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Complement receptor proteins CR2 (CD21) and CR1 (CD35) have been identified as components of the murine B cell co-receptor complex. Gene expression profiles between naive WT, C3−/−, and CD21/35−/− B cells demonstrate enhanced expression of a Ca2+-modulating gene, Pcp4, in WT mice compared to the complement-deficient animals. Increased expression of Pcp4 is also coincident with B cell maturation into end stage phenotypes. Prolonged activation of B cells via crosslinking of the BCR (but not CR1/CR2 alone) leads to increased expression of Pcp4 and suppressed Ca2+ release. In total these data demonstrate that the expression of Pcp4 in naïve resting mature B cells is dependent upon tonic stimulation from the CR1/CR2 proteins via a C3 ligand, and that antigen specific B cell activation can also elevate Pcp4 expression that is coincident with suppression of calcium-dependent responses.
Signals through the B cell antigen receptor regulate B lymphocyte development and function (Tedder et al., 1997). B cell receptor engagement activates signaling pathways by protein tyrosine kinases (PTKs) including members of the Src-family of PTKs, Syk, and Btk, and results in Ca2+ mobilization. After receptor activation, the release of Ca2+ from endoplasmic reticulum (ER) stores into the cytosol is the first detectable increase in cytosolic Ca2+. Once ER stores are depleted, BCR signals are able to sustain intracellular Ca2+ ([Ca2+]i) levels via store-operated calcium entry (SOCE) that allows Ca2+ to pass from the extracellular environment to the cytosol (Cahalan et al., 2001; Engelke et al., 2007). Negative regulation of BCR-induced Ca2+ signaling can occur through co-ligation with either the low-affinity Fc receptor for IgG (FcγRII) or CD22 with the BCR (Nitschke et al., 1997; Sato et al., 1998; Wilson et al., 1987). Modifications of Ca2+ signaling in B cells contribute to B cell activation and maturation, differentiation, and gene expression.
Complement receptor protein CD21 (CR2) along with CD19, CD81, and Leu13 have been identified as components of the human B cell co-receptor complex. The mouse CD35 (CR1) protein is also part of this complex on murine B cells (reviewed in (Jacobson and Weis, 2008). Co-ligation of CD21 and the BCR via complement-bound antigens results in a higher level of cellular activation, elevation of intracellular Ca2+ and activation of mitogen-activated protein kinases than BCR signaling alone (Carter et al., 1988; Luxembourg and Cooper, 1994). The CD21/35 proteins also enhance complex-bound antigen uptake and processing, assist in the localization of the BCR to lipid rafts, and function in transferring immune complexes from marginal zone B cells to follicular dendritic cells (FDCs) (Cherukuri et al., 2001; Whipple et al., 2004). Enhanced B cell activation by CD21/35 cross-linking is primarily considered to be dependent upon its association with CD19. After BCR and CD21/35/CD19 ligation, phosphorylation of CD19 recruits the Src family kinase, Lyn, resulting in amplified Lyn kinase activity (Fujimoto et al., 1999b; Hasegawa et al., 2001). This action facilitates CD19 interactions with phosphoinositide 3-kinase (PI3K) and Vav, initiate downstream signaling events and results in augmentation of [Ca2+]i responses (Buhl et al., 1997; Fujimoto et al., 1999a; Sato et al., 1997).
In the mouse, CD21/35 proteins are encoded by the same Cr2 locus and co-expressed on B cells and FDCs (Kurtz et al., 1990). The role for complement C3 cleavage products, such as the ligands for CD21/35, as regulators of B cell signaling and humoral immunity is well appreciated. As such, animals lacking the CD21/35 proteins generate modest antibody responses to both T cell-dependent and T cell-independent antigens (Ahearn et al., 1996; Haas et al., 2002; Molina et al., 1996). It is important to note that although C3 breakdown products positively regulate B cell activation through the CD19/CD21 complex, excess CD21 ligation has also been shown to attenuate Ca2+ responses (Chakravarty et al., 2002; Lee et al., 2005).
