Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 2000 May;120(2):338–345. doi: 10.1046/j.1365-2249.2000.01208.x

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

C A P Fijen *, R G M Bredius *,†, E J Kuijper *, T A Out *, M De Haas †, A P M De Wit §, M R Daha ‡, J G J Van De Winkel §
PMCID: PMC1905639  PMID: 10792385

Abstract

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 FcγRIIa (CD32) and FcγRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcγRIIa and FcγRIIIb allotypes was assessed in 15 LCCD and in 15 properdin-deficient patients with/without previous meningococcal disease, we found the combination of FcγRIIa-R/R131 with FcγRIIIb-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 FcγRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcγRIIa-R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0·05) less than PMN from FcγRIIa-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 FcγR allotypes. Properdin-deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.

Keywords: Fcγ receptor, Neisseria meningitidis, complement deficiency

INTRODUCTION

In general, host defence against meningococci is provided by mucosal immunity, as well as serum bactericidal and phagocytic activities. Individuals with a complement deficiency either of an alternative pathway component (properdin, factor D or H), a late complement component (C5, C6, C7, C8, C9), or of C3 are at increased risk to contract disease by Neisseria meningitidis[1], frequently due to uncommon (Y and W135) serogroups [1–3]. Late complement component-deficient LCCD persons and properdin-deficient individuals are reported to develop meningococcal disease mainly at age 10–30 years [1], when it is assumed that anti-meningococcal antibodies (either specific or cross-reactive) are present [4]. LCCD individuals have defective serum bactericidal activity due to lack of membrane attack complex (MAC) formation, which is essential for penetration of cell membranes and lysis [5]. Therefore, protection against meningococci in these individuals depends on effective phagocytosis. The increased risk of meningococcal disease among properdin-deficient patients is not completely understood, since classical pathway of complement-mediated bactericidal activity is intact and most of the meningococcal infections occur at an age at which meningococcal antibodies are usually present [6].

Knowledge about antibody-mediated phagocytic functions increased rapidly in the last decade [7,8]. Three classes of receptors for the Fc region of IgG (FcγR) on phagocytes have been identified, FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), encompassing eight subclasses [8]. Polymorphonuclear cells (PMN) constitutively express FcγRIIa and FcγRIIIb, which are both involved in phagocytosis [8]. Both receptors can bind human IgG1 and IgG3 subclasses, but FcγRIIa represents the sole FcR capable of binding IgG2. Interestingly, both FcγRIIa and IIIb exhibit allelic polymorphisms, which affect antibody-mediated processes [8]. The FcγRIIa occurs in two allotypic forms, designated FcγRIIa-R131, and IIa-H131, due to the presence of either an arginine or a histidine residue at amino acid position 131 [8]. Previously, we have shown that PMN from FcγRIIa-R/R131 homozygous individuals phagocytose IgG2-opsonized N. meningitidis serogroup B less effectively than PMN from FcγRIIa-H/H131 donors [9]. The neutrophil-specific FcγRIIIb (CD16b) also exists in two allotypic forms, referred to as neutrophil antigen 1 (NA1) and NA2 [8]. PMN from FcγRIIIb-NA2/2 homozygous individuals exhibited less IgG1-mediated phagocytosis of N. meningitidis serogroup B than PMN of FcγRIIIb-NA1/1 donors [10].

We hypothesize that the basis of the enhanced risk for meningococcal disease in both properdin-deficient and LCCD patients is a reduced phagocytic efficacy. Phagocytosis of bacteria is known to be dependent on both antibody and complement. For the latter factor, the leucocyte-specific complement receptor CR3 (CD11b/CD18) especially seems involved [7,11–13]. Properdin acting as a stabilizer of the alternative pathway C3 convertase has been postulated to increase C3b deposition onto bacterial surfaces [14,15]. Stabilization of the C3 convertase may be essential for meningococci since sialic acid containing capsules and lipopolysaccharides (LPS) of the meningococcal surface are strong down-regulators of the alternative pathway activity [16]

The present study aims to assess whether PMN FcγR allotypes are associated with the risk of contracting meningococcal disease in either properdin-deficient or LCCD persons. In addition, we determined the role of meningococcal C3 deposition in PMN phagocytosis.

SUBJECTS AND METHODS

LCCD individuals

Thirteen C8- and two C6-deficient individuals belonging to four families were studied for FcγR allotypes. Informed consent was obtained from all patients, and permission was obtained for this study from the Medical Ethical Commission of the Academic Medical Centre (Amsterdam, The Netherlands). In each family we studied LCCD individuals with and without previous meningococcal disease. Eight complement-deficient persons had experienced in total 15 episodes of meningococcal disease, of which nine were diagnosed as meningitis, three as meningitis with sepsis, two as sepsis only, and one as chronic meningococcal disease. The meningococcal disease was bacteriologically proven in seven episodes: two episodes were due to serogroup W135, one to serogroup A, one to serogroup B, and one to serogroup C. In one episode the meningococcal isolate was non-groupable, and in another episode the isolate was not serogrouped. In two patients no isolates were obtained; one patient had a typical clinical syndrome of meningococcal meningitis with sepsis and shock and the other patient had a medical history of two episodes of meningitis in 1938 and 1944. Seven LCCD persons had experienced no meningococcal disease. The mean age (42 ± 28 years) and age distribution of the LCCD persons with and without previous meningococcal disease were similar.

