SAGE-Hindawi Access to Research
Volume 2011, Article ID 484936, 6 pages
TransgenicMiceIs FcγRIIB Dependent
Xian-ZhenHu,1TylerT. Wright,1Nicholas R.Jones,1Theresa N.Ramos,2
Gregory A.Skibinski,1Mark A.McCrory,1Scott R.Barnum,2andAlexanderJ. Szalai1
1Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham,
1825 University Boulevard, SHEL 214, Birmingham, AL 35294-2182, USA
2Department of Microbiology, University of Alabama at Birmingham, 1825 University Boulevard, SHEL 214,
Birmingham, AL 35294-2182, USA
Correspondence should be addressed to Alexander J. Szalai, firstname.lastname@example.org
Received 26 August 2010; Accepted 24 September 2010
Academic Editor: Noriko Isobe
Copyright © 2011 Xian-Zhen Hu et al.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
We showed earlier that experimental autoimmune encephalomyelitis (EAE) in human C-reactive protein (CRP) transgenic mice
(CRPtg) has delayed onset and reduced severity compared to wild-type mice. Since human CRP is known to engage Fc receptors
and Fc receptors are known to play a role in EAE in the mouse, we sought to determine if FcγRI, FcγRIIb, or FcγRIII was needed to
manifest human CRP-mediated protection of CRPtg. We report here that in CRPtg lacking either of the two activating receptors,
FcγRI and FcγRIII, the beneficial effects of human CRP are still observed. In contrast, if CRPtg lack expression of the inhibitory
receptor FcγRIIB, then the beneficial effect of human CRP is abrogated. Also, subcutaneous administration of purified human
FcγRIIB. The results reveal that a CRP → FcγRIIB axis is responsible for protection against EAE in the CRPtg model.
C-reactive protein (CRP) is a widely used blood marker
of inflammation , but it is increasingly apparent that
the protein plays a causal role in host defense against
microbial pathogens  and in cardiovascular disease .
Furthermore, in at least three different mouse models,
human CRP has been shown to protect against autoim-
mune disease [4–6]. Importantly, we showed that human
CRP transgenic mice (CRPtg) are resistant to experimental
autoimmune encephalomyelitis (EAE) , an animal model
of multiple sclerosis (MS). Thus in CRPtg compared to
wild-type mice, EAE onset was delayed, its severity was
attenuated, and infiltration of encephalitogenic T-cells and
monocytes/macrophages into the CNS was prevented .
The encephalitogenic cells with which CRP interacts to
CRP on these cells were not identified. Since human CRP
binds both human and mouse Fc receptors [7–10] and
because there is growing evidence that Fc receptors play a
major role in controlling the emergence of EAE and other
autoimmune diseases [11–15], we sought to determine if
FcγRs were required for human CRP-mediated protection
against EAE in the mouse.
Here we show that for CRPtg mice lacking expression
of the activating receptors FcγRI and FcγRIII, expression
of human CRP delays onset and reduces severity of EAE
as well as or better than it does in CRPtg with an intact
FcγR repertoire. In contrast in CRPtg mice that lack
expression of the inhibitory receptor, FcγRIIB, no human
CRP-mediated protection from EAE is observed. Likewise,
administration of purified human CRP to wild-type mice
with ongoing EAE prevented the disease from worsening,
whereas the same treatment failed to halt worsening of EAE
for mice lacking FcγRIIB. The combined data suggest that
human CRP→ mouse FcγRIIB interaction and its presumed
Human CRP (μg/mL)
Figure 1: No effect of FcγR deficiencies on expression of human
CRP by CRPtg mice. Each bar and each whisker are the mean
and standard deviation, respectively, for human CRP serum
concentration measured for n = 5 mice. Blood was obtained 24
hours after i.p. injection of 25μg endotoxin, and human CRP was
measured by ELISA, both as described in .