Even though antibody responses are depressed, B cell activation and maturation occurs in the absence of CD21/35 proteins (Haas et al., 2002; Jacobson et al., 2008). To better understand splenic B cell activation in the presence or absence of CD21/35 co-ligation in-vivo, we analyzed gene expression profiles from splenocytes of naïve and immunized WT and CD21/35−/− mice. Of the various genes differentially expressed, we choose to further analyze one gene of interest, that encoding the purkinje cell protein 4 (Pcp4). Pcp4 (also known as Pep19) belongs to a family of proteins involved in calcium transduction signals. Initially identified in neurons, Pcp4 modulates Ca2+ by binding to calmodulin via an IQ motif (Johanson et al., 2000). More relevant to our studies, gene expression profiling identified Pcp4 as a transcript increased in anergic B cells (Glynne et al., 2000). We have found the expression of Pcp4 was increased 6 fold in splenocytes from immunized WT mice compared to splenocytes from immunized CD21/35−/− animals. These data were consistent with previous gene expression profiles between naïve WT and CD21/35−/− splenocytes (Jacobson et al., 2008) indicating that the altered expression was due to the lack of the CD21/35 proteins, not the effect of immunization. Additionally, transcript analysis from C3-deficient splenocytes also demonstrated decreased levels of Pcp4 expression (compared to wild type), implicating a role for tonic stimulation of the B cell via C3-CD21/35 interaction in-vivo. Finally, in-vitro studies demonstrated that BCR activation independent of CD21/35 crosslinking induced Pcp4 expression coincident with suppression of Ca2+ responses. These data suggest that Pcp4 may be a novel regulator of BCR/CD21 induced Ca2+ signaling in splenic B cells.
Female BALB/c and C57BL/6 mice were obtained from the National Cancer Institute. CD21/35−/− mice were generated as described (Haas et al., 2002) and backcrossed 10 times onto BALB/c or C57BL/6 backgrounds and used for the experiments described. C57BL/6 C3−/− mice (Wessels et al., 1995) were purchased from Jackson Labs. Animals were housed in the Animal Resource Center (University of Utah Health Science Center, Salt Lake City, UT) according to the guidelines of the National Institute of Health for the care and use of laboratory animals.
Mice 8 wk old were immunized by i.p injection of 85 μg of trinitrophenyl keyhole limpet hemocyanin precipitated in aluminum hydroxide gel (Sigma). Spleens were harvested for RNA 4 days post injection.
Equal amounts of total RNA from the spleens of 5 immunized (see above) female 8-wk-old BALB/c or C57BL/6 WT or CD21/35−/− mice were pooled into a sample that was prepared for Affymetrix array hybridization. cDNA was synthesized from 8 μg of total RNA, and each sample was hybridized to a single GeneChip Mouse Genome 430.2 Array (Affymetrix). Data was processed as described in (Jacobson et al., 2008). Gene expression analysis between strains was performed in duplicate.
Total RNA was isolated from spleen tissue using CsCl guanidine extraction. After RBC lysis, single cell populations of splenocytes were labeled with B220 magnetic microbeads (Miltenyi Biotech) and separated according to manufacturer’s protocol. Total RNA was isolated from cells using Amersham RNA mini-spin kit (GE Healthcare). RT-PCR on 2μg of total RNA was preformed using random primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). cDNA levels were assessed using quantitative PCR on the LightCycler (Roche) as in (Jacobson et al., 2008). β-Actin: forward, 5′-gtaacaatgccatgttcaat-3′; reverse, 5′-ctccatcgtgggccgctctag-3′, 135bp. PCP4: forward, 5′-cagccacccctgagcattg-3′; reverse, 5′-ttgtcttttccgttggtcgc-3′, 108bp. CD19: forward, 5′-aggaaaaggaagcgaatgact-3′; reverse, 5′-gcctggccagaggtagatgta-3′, 125bp.
Single-cell suspensions were made from spleens of 6-to 8wk-old mice, and RBCs were lysed with ACK lysing buffer. Total splenocytes or B220+ sorted cells were cultured at 5 X 106 cells/ml in RPMI 1640 supplemented with 10% FBS (Invitrogen Life Technologies), L-glutamine, 50 μM 2-ME, 100U/ml penicillin, and 100 μg/ml streptomycin in the presence or absence of 10 μg/ml Goat F(ab′)2 anti-mouse IgM (Southern Biotech), 5 μg/ml rat anti-mouse CD21/35 (BD Pharmingen), or Rat IgG2b (BD Pharmingen).