Properdin-deficient individuals

Fifteen properdin-deficient individuals belonging to five families were studied for the distribution of FcγR allotypes. In each family we studied persons with and without previous meningococcal disease. Seven properdin-deficient persons had experienced seven meningococcal disease episodes. These were caused by serogroup W135 in three cases, serogroup Y in three cases, and serogroup C in one case. Mean age of the persons with previous meningococcal disease at the time of the study was 24·2 ± 8 years. Eight properdin-deficient individuals had experienced no meningococcal disease and their mean age was 31·7 ± 26·3 years. For the C3 deposition study and phagocytosis assays, we included three additional properdin-deficient patients. Of them, two had had meningococcal disease due to serogroup W135, and one due to serogroup Y.

Relatives as controls

Sera from 20 male relatives of properdin-deficient (n = 9) or LCCD patients (n = 14) with a normal complement system (as determined by CH50 and AP50), and with normal serum properdin levels, served as controls in the C3 deposition assays.

Healthy volunteers

Laboratory personnel with normal complement function served as donors for PMN of specific FcγR allotypes.

Materials

PMN and mononuclear cells

PMN and mononuclear cells were separated from platelets and mononuclear cells by density gradient centrifugation over 1·076 g/ml Percoll (Pharmacia, Uppsala, Sweden) as in [17]. Erythrocytes were removed from PMN fractions by isotonic lysis at 0°C, and purified PMN or mononuclear cells were resuspended in Ca2+-free HEPES.

Complement source

Serum (AGS) obtained from an agammaglobulinaemia patient [9] with very low antibacterial IgG (level <1% of that observed in pooled human serum) and normal haemolytic complement activity was used as a source of complement in phagocytosis assays.

Bacterial strain

Neisseria meningitidis serogroup W135, type 2a, subtype P1.2,1.5 was used. Serogrouping and (sub)typing was performed at the Reference Laboratory as previously described [18].

Purified properdin

Properdin was purified as reported to a standard solution of 1 mg/ml [19]. Reconstitution of properdin-deficient serum with 2·5 μg/ml of purified properdin restored the haemolytic activity of the alternative pathway.

IgG1 and IgG2 anti-N. meningitidis W135 preparations

Polyclonal IgG1 and IgG2 preparations were purified as described previously from sera of two patients containing high levels of anti-N. meningitidis W135 capsular IgG [20].

Buffers

Coating buffer (0·05 m NaHCO3 pH 9·6) served to coat wells of ELISA plates with meningococci W135. Phosphate buffer containing Ca2+ and Mg2+ (PiCM buffer; pH 7·2–7·4) consisted of 137 mm NaCl, 2·7 mm KCl, 8·1 mm Na2HPO4, 1·5 mm KH2PO4, 1·0 mm MgCl2, 0·6 mm CaCl2, 1% (w/v) glucose (all from Merck, Schuchardt, Germany), and 2·5% (v/v) human serum albumin (CLB, Amsterdam, The Netherlands). Ca2+-free HEPES (Merck) contained 20 mm HEPES, 132 mm NaCl, 6 mm KCl and 1·2 mm KH2PO4.

Methods

FcγRIIa allotyping by polymerase chain reaction analysis

Chromosomal DNA was extracted from peripheral blood leucocytes of properdin-deficient persons [21]. FcγRIIa allotypes were analysed via polymerase chain reaction (PCR) typing technique using FcγRIIa-R131- and FcγRIIa-H131-specific oligonucleotides [22].

FcγRIIIb allotyping by radioimmunoassay

FcγRIIIb-NA1/NA2 allotypes of properdin-deficient persons were determined serologically by radioimmunoassay (RIA) as described [23,24].

FcγR allotyping by FACS analysis

The FcγRIIa (CD32) polymorphisms on monocytes and PMN of LCCD persons were determined by an indirect immunofluorescence assay [25] using MoAb 41H16 (generously provided by Dr B. Longenecker, University of Alberta, Edmonton, Canada) and MoAb IV.3 (Medarex, Annandale, NJ). FcγRIIIb (CD16b)-NA1 and NA2 were typed by using CD16 antibody CLBGran11 (CLB; specific for IIIb-NA1) and GRM1 (provided by Dr M. Garrido, Granada, Spain; selective for IIIb-NA2), respectively [26]. Fluorescence was quantified with a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Measurement of C3 deposition onto N. meningitidis W135

