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The Journal of Immunology, 2000, 164: 1514-1520.
Copyright © 2000 by The American Association of Immunologists

C-Reactive Protein Binding to Murine Leukocytes Requires Fc{gamma} Receptors1

Mary-Pat Stein*,{dagger}, Carolyn Mold{ddagger} and Terry W. Du Clos2,*,{dagger}

* Veterans Affairs Medical Center, Albuquerque, NM 87108; and Departments of {dagger} 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
 References
 
Human C-reactive protein (CRP) is an acute phase protein that binds to receptors on human and mouse leukocytes. We have recently determined that the high and low affinity receptors for CRP on human leukocytes are Fc{gamma}RIIa and Fc{gamma}RI, respectively. Previous work by others suggested that CRP receptors on mouse macrophages are distinct from Fc{gamma}R. We have taken advantage of the availability of mice deficient in one or more Fc{gamma}R to reexamine the role of Fc{gamma}R in CRP binding to mouse leukocytes. Three strains of Fc{gamma}R-deficient mice were examined: {gamma}-chain-deficient mice that lack Fc{gamma}RI and Fc{gamma}RIII, Fc{gamma}RII-deficient mice, and mice deficient in both {gamma}-chain and Fc{gamma}RII that lack all Fc{gamma}R. No binding of CRP was detected to leukocytes from double-deficient mice, indicating that Fc{gamma}R are required for CRP binding. CRP binding to leukocytes from {gamma}-chain-deficient and Fc{gamma}RII-deficient mice was reduced compared with binding to leukocytes from wild-type mice. Further analysis of CRP binding to macrophages, neutrophils, and lymphocytes provides direct evidence that Fc{gamma}RIIb1, Fc{gamma}RIIb2, and Fc{gamma}RI are the receptors for CRP on mouse leukocytes. These findings may have important implications in understanding the physiological function of CRP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The C-reactive protein (CRP)3 is an ancient, highly conserved host defense molecule that shares several functional activities with IgG, including specific ligand binding, opsonization of foreign particles (1, 2, 3), activation of complement by binding to C1q (4), and binding to Fc{gamma}R on phagocytic cells (5, 6). CRP is an acute phase protein in humans, with serum levels increasing from less than 1 µg/ml up to 500 µg/ml during an inflammatory response. In contrast, in mice, CRP is present in serum at concentrations of less than 2 µg/ml and does not increase markedly during the acute phase response (7, 8). Because human and rabbit CRP bind to receptors on murine leukocytes and activate mouse complement, injection of human CRP and expression of transgenic human or rabbit CRP in mice have been used to examine the in vivo function of CRP. Human CRP protected mice from a lethal challenge with Streptococcus pneumoniae (9) and altered the sites of clearance of infectious organisms (10). More recently, CRP transgenic mice were shown to be resistant to C5adesArg-induced alveolitis and endotoxemia (8, 11) as well as pneumococcal infection (12). In addition, CRP administration to (NZB x NZW)F1 autoimmune mice prolonged survival and decreased autoantibody levels (13).

CRP binds to receptors on human monocytes, neutrophils, and myeloid cell lines (5, 14). Skatchard analyses suggest that CRP binding to human and mouse macrophages is mediated by both high affinity and low affinity receptors (14, 15). We previously determined that CRP binds to Fc{gamma}RI with low affinity (5, 6). However, until recently, the high affinity receptor for CRP remained unidentified. We have now determined that the major receptor for CRP on human leukocytes is Fc{gamma}RIIa (16). Furthermore, CRP binds preferentially to the R131 and not to the H131 allotype of Fc{gamma}RIIa on human leukocytes (60). This finding suggests that the physiological effects of CRP will vary among human populations based on this differential CRP binding.

Fc{gamma}R provide an important link between humoral and cellular immunity by binding IgG molecules via their Fc regions. Stimulation of cells through Fc{gamma}R results in a wide variety of effector functions, including Ab-dependent cell-mediated cytotoxicity (17), phagocytosis (18, 19), oxidative burst (20, 21, 22), and release of inflammatory mediators (23). Three classes of Fc{gamma}R have been described and reviewed (24, 25, 26). Fc{gamma}RI binds monomeric IgG with high affinity. Fc{gamma}RII and Fc{gamma}RIII are low affinity IgG receptors that bind immune complexes. The ligand-binding domains of Fc{gamma}R are structurally similar, suggesting that the diversity of downstream effector functions associated with IgG binding to Fc{gamma}R results from structural heterogeneity found in the cytoplasmic domains of these receptors (26).