A fraction of purified B cells from 48 hour cultures were analyzed for IgM expression levels using flourochrome-Cy5-conjugated anti-IgM (Biomeda Corp). Cell staining and analysis was performed as described previously (Jacobson et al., 2008). Remaining B cells were incubated for 45 minutes at 37° with the Ca2+-sensitive dye Fluo-4 (3 μg/ml; Molecular Probes) in RPMI medium containing 10% FBS. Lymphocytes were washed and incubated in the dark for 20 minutes at 37° and then analyzed on a FACS Caliber flow cytometer (BD Bioscience). Baseline Ca2+ was measured for 30s, followed by the addition of 10 μg/ml F(ab′)2 anti-mouse IgM or 1μg/ml ionomycin. Ca2+ was measured for 5 minutes.
Data are given as means ± SEM. Student’s t test was used to determine whether there were significant differences between sample means.
The murine CD21/35 proteins are members of the B cell co-receptor complex such that cross-linking the BCR along with CD21/35 results in a higher level of cellular activation than signaling through the BCR alone. To investigate differences downstream of B cell receptor signaling between WT and CD21/35−/− mice during the early events of an immune response, gene expression profiles from total spleen RNA of immunized WT or CD21/35−/− were obtained using Affymetrix GeneChip Microarrays. Analysis of the microarray data from these immunized mice indicated that a variety of genes are differentially expressed between WT and CD21/35−/− splenocytes on both the BALB/c and C57BL/6 strain background. Of the 25 genes with increased expression levels in WT compared to CD21/35−/− on both BALB/c and C57BL/6 background, Pcp4 (increased 2–6 fold) was of particular interest (Table 1). It has been previously reported that Pcp4 expression is increased in anergic B cells compared to naïve B cells (Glynne et al., 2000) suggesting elevated levels of Pcp4 may inhibit B cell activation.
Previous gene expression profile analysis between naïve WT and CD21/35−/− splenocytes also demonstrated elevated expression of Pcp4 in WT cells indicating depressed Pcp4 expression in CD21/35−/− mice was not due to the immunization regimen (Table 1) (Jacobson et al., 2008). Additionally, past analysis of maturing B cells demonstrated increasing Pcp4 expression with differentiation (Debnath et al., 2008). Pcp4 expression differences were confirmed by RT-PCR for both BALB/c and C57BL/6 WT and CD21/35−/− naïve (non-immunized) spleen samples (Fig. 1A). To determine if decreased Pcp4 expression from CD21/35−/− cells could be due to a lack of C3 cross-linking of CD21, Pcp4 transcripts were also analyzed from splenocytes from naïve C3−/− mice. Compared to C57BL/6 WT, Pcp4 mRNA was decreased 2 fold in C3−/− splenocytes (Fig. 1A).
Because the gene expression analysis was preformed with total splenocyte populations, Pcp4 expression was confirmed in B cells by transcript quantification of B220+ and B220− sorted splenocyte populations. As shown in Fig. 1B, Pcp4 expression was predominantly found in B220+ cells compared to B220− cells in the spleen. In total, these data demonstrate that the lack of the CD21 proteins and/or C3 depress expression of Pcp4 in murine splenic B cells obtained from either naïve or immunized animals. Thus tonic stimulation of CD21/35 via C3 intermediates on naïve, resting B cells is required for the normal expression level of Pcp4.
CD21/35 transcript and surface expression levels increase as B cells develop from the bone marrow to mature splenic B cells. Furthermore, our previous gene expression data suggested an increase in Pcp4 expression throughout B cell maturation that paralleled the increased expression of CD21 (Debnath et al., 2008). To determine if Pcp4 transcripts were increased in mature, CD21/35 -expressing B cells, we analyzed Pcp4 expression differences between developing and mature B cells from BALB/c WT or CD21/35−/− mice. Bone marrow B cells from 2-week-old animals, in which all of the B cells are immature and CD21/35 negative, demonstrated barely detectable Pcp4 expression levels (Fig. 2). Transitional, maturing B cells from the spleens of 2wk old animals had a slight increase in Pcp4 mRNA, whereas Pcp4 displayed the highest expression level in mature B cells from the spleens of WT animals (Fig. 2). These data show that Pcp4 expression levels from developing WT B cells correlates with the level of maturation of the B cell. When coupled with the decrease in Pcp4 mRNA from splenocytes of CD21/35−/− and C3 deficient mice, these data further suggest a role for the CD21/35 proteins in regulating Pcp4 expression levels in maturing B cells.
