C-Reactive Protein Triggers Calcium Signalling in Human Neutrophilic Granulocytes via FcγRIIa in an Allele-Specific Way

Authors


  • Jens-Gustav Iversen — Deceased.

Correspondence to: V. Aas, Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences, PO Box 4, St. Olavs plass, N-0130 Oslo, Norway. E-mail: vigdis.aas@hioa.no

Abstract

C-reactive protein (CRP) binds to Fcγ-receptors, FcγRIIa (CD32) with high affinity and to FcγRIa (CD64) with low affinity. The binding to CD32 has been shown to be allele specific, that is, it binds to R/R131 but not to H/H131. Little is known about the cooperation of CRP and neutrophilic granulocytes (PMNs) in inflammatory reactions. The purpose of the present study was to examine CRP signalling in human PMNs, and whether this signalling is also allele specific. Cytosolic calcium of PMN was measured in a single-cell digital imaging system. Receptor expression and polymorphism were studied by real-time RT-PCR, flow cytometry and standard PCR. C-reactive protein induced cytosolic calcium signals in PMNs from homozygote R/R131donors, but not in PMNs from heterozygote R/H131 donors. However, after the heterozygote PMNs had been incubated with IFN-γ (100 U/ml) for 2 h, both the proportion of cells responding and the size of the CRP-induced calcium signals increased. IFN-γ increased mRNA expression of CD64 about fivefold and surface protein expression of CD64 about fourfold. The calcium signal elicited by CRP was augmented by PMN adhesion to fibronectin, but almost totally abrogated by sphingosine kinase inhibitors. The signals were partly dependent on calcium influx. In conclusion, calcium signalling instigated by CRP in human PMN is FcγRIIa allele specific, as R/R131 responded to CRP, whereas R/H131 did not. However, increased expression of FcγRIa (CD64), stimulated by IFN-γ, can augment calcium signalling by CRP in low-responders. This suggests that the state of the PMNs, as well as the genetic origin, affect sensitivity for CRP.

Introduction

C-reactive protein (CRP) is a prototypic acute-phase protein in man. Its concentration can increase up to 1000-fold during the inflammatory response to acute injury or infection. The main role of CRP appears to be in innate immunity responses, the first line of host defence. It activates complement, stimulates phagocytosis and binds to immunoglobulin receptors (FcγRI and FcγRII) [1]. Recently, slightly elevated CRP plasma levels (3–10 μg/ml) have been associated with cardiovascular disease, obesity, metabolic syndrome and colon cancer, clinical conditions involving low levels of chronic inflammation [2-4].

The effect of CRP on human PMNs is of special interest, because these cells are essential participants in innate immunity, and also active in more chronic inflammatory conditions, such as atherosclerosis and coronary inflammation [5]. How CRP affects neutrophil function is apparently controversial, because both inhibitory [6] and stimulatory [7, 8] effects on the PMN oxidative burst have been reported. PMN migration and chemotaxis seem, however, to be inhibited [9, 10], whereas phagocytosis has been shown to increase [11].

C-reactive protein binds to Fc-receptors on neutrophils, to FcγRIa (CD64) with low affinity and to FcγRIIa (CD32) with high affinity [12]. Accordingly, the dominant CRP receptor on neutrophils is FcγRIIa. Furthermore, it has been shown that binding of CRP to neutrophil FcγRIIa is allele specific. A single-nucleotide polymorphism, producing an amino acid difference at position 131, causes the different binding affinity. CRP binding was evident in R/R 131 cells, but minimal in the H/H 131 homozygote variety [13]. Whether this polymorphism has any functional consequence has been questioned [14, 15], but it is associated with several diseases, such as rheumatoid arthritis and systemic lupus erythematosus (for review [16]).

The FcγRIa receptors are expressed at very low levels on resting neutrophils, but can be upregulated by inflammatory cytokines, such as interferon-γ (IFN-γ) [17]. In chronic, low-grade inflammatory conditions, like obesity and coronary artery disease (CAD), inflammatory cytokines are elevated, including IFN-γ [18]. It is not known if this increased expression of FcγRIa receptors affects CRP-mediated effects or signalling events.