Previous studies demonstrated that CRP binds to several murine macrophage-like cell lines as well as to resident and thioglycolate-elicited macrophages (15). CRP binding to murine macrophages has been reported to activate macrophage tumoricidal activity (27) and to induce phagocytosis (28). Binding was shown to be dose dependent, saturable, reversible, and inhibited by excess unlabeled ligand as well as aggregated IgG. mAb 2.4G2, which inhibits IgG binding to murine Fc{gamma}RII and Fc{gamma}RIII, did not inhibit CRP binding and CRP was unable to inhibit the binding of aggregated IgG. Therefore, it was concluded that a CRP-specific non-FcR mediated CRP binding (15).

Identification of a CRP-specific receptor on human and murine cells bearing multiple Fc{gamma}R has been difficult. Our recent findings that CRP binds to human Fc{gamma}RIIa and Fc{gamma}RI suggest that CRP may also bind to murine Fc{gamma}R. However, the expression of Fc{gamma}R in mice and humans differs, in particular with respect to the low affinity Fc{gamma}R. In mice, only one gene for Fc{gamma}RII expression has been identified (Fc{gamma}RIIB), and differential splicing produces three isoforms: Fc{gamma}RIIb1, which is expressed primarily on lymphocytes; Fc{gamma}RIIb2, which is expressed primarily on myeloid cells; and Fc{gamma}RIIb3, which is a soluble form of Fc{gamma}RII released primarily by macrophages (26). In addition, murine Fc{gamma}RII molecules encode an ITIM-containing sequence in their intracellular domains that negatively regulates ITAM-mediated cell activation. In contrast, three distinct genes for human Fc{gamma}RII exist: Fc{gamma}RIIA, Fc{gamma}RIIB, and Fc{gamma}RIIC. Fc{gamma}RIIA and Fc{gamma}RIIC both encode an ITAM-containing motif in their cytoplasmic tails, while Fc{gamma}RIIB encodes an ITIM motif in its cytoplasmic tail. Fc{gamma}RIII expression differs between mice and humans as well. Mice express only a transmembrane form of Fc{gamma}RIII, whereas humans express both a transmembrane-spanning and a GPI-linked Fc{gamma}RIII. We therefore sought to determine which murine Fc{gamma}R were responsible for CRP binding.

The recent production of Fc{gamma}R-deficient mice by targeted gene disruption provides a valuable tool for determining the role of Fc{gamma}R in CRP binding to murine phagocytic cells. Mice deficient in {gamma}-chain, a molecule required for the expression of Fc{gamma}RI, Fc{gamma}RIII, and Fc{epsilon}RI (29, 30, 31), are deficient in phagocytosis, Ab-dependent cell-mediated cytotoxicity, and mast cell-mediated allergic responses (31). In addition, {gamma}-chain-deficient mice are resistant to experimental immune hemolytic anemia and thrombocytopenia (32) and are less susceptible to autoimmune glomerulonephritis (33). In contrast, mice lacking Fc{gamma}RII are more susceptible to immune complex-mediated alveolitis (34) and to type II collagen-induced arthritis (35). Decreased inflammation associated with the loss of the {gamma}-chain, which associates with Fc{gamma}RI and Fc{gamma}RIII, has been attributed to the loss of an activatory signal produced by the {gamma}-chain’s intracytoplasmic ITAM (36). In contrast, increased inflammation in mice lacking Fc{gamma}RIIb has been attributed to the loss of the Fc{gamma}RIIb intracytoplasmic ITIM (37, 38). A strain of mice lacking {gamma}-chain and Fc{gamma}RII that does not express any functional Fc{gamma}R has also been produced.

The current study was undertaken to determine the role of Fc{gamma}R in CRP binding to murine leukocytes. Cells from Fc{gamma}R-deficient mice fail to bind CRP, indicating that in the mouse, as in humans, CRP binding occurs through Fc{gamma}R. Analyses of CRP binding to leukocytes from {gamma}-chain-deficient and Fc{gamma}RII-deficient strains of mice indicate that CRP binds to Fc{gamma}RI and Fc{gamma}RII on macrophages and neutrophils and to Fc{gamma}RII on B cells. These findings may provide insight into the mechanisms by which CRP produced potent anti-inflammatory activity in previous in vivo studies of CRP transgenic and CRP-injected mice. Identification of the receptors to which CRP binds will aid in our understanding of CRP-mediated regulation of infectious, inflammatory, and autoimmune disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Male and female C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and were used as controls for the {gamma}-chain-/- mice. Female and male B6 x 129 F2J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were used as controls for the Fc{gamma}RII-/- and the {gamma}-chain-/-/Fc{gamma}RII-/- mice. Male, 4–8 wk old, {gamma}-chain-/- (31), Fc{gamma}RII-/- (39), and {gamma}-chain-/-/Fc{gamma}RII-/- mice were purchased from Taconic Farms (Westminster, NY). All mice were housed in conventional caging and were used between 2 and 5 mo of age.

