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. 2012 Sep;137(1):48–55. doi: 10.1111/j.1365-2567.2012.03602.x

The CD19/CD81 complex physically interacts with CD38 but is not required to induce proliferation in mouse B lymphocytes

Felipe Vences-Catalán 1, Ranjani Rajapaksa 2, Shoshana Levy 2, Leopoldo Santos-Argumedo 1
PMCID: PMC3449246  PMID: 22564057

Abstract

In B lymphocytes, the cell surface receptor CD38 is involved in apoptosis of immature B cells, proliferation and differentiation of mature B cells. Although CD38 has been establish as a receptor, its signaling has been only partially characterized. As a result of the lack of signaling motifs in the cytoplasmic domain, CD38 must use a co-receptor to induce signaling within the cell. Accordingly, CD38 has been associated with different receptors such as the T-cell receptor/CD3 complex on T cells, CD16 on natural killer cells and MHC class II molecules on monocytes. The CD19/CD81 complex has been proposed as a co-receptor for CD38 in human B lymphocytes, but little or no characterization has been performed in mice. In this study the contribution of the CD19/CD81 complex in murine CD38 signaling was evaluated. Proliferation assays were performed using CD19−/− or CD81−/− deficient mice; CFSE-labeled B lymphocytes from wild-type mice and CD19−/−, CD81−/− and CD38−/− deficient mice were stimulated with agonistic antibodies against CD38. Immunoprecipitation and immunofluorescence were also performed to detect protein–protein interactions. Our results indicate that the CD19/CD81 complex interacts with CD38 but this interaction is not required to induce proliferation in mouse B lymphocytes, suggesting that other receptors may contribute to the proliferation induced by CD38 in B lymphocytes.

Keywords: B cells, CD38, cell surface molecules, signaling

Introduction

CD38 is a cell surface receptor on B lymphocytes and other cells in the immune system. Cross-linking with agonistic antibodies (mimicking a putative natural ligand) induces the redistribution of CD38 to lipid rafts where in proximity with other receptors it triggers signals within the cell.1,2 Lyn, Fyn and Btk are tyrosine kinases that are activated in CD38 signaling in B lymphocytes. These data are supported by several reports in which B lymphocytes from Lyn−/−, Fyn−/− and X-linked immunodeficient mice failed to proliferate in response to CD38.3–7 However, the cytoplasmic tail of CD38 has no canonical motifs or domains that interact with signaling molecules. In addition, it has been shown that the removal or replacement of the cytoplasmic tail does not impair CD38 signaling.8,9 For this reason, it has been suggested that CD38 should be associated with other receptors to signal.

The CD38 cis interactions have been explored mainly in human lymphocytes, where CD38 has been associated with the T-cell receptor (TCR)/CD3 complex in T lymphocytes,10–12 CD16 in natural killer cells 13 and MHC class II molecules on monocytes.14 The CD19/CD21 complex in human B lymphocytes has been proposed as co-receptor of CD38.11 The cytoplasmic tail of CD19 contains tyrosine signaling motifs, which can recruit Lyn and phosphoinositide 3-kinase.15,16 Additional studies have shown that co-engagement of the B-cell receptor (BCR) and CD19/CD21/CD81 complex reduces the threshold of B-cell activation. 17 CD81, a member of the tetraspanin family, is important for the expression of CD19, acting as a chaperone and is also a component of the tetraspanin web that serves as a docking site for other receptors and signaling molecules.

In human B cells co-capping experiments have demonstrated that CD38 co-localizes in lipid rafts with these two receptors. The same studies have also shown that CD81 and CD19 co-immunoprecipitate with CD38,18,19 this interaction with CD81 and CD19 has also been observed in exosomes from human lymphoblastoid B cells.20 Functionally, CD38 cross-linking induces CD19, Lyn, phosphoinositide 3-kinase and c-cbl phosphorylation in human B cell lines. When CD19 is down-regulated with small interfering RNA, calcium flux is inhibited after CD38 cross-linking, which suggests that CD19 is the main co-receptor in human B cells.18,21 However, the interaction of CD38 with the CD19/CD81 complex in mouse B cells has not been explored. We reasoned that the use of mice deficient for CD19, CD38 and CD81 might allow a more direct approach to evaluate these interactions. In this work we confirm that CD81 and CD19 physically interact with CD38 in lipid rafts from mouse B cells; interestingly, this interaction is not necessary for CD38-induced proliferation, suggesting that CD38 might use different co-receptors in human and mouse B lymphocytes.