In the cell line HL-60, which can differentiate to granulocytes, CRP induces tyrosine phosphorylation of FcγRIIa, Syk and phospholipase Cγ2 (PLCγ2) [19]. Both PLCγ2 and phosphatidylinositol 3-kinase (PI-3K) were here shown to translocate to the membrane, followed by transient increases in cytosolic calcium. In THP-1 cells, a monocytic leukaemia cell line, CRP caused a rapid, dose-dependent increase in cytosolic [Ca2+] that was dependent on Syk activity [20]. In fact, all the FcγRs can initiate an increase in cytosolic calcium concentration, but the relevant second messenger, inositol trisphosphate (IP3) or sphingosine-1-phosphate (S1P) may vary according to the particular receptor involved [21]. Complicating the picture further, adherent neutrophils responded in a different way than cells in suspension [22], implying a modulatory role for integrins in ligand-induced calcium signalling.

The aim of this study was to study how CRP initiate calcium signalling in human neutrophils and how relevant polymorphism in FcγRIIa is for calcium signalling, with or without induced expression of FcγRIa. The role of PMN adhesion for CRP signalling was also addressed. Expectantly, the results will contribute to the understanding of the role CRP plays for PMN function.

Materials and methods

Cell preparation and solutions

Blood was drawn from healthy volunteers, and neutrophils were isolated with density-dependent centrifugation (Polymorphprep TM; Axis-Shield PoC AS, Oslo, Norway), with aseptic technique at room temperature.

All cells were used from one to 6 h after blood withdrawal. PMN were suspended in HEPES buffer, containing 136 mm NaCl, 5 mm KCl, 1.18 mm MgCl2·6H2O, 11 mm D-glucose, 1.2 mm CaCl2·2H2O and 10 mm HEPES (all chemicals from Sigma-Aldrich, St. Louis, MO, USA). The wells where the cells were kept during fura-loading and calcium registrations were made from a cylindrical plastic tube glued with Silicone rubber RTV 118 (GE Silicones, Huntersville, NC, USA) onto a glass coverslip. The coverslips were coated the day of the experiments according to the following procedure: The wells were filled with 150 μl 0.1 mg/ml fibronectin or 10 μg/ml RGD (both obtained from Sigma-Aldrich) and then stored at room temperature for one hour and washed twice with distilled water and left to dry before use.

Materials for preincubation treatment of the cells, N-acetylsphingosine (N-Ac-Sp), N,N-dimethylsphingosine (DMS), ethylene glycol-bis-(β-amino-ethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and human interferon-γ (IFN-γ) were from Sigma-Aldrich. Agonists used were pentameric C-reactive protein (CRP) from Calbiochem (Merck KGaA, Darmstadt, Germany) and formyl-methionyl-leucyl-phenylalanine (fMLP) from Sigma-Aldrich; HEPES buffer was used as control.

Measurement of cytosolic calcium in single adherent cells

Cytosolic free Ca2+ in single cells was determined as previously described [23]. In short, the cells were incubated for 45 min at room temperature with HEPES buffer containing 5 μm fura-2/AM (Teflabs, Austin, TX, USA), 0.025% Pluronic F-127 (from Molecular Probes, Eugene, OR, USA) and 0.25% DMSO (obtained from Sigma-Aldrich). The cells were then washed once and incubated in 400 μl HEPES buffer. Agonist applications were performed by injection of 100 μl of volumes into the wells, control and CRP additions were performed in alternating sequence. The Ca2+ imaging and registration software has been developed in our laboratory [24]. The equipment consisted of a PTI Δ-scan excitation device, a Nikon Diaphot-TMD inverted microscope, a Hamamatsu CCD video camera (C3077) and an intensifier head (C2400-8) and a computer controlling a frame grabber, synchronizing the chopper speed with the video camera as well as storing the video recordings. The cytosolic Ca2+ concentration was calculated with the equation: [Ca2+] = Kdβ(R − Rmin)/(Rmax − R) as applied by Grynkiewicz et al. [25]. R is the ratio between fluorescence intensity at 345- and 385-nm excitation. The autofluorescence from non-fura-2 loaded cells was less than 10% of the total fluorescence and was subtracted before calculating the ratio. An indirect intracellular calibration method developed in our laboratory was used to calculate the calibration constants (β, Rmin and Rmax) [26]. The experiments were carried out at 35–37 °C, and moving cells were tracked during the registration [24].