inhibitory consequences are essential for realizing human
CRP-mediated protection against EAE in the CRPtg mouse
2.1. Animals. CRPtg mice have been described in detail
elsewhere [16, 17]. The CRPtg strain (C57BL/6 background)
carries a 31-kb human DNA fragment encoding the CRP
gene, all the known cis-acting CRP regulatory elements (i.e.,
the entire human CRP promoter) and the CRP pseudogene
. Cis-acting regulatory elements within the transgene
are responsible for both tissue specificity and acute phase
inducibility of its expression, and the trans-acting factors
required for its correct regulation are conserved from mouse
to man [16, 17]. Human CRP is expressed as an acute phase
reactant in CRPtg and reaches blood levels comparable to
those observed in humans with inflammatory disease (up to
500μg/mL) . We showed earlier that human CRP level in
the blood of CRPtg was elevated during the course of EAE
CRPtg mice were mated to mutants (also C57BL/6)
lacking functional expression of the genes encoding the α-
chains of FcγRI (FcγRI−/−mice) , FcγRIIB (FcγRIIB−/−
mice) , and FcγRIII (FcγRIII−/−mice) . FcγR-
deficient versus sufficient and CRPtg versus non-CRPtg
progeny were obtained in the expected Mendelian ratios
each genotype appeared phenotypically normal, and none
of the FcγR deficiencies significantly altered expression of
human CRP (Figure 1). To identify the various genotypes,
we used CRP transgene-specific and FcγR mutation-specific
PCRs, as described [12, 16–19]. All mice were fed a standard
chow diet (Ralston Purina Diet) and maintained at constant
humidity (60 ± 5%) and temperature (24 ± 1◦C) with
a 12-hour light cycle (6 AM to 6 PM). All protocols
were approved by the Institutional Animal Care and Use
Committee at the University of Alabama at Birmingham
and were consistent with the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of
Health (NIH publication 96-01, revised 1996).
2.2. Induction of EAE. An immunodominant myelin oligo-
dendrocyte protein (MOG) peptide was used to immunize
10–12-week-old mice, as described in . On days 0 and
7, mice received subcutaneously an injection of 150μg
MOG peptide emulsified in complete Freund’s adjuvant
containing 500μg heat-killed Mycobacterium tuberculosis
(Difco, Detroit, MI). On days 0 and 2, mice received an
intraperitoneal injection of pertussis toxin (500ng) (List
Biological Laboratories, Campbell, CA). Development of
EAE symptoms was monitored daily using a clinical scale
ranging from 0 to 6 as follows: 0, asymptomatic; 1, loss of
tail tone; 2, flaccid tail; 3, incomplete paralysis of one or two
hind limbs; 4, complete hind limb paralysis; 5, moribund
(in which case animals were humanely euthanized); 6, dead.
of at least 2 for more than 2 consecutive days were deemed to
have developed EAE. The maximum clinical score achieved
by each animal during the 30-day observation period was
used to calculate average maximum clinical score (severity)
for each experimental group. To study the time-course of
disease, average clinical scores were calculated and plotted
daily for each group of mice, and cumulative disease index
was calculated by area under the curve. When determining
the average day of onset of EAE, animals that did not
assigned a day of onset of 31.
2.3. Administration of Human CRP to Mice with EAE. EAE
was induced as described above, and the development
of symptoms was monitored. On the day their disease
symptoms achieved or eclipsed a score of 2 (flaccid tail),
each mouse received subcutaneously an injection of 50μg
of highly purified (95%–98%) human CRP (US Biological,
Swampscott, MA). The disease course was then followed
for an additional 10 days. The CRP preparation was
sodium azide-free, contained <0.4ng endotoxin/mg protein
by Limulus amebocyte assay, and had pentameric integrity
as judged by overloaded native polyacrylamide gel elec-
trophoresis (data not shown). Control animals received
200μg of heat-denatured (boiled for 5 minutes) human CRP.
day of onset and maximum clinical score (mean ± sem)
7 1421 28
7 14 2128
7 14 21
expression of FcγRI (b), FcγRIII (c), or FcγRIIB (d) were injected with MOG peptide, and EAE symptoms were monitored. Presence of the
CRP transgene (closed circles in each panel) delayed onset of EAE in mice with intact FcγRs (a) and delayed onset and reduced severity of
EAE in mice lacking FcγRI (b) or FcγRIII (c). In contrast in mice lacking FcγRIIB (d), expression of human CRP had no beneficial effect.