The strength of the B cell signal throughout the course of development is important for B cell survival and selection. The finding that anergic B cells demonstrate elevated levels of Pcp4 transcripts suggests that the Pcp4 protein may serve to suppress B cell activation. These data led us to hypothesize that Pcp4 expression may also be influenced by BCR signaling. Splenic B cells were held in culture for either 5 min or 48hr prior to RNA isolation. The expression level of Pcp4 drops dramatically to about the same level as the CD21/35−/− cells when they are held culture without any additional stimulation (WT media controls) (Fig. 3). When the BCR was cross-linked with F(ab′)2 anti-IgM in culture, Pcp4 expression was increased compared to un-treated splenocyte cultures (Fig. 3). Cross-linking the BCR with CD21/35 led to a slight increase in Pcp4 transcription over BCR cross-linking alone suggesting that, in vitro, B cell receptor activation dependent induction of Pcp4 transcription may be augmented with CD21/35 signaling. Cross-linking the BCR with anti-IgM maintained Pcp4 expression in CD21/35−/− B cells as well (Fig. 3), indicating that there is not a developmental or intrinsic defect in these cells to induce Pcp4 transcripts upon BCR mediated activation.
To date, the identified function for Pcp4 is as a calmodulin(CaM)-binding protein in neurons, acting as an modulator of Ca2+-dependent activation of CaM kinase II (Johanson et al., 2000). The known role for Pcp4 in regulating calcium signaling (in neurons) and its induction downstream of BCR cross-linking (see above) prompted us to determine if Pcp4 is a modulator of BCR-dependent calcium signaling in B cells. We induced Pcp4 expression in B cell cultures by BCR cross-linking with anti-IgM, similar to above, except 24 hrs after induction, cells were placed in fresh media in the absence of stimulus for the remaining 24hrs of culture. RT-PCR analysis of cultured B cells again showed an increase in Pcp4 expression 48 hours after BCR cross-linking (Fig. 4A). To determine if Pcp4 expression levels altered BCR-induced intracellular calcium ([Ca2+]i) flux, we loaded media-treated (low Pcp4) and BCR cross-linked (high Pcp4) B cells with a Ca2+- sensitive dye, stimulated the cells with anti-IgM and measured [Ca2+]i flux by flow cytometry. B cells possessing increased levels of Pcp4 responded to BCR stimulation with a lower [Ca2+]i flux than those without a prior BCR stimulation (and thus lower expression levels of Pcp4) (Fig. 4C, upper panel). Ionomyocin stimulation yielded similar [Ca2+]i release from both cell populations controlling for equivalent dye loading Fig. 4C, lower panel). Furthermore, cells stimulated with anti-IgM demonstrated increased amounts of IgM on the cell surface, indicating that the decrease in [Ca2+]i flux was not due to unavailability of BCR to stimulate (Fig. 4B). These data suggest that previous BCR stimulation leads to Pcp4 induction and dampened [Ca2+]i release after BCR cross-linking.
In this report, we have identified a novel, potential regulator of BCR-induced Ca2+ responses. Gene expression analysis between immunized and resting, naïve WT and CD21/35−/− splenocytes indicated elevated Pcp4 expression in WT spleens, which we demonstrate was produced in mature B cells. A similar depression of expression of Pcp4 in naïve, resting B cells was also found in animals lacking C3 suggesting tonic stimulation of mature, naïve B cells via CD21/35 is required for normal expression of the Pcp4 gene. Interestingly, expression of Pcp4 could be elevated over WT base line levels by crosslinking the BCR. This elevated expression was slightly enhanced with additional CD21 crosslinking although CD21 crosslinking alone did not maintain Pcp4 transcription to the same level as BCR activation. These data suggest two thresholds of Pcp4 expression may exist in mature B cells: the first level, dictated by tonic stimulation of resting B cells by the CD21/35 proteins (via a C3 ligand) and a second level within activated B cells that is dependent upon BCR signaling. Finally, consistent with its role as a calmodulin-binding protein in neurons, Pcp4 expression may have a negative influence on [Ca2+]i flux as cells with increased Pcp4 expression levels via BCR-cross-linking mounted decreased [Ca2+]i responses in response to additional anti-IgM stimuli.
