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Infection and Immunity, April 2007, p. 2075-2078, Vol. 75, No. 4
0019-9567/07/$08.00+0 doi:10.1128/IAI.01920-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Mice Lacking CD21 and CD35 Proteins Mount Effective Immune Responses against Borrelia burgdorferi Infection
Amanda C. Jacobson,1
Ying Ma,1
James F. Zachary,2
Janis J. Weis,1 and
John H. Weis1*
Division of Cell Biology and Immunology, Department of Pathology, 15 North Medical Drive East, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650,1
Department of Veterinary Pathobiology, University of Illinois, Urbana, Illinois 618022
Received 5 December 2006/
Returned for modification 2 January 2007/
Accepted 25 January 2007

ABSTRACT
CD21/35
/ mice, deficient in CD21 and CD35 (complement
receptors 2 and 1, respectively), were infected with
Borrelia burgdorferi to assess the role of these receptors in a chronic
bacterial infection. Although CD21/35
/ mice on
both C57BL/6 and BALB/c backgrounds produced less
B. burgdorferi-specific
antibodies than did wild-type mice, spirochete levels and arthritis
severity were similar.

TEXT
Complement plays an important part in the clearance of bacteria.
The decoration of pathogen antigens with complement fragments
provides signals important for the opsonization of bacteria
and induction of inflammatory responses. In the mouse, CD21
and CD35 (complement receptors 2 and 1, respectively; CD21/35)
are spliced products from the
Cr2 gene and serve as receptors
for complement breakdown products. On B cells, CD21/35 functions
as the B-cell coreceptor and augments B-cell signaling (
5).
Mice lacking CD21/35 (CD21/35
/) mount decreased
antibody responses to T-cell-dependent and T-cell-independent
antigens, with an impairment in the immunoglobulin G3 (IgG3)-specific
antibody response (
5). Consequently, CD21/35
/ mice were more susceptible to lethal
Streptococcus pneumoniae infection (
6). These studies reveal an important role for CD21/35
in protective antibody responses during acute bacterial infection;
however, the role of these proteins during a chronic bacterial
infection has not been addressed.
Infection of mice with the spirochete Borrelia burgdorferi provides an excellent model for understanding host-pathogen interactions involved in chronic infection. Arthritis caused by persistent infection of the spirochete within joint tissue appears to result from the sustained inflammatory challenges to the host from the bacterial components (18). Bacterium-specific antibodies produced during infection are not causative of or protective against acute Lyme arthritis in mice; however, they are important for controlling spirochete growth and resolution of disease (2, 14).
B. burgdorferi has evolved mechanisms to evade the host immune response, including resistance to host complement. B. burgdorferi is able to bind host complement regulatory proteins factor H and factor H-like 1, which permits the bacteria to evade complement-mediated killing (1, 4, 7-9, 13, 17). Additionally, higher spirochete levels and generally higher histopathology scores are found in joint tissue of C3/ mice (10). In these mice, anti-B. burgdorferi IgG levels were reduced compared to those in the wild type. These data suggest a role for complement proteins and their receptors in the immune response against B. burgdorferi.
In this study, we used B. burgdorferi infection to assess the role of complement receptor proteins CD21 and CD35 in a chronic bacterial infection. CD21/35/ mice were generated as previously described (6) and used at the 10th generation backcross to both C57BL/6 and BALB/c mice.
To assess the role of complement receptor proteins CD21 and CD35 in B. burgdorferi-specific antibody production, wild-type and CD21/35/ animals on both the BALB/c and C57BL/6 backgrounds were infected intradermally with a cloned N40 isolate of B. burgdorferi that had been passaged three times (provided by S. Barthold, University of California, Davis). To achieve equivalent disease severity on both backgrounds, mice on the BALB/c background received 2 x 104 bacteria, whereas C57BL/6 mice received 2 x 103 bacteria (12). C3H mice were infected with 2 x 103 bacteria as positive controls for infection. Bacterium-specific and total antibody titers were measured (12) at 2 and 4 weeks postinfection. B. burgdorferi-specific IgG3 antibodies were significantly decreased at 4 weeks postinfection in CD21/35/ animals on the C57BL/6 background, whereas a less drastic decrease was observed on the BALB/c background (Table 1). CD21/35/ mice produced increased bacterium-specific IgM antibodies on the BALB/c background. Knockout (KO) animals on both strain backgrounds produced significantly less B. burgdorferi-specific IgG2b (Table 1). Collectively, CD21/35/ mice had varied abilities to produce bacterium-specific antibodies important for immediate (IgM and IgG3) and long-term (IgG2b) protection. Total isotype-specific antibody titers in infected CD21/35/ mice were similar to those of infected wild-type mice and increased greater than twofold relative to those of uninfected mice (data not shown). This demonstrates that the CD21/35/ mice do not have an overall defect in the production of Ig against B. burgdorferi.
