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The Journal of Immunology, 2006, 176: 7598-7604.
Copyright © 2006 by The American Association of Immunologists

C-Reactive Protein Increases Cytokine Responses to Streptococcus pneumoniae through Interactions with Fc{gamma} Receptors1

Carolyn Mold2,{ddagger} and Terry W. Du Clos*,{dagger}

* Veterans Affairs Medical Center, Albuquerque, NM 87108; and Departments of {dagger} Internal Medicine and {ddagger} Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Streptococcus pneumoniae is the most common organism responsible for community acquired pneumonia and meningitis. In pneumococcal pneumonia, a strong local inflammatory cytokine response reduces the frequency of bacteremia and increases survival. The initiation of this cytokine response by innate recognition of bacterial cell wall components through TLR has been described, but the role of soluble innate mediators has received limited attention. C-reactive protein (CRP) is an acute phase protein that binds phosphocholine residues on S. pneumoniae cell walls. CRP interacts with phagocytic cells through Fc{gamma}RI and Fc{gamma}RII and activates the classical complement pathway. CRP is protective in mouse pneumococcal bacteremia by increasing complement-dependent clearance and killing of bacteria. We studied the cytokine response of PBMC stimulated with CRP-opsonized S. pneumoniae to determine the effect of CRP interaction with Fc{gamma}R. CRP dramatically increased the production of TNF-{alpha} and IL-1beta in response to S. pneumoniae. These increases were blocked by phosphocholine, which inhibits CRP binding to S. pneumoniae, by inhibitors of Fc{gamma}R signaling, and by mAb to Fc{gamma}RI and Fc{gamma}RII. A mutated rCRP with decreased Fc{gamma}R binding had a decreased ability to stimulate TNF-{alpha} release, compared with wild-type CRP. Individuals who were homozygous for the R-131 allele of Fc{gamma}RIIA, which has a higher affinity for CRP, showed higher responses to CRP-opsonized bacteria than did individuals homozygous for the H-131 allele, further implicating this receptor. The results indicate that CRP recognition of S. pneumoniae and binding to Fc{gamma}R may enhance the early protective cytokine response to infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is associated with a high mortality rate among hospitalized patients (1, 2). Exposure to S. pneumoniae without infection is common, and as many as 40% of individuals in the general population are asymptomatic carriers. Infection occurs when colonizing organisms spread to the lungs, brain, or other sites. If the infection is not effectively cleared, bacteremia and sepsis may result. The risk of invasive pneumococcal disease is greatest among the very young, the elderly, and immunocompromised individuals. The innate immune system provides the stimulus for an early inflammatory response, which is essential in preventing invasive infection with this ubiquitous pathogen, but also contributes to morbidity and mortality in pneumococcal meningitis and sepsis (3).

C-reactive protein (CRP)3 is a component of innate immunity, which was discovered 75 years ago in the serum of patients acutely ill with pneumococcal pneumonia (4). CRP was shown to precipitate the C-polysaccharide of the pneumococcal cell wall. The reactivity of CRP for C-polysaccharide was later found to be calcium dependent and specific for phosphocholine (PC) groups on the teichoic acid and lipoteichoic acid of all S. pneumoniae (5). CRP is an acute phase reactant that is synthesized rapidly by the liver following injury, infection, or trauma (6, 7). CRP is normally present in the blood at <5 µg/ml, but acute phase levels as high as 100–500 µg/ml may be reached. Although the liver is the major source of serum CRP, there is evidence for local production by epithelial cells in the upper respiratory tract and release into nasal secretions and sputum (8).

Human CRP, either injected or synthesized from a transgene, is protective in mouse models of pneumococcal bacteremia (9, 10, 11). Based on these studies, the primary mechanism of CRP-mediated protection in S. pneumoniae bacteremia is believed to be more efficient complement-dependent clearance and killing of S. pneumoniae (12, 13, 14). Effects of CRP on cytokine responses to pneumococcal infection have not been reported. In addition to its ability to activate complement, CRP interacts with phagocytic cells directly through Fc{gamma}RI and Fc{gamma}RII (15, 16, 17, 18).

