Properdin-deficient Serum Research Articles

Complement activation by tubular cells is mediated by properdin binding

Activation of filtered complement products on the brush border of the tubular epithelium is thought to be a key factor underlying proteinuria-induced tubulointerstitial injury. However, the mechanism of tubular complement activation is still unclear. Recent studies on mechanisms of complement activation indicate a key role for properdin in the initiation of an alternative pathway. We hypothesized that properdin serves as a focal point for complement activation on the tubulus. We observed a strong staining for properdin on the luminal surface of the tubules in kidney biopsies from patients with proteinuric renal disease. In vitro experiments revealed dose-dependent binding of properdin to proximal tubular epithelial cells (PTEC), whereas no significant binding to endothelial cells was detected. Exposure of PTEC with normal human serum as a source of complement resulted in complement activation with deposition of C3 and generation of C5b-9. These effects were virtually absent with properdin-deficient serum. Preincubation of PTEC with properdin before addition of properdin-depleted serum fully restored complement activation on the cells, strongly suggesting a key role for properdin in the activation of complement at the tubular surface. In proteinuric renal disease, filtered properdin may bind to PTEC and act as a focal point for alternative pathway activation. We propose that this contribution of properdin is pivotal in tubular complement activation and subsequent damage. Interference with properdin binding to tubular cells may provide an option for the treatment of proteinuric renal disease.

Properdin Plays a Protective Role in Polymicrobial Septic Peritonitis

Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5alpha. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5alpha. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.

The role of Fcgamma receptor polymorphisms and C3 in the immune defence against Neisseria meningitidis in complement-deficient individuals.

Individuals with either a late (C5-9) complement component deficiency (LCCD) or properdin deficiency are at increased risk to develop meningococcal disease, often due to serogroups W135 and Y. Anti-meningococcal defence in both LCCD persons and properdin-deficient individuals without bactericidal antibodies depends mainly on phagocytosis. Three types of opsonin receptors are involved in phagocytosis by polymorphonuclear cells (PMN). These represent the polymorphic FcgammaRIIa (CD32) and FcgammaRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcgammaRIIa and FcgammaRIIIb allotypes was assessed in 15 LCCD and in 15 properdin-deficient patients with/without previous meningococcal disease, we found the combination of FcgammaRIIa-R/R131 with FcgammaRIIIb-NA2/NA2 allotypes to be associated with previous meningococcal disease (odds ratio 13.9, Fisher's test P = 0.036). No such relation was observed in the properdin-deficient patients. The importance of FcgammaRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcgammaRIIa-R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0.05) less than PMN from FcgammaRIIa-H/H131 donors. When properdin-deficient serum was tested, it was observed that reconstitution with properdin resulted in enhanced PMN phagocytosis of the W135 meningococci (P = 0.001). This enhanced phagocytosis was parallelled by an increase in C3 deposition onto the opsonized meningococci W135 (r = 0.6568, P = 0. 01). We conclude that the occurrence of meningococcal disease in LCCD patients is associated with certain FcgammaR allotypes. Properdin-deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.

Complement-mediated serum sensitivity among spirochetes that cause Lyme disease.

Borrelia burgdorferi-related isolates were tested for their sensitivity to normal human serum (NHS) and their ability to activate complement. By dark-field microscopy, electron microscopy, and subsurface plating, it was shown that exposure of a Borrelia garinii isolate to 10% or more NHS resulted in immobilization, blebbing, and killing of the spirochetes. These effects were mediated by complement, since they were not seen after heat treatment of NHS, in the presence of EDTA, or in an agammaglobulinemic serum. All seven B. garinii type 5 or 6 and all four VS116/M19 strains were serum sensitive, whereas all eight Borrelia afzelii, five of eight B. garinii type 4, and three of seven B. burgdorferi sensu stricto isolates were serum resistant. The other isolates were partially serum sensitive. Four serum-sensitive B. garinii isolates had been isolated from human cerebrospinal fluid. Most likely, activation of both the alternative pathway and the classical pathway of complement was involved, since bactericidal activity was diminished in properdin-deficient sera as well as in a C1q-depleted serum and in a C4-deficient serum. Bactericidal activity could be restored when a serum lacking C1q or C4 was mixed with a properdin-deficient serum. Isolates with various genetic backgrounds were equally able to activate C3 as measured by enzyme-linked immunosorbent assay. In the presence of Mg-EGTA, C3 was activated by all isolates after exposure to > or = 10% NHS. This study shows that B. burgdorferi-related spirochetes can be either serum sensitive or serum resistant in vitro and that this characteristic is associated with their genetic background.