The decoration of bacteria with complement fragments provides
signals for the opsonization, lysis, and clearance of the bacteria.
To determine if the antigens recognized by CD21/35
/ mice were altered relative to wild-type mice, Western blot analysis
was performed comparing the antigens recognized by sera (
3)
from infected CD21/35
/ mice and wild-type controls.
IgG and IgG3 in sera from CD21/35
/ and wild-type
mice recognized similar proteins in
B. burgdorferi lysates (Fig.
1). Sera from infected CD21/35
/ and wild-type
mice were also analyzed for antibody against the C6 peptide
of the VlsE variable lipoprotein of
B. burgdorferi, which has
been used diagnostically for Lyme borreliosis in humans (
11).
There were no significant differences in anti-C6 peptide antibody
levels between wild-type and CD21/35-deficient mice on both
the C57BL/6 and BALB/c backgrounds (data not shown). In brief,
CD21/35
/ mice appear to recognize and mount antibody
responses against a variety of
B. burgdorferi antigens, including
lipoproteins, albeit producing lower levels of
B. burgdorferi-specific
antibodies.
Bacterium-specific antibodies produced during infection play
a role in controlling bacterial numbers and resolution of disease
(
14). To consider the effect of decreased
B. burgdorferi-specific
antibody responses in the CD21/35
/ mouse, spirochete
numbers and arthritis severity of the rear ankle joints of infected
animals were assessed by quantitative PCR as described in detail
in references
15 and
19. Deficiency in CD21/35 did not have
an effect on
B. burgdorferi numbers in ankle tissue 4 weeks
postinfection (Fig.
2), indicating that the antibodies produced
by a CD21/35
/ animal are sufficient to control
spirochete numbers. Spirochete numbers in the joint tissue of
BALB/c mice correlates with arthritis severity (
12). Arthritis
severity within the ankle joints of infected mice did not differ
significantly between wild-type and CD21/35-deficient mice as
seen by ankle swelling and histological analysis (Table
2).
Converse to the acute bacterial infection with
S. pneumoniae,
CD21/35
/ mice demonstrated the ability to control
B. burgdorferi infection to wild-type levels regardless of an
altered repertoire of antibodies against the bacteria. This
may be best explained by the role of antibodies in protecting
the host against bacterial infections. IgG3 is the major mouse
IgG isotype produced in response to T-cell-independent type
2 antigens like pneumococcal polysaccharides (
16). Conversely,
the immune response against
Borrelia antigens uses both innate
antibodies, like IgG3 and IgM, and those isotypes which require
T-cell help, like IgG2a/b (
14). Complement proteins may be more
critical adjuvants for immune responses against
S. pneumoniae polysaccharides than
B. burgdorferi lipoproteins. The evasion
of host complement activation by the more recently described
B. burgdorferi CRASP proteins may be more critical for bacterial
survival during the initial infection. Complement receptors
CD21/35, on the other hand, would have a more significant role
in complement-mediated immune responses to the bacteria within
the splenic environment, like antibody production. The data
presented here suggest that complement receptors CD21 and CD35
do not have a direct role in regulating spirochete numbers,
and the ability of CD21/35
/ mice to control
B. burgdorferi infection suggests that
B. burgdorferi does not
have an advantage in a host lacking complement receptors 1 and
2.

ACKNOWLEDGMENTS
This work was supported by U.S. Public Service grants AI-24158
to J.H.W. and AI-32223 to J.J.W. from the National Institutes
of Health.

FOOTNOTES
* Corresponding author. Mailing address: Department of Pathology, University of Utah School of Medicine, 15 North Medical Drive East, Salt Lake City, UT 84112-5650. Phone: (801) 581-7054. Fax: (801) 585-7376. E-mail:
john.weis{at}path.utah.edu.

Published ahead of print on 5 February 2007. 
Editor: W. A. Petri, Jr.

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Infection and Immunity, April 2007, p. 2075-2078, Vol. 75, No. 4
0019-9567/07/$08.00+0 doi:10.1128/IAI.01920-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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