Complement Factor D as a Strategic Target for Regulating the Alternative Complement Pathway.

The complement system is central to first-line defense against invading pathogens. However, excessive complement activation and/or the loss of complement regulation contributes to the development of autoimmune diseases, systemic inflammation, and thrombosis. One of the three pathways of the complement system, the alternative complement pathway, plays a vital role in amplifying complement activation and pathway signaling. Complement factor D, a serine protease of this pathway that is required for the formation of C3 convertase, is the rate-limiting enzyme. In this review, we discuss the function of factor D within the alternative pathway and its implication in both healthy physiology and disease. Because the alternative pathway has a role in many diseases that are characterized by excessive or poorly mediated complement activation, this pathway is an enticing target for effective therapeutic intervention. Nonetheless, although the underlying disease mechanisms of many of these complement-driven diseases are quite well understood, some of the diseases have limited treatment options or no approved treatments at all. Therefore, in this review we explore factor D as a strategic target for advancing therapeutic control of pathological complement activation.

Adipsin deficiency does not impact atherosclerosis development in Ldlr-/- mice.

Obesity is a potent risk factor for atherosclerotic morbidity and mortality. Cytokines secreted from adipose tissue, namely, adipokines, have been suggested to be actively involved in atherosclerosis. One of the most abundant adipokines, adipsin, is downregulated in obesity. It catalyzes the rate-limiting step of alternative complement activation, which is one of the three complement pathways potentially involved in inflammation in atherosclerosis. Interestingly, adipsin has been identified as a novel biomarker in human coronary artery disease. However, its role in the development of atherosclerosis remains unexplored. We crossed adipsin-/- mice onto an Ldlr-/- background [double-knockout (DKO) mice] and induced atherogenesis by high-fat and high-cholesterol feeding. Metabolic profiles were systemically characterized, and atherosclerotic plaques were measured at both aortic root and arch regions. Western blotting was conducted to assess adipsin level and complement activity. The DKO mice exhibited similar sizes of atherosclerotic lesions as Ldlr-/- control mice at both the aortic root and arch regions. Accordingly, they displayed comparable metabolic parameters, including body weight, insulin sensitivity, and lipid profiles, along with compensated complement activity. Adipsin deficiency does not impact the development of atherosclerosis in Ldlr-/- mice despite its crucial function in alternative complement activation. Therefore, it is unlikely to play an important role in mediating the risk of atherosclerotic complications in obesity.NEW & NOTEWORTHY Adipsin deficiency does not impact the development of atherosclerosis in Ldlr-/- mice despite its crucial function in alternative complement activation. Therefore, it is unlikely to play an important role in mediating the risk of atherosclerotic complications in obesity.

HFD-induced hepatic lipid accumulation and inflammation are decreased in Factor D deficient mouse.

Excessive intake of fat causes accumulation of fat in liver, leading to non-alcoholic fatty liver disease (NAFLD). High-fat diet (HFD) upregulates the expression of Factor D, a complement pathway component, in the liver of mice. However, the functions of Factor D in liver are not well known. Therefore, the current study investigated the relationship between Factor D and hepatic lipid accumulation using CRISPR/Cas9-mediated Factor D knockout (FD-KO) mice. Factor D deficiency downregulated expression of genes related to fatty acid uptake and de novo lipogenesis in the liver. Furthermore, Factor D deficiency reduced the expression of inflammatory factors (Tnf and Ccl2) and fibrosis markers and decreased accumulation of F4/80-positive macrophages. These data suggest that the Factor D deficiency improved hepatic lipid accumulation and hepatic inflammation in HFD-fed mice.

Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut.

The expansion of Enterobacteriaceae, such as E. coli is a main characteristic of gut inflammation and is related to multiple human diseases. However, how to control these E. coli overgrowth is not well understood. Here, we demonstrate that gut complement factor D (CFD) plays an important role in eliminating E. coli. Increased E. coli, which could stimulate inflammatory macrophages to induce colitis, were found in the gut of CFD deficient mice. We also showed that gut Reg4, which is expressed in gut epithelial cells, stimulated complement-mediated attack complexes to eliminate E. coli. Reg4 deficient mice also had increased E. coli. The dominant E. coli were isolated from colitis tissues of mice and found to be sensitive to both CFD- and Reg4-mediated attack complexes. Thus, gut Reg4- and CFD-mediated membrane attack complexes may maintain gut homeostasis by killing inflammatory E. coli.