The data presented in this report suggest two, if not three, mechanisms regulating Pcp4 expression: tonic via CD21/35 crosslinking, increased expression with increased maturation of the B cell (which is coincident with increased expression levels of CD21/35), and the elevated expression levels after BCR crosslinking. CD21/35−/− B cells produced Pcp4 transcripts in similar quantity to WT after BCR stimulation, indicating that cells from CD21/35-deficient animals do not have an intrinsic defect in Pcp4 transcription or signaling via the BCR. Other B cell stimuli, such as LPS through toll-like receptor 2, or CD40L did not enhance Pcp4 transcripts alone, or in conjunction with BCR cross-linking (data not shown). Collectively, these data suggest that signaling through the B cell receptor alone is sufficient to induce the elevated levels of Pcp4 transcripts.
The observations that Pcp4 levels are depressed (compared to WT) in naïve, resting B cells obtained from both CD21/35−/− and C3- strains of mice suggests that tonic stimulation via C3 ligands through the CD21/35 signal transduction pathway is required for normal Pcp4 expression. However an alternative model for the role of CD21/35 and C3 proteins in inducing Pcp4 expression in B cells in-vivo implicates the function of these proteins on FDCs. The role for FDCs and complement receptors in antigen acquisition in the spleen is well appreciated (reviewed in (Roozendaal and Carroll, 2007)). For example, complement-coated antigen bound by CD21/35 on FDCs is required for efficient maintenance of antibody producing cells (Barrington et al., 2002; Fang et al., 1998). Within follicles of lymphoid organs, C3-coated immune complexes bound by CD21/35 on FDCs can serve as means of cell-to-cell interaction with B cells. Thus, a function for C3/CD21/35 in regulating steady state Pcp4 expression by B cells in-vivo may be via a FDC interaction of B cells with complement-coated antigen complexes. This is an environment not reproduced in our culture system. Furthermore, unstimulated CD21/35−/− B cells produced similar levels of Pcp4 transcripts as WT B cells after 48 hrs in culture, compared to decreased relative transcript levels after 5 min in culture (Fig. 3). These data suggest that after 48 hours of culture signals, such as low level BCR-activation, required for normal induction of Pcp4 are absent and that the B cell micro-environment may influence Pcp4 expression.
Our data indicated that cells with increased Pcp4 expression responded with decreased [Ca2+]i responses to BCR ligation. From these data we can infer a role for negative regulatory role of PCP4 in B cell [Ca2+]i responses. The increased expression of Pcp4 observed in anergic B cells (and the link of Pcp4 expression to C3/CD21/CD35 in WT B cells) is interesting in light of the report that B cell anergy can be broken by the crosslinking of the CD21 proteins on anergic B cells (Lyubchenko et al., 2007). Putkey and colleagues have defined a role for Pcp4 (and similar IQ motif proteins) to accelerate the rates of association and dissociation of Ca2+ to sites II and IV in the C-domain of calmodulin (Putkey et al., 2003; Putkey et al., 2008). Additionally, Pcp4 can alter the function of free calmodulin as well as that bound to calmodulin-dependent protein kinase II. These data suggest that modulation of Pcp4 expression in B cells could alter the function of calmodulin in such cells, altering their steady state and activation dependent Ca2+ responses. Modulating the expression of Pcp4 in naïve and anergic B cells should help define the role of this protein in regulating B cell activation and anergy, and autoimmunity.
The authors would like to thank members of both labs for their insightful and stimulating critiques of this work. This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI-24158, JHW: AI-32223, JJW). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Institute of Allergy and Infectious Diseases or the National Institutes of Health. A.C.J. was supported by the Training Program in Microbial Pathogenesis, 5T32-AI-055434.
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