See Table 1 for sample sizes and statistical analyses.
4 Autoimmune Diseases
No treatment (n = 10)
50μg human CRP (n = 4)
200μg heat denatured CRP (n = 7)
No treatment (n = 10)
50μg human CRP (n = 10)
200μg heat denatured CRP (n = 7)
7 14 21
Figure 3: CRP treatment stalls progression of EAE in wild-type mice but not in FcγRIIB−/−mice. Wildtype (a) versus FcγRIIB−/−(b) with
ongoing EAE were injected with 50μg purified CRP s.c. when their clinical scores reached 2 (horizontal line), and EAE symptoms were
monitored for 10 days. Controls received heat-denatured CRP.
were evaluated by one-way ANOVA and posthoc Neuman-
Keul’s multiple comparison tests. A P value less than .05 was
∼1 week for CRPtg compared to wild type mice (Figure 2(a)
and Table 1; P < .001, t-test), and this delay led to reduced
cumulative disease index (Table 1; 32.8 versus 46.55) even
though average disease severity was not significantly lowered
(Table 1 and Figure 2(a)). In comparison, for CRPtg that
lacked expression of either FcγRI or FcγRIII (Figures 2(b)
and 2(c), resp.), human CRP-mediated protection included
not only a delay in EAE onset and a reduced cumulative
disease index but also a significant reduction in disease
severity (Table 1). In contrast, for CRPtg mice lacking the
inhibitory receptor FcγRIIB, expression of human CRP
conferred no protective benefit (Table 1 and Figure 2(d)).
Other groups showed that human CRP administered
subcutaneously to mice can reverse autoimmune- and
antibody-induced inflammation [5, 20], a beneficial effect
that reportedly requires certain FcγRs . To test if human
CRP administration might likewise protect mice from EAE
and to test if FcγRIIB was required, we administered purified
human CRP to wildtype versus FcγRIIB−/−mice with
ongoing disease. The results are summarized in Figure 3.
We observed that for wildtype mice (Figure 3(a)) treatment
with human CRP, but not treatment with heat-denatured
CRP, halted progression of EAE. In contrast, no protective
influence of CRP therapy was observed for FcγIIB−/−mice
of human CRP likely result (directly or indirectly) from the
proteins ability to bind FcγRs [20, 21]. FcγRs are a family
of receptors of which most mammals express four main
types: FcγRI, FcγRII, FcγRIII, and FcγRIV [14, 22, 23]. Each
of FcγRI, FcγRIII, and FcγRIV is comprised of a ligand
binding α-chain paired with a common γ-chain (FcRγ)
that encodes an immunoreceptor tyrosine-based activation
motif (ITAM) essential to propagate cell activating signals.
FcγRIIB on the other hand is comprised of a single α-
chain and it carries a cytoplasmic tyrosine-based inhibitory
motif (ITIM) that propagates cell inhibiting signals. Various
investigators have reported that human CRP binds to one
or more isoforms of FcγRI, FcγRII, and FcγRIII, in both
mouse and man [7–10], and FcγRs reportedly influence
EAE in the mouse [14, 24]. Thus in CRPtg, human CRP
inflammation-promoting FcγRs on encephalitogenic cells or
dampen EAE by binding FcγRIIB. Using CRPtg mice with
selective deletion of FcγRs, we were able to investigate if
either capacity is realized in vivo.
Compelling evidence was obtained that the beneficial
action of human CRP in mouse EAE depends mainly on
Table 1: Effect of transgenic expression of human CRP on the outcome of EAE in mice lacking various Fcγ receptorsa.
Strain No. mice
Clinical measure of disease symptoms
Day of onset
aEAE was induced with MOG peptide as described in Section 2.
bCumulative disease index (area under the curve: arbitrary units as described in Section 2).
cResults of Neuman-Keuls multiple comparison test comparing indicated genotype to wildtype
dResults of Neuman-Keuls multiple comparison test comparing indicated CRPtg genotype to its non-CRPtg littermates.