In human pneumococcal pneumonia and in mice infected intranasally with S. pneumoniae, a strong local and systemic inflammatory cytokine response occurs and this early response is associated with increased survival (19, 20, 21). Experiments using cytokine- or cytokine receptor- deficient mice have implicated TNF-{alpha} and IL-6, and to a lesser extent, IL-1beta, as important mediators of this protective inflammatory response (22, 23, 24, 25). In contrast, deficiency of IFN-{gamma} did not affect survival from S. pneumoniae infection (26), and inhibition of IL-10 enhanced host defense (27). Coinjection of TNF-{alpha}, LPS or heat-killed S. pneumoniae along with an intranasal inoculum of virulent pneumococci enhanced recruitment of neutrophils into the lung and increased bacterial clearance and survival (20). Other experiments have found that, although S. pneumoniae induce a strong IL-1beta response, they are poor inducers of TNF-{alpha} in human PBMC (28). Thus, agents that would increase the TNF-{alpha} response to S. pneumoniae infection could potentially improve survival.

The importance of TLR2 in the cytokine response to S. pneumoniae cell wall components has been established (29). However, less is known about the role of soluble innate recognition molecules, which also mediate pathogen recognition. A recent report identified the acute phase protein, LPS binding protein (LBP) as a recognition molecule for pneumococcal cell walls and an important stimulus for the inflammatory response in pneumococcal meningitis (30). CRP binds to the teichoic acid and lipoteichoic acid components of the S. pneumoniae cell wall. CRP interacts with monocytes, macrophages, and neutrophils through Fc{gamma}R (17, 18), protects mice from pneumococcal infection and facilitates S. pneumoniae clearance from the bloodstream (9, 12). Because cytokine responses, including TNF-{alpha} also may be triggered through Fc{gamma}R (31), we hypothesized that the binding of the innate opsonin, CRP, to pneumococci might alter the cytokine response as well as enhance clearance of the bacteria. CRP is likely to be available for early host defense in pneumococcal pneumonia, because it is not only rapidly synthesized in the liver (6, 7), but also is locally synthesized (8).

In the present study, we examined the effect of CRP on human mononuclear cell cytokine responses to S. pneumoniae. We found that CRP significantly enhances the TNF-{alpha} and IL-1beta responses to these bacteria and that this effect is mediated by its binding to the bacteria and interaction with Fc{gamma}RI and Fc{gamma}RII. These results suggest another mechanism by which CRP could protect against invasive pneumococcal infection. In addition, this report is the first to directly demonstrate that Fc{gamma}R mediate the cytokine response elicited in human PBMC by CRP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Human CRP was purified from human pleural fluid as described previously (32). All preparations were examined on overloaded SDS-PAGE gels to ensure purity. No additional bands other than the major band at ~25 kDa were seen. The preparations were examined for endotoxin by a quantitative chromogenic Limulus amebocyte lysate assay (Cambrex). If needed, endotoxin was removed on an Etox Acticlean column (Sterogene) so that final CRP preparations contained <0.3 ng endotoxin/mg protein. In addition, 10–20 µg/ml polymyxin B was added to all in vitro assays to block possible cytokine stimulation by endotoxin contaminating any of the other reagents.

Recombinant wild-type and mutant human CRP were prepared using a baculovirus expression system as described previously (33). The expressed proteins were isolated from Trichoplusia ni larvae by PC-Sepharose affinity chromatography, and their structural integrity was confirmed by retention of binding to pneumococcal C-polysaccharide and PC-BSA and lack of reactivity with mAb to denatured determinants. The T173A mutant protein used here fails to bind to Fc{gamma}RI or C1q and shows greatly reduced binding to Fc{gamma}RII (33). The 4-fold-enhanced TNF-{alpha} response to R36a seen with wild-type rCRP was similar to that observed with purified human CRP (range 1.8- to 4.2-fold).

Piceatannol and wortmannin were purchased from Sigma-Aldrich. Heat-aggregated IgG (aggIgG) was prepared from human IgG (Sigma-Aldrich) by incubation at 63° for 30 min at 10–12 mg/ml. Purified small nuclear ribonucleoprotein (snRNP) particles was generously provided by R. Burlingame (Sm/RNP Ag; Inova Diagnostics). In the experiment shown, CRP complexes with snRNP were prepared using 200 µg/ml CRP and 120 µg/ml snRNP particles.