FcγRIIA polymorphism and FcγRI expression

Standard polymerase chain reaction (PCR) performed to determine FcγRIIA polymorphism was carried out as described by Rodriguez et al. [14]. Primers for H-mix and R-mix were bought from MWG-BIOTECH AG. DNA and RNA were extracted from isolated PMNs using Tri-reagent (Sigma-Aldrich). PCR was performed on an eppendorf thermal cycler. Before extraction of RNA, the purity of the cell suspension was examined by microscopy of Colorrapid (Lucerna-Chem AG, Luzern, Switzerland) stained smears. Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7900 with Taqman assays and TATA-binding protein (TBP) and 18s rRNA (Applied Biosystems, Paisley, UK) as internal controls. Primers and probes were designed using Primer Express software (Applied Biosystems). cDNA synthesis and qRT-PCR was performed with a two-step qRT-PCR kit from Invitrogen (Paisley, UK). The reaction was optimized on standard PCR for temperature, template volume and primer concentration before qRT-PCR. Amplification products were visualized with ethidium bromide to check for unspecific products or primer dimers. Results from qRT-PCR were analysed with the comparative Ct method [27].

Flow cytometry

Isolated PMN were incubated with IFN-γ (100 IU/ml) for 2 h at room temperature and washed with PBS containing 0.1% sodium azide. For staining of FcγRs, the cells were incubated with human IgG (10 mg/ml, BD Pharmingen and R&D Systems) for 30 min and then incubated with 1 μl FITC-conjugated monoclonal antibodies against CD64 from R&D Systems (Minneapolis, MN, USA) and AbD Serotec (Oxford, UK), 2 μl FITC-conjugated monoclonal antibodies against CD32 from Abcam (Cambridge, UK) and BD Pharmingen (Franklin Lakes, NJ, USA) or 1 μl control isotypes from R&D Systems and BD Pharmingen for 45 min on ice. After washing twice with PBS, cells were analysed by flow cytometry (FACSCalibur, Becton Dickinson Biosciences, San Jose, CA, USA). Markers were set according to the isotype control FITC-conjugated mouse IgG. Geometric mean fluorescence intensity was calculated.

Detection of CRP and IFN-γ

Plasma samples from all blood donors were stored at −20 °C for determination of CRP and IFN-γ content. hsCRP analyses were performed with the Tina-quant C-reactive protein (latex) high-sensitivity immunoturbidimetric assay applied on the Modular P analyzer (Roche Diagnostics, Mannheim, Germany). IFN-γ concentrations were measured spectrophotometrically with an immunoassay kit (Quantikine, R&D Systems).

Quantitative and statistical analysis

The fluorescence data were stored on a DVD disc and analysed with a program (LICS) written for this purpose in our laboratory [26]. We smoothed the fluorescence signals and the calculated Ca2+ signals, with a Hamming window low pass filter, excluding higher frequencies than 0.33 and 0.20 Hz, respectively. Baseline cytosolic calcium concentration was calculated as the average calcium concentration the first 30 s of the registration period. Responsive cells were defined as cells showing more than 50 nm increase in cytosolic calcium concentration above baseline within 70 s after stimulation. Cells with delayed or slight increases in cytosolic calcium (less than 50 nm) were classified as low-responders. There was some spontaneous activity in cells before any stimulation. If the signals rose above 300 nm within 30 s, the cells were omitted from further analysis. Omitted or invalid cells counted less than 1.5% of total cells in every group.

The Ca2+ data were exported to GraphPad Prism v. 4.0 where graphs were made, and baseline and stimulated increases in cytosolic calcium were calculated. Non-cytosolic fura-2 was present in cells up to about 10% of total intracellular content. This will lead to a systematic overestimation of the numeric value of the cytosolic Ca2+ concentration, but we assume that this will not invalidate conclusions drawn on the basis of changes in Ca2+ concentration.