Reagents and Abs

Human CRP was purified from pleural fluid by affinity chromatography and ion-exchange chromatography, as previously described (40). Sodium azide and essentially Ig-free BSA were purchased from Sigma (St. Louis, MO). PE-conjugated F(ab')2 goat anti-mouse IgG (PE-GAM) and PE-conjugated F(ab')2 goat anti-rat IgG (PE-GAR) were purchased from Caltag Laboratories (Burlingame, CA). PE-conjugated rat anti-mouse CD16/32 (PE-2.4G2), PE-conjugated rat anti-mouse CD2, PE-conjugated rat anti-mouse CD45R/B220, rat anti-mouse CD90.2, and rat anti-mouse Ly-6G (Gr-1) Abs were purchased from PharMingen (San Diego, CA). Rat anti-mouse F4/80 Ag was purchased from Serotec (Raleigh, NC). Abs were used at the manufacturer’s suggested working concentrations. The mAb 2C10, a murine IgG1 anti-human CRP, was the generous gift of Dr. Larry Potempa (ImmTech International, Evanston, IL) and was purified and FITC conjugated in our laboratory.

Cells

Elicited peritoneal exudate cells (PEC) were obtained by peritoneal lavage of mice injected with 1 ml of 3% thioglycolate medium (Difco Laboratories, Detroit, MI) 5 days (for macrophages) or 16–24 h (for neutrophils) before harvest. Cells were collected in 10 ml of HBSS without calcium and magnesium (HBSS-). Cells were washed twice in ice-cold PBS containing 0.05% sodium azide and 0.1% BSA (PAB). Cells were counted and then distributed into tubes for staining. An aliquot of cells was removed, centrifuged onto slides, and stained with the Diff-Quik Stain Set (Dade International, Miami, FL). Greater than 50% of the elicited PEC were monocytes/macrophages after a 5-day elicitation, whereas greater than 60% of the PEC were neutrophils after a 16–24-h elicitation.

To examine CRP binding to NK cells, B cells, and T cells, spleens were removed from mice. Single cell suspensions were obtained by passing spleens through a 200-µm wire mesh screen. Cells were washed in HBSS-, and RBC were lysed by incubation in 0.15 M ammonium chloride, 1 mM potassium carbonate, and 0.1 mM disodium EDTA, pH 7.4, for 5 min at room temperature. Cells were washed twice in ice-cold PAB before analysis of CRP binding. Greater than 95% of the cells were lymphocytes, as determined by Diff-Quik staining.

Bone marrow cells obtained from femurs of mice were washed in HBSS- and incubated at 5 x 105 cells/ml in HBSS- in the presence or absence of 50 µg/ml of pronase E (Sigma) for 30 min at 37°C with gentle swirling every 10 min. After pronase treatment, cells were washed three times in ice-cold PAB. CRP binding was performed as described below. Bone marrow cells were ~50% neutrophils, 30% lymphocytes, and 20% mononuclear cells, as determined by Diff-Quik staining.

CRP-binding assay

PEC and bone marrow neutrophils were incubated in the presence of human CRP at the concentrations indicated for 1 h on ice. Cells were then washed twice with 1 ml of PAB and incubated in the presence of mAb 2C10, rat anti-mouse F4/80, or rat anti-mouse Ly-6G. Following a 30-min incubation at 4°C, cells were washed twice with 1 ml PAB. Cells were then incubated for 30 min with PE-GAM, PE-GAR, or PE-2.4G2. Cells were washed twice with 1 ml PAB and then resuspended in 0.25–0.5 ml PAB for analysis by flow cytometry.

CRP binding to splenic NK cells, B cells, and T cells was analyzed as described above, except that the mAb 2C10 was directly conjugated with FITC and was used at a final concentration of 10 µg/ml. Two-color analyses of CRP binding to NK cells, B cells, and T cells were performed by incubating spleen cells with 400 µg/ml CRP alone or in combination with rat anti-CD90.2 for 1 h on ice. Unbound CRP or anti-CD90.2 was removed by two washes with ice-cold PAB. Cells were then incubated in PAB containing FITC-2C10/PE-NK1.1, FITC-2C10/PE-CD45R, or FITC-2C10/PE-GAR for 30 min on ice before analysis by flow cytometry.

Flow cytometry

Cells were analyzed on a Becton Dickinson FACSCalibur flow cytometer. Cells were collected, and dead cells were excluded from fluorescence analysis based on forward and side scatter characteristics. Cell fluorescence was analyzed using CellQuest software (Becton Dickinson, Mountain View, CA). All fluorescence data were collected on a log scale, and data are reported as geometric mean fluorescence intensities (GMFI). CRP binding is calculated as the change in GMFI between cells incubated with CRP and secondary Abs compared with Abs alone.