Deposition of complement C3 onto meningococci W135 was assessed by ELISA, as reported previously [27]. In short, 96-well ELISA flat-bottomed plates (Greiner, Langerthal, Germany) were coated (150 μl/well; room temperature, 2 h) with approximately 5 × 108 colony-forming units (CFU)/ml meningococci W135 in coating buffer. Free binding sites were blocked with 2·5% human serum albumin (HSA) (CLB) in 150 μl PiCM buffer. Patient serum (100 μl; 1% v/v in PiCM) was added to each well, in the presence or absence of 2·5 μg/ml purified properdin and incubated at 37°C for 1 h. Plates were washed with PBS containing 0·05% (v/v) Tween-20 (PBS–T), and incubated at 37°C for 2 h with horseradish peroxidase (HRP)-conjugated rabbit anti-human C3c (Dako, Glostrup, Denmark). The anti-C3c reagent recognizes the C3c part of native C3 and C3b, including C3bi. After washing with PBS–T, and incubation with 100 μl substrate the reaction was stopped by addition of 100 μl 2 m H2SO4. The optical density (OD) was then measured at Å450nm with a Titertek Multiscan MC (Flow Labs, Irvine, UK). Results are presented as ratio of the OD-Å450nm in the serum reconstituted with purified properdin and the OD-Å450nm of serum without addition of purified properdin. Tests were performed at least in duplicate. Blocking of classical pathway of complement activation in properdin-deficient sera was performed by addition of 10 mm MgEGTA.

Phagocytosis assay

PMN phagocytic capacity was analysed using methods reported [28]. Briefly, meningococcal strain W135 suspended in PiCM was labelled with FITC (0·015 mg/ml; Sigma, St Louis, MO) for 15 min at 37°C. Unbound FITC was removed by washing three times. Opsonization of 2 × 108 CFU FITC-labelled meningococci W135 was performed with purified IgG1 or IgG2 preparations in the presence or absence of AGS as complement source (2% v/v), or in properdin-deficient sera in the presence or absence of 10 μg/ml purified properdin. After opsonization at 37°C for 20 min, bacteria were washed with PiCM buffer, and resuspended in calcium-free HEPES buffer. PMN and opsonized bacteria (2 × 108/ml) were added together at a 1:10 ratio and incubated in a shaking water bath at 37°C. At selected time intervals, samples were removed and diluted in ice-cold HEPES buffer to stop phagocytosis. Fluorescence of adherent or ingested FITC-labelled bacteria was quantified by flow cytometry. Phagocytosis was expressed as the percentage of total PMN that were green fluorescent. In each experiment controls without antibody and/or complement were included. Trypan blue (0·064% (w/v); Flow Labs) was used to quench fluorescence from adherent FITC-labelled bacteria [28]. The intra-assay coefficient of variation of the flow cytometric measurement was < 10%.

Statistical analysis

The Fisher's exact test and odds ratios (OR) were calculated to assess the significance of the FcγR allotype distributions with StatXactTM, Cytel 1989. The Mann–Whitney test was used to determine the differences in C3 deposition and phagocytosis of meningococci. The phagocytosis ratio expresses the ratio of phagocytosis of N. meningitidis W135 opsonized in sera with added properdin/the phagocytosis of meningococci opsonized in sera without added properdin. The C3 deposition ratios onto N. meningitidis W135 representing the C3 deposition in sera with/without properdin added, were correlated with the phagocytosis ratios. Statgraph version 3.0 was used for calculations.

RESULTS

LCCD individuals

The combined FcγRIIa-R/R131 and FcγRIIIb-NA2/2 phenotype was found in six out of eight LCCD individuals with meningococcal disease, as determined by FACS analysis, and in only one (26-year-old) LCCD male of the seven LCCD relatives without meningococcal disease (Table 1). Other combinations of FcγR allotypes were observed in only two persons with, and six persons without meningococcal disease. Statistical analyses revealed a significant association between the combined FcγRIIa-R/R131 and FcγRIIIb-NA2/2 phenotype, and previous meningococcal disease (Fisher's test P = 0·036; OR 13·9; 95% confidence interval (CI) 1·2–478). Distribution of FcγR allotypes was associated neither with the recurrence of meningococcal disease, nor with the age of the subjects at the time of their first disease episode. The only case of chronic meningococcaemia with serogroup B4:P1.14,15 occurred in the individual with the FcγRIIa-R/R131 and IIIb-NA1/2 phenotype (Table 1).

Table 1.

Distribution of FcγR allotypes in members of four families with late component of complement deficiency (LCCD) and five families with properdin deficiency

Number of
C6- or C8-deficient individuals (n = 15) Properdin-deficient individuals (n = 15)
FcγR allotypes
With NmD* Without NmD† With NmD Without NmD
FcγRIIa FcγRIIIb (n = 8) (n = 7) (n = 7) (n = 8)
IIa-R/R131 IIIb-NA2/2 6 1
IIa-R/R131 IIIb-NA1/2 1 2 1
IIa-R/R131 IIIb-NA1/1 1
IIa-R/H131 IIIb-NA2/2 1 4 2 2
IIa-R/H131 IIIb-NA1/2 1 4
IIa-H/H131 IIIb-NA2/2 1
IIa-H/H131 IIIb-NA1/2 1 2
Open in a new tab
*

Previous Neisseria meningitidis disease (NmD) episode(s).

†

Medical history without disease episode suspect for NmD.

Properdin-deficient individuals

Because both genomic DNA and serum were available, allotyping of FcγRIIa was performed by PCR and of FcγRIIIb by RIA. FcγR allotypes were determined in 15 properdin-deficient persons, of whom seven had experienced meningococcal disease. No FcγR phenotype among properdin-deficient persons was significantly associated with the occurrence of meningococcal disease (Table 1).

