB), factor properdin (fP), mannose-binding lectin, C3aR, C5aR, or Ig and assessed renal IRI in the resulting mutant strains. We found that deletion of C3, fB, fP, C3aR, or C5aR significantly ameliorated renal IRI in DAF(-/-)CD59(-/-) mice, whereas deficiency of C4, Ig, or mannose-binding lectin had no effect. Treatment of DAF(-/-)CD59(-/-) mice with an anti-C5 mAb reduced renal IRI to a greater degree than did C5aR deficiency. We also generated and tested a function-blocking anti-mouse fP mAb and showed it to ameliorate renal IRI when given to DAF(-/-)CD59(-/-) mice 24 h before, but not 4 or 8 h after, ischemia/reperfusion. These results suggest that complement is activated via the alternative pathway during the early phase of reperfusion, and both anaphylatoxin-mediated inflammation and the membrane attack complex contribute to tissue injury. Further, they demonstrate a critical role for properdin and support its therapeutic targeting in renal IRI.

Properdin is critical for antibody-dependent bactericidal activity against Neisseria gonorrhoeae that recruit C4b-binding protein.

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, is an important cause of morbidity worldwide. A safe and effective vaccine against gonorrhea is needed because of emerging resistance of gonococci to almost every class of antibiotic. A gonococcal lipooligosaccharide epitope defined by the mAb 2C7 is being evaluated as a candidate for development of an Ab-based vaccine. Immune Abs against N. gonorrhoeae need to overcome several subversive mechanisms whereby gonococcus evades complement, including binding to C4b-binding protein (C4BP; classical pathway inhibitor) and factor H (alternative pathway [AP] inhibitor). The role of AP recruitment and, in particular, properdin in assisting killing of gonococci by specific Abs is the subject of this study. We show that only those gonococcal strains that bind C4BP require properdin for killing by 2C7, whereas strains that do not bind C4BP are efficiently killed by 2C7 even when AP function is blocked. C3 deposition on bacteria mirrored killing. Recruitment of the AP by mAb 2C7, as measured by factor B binding, occurred in a properdin-dependent manner. These findings were confirmed using isogenic mutant strains that differed in their ability to bind to C4BP. Immune human serum that contained bactericidal Abs directed against the 2C7 lipooligosaccharide epitope as well as murine antigonococcal antiserum required functional properdin to kill C4BP-binding strains, but not C4BP-nonbinding strains. Collectively, these data point to an important role for properdin in facilitating immune Ab-mediated complement-dependent killing of gonococcal strains that inhibit the classical pathway by recruiting C4BP.

Genetic and therapeutic targeting of properdin in mice prevents complement-mediated tissue injury.

The alternative pathway (AP) of complement activation is constitutively active and must be regulated by host proteins to prevent autologous tissue injury. Dysfunction of AP regulatory proteins has been linked to several human inflammatory disorders. Properdin is a positive regulator of AP complement activation that has been shown to extend the half-life of cell surface­bound C3 convertase C3bBb; it may also initiate AP complement activation. Here, we demonstrate a critical role for properdin in autologous tissue injury mediated by AP complement activation. We identified myeloid lineage cells as the principal source of plasma properdin by generating mice with global and tissue-specific knockout of Cfp (which encodes properdin) and by generating BM chimeric mice. Properdin deficiency rescued mice from AP complement­mediated embryonic lethality caused by deficiency of the membrane complement regulator Crry and markedly reduced disease severity in the K/BxN model of arthritis. Ab neutralization of properdin in WT mice similarly ameliorated arthritis development, whereas reconstitution of properdin-null mice with exogenous properdin restored arthritis sensitivity. These data implicate systemic properdin as a key contributor to AP complement­mediated injury and support its therapeutic targeting in complement-dependent human diseases.

Mechanism of complement inactivation by glycoprotein C of herpes simplex virus.