Analysis of C3 deposition and degradation on Neisseria meningitidis and Neisseria gonorrhoeae

The deposition and degradation of human complement component C3 on the cell surfaces of Neisseria meningitidis and Neisseria gonorrhoeae were studied. Bacteria were incubated in human serum, and ester-linked C3 fragments were analyzed by hydroxylamine release and immunoblot detection. Similar patterns of C3 degradation were found for both serum-resistant and serum-sensitive meningococcal strains of serogroups A, B, C, Y, and W135, as well as for serum-sensitive gonococcal strains and their sialylated serum-resistant variants. The predominant fragments in all cases were the 40-kDa alpha' 2 chain of iC3b and the 75-kDa beta chain common to both C3b and iC3b. The 67-kDa alpha' 1 chain of iC3b was also detected. The 105-kDa alpha' chain of intact C3b represented a minor proportion of deposited C3. Capsule-specific immunoglobulin G or immunoglobulin A1 did not alter the observed degradation patterns, nor did incubation of meningococci in properdin-deficient serum. The degradation of C3 in C5-, C6-, or C8-deficient serum was the same as that in normal serum, although the deposition of C3 was severely limited, based as indicated by the intensity of the fragments. With the use of an enzyme-linked immunosorbent assay that measured total iC3b and C3, I found that both iC3b deposition and C3 deposition varied among meningococcal and gonococcal strains and that the amounts of iC3b and C3 were independent of the relative quantities of cell surface sialic acid and of serum sensitivity for meningococci but not for gonococci. I conclude that complement activation on neisserial cell surface results in the formation of an identical repertoire of predominantly iC3b fragments of ester-linked C3b molecules regardless of the presence of sialic acid in either the capsule or the lipooligosaccharide or of the sensitivity of the organism to complement-mediated lysis but that the quantities of both ester- and amide-linked iC3b molecules deposited exhibit strain variability.

Human IgA1 blockade of IgG-initiated lysis of Neisseria meningitidis is a function of antigen-binding fragment binding to the polysaccharide capsule.

We have recently shown that human IgA1 can initiate lysis of group C Neisseria meningitidis via the classical C pathway when bound to specific outer membrane proteins, but that IgA1 can also function as a blocking antibody when bound to the polysaccharide capsule of meningococci. In this report, we further characterized IgA1 blockade by examining the effect of IgA1 on IgG-initiated immune lysis of group C meningococci. We purified IgG and monomeric IgA1 from either convalescent group C meningococcal case sera or tetravalent (A, C, Y, W135) polysaccharide vaccinate sera. In the absence of IgA1, IgG initiated complete lysis (greater than 99%) of strains 118V (C:P3,4:L2,4) 126E (C:P3:L1,8), and 35E (C:P5:L2). Addition of IgA1 to the bactericidal reaction mixture completely blocked the lytic function of IgG. Removal of the Fc portion of IgA1 with either pepsin or IgA1 protease did not affect blockade. Both the F(ab')2 and Fab derivatives of IgA1 blocked lysis quantitatively as well as intact IgA1. The Fc fragment produced by IgA1 protease cleavage neither increased nor decreased Fab-mediated blockade. IgA1 and its Fab and F(ab')2 fragments blocked IgG-initiated lysis via either the classical pathway in factor B-depleted and in properdin-deficient serum, the alternative pathway in MgEGTA-chelated serum, or both pathways combined. Absorption of the IgA1 and IgG with alum-bound group C polysaccharide completely removed blocking and lytic activity, respectively, indicating that both the blocking IgA1 and the lytic IgG were specific for the group C capsule. Blocking by IgA1 was a linear function of the polysaccharide Ag-binding capacity (ABC) ratio of blocking IgA1 to lytic IgG. Complete blockade was observed at an ABC ratio of 5.5. At ABC ratios of 3.3 and 4.4, IgA1 affected significant blockade whether added previous to, concurrent with, or subsequent to sensitization of the organisms with IgG. With the use of a C polysaccharide ELISA, we found that the binding of IgA1 to the group C capsule in the presence of IgG exhibited positive cooperativity and therefore that blockade was independent of the ability of IgA1 to directly compete with IgG for binding to epitopes within the group C capsule. We conclude that IgA1, when bound to the group C polysaccharide capsule, can block IgG-initiated lysis of group C meningococci through either the classical or the alternative pathway before or after the organism is exposed to IgG, and that blockade is an Fc-independent event.