Role of FcγRIIa allotypes in PMN phagocytosis of opsonized N. meningitidis W135

PMN from two healthy volunteers, homozygous for the FcγRIIa allotypes H131 or R131 and matched for their FcγRIIIb allotypes, were used as phagocytic effectors. Phagocytosis of IgG2-opsonized N. meningitidis W135 was consistently higher with PMN from FcγRIIa-H/H131 volunteer than from FcγRIIa-R/R131 volunteer (Fig. 1). In contrast, IgG1-opsonized meningococci were phagocytozed at the same level by PMN of both phenotypes. Overall, levels of N. meningitidis W135 phagocytosis were higher in the presence than in the absence of 2% complement.

Fig. 1.

Fig. 1

Open in a new tab

Phagocytosis of IgG2- (a) and IgG1 (b)-opsonized Neisseria meningitidis W135 in the presence (+ C) or absence (− C) of an exogenous source of complement by polymorphonuclear cells (PMN) homozygous for the FcγRIIa-H131 or FcγRIIa-R131 allotypes. PMN were obtained from two volunteers matched for their FcγRIIIb allotypes. The data represent results from two independent experiments.

Addition of purified properdin to sera and subsequent C3 deposition on N. meningitidis W135

We tested the effect of properdin supplementation on C3 deposition of N. meningitidis W135 in sera of eight properdin-deficient patients (without previous meningococcal disease), 10 properdin-deficient persons (with previous meningococcal disease), six LCCD persons (without previous meningococcal disease), and 20 relatives as controls (no meningococcal disease, normal complement). Following addition of properdin, C3 deposition on N. meningitidis W135 increased in all sera, reflected by the ratios between sera with/without addition of properdin (which was always >1; Fig. 2). Addition of properdin to properdin-deficient sera of persons without meningococcal disease resulted in significantly increased C3 deposition, compared with the sera of the properdin-deficient persons with previous meningococcal disease (Mann–Whitney test; Z = 2·5, P = 0·01), or relatives as controls (Mann–Whitney test; Z = 3·6, P = 0·0003) (Fig. 2). Comparing both properdin-deficient sera and sera of control relatives without addition of properdin, C3 deposition did not differ significantly (Mann–Whitney test; Z = 0·5, P = 0·57). Importantly, no increased IgG deposition was observed in sera to which properdin was added, as measured by ELISA. In sera from control relatives, C3 deposition upon properdin addition was significantly increased (Mann–Whitney test; Z = 2·9, P = 0·004) compared with LCCD sera (Fig. 2). The ratios of C3 deposition after properdin addition in sera from controls, and properdin-deficient patients with previous meningococcal disease were not significantly different (P = 0·6). To test whether the increase in C3 deposition upon supplementation with properdin (of 1% and 10% properdin-deficient sera) onto meningococci was classical pathway-mediated we performed the assay in the presence of 10 mm MgEGTA (classical pathway of complement activation blocked). No increase in C3 deposition occurred (Fig. 2). Blocking complement activation completely by the presence of 10 mm EDTA in a properdin-deficient and a complement normal serum, no C3 deposition was detectable, as in C3-deficient sera. Finally, C3 deposition onto N. meningitidis W135 did not occur in agammaglobulinaemic serum (with/without added properdin), showing complement activation to be antibody-dependent.

Fig. 2.

Fig. 2

Open in a new tab

Enhancement of C3 deposition onto Neisseria meningitidis W135 upon properdin addition to various sera during opsonization. The meningococci were incubated with sera of either eight properdin-deficient persons without previous meningococcal disease (– NmD), or 10 properdin-deficient persons with previous NmD (+ NmD), or 20 controls or six late complement component deficiency (LCCD) persons (all without previous NmD) in the presence or absence of 2·5 μg/ml purified properdin. The increase in C3 deposition onto meningococci is presented as ratio of the OD450nm of sera reconstituted with purified properdin relative to that of unreconstituted sera. The differences between the group of properdin-deficient persons without previous NmD and the properdin-deficient persons with previous NmD and controls were significant (P = 0·01, and P = 0·0003, respectively) as well as the differences between the LCCD group and controls (P = 0·004). Blocking the classical complement pathway by addition of MgEGTA during the incubation abolished the enhancing effect of properdin supplementation. Symbols represent the mean of duplicate measurements from serum of individual persons within one experiment; means of groups are indicated by horizontal bars.

Role of C3 deposition in phagocytosis of opsonized N. meningitidis W135

The impact of the properdin-induced C3 deposition onto N. meningitidis W135 on PMN phagocytosis was assessed in the phagocytosis assay using properdin-deficient sera of 13 properdin-deficient individuals (eight with previous meningococcal disease, five without previous meningococcal disease) for opsonization of FITC-labelled meningococci W135. Sera were used either directly, or upon reconstitution with 10 μg/ml purified properdin. In all 13 patients the percentage of FITC-labelled PMN increased significantly (Mann–Whitney test; Z = 3·3, P = 0·001) upon addition of properdin (Fig. 3). The effect was more pronounced in patients without meningococcal disease (Fig. 3). Notably, upon reconstitution of the properdin-deficient sera, the increase in C3 deposition in the ELISA assay correlated with enhancement of phagocytosis (r = 0·66, P = 0·01). In all assays PMN phagocytosis was complement- and antibody-dependent.

