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Department of Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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36% higher cell surface CD19
expression, whereas CD21/35 expression was increased by 45% on B
cells from C3-deficient mice. Negative regulation of CD19 and CD21
expression by CD21 and C3, respectively, may be functionally
significant because small increases in cell surface CD19 overexpression
can predispose to autoimmunity. Otherwise, B cell development and
function in CD19-deficient and -overexpressing mice were not
significantly affected by a simultaneous loss of CD21 expression.
Although CD21-deficient mice were found to express a hypomorphic cell
surface CD21 protein at low levels that associated with mouse CD19, C3
deficiency did not significantly affect B cell development and function
in CD19-deficient or -overexpressing mice. These results, and the
severe phenotype exhibited by CD19-deficient mice compared with CD21-
or C3-deficient mice, collectively demonstrate that CD19 can regulate B
cell signaling thresholds independent of CD21 engagement and complement
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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240-aa
cytoplasmic domain that is critical for CD19-CD21 complex signaling
(8, 11, 12). Specifically, CD19 functions as a specialized
adapter protein for the amplification of Src family kinases and as an
interaction molecule for multiple signaling pathways crucial for
modulating intrinsic and Ag receptor-induced signals
(13, 14, 15, 16, 17, 18). The cytoplasmic domains of human CD19 (hCD19)
and mouse CD19 (mCD19) are highly homologous (19). In
fact, hCD19 can replace mCD19 function when expressed at the
appropriate site density in CD19-/- mice
(20). Cell surface CD19 is present in molar excess of CD21
at all stages of B cell development, although CD19-CD21 complexes are
presumed to represent 1:1 complexes (4, 21). CD21 is expressed on FDCs and mature B cells, with expression first by IgMhighIgDlow transitional B cells (3, 22). CD21 is composed of an extracellular domain containing 15 or 16 repeating structural elements termed short consensus repeats (SCRs), a transmembrane region, and a 34-aa cytoplasmic domain (23, 24). CD21 and CD35 (complement receptor 1) are alternative splice products of the same Cr2 gene in mice, but are encoded by different genes in humans (25, 26). In mice, CD35 is generated by the addition of six SCRs to the amino-terminal end of the CD21 protein. Well-characterized ligands for CD21 are the iC3b/C3d,g cleavage fragments of complement. These C3 cleavage products form covalent bonds with foreign Ags or immune complexes to generate C3d-Ag complexes that are proposed to bind CD21 and regulate B cell function by signaling through the CD19 complex (1, 2, 7). CD35 binds C3b and C4b, and serves as a cofactor for the hydrolysis of C3b-Ag complexes into C3d-Ag complexes (10, 27). This process is important for the processing of Ag-Ab complexes and the final deposition of C3d-Ag complexes on the surface of B cells and FDCs through CD21. A recent study indicates that human CD21 can physically associate with mCD19, and can restore humoral immune response in CD21/35-/- mice (28).
Studies using mice that lack or overexpress CD19 indicate that CD19
and/or CD19-CD21 complexes regulate signal transduction thresholds
governing humoral immunity. B cells from
CD19-/- mice are hyporesponsive to a variety of
transmembrane signals, which leads to significant defects during the
later stages of B cell maturation, clonal expansion, and
differentiation (29, 30, 31). By contrast, transgenic mice
that overexpress hCD19 (hCD19TG+/+) are
hyperresponsive to transmembrane signals and display severe alterations
during early stages of B cell development, which leads to diminished
numbers of B cells in the peripheral pool (9, 29, 32). The
development of B1 cells is severely decreased in
CD19-/- mice, whereas there is an increased
frequency of B1 cells within the peritoneum and spleen of
hCD19TG+/+ mice (9, 29, 30). In two
independent lines of CD21/35-/- mice,
lymphocyte development, phenotypes, and numbers are normal (33, 34). A 40% reduction in the frequency of peritoneal B1 cells
has been observed in one line of CD21/35-/-
mice (33). Both lines of
CD21/35-/- mice exhibit markedly impaired
primary and secondary humoral immune responses and germinal center
formation, especially IgG responses to T cell-dependent (TD) Ags
(33, 34, 35). This is not due to a defect in B cell Ag
receptor signaling in CD21/35-/- mice because
their B cells respond normally to IgM and/or CD40 cross-linking
(33). Pretreatment of mice with either a CD21/35-specific
mAb or a CD21-IgG fusion protein also blocks secondary humoral
responses (36, 37, 38). C3-/- mice
have a normal phenotype and serum Ig levels, whereas mice that lack C4,
which is required for C3 activation, have decreased IgG1, IgG2a, and
IgG3 levels (39, 40). Both C3-/-
and C4-/- mice have modest TD immune responses
with defects in germinal center formation. In addition, complement and
CD21/35 regulate the elimination of self-reactive B cells because
systemic lupus erythematosus-prone
C57BL/6lpr/lpr mice with CD21/35 or C4
deficiencies have exacerbated disease and increased autoantibody
production (41, 42). Spontaneous autoimmunity has been
observed in C4-/- mice, but not in
CD21/35-/- mice, presumably due to an impaired
clearance of immune complexes (43).
