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Receptors1
*
Veterans Affairs Medical Center, Albuquerque, NM 87108; and Departments of
Medicine and
Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIa and FcRI, respectively. Previous work by others
suggested that CRP receptors on mouse macrophages are distinct from
FcR. We have taken advantage of the availability of mice deficient
in one or more FcR to reexamine the role of FcR in CRP binding to
mouse leukocytes. Three strains of FcR-deficient mice were examined:
-chain-deficient mice that lack FcRI and FcRIII,
FcRII-deficient mice, and mice deficient in both -chain and
FcRII that lack all FcR. No binding of CRP was detected to
leukocytes from double-deficient mice, indicating that FcR are
required for CRP binding. CRP binding to leukocytes from
-chain-deficient and FcRII-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 FcRIIb1, FcRIIb2, and FcRI are the receptors for
CRP on mouse leukocytes. These findings may have important implications
in understanding the physiological function of
CRP. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 FcRI 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 FcRIIa
(16). Furthermore, CRP binds preferentially to the R131
and not to the H131 allotype of FcRIIa on human leukocytes
(60). This finding suggests that the
physiological effects of CRP will vary among human populations based on
this differential CRP binding.
FcR provide an important link between humoral and cellular immunity
by binding IgG molecules via their Fc regions. Stimulation of cells
through FcR 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 FcR have been described and
reviewed (24, 25, 26). FcRI binds monomeric IgG with high
affinity. FcRII and FcRIII are low affinity IgG receptors that
bind immune complexes. The ligand-binding domains of FcR are
structurally similar, suggesting that the diversity of downstream
effector functions associated with IgG binding to FcR 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
FcRII and FcRIII, 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 FcR has been difficult. Our recent findings that
CRP binds to human FcRIIa and FcRI suggest that CRP may also bind
to murine FcR. However, the expression of FcR in mice and humans
differs, in particular with respect to the low affinity FcR. In
mice, only one gene for FcRII expression has been identified
(FcRIIB), and differential splicing produces three isoforms:
FcRIIb1, which is expressed primarily on lymphocytes; FcRIIb2,
which is expressed primarily on myeloid cells; and FcRIIb3, which is
a soluble form of FcRII released primarily by macrophages
(26). In addition, murine FcRII molecules encode an
ITIM-containing sequence in their intracellular domains that negatively
regulates ITAM-mediated cell activation. In contrast, three distinct
genes for human FcRII exist: FcRIIA, FcRIIB, and FcRIIC.
FcRIIA and FcRIIC both encode an ITAM-containing motif in their
cytoplasmic tails, while FcRIIB encodes an ITIM motif in its
cytoplasmic tail. FcRIII expression differs between mice and humans
as well. Mice express only a transmembrane form of FcRIII, whereas
humans express both a transmembrane-spanning and a GPI-linked
FcRIII. We therefore sought to determine which murine FcR were
responsible for CRP binding.
The recent production of FcR-deficient mice by targeted gene
disruption provides a valuable tool for determining the role of FcR
in CRP binding to murine phagocytic cells. Mice deficient in -chain,
a molecule required for the expression of FcRI, FcRIII, and
Fc
RI (29, 30, 31), are deficient in phagocytosis,
Ab-dependent cell-mediated cytotoxicity, and mast cell-mediated
allergic responses (31). In addition, -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 FcRII 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 -chain, which associates with FcRI and FcRIII, has been
attributed to the loss of an activatory signal produced by the
-chain’s intracytoplasmic ITAM (36). In contrast,
increased inflammation in mice lacking FcRIIb has been attributed to
the loss of the FcRIIb intracytoplasmic ITIM (37, 38).
A strain of mice lacking -chain and FcRII that does not express
any functional FcR has also been produced.
The current study was undertaken to determine the role of FcR in CRP
binding to murine leukocytes. Cells from FcR-deficient mice fail to
bind CRP, indicating that in the mouse, as in humans, CRP binding
occurs through FcR. Analyses of CRP binding to leukocytes from
-chain-deficient and FcRII-deficient strains of mice indicate
that CRP binds to FcRI and FcRII on macrophages and neutrophils
and to FcRII 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male and female C57BL/6 mice were purchased from the National
Cancer Institute (Frederick, MD) and were used as controls for the
-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
FcRII-/- and the
-chain-/-/FcRII-/-
mice. Male, 4–8 wk old, -chain-/-
(31), FcRII-/-
(39), and
-chain-/-/FcRII-/-
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,
-chain-/-, and
FcRII-/- 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R expression
To determine whether FcR 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
-chain-/- mice, while B6 x 129 F2J
mice provided controls for both the FcRII-/-
and the
-chain-/-/FcRII-/-
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 FcRII and FcRIII expression.