The data are presented as means with their standard error (±SEM), and differences between groups were evaluated with two-tailed Student's t-test. Number of responders in each group was tested with chi-squared test. P-values <0.05 were considered significant.

Results

CRP-induced calcium signals in adherent neutrophils

From our previous work, we know that calcium signalling in adherent PMNs depends on the surface to which the cells adhere [22]. In particular, we have shown that adherence to fibronectin (FN) evokes optimal calcium signalling in response to IFN-γ [22]. Whether CRP could induce a cytosolic calcium signal in adherent neutrophils was therefore studied, in cells isolated from six different blood donors. Initially, CRP-responses were studied in PMNs both on uncoated and fibronectin (FN)-coated glass coverslips. For comparison, IFN-γ (100 U/ml) stimulation was included (Table 1). As a control, HEPES buffer (HSS) was used, for both uncoated and FN-coated surfaces. Spontaneous responses were observed in 9% (±1) and 14% (±2) of the cells, respectively. FN-coating increased the number of CRP-responding cells by 23% and the size of the calcium responses by 19 nM. The average responses to CRP were about 60% smaller than the responses to IFN-γ. However, when examining the single-cell responses more closely, we discovered that three of the six donors were responsive to CRP, whereas the other three were low-responders (Fig. 1A), and the proportion of CRP-responding cells was significantly higher in responders than low-responders, which was equal to HSS control (Fig 1B). When we were examining responding cells only (n = 107 for responders and n = 36 for low-responders), the sizes of the Ca2+-signal were very similar, 147 (±14) nm for low-responders and 184 (±9) nm for responders. For further experiments on calcium signalling, PMNs from responsive donors only were studied.

Table 1. Calcium signals in CRP and IFN-γ stimulated PMNs
StimuliSubstrate coatingNumber of cells scoredBasal [Ca2+] (nm)[Ca2+] response (nm) above baselineResponding cells (%)
  1. CRP, C-reactive protein; IFN-γ, interferon- γ, FN, fibronectin. Mean ± SEM are shown.

  2. a

    Significantly different from cells on uncoated glass coverslips (P < 0.05).

CRP 50 μg/ml

184150 (±8)79 (±8)15 (±2)
FN169168 (±12)98 (±5)a38 (±1)a
IFN-γ 100 U/ml187139 (±4)63 (±4)16 (±2)
FN227140 (±5)229 (±8)a44 (±3)a
Figure 1.

(A) C-reactive protein (CRP) (50 μg/ml) induced cytosolic calcium signals in PMN from three blood donors (responders, 202 cells), but not in the remaining three (low-responders, 138 cells). All donors responded to fMLP (100 nm) added 90 sec after CRP. Average responses from all cells are shown. (B) The fraction of CRP-responding cells in responders and low-responders versus fraction of spontaneous responders (HEPES buffer – HSS) and responders to fMLP. Means ± SEM are shown; same experiments in A and B. *Significantly different from low-responders (P < 0.05).

Dose dependence of CRP

In responders, both the relative number of responding cells and the amplitude of the Ca2+-signals increased with increasing CRP concentration up to 50 μg/ml (Table 2). FMLP was included as a positive control – added after the CRP or buffer – and the number of cells responding to fMLP was similar in control cells and cells already stimulated with CRP (90% and 91%, respectively). In addition, there was no correlation between the amplitudes of the CRP-responses and the subsequent fMLP-responses.

Table 2. Dose-response CRP versus cytosolic calcium signalling
Concentration of CRP (μg/ml)Number of cells% CRP respondersBasal [Ca2+] (nm)CRP-response (nm)fMLP-response (nm)
  1. CRP, C-reactive protein; fMLP, formyl-methionyl-leucyl-phenylalanine. See Table 1 for more explanations.

  2. a

    Significantly different from unstimulated cells (P < 0.05).