Abs were purchased as follows: anti-human CD64 (clone 10.1 and its F(ab')2), mouse IgG1 and IgG2a from Ancell, anti-human CD32 (clone FLI8.26), and mouse IgG2b from BD BioSciences; anti-human CD32 (clone AT10) from Serotec. Anti-human CD32 (clone IV.3) was purified from tissue culture supernatants of the hybridoma cell line (American Type Culture Collection (ATCC)) on protein G-Sepharose.

Bacteria

S. pneumoniae R36a (a nonencapsulated variant of type 2 S. pneumoniae) was purchased from the ATCC. R36a were grown to mid-log phase in Todd-Hewitt broth containing 0.5% yeast extract. The concentration of R36a was estimated by absorbance at 600 nm and verified by plate counts. R36a were washed into PBS and heated at 60°C for 60 min. Heat treatment kills the bacteria and also destroys pneumolysin activity. For CRP opsonization, R36a were preincubated for 20 min at room temperature with 100 µg/ml CRP. The final concentration of CRP added to the wells was 25 µg/ml. In experiments using inhibitors of signaling, mAb to Fc{gamma}R and rCRP, the bacteria were washed after treatment with CRP. Washing did not affect the cytokine responses observed.

Peripheral blood cells

Blood from normal volunteers was drawn into heparinized tubes. Genomic DNA extracted from whole blood was used to determine the presence or absence of the H-131/R-131 alleles of Fc{gamma}RIIA as described previously (17). Donors heterozygous for Fc{gamma}RIIA alleles were used in all experiments, except where otherwise indicated. PBMC were obtained by gradient separation using Mono-Poly Resolving Medium (ICN). PBMC were washed three times in RPMI 1640 medium and cultured in 96-well plates at a concentration of 0.5 x 106 cells per well in 200 µl of complete RPMI 1640 medium (containing 10% heat-inactivated FBS and 10–20 µg/ml polymyxin B). PBMC were incubated for 2 h with or without inhibitors before the addition of stimuli. Unless otherwise indicated, PBMC were cultured an additional 24 h after the addition of stimuli. After 24 h, supernatants were collected, centrifuged to remove any cells, and stored at –80°C for cytokine determinations.

Cytokine determinations

Cell culture supernatants were assayed for cytokines by flow cytometry using the human inflammation Cytometric Bead Array (CBA) kit from BD BioSciences. TNF-{alpha}, IL-1beta, IL-10, and IL-8 proteins were also determined individually by ELISA using BD BioSciences reagents.

Levels of cytokine mRNA were determined by RNase protection assay (RPA) using cells collected after 4-h incubation with stimuli. Samples were prepared and analyzed using the hCK-2 or hCK-3 MultiProbe template sets and developing reagents from BD BioSciences.

Data analysis

Graphical and statistical analyses were performed using GraphPad Prism software (GraphPad). For experiments testing the inhibition of TNF-{alpha} and IL-1beta, cytokine levels are presented as a percentage of the amount observed in cultures stimulated with R36a alone. This was done to allow multiple experiments to be combined. Paired t tests were used to compare effects of inhibitors on cytokine synthesis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CRP increases the PBMC cytokine response to S. pneumoniae R36a

Protein concentrations of six cytokines were determined by CBA in 24-h culture supernatants from PBMC stimulated with different concentrations of R36a or CRP and R36a. The results are shown in Fig. 1. The addition of CRP greatly enhanced the production of the early proinflammatory cytokines, TNF-{alpha} and IL-1beta, in response to R36a across the entire dose curve (Fig. 1, A and B). CRP also increased the IL-10 and IL-6 responses at low concentrations of bacteria (Fig. 1, C and D). No significant IL-12 production was detected in any of the samples (<10 pg/ml) (Fig. 1E). IL-8 concentrations in the culture supernatants were too high to measure in the undiluted supernatants by the CBA, so IL-8 was determined by ELISA after appropriate dilution (Fig. 1F). CRP had no detectable effect on the IL-8 response to R36a. The data presented are representative of dose-response curves repeated with additional donors using ELISA to measure TNF-{alpha}, IL-1beta, IL-8, and IL-10. TNF-{alpha} and IL-1beta were increased by CRP at all concentrations of bacteria, IL-8 was unaffected, and IL-10 was increased at low concentrations of bacteria. We also evaluated the concentrations of these cytokines in supernatants from PBMC incubated with CRPalone (10–100 µg/ml) (Table I). PBMC incubated with CRP alone produced IL-6 and IL-8, but at levels much lower than those seen in the presence of R36a. In additional experiments using PBMC incubated with CRP in the absence of bacteria, we have found significant induction of IL-1beta, IL-10, and IL-1RA by CRP concentrations ≥100 µg/ml, but low responses to the 25 µg/ml CRP used in the experiments with R36a. These results are consistent with previous reports of CRP-induced cytokine responses in PBMC but do not represent a significant contribution to the levels of cytokines produced in the presence of R36a (34).