In Fig. 1
, 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
-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 FcRII-/- mice was similar to CRP
binding to the -chain-/- PEC in that two
distinct populations of cells were present. Approximately 70% of the
F4/80-positive PEC from FcRII-/- 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
-chain-/-/FcRII-/-
double-deficient mice was observed, suggesting that FcR are required
for CRP binding to murine peritoneal macrophages.
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R, we
analyzed FcRII and FcRIII expression on elicited macrophages. The
levels of CD32 (FcRII) and CD16 (FcRIII) on F4/80-positive PEC,
as determined by mAb 2.4G2 reactivity, were compared (Fig. 1
).
F4/80-positive PEC from wild-type C57BL/6 and B6 x 129 F2J mice
display similar 2.4G2 reactivity. Interestingly, the
-chain-/- and
FcRII-/- 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
-chain-/-/FcRII-/-
mice. CRP binding to macrophages is dose dependent and saturable
To further assess the relative contributions of FcR to CRP
binding to murine F4/80-positive cells, dose-response curves were
generated (Fig. 2). CRP binding to
thioglycolate-elicited F4/80-positive PEC from wild-type C57BL/6 mice
and -chain-/- mice was compared. CRP binding
to both the wild-type and -chain-/-
macrophages was dose dependent and saturable, with saturation reached
at 100 µg/ml. However, macrophages from
-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 -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,
FcRII-/-, and
-chain-/-/FcRII-/-
mice (Fig. 2). Similar to the binding of CRP to C57BL/6 and
-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 FcRII-/-
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
FcRII-/- macrophages, respectively. These
Kd values demonstrate a 2-fold
decrease in affinity compared with the C57BL/6 and
-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
-chain-/-/FcRII-/-
macrophages was not detected. These data suggest that not only are
FcR required for CRP binding to murine macrophages, but to attain
maximum CRP binding, expression of more than one class of FcR is
required.
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RIII on splenic NK cells
As all three classes of FcR may be present on elicited murine
macrophages (25), determining which classes of FcR are
responsible for CRP binding to murine macrophages was difficult. As no
CRP binding was detected on macrophages from mice deficient in FcR
expression, CRP binding to macrophages from
-chain-/- mice was therefore mediated by
binding to murine FcRIIb. However, macrophages from
FcRII-/- mice, which express both FcRI
and FcRIII, also bound CRP. To determine whether CRP binds to murine
FcRIII, CRP binding to murine splenic NK cells was examined (Fig. 3, 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. 3). These data suggest that CRP
does not bind to mouse FcRIII, and that mouse FcRI may be
responsible for CRP binding to FcRII-/-
macrophages.
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CRP binding to splenic T cells and B cells from C57BL/6 wild-type
mice was examined (Fig. 3). 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 FcRIIb1, the
only FcR present on B cells.
To compare CRP binding to FcRIIb2 on macrophages with CRP binding to
FcRIIb1 on splenic B cells, CRP binding to murine B cells was
analyzed by generating dose-response binding curves by two-color
fluorescence (Fig. 4). 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
-chain-/- splenic B cells was also dose
dependent and saturated at 100 µg/ml. Unexpectedly, total binding
to the -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
FcRII-/- or
-chain-/-/FcRII-/-
splenic B cells was detected at any concentration of CRP tested. B
cells from C57BL/6, -chain-/-, and B6
x 129 F2J mice displayed similar 2.4G2 binding, while B cells from
FcRII-/- and
-chain-/-/FcRII-/-
bound significantly less 2.4G2 (data not shown).
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Mouse and human neutrophils express FcRII and FcRIII.
Because CRP binds to human neutrophils through FcRII, 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. 5). 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 -chain-/-
neutrophils. However, no increase in CRP binding was detected for
either the FcRII-/- or the
-chain-/-/FcRII-/-
neutrophils. These data further support the finding that in the absence
of all FcR, no CRP binding is detectable. These data also suggest
that the primary receptor for CRP on murine neutrophils is
FcRIIb.