The exact mechanisms by which complement and complement receptors affect humoral immune responses remain uncertain. C3d binding to CD21 supplies an already characterized ligand for the CD19 complex, thereby linking complement activation and B cell function. However, CD19 may also have signaling roles and ligand-binding activities independent of CD21 (1, 8). Alternatively, FDC expression of CD21/35 may be required for the generation of humoral immunity. In one study, normal humoral immune responses require CD21/35 expression on FDCs (44). Another study has reported that B cell but not FDC expression of complement receptors is required for humoral immune responses (45). In a third study, optimal humoral immune responses required a combination of complement-derived CD21 ligands on FDCs and CD21 on B cells (46). Furthermore, CD21 may provide an Ag-independent signal required for the survival of B cells in germinal centers (47). To determine the extent that CD19 function is controlled by complement activation, CD21/35-/- and C3-/- mice were generated that either lack or overexpress CD19. The phenotypes of these mice demonstrate that CD19, CD21, and C3 expression are interrelated and may thus form a regulatory loop that influences B cell function. In addition, the functional properties of these mice demonstrate that CD19 can regulate B cell signaling thresholds independent of complement activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD21/35-/- (129 x C57BL/6), C3-/- (129 x C57BL/6), CD19-/- (C57BL/6, >6 generations), and hCD19TG+/+ (C57BL/6, >12 generations) mice were generated as described (29, 32, 33, 40). Specifically, the hCD19TG+/+ mice used were from the TG-1 line, which expresses 2.6-fold higher levels of CD19 (20). CD19-/-CD21/35-/-, CD19-/-C3-/-, hCD19TG+/+CD21/35-/-, and hCD19TG+/+C3-/- littermates were generated through breedings of homozygous single-mutant mice to generate heterozygous offspring at each locus. Heterozygous offspring were crossed to generate littermates homozygous at each locus and wild-type control offspring. In all cases, results with wild-type littermates from each breeding group (CD19-/-, hCD19TG+/+, CD21/35-/-, and C3-/-) were similar and were therefore pooled. Thereby, any potential background genetic effects were distributed throughout the test population without regard to the disrupted gene loci. All mice used were 2–3 mo of age unless indicated otherwise, and were housed in a specific pathogen-free barrier facility. All studies and procedures were approved by the Animal Care and Use Committee of Duke University.
Antibodies
Abs used in this study included: mouse IgA anti-mCD19 (MB19–1; Ref. 9), mouse anti-hCD19 (HB12b; Ref. 48), rat anti-mouse CD21/35 (7E9: IgG2a, 7G6: IgG2b; provided by Dr. T. Kinoshita, Osaka University, Osaka, Japan: Ref. 49), FITC-conjugated anti-mouse CD21/35 (7G6; BD PharMingen, San Diego, CA), PE -conjugated anti-mCD19 (1D3; BD PharMingen), PE-conjugated anti-hCD19 (B4; Coulter, Miami, FL), PE-conjugated anti-CD5 (53-7.3; BD PharMingen), biotinylated or FITC-conjugated anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL), biotinylated or FITC-conjugated anti-B220 (CD45RA, RA3-6B2; provided by Dr. R. Coffman, DNAX Research Institute, Palo Alto, CA), and biotinylated anti-mouse IgD (Southern Biotechnology Associates) Abs. Anti-CD21/35 Ab binding (7E9) was visualized using FITC- or PE-conjugated goat anti-rat IgG (H + L) Abs (Caltag Laboratories, Burlingame, CA) diluted to the appropriate concentration for optimal immunostaining. PE-conjugated streptavidin (Southern Biotechnology Associates) was used to reveal biotin-coupled Ab staining.
Immunofluorescence analysis
Single-cell suspensions of lymphocytes from spleen, bone marrow, peritoneal lavage, and peripheral lymph nodes were isolated before two-color immunofluorescence analysis. Leukocytes (0.5–1 x 106) were stained at 4°C using predetermined optimal concentrations of Abs for 20 min. Blood erythrocytes were lysed after staining using FACS lysing solution (BD Biosciences, San Jose, CA). Cells with the forward and side light scatter properties of lymphocytes were analyzed on a FACScan flow cytometer (BD Biosciences) with fluorescence intensity shown on a 4-decade log scale. Fluorescence contours for 5000 cells/sample are shown as 50% log density plots. Positive and negative populations of cells were determined using unreactive isotype-matched mAbs (Caltag Laboratories) as controls for background staining. Background levels of staining were delineated using gates positioned to include >98% of the control cells.