Figure 1
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  		FIGURE 1. CRP increases PBMC cytokine responses to S. pneumoniae R36a. PBMC were incubated for 24 h with the indicated concentrations of R36a or R36a and 25 µg/ml CRP. A–E, Cytokine production in culture supernatants was determined by CBA. F. IL-8 levels were determined by ELISA.

 

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  		Table I. Cytokine responses of PBMC to CRP

 
The effect of CRP on mRNA levels of these cytokines was examined by RPA (Fig. 2). The combined results of four experiments are shown for a single concentration of R36a (4 x 107 CFU) in the presence or absence of CRP and for cells treated with the equivalent concentration of CRP alone (25 µg/ml). The results are consistent with the protein analysis with significant increases in IL-1beta and IL-6 in the cells treated with CRP and R36a, compared with R36a alone. Treatment with CRP alone increased mRNA for IL-1, IL-6, IL-1RA, and IFN-{gamma}. The probe for TNF-{alpha} was not included in the data shown in Fig. 2 but was analyzed in three additional experiments using a different probe set. CRP did not significantly affect the level of TNF-{alpha} mRNA, compared with R36a alone at any concentration of bacteria at the 4-h time point (not shown). This may indicate rapid and transient TNF-{alpha} transcription, as seen in mast cells stimulated through Fc{epsilon}RI (35), or may indicate that some or all of the increased TNF-{alpha} in the 24-h supernatants is released from preformed stores.


Figure 2
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  		FIGURE 2. CRP increases PBMC cytokine mRNA in response to S. pneumoniae R36a. PBMC were incubated for 4 h with CRP (25 µg/ml) or with 4 x 107 CFU R36a or R36a and CRP. RNA was extracted from cells and analyzed for cytokine mRNA by RPA using the hck2b Multiprobe Panel. Values are relative units using the L32 standard to correct for loading. Results from four separate experiments have been combined. mRNA levels in cultures with and without CRP were compared by paired t tests, and statistically significant differences are indicated by *, p < 0.05.

 
CRP binding to ligand is required for the enhanced PBMC TNF-{alpha} response to R36a

Additional experiments focused on the ability of CRP to enhance the TNF-{alpha} and IL-1beta responses to R36a, because these cytokines were most affected by CRP opsonization and both contribute to resistance to pneumococcal infection in vivo (19, 20, 21, 22, 24). To determine whether CRP binding to R36a was required for increased TNF-{alpha} secretion, CRP was incubated with R36a in the presence of 0.1 mM PC, which blocks CRP binding to S. pneumoniae (Fig. 3A). In the presence of PC, CRP had no effect on the TNF-{alpha} response to R36a. In addition, when soluble CRP was added in different concentrations to PBMC with 108 CFU/ml R36a, the increased TNF-{alpha} response saturated at ~25 µg/ml CRP (Fig. 3B). The binding of CRP to R36a at saturation is 20 µg of CRP/108 CFU R36a (36). These results indicate that only CRP bound to R36a was effective in stimulating TNF-{alpha} release.


Figure 3
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  		FIGURE 3. CRP binding to ligand is required for the enhanced PBMC TNF-{alpha} response to R36a. A, PBMC were incubated for 24 h with the indicated concentrations of R36a, R36a and CRP, or R36a and CRP in the presence of 0.1 mM PC to inhibit binding. B, PBMC were incubated for 24 h with 5 x 107 CFU R36a or R36a and the indicated concentrations of CRP. The concentration of CRP in the other experiments was 25 µg/ml. C, PBMC were incubated for 24 h with the indicated concentrations of CRP, aggIgG, complexes of CRP and snRNP, or snRNP alone. TNF-{alpha} concentrations in culture supernatants were measured by ELISA. The experiments shown were done with PBMC used a donor homozygous for the R-131 allele of Fc{gamma}RIIA.

