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. 2004 Nov;138(2):271–277. doi: 10.1111/j.1365-2249.2004.02603.x

Neutrophil responses to CRP are not dependent on polymorphism of human FcγRIIA (R131H)

J A RODRÍGUEZ *, K B BODMAN-SMITH *, J G RAYNES *
PMCID: PMC1809209  PMID: 15498037

Abstract

IgG2a mediated in vitro phagocytosis is less effective for individuals homozygous for FcγRIIaR131 allele and such individuals are also more susceptible to certain infections. It has been reported that CRP binds to FcγRIIaR131 but not FcγRIIaH131 and since FcγRIIa is also a major Fc receptor on neutrophils it would be expected that normal healthy donors who did not have at least one copy of FcγRIIaR131 would not respond to CRP. We examined responses reported to be dependent on FcγRIIa but no difference between groups was observed in CRP mediated phagocytosis of S. pneumoniae, reactive oxygen production, or IL-8 synthesis. This suggests that either neutrophil receptors other than FcγRIIa are responsible for CRP mediated responses or differences in CRP binding to the forms of FcγRIIa are comparatively minor.

Keywords: CRP, Fc receptor, neutrophils, Streptococcus pneumoniae, reactive oxygen, phagocytosis, interleukin 8

INTRODUCTION

CRP is a pentraxin of 110 kD composed of five identical, noncovalently associated subunits, arranged in pentameric radial symmetry [1]. Originally described as a serum precipitin for the C-polysaccharide found in pneumococcal cell wall, the binding is related to the presence of phosphorylcholine (PCh). Individual subunits can bind with moderate affinity to such ligands; multimeric interactions giving rise to higher avidities. CRP binds in a calcium-dependent way to a widely distributed group of ligands including not only PCh, but also polyelectrolytes [2] and certain phosphorylated polysaccharides [3]. The main interaction between PCh and CRP occurs between the phosphate group of PCh and calcium molecules of CRP in a shallow pocket of each subunit [4]. CRP also binds to C1q leading to activation of the classical complement cascade which requires binding to an appropriate multivalent ligand [5]. CRP binds to apoptotic cells [6] through binding of PCh [7]. In experimental and disease-caused lesions CRP has been localized to neutrophils [8,9].

CRP is an acute phase protein with an upper limit of normal of about 10 µg/ml although concentrations can increase by up to 1000 times upon initiation of severe systemic inflammation [10]. Following early studies that showed IgG in various forms inhibited CRP binding to neutrophils to various extents, monomeric CRP was shown to bind to FcγRI transfected cos cells [11] and subsequently to bind directly to immobilized receptor [12]. Functional responses consistent with interaction with FcγRI include CRP mediated phagocytosis by cos cells transfected with this receptor and gamma chain [12]. Interaction with the low affinity IgG receptor FcγRIIa was shown to only the allelic form that contains arginine at position 131 [13]. Although this data is controversial [14] CRP binding to FcγRIIa was confirmed in our lab by demonstrating binding and phagocytosis of CRP-opsonized PCh-labelled SRBC to FcγRIIaR131 transfected-Cos cells, a phenomenon that can be inhibited with F(ab′)2 anti-CRP [15]. Signalling through CRP binding to FcγRIIa including phosphorylation of the receptor has been reported for HL60 cells which express R allele [16].

FcγRIIa and FcγRIIIb are the Fc receptors found predominantly on resting neutrophils and the FcγRI present at low levels on resting neutrophils is up-regulated on activation. FcγRI and FcγRIIIb both signal via ITAMs present in associated γ chain although recent evidence is presented that FcγRI can also recruit FcγRIIa [15,17,18]. The FcγRIIa polymorphism is known to influence the binding of IgG2a which is generated for protection against encapsulated bacteria. The H allele binds IgG2a more strongly than the R allelle [19,20] and this has been shown to be correlated with in vitro phagocytosis efficiency [21]. Individuals homozygous for R131 are more susceptible to bacterial infections such as Neisseria meningitidis and S. pneumoniae [22–24]

CRP has been reported to directly instigate a number of different neutrophil responses which include the induction of the respiratory burst [25] although not all responses at all CRP concentrations are activatory. Recently indeed the number of reports of effects of CRP in various cell types has increased greatly but most do little to provide any mechanistic understanding of the response. We sought to confirm the ability of CRP to cause responses through interaction with FcγRIIa R131 but not H131 in neutrophils because of their high level of FcγRIIa expression. In deciding what conditions to use for investigation of such interactions we chose to exclude serum because of potential effects of IgG or complement. The interactions of CRP with FcγRIIa receptors are likely to be relatively low affinity as for IgG therefore CRP was investigated not only as a free soluble protein but also in combination with S. pneumoniae to which CRP binds. Such surface complexed CRP would allow interaction with lower affinity receptors. S. pneumoniae was also a relevant choice for stimulus because as stated above this pathogen is associated with increased incidence of infection.