Materials and methods

Mice

C57BL/6 wild-type, CD19−/− and CD38−/− mice were bred and maintained in the CINVESTAV-IPN animal facility. BALB/c wild-type and CD81−/− mice were maintained at Stanford University according to the Public Health Service Policy for Humane Care and Use of Laboratory Animals. All experiments were approved by the Animal Care and Use Committee of CINVESTAV.

Antibodies

Goat and rabbit anti-mouse CD38 polyclonal antibodies were produced in our laboratory. Anti-CD81 (104907) and anti-CD19 (115513) were purchase from Biolegend (San Diego, CA), anti-B220-FITC (553088) and anti-CD9 (558749) were purchase from BD Pharmingen (San Diego, CA); and anti-CD63 (sc15363) from Santa Cruz Biotechnology, Inc (CA) and secondary antibodies anti-rabbit-Cy3 (Caltag Laboratories, Burlingame, CA; L42010), anti-hamster-FITC (BD Pharmingen; 554011), anti-rabbit-horseradish peroxidase (HRP) (Thermo Scientific, Pittsburgh, PA; 1858415), anti-hamster-HRP (BD Pharmingen; 554012), anti-rat-HRP (ZyMax, San Francisco, CA; 81-9520) and anti-mouse-HRP (Thermo Scientific; 1858413). Anti-Lyn (sc-15) was kindly provided by Dr Claudia González Espinoza. Rat anti-mouse CD38 (NIM-R5) (Southern Biotechnology, Birmingham, AL) was used for immunoprecipitation and rat IgG2a as an isotype control.

Capping experiments

Splenocytes from C57BL/6 mice (1 × 106 cells) were incubated with anti-CD38 (30 min on ice), washed, and reacted with secondary antibody anti-rabbit-Cy3 for 20 min on ice. Samples were then moved for 1 hr to 37° to induce capping and then stopped by blocking with ice-cold PBS containing 0·5% BSA and 0·1% NaN3. Counterstaining was performed in ice-cold PBS containing 0·5% BSA and 0·1% NaN3 with anti-CD81, anti-CD63, anti-CD9 or anti-B220 antibodies, followed by FITC-labeled secondary antibodies. After washing, cells were fixed (4% paraformaldehyde), placed on poly-l-lysine-coated coverslips and analysed using confocal microscopy (Olympus IX71 Center Valley, PA) at 60× magnification. Confocal images were analysed with imagej software (http://rsbweb.nih.gov/ij).

Lipid rafts isolation

Briefly, splenocytes from C57BL/6 and CD19−/− mice (5 × 108 to 10 × 108 cells) were lysed in lysis buffer containing 1 mm Tris–HCl, 15 mm NaCl, 0·5 mm EDTA, 10% Brij-98, 2 mm Na3VO4 (Sigma-Aldrich, St. Louis, MO), 10 mm NaF (Sigma-Aldrich), 1 mm PMSF (Sigma-Aldrich), 2 μg/ml aprotinin (Sigma-Aldrich) and 2 μg/ml leupeptin (Roche Diagnostics, Indianapolis, IN), pH 7·5, for 30 min on ice. Lysates were homogenized with 10 strokes of a loose-fit Dounce homogenizer. Then the lysates were centrifuged at 900 g for 11 min at 4° to remove mitochondria, nuclei and other large debris. The cleared lysates were mixed 1 : 1 with 85% sucrose in Beckman centrifuge tubes. Then the 35% and 5% sucrose were added dropwise. Samples were centrifuged at 200 000 g for 16–20 hr at 4°. In total, 10 fractions of 1 ml each were collected on ice from the top to the bottom of the gradient. Fractions 3, 4 and 5 correspond to lipid rafts. Aliquots of each fraction were resolved by 10–15% SDS-PAGE and transferred to nitrocellulose membranes and probed with unlabeled anti-Lyn or anti-CD38 followed by secondary antibody anti-rabbit-HRP (Pierce, Thermo Scientific) and revealed by chemiluminescence system (Thermo Scientific).

Immunoprecipitation and Western blot

Lipid raft fractions 3, 4 and 5 were immunoprecipitated with 5 μg monoclonal antibody anti-CD38 (NIM-R5) or IgG2a isotype control (overnight at 4°) and 50 μl rec-Protein G–Sepharose 4B conjugate (Invitrogen, Carlsbad, CA). The complex was eluted in Laemmli sample buffer containing 5%β-mercaptoetanol. The immunoprecipitates were resolved by 10–15% SDS–PAGE and transferred to nitrocellulose membranes. Blots were blocked and incubated with primary (for CD38 detection we use rabbit polyclonal anti CD38 antibody) and secondary antibodies, as indicated by the manufacturer’s instructions and detected by chemiluminescence (Thermo Scientific).