Immunization of mice
Two-month-old littermates were immunized i.p. with 50 µg of T cell-independent type 1 Ag, 2,4,6-trinitrophenol-conjugated LPS (TNP-LPS; Sigma, St. Louis, MO) in saline. Other littermates were immunized i.p. with 100 µg of the TD Ag, DNP-keyhole limpet hemocyanin (DNP-KLH; Calbiochem-Novabiochem, La Jolla, CA) in CFA and were boosted 21 days later with DNP-KLH in adjuvant. Serum was collected before and after immunization.
Mouse Ig isotype-specific ELISAs
IgM and IgG1 concentrations in sera were determined by
ELISA using affinity-purified mouse IgM and IgG1 (Southern
Biotechnology Associates) to generate standard curves as described
(29). Relative Ig concentrations in individual samples
were determined by comparing the mean OD values obtained for duplicate
wells to a semilog standard curve of titrated standard Ab using linear
regression analysis. TNP- and DNP-specific Ab titers of sera were
measured as described (31), using 96-well microtiter ELISA
plates (Costar, Cambridge, MA) coated with 5 µg/ml TNP-BSA (Biosearch
Technologies, San Rafael, CA) and DNP-BSA (Calbiochem-Novabiochem).
ELISA color development was allowed to progress until the wells
containing the highest Ab levels reached OD values of 2.0. These OD
values were determined to be within the linear range of the ELISA using
sera over multiple dilutions. Serum IgM and IgG anti-dsDNA levels
were determined by ELISA using 96-well microtiter plates coated with 5
µg/ml calf thymus dsDNA (Sigma), as described (9).
Immunoprecipitation and Western blot analysis
Splenic B cells were purified by removing T cells with anti-Thy 1.2 Ab-coated magnetic beads (Dynal, Lake Success, NY). Purified (wild-type, >95%; CD21/35-/-, >93%; hCD19TG+/+, >89% B220+) splenic B cells were lysed in buffer containing 1% digitonin, 10 mM triethanolamine, 150 mM NaCl, 5 mM EDTA, and protease inhibitors (pH 7.8). For immunoprecipitations, the cell lysates were precleared twice with mouse IgG plus protein G-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden), followed by incubation with protein G beads plus 7G6 Ab for 3 h at 4°C. CD19 was immunoprecipitated using anti-mCD19 (MB19-1) or anti-hCD19 (HB12b) Abs covalently attached to Affigel 10 beads (Bio-Rad, Hercules, CA). After washing the beads with lysis buffer four times, immunoprecipitates were subjected to SDS-PAGE, with subsequent electrophoretic transfer to nitrocellulose membranes. The membranes were incubated with goat anti-mouse CD21 polyclonal antisera (D-19 or M-19; Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with HRP-conjugated donkey anti-rabbit IgG Abs (Jackson ImmunoResearch Laboratories, West Grove, PA). Blots were developed using an ECL kit (Pierce, Rockford, IL).
PCR amplification and sequencing of CD21 transcripts
Cytoplasmic RNA free of DNA contamination was isolated from splenocytes of wild-type and CD21/35-/- littermates using a RNeasy Mini kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. Equal amounts of RNA were used for cDNA synthesis and PCR amplification. The region spanning exons 9/10 in the cDNA (25) was amplified using a sense primer identical with sequence in exon 8 (5'-GGA CAG CTG TTA ATT CTT CTT GTG-3') and an antisense primer identical with sequence in exon 11 (5'-TCA TAA GTA TAT CCA GTC AAC TGG-3') to generate a 622-bp fragment in wild-type cDNA. The conditions used for PCR amplification were: 94°C for 3 min, then 30 cycles at 94°C for 1 min, 58°C for 1 min, followed by 72°C for 1 min. The PCR products were electrophoresed and visualized by ethidium bromide staining. Amplified PCR products were purified from agarose gels using the QIAquick gel purification kit (Qiagen) and were sequenced directly in both directions using an ABI 377 PRISM DNA sequencer after amplification using the PerkinElmer Dye Terminator Sequencing system with AmpliTaq DNA polymerase and the same primers that were used for the initial PCR amplification.