0879 (±1)192 (±6)149 (±5)345 (±12)
107825 (±1)a184 (±5)157 (±8)293 (±9)
5042954 (±3) a199 (±4)177 (±4)a315 (±8)
10032534 (±2)a197 (±7)173 (±6)a279 (±10)

Effect of different coating

Based on previous work, PMNs respond differently when adherent to different coatings [22].We therefore investigated CRP signalling in responsive cells on uncoated, FN-coated and RGD-coated glass coverslips (Table 3). Adherent PMNs were stimulated with CRP (50 μg/ml) or with HEPES buffer as a negative control. The responses to CRP were significantly increased in both RGD- and FN-coated glass compared with uncoated glass (P < 0.05). Spontaneous responses to addition of HEPES buffer were seen in 11% of the cells on glass, 16% of the cells on FN and 36% of the cells on RGD. FN-coating was therefore chosen for the following experiments.

Table 3. Responses to CRP (50 μg/ml) in PMN adherent to different coatings
CoatingNumber of cells% CRP respondersBasal [Ca2+] (nM)CRP-response (nM)fMLP-response (nM)
  1. CRP, C-reactive protein; fMLP, formyl-methionyl-leucyl-phenylalanine; RGD, Arginine-Glycine-Aspartic Acid.

  2. a

    Significantly different from uncoated glass P < 0.05. See Table 1 for more explanations.

Uncoated18011 (±0.3)195 (±6)161 (±7)347 (±22)
RGD32566 (±1)a190 (±4)257 (±8)a380 (±8)
Fibronectin (FN)29261 (±1)a195 (±4)263 (±8)a333 (±9)

Characterization of the CRP-induced Ca2+-signals

To evaluate whether influx of calcium from the extracellular media is part of the CRP-induced calcium signal, stimulation with CRP was performed in presence of Ca2+-binding EGTA (2 mm), added 10 minutes prior to registration. As can be seen from Fig. 2, the responses to CRP are clearly reduced in the presence of EGTA. The calcium increase was reduced by 15% by EGTA (P < 0.05). Notably, the baseline [Ca2+] was also significantly decreased by EGTA, from 194 (±7) to 166 (±5) nm.

Figure 2.

(A) C-reactive protein (CRP)-induced (50 μg/ml) calcium signals were reduced by addition of EGTA (2 mm) to the extracellular medium. FMLP (100 nm) was added 90 s after CRP. Average responses from 402 control cells and 495 EGTA-treated cells are shown. (B) The fractions of responding cells in control medium and in EGTA-medium. Mean ± SEM are shown. The basal calcium concentration was significantly lower in EGTA-treated cells (P < 0.001), and both the size of the calcium signals and the number of responders to CRP were significantly reduced (P < 0.05). *Significantly different from control (P < 0.05).

Sphingosine 1-phosphate is known to release calcium from intracellular stores by a non-IP3-dependent mechanism [28]. The sphingosine kinase inhibitors DMS (30 μm) and N-Ac-SP (30 μm) significantly reduced the number of responding cells and the size of the CRP-responses (Table 4). DMS also increased the delay to response. Spontaneous responses to HEPES were observed in 9% of the cells in these experiments (data not shown).

Table 4. Effect of sphingosine kinase inhibitors on CRP-induced calcium signals
TreatmentNumber of cells% CRP respondersBasal [Ca2+] (nM)CRP-response (nM)Response delay (sec)
  1. CRP, C-reactive protein; DMS, dimethylsphingosine; N-Ac-Sp, N-acetylsphingosine.

  2. a

    Significantly reduced from control (P < 0.05).

  3. b

    Significantly increased from control (P < 0.05). See Table 1 for more explanations.

Control25451 (±1)148 (±5)178 (±6)41 (±6)
DMS27713 (±2)a151 (±6)132 (±10)a55 (±8)b
N-Ac-SP26617 (±3)a151 (±6)151 (±8)a43 (±7)

Characterization of the CRP-responding neutrophils

The binding of CRP to the receptor FcγRIIa (CD32) depends on polymorphism in the 131 position [13]. Receptors with arginine (R/R131) in this position bind CRP with high affinity, whereas receptors with histidine (H/H131) in this position has very low or no affinity for CRP. We therefore tested this allele polymorphism in our cell donors. The responding cells all expressed the FcγRIIa R/R131 allele (n = 3), and all the low-responders were heterozygote, expressing the FcγRIIa R/H131 allele (n = 3) (Fig. 3). Previously, it has been shown that the H-allele coded receptor dominates over R in heterozygotes when they simultaneously compete for the same ligand; however, in these experiments, IgG was the ligand, not CRP [29].