Fig. 3.

Fig. 3

Open in a new tab

Effect of properdin reconstitution on Neisseria meningitidis W135 phagocytosis by polymorphonuclear cells (PMN). Sera of eight properdin-deficient persons without previous meningococcal disease (− NmD) and five properdin-deficient patients with previous NmD due to meningococci W135 (+ NmD W135) were individually used to opsonize FITC-labelled meningococci W135. Sera were used as such (properdin −) or upon reconstitution with 10 μg/ml purified properdin (properdin +). The percentages of PMN phagocytozing opsonized N. meningitidis W135 are represented for individual sera after incubation for 25 min at 37°C. Sera of one individual are linked. The statistical analysis (Mann–Whitney test) was performed on paired sera from all 13 patients. The subgroups (with and without meningococcal disease) were not analysed separately.

Role of CR3 (CD11b/CD18) in phagocytosis

In order to evaluate the significance of the presence of properdin and, thus, C3 deposition onto meningococci (Fig. 2) for PMN phagocytic activity against meningococci, we tested the role of the PMN C3 receptor CR3 in phagocytosis of the opsonized meningococcus W135 strain. Blockade of PMN CR3 by IgM anti-CR3 MoAb B2.12 (CLB; an antibody isotype not interfering with FcγRs [8]) resulted in a reduced uptake by PMN of the N. meningitidis W135 opsonized by the properdin serum alone (Fig. 4, −properdin). However, a greater reduction was seen upon addition of properdin (Fig. 4, +properdin) during opsonization. In control experiments, CR3 blockade had no effect on phagocytosis of unopsonized or antibody-only-opsonized bacteria (data not shown).

Fig. 4.

Fig. 4

Open in a new tab

Role of complement receptor 3 (CD11b/CD18) interaction in polymorphonuclear cell (PMN) phagocytosis of opsonized FITC-labelled Neisseria meningitidis W135. Meningococci were opsonized with sera of five properdin-deficient persons, either used directly (– properdin) or upon reconstitution with purified properdin (+ properdin). PMN phagocytosis was performed in the presence (PMN + anti-CR3; ▪) or absence (PMN control; □) of a CR3 blocking antibody (MoAb B2.12), and measured after incubation for 30 min at 37°C. Range and mean are given. Results represent duplicate data.

DISCUSSION

The incidence of meningococcal disease is highest in early childhood and decreases gradually until the age of 5–10 years, when protective antibodies to meningococci develop. Individuals with a defect of the complement system are at an increased risk of developing meningococcal disease, especially after the age of 5 years when the incidence and circulation of nasopharyngeal meningococcal carriership increases [1,2,6]. It is assumed that anti-meningococcal antibodies are then present and that the development of meningococcal disease depends on the effectiveness of antibody-mediated bactericidal activity and antibody-mediated phagocytic activity [4]. IgG2 subclasses dominate in the immune response to encapsulated bacteria and polymorphisms of FcγRIIa affect the susceptibility to infections considerably [29]. The aim of this study was to elucidate the role of C3 and FcγRIIa allotypes in the immune defence against meningococcal disease in complement-deficient individuals in more detail.

Our results indicate that certain FcγRIIa allotypes are associated with the development of meningococcal disease in LCCD patients. We typed 10 out of 15 LCCD relatives (i.e. 60% of four LCCD families studied) as FcγRIIa-R/R131 and 12 out of 15 as FcγRIIIb-NA2/2 (80%). The high percentages of LCCD persons homozygous for either FcγRIIa-R131 or FcγRIIIb-NA2/2 may be due to the restricted number of families studied. However, a significant association was noted between the combined FcγRIIa-R/R131 and FcγIIIb-NA2/2 phenotypes, and previous meningococcal disease. In the Dutch (Caucasian) population, the frequency of FcγRIIa-H/H131, IIa-H/R131, and IIa-R/R131 is estimated to be 29%, 48% and 23%, respectively [24]. For FcγRIIIb-NA1/1, IIIb-NA1/2, and IIIb-NA2/2 the frequencies are 17·4%, 52·2% and 30·4%, respectively [30]. Our results are in agreement with the study of Platonov, who found an association of IIa-R/R131 and IIa-R/H131 allotypes with the development of meningococcal disease in 29 LCCD patients above the age of 10 years [31]. Interestingly, he found that Russian LCCD patients homozygous for IIa-H/H131 developed less severe meningococcal disease above the age of 10 years when compared with heterozygous IIa-R/H131 or homozygous IIa-R/R131 [31]. The association between FcγRIIa-R/R131 and severe meningococcal disease is consistent with data from two recent studies showing a significantly higher frequency of the FcγRIIa-R/R131 phenotype among children (without complement deficiency) suffering from fulminant meningococcal shock or severe meningococcal disease [9,32]. The numbers of patients and disease episodes included in the study we present are too small to draw conclusions on the severity of disease, age distribution or recurrent meningococcal disease.