In vivo protective effect of adipsin-deficiency on spontaneous knee osteoarthritis in aging mice.

The adipokine adipsin is an emerging mediator of human osteoarthritis (OA) progression. Here, we investigated its in vivo role in the development of spontaneous OA in aging mice. We compared articular knee joint morphology, histology in knee cartilage, synovial membrane, subchondral bone, meniscus, and anterior cruciate ligament (ACL); and chondrogenesis in the ACL from adipsin-deficient (Df-/-) and wild-type (Df+/+) 20-week- and 20-month-old mice. Serum levels of a panel of adipokines, inflammatory factors, and metalloproteases known to be implicated in OA were investigated. Data first revealed that the early manifestation of OA appeared in the ACL of 20-week-old mice, progressing to severe alterations in the 20 month-old wild-type mice. Further results demonstrated that adipsin-deficiency protected the articular tissues from spontaneous OA progression and triggered significantly higher serum levels of the adipokines adiponectin and FGF-21 while lowering levels of the inflammatory factor interleukin 6 (IL-6) in both young and old mice. This work further underlines the clinical relevance of adipsin as a novel therapeutic approach of human OA. Moreover, this study shows the potential beneficial effect of the adipokine FGF-21 against OA, and provides support for this factor to be a new biomarker and/or target of primary OA therapeutic avenues.

Alternative complement pathway component Factor D contributes to efficient clearance of tissue debris following acute CCl4-induced injury.

Complement, part of the innate immune system, is involved with immune protection against invading pathogens as well as cell survival and tissue regeneration. It is known that complement activation is required for timely hepatocyte recovery following an acute toxic injury, but which pathway of complement activation is involved in response to hepatocyte injury has not been identified. In these studies we utilize mice deficient in C1qa, C4 and Factor D, lacking the classical, classical/MBL, and alternative pathways of complement activation, respectively, to identify an essential role for Factor D in the ability of the liver to recover from acute toxic injury. Here we demonstrate that following an acute CCl4-induced injury, the involvement of the alternative complement pathway is essential for efficient liver recovery.

Distinct different contributions of the alternative and classical complement activation pathway for the innate host response during sepsis.

Complement activation represents a crucial innate defense mechanism to invading microorganisms, but there is an eminent lack of understanding of the separate contribution of the different complement activation pathways to the host response during sepsis. We therefore investigated different innate host immune responses during cecal ligation and puncture (CLP)-induced sepsis in mice lacking either the alternative (fD(-/-)) or classical (C1q(-/-)) complement activation pathway. Both knockout mice strains showed a significantly reduced survival and increased organ dysfunction when compared with control mice. Surprisingly, fD(-/-) mice demonstrated a compensated bacterial clearance capacity as control mice at 6 h post CLP, whereas C1q(-/-) mice were already overwhelmed by bacterial growth at this time point. Interestingly, at 24 h after CLP, fD(-/-) mice failed to clear bacteria in a way comparable to control mice. However, both knockout mice strains showed compromised C3 cleavage during sepsis. Investigating potential causes for this discrepancy, we were able to demonstrate that despite normal bacterial clearance capacity early during the onset of sepsis, fD(-/-) mice displayed increased inflammatory cytokine generation and neutrophil recruitment into lungs and blood when compared with both control- and C1q(-/-) mice, indicating a potential loss of control over these immune responses. Further in vitro experiments revealed a strongly increased Nf-κB activation capacity in isolated neutrophils from fD(-/-) mice, supporting this hypothesis. Our results provide evidence for the new concept that the alternative complement activation pathway exerts a distinctly different contribution to the innate host response during sepsis when compared with the classical pathway.

Eradication of Helicobacter spp. by using medicated diet in mice deficient in functional natural killer cells and complement factor D.