 
To determine whether CRP bound to a nonbacterial ligand would also induce TNF-{alpha} release, complexes of CRP and snRNP particles were tested for stimulation of PBMC. CRP has been shown to precipitate snRNP particles from nuclear extracts, to bind to purified snRNP through the 70 K and D proteins and to react with snRNP particles in cell nuclei (32, 37). The binding of CRP to snRNP particles is similar to binding to S. pneumoniae in that it is calcium-dependent and can be inhibited by PC. PBMC incubated with CRP-snRNP complexes released TNF-{alpha} in a dose-dependent manner, although the response was lower than that observed with R36a (Fig. 3C). The release of TNF-{alpha} in response to aggIgG is shown in comparison. These results suggest that aggregation of CRP by ligand is sufficient for the induction of TNF-{alpha} release.

Inhibitors of Fc{gamma}R signaling block the enhanced TNF-{alpha} and IL-1beta responses to CRP-opsonized R36a

To determine the role of Fc{gamma}R in CRP enhancement of the TNF-{alpha} and IL-1beta responses to R36a, two inhibitors of Fc{gamma}R signaling pathways were used. Pretreatment of PBMC with 10 ng/ml wortmannin, a PI3K inhibitor, or 10 µg/ml piceatannol, a Syk inhibitor, substantially blocked the effect of CRP on TNF-{alpha} and IL-1beta release in the presence of 4 x 107 CFU R36a (Fig. 4). We determined previously that these inhibitor concentrations block CRP-mediated phagocytosis (38). The IL-8 response to R36a or CRP-treated R36a in the same supernatants was not affected by wortmannin or piceatannol (not shown).


Figure 4
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  		FIGURE 4. Treatment of PBMC with inhibitors of Fc{gamma}R signaling decreases TNF-{alpha} and IL-1beta responses to CRP-treated R36a. PBMC were incubated for 24 h with 4 x 107 CFU R36a or CRP-treated R36a. Wortmannin (10 ng/ml) was added to inhibit PI3K and piceatannol (10 µg/ml) was added to inhibit Syk. TNF-{alpha} concentrations (A) and IL-1beta concentrations(B) in culture supernatants were measured by ELISA. Values are normalized to R36a alone (n = 3). Significant differences are indicated by *, p < 0.05, or **, p < 0.01, compared with the R36a + CRP control.

 
Monoclonal Ab to Fc{gamma}RI and Fc{gamma}RII block the enhanced TNF-{alpha} and IL-1beta responses to CRP-opsonized R36a

CRP binds to Fc{gamma}RI and Fc{gamma}RIIa on human monocytes (17). To determine the roles of these two receptors in enhanced cytokine release by CRP-R36a, we tested the ability of mAb to each receptor to inhibit the response. We found that the anti-Fc{gamma}RI (CD64) mAb 10.1, but not an IgG1 isotype control, when added to the cultures at 5 µg/ml inhibited the CRP-dependent increase in TNF-{alpha} and IL-1beta without affecting the baseline response to R36a (Fig. 5, A and B). However, an F(ab')2 of this mAb was not inhibitory at either 5 µg/ml (Fig. 5A) or 10 µg/ml (data not shown). This suggested that an additional interaction with Fc{gamma}RIIa might be involved, and that the whole anti-CD64 might be inhibiting both receptors by binding to Fc{gamma}RIIa through its Fc region.


Figure 5
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  		FIGURE 5. Treatment of PBMC with mAb to Fc{gamma}RI (CD64) blocks the enhanced TNF-{alpha} and IL-1beta responses to CRP-treated R36a. PBMC were incubated for 24 h with 4 x 107 CFU R36a or CRP-treated R36a. The anti-CD64 mAb 10.1, an IgG1 control mAb or the F(ab')2 of mAb 10.1 was added to cultures at 5 µg/ml. TNF-{alpha} concentrations (A) and IL-1beta concentrations (B) in culture supernatants were measured by ELISA. Values are normalized to R36a alone (n = 7–8). Significant differences are indicated by *, p < 0.05, compared with the R36a + CRP control. These experiments included donors of all three Fc{gamma}RIIA genotypes.