Data analysis

Dose-dependent binding curves of CRP to C57BL/6, {gamma}-chain-/-, and Fc{gamma}RII-/- cells were generated using GraphPad PRISM software (GraphPad Software, San Diego, CA). Data shown are representative of at least two experiments. Binding of CRP was analyzed by nonlinear regression analysis using GraphPad Prism software to generate a best-fit curve by minimizing the sum of the squares of the vertical distances of the data points from the curve (using the method of Levenberg and Marquardt (41)). Kd values were then derived from the generated binding curves.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CRP binding to PEC requires Fc{gamma}R expression

To determine whether Fc{gamma}R are required for CRP binding to murine macrophages, CRP binding to F4/80-positive PEC from five strains of mice was examined. C57BL/6 mice served as controls for the {gamma}-chain-/- mice, while B6 x 129 F2J mice provided controls for both the Fc{gamma}RII-/- and the {gamma}-chain-/-/Fc{gamma}RII-/- mice. Peritoneal macrophages were identified in total PEC suspensions by reactivity with a rat mAb to F4/80, a plasma membrane glycoprotein found on mature macrophages (42). An F4/80-positive population of cells was identified by forward and side scatter characteristics, and these cells were analyzed for CRP binding. F4/80-positive PEC were also examined for reactivity with 2.4G2 to determine the levels of Fc{gamma}RII and Fc{gamma}RIII expression.

In Fig. 1Go, CRP binding is compared with background binding of detecting Abs alone. Approximately 95% of the F4/80-positive PEC from wild-type C57BL/6 mice bound CRP with a change in GMFI of 105. In contrast, 43% of the F4/80-positive PEC from the {gamma}-chain-/- mice bound CRP with a change in GMFI of 423. CRP bound to ~95% of wild-type B6 x 129 F2J F4/80-positive PEC with a change in GMFI of 131, similar to that described for C57BL/6 wild-type macrophages. Interestingly, CRP binding to the Fc{gamma}RII-/- mice was similar to CRP binding to the {gamma}-chain-/- PEC in that two distinct populations of cells were present. Approximately 70% of the F4/80-positive PEC from Fc{gamma}RII-/- mice bound CRP with a change in GMFI of 92, while the remaining 30% showed no detectable CRP binding. No CRP binding to F4/80-positive cells from {gamma}-chain-/-/Fc{gamma}RII-/- double-deficient mice was observed, suggesting that Fc{gamma}R are required for CRP binding to murine peritoneal macrophages.



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  		FIGURE 1. CRP binding to F4/80-positive PEC from wild-type and Fc{gamma}R-deficient mice. Five-day thioglycolate-elicited PEC were harvested from wild-type C57BL/6 (A and F), {gamma}-chain-/- (B and G), wild-type B6 x 129 F2J (C and H), Fc{gamma}RII-/- (D and I), and {gamma}-chain-/-/Fc{gamma}RII-/- (E and J) mice. Cells were incubated with 200 µg/ml CRP for 1 h on ice. CRP binding to F4/80-positive PEC was detected using mAb 2C10 and PE-GAM. Histograms in A–E compare the background binding of detecting Abs alone (dotted lines) with binding of CRP (solid lines). Histograms in F–J compare the autofluorescence of F4/80-positive cells (dotted lines) with binding of PE-2.4G2 (solid lines).

 
As total CRP binding may reflect binding to all three Fc{gamma}R, we analyzed Fc{gamma}RII and Fc{gamma}RIII expression on elicited macrophages. The levels of CD32 (Fc{gamma}RII) and CD16 (Fc{gamma}RIII) on F4/80-positive PEC, as determined by mAb 2.4G2 reactivity, were compared (Fig. 1Go). F4/80-positive PEC from wild-type C57BL/6 and B6 x 129 F2J mice display similar 2.4G2 reactivity. Interestingly, the {gamma}-chain-/- and Fc{gamma}RII-/- F4/80-positive PEC both display two populations of 2.4G2-positive cells; one population of highly 2.4G2-reactive cells, with GMFIs equivalent to those expressed by wild-type macrophages, and a second population of cells that display significantly less 2.4G2 reactivity. As expected, virtually no reactivity with 2.4G2 is detected on macrophages from the {gamma}-chain-/-/Fc{gamma}RII-/- mice.