Figure 3.

Donor polymorphism of FcγRIIA. The upper bands show amplification of FcγRIIA. DNA was extracted from isolated PMN using Tri-reagent (Sigma-Aldrich) and amplified. The lanes marked H show amplification with the H131-specific primer, and lanes marked R show amplification with the R131-specific primer. Four of the six donors are shown, donor one and two are homozygote R/R131 and responders, whereas donors three and four are heterozygote R/H131 and low-responders. Primers were made according to Rodriguez et al. [14].

Effect of pre-treatment with IFN-γ

Expression of the CRP low-affinity receptor FcγRIa (CD64) can be upregulated by interferon-γ [17]. To examine whether this receptor might contribute to calcium signalling induced by CRP, we pre-treated neutrophils with IFN-γ (100 IU/ml) for 1–4 h. After 2 h with IFN-γ, the FcγRIa mRNA was increased 5.7-fold (range, 3.6–8.8) compared with untreated control cells. This was assessed by RT-PCR with R18S and TBP as reference genes. The increased expression of FcγRIa after IFN-γ pre-treatment was confirmed with flow cytometry (Fig. 4). Surface expression of FcγRIa (CD64) increased about fourfold after IFN-γ for 2 h in heterozygote R/H cells from 6.95 (±0.26) to 28.13 (±2.98) (Fig. 4A), whereas there was no significant increase in homozygote R/R cells, from 5.11 (±1.33) to 5.24 (±1.29) (Fig. 4B). The surface expression of FcγRIIa (CD32) was unaffected by IFN-γ both in R/H (Fig. 4C) and R/R cells (Fig. 4D).

Figure 4.

Surface expression of FcγRIa (CD64) (A, B) and FcγRIIa (CD32) (C, D) on PMNs after pre-treatment with IFN-γ. PMN from low-responders (R/H) (A, C) and responders (R/R) (B, D) were incubated with IFN-γ (100 U/ml) for 2 h. FITC-conjugated antibodies against CD64 and CD32 were added and flow cytometry performed. A representative experiment is shown, and the experiment was performed with 3 RR and 3 RH donors. The increase in CD64 expression after IFN-γ in low-responders was statistically significant (P = 0.01).

Both the proportion of responding cells and the size of the calcium signals were increased after IFN-γ pre-treatment (Table 5). Surprisingly, the responses to fMLP were decreased by IFN-γ pre-treatment. All cells responded to fMLP, but the amplitude of the responses was slightly reduced. We also observed a dose-dependent increase in the relative number of CRP-responding cells after IFN-γ pre-treatment (Fig. 5).

Table 5. Effect of pre-treatment of low-responding PMNs with IFN-γ (100 IU/ml) on CRP-induced (50 μg/ml) calcium signals
Pre-treatmentNumber of cells% CRP respondersBasal [Ca2+] (nM)CRP-response (nM)fMLP-response (nM)
  1. CRP, C-reactive protein; fMLP, formyl-methionyl-leucyl-phenylalanine.

  2. a

    Significantly increased from no pre-treatment (P < 0.05).

  3. b

    Significantly decreased from no pre-treatment (P < 0.05). See Table 1 for more explanations.

None13824 (±1)165 (±4)156 (±37)549 (±17)
IFN-γ 1 h15334 (±2)162 (±4)231 (±15)a481 (±22)b
IFN-γ 2 h13348 (±1)a161 (±4)268 (±14)a478 (±16)b
IFN-γ 4 h14541 (±2)a157 (±4)219 (±14)507 (±24)b
Figure 5.

The fraction of cells responding to CRP (10 or 50 μg/ml) after 2 h pre-treatment with IFN-γ (100 IU/ml). Mean ± SEM are shown. *Significantly different from control (P < 0.05).

The endogenous level of IFN-γ was below detection limit in blood of all the cell donors on the day of blood sampling (data not shown). The average plasma concentration of CRP was 1.8 (range, 0.9–3.6) μg/ml.