 
We tested several different mAb specific for Fc{gamma}RIIa (CD32) and the corresponding isotype controls. As shown in Fig. 6, mAb IV.3 (an IgG2b that binds to Fc{gamma}RIIa), inhibited TNF-{alpha} and IL-1beta responses to CRP-R36a, but not the responses to R36a alone. mAb AT10 (an IgG1) did not inhibit, and mAb FLI8.26 (an IgG2b which binds to both Fc{gamma}RIIa and Fc{gamma}RIIb) inhibited the cytokine responses to both R36a and CRP-R36a (data not shown). Isotype control Abs of the IgG1 (Fig. 5A), IgG2b, and IgG2a subclasses were tested at 5 µg/ml and did not inhibit the TNF-{alpha} response to R36a or CRP-R36a. These results suggest that CRP binding to both Fc{gamma}RI and Fc{gamma}RIIa contributes to the enhanced cytokine response. The addition of mAb 10.1 and IV.3 together had the same inhibitory effect as either added separately (data not shown).


Figure 6
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  		FIGURE 6. Treatment of PBMC with mAb to Fc{gamma}RII (CD32) blocks the enhanced TNF-{alpha} and IL-1beta responses to CRP-treated R36a. PBMC were incubated for 24 h with 4 x 107 CFU R36a or CRP-treated R36a. The anti-CD32 mAb IV.3 or an IgG2 isotype control mAb was added to cultures at 5 µg/ml. TNF-{alpha} concentrations (A) and IL-1beta concentrations (B) in culture supernatants were measured by ELISA. Values are normalized to R36a alone (n = 3). Significant differences are indicated by *, p < 0.05, or **, p < 0.01, compared with the R36a + CRP control.

 
Fc{gamma}RIIa allelic polymorphisms affect CRP-enhanced TNF-{alpha} release

We have reported previously (17), and others have confirmed (39, 40), that CRP binds preferentially to one of two common allelic variants of Fc{gamma}RIIA, the form with arginine (R) rather than histidine (H) at amino acid position 131. We have used individuals heterozygous for these two Fc{gamma}RIIA alleles for the majority of the experiments shown here. However, we wished to determine whether individuals who were homozygous for the high CRP-binding R allele would show a greater response to CRP-opsonization than individuals who were homozygous for the low CRP-binding H allele. Six different homozygous R-131 donors and three different homozygous H-131 donors were tested for the effect of CRP on the TNF-{alpha} response to R36a (Fig. 7). Although only a small number of individuals were tested, greater TNF-{alpha} responses were observed in the R-131 individuals. These results further implicate Fc{gamma}RIIa in this response.


Figure 7
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  		FIGURE 7. Fc{gamma}RIIa allelic polymorphisms affect the enhanced TNF-{alpha} response to CRP-treated R36a. PBMC from different individual donors who were homozygous for either the H-131 allele (HH, n = 3) or the R-131 (RR, n = 6) allele of Fc{gamma}RIIa were incubated for 24 h with 4 x 107 CFU R36a or CRP-treated R36a. TNF-{alpha} concentrations in culture supernatants were measured by ELISA and responses of individual donors to R36a and CRP-treated R36a are shown.

 
Mutagenesis of CRP to decrease Fc{gamma}R binding decreases its ability to enhance the TNF-{alpha} response to R36a

We have recently identified several amino acid residues on CRP that are important in its ability to bind to human Fc{gamma}RI and Fc{gamma}RIIa. To further test the role of CRP binding to Fc{gamma}R, we compared the ability of the T173A mutant and wild-type rCRP to enhance the TNF-{alpha} response to R36a. The T173A mutant has reduced binding to Fc{gamma}RI- and Fc{gamma}RIIa-transfected COS cells as well as to Fc{gamma}RIIa on the K562 cell line, compared with wild-type CRP. In Fig. 8, the TNF-{alpha} responses to CRP-treated R36a are shown relative to the baseline response to R36a. There was a strong enhancement of the TNF-{alpha} response by wild-type rCRP but a greatly reduced response to the mutant CRP.