MATERIALS AND METHODS

C-reactive protein

CRP was extracted from human acute serum using an affinity chromatography protocol [26]. CRP was quantified assuming OD280 1 mg/ml = 1·98. The purity was assessed by SDS-PAGE and absence of significant IgG contamination was confirmed by Western blotting with antihuman IgG. Human recombinant CRP (rCRP) [27], was kindly provided by Dr Toshio Tanaka (Nagahama Institute for Biochemical Science, Oriental Yeast Co., Japan). The preparation contained 9 ng of LPS per 1 mg of rCRP.

Isolation of human neutrophils

Human neutrophils were isolated according to the method described by Gotnrie et al. [28]. Briefly, ACD anticoagulated blood was transferred to a tube containing dextran solution: 3% dextran T 500 (Pharmacia, Milton Keynes, UK) + 0·9% (w/v) NaCl in a ratio 1 : 2 (dextran: blood). Then, the leucocyte enriched supernatant was carefully layered onto 15 ml of Histopaque 1·070 and centrifuged for 30 min at 400 g at 4°C to remove PBMCs. Distilled water for 30 s was used to eliminate contaminant red blood cells. Neutrophils were counted and resuspended in CD-hybridoma medium (Gibco, Paisley, UK). More that 95% were confirmed as polymorphonuclear cells and viability was more that 97%.

PCR to determine FcγRIIA polymorphism

PCR was performed to amplify specific FcγRIIA sequence of DNA based on a method described elsewhere [29]. A PCR mix was prepared containing 200 µm of each dNTP (Bioline, London, UK), 0·5 U of Taq polymerase (Bioline), 0·125 µm of primers for human growth hormone gene as an internal control (CAGTGC CTTCCCAACCATTCCCTTA and ATCCACTCACGGATT TCTGTTGTGTTTC), 0·5 µm common antisense primer from an intron shared by the sequences for FcγRIIA, FcγRIIB and FcγRIIC CAATTTTGCTGCTATGGGC, and 0·5 µm of H131-specific sense primer ATCCCAGAAATTCTCCCA (Hmix) or R131-specific sense primer ATCCCAGAAATTCTCCCG (Rmix) in 10X buffer and 15 mm MgCl2 (Bioline). All primers were from Sigma-Genosys Ltd (Cambridgeshire, UK). 22·5 µl of either Hmix or Rmix were used in combination with 2·5 µl of DNA sample. PCR was performed on a thermal cycler (Omnigen, Hybaid Ltd, UK) as follows: 10 min at 95°C, 10 cycles of 1 minute at 96°C, 2 min at 54°C and 1 minute at 72°C; thereafter, to enhance the sensitivity, 22 cycles of 1 minute 95°C, 2 min at 54°C, and 1 minute at 72°C and final extension step for 10 min at 72°C. The PCR amplification products and a 1kb DNA ladder (Gibco/BRL) were separated on 1·5% agarose and visualized using ethidium bromide.

Culture and FITC labelling of Streptococcus pneumoniae

Encapsulated S. pneumoniae serotype 3 was obtained from clinical isolates kindly provided by Prof S. Gillespie, Department Microbiology, Royal Free Hospital, London, UK). Approximate concentrations were calculated on the basis that OD600 of 1·0 is equivalent to 5 × 108 organisms/ml. S. pneumoniae was incubated with 3% (v/v) formaldehyde at 37°C for 1 h, then washed twice with PBS at 2000 g for 5 min and then FITC-labelled in 1 ml of 0·5 mg/ml FITC (Sigma, Poole, UK) for 1 h at 37°C and washed extensively with PBS.

Determination of IL-8

Immulon 2HB 96 well ELISA plates (Dynex technologies, Southampton, UK) were coated with 50 µl of 2 µg/ml mouse IgG2b antihuman IL-8 monoclonal antibody (BD Biosciences, Oxford, UK) diluted in 0·1 m Na2HPO4 pH 9·0 and incubated overnight at 4°C. Plates were blocked with PBST 10% (v/v) FCS for 1 h at RT. Standard curves were performed in duplicates by adding 50 µl of rIL-8 at 2 ng/ml to 31 pg/ml (National Institute for Biological Standards and Control, South Mimms, UK), samples were also added in duplicates and left overnight at 4°C. After washes, 50 µl of biotinylated mouse anti-IL-8 monoclonal antibody (BD Biosciences, Oxford, UK) was added and left for 2 h at RT. After washing 50 µl of HRP-Streptavidin (Dako SA, Glostrup, Denmark) was added and following washing TMB was used as a substrate using standard detection procedures.