Statistical analysis
ANOVA was used to analyze the data, with Student’s t test used to determine the level of significance for differences between sample means.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because CD19 and CD21/35 physically associate on the cell surface,
it was assessed whether loss of CD21/35 expression affected CD19
expression. As determined by immunofluorescence staining, CD19
expression on B220high mature bone marrow B cells
from CD21/35-/- mice was significantly
increased (19% higher) relative to wild-type littermates (Fig. 1
, A and B). CD19
expression was similar on immature B220low B
cells from CD21/35-/- and wild-type littermates
(Fig. 1A), consistent with a lack of or low-level CD21/35
expression by immature bone marrow B cells (50). CD19
expression was also increased on CD21/35-/- B
cells from blood, spleen, and lymph nodes by 16, 36, and 31%,
respectively (Fig. 1). By contrast, mCD19 expression by mature B cells
from bone marrow, blood, spleen, and lymph nodes of
C3-/- mice was not significantly different from
wild-type levels (Fig. 1B). Thus, CD21/35 expression
regulates cell surface CD19 expression levels, whereas C3 deficiency
does not affect CD19 expression by the majority of B cells.
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), as previously reported
(20). In the absence of CD21/35 expression, mCD19 was
expressed at wild-type levels in hCD19TG+/+ mice
in these tissues (Fig. 1). C3 deficiency did not significantly alter
mCD19 expression levels in hCD19TG+/+ mice. Thus,
CD21/35 expression negatively influenced endogenous mCD19 expression
levels, but did not influence cell surface hCD19 expression. CD19 expression influences CD21/35 expression
Because CD21/35 expression negatively influences cell surface
mCD19 levels, the effect of CD19 deficiency on CD21/35 expression was
assessed. CD21/35 expression levels on blood B cells from
CD19-/- mice were 25% lower than on B cells
from wild-type littermates (Fig. 2).
Nonetheless, CD19 deficiency did not significantly affect CD21/35
expression by B cells in spleen or lymph nodes (Fig. 2). Similar
results were obtained when using either the 7E9 or 7G6 mAbs (data not
shown) that bind distinct regions of the CD21/35 molecule
(49). Thus, basal mCD19 expression levels do not
significantly modulate CD21/35 expression levels. By contrast, mature B
cells from hCD19TG+/+ mice expressed 57% less
CD21/35 than wild-type B cells (Fig. 2). That CD19 overexpression
results in a significant decrease in CD21/35 expression may relate to
the hyperresponsive status of these B cells.
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Because C3 cleavage fragments are ligands for CD21/35, the effect
of C3 deficiency on CD21/35 expression was assessed. Blood, spleen, and
lymph node B cells from C3-/- mice had
significantly increased CD21/35 expression (46, 33, and 31%,
respectively) when compared with wild-type B cells (Fig. 2). Similar
results were obtained when using either the 7E9 or 7G6 mAbs (data not
shown). Similarly, CD19-/- littermates that
were C3-deficient had increased CD21/35 expression (Fig. 2B). CD21/35 expression was higher on B cells from
hCD19TG+/+C3-/- mice than
hCD19TG+/+ littermates, although the levels of
CD21/35 expression remained below those of wild-type littermates (Fig. 2B). Thus, C3 deficiency resulted in significantly increased
CD21/35 expression by mature B cells.
CD19 regulates B cell development independent of CD21/35 or C3 expression
In the current studies using CD19-/-,
hCD19TG+/+, CD21/35-/-,
and C3-/- littermates, CD19 deficiency and
overexpression had significant effects on the development and expansion
of peripheral B cells, whereas CD21/35 and C3 deficiencies had modest
effects (Fig. 3). In all cases, combined
CD19 deficiency or overexpression with CD21/35 or C3 deficiencies did
not have additive influences on B cell development in the bone marrow,
blood, spleen, or peritoneal cavity (Fig. 3 and data not shown).
Although the frequency and number of
B220lowCD5+ B1 cells is
significantly inhibited or augmented by CD19 deficiency or
overexpression, respectively, the frequency and number of
B220lowCD5+ B cells was not
significantly affected by CD21/35 or C3 deficiencies (Fig. 3). Thus,
the effects of CD19 deficiency or overexpression on B cell development
were not significantly influenced by CD21/35 or C3 expression.
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Consistent with CD19 regulating intrinsic signaling thresholds,
mature B cells from CD19-deficient or -overexpressing mice have
significantly altered surface IgM and IgD levels (Fig. 4). By contrast, blood, spleen, and lymph
node B cells from CD21/35-/- or
C3-/- littermates had wild-type levels of IgM
and IgD expression (Fig. 4). In all cases, combined CD19 deficiency or
overexpression with CD21/35 or C3 deficiencies did not have significant
additive influences on IgM or IgD expression by B cells from blood,
spleen, or lymph nodes (Fig. 4). Therefore, the effects of CD19
deficiency or CD19 overexpression on B cell Ag receptor expression were
not significantly influenced by CD21/35 or C3 expression. Thus,
although CD19 expression levels regulate signaling thresholds that
influence cell surface IgM and IgD density, the loss of CD21 or C3 does
not alter thresholds to an extent that affects Ag receptor
expression.