A commercial 4-drug diet has shown promise in eradicating Helicobacter spp. from rodents; however, its effectiveness in immunocompromised mice is unknown. This study evaluated the efficacy of this treatment in eradicating Helicobacter spp. from mice deficient in functional natural killer cells (Cd1(-/-)) or complement factor D (Df(-/-)). Cd1(-/-) mice naturally infected with H. hepaticus with or without H. rodentium were fed either control or medicated diet for 8 wk followed by 4 wk on control diet. Fecal samples were PCR-evaluated for Helicobacter spp. before mice began treatment and then every 2 wk thereafter for 12 wk. The same experimental design was repeated for eighteen 9- to 21-wk-old Df(-/-) mice naturally infected with H. bilis with or without H. rodentium. All Df(-/-) mice and 8- to 21-wk-old Cd1(-/-) mice ceased shedding Helicobacter spp. after 2 wk of treatment and remained negative throughout the study. In contrast, the Cd1(-/-) mice that were 24 wk or older shed Helicobacter spp. for the first 8 wk but tested negative at 10 and 12 wk. All treated animals had enlarged ceca and gained less weight than control untreated mice, and 6 of 7 treated Cd1(-/-) male mice developed mild portal fibrosis. These findings show that within 2 wk of treatment, the 4-drug diet eradicated H. hepaticus and H. rodentium from young Cd1(-/-) mice and H. bilis and H. rodentium from Df(-/-) mice, but eradication of established infections in Cd1(-/-) mice required 8 wk of treatment.

Initiation of the alternative pathway of murine complement by immune complexes is dependent on N-glycans in IgG antibodies.

OBJECTIVE: Collagen antibody-induced arthritis in mice exhibits a requirement for amplification by the alternative pathway of complement. Although the alternative pathway is activated by spontaneous hydrolysis, it is not known whether this pathway can also be initiated directly by IgG antibodies in immune complexes (ICs). IgG lacking terminal sialic acid and galactose (G0 IgG) can activate the lectin pathway of complement, but it is not known if G0 IgG can also activate the classical or alternative pathway. The purpose of this study was to examine the mechanism of initiation of the alternative pathway of complement by ICs. METHODS: We used adherent ICs containing bovine type II collagen (CII) and 4 monoclonal antibodies (mAb) to CII (adCII-IC). C3 activation was measured in the presence of sera from wild-type C57BL/6 mice or from mice deficient in informative complement components. The mAb were used intact or after enzyme digestion to create G0 IgG or to completely remove the N-glycan. RESULTS: Both the classical and alternative pathways, but not the lectin pathway, mediated C3 activation induced by the adCII-IC. Mannose inhibited the alternative pathway-mediated C3 activation but had no effect on the classical pathway, and N-glycans in IgG were required by the alternative pathway but not the classical pathway. Both the classical and alternative pathways mediated C3 activation induced by G0 IgG. Mannose-binding lectin bound avidly to G0 IgG, but lectin pathway-mediated C3 activation was only slightly increased by G0 IgG. CONCLUSION: The alternative pathway of complement is capable of initiating C3 activation induced by adCII-IC and requires the presence of N-glycans on the IgG. G0 IgG activates both the classical and alternative pathways more strongly than the lectin pathway.

Acylation-stimulating protein deficiency and altered adipose tissue in alternative complement pathway knockout mice.

Acylation-stimulating protein (C3adesArg/ASP) is an adipokine that acts on its receptor C5L2 to stimulate triglyceride (TG) synthesis in adipose tissue. The present study investigated ASP levels in mouse models of obesity and leanness and the effect of ASP deficiency in C3 knockout (C3KO) mice on adipose tissue morphology. Plasma ASP levels in wild-type (WT) mice correlated positively with plasma nonesterified fatty acids (NEFA) (R = 0.664, P < 0.001) and total cholesterol (R = 0.515, P < 0.001). Plasma ASP was increased by 85% in obese ob/ob leptin-deficient mice and decreased in lean diacylglycerol acyltransferase 1 (DGAT1) KO mice (-54%) and C/EBPalpha(beta/beta) transgenic mice (-70%) compared with WT. Mice lacking alternative complement factor B or adipsin (FBKO or ADKO), required for ASP production, were also ASP deficient. Both FBKO and C3KO mice had delayed postprandial TG and NEFA clearance on low-fat (LF) and high-fat (HF) diets, suggesting that lack of ASP, not C3, drives the metabolic phenotype. Adipocyte size distribution in C3KO mice was polarized (increased number of both small and large cells), with decreased adipsin expression (-33% gonadal HF), DGAT1 expression (-31% to -50%) and DGAT activity (-41%). Overall, a reduction/deficiency in ASP is associated with an antiadipogenic state and ASP may provide a target for controlling fat storage.

Mesangial immune complex glomerulonephritis due to complement factor D deficiency.