B-cell proliferation

Splenocytes from C57BL/6 wild-type, CD19−/−, CD38−/− and CD81−/− mice (1 × 106 cells) were incubated with 1 μm carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Invitrogen, Carlsbad, CA) for 10 min in serum-free medium at 37°. Cells were then washed in cold RPMI medium containing 10% FCS. The CFSE-labeled cells were plated in flat-bottomed 48-cell plates and were incubated with anti-IgM F(ab′)2 fragments (10 μg/ml; Cappel Laboratories, Durham, NC), lipopolysaccharide (LPS, from Escherichia coli 055; B5; 10–20 μg/ml) and with 10, 50 and 100 μg/ml of goat or rabbit polyclonal anti-CD38 in the presence of 5–10 ng/ml interleukin-4 (R&D Systems, Minneapolis, MN) for 3 and 6 days at 37°. Cells were washed once with PBS and stained with anti-B220 antibody. Data were analyzed by flow cytometery (Cyan™ADP Beckman Coulter) using flowjo software (http://www.flowjo.com).

Results

CD19/CD81 co-localizes with CD38

In human B lymphocytes and B-cell lines, CD38 is proposed to interact with the CD19/CD21/CD81 complex and with the BCR/Igα/Igβ complex.22 It has been shown that CD38 co-localizes with CD19, CD81 and the BCR complex by confocal microscopy. However, in mouse B lymphocytes such interactions have not been analyzed. In this study we explored the interactions of CD19 and CD81 with CD38. First, we analyzed colocalization with these receptors after CD38 cross-linking in capping and co-capping experiments. Splenocytes from C57BL/6 mice were incubated with rabbit polyclonal anti-CD38 antibody to induce capping for 1 hr, then analyzed to determine if CD81 redistributes to the capping area. The results indicate that in resting conditions CD38, CD19 and CD81 were homogeneously distributed in the membrane (Fig. 1a), but after capping, CD38 was polarized in association with CD81 and CD19 (Fig. 1a,c); in contrast, B220 did not co-cap and was used as a negative control in subsequent studies (Fig. 1a). Moreover, when we induced capping with CD81 and then analyzed CD38 co-capping we observed the same redistribution (Fig. 1b). This demonstrated that CD38 is in proximity with CD81 and CD19; we therefore investigated the functional consequences of this proximity.

Figure 1.

Figure 1

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Co-capping of CD38 with CD81 and CD19. (a) Splenocytes from C57BL/6 mice were incubated with rabbit polyclonal anti-CD38 antibody for 30 min on ice, washed and reacted with secondary antibody anti-rabbit-Cy3 for 1 hr at 37°. Counterstaining was performed at 4° with anti-CD81 and anti-CD19 (top and middle) or B220 (bottom) and DAPI was used for staining of nuclei. (b) Capping with anti-CD38 (left panel) or anti-CD81 (right panel) for 30 min on ice and subsequently with anti-hamster-FITC. Counterstaining was performed with anti-CD81 (left panel) and anti-CD38 (right panel). (c) The number of co-caps is presented as percentage of cells analyzed. Confocal sections for the merged images of representative cells are shown. Coefficient of correlation (CC) for colocalization was calculated using imagej software.

CD19 and CD81 are associated with CD38 in lipid rafts

Lipid rafts are membrane microdomains that function as platforms for recruitment of receptors and signaling molecules. They play an important role in the early stages of lymphocyte signaling where Lyn, a resident lipid raft protein, is required for CD38 signaling, because B cells from Lyn-deficient mice were shown to have impaired proliferation and differentiation in response to CD38 stimulation.3 Moreover, after cross-linking, CD38 is mobilized to lipid rafts where it is found in close contact with other receptors, such as CD19 and CD81, which are also redistributed to these membrane domains.23 To determine if CD38 physically interacts with CD19 and CD81 in lipid rafts we isolated lipid rafts from unstimulated C57BL/6 splenocytes by sucrose gradient. First, we analyzed the 10 fractions from the sucrose gradient with the lipid raft marker Lyn, and then with CD38. Fractions 3, 4 and 5 correspond to lipid rafts as is described elsewhere24 (Fig. 2a; top). CD38 was localized not only in lipid rafts but also in the non-rafts fractions (Fig. 2a; bottom). We then immunoprecipitated CD38 from lipid rafts and searched for CD19 and CD81 interaction. The precipitated proteins were then analyzed by SDS–PAGE and Western blotting with anti-CD38, anti-CD19 and anti-CD81. As shown in Fig. 2(b), CD19 and CD81 were present in the CD38 immunoprecipitates. Neither CD81 nor CD19 were found in the immunoprecipitates when an isotype-matched irrelevant antibody was used. As CD81 physically interacts with CD19, we performed the same immunoprecipitation scheme using the CD19-deficient mice, to see if the interaction with CD81 was directly with CD38 or required CD19 to form the complex. We observed that CD81 is directly associated with CD38, because the absence of CD19 did not impair this interaction (Fig. 2c). The presence of CD38 in lipid rafts might suggest that it is functionally active because is interacting with CD81, CD19 and probably with Lyn. Little co-immunoprecipitation was observed in the non-raft fraction (data not shown).