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The effects of CD21/35 or C3 deficiency on B cell differentiation
in CD19-/- and hCD19TG+/+
littermates were assessed (Fig. 5A). Although serum IgM levels
were decreased by 70% in CD19-/- mice and
increased by 96% in hCD19TG+/+ littermates,
CD21/35-/- and C3-/-
littermates had wild-type IgM levels (Fig. 5A). Serum
IgM levels in CD19-/-CD21/35-/- and
CD19-/-C3-/- littermates were
similar to those of CD19-/- littermates. Likewise, IgM
levels of
hCD19TG+/+CD21/35-/- and
hCD19TG+/+C3-/-
littermates were comparable to those of
hCD19TG+/+ littermates.
CD19-/- and hCD19TG+/+
littermates had 89% decreased and 132% increased IgG1 levels,
respectively. Unexpectedly, C3-/- mice had 98%
higher IgG1 levels than CD21/35-/- or wild-type
littermates (Fig. 5A). However, combining CD21/35 and C3
deficiencies with alterations in CD19 expression did not significantly
alter IgG1 levels beyond the CD19-induced changes.
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Because hCD19TG+/+ mice produce autoantibodies
including anti-dsDNA Abs (9, 51), the effects of
CD21/35 or C3 loss on anti-dsDNA Ab production were assessed in
5-mo-old littermates (Fig. 5B). IgM and IgG anti-dsDNA Abs
were increased by 510 and 340% in hCD19TG+/+ mice
compared with wild-type littermates, respectively. By
contrast, anti-dsDNA Ab levels were not significantly
different in wild-type, CD19-/-,
CD21/35-/-, or C3-/- littermates.
Anti-dsDNA Ab levels were also comparable in hCD19TG+/+,
hCD19TG+/+CD21/35-/-, and
hCD19TG+/+C3-/- littermates. Thus, CD21/35 or
C3 loss did not significantly affect autoantibody production in
hCD19TG+/+ littermates.
CD19 function dominates during humoral immune responses
The influence of CD19, CD21/35, and C3 expression on humoral
immune responses was assessed by immunizing littermates with a T
cell-independent type 1 Ag, TNP-LPS, and a TD Ag, DNP-KLH. Following
immunization with TNP-LPS, wild-type,
CD21/35-/-, and C3-/-
littermates generated comparable primary IgM responses (Fig. 6A).
CD19-/- littermates demonstrated modest IgM
responses to TNP-LPS that were not significantly affected by the
additional loss of CD21 or C3 expression. Given the significantly
reduced numbers of peripheral B cells in
hCD19TG+/+ mice relative to wild-type littermates
(Fig. 3), hCD19TG+/+ mice generate significant
humoral immune responses (31). IgM responses by
CD19TG+/+,
hCD19TG+/+CD21/35-/-, and
hCD19TG+/+C3-/-
littermates were comparable, but elevated compared with responses of
wild-type littermates.
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B). By contrast, primary and secondary IgM responses to
DNP-KLH were modest in CD19-/- mice and their
CD19-/-CD21/35-/- and
CD19-/-C3-/-
littermates. CD19-/-,
CD19-/-CD21/35-/-, and
CD19-/-C3-/-
littermates also showed similar profound defects in their primary and
secondary IgG1 responses to DNP-KLH. Primary IgG1 responses to DNP-KLH
were similarly delayed in hCD19TG+/+,
CD21/35-/-, and C3-/-
littermates (Fig. 6B). These effects were not
additive because primary IgG1 responses were similar in
hCD19TG+/+, hCD19TG+/+CD21/35-/-,
and hCD19TG+/+C3-/- littermates. Nonetheless,
secondary IgG1 responses were comparable in
hCD19TG+/+,
hCD19TG+/+CD21/35-/-, and
hCD19TG+/+C3-/- littermates. Thus,
humoral immune responses were largely regulated by
expression of CD19 with minimal contributions from CD21/35 or
C3. CD21 preferentially associates with mCD19 in hCD19TG+/+ B cells
Expression of CD21/35 on hCD19TG B cells selectively affected
mCD19 but not hCD19 expression (Fig. 1). Because we have previously
stated that hCD19 can associate with mouse CD21/35 at detectable levels
when expressed in mouse B cells that are
mCD19-/- (20), it was assessed
whether CD21/35 preferentially associates with mCD19 when both hCD19
and mCD19 are expressed. CD19-associated proteins were
immunoprecipitated from digitonin-lysed
hCD19TG+/+ and wild-type B cells, transferred to
nitrocellulose, and probed using two different antisera reactive with
either the amino- or carboxyl-terminal regions of CD21. Precipitated
mCD19 coimmunoprecipitated CD21/35 proteins of 190, 165 (Fig. 7A, upper arrow), 157, 147, 141, and 120 kDa from
wild-type B cells (Fig. 7A).