Activity of NADPH oxidase in neutrophils

Oxidation of dihydrorhodamine 123

This procedure is based on a previously described technique for NADPH oxidase analysis using human neutrophils by FACS [30]. Isolated neutrophils were resuspended at 2 × 106 cells/ml in CD hybridoma medium (Gibco) then cultured overnight with or without IFNγ (100 IU/ml). 100 µl of cells were incubated in plastic Falcon tubes with 100 µl of stimuli as indicated or 1 µm (PMA) diluted in PBS for 20 min at 37°C. Then, 50 µl of 100 µm of Dihydrorhodamine 123 (DHR; Calbiochem, Nottingham, UK) PBS was added and incubated for a further 10 min. The reaction was stopped with 200 µl PBS-0·02% (w/v) EDTA and washed once with PBS. The cells were fixed with 200 µl of cold 1% (w/v) PFA at 4°C and stored at 4°C until read. Cells were acquired by flow cytometry (FACS SCALIBUR, Becton-Dickinson, Oxford, UK) and analysed by Cell Quest software. At least 30000 events were counted per sample.

Superoxide production by neutrophils

0·5 × 106 neutrophils were incubated with stimuli or 5 µm PMA with or without 300 U/ml superoxide dismutase (SOD) (Sigma) and 3·75 µm cytochrome c (Sigma) a total volume of 250 µl CD–hybridoma medium. Absorbances were determined each 15 min up to 2 h at 550 nm in a microplate spectrofluorometer reader (Spectra MAX Gemini, Sunnyvale, USA). The amount of reduction of cytochrome c was calculated by subtracting the background OD obtained with SOD, using a molar extinction coefficient for cytochrome c of 21·1 × 10−3m.

Phagocytosis by neutrophils

Isolated neutrophils from both HH and RR donors were resuspended in CD-hybridoma medium at 2 × 106 cells/ml, were incubated with FITC-labelled pneumococcus at a ratio 30 : 1 (bacteria : cells) with or without rCRP at stated concentrations in the presence or absence of 1 µm Cytochalasin D (Sigma) at 37°C for 30 min. In all experiments, phagocytosis was stopped with cold PBS; after two washes with cold PBS at 300 g for 5 min, cells were fixed with 1% (w/v) PFA for 30 min and at least 20 000 events were acquired by Flow cytometry (FACS SCALIBUR, Becton-Dickinson). Phagocytosis was expressed as the difference between the mean fluorescent intensity (MFI) of all gated neutrophils incubated under each condition at 37°C and neutrophils treated similarly but including cytochalasin D.

Statistical analysis

The software GraphPad Prism® 3·03 version was used for statistical analysis using an unpaired Mann–Whitney test.

RESULTS

FcγRIIA polymorphisms were determined using PCR (as shown in Fig. 1) in 34 adult healthy volunteers, 16 of them were heterozygous, whereas 9 were homozygous for R131 and 9 were homozygous for H131. This data therefore followed Hardy–Weinberg's equilibrium.

Fig. 1.

Fig. 1

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Donor polymorphism of FcγRIIA The bigger arrow shows the amplification of human growth hormone (439 bp) as an internal control, whereas the smaller arrow shows amplification of FcγRIIA (253 bp). Lanes 1, 3, 5 show amplification with H131 specific primer and 2,4,6 show amplification with R131 specific primer. Tracks 1 and 2: DNA from donor a; 3 and 4 donor b and 5 and 6 donor c. The gel shows that a is homozygous H; b is homozygous R and c is heterozygous

CRP induces IL-8 production from isolated neutrophils regardless of FcγRIIA H131R polymorphism