In vitro data consequently show that optimal phagocytosis of meningococci is IgG antibody subclass- and complement-dependent [13,28,32]. Additionally, we observed a functional difference between the two FcγRIIa polymorphic forms affecting the IgG2-mediated interaction between PMN and meningococcus W135. This finding confirms previous reports demonstrating poor phagocytosis of IgG2-opsonized bacteria by FcγRIIa-R/R131-PMN, compared with IgG1 opsonized bacteria [10,28]. The relevant anti-meningococcal IgG subclass for defence against meningococcal disease is not known, albeit that our results are consistent with a significant role for IgG2 antibodies in phagocytosis of meningococci.

The absence of MAC formation in LCCD individuals may be relevant to the amount of C3 deposited on meningococci during opsonization. MAC formation enhances decay of the alternative pathway C3 convertase (C3bBb/P). Properdin functionally counterbalances this C3 deposition limiting effect of the MAC formation. The observed low increase in C3 deposition upon properdin addition to LCCD sera (Fig. 2) is consistent with this function of the MAC, and indicates that C3 deposition onto meningococci in LCCD patients is not curtailed by a relative defect of properdin.

We found no association between FcγRIIa allotypes and previous meningococcal disease in properdin-deficient patients and concluded that FcγRIIa allotypes are not associated with an increased risk of meningococcal disease in these patients. However, these data need to be confirmed in a study encompassing more properdin-deficient patients, since this study included only 15 patients. We hypothesized that impaired C3 deposition onto meningococci in properdin-deficient serum may underlie inefficacious phagocytosis. C3 deposition to opsonized bacterial surfaces is the result of complement-activating capacity of these surfaces, the presence of antibodies (and other complement-activating proteins/lectins) binding to bacteria, and the attraction of down-regulators of complement activation (factor H, MAC) by bacterial surfaces. To circumvent the antibody level of sera as a variable, we measured in each serum the increase in C3 deposition upon addition of purified properdin during opsonization. Our hypothesis was supported by these experiments showing that addition of properdin to properdin-deficient (and normal) sera increased C3 deposition onto the surface of N. meningitidis W135 in contrast with agammaglobulinaemic serum supplemented with properdin. This identifies properdin as a limiting factor for C3 deposition under conditions in which alternative pathway activation does not occur. Our observation of low/absent alternative pathway activation by meningococci is in agreement with the findings of Fredlund et al. demonstrating alternative pathway of complement activation by meningococci W135 in post-vaccination sera with very high levels of anti-capsular W135 antibodies only [12].

The increase of C3 deposition on N. meningitidis by properdin added to sera from properdin-deficient persons without previous meningococcal disease, compared with this effect of properdin supplementation to sera of persons that experienced meningococcal disease, suggests that anti-meningococcal antibodies may allow C3 deposition independent of properdin. The increased phagocytosis of N. meningitidis W135 after opsonization with reconstituted properdin-deficient serum was highly dependent on C3 deposition. This was supported by the effect of blocking complement receptor 3, the main receptor for C3-mediated phagocytosis on PMN [33]. The relevance of C3 deposition by properdin is further supported by the correlation found between C3 deposition and phagocytic activity induced by (added) properdin.

In conclusion, we observed the combined FcγRIIa-R/R131 and FcγRIIIb-NA2/2 phenotype to be over-represented in LCCD patients who experienced meningococcal disease. Since vaccination in such patients has been recommended [1,34], the efficacy of such a preventive approach may depend on the interaction of their PMN FcγR allotypes with specific IgG antibody subclasses. Evaluation of the PMN allotypes and IgG subclasses may be valuable as a predictive factor for vaccination protective effects. A critical role of properdin in classical pathway-induced C3 deposition onto meningococci was found. The increased risk of properdin-deficient persons for meningococcal disease warrants the search for factors interfering with C3 deposition onto meningococci in these individuals. Since phagocytosis is of importance in the defence against meningococci, C3 deposition may be relevant also in complement-normal patients as a risk factor for the onset of meningococcal disease.

Acknowledgments

The authors thank all patients, general practitioners and medical specialists for their co-operation, and Nomdo Westerdaal for excellent technical support. M.d.H. was financially supported by the Netherlands Foundation for Scientific Research (NWO), grant number 900-512-092. FcγR typing was financially supported by INTAS, contract number 97-01-0108.