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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 FcR-deficient mice
following pronase treatment (Fig. 5). CRP binding to Ly-6G-positive
bone marrow cells from C57BL/6, B6 x 129 F2J,
-chain-/-, and
FcRII-/- mice was significantly enhanced by
pronase treatment. Pronase treatment, however, did not increase CRP
binding to Ly-6G-positive cells from
-chain-/-/FcRII-/-
mice. These data indicate that CRP binding to murine bone marrow
neutrophils is enhanced by proteolytic activation similar to CRP
binding to human FcR. However, CRP binding to
FcRII-/- neutrophils was also enhanced,
suggesting that in mice, an FcR other than FcRIIb 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. 6). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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105
binding sites per cell present, while the second was a lower affinity
receptor. Both CRP receptors were thought to be distinct from FcR
based on the inability of mAb 2.4G2, which blocks IgG binding to
FcRII and FcRIII, to inhibit CRP binding. Recent work in our
laboratory has demonstrated that the high and low affinity receptors
for CRP on human leukocytes are FcRIIa and FcRI, respectively
(5, 6, 16). Therefore, we wanted to determine whether CRP
binds to murine FcR and whether receptors other than FcR 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 FcR on leukocytes. Although a family
of individuals that are FcRI deficient has been reported
(44), no humans devoid of all FcR expression have been
identified. The availability of mice that do not express subsets of
murine FcR has allowed us to examine CRP binding to murine cells in
the presence or absence of specific FcR. Peritoneal macrophages and
neutrophils, splenic lymphocytes, and bone marrow-derived neutrophils
were isolated from C57BL/6, B6 x 129 F2J,
-chain-/-,
FcRII-/-, and
-chain-/-/FcRII-/-
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 FcR-deficient mice
(-chain-/-/FcRII-/-)
was observed. Similarly, no CRP binding to splenic B cells or bone
marrow-derived neutrophils from FcR-deficient mice was detected.
This work demonstrates for the first time that the expression of FcR
is required for CRP binding to murine leukocytes.
Furthermore, results using mice deficient in subsets of FcR suggest
that in the mouse, as in humans, CRP binds to more than one class of
FcR. CRP binding to -chain-/- macrophages
and neutrophils that do not express functional FcRI or FcRIII was
mediated by binding to FcRIIb2. CRP binding to
FcRII-/- macrophages and neutrophils was
also observed, suggesting that CRP binds to either FcRI or
FcRIII. No CRP binding to NK cells, which express FcRIII as their
sole FcR, was detected, even though murine FcRIII receptors share
95% amino acid identity in their extracellular domains with murine
FcRIIb receptors (26). Thus, CRP binding to macrophages
from FcRII-/- mice is most likely mediated
by binding to FcRI. These findings are consistent with results using
human NK cells in which CRP binding to FcRIII was not detected
(60). Interestingly, the apparent affinities for CRP
binding by macrophages from FcRII-/- mice
and -chain-/- mice did not differ
significantly from each other. However, total binding of macrophages
from FcR-deficient strains was reduced compared with their
respective controls, suggesting that expression of multiple FcR
increased the number of available CRP receptors.
Because protease treatment of cells results in enhanced IgG binding to
human FcRIIa (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 FcRIIb1 on B cells was not affected (data
not shown). Because FcRII is the only FcR known to be
proteolytically activated, the finding that pronase treatment of
FcRII-/- neutrophils enhanced CRP binding
was unexpected. Because no increase in CRP binding was detected on
neutrophils in the absence of FcR, and because murine bone marrow
neutrophils may express both FcRI and FcRIII, the data from the
FcRII-/- neutrophils suggest that an
additional murine FcR is activated by pronase treatment.
The proteolytic enhancement of CRP binding to FcRIIb2 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
FcR 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
FcR 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 FcRIIb1 on splenic B cells was also demonstrated.
Because FcRIIb1 is an important regulator of inflammatory responses
of mice (37), this result may also have important
implications in vivo. FcRIIb1 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-
(8). A possible mechanism
for these in vivo activities is that CRP binds to murine FcRIIb,
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 (FcRI) as well as an
ITIM-containing receptor (FcRIIb1) 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.
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Acknowledgments |
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Footnotes |
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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:
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.
Received for publication September 3, 1999. Accepted for publication November 18, 1999.
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