CRP binding to macrophages is dose dependent and saturable

To further assess the relative contributions of Fc{gamma}R to CRP binding to murine F4/80-positive cells, dose-response curves were generated (Fig. 2Go). CRP binding to thioglycolate-elicited F4/80-positive PEC from wild-type C57BL/6 mice and {gamma}-chain-/- mice was compared. CRP binding to both the wild-type and {gamma}-chain-/- macrophages was dose dependent and saturable, with saturation reached at ~100 µg/ml. However, macrophages from {gamma}-chain-/- mice bound less CRP than macrophages from wild-type mice. The calculated Kd value for wild-type macrophages was 3.8 x 10-7 M, while that for macrophages from {gamma}-chain-/- mice was 1.9 x 10-7 M. Dose-response curves were also generated for CRP binding to macrophages from B6 x 129 F2J, Fc{gamma}RII-/-, and {gamma}-chain-/-/Fc{gamma}RII-/- mice (Fig. 2Go). Similar to the binding of CRP to C57BL/6 and {gamma}-chain-/- macrophages, CRP binding to wild-type B6 x 129 F2J F4/80-positive PEC was dose dependent and saturable, although saturation occurred at ~200 µg/ml CRP. In addition, wild-type B6 x 129 F2J F4/80-positive PEC bound significantly more CRP than the Fc{gamma}RII-/- F4/80-positive PEC, and the calculated Kd values were 9.1 x 10-7 M vs 7.7 x 10-7 M for the B6 x 129 F2J and Fc{gamma}RII-/- macrophages, respectively. These Kd values demonstrate a 2-fold decrease in affinity compared with the C57BL/6 and {gamma}-chain-/- macrophages. When directly compared, the amount of CRP binding to C57BL/6 macrophages was always greater than CRP binding to B6 x 129 F2J macrophages (data not shown). Thus, genetic differences between mice may affect the level of CRP binding to murine macrophages. Finally, CRP binding to {gamma}-chain-/-/Fc{gamma}RII-/- macrophages was not detected. These data suggest that not only are Fc{gamma}R required for CRP binding to murine macrophages, but to attain maximum CRP binding, expression of more than one class of Fc{gamma}R is required.



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  		FIGURE 2. Dose-dependent CRP binding to F4/80-positive 5-day thioglycolate-elicited macrophages. A population of cells that was positive for F4/80 staining was identified by forward and side scatter characteristics and gated on for analysis of CRP binding. The background fluorescence of cells due to binding of the detecting Abs alone, mAb 2C10 and PE-GAM, was subtracted from cells positive for CRP binding, resulting in a net change in GMFI for each concentration of CRP tested. Data were analyzed using GraphPad PRISM software, and binding curves were generated by nonlinear regression analysis. A, CRP binding to macrophages from C57BL/6 and {gamma}-chain-/- mice. B, CRP binding to macrophages from B6 x 129 F2J, Fc{gamma}RII-/-, and {gamma}-chain-/-/Fc{gamma}RII-/- mice.

 
CRP does not bind to Fc{gamma}RIII on splenic NK cells

As all three classes of Fc{gamma}R may be present on elicited murine macrophages (25), determining which classes of Fc{gamma}R are responsible for CRP binding to murine macrophages was difficult. As no CRP binding was detected on macrophages from mice deficient in Fc{gamma}R expression, CRP binding to macrophages from {gamma}-chain-/- mice was therefore mediated by binding to murine Fc{gamma}RIIb. However, macrophages from Fc{gamma}RII-/- mice, which express both Fc{gamma}RI and Fc{gamma}RIII, also bound CRP. To determine whether CRP binds to murine Fc{gamma}RIII, CRP binding to murine splenic NK cells was examined (Fig. 3Go, A and D). Spleen cells from wild-type C57BL/6 mice were isolated and stained with a PE-conjugated rat mAb to NK1.1, a marker for NK cells. FITC-2C10 was used to analyze CRP binding by two-color analysis of the NK1.1-positive spleen cells. In the presence of CRP, NK1.1-positive cells did not increase in FITC fluorescence (Fig. 3Go). These data suggest that CRP does not bind to mouse Fc{gamma}RIII, and that mouse Fc{gamma}RI may be responsible for CRP binding to Fc{gamma}RII-/- macrophages.



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  		FIGURE 3. CRP binding to spleen cells from C57BL/6 wild-type mice. Two-color fluorescence was used to examine CRP binding. PE-conjugated rat mAb to NK1.1 (A and D), CD90.2 (B and E), and CD45R/B220 (C and F) were used to identify NK, T, and B cells, respectively. CRP binding was analyzed by incubating cells in the presence or absence of 400 µg/ml CRP, followed by staining with FITC-2C10. A–C, Cells incubated with mAb for cell surface markers and FITC-2C10. D–F, Staining with mAb for cell surface markers, CRP and FITC-2C10.

 
Splenic B cells, but not T cells, bind CRP

CRP binding to splenic T cells and B cells from C57BL/6 wild-type mice was examined (Fig. 3Go). Approximately 55% of the spleen cells were identified as T cells by mAb CD90.2 binding. No detectable binding of CRP to T cells was seen. In contrast, 45% of the spleen cells stained positive for B220, a B cell marker. These cells bound CRP, with a shift in GMFI from 8.5 in the absence of CRP to 65.5 in the presence of 400 µg/ml CRP. These data demonstrate that CRP binds to Fc{gamma}RIIb1, the only Fc{gamma}R present on B cells.