Figure 8
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  		FIGURE 8. Mutagenesis of CRP to decrease Fc{gamma}R binding decreases its ability to enhance the TNF-{alpha} response to R36a. PBMC were incubated for 24 h with 4 x 107 CFU R36a or R36a treated with 100 µg/ml CRP and washed. Recombinant wild-type (wt) and T173 mutant CRP purified from T. ni larvae were used. Values from two experiments are combined. *, p < 0.05 for mutant vs wt CRP.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results presented here characterize the cytokine response of PBMC to CRP and demonstrate the dependence of this response on Fc{gamma}R. The ability of CRP to induce proinflammatory cytokines like TNF-{alpha} and IL-1beta has been previously established by several investigators (34, 41, 42, 43). Similar to results presented here, CRP in the absence of ligand produces relatively low levels of TNF-{alpha}, IL-1beta, and IL-6 (34). In some studies, CRP also induced the anti-inflammatory cytokines IL-1RA and IL-10 (41, 44, 45). CRP also has been shown to markedly up-regulate the IL-1beta and IL-1RA response to LPS (41, 46). Unlike previous studies, we have determined that CRP binding to a natural ligand, S. pneumoniae, can substantially up-regulate the production of proinflammatory cytokines. The levels of cytokines produced are much higher than those found with CRP alone. Because CRP also increases LPS-induced responses, we carefully removed LPS from CRP and added polymyxin B to cultures. CRP also was shown to induce a proinflammatory cytokine response to an unrelated multivalent ligand, the snRNP particle, to which CRP binds with high affinity. Similar results were obtained with CRP bound to a polyvalent cation, poly-L-lysine (data not shown). Thus, it seems likely that optimal interaction of CRP with Fc{gamma}R to induce cytokine responses requires interactions with multiple receptors and therefore requires binding to a multivalent ligand. The ability of soluble PC to inhibit the response to CRP-treated R36a supports this model, which also is consistent with the requirement for cross-linking in Fc{gamma}R signaling (47).

This study demonstrates that the induction of proinflammatory cytokine synthesis (TNF-{alpha}, IL-1beta) in PBMC cultures by CRP is dependent on Fc{gamma}R activation. CRP binds to both Fc{gamma}RI and Fc{gamma}RIIa on human monocytes (17), and these receptors share signaling pathways that include Syk and PI3K (47). Treatment of cultures with inhibitors of these kinases decreased the cytokine response to CRP-opsonized R36a back to the level of bacteria alone. We confirmed the requirement for Fc{gamma}R binding by showing that a CRP mutant that lacks receptor binding also decreased TNF-{alpha} stimulating ability (33). The wild-type rCRP was fully active in inducing TNF-{alpha} further demonstrating that other human proteins that could potentially contaminate the purified CRP were not required.

CRP-dependent cytokine responses were inhibited by mAb to both Fc{gamma}RI and Fc{gamma}RIIa. Inhibition was equally effective using mAb to either one or both receptors, suggesting a cooperative response. Others have demonstrated cooperation between Fc{gamma}RI and Fc{gamma}RIIa in the uptake of CRP-opsonized particles (39). It is likely that multiple factors determine which of these receptors plays a dominant role in CRP-mediated responses. Fc{gamma}RI expression is increased by cytokines such as IFN-{gamma}, and this treatment dramatically increases CRP binding to monocytes (17). Other factors such as protease activation of the receptors increase the ability of CRP to bind to Fc{gamma}RII (18). CRP also shows higher affinity binding to the R-131, compared with the H-131 allelic form of Fc{gamma}RIIa (39) and neutrophil responses to CRP as measured by calcium influx studies required the R-131 allele (17, 39). Other investigators did not find a difference in responses to CRP between donors homozygous for the R-131 and H-131 allele (48). That study examined neutrophil IL-8 release, phagocytosis, and respiratory burst to CRP-opsonized S. pneumoniae, which may account for the different results. The preferential binding of CRP to the R-131 allele is a difference in affinity and not an absolute difference between the two forms of the receptor. The higher response of donors homozygous for the R-131 allele to CRP-opsonized R36a is consistent with a role for Fc{gamma}RIIa in the TNF-{alpha} response of monocytes to S. pneumoniae.