Figure 2.

Figure 2

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CD38 in lipid rafts forms a complex with CD19/CD81. Lipids rafts were isolated by sucrose gradient centrifugation of splenocyte lysates from C57BL/6 or CD19−/− mice. (a) Ten fractions were collected and analyzed by Western blot with the indicated antibodies. Fractions corresponding to lipid rafts from C57BL/6 wild-type (b) or CD19−/− mice (c) were used for immunoprecipitation with monoclonal anti-CD38 (NIM-R5) or isotype-matched control antibody. Precipitates were resolved in 10–15% SDS–PAGE and transferred onto nitrocellulose membrane and incubated with the indicated antibodies. IC: isotype control.

CD19/CD81 complex is not required for CD38-induced proliferation

Once we had observed that CD38 interacts with CD19 and CD81 in lipid rafts, we next explored the functionality of these interactions. It is well established that CD38 stimulation can induce proliferation and differentiation of mouse mature B lymphocytes.3,25 For this reason, we performed proliferation assays using CD19-deficient and CD81-deficient mice. Splenocytes from wild-type (as positive control), CD19−/−, CD81−/− and CD38−/− (as negative control) mice were CFSE-labeled and stimulated with anti-IgM, LPS and with goat or rabbit polyclonal anti-CD38 antibodies plus interleukin-4 for 6 days. B lymphocytes proliferated to anti-IgM and LPS as has been previously described for all these mice (Fig. 3; left panels); surprisingly, when we analyzed the proliferation of B lymphocytes from CD19-deficient and CD81-deficient mice we did not observe impaired proliferation (Fig. 3; right panels). Indeed, both CD19-deficient and CD81-deficient mice responded in a similar way to the wild-type mice, in contrast to CD38−/− mice (Fig. 3; bottom panels). These results differ from what has been described in humans, where CD81 and CD19 were reported to be important in CD38 signaling in B lymphocytes.21 Additionally we did not observed differences in plasma cell differentiation and apoptosis after CD38 stimulation in B cells from wild-type and CD19-deficient mice (data not shown).

Figure 3.

Figure 3

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CD19−/− and CD81−/− mice proliferate in response to CD38. Splenocytes from wild-type, CD19−/− and CD38−/− mice were labeled with 1 μm CFSE and cultured for 6 days in the presence of interleukin-4 (IL-4; 5–10 ng/ml) and anti-IgM F(ab′)2, lipopolysaccharide (LPS; 10–20 μg/ml), rabbit or goat polyclonal anti-CD38 (100 μg/ml). CFSE experiments with CD81−/− mice were performed separately and 10 μg/ml of either rabbit or goat polyclonal anti-CD38 and IL-4 (10 ng/ml) were used. Proliferation was analyzed by flow cytometry. CFSE dilution in gated live and B220+ cells is shown. Representative histograms from six independent experiments are shown.

CD38 associates with the tetraspanin web in B lymphocytes

Because we did not observe a functional interaction between CD38 and the CD19/CD81 complex, we next examined if CD38 was able to interact with other tetraspanin members. It has been described that B lymphocytes expressed several members of the tetraspanin family including CD9, CD37, CD53, CD63, CD82 and CD151.26 Some tetraspanins have been shown to associate with B-cell surface markers, such as BCR, MCH-II, CD20, integrins and signaling molecules like phosphatidylinositol 4-kinase and Lyn.27,28 Therefore, tetraspanins have been suggested to participate in B-lymphocyte signaling. We performed capping experiments to determine if engagement of CD38 leads to colocalization with other tetraspanins like CD9 and CD63 (Fig. 4). We observed that both CD63 and CD9 co-localize with CD38, suggesting that CD38 is part of the tetraspanin web.

Figure 4.

Figure 4

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Co-capping of CD38 with CD63 and CD9. (a) Splenocytes from C57BL/6 mice were incubated with rabbit polyclonal anti-CD38 antibody for 30 min on ice, washed and reacted with a secondary anti-rabbit-Cy3 antibody for 1 hr at 37°. Counterstaining was performed at 4° with anti-CD63 (a) or CD9 (b) and DAPI was used for staining of nuclei. Confocal sections for the merged images of representative cells are shown. Coefficient of correlation (CC) for colocalization was calculated using imagej software. (b) The number of co-caps is presented as percentage of cells analyzed. Confocal sections for the merged images of representative cells are shown.