Complement factor D is a serine protease essential for the activation of the alternative pathway and is expressed in the kidney, adipocytes, and macrophages. Factor D is found at relatively high levels in glomeruli suggesting that this component of the complement cascade could influence renal pathophysiology. In this study, we utilize mice with a targeted deletion of the activating complement factor D gene and compare these results to mice with targeted deletion of the inhibitory complement factor H gene. Eight-month-old mice with a deleted factor D gene spontaneously develop albuminuria and have reduced creatinine clearance due to mesangial immune complex glomerulonephritis. These mesangial deposits contain C3 and IgM. In contrast to the mesangial location of the immune deposits in the factor D-deficient mice, age-matched factor H-deficient mice develop immune deposits along the glomerular capillary wall. Our observations suggest that complement factor D or alternative pathway activation is needed to prevent spontaneous accumulation of C3 and IgM deposits within the mesangium. Our studies show that the complement factor D gene knockout mice are a novel model of spontaneous mesangial immune complex glomerulonephritis.

Complement activation contributes to both glomerular and tubulointerstitial damage in adriamycin nephropathy in mice.

Adriamycin nephropathy is a model of focal segmental glomerulosclerosis, characterized by proteinuria and progressive glomerulosclerosis and tubulointerstitial damage. In this study, we examined the role of complement in the etiology of adriamycin nephropathy in mice. We used mice deficient in C1q, factor D, C3, and CD59, and compared them with strain-matched controls. C3 deposition occurred in the glomeruli of wild-type mice as early as 48 h following a single i.v. injection of adriamycin. C3-deficient mice developed significantly less proteinuria and less podocyte injury at day 3 postadriamycin than controls, suggesting that complement is important in mediating the early podocyte injury. At later time points, C3-deficient mice were protected from glomerulosclerosis, tubulointerstitial injury, and renal dysfunction. Factor D-deficient mice were also protected from renal disease, confirming the importance of alternative pathway activation in this model. In contrast, C1q-deficient mice developed similar disease to controls, indicating that the complement cascade was not activated via the classical pathway. CD59-deficient mice, which lack adequate control of C5b-9 formation, developed significantly worse histological and functional markers of renal disease than controls. Interestingly, although more C9 deposited in glomeruli of CD59-deficient mice than controls, in neither group was tubulointerstitial C9 staining apparent. We have demonstrated for the first time that alternative pathway activation of complement plays an important role in mediating the initial glomerular damage in this in vivo model of focal segmental glomerulosclerosis. Lack of CD59, which regulates the membrane attack complex, led to greater glomerular and tubulointerstitial injury.

Deficient alternative complement pathway activation due to factor D deficiency by 2 novel mutations in the complement factor D gene in a family with meningococcal infections.

The complement system is an essential element in our innate defense against infections with Neisseria meningitidis. We describe 2 cases of meningococcal septic shock, 1 of them fatal, in 2 children of a Turkish family. In the surviving patient, alternative pathway activation was absent and factor D plasma concentrations were undetectable. Concentrations of mannose-binding lectin (MBL), C1q, C4 and C3, factor B, properdin, factor H, and factor I were normal. Mutation analysis of the factor D gene revealed a T638 > G (Val213 > Gly) and a T640 > C (Cys214 > Arg) mutation in the genomic DNA from the patient, both in homozygous form. The consanguineous parents and an unaffected sister had these mutations in heterozygous form. In vitro incubation of factor-D-deficient plasma of the boy with serogroup B N meningitidis showed normal MBL-mediated complement activation but no formation of the alternative pathway C3-convertase C3bBbP, and severely decreased C3bc formation and terminal complement activation. The defect was restored after supplementation with factor D. In conclusion, this is the second report of a factor D gene mutation leading to factor D deficiency in a family with meningococcal disease. This deficiency abolishes alternative-pathway dependent complement activation by N meningitidis, and leads to an increased susceptibility to invasive meningococcal disease.

Mannan-binding lectin (MBL)-mediated opsonization is enhanced by the alternative pathway amplification loop.