This study also demonstrates an important role for soluble pattern recognition molecules such as CRP, LBP, and mannose binding protein in the innate response of monocytes and macrophages to bacteria. S. pneumoniae infection induces a strong cytokine response by the host. This response is inflammatory and is both required for host defense and responsible for many of the clinical characteristics of pneumococcal infection. Peptidoglycan and lipoteichoic acid are the primary inflammatory S. pneumoniae cell wall components that interact with TLR2 to induce cytokine release (29). The intracellular protein pneumolysin, which is released following bacterial lysis, also is inflammatory and has been reported to stimulate responses through TLR4 (49). Recently, the acute phase protein, LBP, was shown to bind to the peptidoglycan component and enhance the response to S. pneumoniae cell walls through TLR2 (30). Human monocytes respond to S. pneumoniae by producing TNF-{alpha}, IL-1beta, IL-8, and NO, but the amount of TNF-{alpha} produced in response to pneumococcal cell wall components is low, compared with the response to LPS (28). This cytokine response is crucial for mobilization of the innate immune response to the infection. TNF-{alpha} increases cellular infiltration into tissue sites of infection, increases bactericidal activity of phagocytic cells, and may protect against systemic damage in pneumococcal infection (50). Agents that enhance the early production of these danger signals are expected to increase the ability to fight the infection. CRP is a part of the innate immune response and is secreted rapidly in response to IL-6 and IL-1. The findings presented here indicate that CRP is capable of ramping up this early innate response. A protective role for CRP in S. pneumoniae bacteremia has been described previously by our laboratory and others. In these infection models, CRP protection is, for the most part, complement dependent and Fc{gamma}R independent. Based on the current study and studies of the role of TNF in S. pneumoniae infection, we propose that CRP interaction with Fc{gamma}R may be beneficial in a pulmonary infection model. However, this type of response also would be expected to have deleterious effects in pneumococcal meningitis where the inflammatory response is a major cause of morbidity and mortality.

CRP binds to PC moieties of lipoteichoic acid and teichoic acid of the pneumococcal cell wall. These PC residues are important binding sites for several choline-binding proteins that are virulence factors for the organism and also may interact with platelet-activating factor receptors on cells to facilitate transport of bacterial across cell barriers (51). Lipoteichoic acid lacking PC is less stimulatory for monocytes (28). However, the effect of LBP on cytokine responses to pneumococcal cell walls was independent of PC (30). Thus, the effects of the two acute phase reactants, CRP and LBP, are likely to be additive because they act through different binding sites on the bacterial cell wall and different receptors on the responding cells.

S. pneumoniae produces an acute, fulminant systemic disease in man. The induction of a rapid response by the innate immune system is crucial for the protective, lifesaving response by the host. Recognition of S. pneumoniae by CRP in a pattern recognition manner before the adaptive immune response may provide for a more rapid and intense response by the host, laying the foundation for the full response by the immune system. CRP binding to the organism, at concentrations below those found during the full acute phase response can trigger Fc{gamma}R-bearing cells to produce protective, inflammatory cytokines. Studies from our laboratory and others indicate that, once acute phase levels of CRP are achieved, it plays a regulatory role in the inflammatory response (45, 52, 53).


   Acknowledgments
 
We thank Rufus Burlingame (Inova Diagnostics) for providing purified snRNP and Jeffrey Edberg (University of Alabama at Birmingham, Birmingham, AL) for determining Fc{gamma}RIIa genotypes on the donors.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Department of Veterans Affairs and National Institutes of Health Grant AI28358. Back

2 Address correspondence and reprint requests to Dr. Carolyn Mold, Department of Molecular Genetics and Microbiology, 1 University of New Mexico, MSC08 4660, Albuquerque, NM 87131. E-mail address: cmold{at}salud.unm.edu Back

3 Abbreviations used in this paper: CRP, C-reactive protein; PC, phosphocholine; snRNP, small nuclear ribonucleoprotein; aggIgG, heat-aggregated IgG; CBA, cytometric bead array; RPA, RNase protection assay. Back

Received for publication September 20, 2005. Accepted for publication March 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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