Discussion

The present study has shown that CRP can induce cytosolic calcium signals in PMNs expressing the homozygote R/R131 allele of FcγRIIa. Heterozygote H/R131 FCγRIIa was associated with low calcium responses to CRP. However, pre-incubation of these low-responsive cells with IFN-γ improved sensitivity towards CRP in conjunction with increased expression of the low-affinity CRP-receptor FcγRIa. The calcium signalling was strengthened by cell adhesion to fibronectin, and the calcium signals were composed of both calcium influx and calcium release from cytoplasmatic stores.

That calcium signalling instigated by CRP in PMN is allele specific is in line with previous findings that binding of CRP to FcγRIIa is dependent on the genotype. R/R131 homozygotes bind CRP with high affinity, H/H131 homozygotes with low affinity, and H/R131 heterozygotes with intermediate affinity [13]. Recently, it was shown that the H-allele outcompeted the R-allele when their protein products simultaneously competed for IgG as a ligand [29]. In contrast to CRP, IgG2-binding to FcγRIIa is lower in the R131 than in the H131 allotype [30]. We therefore cannot exclude that CRP acts differently from IgG as a ligand, but these observations suggest that the H-allele is dominant and that calcium signalling by Fcγ-RIIa is markedly influenced by allele type.

Pre-treatment of low-responder PMNs with IFN-γ upregulated expression of the low-affinity receptor FcγRIa, assessed by both real-time PCR and flow cytometry. Concomitantly, calcium signalling stimulated by CRP increased. IFN-γ had no effect on FcγRIa expression in responders, or on FcγRIIa expression in either responders or low-responders. The lack of effect of IFN-γ on FcγRIIa expression has been seen before [31]. However, the lack of effect of IFN-γ on FcγRIa expression in R/R131 FcγRIIa homozygote cells is to our knowledge a new finding. A donor-dependent effect of IFN-γ on HLA-DR expression has however been reported [32], and we have shown that PMN responses to IFN-γ depend on the adherence state of the cells. The divergent responses to IFN-γ depended on whether the cells were adherent or not, and to what surface they adhered. Actually, how IFN-γ-induced expression of FcγRI depends on the FcγRII polymorphism is not known. We suggest that inflammatory mediators, such as IFN-γ, can thus increase PMN sensitivity towards CRP and turn low-responders into responders. This finding, together with the observation that fibronectin adherent cells respond better to CRP, suggests that an existing (pro-)inflammatory state may affect the sensitivity to CRP and, consequently, the PMN responses generated by CRP. However, we cannot exclude that another mechanism than increased FcγRIa expression could explain or contribute to the effect of IFN-γ on CRP signalling.

The CRP-induced calcium signals were clearly different from classical fMLP-induced signals, which are mediated by IP3-stimulated calcium release from internal stores, as well as calcium influx from the extracellular medium [33]. However, the Ca2+-signals we observed seemed to be similar to CRP-induced Ca2+-signals in hemic cell lines, such as THP-1 and HL-60 cells [19, 20]. The increase in cytosolic calcium was in the range 100–150 nm, appeared rapidly and returned to baseline within few minutes. In agreement with our results, the calcium signals evoked by CRP in HL-60 cells were attenuated by extracellular EGTA [19]. In THP-1 cells, the CRP-induced calcium signals were totally abrogated by the Syk-specific inhibitor piceatannol [20]. It is known that activation of all Fc-receptors can trigger calcium signals [21]. It has also been reported that ligation of FcR in neutrophils causes calcium release that is independent of IP3 and pertussis toxin [34]. The mechanism, however, is not clear. The signals, evoked by aggregated IgG, were very similar to the CRP-triggered Ca2+-signals observed here.