The complement system is a humoral effector in the innate immune system. Three activation pathways exist in the complement system, known as the classical pathway, the lectin pathway and the alternative pathway. Dysfunction of lectin pathway activation is caused by MBL deficiency. MBL deficiency in a cohort of healthy Caucasian blood bank donors was investigated with MBL genotyping and MBL plasma concentration. Recognition of the yeast-derived zymosan by MBL was investigated with Western blot. The involvement of the alternative pathway amplification loop in enhancing MBL-mediated opsonization of zymosan was investigated in a novel opsonophagocytosis assay for flow cytometry. Sera deficient for MBL, factor D or properdin were tested, and purified MBL, factor D or properdin were used to recover opsonization. The optimal receiver-operator characteristic (ROC) cut-off value for dividing the Caucasian cohort in MBL-sufficient and MBL-deficient was calculated at 0.7 microg/ml. Thirty-eight percent of the group had concentrations below 0.7 microg/ml. Zymosan eluates opsonized with MBL-sufficient sera contain high oligomers of MBL, while eluates from MBL-deficient donors contained hardly any MBL. The MBL-, factor D- and properdin-deficient sera showed reduced opsonophagocytosis by human control neutrophils, as compared to normal MBL-sufficient sera. This reduction in opsonization was restored to normal levels by addition of purified MBL, factor D and properdin. The absence of opsonization in the factor D- and properdin-deficient sera, but presence in normal serum after blocking with anti-C1q-F(ab)2 and anti-MBL-F(ab)2, demonstrates the involvement of the amplification loop in MBL-initiated zymosan opsonization, even at very low serum concentrations (up to 3%, v/v). In conclusion, our data demonstrate that the MBL-mediated route of complement activation depends on the alternative pathway amplification loop for optimal opsonization of zymosan.

Mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury.

The mannose-binding lectin (MBL), a circulating pattern recognition molecule, recognizes a wide range of infectious agents with resultant initiation of the complement cascade in an Ab-independent manner. MBL recognizes infectious non-self and altered self in the guise of apoptotic and necrotic cells. In this study, we demonstrate that mice lacking MBL, and hence are devoid of MBL-dependent lectin pathway activation but have fully active alternative and classical complement pathways, are protected from cardiac reperfusion injury with resultant preservation of cardiac function. Significantly, mice that lack a major component of the classical complement pathway initiation complex (C1q) but have an intact MBL complement pathway, are not protected from injury. These results suggest that the MBL-dependent pathway of complement activation is a key regulator of myocardial reperfusion ischemic injury. MBL is an example of a pattern recognition molecule that plays a dual role in modifying inflammatory responses to sterile and infectious injury.

The virulence function of Streptococcus pneumoniae surface protein A involves inhibition of complement activation and impairment of complement receptor-mediated protection.

Complement is important for elimination of invasive microbes from the host, an action achieved largely through interaction of complement-decorated pathogens with various complement receptors (CR) on phagocytes. Pneumococcal surface protein A (PspA) has been shown to interfere with complement deposition onto pneumococci, but to date the impact of PspA on CR-mediated host defense is unknown. To gauge the contribution of CRs to host defense against pneumococci and to decipher the impact of PspA on CR-dependent host defense, wild-type C57BL/6J mice and mutant mice lacking CR types 1 and 2 (CR1/2(-/-)), CR3 (CR3(-/-)), or CR4 (CR4(-/-)) were challenged with WU2, a PspA(+) capsular serotype 3 pneumococcus, and its PspA(-) mutant JY1119. Pneumococci also were used to challenge factor D-deficient (FD(-/-)), LFA-1-deficient (LFA-1(-/-)), and CD18-deficient (CD18(-/-)) mice. We found that FD(-/-), CR3(-/-), and CR4(-/-) mice had significantly decreased longevity and survival rate upon infection with WU2. In comparison, PspA(-) pneumococci were virulent only in FD(-/-) and CR1/2(-/-) mice. Normal mouse serum supported more C3 deposition on pneumococci than FD(-/-) serum, and more iC3b was deposited onto the PspA(-) than the PspA(+) strain. The combined results confirm earlier conclusions that the alternative pathway of complement activation is indispensable for innate immunity against pneumococcal infection and that PspA interferes with the protective role of the alternative pathway. Our new results suggest that complement receptors CR1/2, CR3, and CR4 all play important roles in host defense against pneumococcal infection.

Effects of complement factor D deficiency on the renal disease of MRL/lpr mice.