REFERENCES

  • 1.Figueroa JE, Densen P. Infectious disease associated with complement deficiencies. Clin Microbiol Rev. 1991;4:359–95. doi: 10.1128/cmr.4.3.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Fijen CAP, Kuijper EJ, Hannema AJ, Sjöholm AG, van Putten JPM. Complement deficiencies in patients over ten years old with meningococcal disease due to uncommon serogroups. Lancet. 1989;ii:585–8. doi: 10.1016/s0140-6736(89)90712-5. [DOI] [PubMed] [Google Scholar]
  • 3.Orren A, Caugant DA, Fijen CAP, Dankert J, van Schalkwyk EJ, Poolman JT, Coetzee GJ. Characterization of strains of Neisseria meningitidis recovered from complement-normal and complement-deficient patients in the Cape, South Africa. J Clin Microbiol. 1994;32:2185–91. doi: 10.1128/jcm.32.9.2185-2191.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Goldschneider I, Gotschlich EC, Artenstein MS. Human immunity to the meningococcus. I. The role of humoral antibodies. J Exp Med. 1969;129:1307–48. doi: 10.1084/jem.129.6.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Würzner R, Orren A, Lachmann PJ. Inherited deficiencies of the terminal components of human complement. Immunodef Rev. 1992;3:123–47. [PubMed] [Google Scholar]
  • 6.Sjöholm AG. Inherited complement deficiency states: implications for immunity and immunological disease. Acta Pathol Microbiol Immunol Scand. 1990;98:861–74. doi: 10.1111/j.1699-0463.1990.tb05008.x. [DOI] [PubMed] [Google Scholar]
  • 7.Griffin FM. Opsonisation, phagocytosis and intracellular microbial killing. In: Rother K, Till GO, editors. The complement system. Berlin: Springer Verlag; 1988. pp. 395–418. [Google Scholar]
  • 8.Deo YM, Graziano RF, Repp R, van de Winkel JG. Clinical significance of IgG Fc receptors and Fc gamma R-directed immunotherapies. Immunol Today. 1997;18:127–35. doi: 10.1016/s0167-5699(97)01007-4. [DOI] [PubMed] [Google Scholar]
  • 9.Bredius RGM, Derkx BHF, Fijen CAP, de Wit TPM, de Haas M, Weening RS, van de Winkel JGJ, Out TA. FcγRIIa (CD32) polymorphism in fulminant meningococcal septic shock in children. J Infect Dis. 1994;170:848–53. doi: 10.1093/infdis/170.4.848. [DOI] [PubMed] [Google Scholar]
  • 10.Bredius RGM, Fijen CAP, de Haas M, Kuijper EJ, Weening RS, van de Winkel JGJ, Out TA. Role of neutrophil FcγRIIa (CD32) and FcγRIIIb (CD16) polymorphic forms in phagocytosis of human IgG1- and IgG3-opsonized bacteria and erythrocytes. Immunology. 1994;83:624–30. [PMC free article] [PubMed] [Google Scholar]
  • 11.Edberg JC, Kimberley RP. Modulation of Fcγ and complement receptor function by the glycosyl-phosphotidylinositol-anchored form of FcγRIII. J Immunol. 1994;152:5826–35. [PubMed] [Google Scholar]
  • 12.Edwards KM, Gewurz H, Sokalski SJ, Chan L, Lint TF. Antibody and complement in the stimulation of neutrophil chemiluminescence by Neisseria meningitidis: studies in a patient with complete deficiency of C7. J Infect Dis. 1982;145:65–71. doi: 10.1093/infdis/145.1.65. [DOI] [PubMed] [Google Scholar]
  • 13.Zhou M, Brown EJ. CR3(Mac-1,αMß2, CD11b/CD18) and FcγRIII cooperate in generation of a neutrophil respiratory burst: requirement for FcγRII and tyrosine phosphorylation. J Cell Biol. 1994;125:1407–16. doi: 10.1083/jcb.125.6.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Mak LW, Lachman PJ, Majewski J. The activation of the C3b feedback cycle with human complement components. Clin Exp Immunol. 1977;30:200–21. [PMC free article] [PubMed] [Google Scholar]
  • 15.Maves KK, Weiler JM. Properdin: approaching four decades of research. Immunol Res. 1993;12:233–43. doi: 10.1007/BF02918255. [DOI] [PubMed] [Google Scholar]
  • 16.Sim RB, Kölbe K, McAleer MA, Dominguez O, Dee VM. Genetics and deficiencies of the soluble regulatory proteins of the complement system. Intern Rev Immunol. 1993;10:65–86. doi: 10.3109/08830189309051172. [DOI] [PubMed] [Google Scholar]
  • 17.Kuypers TW, Eckmann CM, Weening RS, Roos D. A rapid turbidometric assay of phagocytosis and serum opsonizing capacity. J Immunol Methods. 1989;124:85–94. doi: 10.1016/0022-1759(89)90189-0. [DOI] [PubMed] [Google Scholar]
  • 18.Scholten RJPM, Bijlmer HA, Poolman JT, Kuipers B, van Alphen L, Dankert J, Valkenburg HA. Meningococcal disease in the Netherlands, 1958–90: a steady increase in the incidence since 1982 partially caused by new serotypes and subtypes of Neisseria meningitidis. Clin Infect Dis. 1993;16:237–46. doi: 10.1093/clind/16.2.237. [DOI] [PubMed] [Google Scholar]