These proteins were also immunoprecipitated using the anti-CD21/35
mAb 7G6 (Fig. 7A). The 190-kDa band most likely represents
CD35, whereas the other forms of CD21/35 protein are likely to result
from the differential use of exons by CD21 and CD21/35 protein isoforms
as described (25, 26). Although coprecipitated at lower
levels, a similar spectrum of CD21/35 proteins was immunoprecipitated
from hCD19TG B cells using anti-mCD19 mAb when the autoradiographs
were visualized after further exposure. Reduced levels of
coprecipitated CD21/35 protein are most likely to reflect reduced
CD21/35 expression by hCD19TG B cells (Fig. 2). By contrast,
coprecipitation of CD21/35 was never detected with hCD19
immunoprecipitated from wild-type or hCD19TG+/+ B
cells, although the anti-hCD19 Ab efficiently precipitated hCD19
(data not shown). Coprecipitation of CD21/35 was never detected with
hCD19 immunoprecipitated from wild-type or
hCD19TG+/+ B cells even when the autoradiographs
for mCD19 immunoprecipitations were significantly overexposed.
Therefore, CD21/35 may associate with hCD19 at detectable levels in the
absence of mCD19 expression, but CD21/35 preferentially associates with
mCD19 when both hCD19 and mCD19 are expressed. This suggests that hCD19
regulates signaling thresholds in hCD19TG+/+ B
cells independent of an association with CD21/35.
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A surprising observation was that CD19 coprecipitated a number of
proteins from CD21/35-/- B cells, although
these proteins were consistently 14 kDa smaller than those
coprecipitated from wild-type B cells (Fig. 7A, lower
arrow). These proteins were CD21 because they reacted with two
different antisera specific for either the amino-or carboxyl-terminal
regions of CD21 and were precipitated by the 7G6 Ab (Fig. 7A
and data not shown). In four different immunoprecipitations, the
CD21-like proteins were abundant and were readily coprecipitated with
mCD19 from CD21/35-/- B cells. Thus,
CD21/35-/- B cells appear to express a
hypomorphic CD21/35 protein product.
The genotype of the CD21/35-/- mice was
verified using Southern blot analysis (Fig. 7B) as described
in the original paper characterizing these mice (33).
Immunofluorescence staining of B cells from
CD21/35-/- mice using two independent
anti-CD21/35 mAbs verified surface expression of the hypomorphic
CD21/35 protein (Fig. 7C). When assessed using the 7E9 mAb
in two-color immunofluorescence staining experiments, overall CD21/35
expression by B220+ blood and spleen B cells from
CD21/35-/- mice was 37 ± 6% of wild-type
levels (n = 3, data not shown). When assessed using the
7G6 mAb, blood and spleen B220+ B cells from
CD21/35-/- mice expressed CD21/35 at 24 ±
2% of wild-type levels (n = 5, data not shown), which
was consistent with the immunoprecipitation results (Fig. 7A). Because CD21/35-/- mice were
generated by targeting exons encoding SCRs 9–10 of CD21, cDNA
generated from CD21/35-/- B cells was RT-PCR
amplified using a forward primer specific for the exon encoding SCR 8
and a reverse primer specific for SCR 11. CD21/35 transcripts were
readily detected in wild-type and CD21/35-/- B
cells, although exon 9/10 appeared to be selectively spliced out in
transcripts from CD21/35-/- mice (Fig. 7D). Sequence analysis of the PCR products verified that
CD21/35-/- mice generated CD21/35 transcripts
splicing out the exon encoding SCRs 9–10, but with in-frame coding
sequence (Fig. 7E). Thus, CD21/35-/-
mice produce hypomorphic CD21/35-/- proteins
lacking the SCRs encoded by Cr2 exons 9/10
(25).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). More important is that hCD19 expression by mouse B cells
results in CD19 overexpression and a dramatic hyperresponsive phenotype
(29). However, the hallmark characteristics of
hyperresponsive B cells in hCD19TG mice were not significantly altered
when combined with CD21/35 or C3 deficiencies (
Figs. 3–6). Therefore,
the hyperresponsive phenotype of B cells that overexpress CD19 is
unlikely to result from increased sensitivity to complement activation.
In addition, biochemical studies revealed that little if any hCD19
associates with CD21/35 in hCD19TG B cells when mCD19 is expressed
(Fig. 7A). Thus, hCD19 is not equivalent to mCD19 in
generating CD19-CD21 complexes in mouse B cells, although hCD19
expression at wild-type levels can restore normal function in
CD19-/- mice (20). These findings
do not rule out the possibility that complement activation and CD21/35
engagement generate transmembrane signals through CD19, but suggest
that CD19 predominantly regulates signal transduction through
complement-independent mechanisms.