IL-8 is one of the major chemokines synthesized by neutrophils as an autocrine way to attract cells to the site of inflammation. Fc receptor cross linking has been reported to induce IL-8 at least in macrophages [31] but to our knowledge no previous studies had examined whether CRP altered its production. We assessed the ability of CRP at 10 µg/ml and 100 µg/ml in the presence or absence of S. pneumoniae (at a ratio of bacteria to neutrophils that gave sub maximal responses) to induce production of IL-8 by neutrophils from HH and RR homozygous donors. S. pneumoniae type 3 induced IL-8 production from both HH and RR donors but was not further increased by the presence of CRP. CRP on its own was able to induce the synthesis of IL-8 in neutrophils from both groups. S. pneumoniae R36A induced lower amounts of IL-8 compared with S. pneumoniae type 3 and CRP increased IL-8 responses in the presence of the organism in the same way as CRP alone (Fig. 2). Again no significant difference in response of RR or HH donors was detected. Data from heterozygous donors gave similar IL-8 levels (data not shown). Intracellular IL-8 in isolated neutrophils measured on a limited number of donors by FACS also demonstrated strong induction by S. pneumoniae strain 3 and an increase in response to CRP alone consistent with data on secreted IL-8 (data not shown). S. pneumoniae increased apoptosis of neutrophils but this was not altered by the presence of CRP nor did CRP alone alter apoptosis (data not shown).

Fig. 2.

Fig. 2

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Effect of S. pneumoniae on IL-8 production by isolated neutrophils. Isolated neutrophils from either FcγRIIa 131HH (□) or 131RR (▴) homozygous individuals were cultivated overnight at 37°C with CRP at 0,10 and 100 µg/ml (a) alone or in the presence of S. pneumoniae types R36A (b) or strain 3 (c). IL-8 concentration in supernatants (pg/ml) was determined by ELISA. Graphs show means ± s.e.m of IL-8 produced by 6 HH and 6 RR donors for (a) and (b) and 4 donors of each type for (c).

CRP induction of NADPH oxidase activity in isolated neutrophils is not altered by FcγRIIA polymorphism

We tested the ability of CRP to activate neutrophils using dihydrorhodamine which is oxidized by intracellular hydrogen peroxide. We analysed DHR oxidation in response to CRP with or without S. pneumoniae in both HH- and RR-donors since FcγRIIa activates reactive oxygen [32]. CRP was used at several concentrations but maximal responses were obtained at 10 µg/ml. In Fig. 3 it can be seen that again the response was independent of the polymorphism. If cells were pretreated overnight with IFNγ there was also no difference in DHR oxidation between homozygous HH and RR donors (data not shown). Recombinant CRP behaved in a similar manner to purified CRP. For instance, in the presence of S. pneumoniae reactive oxygen MFI increased from 34 ± 10 in the absence of rCRP to 78 ± 18 in the presence of 10 µg/ml rCRP for HH donors and 28 ± 6–65 ± 9 for RR donors. The assayed LPS content of CRP had no effect on DHR (data not shown).

Fig. 3.

Fig. 3

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CRP induces oxidation of DHR 123 in isolated neutrophils from both HH and RR donors. Isolated neutrophils (2 × 106/ml) from HH (a, c) and RR (b, d) were incubated overnight with (c, d) or without (a, b) IFNγ (100 IU/ml) and then treated with (Inline graphic) or without (▪) S. pneumoniae (ratio 30 : 1; bacterium: neutrophil) in the presence or absence of 10 µg/ml CRP for 30 min. Oxidation of DHR 123 was determined by FACS. Mean and standard error for fold increase in MFI from 4 different donors are shown.

We also examined secreted free radicals both H2O2 and superoxide release by neutrophils since these have been reported to demonstrate differences in requirement for signalling pathways although both are induced by FcγRIIa [32,33]. No differences were observed (data not shown).

CRP induction of phagocytosis of S. pneumoniae by human neutrophils is identical in HH and RR homozygous donors

Direct (non complement mediated) phagocytosis in response to CRP was investigated because this has been reported to be largely mediated through FcγRIIa in humans [25]. For these experiments we used rCRP which gave similar responses to purified CRP in previous assays. Maximal binding of CRP to S. pneumoniae occurred at approximately 10 µg/ml. At this concentration the CRP induced significant phagocytosis of S. pneumoniae seroype 3. Phagocytosis under each condition was expressed as the difference between the fluorescence without and with cytochalasin D. There was however, no statistical difference between phagocytosis of fluorescently labelled bacteria by neutrophils from HH or RR donors (Fig. 4).

Fig. 4.

Fig. 4

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Phagocytosis of S. pneumoniae is similar in HH and RR individuals. Phagocytosis of S. pneumoniae serotype 3 at a ratio 1 : 30 (cells : bacterium) was performed using 2 × 106 neutrophils/ml over 30 min incubation at 37°C in the presence or absence of CRP. (a) FACS analysis shows neutrophils in the absence of bacteria (filled histogram), presence of fluorescently labelled S. pneumoniae (thick line); bacteria and CRP (dotted line) and bacteria and CRP with cytochalasin D (thin line). (b) Phagocytosis of pneumococci in presence of different amounts of CRP is shown for 4 HH (Inline graphic) and 4 RR (▪) individuals. Uptake of fluorescent pneumococci is expressed as the mean ± s.e.m. of the difference in MFI between sample and Cytochalasin D control. The difference between cytochalasin control with bacteria and no bacteria was less than 20% of the phagocytosis and was not increased in the presence of CRP.