To compare CRP binding to Fc{gamma}RIIb2 on macrophages with CRP binding to Fc{gamma}RIIb1 on splenic B cells, CRP binding to murine B cells was analyzed by generating dose-response binding curves by two-color fluorescence (Fig. 4Go). Splenic B cells from C57BL/6 wild-type mice demonstrated dose-dependent binding of CRP. CRP binding saturated at ~200 µg/ml, with an apparent affinity of 75 µg/ml (6.5 x 10-7 M). CRP binding to {gamma}-chain-/- splenic B cells was also dose dependent and saturated at ~100 µg/ml. Unexpectedly, total binding to the {gamma}-chain-/- mice was approximately one-half that of binding to C57BL/6 B cells, with an apparent affinity of 33 µg/ml (2.8 x 10-7 M). CRP binding to B cells from B6 x 129 F2 was dose dependent and saturated at ~100 µg/ml. No CRP binding to Fc{gamma}RII-/- or {gamma}-chain-/-/Fc{gamma}RII-/- splenic B cells was detected at any concentration of CRP tested. B cells from C57BL/6, {gamma}-chain-/-, and B6 x 129 F2J mice displayed similar 2.4G2 binding, while B cells from Fc{gamma}RII-/- and {gamma}-chain-/-/Fc{gamma}RII-/- bound significantly less 2.4G2 (data not shown).



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  		FIGURE 4. Dose-dependent CRP binding to CD45R/B220-positive splenic B cells. B220-positive spleen cells from wild-type and Fc{gamma}R-deficient mice were analyzed for CRP binding using FITC-2C10. The change in GMFI for each concentration of CRP tested was determined by subtracting the GMFI of cells in the presence of FITC-2C10 without CRP. Data were analyzed using GraphPad PRISM software, and binding curves were generated by nonlinear regression analysis. A, CRP binding to B cells from C57BL/6 and {gamma}-chain-/- mice. B, CRP binding to B cells from B6 x 129 F2J, Fc{gamma}RII-/-, and {gamma}-chain-/-/Fc{gamma}RII-/-.

 
CRP binding to bone marrow neutrophils is enhanced by pronase treatment

Mouse and human neutrophils express Fc{gamma}RII and Fc{gamma}RIII. Because CRP binds to human neutrophils through Fc{gamma}RII, CRP binding to murine neutrophils was analyzed. First, CRP binding to 1-day elicited PEC was examined. CRP bound to Ly-6G (a GPI-linked cell surface protein found on mature granulocytes)-positive cells; however, the shift in GMFI was minimal (data not shown). Bone marrow-derived neutrophils were also examined for CRP binding. Neutrophils constitute ~50% of total bone marrow-derived cells, as determined by cytospin staining. A Ly-6G-positive population was identified by forward and side scatter characteristics, and these cells were analyzed for CRP binding (Fig. 5Go). A small but significant shift in fluorescence from background binding of Abs alone to binding of 400 µg/ml CRP is detected in wild-type C57BL/6 and B6 x 129 F2J neutrophils. A smaller, but still significant increase in CRP binding was also detected on the {gamma}-chain-/- neutrophils. However, no increase in CRP binding was detected for either the Fc{gamma}RII-/- or the {gamma}-chain-/-/Fc{gamma}RII-/- neutrophils. These data further support the finding that in the absence of all Fc{gamma}R, no CRP binding is detectable. These data also suggest that the primary receptor for CRP on murine neutrophils is Fc{gamma}RIIb.



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  		FIGURE 5. CRP binding to bone marrow neutrophils. Bone marrow cells were harvested from wild-type C57BL/6 (A and F), {gamma}-chain-/- (B and G), wild-type B6 x 129 F2J (C and H), Fc{gamma}RII-/- (D and I), and {gamma}-chain-/-/Fc{gamma}RII-/- (E and J) mice. Cells were washed in HBSS- and RBCs were lysed. Cells were incubated in HBSS- (A–E) or 50 µg/ml pronase in HBSS- (F–J) for 30 min at 37°C. Cells were washed three times in ice-cold PAB, and CRP binding was determined, as described previously. Cells staining for Ly-6G, a murine granulocyte marker, were gated based on forward and side scatter characteristics. Histograms shown in all panels compare the background binding of mAb 2C10 and PE-GAM (dotted lines) with binding of 200 µg/ml CRP (solid lines) to bone marrow neutrophils.

 
Van de Winkel et al. (43) previously demonstrated that proteolytic treatment of cells and cell lines bearing Fc{gamma}RIIa results in enhanced binding of IgG (43). Recent work in our laboratory has demonstrated that proteolytic treatment of human monocytes and neutrophils also enhances CRP binding (60). Human neutrophils demonstrated a significant increase in CRP binding following pronase treatment. Therefore, we examined CRP binding to murine bone marrow neutrophils from wild-type and Fc{gamma}R-deficient mice following pronase treatment (Fig. 5Go). CRP binding to Ly-6G-positive bone marrow cells from C57BL/6, B6 x 129 F2J, {gamma}-chain-/-, and Fc{gamma}RII-/- mice was significantly enhanced by pronase treatment. Pronase treatment, however, did not increase CRP binding to Ly-6G-positive cells from {gamma}-chain-/-/Fc{gamma}RII-/- mice. These data indicate that CRP binding to murine bone marrow neutrophils is enhanced by proteolytic activation similar to CRP binding to human Fc{gamma}R. However, CRP binding to Fc{gamma}RII-/- neutrophils was also enhanced, suggesting that in mice, an Fc{gamma}R other than Fc{gamma}RIIb is also susceptible to proteolytic activation.