BACKGROUND: The alternative complement pathway (AP) is activated in individuals with lupus nephritis and in murine models of systemic lupus erythematosus, including MRL/lpr mice. A previous study from our laboratory evaluated the development of renal disease in MRL/lpr mice genetically deficient in factor B (Bf-/-), a protein necessary for AP activation. MRL/lpr Bf-/- mice developed less renal disease and had improved survival; however, these mice were also a different major histocompatibility complex (MHC) haplotype (H-2b) than their wild-type littermates (H-2k) due to the gene for Bf being located in the MHC gene complex. We undertook the current study to determine if the decreased renal disease in MRL/lpr Bf-/- mice was due to the lack of AP activation or the H-2b haplotype by studying the effects of factor D (Df) deficiency, a critical protein for AP activation, on disease development in MRL/lpr mice. METHODS: Df-deficient mice were backcrossed with MRL/lpr mice for four to nine generations. MRL/lpr H-2k Df-/-, Df+/-, and Df+/+ littermates were evaluated for disease development. Lack of AP activation in MRL/lpr Df-/- mice was determined by the zymosan assay. Serum creatinine levels were measured using a creatinine kit. Proteinuria and autoantibody levels were determined by enzyme-linked immunosorbent assay (ELISA). Sections from one kidney were stained with fluorescein isothiocyanate (FITC) alpha-murine C3 or alpha-murine IgG to detect C3 and IgG deposition. The remaining kidney was cut in half with one half fixed, sectioned, and stained with hematoxylin and eosin and periodic acid-Schiff (PAS) to evaluate pathology and another half fixed in glutaraldehyde and examined via electron microscopy. RESULTS: MRL/lpr Df-/- mice had similar glomerular IgG deposition, proteinuria and autoantibody levels, as Df+/+ and Df+/- littermates. However, glomerular C3 deposition, serum creatinine levels, and pathologic renal disease were significantly reduced in Df-/- mice. Despite the lack of renal disease in Df-/- mice, life span was not impacted by factor D deficiency. CONCLUSION: The absence of Df and AP activation is protective against the development of proliferative renal disease in MRL/lpr mice suggesting the similar effect of Bf deficiency in MRL/lpr mice was also due to the lack of AP activation.

Role for the alternative complement pathway in ischemia/reperfusion injury.

The terminal complement components play an important role in mediating tissue injury after ischemia and reperfusion (I/R) injury in rats and mice. However, the specific complement pathways involved in I/R injury are unknown. The role of the alternative pathway in I/R injury may be particularly important, as it amplifies complement activation and deposition. In this study, the role of the alternative pathway in I/R injury was evaluated using factor D-deficient (-/-) and heterozygote (+/-) mice. Gastrointestinal ischemia (GI) was induced by clamping the mesenteric artery for 20 minutes and then reperfused for 3 hours. Sham-operated control mice (+/- versus -/-) had similar baseline intestinal lactate dehydrogenase activity (P = ns). Intestinal lactate dehydrogenase activity was greater in -/- mice compared to +/- mice after GI/R (P = 0.02) thus demonstrating protection in the -/- mice. Intestinal myeloperoxidase activity in +/- mice was significantly greater than -/- mice after GI/R (P < 0.001). Pulmonary myeloperoxidase activity after GI/R was significantly higher in +/- than -/- mice (P = 0.03). Addition of human factor D to -/- animals restored GI/R injury and was prevented by a functionally inhibitory antibody against human factor D. These data suggest that the alternative complement pathway plays an important role in local and remote tissue injury after GI/R. Inhibition of factor D may represent an effective therapeutic approach for GI/R injury.

Complement activation in factor D-deficient mice.

To assess the contribution of the alternative pathway in complement activation and host defense and its possible role in the regulation of systemic energy balance in vivo, factor D-deficient mice were generated by gene targeting. The mutant mice have no apparent abnormality in development and their body weights are similar to those of factor D-sufficient littermates. Complement activation could not be initiated in the serum of deficient mice by the alternative pathway activators rabbit erythrocytes and zymosan. Surprisingly, injection of cobra venom factor (CVF) caused a profound and reproducible reduction in serum C3 levels, whereas, as expected, there was no C3 reduction in factor B-deficient mice treated similarly. Studies of C3 and factor B activation in vitro by CVF demonstrated that in factor D-deficient serum the alpha chain of C3 was cleaved gradually over a period of 60 min without detectable cleavage of factor B. CVF-dependent C3 cleavage in the deficient serum required the presence of Mg(2+), whereas in normal mouse serum the presence of divalent cations was not required. These results suggest that in mouse proteolytic cleavage of factor B by factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bound factor B. Kinetics of opsonization of Streptococcus pneumoniae by C3 fragments was much slower in factor D-deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host defense early after infection.