Granulocyte functions and Neisseria meningitidis: influence of properdin‐deficient serum

Granulocyte-mediated reactions such as opsonization, chemotaxis, and release of granulocyte myeloperoxidase and lactoferrin were studied in properdin-deficient and normal human serum incubated with serogroup A and W-135 meningococci. There were no differences between the sera when serogroup A meningococci were studied. Opsonic and chemotactic activity were impaired against serogroup W-135 meningococci in properdin-deficient serum. Restitution with properdin restored both activities. We found similar release of myeloperoxidase and lactoferrin from granulocytes challenged with serogroup A or W-135 meningococci in either sera. These findings are in accordance with the clinical observations of meningococcal infections caused by serogroup W-135 in properdin-deficient patients as well as the absence of infections caused by serogroup A meningococci.

Bactericidal activity for Neisseria meningitidis in properdin-deficient sera.

We investigated serum bactericidal reactions against Neisseria meningitidis (serogroups A, B, C, D, Y, W-135, 29E, X, and Z) in the sera of two healthy adults with properdin deficiency. Bactericidal reactions mediated via the classic complement pathway (unchelated system) were not impaired in properdin-deficient serum. The properdin-deficient sera supported alternative pathway-mediated killing (Mg++EGTA-chelated system) of some, but not all, of the strains investigated. Vaccination of the properdin-deficient individuals with serogroup A and C polysaccharide clearly increased the concentrations of antibody to meningococci. At least some of the antibodies induced by vaccination supported the bactericidal activity of properdin-deficient serum. Some antibodies to meningococci, probably of the IgM class, promoted alternative pathway-mediated bactericidal reactions in the absence of properdin. By contrast, presensitizing meningococci with IgG enhanced the alternative pathway-mediated reactions, but this was strictly a properdin-dependent effect.

Formation of soluble immune complexes by complement in sera of patients with various hypocomplementemic states. Difference between inhibition of immune precipitation and solubilization.

To examine whether the ability of complement to form soluble immune complexes plays a role in preventing immune complex-mediated diseases, we analyzed the capacity of complement to inhibit immune precipitation (IIP) and to solubilize preformed immune aggregates (SOL) in 23 sera of patients with various hypocomplementemic states, and we correlated the results of these studies with the clinical syndromes found in the various patients. In sera with deficiency or depletion of early classical pathway components, IIP was profoundly altered, whereas SOL was delayed but in the normal range. In contrast, in sera with C3 depletion but intact classical pathway and in properdin-deficient serum, IIP was initially preserved, whereas SOL was abolished. Since the incidence of immune complex diseases in various hypocomplementemic states correlates with the severity of IIP defects, but not with reduced SOL, it is suggested that IIP is an essential biological function of complement that prevents the rapid formation of insoluble immune complexes in vivo.

Leukocyte chemotaxis induced by Streptococcus pneumoniae and serum.

The effects on normal polymorphonuclear leukocytes (PMNs) of chemotactic factors in sera incubated with five types of S. pneumoniae commonly encountered in otitis media and other respiratory infections (types 3, 6A, 14, 19F and 23F) and five types that rarely cause infections (types 31, 35, 36, 42 and 43) were compared by the means of migration under agarose technique. All the types frequent in otitis media and some of the types that are less frequent, viz. 31 and 36, showed a good chemotactic activation in pooled serum. No correlation between tendency to cause otitis media and ability to regenerate chemotactic activity in serum was found. Impaired generation of chemotactic activity was found in C2-deficient serum, Mg2 + EGTA-chelated serum and properdin-deficient serum, indicating the significance of both the classical and the alternative complement pathways for production of chemotactic factors by pneumococcal types.