P < .001c,d
P < .001d
P < .05d
P < .01d
P < .05d
expression of the inhibitory receptor FcγRIIB. Thus in
FcγRIIB−/−mice, EAE is neither delayed nor dampened by
transgenic expression of human CRP. In fact the tempo and
severity of EAE in CRPtg/FcγRIIB−/−was not significantly
different from that seen in wild type mice. In contrast, the
human CRP-associated delay in EAE onset and attenuation
activating receptors: FcγRI or FcγRIII. We did not formally
rule out the possibility that FcγRIV might play a role, as
FcγRIV−/−mice are not available to us, but we did perform
experiments with mice that lack the FcR common gamma
chain FcRγ, which are predicted to lack expression of FcγRI,
FcγRIII, and FcγRIV . Human CRP transgenic FcRγ−/−
were obviously more resistant than wildtype mice (data not
shown), nevertheless the contribution of FcRγ (and thus
FcγRIV) to human CRP-mediated resistance to EAE remains
unclear because FcRγ−/−mice per se are intrinsically very
resistant to EAE . Thus in their sum the data suggest that
depends largely on the availability/expression of FcγRIIB.
Presumably by binding FcγRIIB, human CRP expressed
endogenously during the course of disease dampens the
activation state of encephalitogenic (FcγRIIB-expressing)
cells in CRPtg. Likewise, in nontransgenic mice, exogenously
administered human CRP has the same effect as long as
FcγRIIB is present.
For CRPtg mice, transgene-expressed human CRP inhibits
EAE, and this beneficial action requires FcγRIIB. If as in
CRPtg with EAE, a protective CRP→FcγRIIB axis exists
in humans with MS, then CRP administration might be
beneficial in the clinical treatment of patients with MS.
Ongoing efforts in our laboratory are aimed at identifying
the CRP-responsive FcγRIIB-expressing encephalitogenic
cell(s) involved in this action, which we posit to be dendritic
The paper described herein was funded by a research Grant
from the National Multiple Sclerosis Society (RG 3216-A-
5 to AJS and SRB), by training Grants from the National
Institutes of Health (T32 AI07051 to TNR and T32 AR07450
to NRJ), and the Howard Hughes Med-Grad Fellowship
Program at UAB (to TTW).
 C. Gabay and I. Kushner, “Acute-phase proteins and other
systemic responses to inflammation,” New England Journal of
Medicine, vol. 340, no. 6, pp. 448–454, 1999.
 A. J. Szalai, “The antimicrobial activity of C-reactive protein,”
Microbes and Infection, vol. 4, no. 2, pp. 201–205, 2002.
 R. J. Bisoendial, S. M. Boekholdt, M. Vergeer, E. S. G. Stroes,
and J. J. P. Kastelein, “C-reactive protein is a mediator of
cardiovascular disease,” European Heart Journal, vol. 31, no.
17, pp. 2087–2095, 2010.
 A. J. Szalai, C. T. Weaver, M. A. McCrory et al., “Delayed
lupus onset in (NZB × NZW)F1 mice expressing a human C-
reactive protein transgene,” Arthritis and Rheumatism, vol. 48,
no. 6, pp. 1602–1611, 2003.
 W. Rodriguez, C. Mold, L. L. Marnell et al., “Prevention and
reversal of nephritis in MRL/lpr mice with a single injection of
C-reactive protein,” Arthritis and Rheumatism, vol. 54, no. 1,
pp. 325–335, 2006.
tal allergic encephalomyelitis is inhibited in transgenic mice
expressing human C-reactive protein,” Journal of Immunology,
vol. 168, no. 11, pp. 5792–5797, 2002.
 L. L. Marnell, C. Mold, M. A. Volzer, R. W. Burlingame, and T.
W. Du Clos, “C-reactive protein binds to FcγRI in transfected
COS cells,” Journal of Immunology, vol. 155, no. 4, pp. 2185–
 M.-P. Stein, C. Mold, and T. W. Du Clos, “C-reactive protein
binding to murine leukocytes requires Fcγ receptors,” Journal
of Immunology, vol. 164, no. 3, pp. 1514–1520, 2000.
 C. Mold, H. D. Gresham, and T. W. Du Clos, “Serum amyloid
P component and C-reactive protein mediate phagocytosis
through murine FcγRs,” Journal of Immunology, vol. 166, no.