To compare CRP binding to neutrophils with the binding to elicited macrophages and splenic B cells, dose-response binding curves for CRP binding to C57BL/6 bone marrow-derived Ly-6G-positive cells before and after pronase treatment were generated (Fig. 6Go). CRP binding to untreated bone marrow neutrophils was minimal at all concentrations tested. However, following pronase treatment, CRP binding was dramatically increased. CRP binding to pronase-treated cells reached saturation at ~200 µg/ml with an apparent affinity for CRP of 117 µg/ml (1 x 10-6 M). Thus, pronase-treated neutrophils bind CRP with a lower affinity than elicited macrophages (3.8 x 10-7 M) or splenic B cells (6.5 x 10-7 M).



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  		FIGURE 6. Dose-dependent CRP binding to C57BL/6 bone marrow-derived neutrophils with and without pronase treatment. Ly-6G-positive bone marrow cells from C57BL/6 mice were analyzed for CRP binding using mAb 2C10 and PE-GAM. The change in GMFI for each concentration of CRP tested was determined by subtracting the GMFI of cells in the presence of 2C10 and PE-GAM without CRP. Data were analyzed and binding curves were generated by nonlinear regression analysis using GraphPad PRISM software.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Identification of the receptor for CRP on human and murine leukocytes has been the subject of intense research for many years. Work by Zahedi and Mortensen (15) demonstrated that CRP binding to murine macrophages was mediated by two distinct receptors. One receptor bound CRP with an apparent affinity of 1.25 x 10-8 M, with ~105 binding sites per cell present, while the second was a lower affinity receptor. Both CRP receptors were thought to be distinct from Fc{gamma}R based on the inability of mAb 2.4G2, which blocks IgG binding to Fc{gamma}RII and Fc{gamma}RIII, to inhibit CRP binding. Recent work in our laboratory has demonstrated that the high and low affinity receptors for CRP on human leukocytes are Fc{gamma}RIIa and Fc{gamma}RI, respectively (5, 6, 16). Therefore, we wanted to determine whether CRP binds to murine Fc{gamma}R and whether receptors other than Fc{gamma}R are required for CRP binding to murine leukocytes.

Analysis of CRP binding to human and murine cells has been complicated by the expression of multiple Fc{gamma}R on leukocytes. Although a family of individuals that are Fc{gamma}RI deficient has been reported (44), no humans devoid of all Fc{gamma}R expression have been identified. The availability of mice that do not express subsets of murine Fc{gamma}R has allowed us to examine CRP binding to murine cells in the presence or absence of specific Fc{gamma}R. Peritoneal macrophages and neutrophils, splenic lymphocytes, and bone marrow-derived neutrophils were isolated from C57BL/6, B6 x 129 F2J, {gamma}-chain-/-, Fc{gamma}RII-/-, and {gamma}-chain-/-/Fc{gamma}RII-/- mice, and CRP binding was analyzed by flow cytometry. The flow-cytometric assay allowed us to analyze CRP binding to subpopulations of cells and to use unmodified CRP as a ligand. Differences between Kd values reported in this work and those reported previously (15) might be due to the methods used to analyze CRP binding as prior studies used radiolabeled CRP.

No detectable CRP binding to resident (data no shown) or elicited macrophages from Fc{gamma}R-deficient mice ({gamma}-chain-/-/Fc{gamma}RII-/-) was observed. Similarly, no CRP binding to splenic B cells or bone marrow-derived neutrophils from Fc{gamma}R-deficient mice was detected. This work demonstrates for the first time that the expression of Fc{gamma}R is required for CRP binding to murine leukocytes.

Furthermore, results using mice deficient in subsets of Fc{gamma}R suggest that in the mouse, as in humans, CRP binds to more than one class of Fc{gamma}R. CRP binding to {gamma}-chain-/- macrophages and neutrophils that do not express functional Fc{gamma}RI or Fc{gamma}RIII was mediated by binding to Fc{gamma}RIIb2. CRP binding to Fc{gamma}RII-/- macrophages and neutrophils was also observed, suggesting that CRP binds to either Fc{gamma}RI or Fc{gamma}RIII. No CRP binding to NK cells, which express Fc{gamma}RIII as their sole Fc{gamma}R, was detected, even though murine Fc{gamma}RIII receptors share ~95% amino acid identity in their extracellular domains with murine Fc{gamma}RIIb receptors (26). Thus, CRP binding to macrophages from Fc{gamma}RII-/- mice is most likely mediated by binding to Fc{gamma}RI. These findings are consistent with results using human NK cells in which CRP binding to Fc{gamma}RIII was not detected (60). Interestingly, the apparent affinities for CRP binding by macrophages from Fc{gamma}RII-/- mice and {gamma}-chain-/- mice did not differ significantly from each other. However, total binding of macrophages from Fc{gamma}R-deficient strains was reduced compared with their respective controls, suggesting that expression of multiple Fc{gamma}R increased the number of available CRP receptors.