Screening for deficiencies in the classical and alternative pathways of complement by hemolysis in gel.

Two assays based on hemolysis in gel were assessed for screening complement (C) component deficiencies. In one assay sensitized sheep erythrocyte (EA) were incorporated in agarose gel containing Ca2+ and Mg2+, in the other guinea pig erythrocytes (GpE) were used in the presence of Mg2+and EGTA. With few exceptions, fresh samples from healthy individuals produced homogeneous areas of complete hemolysis in both assays. Clearly aberrant patterns were observed in approximately 4% of healthy blood donors. Sera from patients having complete deficiencies of Clq, C2 or C4 produced clear lysis of GpE only, whereas in sera lacking C3 or C8 lysis was grossly impaired in both assays. Properdin deficient serum produced very slight lysis of GpE but normal lysis of EA. Reconstitution of these C-deficient sera gave normal lysis. Together, the two assays supplement immunochemical C3 and C4 determinations for screening out C disorders.

Properties of Highly Purified Human Properdin

Summary Partially purified human properdin was further purified by column chromatography on diethylaminoethyl (DEAE)-cellulose and carboxymethyl-Sephadex. The most highly purified preparation was electrophoretically and ultracentrifugally homogeneous. The diffusion coefficient D20,w was 2.2 × 10-7 cm2/sec; the sedimentation constant S20,w was 5.2 S; and the molecular weight was 223,000. Highly purified properdin formed a single characteristic line in the β-globulin region on immunoelectrophoresis with rabbit anti-properdin serum and failed to react with rabbit antisera to whole human serum, to γG-, γM- or γA- globulins, or to type K or L light chains of human immunoglobulins. Highly purified properdin retained the property of restoring activity to properdin-deficient serum in the zymosan assay, in the ability to cause lysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria and in the killing of Shigella dysenteriae. Properdin activity in both biologic test systems correlated well with properdin activity as measured in the zymosan assay. Properdin did not restore bactericidal activity of serum specifically absorbed with homologous bacteria, but specific bactericidal antibody did not function in this system without properdin. Highly purified properdin neither combined with nor caused agglutination of zymosan. It was without effect on the hemolytic activity of whole complement, had little or no influence on the hemolytic activity of any purified component of complement tested, and was functionally and antigenically unrelated to such complement components. These data demonstrated the existence of properdin as a unique serum protein, distinct from known immunoglobulins or complement components, which participates in certain immunologic reactions of human serum.

The properdin system and immunity. V. The bactericidal activity of the properdin system.

Methods for the preparation and standardization of reagents suitable for studies on the bactericidal action of the properdin system are described. The preparation and properties of serum free of properdin (RP(b)) are presented in detail because of the necessity for a suitable RP(b) in these studies. The properdin system is responsible for the bactericidal action of normal human serum against a variety of microorganisms. The present work shows that the removal of properdin from serum also removes bactericidal activity. Addition of properdin to properdin-deficient serum restores bactericidal activity. A quantitative relationship exists between the final properdin concentration and bactericidal activity against sensitive organisms. The possibilities of a bactericidal assay for properdin are discussed. It is demonstrated that, in addition to properdin, the four components of complement (present in RP(b)) are necessary for the destruction of properdinsensitive bacteria. If any component is missing, bactericidal activity is lost; when the component is replaced, bactericidal activity is restored. Magnesium is also necessary for the bactericidal activity of the properdin system. Maximal bactericidal activity is obtained with magnesium concentrations similar to that of normal human serum (10(-3) to 10(-4)M). The bactericidal activity of the properdin system occurs only at temperatures above 15 degrees . Resistant strains have been encountered in species of bacteria sensitive to the properdin system. Resistance or sensitivity is a characteristic of the individual strain and not of the species. The widespread occurrence of the properdin system in normal mammalian serum and the variety of bacteria destroyed by it suggest that the properdin system is a factor in natural resistance.