2, pp. 1200–1205, 2001.
 J. Lu, L. L. Marnell, K. D. Marjon, C. Mold, T. W. Du Clos, and
P. D. Sun, “Structural recognition and functional activation
of FcγR by innate pentraxins,” Nature, vol. 456, no. 7224, pp.
 S. Kleinau, P. Martinsson, and B. Heyman, “Induction and
suppression of collagen-induced arthritis is dependent on
distinct Fcγ receptors,” Journal of Experimental Medicine, vol.
191, no. 9, pp. 1611–1616, 2000.
 N. Barnes, A. L. Gavin, P. S. Tan, P. Mottram, F. Koentgen,
ations to inflammatory and immune responses,” Immunity,
vol. 16, no. 3, pp. 379–389, 2002.
 K. G. C. Smith and M. R. Clatworthy, “FcγRIIB in autoim-
munity and infection: evolutionary and therapeutic implica-
tions,” Nature Reviews Immunology, vol. 10, no. 5, pp. 328–
 A. J. Szalai and S. R. Barnum, “Fc receptors and the common
γ-chain in experimental autoimmune encephalomyelitis,”
Journal of Neuroscience Research, vol. 75, no. 5, pp. 597–602,
 M. I. Iruretagoyena, C. A. Riedel, E. D. Leiva, M. A. Guti´ errez,
S. H. Jacbobelli, and A. M. Kalergis, “Activating and inhibitory
Fcγ receptors can differentially modulate T cell-mediated
autoimmunity,” European Journal of Immunology, vol. 38, no.
8, pp. 2241–2250, 2008.
 G. Ciliberto, R. Arcone, E. F. Wagner, and U. R¨ uther,
“Inducible and tissue-specific expression of human C-reactive
protein in transgenic mice,” EMBO Journal, vol. 6, no. 13, pp.
 A. J. Szalai and M. A. McCrory, “Varied biologic functions
of C-reactive protein: lessons learned from transgenic mice,”
Immunologic Research, vol. 26, no. 1–3, pp. 279–287, 2002.
 T. Takai, M. Ono, M. Hikida, H. Ohmori, and J. V. Ravetch,
“Augmented humoral and anaphylactic responses in FcγRII-
deficient mice,” Nature, vol. 379, no. 6563, pp. 346–349, 1996.
 W. L. W. Hazenbos, J. E. Gessner, F. M. A. Hofhuis et al.,
“Impaired IgG-dependent anaphylaxis and Arthus reaction in
FcγRIII (CD16) deficient mice,” Immunity, vol. 5, no. 2, pp.
 W. Rodriguez, C. Mold, M. Kataranovski et al., “C-reactive
protein-mediated suppression of nephrotoxic nephritis: role
of macrophages, complement, and Fcγ receptors,” Journal of
Immunology, vol. 178, no. 1, pp. 530–538, 2007.
 D. Xing, F. G. Hage, Y.-F. Chen et al., “Exaggerated neointima
formation in human C-reactive protein transgenic mice is IgG
Fc receptor type I (FcγRI)-dependent,” American Journal of
Pathology, vol. 172, no. 1, pp. 22–30, 2008.
 F. Nimmerjahn, P. Bruhns, K. Horiuchi, and J. V. Ravetch,
“FcγRIV: a novel FcR with distinct IgG subclass specificity,”
Immunity, vol. 23, no. 1, pp. 41–51, 2005.
 F. Nimmerjahn and J. V. Ravetch, “Fcγ receptors: old friends
and new family members,” Immunity, vol. 24, no. 1, pp. 19–
 A. J. Szalai, X. Hu, C. Raman, and S. R. Barnum, “Require-
ment of the Fc receptor common γ-chain for γδ T cell-
mediated promotion of murine experimental autoimmune
encephalomyelitis,” European Journal of Immunology, vol. 35,
no. 12, pp. 3487–3492, 2005.
 R. Zhang, L. Becnel, M. Li, C. Chen, and Q. Yao, “C-reactive
protein impairs human CD14+ monocyte-derived dendritic
cell differentiation, maturation and function,” European Jour-
nal of Immunology, vol. 36, no. 11, pp. 2993–3006, 2006.