Glycoprotein C (gC) of both herpes simplex virus type 1 (HSV-1) and HSV-2 interacts with complement C3b and protects the virus from complement-mediated neutralization. To study the mechanism by which gC modulates complement activation, we expressed both gC-1 and gC-2 in a baculovirus expression system. Baculovirus recombinants containing gC genes spanning the entire gC-1 sequence (gC-1-TMR) or only the extracellular domain(s) of gC-1, gC-2, or a deletion mutant of gC-1 lacking residues 33 through 123 were expressed in sf9 insect cells. Binding of the expressed proteins to human C3 and C3 fragments was assessed by direct and competition ELISA. All four expressed proteins bound to C3, C3b, and C3c but not to C3d, suggesting 1) that the binding sites for these proteins are located in the C3c region of C3; and 2) that gC, in contrast to other C3-binding proteins, interacts with native C3. We have also examined the interaction of native C3 with gC-1 expressed on the HSV-1-infected cells. Analogous to recombinant proteins, gC-1 expressed on the infected cells also bound to native C3. The ability of baculovirus-expressed gCs to inhibit the interaction of C3b with its ligands was also analyzed. We found that gC-1, but not gC-2, inhibited the binding of C5 and properdin to C3b and also inhibited the alternative pathway-mediated lysis of rabbit erythrocytes. Inhibition of alternative pathway-mediated lysis and properdin binding to C3b, but not of C5 binding to C3b, required the transmembrane segment of the gC-1. The specificity of gC interactions was examined by studying the interaction of gC with C3 from various species. In contrast to properdin, both gCs bound to cobra C3; this finding suggests that gC-1 and properdin bind to different sites on C3b. Further analyses suggested that gC-1 sterically hindered access of C5 and properdin to C3b.

Inhibition of the function of activated properdin by squid chondroitin sulfate E glycosaminoglycan and murine bone marrow-derived mast cell chondroitin sulfate E proteoglycan.

We compared the relative capacities of two over-sulfated glycosaminoglycans, heparin and chondroitin sulfate E, to alter the function of native properdin (nP) and activated properdin (aP) in the formation and stabilization of the amplification C3 convertase (C3b,Bb). Heparin was more active on a weight basis than chondroitin sulfate E in inhibiting the formation of C3b,Bb without or with nP, but had no influence on the decay of a pre-formed convertase, either unstabilized or stabilized with nP or aP. In contrast, chondroitin sulfate E was over 10-fold more active than heparin in preventing the formation of C3b,Bb in the presence of aP, and gave dose-related acceleration of decay of pre-formed C3b,Bb,aP but not of unstabilized or nP-stabilized pre-formed convertase. The inhibitory effect of both glycosaminoglycans on the formation of C3b,Bb in the presence of nP or aP was less when the number of C3b sites per target cell was increased. The preferential action of chondroitin sulfate E on the function of aP during the formation and decay of C3b,Bb,aP as compared to C3b,Bb,nP implies functional differences in the two forms of P even when they have been incorporated into C3b,Bb. The equal potency, when adjusted for uronic acid content, of chondroitin sulfate E proteoglycan isolated from the T cell-dependent, bone marrow-derived murine mast cell and of chondroitin sulfate E glycosaminoglycan from squid reveals that the linkage of the glycosaminoglycan to a peptide core does not diminish its regulatory action on the alternative complement pathway.

[Anticomplement activity of a polyanion: pentosan sulfuric polyester. III. Mechanism of functional inactivation of the different properdin and complement system fractions]. / Activité anticomplémentaire d'un polyanion: le polyester sulfurique de pentosane III. - Mécanisme d'inactivation fonctionnelle des diverses fractions des systèmes complément et properdine

In vitro, the drug pentosan-poly-sulfoester (PPS) changes the electrophoretic migration of different proteins from the complement and properdin systems, such as native C3 (beta 1C globulin), C3c (beta-1A globulin), C3d (alpha-2D globulin), C1s inactivator (ClsINA), Clq and properdin factor B (B). Their more anodal migration is the consequence of a molecular alteration and persists after prolonged dialysis. These structural changes, yet undefined, explain the loss of functional activity of these proteins, and the anticomplementary activity of this drug. Moreover, PPS is able to block the alternate pathway activation by its action on properdin factor B (C3 proactivator). In fact, in presence of PPS, the activators of the properdin systems such as C3 nephritic factor are inactive. These altered mobilities are also responsible for the overestimation in the antigenic concentration of B (+ 45%), C4 (+27%) and C3/C3c (+ 14%), found in human serum containing 50 mg/ml of PPS. PPS has an original action upon the complement and properdin systems, which allows its clinical use as a potent inhibitor of the humoral mediators of inflammation.