Discussion

CD38 was initially described as a B-cell activation molecule and was subsequently characterized as an enzyme and as a receptor.29,30 The structural characterization of CD38 revealed that the cytoplasmic tail was not necessary for the signal induction after B-lymphocyte stimulation.8 For this characteristic it has been established that CD38 is a parasite receptor that uses the signaling machinery of other molecules. The function of CD38 as a receptor has been observed in various cells from the immune system, where according to the cell type, the association with other receptors has been explored. In human T lymphocytes, CD38 was found associated with the TCR/CD3 complex.10 The signal induction by CD38 in T lymphocytes is similar to the signaling induced by the TCR/CD3, in which phospholipase C-γ1, ZAP-70 and Lck are phosphorylated leading to calcium mobilization and cytokine production.31 In human natural killer cells, CD16 and CD38 form a complex that is physically and functionally related. After stimulation, CD38 induces phosphorylation of ZAP-70 and extracellular signal-regulated kinase, which results in more efficient cytotoxicity from natural killer cells.13

In human B lymphocytes, CD38 co-localizes with CD19/CD21/CD81 co-receptors, but this interaction has been explored mainly by confocal microscopy and immunoprecipitation; 18,21 few additional reports also evaluated the function of this interaction. In this work we explored the contribution of CD19/CD81 complex in mouse CD38 signaling because CD38 expression and signaling was shown to differ between humans and mice.32,33 Our results confirm that CD38 is physically associated with these two receptors in lipid rafts, as has been described for human B lymphocytes. However, when we analyzed the functional relevance of this interaction we found that neither CD81 nor CD19 were necessary for CD38-induced proliferation of mouse B lymphocytes. Importantly, CD81-deficient mice have reduced expression of CD19,34 which reinforces the data obtained using only the CD19-deficient mice. A previous study that used CD19-deficient mice and a mutant lacking the cytoplasmic domain of CD19, observed reduced proliferation after CD38 stimulation.35 What we observed is not necessarily contradictory, perhaps because we use different agonistic antibodies and CFSE proliferation instead of [3H]thymidine incorporation. Importantly, we used two different polyclonal antibodies at different concentrations (data not shown) and both gave similar results to those presented in Fig. 3. What is clear is that whereas in humans, the CD19/CD81 complex seems to be important for CD38 signaling; in mice this might not be the case. Differences between human and murine CD38 expression in B lymphocytes is likely to be reflected in interaction with other receptors.

Lyn is a key component of CD38 signaling and is activated by other receptors, such as the BCR. Indeed there is evidence that CD38 signaling is somehow connected to the BCR. The A20 B-cell clone lacking BCR/Igα/Igβ, did not respond to cross-linking of CD38 and the response was reconstituted after transfection with Igα/Igβ.22 Another example of this relationship comes from studies in peritoneal B1 cells and immature Transitional 1 cells that are unresponsive to engagement by either anti-IgM or CD38.3,36,37 However, cross-linking of CD38 does not lead to phosphorylation of Igα/Igβ.18,22 Moreover, Ba/F3, a pro-B-cell line lacking both the BCR and CD38 becomes responsive to CD38 cross-linking upon transfection with CD38.9

Here we explored the possibility that CD38 may use the tetraspanin web as a platform to interact with additional cell surface receptors and with signaling molecules. We observed colocalization of CD38 with several tetraspanins. CD63 was shown to associate with Lyn27 and CD9 with the BCR28 and with CD38.14 As tetraspanins are associated with each other and with additional receptors in the tetraspanin web it might be possible that in CD81-deficient and CD19-deficient mice other receptors participate in CD38 signaling and use this web to interact with additional partners. For this reason, it would be interesting to evaluate the contribution of Igα and Igβ and tetraspanins in mouse B lymphocytes and, more importantly, to decipher how CD38 is capable of using different receptors in different types of cells. In any case, these results presented in this work show, as a whole, that CD38 is using still undefined molecules to induce its signals.

Acknowledgments

We thank Héctor Romero-Ramirez for his help and technical assistance, Ricardo Gaxiola Centeno for taking care of the mice and Leticia Aleman Lazarini for confocal microscopy assistance. This work was supported by grant from CONACyT (56836). Felipe Vences Catalán was fellow 203621 from CONACyT.

Disclosures

The authors have no financial conflicts of interest.

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