We have shown that the CRP-induced calcium signals were almost completely abolished by sphingosine kinase inhibitors, implying that sphingosine-1-phosphate can be the second messenger involved in calcium release from intracellular stores. To our knowledge, this is the first report of involvement of sphingosine-1-phosphate in CRP signalling. Ligation of FcRs by other agonists has been shown to involve sphingosine kinase and sphingosine-1-phosphate, and calcium signals induced by cross-linking of FcγRIIa and FcγRIIIb in human PMNs were suppressed by the sphingosine kinase inhibitor DMS [35]. FcγRI stimulation of U937 macrophages by cross-linking antibodies mobilized calcium from intracellular stores by activating sphingosine kinase [36]. Moreover, in mast cells, activation of FcεRI generated Ca2+-signals that depended on sphingosine-1-phosphate production [37, 38]. A contradictory report also exists, as FcγRII-mediated calcium signalling in neutrophils allegedly was not inhibited by the sphingosine kinase inhibitor DMS [39].

Sphingosine-1-phosphate (S1P) is a signalling lipid with both extracellular and intracellular actions. The physiological effects of S1P are mediated by the G-protein coupled receptors (S1PR1-5) or by S1P acting as a second messenger on intracellular targets. Thapsigargin-sensitive calcium channels might be an intracellular target of S1P [40]. Sphingosine-1-phosphate is generated by sphingosine kinase (SK), either SK1 or SK2. SK1 is activated by ERK1/2-mediated serine (Ser225) phosphorylation [41]. FcR-mediated activation of ERK1/2 has been reported in neutrophils. We find it most likely that S1P acts as an intracellular messenger in our cells, although release of S1P and an extracellular effect cannot be excluded.

The CRP-induced calcium signals are improved by FN and RGD coating. This is similar to what we have seen in PMN stimulated with IFN-γ [22]. FN and RGD bind to integrins on the PMN surface. It is well established that integrins generate intracellular signals that can regulate various cellular functions. Ligand-induced integrin clustering and conformational changes probably contributes to efficient recruitment of protein tyrosine kinases [42]. In neutrophils, the tyrosine kinase Syk has an essential role in integrin signalling, particularly in integrin-stimulated respiratory burst, degranulation and spreading [43]. It has also been shown that both integrins and FcγRs activate the adaptor protein SLP-76 and that SLP-76 is essential for calcium signalling in neutrophils [44]. SLP-76 is also involved in Syk family kinases signalling, and could therefore be a link between signalling pathways from integrins and FcγRs. Possibly, PMN binding to an FN-coated surface engages integrins, recruits proteins such as Syk and SLP-76 that cluster nearby, and primes the PMNs for final activation by soluble ligands like CRP.

C-reactive protein is a universal inflammatory mediator, and calcium signalling is involved in activating a wide range of cellular functions. Increased PMN sensitivity towards CRP might be clinically relevant, especially in conditions with slightly increased CRP levels, as in many chronic inflammatory diseases.

Several polymorphisms of the FcRs have been described, and many of them are associated with diseases (for review [16]). The actual R131H-polymorphism of FcγRIIa has been found relevant to receptivity to infectious diseases, such as dengue fever [45] and periodontitis [46], as well as autoimmune Grave's disease [47], systemic lupus erythematosus [48], rheumatoid arthritis [49] and type 1 diabetes [49]. It seems like the R/R131 allele increases the susceptibility to both bacterial infections and autoimmune diseases, whereas the H/H131 allele does not. The heterozygote R/H131 is phenotypically similar to H/H131 [29]. The R/R131 genotype binds CRP with high affinity, IgG with low affinity. Taken together, the association of this polymorphism to several diseases and the allele characteristic binding and signalling might be important for understanding susceptibility to disease. Finally, there are many more cellular participants in inflammatory and immune responses than the PMNs, which should also be subjected to CRP studies.

In conclusion, we have shown that calcium signalling instigated by CRP in human PMN is FcγRIIa allele specific, as R/R131 responded to CRP whereas R/H131 did not. However, increased expression of FcγRIa, stimulated by IFN-γ, can augment calcium signalling by CRP in low-responders. This suggests that the state of the PMNs, as well as the genetic origin, may affect their sensitivity for CRP.

Acknowledgment

We thank professor Stein Bergan at Department of Medical Biochemistry, Oslo University Hospital for doing the CRP measurement, and Professor Terje Einar Michaelsen at The Norwegian Institute of Public Health and PhD Jan-Terje Andersen at Centre for Immune Regulation, Oslo University Hospital, for providing the monoclonal antibodies against CD32 and CD64.

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