Cell surface CD21/35 expression levels were significantly affected by
C3 and CD19 expression. Specifically, CD21/35 expression was increased
by 31–46% on peripheral B cells from C3-deficient littermates (Fig. 2). This suggests that ongoing C3d,g fragment generation may
chronically engage CD21/35, resulting in receptor internalization.
During inflammatory responses or in patients with systemic autoimmune
disease, serum immune complexes can be loaded with C3d. In fact, B
cells from patients with systemic lupus erythematosus have decreased
CD21 (51, 52, 53) and CD19 expression levels
(51), presumably due to the continuous presence of CD21/35
ligands that induce CD21 internalization (54). In
lupus-prone MRLlpr/lpr mice, CD21/35 expression
levels are also specifically decreased on B cells, even before the
development of clinical or serological manifestations of disease
(50). The 44–58% decrease in CD21/35 expression (Fig. 2)
and 20–29% decrease in mCD19 expression (Fig. 1) by B cells from
hCD19TG+/+ mice may also result from increased
CD21/35 receptor internalization because these mice produce
autoantibodies that may activate complement. Furthermore, CD21/35
expression in autoimmune Lyn-/- mice
(55, 56, 57) is reduced by 60% (our unpublished data).
Thus, C3, CD21/35, and CD19 expression may be components of a common
regulatory loop that influences B cell signaling thresholds. CD21/35
engagement may result in increased CD19 turnover, which may limit
signal transduction or partially desensitize B cells chronically
stimulated through the CD19-CD21 complex.
CD19 expression levels were significantly influenced by CD21/35 and C3
expression (Fig. 1). CD19 expression levels were 16–36% higher on
peripheral B cells of CD21/35-/- mice (Fig. 1),
whereas CD21/35 deficiency did not influence cell surface IgM or IgD
expression levels (Fig. 4). Human CD19 expression by hCD19TG B cells
was not affected by CD21/35 deficiency, consistent with a lack of
detectable physical interaction between these xenogeneic molecules
(Fig. 7A). Therefore, CD21/35 may influence mCD19 expression
by regulating cell surface complex turnover. This contrasts with
CD81-deficient mice where cell surface CD19 expression is reduced by
half (58, 59, 60). Increasing CD19 expression by 16–36% on
CD21/35-/- B cells may be functionally
significant because 20% increases in CD19 expression predispose
mice to autoimmunity, and similar increases in CD19 expression
correlate with autoantibody production in humans (51). In
general, CD19 overexpression increases endogenous levels of activated
Lyn, dysregulates tolerance, and results in autoantibody production
(9, 51, 61). This may explain in part why CD21/35
deficiency contributes to autoimmunity (41, 42). Normally,
CD19 expression levels are developmentally regulated and tightly
controlled (10, 20). In mice, the majority of early B
lineage cells express mCD19 at relatively high levels in the bone
marrow, with a 2.5-fold increase in expression during the transition
from an immature B220low to a mature
B220high CD21+ B cell
(9, 25). Therefore, it is interesting that peritoneal B1
cells express lower levels of CD21 (50), yet express
incrementally higher levels of CD19 than conventional B cells
(9). Thus, regulated CD19 and CD21/35 expression levels
may balance intrinsic signal transduction thresholds and B cell
responsiveness to transmembrane signals.
B cell development, surface IgM expression, and serum Ig levels were
almost normal in CD21/35-/- and
C3-/- littermates (
Figs. 3–5), as previously
described (33, 34, 39, 40). Although a decrease in
peritoneal B1 cell numbers has been reported in only one line of
CD21/35-/- mice (33, 34), a
significant difference in numbers or frequency of peritoneal B1 cells
was not observed in this study among the 17 littermate pairs assessed
(Fig. 3). This discrepancy may be due to the different genetic
backgrounds as described (62) or the housing of mice used
in each study, because we generated CD21/35-/-
littermates through paired breedings of
CD19+/-CD21/35+/-
littermates or
hCD19TG+/-CD21/35+/-
littermates. The frequencies of peritoneal B1 cells were also normal in
C3-/- mice (Fig. 3) as previously shown for
both C3-/- and C4-/-
mice (39). Therefore, a decreased frequency of peritoneal
B1 cells in CD21/35-/- mice may be a variable
phenotypic trait. Nonetheless, CD19-/- mice had
significantly decreased peripheral B cells and peritoneal B1 cells,
increased surface IgM expression, and decreased serum Ig levels,
regardless of CD21/35 or C3 expression (
Figs. 3–5). Likewise,
hCD19TG+/+ mice had significantly decreased
numbers of mature B cells in the bone marrow and peripheral tissues,
increased peritoneal B1 cells, down-regulated surface IgM expression,
and increased serum Ig levels, regardless of CD21/35 or C3 expression
(
Figs. 3–5). Increased or down-regulated surface IgM expression is a
consequence of impaired or augmented transmembrane signaling through
the B cell Ag receptor complex, respectively (1, 63). Each
of these results is consistent with the conclusion that CD19 can
function independently of CD19/CD21 complex expression, although
CD21/35 uses the CD19 complex to generate transmembrane signals through
the CD19 cytoplasmic domain (11).