Because protease treatment of cells results in enhanced IgG binding to human Fc{gamma}RIIa (43, 45), we sought to determine whether murine receptors could also be proteolytically activated. Increased CRP binding to bone marrow neutrophils following pronase treatment of cells was observed similar to results reported for human neutrophils (60). Elicited peritoneal macrophages demonstrated only a modest enhancement of CRP binding following pronase treatment (data not shown), and CRP binding to Fc{gamma}RIIb1 on B cells was not affected (data not shown). Because Fc{gamma}RII is the only Fc{gamma}R known to be proteolytically activated, the finding that pronase treatment of Fc{gamma}RII-/- neutrophils enhanced CRP binding was unexpected. Because no increase in CRP binding was detected on neutrophils in the absence of Fc{gamma}R, and because murine bone marrow neutrophils may express both Fc{gamma}RI and Fc{gamma}RIII, the data from the Fc{gamma}RII-/- neutrophils suggest that an additional murine Fc{gamma}R is activated by pronase treatment.

The proteolytic enhancement of CRP binding to Fc{gamma}RIIb2 described in this work may have important in vivo implications. Although the mechanism of enhanced IgG binding following protease treatment has not yet been determined, recent work suggests that increased mobility of Fc{gamma}R on the cell surface may lead to increased binding by clustering receptors (46). At sites of tissue damage and inflammation, neutrophils and macrophages release many proteases such as elastase and cathepsin G (47) that may enhance CRP binding in vivo. Enhanced CRP binding to macrophages and neutrophils at sites of tissue damage or infection may result in enhanced clearance of pathogenic bacteria or cell debris from these sites. CRP binding to Fc{gamma}R on macrophages may also result in downstream events such as the production of reactive oxygen intermediates, proteases, or cytokines that may alter the inflammatory response. Further analysis of downstream signaling events following CRP binding to murine macrophages and neutrophils will be the focus of future studies.

CRP binding to Fc{gamma}RIIb1 on splenic B cells was also demonstrated. Because Fc{gamma}RIIb1 is an important regulator of inflammatory responses of mice (37), this result may also have important implications in vivo. Fc{gamma}RIIb1 binding activates an ITIM (48) that recruits an inositol phosphatase, SH2-containing inositol phosphatase (SHIP) (49). SHIP inhibits ITAM-mediated cell activation (50, 51, 52). CRP has antiinflammatory activity in vivo in transgenic mice challenged with endotoxin, platelet-activating factor, and other inflammatory stimuli, including IL-1ß and TNF-{alpha} (8). A possible mechanism for these in vivo activities is that CRP binds to murine Fc{gamma}RIIb, leading to down-regulation of the inflammatory response.

Although the ability of CRP to bind to a variety of ligands has been well described, the precise in vivo functions of CRP remain unknown. CRP has been localized in vivo to inflammatory lesions in rheumatoid arthritis (53), experimental allergic encephalomyelitis (54), and myocardial infarction (55, 56), while in a mouse model of infection, CRP protects mice from a lethal challenge with S. pneumoniae (9, 57). Although previous work described CRP as a proinflammatory agent (reviewed in Ref. 58), more recent work describes the anti-inflammatory properties of CRP (8, 11, 59). Identification of ITAM-containing receptors (Fc{gamma}RI) as well as an ITIM-containing receptor (Fc{gamma}RIIb1) as CRP receptors may help to explain the conflicting observations made previously. Further analysis of the interaction of CRP with these receptors will aid in our understanding of the mechanisms by which CRP influences the inflammatory response.


   Acknowledgments
 
We thank Dr. Larry Potempa of ImmTech for the mAb 2C10, and Ruobing Xiao for excellent technical assistance.


   Footnotes
 
1 This work was supported in part by the Veterans Administration and by National Institutes of Health Grant AI28358. Back

2 Address correspondence and reprint requests to Dr. Terry W. Du Clos, VA Medical Center, Research Service 151, 1501 San Pedro S.E., Albuquerque, NM 87108. E-mail address: Back

3 Abbreviations used in this paper: CRP, C-reactive protein; GAM, goat anti-mouse; GAR, goat anti-rat; GMFI, geometric mean fluorescence intensity; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; PEC, peritoneal exudate cell. Back

Received for publication September 3, 1999. Accepted for publication November 18, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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