DISCUSSION

Since CRP is synthesized at early stages (days 1–2) following an inflammatory response, its ability to influence neutrophil function could be crucial in the immune response to infection. Although neutrophils were once thought of purely as phagocytic cells, it has been shown that they are very active in the immune response. Pre-treatment of mice with human CRP reduces mortality due to pneumococcal infection serotype 3, an effect that was seen when concentrations reached 9 µg/ml or more [34]. In this model the mechanism by which such protection occurs has been shown to be at least partially through complement [35]. The ability to test CRP actions in a mouse system is problematical because the mouse only expresses FcγRIIb and not FcγRIIa and the mouse does not regulate its CRP concentration in the same way as man and it is unclear if mouse and human CRP behave in similar ways with regard to Fc receptor interaction. In addition to mechanisms that involve complement direct Fc interactions of CRP opsonized organism may be important. Many other microorganisms have PCh on their surfaces and potentially CRP might protect against their infections: Clostridium spp., Lactobacillus spp., Bacillus spp., Haemophilus influenza, Neisseria meningitidis and Pneumocystis carinii [36].

We found that CRP is able to induce IL-8 production by neutrophils cocultured with nonencapsulated type 2 strain R36A S. pneumoniae. Unexpectedly, CRP alone appeared to induce production of IL-8 by neutrophils, which to our knowledge is novel. In these studies to avoid antibody mediated responses we used no serum purely to examine direct CRP specific effects. Under these conditions individual pentamers of CRP would have had the opportunity to interact with receptors and cross link which that might otherwise be prevented by the high levels of monomeric IgG in serum. Indeed in other experiments on DHR responses using whole blood assays CRP alone had little or no effect. Interestingly the presence of the serotype 3 generated a higher IL-8 response when compared with unencapsulated R36A in the absence of CRP, which may be due to capsule or higher production of pneumolysin by serotype 3 which was shown to induce IL-8 production by human neutrophils [37].

Intracellular reactive oxygen in the presence or absence of S. pneumoniae and phagocytosis of organism were increased in the presence of CRP. We have shown that responses to CRP were not restricted to individuals with FcγRIIa R131 nor were they greater in such individuals. It is possible that small differences might be observed if very large numbers of individuals were studied but this would not alter the essential finding that individuals homozygous for FcγRIIa H131 can respond to CRP. Whether FcγRIIa has an important role in phagocytosis in neutrophils is uncertain since neutrophils express FcγRI which can be up-regulated during inflammation as well as FcγRIIIb which are also involved in the responses measured here through γ chain. FcγRIIa is present as a major receptor on neutrophils but the presence of FcγRIIb is unclear. At the simplest level if we assume that CRP does indeed bind to FcγRIIaR131 but not H131 then the implication is that FcγRIIa has little or no importance for neutrophil responses to CRP which may proceed through FcγRI or another as yet undefined receptor. A further implication is that FcγRIIa would also have no role in such responses to IgG. However we see no failure of HH individuals to respond to CRP as was reported previously [13]. The second possibility is therefore that CRP binds preferentially to FcγRIIa R131 but binds both weakly but despite this the recruitment of the receptors in a complex with other receptors and is unaffected by the difference in avidities. The ability of antibodies to inhibit the response may shed light on which of the possibilities is true. It is however, unclear which antibodies might inhibit CRP binding, for instance IV.3 which recognizes FcγRII and inhibits IgG binding was shown to inhibit responses to aggregated CRP but apparently did not CRP inhibit binding to neutrophils [38]. The problem is also confused by likely presence of FcγRIIb on human neutrophils which is at present not readily quantifiable. Finally there are also reports that FcγRII may also adopt different conformation or arrangement in the membrane which altered immune complex binding to this receptor and which may also influence CRP binding [39]. There is thus a requirement for further studies on purified Fc receptors and CRP of the same species to confirm validity of previously reported transfection studies.

Acknowledgments

We would like to thank Universidad Surcolombiana for support of JR. We thank the Wellcome Trust for support of KB. We also thank Carolyn Stanley and the blood donors at LSHTM and Martin Taylor for his help and advice

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