Primary and secondary humoral immune responses to TD and T-independent
Ags were severely compromised in CD19-/- mice,
particularly IgG1 responses to TD Ags (Fig. 6), as previously described
(30, 31). By contrast, serum IgG1 responses to a TD Ag
were only delayed in CD21/35-/- and
C3-/- littermates, and these mice generated
normal primary IgM responses and secondary responses (Fig. 6). Delayed
IgG1 responses to DNP-KLH in CD21/35-/- and
C3-/- littermates did not hinder the IgG1
responses of hCD19TG+/+ littermates when these
genetic alterations were combined. Therefore, there were dramatic
quantitative and qualitative differences between the immune responses
of CD19-deficient mice and CD21/35-/- or
C3-/- littermates. Although CD19 deficiency
always has profound effects on humoral immune responses, TD-immune
responses and affinity maturation in CD21/35-/-
mice vary depending on the Ag dose and use of adjuvants (39, 64, 65, 66). Thus, alterations in CD19 expression can influence Ab
production irregardless of CD21/35 or C3 expression.
Although the CD21/35-/- mice used in the
current studies have been well characterized, these mice expressed low
levels of a hypomorphic CD21/35-/- molecule
(Fig. 7). An appropriate genotype was verified in
CD21/35-/- mice by Southern blot analysis (Fig. 7B), as originally described (33). Cell surface
CD21/35 expression was verified using two mAbs that react with
different extracellular regions of the protein, with one Ab binding to
the ligand-binding site in SCR domains 1–2 (Fig. 7C). PCR
and nucleotide sequence analysis indicate that CD21/35 transcripts in
the CD21/35-/- mice had spliced out exons 9/10
of CD21 (Fig. 7, D and E), which is the
Neor insertion site that generates the
targeted mutation (33). Thereby,
CD21/35-/- mice produce a 14-kDa smaller cell
surface CD21 protein that retains its ability to associate with mCD19
(Fig. 7A) and retains SCR domains 1 and 2 that normally
mediate CD21 interactions with ligands (67, 68). In many
cases, exon length can affect RNA splicing in transcripts initiated
from the endogenous promoter (69). An artificially large
exon with an inserted selective marker may not be recognized by the
splicing mechanism and may be skipped, thereby deleting the mutated
exon from the mRNA transcript. With CD21/35, this allowed the
inappropriate splicing of exons 8 and 11, which does not induce
frame-shift mutations or a null allele. A similar strategy was used to
generate a second line of CD21/35-/- mice,
where exons encoding SCR 8 of CD21 (or SCR 14 of the CD21/35 protein)
were targeted (34). An absence of detectable C3 binding by
FDC or marginal zone B cells has been demonstrated for both lines of
CD21/35-/- mice (33, 66, 70). In
addition, both C3- and CD21/35-deficient mice share similar phenotypic
characteristics. Nonetheless, it remains to be established whether the
hypomorphic CD21/35 molecules expressed at low levels by
CD21/35-/- mice are functionally relevant
because there is a threshold effect for CD21/C3d,g interactions
whereby a minimum concentration of cell surface CD21 is necessary
to bind C3d,g-containing particles (71).
That CD21 expression influences CD19 density on the cell surface provides another mechanism through which the complement system may regulate B cell signaling thresholds. Alterations in CD19 expression are important because CD19 cell surface density intrinsically regulates Src family protein tyrosine kinase activity in B cells (14, 72). CD21 engagement may further modulate B cell activation by cross-linking and further augmenting CD19 function. Although the identity and biological significance of CD19 ligands remains to be demonstrated (1, 3), CD21-independent ligands may also regulate the intrinsic signaling function of CD19. That CD19 can function and regulate B cell signaling thresholds independent of CD21/35 and C3 expression affirms an independent role for CD19 as an intrinsic regulator of B lymphocyte signal transduction that may be influenced by complement activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas F. Tedder, Department of Immunology, Duke University Medical Center, Box 3010, Durham, NC 27710. E-mail address: thomas.tedder{at}duke.edu
3 Abbreviations used in this paper: FDC, follicular dendritic cell; KLH, keyhole limpet hemocyanin; hCD19, human CD19; hCD19TG, transgenic mice that overexpress hCD19; mCD19, mouse CD19; SCR, short consensus repeat unit; TD, T cell-dependent; TNP-LPS, 2,4,6-trinitrophenol-conjugated LPS.
Received for publication May 3, 2001. Accepted for publication July 19, 2001.
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