  • 19.Fearon DT, Austen F, Ruddy S. Properdin factor D. J Exp Med. 1974;140:426–36. doi: 10.1084/jem.140.2.426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Bredius RGM, Driedijk PC, Schouten MFJ, Weening RS, Out TA. Complement activation by polyclonal immunoglobulin G1 and G2 antibodies against Staphylococcus aureus, Haemophilus influenzae type b and tetanus toxoid. Infect Immun. 1992;60:4838–47. doi: 10.1128/iai.60.11.4838-4847.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Mannens M, Slater RM, Heyting C, Geurts van Kessel A, Goedde-Salz E, Frants RR, van Ommen GJB, Pearson PL. Regional localization of DNA probes on the short arm of chromosome 11 using aniridia-Wilms' tumor-associated deletions. Hum Genet. 1987;75:180–7. doi: 10.1007/BF00591083. [DOI] [PubMed] [Google Scholar]
  • 22.Carlsson LE, Santoso S, Baurichter G, et al. Heparin-induced thrombocytopenia: new insights into the impact of the FcgammaRIIa-R-H131 polymorphism. Blood. 1998;92:1526–31. [PubMed] [Google Scholar]
  • 23.Huizinga TWJ, de Haas M, Kleijer M, Nuijens JH, Roos D, von dem Borne AEGKR. Soluble Fcγ receptor III (CD16) in human plasma originates from release by neutrophils. J Clin Invest. 1990;86:416–23. doi: 10.1172/JCI114727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Sanders L, van de Winkel JGJ, Rijkers GT, Voorhorst-Ogink MM, de Haas M, Capel PJA, Zegers BJM. FcγRIIa (CD32) heterogeneity in patients with recurrent bacterial respiratory tract infections. J Infect Dis. 1994;170:854–61. doi: 10.1093/infdis/170.4.854. [DOI] [PubMed] [Google Scholar]
  • 25.Parren PWHI, Warmerdam PAM, Boeije LCM, Capel PJA, van de Winkel JGJ, Aarden LA. Characterization of IgG FcR-mediated proliferation of human T cells induced by mouse and human anti-CD3 monoclonal antibodies: identification of a functional polymorphism to human IgG2 anti-CD3. J Immunol. 1991;148:695–701. [PubMed] [Google Scholar]
  • 26.Huizinga TWJ, Kleijer M, von Roos D, dem Borne AEGKR. Differences between FcRIII of human neutrophils and human K/NK lymphocytes in relation to the NA antigen system. In: Knapp W, Gilks WR, Rieber EP, Schmidt RE, Stein H, Von Dem Borne AEGKR, editors. Leukocyte typing IV. 4. Oxford: Oxford University Press; 1989. pp. 582–5. [Google Scholar]
  • 27.Geelen SPM, Fleer A, Bezemer AC, Gerards LJ, Rijkers GT, Verhoef J. Deficiencies in opsonic defense to pneumococci in the human newborn despite adequate levels of complement and specific IgG antibodies. Pediatr Res. 1990;27:514–8. doi: 10.1203/00006450-199005000-00020. [DOI] [PubMed] [Google Scholar]
  • 28.Bredius RGM, de Vries CEE, Troelstra A, van Alphen L, Weening RS, van de Winkel JGJ, Out TA. Phagocytosis of Staphylococcus aureus and Haemophilus influenzae type b opsonized with polyclonal human IgG1 and IgG2 antibodies: functional hFcγRIIa polymorphism to IgG2. J Immunol. 1993;151:1463–72. [PubMed] [Google Scholar]
  • 29.Siber GR, Schur PH, Aisenberg AC, Wietzman SA, Shiffman G. Correlation between IgG2 serum concentrations and the antibody response to bacterial polysaccharide antigens. N Eng J Med. 1980;303:178–82. doi: 10.1056/NEJM198007243030402. [DOI] [PubMed] [Google Scholar]
  • 30.Fromont P, Bettaieb A, Skouri H, Floch C, Poulet E, Duedari N, Bierling P. Frequency of the polymorphonuclear neutrophil Fcgamma receptor III deficiency in the French population and its involvement in the development of neonatal alloimmune neutropenia. Blood. 1992;79:2131–4. [PubMed] [Google Scholar]
  • 31.Platonov AE, Kuijper EJ, Vershinina IV, et al. Meningococcal disease and polymorphism of FcγRIIa (CD32) in late complement component-deficient individuals. Clin Exp Immunol. 1998;111:97–101. doi: 10.1046/j.1365-2249.1998.00484.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Platonov AE, Shipulin GA, Vershinina IV, Dankert J, van de Winkel J, Kuijper EJ. Association of human FcgammaRIIa (CD32) polymorphism with susceptibility to and severity of meningococcal disease. Clin Infect Dis. 1998;27:746–50. doi: 10.1086/514935. [DOI] [PubMed] [Google Scholar]
  • 33.Fredlund H, Sjöholm AG, Selander B, Holmström E, Olcén P, Danielson D. Serum bactericidal activity and induction of chemiluminescence of polymorphonuclear leukocytes: complement activation pathway requirements in defence against Neisseria meningitidis. Int Arch Allergy Immunol. 1993;100:135–43. doi: 10.1159/000236400. [DOI] [PubMed] [Google Scholar]
  • 34.Brown EJ. Complement receptors and phagocytosis. Curr Opin Immunol. 1991;3:76–82. doi: 10.1016/0952-7915(91)90081-b. [DOI] [PubMed] [Google Scholar]
  • 35.Fijen CAP, Kuijper EJ, Drogari-Apiranthitou M, van Leeuwen Y, Daha MR, Dankert J. Protection against meningococcal serogroup ACYW135 disease in complement-deficient individuals vaccinated with the tetravalent meningococcal capsular polysaccharide vaccine. Clin Exp Immunol. 1998;114:362–9. doi: 10.1046/j.1365-2249.1998.00738.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES