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. Author manuscript; available in PMC: 2014 May 1.
Published in final edited form as: J Immunol. 2011 Oct 12;187(10):5114–5122. doi: 10.4049/jimmunol.1102172

ADAM10 regulates antibody production and maintenance of lymphoid architecture1

Natalia S Chaimowitz 1,*, Rebecca K Martin 1,*, Joanna Cichy 2, David R Gibb 1, Pooja Patil 1, Dae-Joong Kang 1, Julie Farnsworth 3, Eugene C Butcher 4, Brent McCright 5, Daniel H Conrad 1
PMCID: PMC4006936  NIHMSID: NIHMS325696  PMID: 21998451

Abstract

A disintegrin and metalloproteinase 10 (ADAM10) is a zinc dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B-cell specific ADAM10 deficient mice (ADAM10B−/−). In the current study, we show that ADAM10 levels are significantly enhanced on GC B-cells. Moreover, ADAM10B−/− mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10B−/− displayed impaired germinal center formation, had fewer follicular helper T-cells, decreased follicular dendritic cell networks and altered chemokine expression in draining lymph nodes. Interestingly, when spleen and lymph node structures from immunized mice were analyzed for B- and T-cell localization, tissues structure was aberrant in ADAM10B−/−mice. Importantly, when ADAM10-deficient B-cells were stimulated in vitro, they produced comparable Ab as wild type B-cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B-cell activation, likely due to the decrease in follicular helper T-cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure following antigen challenge.

Introduction

Germinal centers (GCs) are critical for humoral immunity and the establishment of immunological memory. GC formation requires interactions between antigen-specific T cells, B cells and follicular dendritic cells (FDCs)(1). Activated T cells upregulate the chemokine receptor CXCR5 and downregulate CCR7(2, 3). These CD4+CXCR5+CCR7lo T cellsmigrate toward B cell follicles. Simultaneously, antigen-activated B cells increase their CCR7 expression and migrate toward the T cell zone(4, 5). Within the T-B border, Follicular helper T (Tfh) cells interact with activated B cells presenting cognate antigen and provide B cell help via CD40L and IL-21. B cells can then differentiate into short-lived plasmablasts outside of the follicle or can enter GCs(6). The generation of high affinity-secreting plasma cells (PC) and memory B cells within GCs depends on B cell interaction with FDCs and further interaction with Tfh(7). While FDCs protect GC B cells from apoptosis and support their proliferation, Tfh promote their the growth, differentiation and class switching(8).

The precise localization of B cells, T cells and FDCs during an immune response is critical for GCs(9). Chemokine receptors CXCR5 and CXCR4, lymphotoxin (LT), and tumor necrosis factor (TNF) are critical for B cell follicle organization, and as such, play a crucial role in GC formation(10). Dysregulation of GC formation has been linked with leukemias, lymphomas and autoimmune diseases(11). Therefore, better understanding of GC formation will shed light on new therapeutic targets for the treatment of B cell or antibody-mediated pathologies.

A disintegrin and metalloproteinases (ADAMs) are zinc dependent proteinases related to matrix metalloproteinases (MMPs). ADAMs can mediate ectodomain shedding and regulated intramembrane proteolysis (RIP) of transmembrane proteins. Ectodomain shedding releases soluble fragments into extracellular space, possibly down-regulating events that depend on transmembrane receptor expression or activating paracrine signaling by soluble products derived from ADAM substrates, such as soluble CD23(12). Although many ADAMs have been identified, ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands(13). A recent study demonstrated that ADAM10 can mediate trans-cleavage of Ephrin-A2(14). Studies have identified a continuously growing list of putative ADAM10 substrates, such as Notch receptors (1–4), Delta-like 1 (Dll1), IL-6R, CXCL16 and CD23 (15, 16). Moreover, ADAM10 has been identified as a key regulator of lymphocyte development. We and others have demonstrated that ADAM10 is critical for T cell and marginal zone B cell (MZB) development(17, 18). However, the role of ADAM10 in mature B cell function remains unknown and warrants further studies.

In this study, we show that Peyer’s Patches GCs have elevated ADAM10 expression. Recently, we generated mice with a B cell-specific deletion of ADAM10 (ADAM10B−/−)(18) and showed that MZBs were missing due to defective Notch2 cleavage. Here we demonstrate that ADAM10B−/− mice have impaired GC formation and markedly diminished antibody responses to T-dependent antigens. Overexpression of the Notch2 intracellular domain (N2ICD) did not correct the defect. Interestingly, ADAM10-deficient B cells can class switch normally when stimulated in vitro, suggesting that the phenotype observed in vivo results from insufficient B cell activation, likely due to decreased T cell help. Consistent with this hypothesis, Tfh numbers were also diminished. Furthermore, clear B and T cell segregation was observed in secondary lymphoid tissues prior to challenge. The defective humoral response seen in ADAM10B−/− mice was associated with altered chemokine expression, loss of FDC networks and changes in T and B cell localization within secondary lymphoid organs. These results demonstrate that B cell expressed ADAM10 is critical for the maintenance of lymphoid architecture and proper positioning of T cells and B cells during immune responses.

Methods and materials

Mice and immunizations

ADAM10B−/− mice were previously described(18). To generate ADAM10B−/−N2ICD-TgB+, ADAM10B−/− mice were bred with N2ICD-Tgflox/flox mice(19). CD19-cre− mice were wild type littermates of ADAM10B−/− and ADAM10B−/−N2ICD-TgB+ mice. CD23Tg mice have been previously described. Balb/CJ mice were used as littermate controls of CD23Tg mice(20). All mouse protocols were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee. Immunization comprised a single intraperitoneal or footpad injection of 10 μg 4-Hydroxy-3-nitrophenylacetyl coupled to keyhole limpet hemocyanin (NP-KLH) at a ratio of 27:1 (Biosearch Technologies) in 4mg of alum, unless noted otherwise.

In vitro activation

Splenocytes were cultured (12.5×106 cells/well) with CD40L-transfected Chinese hamster ovary (CHO) cells as a B cell stimulant (6×104 cells/well) and IL-4 (10,000 units/mL) for seven days. Supernatants were then harvested and analyzed for IgG1 expression by ELISA.

Flow cytometry

Cell isolation and labeling was conducted as described previously(18, 21). Cells were labeled following RBC lysis and filtration through 40 μm cell strainers. Abs included anti-mouse unlabeled 2.4G2, biotinylated CXCR5 (RF8B2); PE-conjugated B220 (RA3-6B2), IgD (11-2C.2a), CD21/CD35 (7G6); allophycocyanin-labeled IgG1 (A85-1) and PE-Cy7 conjugated-streptavidin from BD Bioscience; FITC-conjugated B220 (RA3-6B2), CD4 (RM4-5), F4/80 (BM8); PerCP/Cy5.5-conjugated IgD (11-2c.2a), IgM (RMM-1); APC-conjugated CD4 (RM4-5); PE-conjugated CD4 (H129.19). Alexafluor 700 and Alexafluor 647-conjugated B220 (RA3-6B2); PE/Cy7-conjugated CD38; from BioLegend; and FITC-conjugated Thy1.2 (30-H12) from Southern Biotech. Flow cytometric analysis was performed using a Canto or AriaII (BD Biosciences), and data analysis was conducted with FlowJo (Tree Star).

Quantitative PCR

Total RNA was extracted and purified from draining and non-draining mouse lymph nodes using TRIZOL reagent (Invitrogen). Samples were treated with DNase (Takara Bio Inc.), mixed with phenol/chloroform/isoamyl alcohol solution (25:24:1; USB), and precipitated with ethyl alcohol. The purity of RNA was quantified by a spectrophotometer (ND-100; NanoDrop). 1 μg RNA was reverse transcribed using the High Capacity cDNA RT kit (Applied Biosystems). Real-time quantitative PCR was performed with a real-time PCR machine (iQ5; Bio-Rad Laboratories). Primers and probes for running a TaqMan quantitative PCR assay were purchased from Applied Biosystems. TaqMan gene expression assays included CCL19: Mm00839967_g1 and IL-21: Mm00517640_m1. Primers for CCL21 were synthesized by IDT as previously described(22). PCR products, labeled with 6-FAM–conjugated probes, were amplified with 18S as an internal control. Reaction parameters were as follows: hold at 48°C for 30 min and hold at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Results were analyzed with iQ5 real-time PCR software (version 2.0).

ELISA and ELISPOT

For ELISA, serum was collected from mice at different time points after immunization. Samples were diluted serially and added to 96-well plates (50 μL per well) precoated with NP-14-BSA (Biosearch Technologies; 15 μg/mL). After incubation at 37 °C for 1 h, biotinylated anti-mouse Ig (Southern Biotech) was added to plates. After a second incubation at 37 °C for 1 hour, bound antibodies were revealed by Streptavidin-AP (Southern Biotech). For measurement of high affinity antibodies, protocol was carried out as described except that plates were coated with NP-4-BSA (Biosearch Technologies; 15 μg/mL). Standard curves were performed by coating with anti-IgM or anti-IgG and adding known amounts of IgM or IgG, respectively. The value for experimental samples are reflected as relative units (RU) since serum Ag specific antibodies were capture with Ag. For ELISPOT, 96-well MultiScreen filtration plates (Millipore) were coated with NP-4-BSA or NP-14-BSA. While only high affinity antibodies can efficiently bind NP-4-BSA, and both low and high affinity antibodies bind NP-14-BSA, this ELISA system allows for the determination of high vs. low affinity antibodies(23). Total splenic cells were seeded into the plate in duplicate and cultured for 18 hours at 37 °C. Plates were developed as previously described(24).

Immunofluorescence and confocal microscopy

Spleens were frozen on dry ice in OCT compound (Tissue-Tek; Sakura). Serial 10-μm sections were cut from frozen blocks using a cryostat (Frigocut 2800E; Jung), fixed in absolute acetone, and air-dried. Sections were blocked with 10% BSA in PBS to prevent background staining and then washed and incubated for 60 min with different combinations of 2–5 μg/ml anti–mouse antibodies. Antibodies included PE-conjugated B220 (RA3-6B2) and IgD (11-26c.2a), FITC-conjugated Thy1.2 (53-2.1), CD21/CD35 (7E9) and CD3 (17A2) from biolegend; FITC conjugated GL7 (GL7) from eBioscience. Sections were washed, mounted with antifade mounting medium (Vectashield; Vector Laboratories), cover slipped, and examined with a confocal laser scanning microscope (TCS-SP2 AOBS; Leica) fitted with an oil Plan-Apochromat 40× objective. Two lasers were used: argon for FITC and PE and HeNe for allophycocyanin. Parameters were adjusted to scan at a 512 × 512 pixel density for figure 1 or 1024 × 1024 pixel density for remainder of confocal images. Emissions were recorded in three separate channels. Digital images were captured, overlaid, and processed with the Confocal and LCS Lite programs (Leica).

Figure 1. ADAM10 expression on germinal center B cells.

Figure 1

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(A–B) Expression of ADAM10 on gated germinal center PNAhiIgDlo (blue) and naive PNAloIgDhi (red) B (CD19+) cells isolated from Peyer’s patches. Flow cytometry gates used to define the germinal center and naïve B cell populations are indicated in left. To demonstrate specificity of staining, isotype-matched mAb were used (isotype). (A) Gating protocol is shown. (B) Percentage of ADAM10+ cells in naive and GC B cells. (C) Frozen serial sections of Peyer’s patches were stained to detect B cell follicles (B220+, blue), germinal centers (PNA+, green) and ADAM10 (red) (right panel). To demonstrate specificity of staining, isotype-matched mAb were used instead of mAb against ADAM10 (left panel). Original magnification: x20. The mucosal epithelium (E) also reacts with PNA and anti-ADAM10.

Statistical analysis

p-values were calculated using unpaired two-tailed Student’s t tests in Graphpad Prism. Error bars represent the SEM between samples. p<0.05 is considered significant.

Results

ADAM10 is highly expressed in GC B cells

Delineation of ADAM10 expression patterns in B cells is an important step toward understanding the potential role of the proteinase in humoral immunity. Flow cytometry analysis demonstrated that, within Peyer’s patches, while less than 2% of naïve B cells expressed ADAM10, over 80% of GCB cells were ADAM10+ (Fig. 1A,B). The flow cytometric results were confirmed by immunohistochemistry of Peyer’s patches stained for ADAM10, PNA and IgD. Consistent with our flow cytometry results, ADAM10 was highly expressed by cells within GCs (Fig. 1C). Peyer’s patches were chosen due to the expected high level of GC activation, however, in other experiments (data not shown), elevated levels of ADAM10 was also seen on B cells from the draining lymph nodes (LN) of immunized mice.

Humoral immune responses in ADAM10B−/−deficient mice

Given the significant expression of ADAM10 in GC B cells, we investigated its role in humoral responses by using B cell specific ADAM10-deficient mice (ADAM10B−/−). Basal levels of serum IgM, IgG1, IgG2a and IgG2b were significantly reduced in ADAM10B−/− mice when compared to littermate or heterozygous (not shown) controls, suggesting a defect in antibody production (Fig. 2A). Since cre expression reduces the CD19 expression by about one-half, this result also shows that the decrease in Ab responses is not secondary to decreased CD19 expression. To directly assess antigen-specific Ab production, ADAM10B−/−mice were immunized i.p. with 4-Hydroxy-3-nitrophenylacetyl coupled to keyhole limpet hemocyanin (NP-KLH) and we examined serum levels of NP-specific Ab. ADAM10B−/− mice showed reduced levels of NP-specific IgM 7 days post-immunization, however, levels reached control values by day 14 and through the remainder of the experiment (Fig. 2B). In contrast, NP-specific IgG levels, both total and high affinity, was reduced for 4 weeks post-immunization (Fig. 2B). This reduction in IgG was not specific to a particular isotype, as antigen-specific IgG1, IgG2a and IgG2b were dramatically reduced in ADAM10B−/− mice (Supplemental Fig. 1A). Similar results were obtained when mice were immunized subcutaneously (results not shown). Furthermore, even when immunization with a high antigen dose, 1mg of NP-KLH, was performed, ADAM10B−/− mice failed to mount a normal Ab response, demonstrating that the defect in Ab production cannot be overcome by simply providing more antigen (Supplemental Fig. 1B).

Figure 2. ADAM10B−/− mice have impaired humoral responses.

Figure 2

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(A) Serum IgM, IgG1, IgG2a and IgG2b were measured by capture ELISA from non-manipulated 8–12 week old mice. (B) ADAM10B−/− mice and wild type littermate controls were immunized with 10μg NP-KLH emulsified in alum. At the indicated times, serum samples were collected and total NP-specific anti-IgM, total IgG and high affinity IgG antibody titers were determined by ELISA with NP16-BSA as capture antigen for total and NP4-BSA for high affinity ELISA. (C) Mice were immunized with NP-KLH emulsified in alum, rested for 42 days, and boosted with 10 μg NP-KLH for 5 days. Mice were bled weekly throughout the course of the experiment. Total and high affinity IgG levels were measured by ELISA at each time point. The relative unit (RU) values for alum-injected mice were less than 0.001. Bars represent the mean ± SE of 5–9 mice per group (*p < 0.05, **p<0.01, ***p<0.001). Data represent results obtained in at least two independent experiments.

To test if memory responses were also impaired in ADAM10B−/− mice. Littermate controls and ADAM10B−/−mice were immunized and boosted 42 days later. Consistent with data shown in Figure 2B, ADAM10B−/−mice had decreased levels of antigen-specific antibodies after primary immunization. While littermate controls exhibited a 12-fold increase in anti-NP IgG antibodies five days after secondary immunization (from 38.34 +/− 25 to 461.5 +/− 72.45), ADAM10B−/−mice had a 4-fold increase(from 3.133 +/− 2.888 to 12.36 +/− 6.4460). Five days after boost, control mice produced 16 times more high affinity antigen-specific IgG and 37 times more total NP-specific IgG than ADAM10B−/−mice (Fig. 2C). These data demonstrate that in ADAM10B−/− mice, B cell memory responses are impaired.

Plasma cell development is impaired in ADAM10B−/− mice

Examination of plasma cell (PC) generation by ELISPOT assays showed that ADAM10B−/−mice that had been immunized had drastically reduced numbers of NP-specific ASCs in the spleens and BM 21 days after immunization (Fig. 3A,B). Similar results were obtained at 14 and 28 days after immunization (results not shown). These results demonstrate that the decrease in splenic ASCs is not due to altered migration or preferential localization to the bone marrow, but likely due to impairments in PC differentiation and/or survival.

Figure 3. ADAM10B−/− mice have decreased numbers of Ag-specific ASCs.

Figure 3

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ELISPOT analysis of cells producing (A) total (both low and high affinity), (B) high affinity NP-specific IgG and (C) high vs. low affinity ratio present in the spleen (top) and bone marrow (bottom) isolated from WT and ADAM10B2/2 Q:20 mice 21 d postimmunization with 10 mg NP-KLH emulsified in alum. (*p<0.05, ***p<0.001). Bars represent the mean 6 SE of five mice per group. Data represent results obtained in two independent experiments.

A critical component of the humoral response is affinity maturation. Thus, we next examined affinity maturation in immunized ADAM10B−/− mice. While 21 days after immunization most ASCs were secreting high affinity antibodies in littermate controls, the few ASCs that were present in ADAM10B−/−mice were secreting low affinity Ab. Littermate controls had 12-fold higher number of NP-specific ASCs and 20-fold higher number of NP-specific ASCs producing high affinity antibodies when compared to ADAM10B−/− mice. These results suggest a defect in affinity maturation in ADAM10B−/−mice (Fig. 3C).

GC formation is impaired in ADAM10B−/−mice

Figure 3 demonstrated that antigen-specific ASCs were reduced in immunized ADAM10B−/−. Being that long-lived PCs and memory cells are generated within GCs(9), GC formation in ADAM10B−/−mice was examined. GC B cells, defined as IgMloIgDloB220+IgG1+CD38lo were enumerated by flow cytometry(25)(Fig. 4A). Remarkably, the number and percentage of GC B cells within the spleen was dramatically decreased in ADAM10B−/−mice (Fig. 4B,C). Similar results were obtained when draining LNs were analyzed (data not shown).

Figure 4. GC formation and following T- dependent immunization.

Figure 4

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ADAM10B−/− and WT mice were immunized with 10 μg NP- KLH emulsified in alum. Fourteen and twenty one days post immunization flow cytometry was carried out and the presence of GC B cells (IgMloIgDloB220+IgG1+CD38-) in the spleen of WT and ADAM10B−/− was assessed. Staining protocol is depicted (A). Both percentage (B) and total number (C) of GCs were enumerated. Bars represent the mean ± SE of 8 mice per group. Data are representative of 3 independent experiments.

ADAM10B−/−mice have altered splenic and lymph node architecture as well as dysregulated chemokine expression following antigen challenge

In order to better understand the relationship between decreased GC B cells and the defects in Ab production depicted in Figure 2, GC formation in immunized mice was assessed by immunohistochemistry. As depicted in Figure 5, while several GCs could be detected in littermate controls, as determined by GL-7+ positive cells, draining LN of ADAM10B−/−mice contained a paucity of GL7+ clusters. Interestingly, GL7+ cells were scattered throughout the LN of ADAM10B−/− mice (Fig. 5A). Given the unusual pattern of GL-7 expression, we then analyzed B- and T cell localization within the LN. Surprisingly, the distribution of B cells and T cells was aberrant, suggesting that B cell specific ADAM10 deletion leads to changes in LN structure (Fig. 5B). In contrast, LN structure in non-draining nodes or unimmunized mice were relatively normal with regards to B- and T cell segregation (Fig. 5D, and data not shown). Unlike the draining LN, GL-7 staining was not detected in spleen of ADAM10B−/− mice (Fig. 6A). However, although normal in unimmunized mice, splenic architecture was also clearly altered post-immunization (Fig. 6B, C).

Figure 5. Immohistochemisty analysis of GC formation and lymph node architecture following T-dependent immunization.

Figure 5

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ADAM10B−/− and WT mice were immunized with 10 μg NP-KLH emulsified in alum. Fourteen days after immunization, draining lymph node sections and stained for (A) GL7 (green) and IgD (red); (B) Thy1.2 (green) and B220 (red); (C) CD21/35 (green) and B220(red). (D) Lymph nodes non-draining lymph nodes were also stained with CD3 (green) and B220 (red). Pictures are representative of 3 independent experiments. Magnification displayed is 20x.

Figure 6. Immunohistochemistry analysis of GC formation and splenic architecture after T-dependent immunization.

Figure 6

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ADAM10B−/− and WT mice were immunized with 10μg NP-KLH emulsified in alum. Fourteen days after NP-KLH immunization, spleens were isolated and sectioned. Sections stained for (A) GL7 (green) and IgD (red). Photomicrographs are representative of three independent experiments. (B) Spleens isolated from naïve animals or (C) isolated 14 d postimmunization were stained with CD3 (red) and B220 (green). Photomicrographs are representative of three independent experiments. Original magnification X20 (A,B); x10 (C).

Given that FDCs play a crucial role in GC formation and lymphoid tissue structure(5, 8, 26), we stained draining LN sections for activated FDC markers. Consistent with an absence in GCs and disorganized lymphoid architecture, relatively few FDC networks were evident in LN and spleen from ADAM10B−/− mice (Fig. 5C, not shown)

The organization of secondary lymphoid tissues is highly regulated by chemokine signaling. To determine if changes the changes in structure observed where due to altered chemokine expression, draining and non-draining LN chemokine expression was assessed by qPCR. As expected, non-draining LN from control and ADAM10B−/− mice had similar CXCL13, CCL19 and CCL21 expression (Fig. 7A). In sharp contrast, draining LN isolated from ADAM10B−/−mice exhibited increased CCL21 expression, while CXCL13 levels were comparable. CCL19 levels trended higher, but the increase was not statistically significant (Fig. 7B). Analysis of CCL21 expression by immunohistochemistry revealed diffuse staining in the ADAM10B−/− LN (results not shown), consistent with the localization of T cells within draining lymph nodes (Fig. 5B). These results demonstrate that B cell expressed ADAM10 is important for maintenance of lymphoid architecture by regulating chemokine expression during active immune responses.

Figure 7. Chemokine expression in draining lymph nodes.

Figure 7

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Mice were immunized in the footpad with 10 μg of NP-KLH. Fourteen days after immunization, draining and non-draining lymph nodes were isolated and chemokine expression was analyzed by quantitative PCR for CCL19, CCL21 and CXCL13. Non-draining (A) and draining (B) lymph nodes are shown. (*p<0.05). Bars represent the mean ± SE of 8 mice per group. Data are representative of 2 independent experiments.

Defect in humoral responses in ADAM10B−/− mice is Notch2 and CD23-independent

Recent studies have suggested that Notch signaling might be involved in Ab production and differentiation of Ab secreting cells (ASC)(27, 28). ADAM10 is critical for Notch signaling. To determine if the Ab production defects were due to impaired Notch signaling, ADAM10B−/− mice that have constitutively active Notch2 signaling (ADAM10B−/−N2ICD-TgB+) were immunized and antibody production, germinal center formation and lymph node architecture was examined. As expected, expression of N2ICD recovered MZB development in these mice, demonstrating that the transgene was indeed active (Supplemental Fig. 2A). Littermate controls, ADAM10B−/−and ADAM10B−/−N2ICD-TgB+ mice were immunized. Consistent with previous experiments, ADAM10B−/−mice had significantly reduced levels of NP-specific antibodies. Importantly, introduction of N2ICD transgene failed to rescue the defect in antibody production and germinal center formation and did not prevent changes in lymph node architecture(Supplemental Fig. 2B,C). These results demonstrate that increased Notch signaling cannot rescue the defects in humoral responses.

We recently reported that ADAM10 is responsible for the cleavage of the low affinity IgE receptor, CD23(29), and consequently, ADAM10B−/− B cells have increased CD23 expression(18). Studies have revealed that CD23 overexpression led to decreased IgG1 and IgE production(20). However, when CD23Tg mice were immunized with NP-KLH analysis of NP-specific IgG indicated that CD23Tg mice had a normal Ab response to NP-KLH (Supplemental Fig. 3). Furthermore, it has been previously reported that CD23Tg mice form germinal centers following immunization with T-dependent antigens(30). These results suggest that the humoral defect observed in ADAM10B−/− mice is not secondary to CD23 overexpression.

Defective Ab production seen in ADAM10B−/− mice can be explained by decreased B cell help

We have demonstrated that ADAM10B−/− mice have impaired Ab responses to T-dependent antigens as well as changes in splenic and LN architecture. In order to assess whether the decreased Ab levels result from a B cell intrinsic defect, splenocytes were stimulated with CD40L and IL-4in vitro. Interestingly, wild type and ADAM10-null B cells made comparable amounts of IgG1 (Fig. 8A), demonstrating that when adequately stimulated, ADAM10-null B cells are able to class switch normally.

Figure 8. Normal responses of ADAM10 deficient B cells in vitro, but impaired Tfh cell in vivo.

Figure 8

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(A) Splenocytes were cultured (12.5×106 cells/well) with CD40L-transfected Chinese hamster ovary (CHO) cells as a B cell stimulant (6×104 cells/well) and IL-4 (10,000 units/mL) for seven days. Supernatants were then harvested and analyzed for IgG1 expression by ELISA. (B-D) Mice were immunized with NP-KLH. Fourteen days post immunization flow cytometry was carried out and the presence of Tfh (B220-CD4+CXCR5hiPD-1hi) in the spleen of WT and ADAM10B−/− was assessed. Staining protocol is depicted (B). Percentage of Tfh out of CD4+ was enumerated (C). Draining lymph nodes were analyzed for IL-21 mRNA by quantitative PCR (D). (*p<0.05). Bars represent the mean ± SE of 6 mice per group. Data are representative of 2 independent experiments.

Tfh cells provide B cell help via CD40L, IL-21 and IL-4 and play important roles in GC B cell survival, affinity maturation and terminal differentiation to PCs(31). Tfh cells are characterized by high expression of CXCR5 and PD-1, and IL-21 production(31).Given our in vitro findings and the changes in architecture observed in ADAM10B−/− mice, we hypothesized that the defects in humoral responses observed resulted from inadequate B cell stimulation and decreased T cell help. Results revealed that Tfh development and/or maintenance was impaired in ADAM10B−/−mice, fewer Tfh cells were present in draining lymph nodes of ADAM10B−/− mice(Fig. 8B). Consistent with decreased number of Tfh cells, IL-21 message levels were also reduced (Fig. 8C). These results demonstrate that while ADAM10 deficient B cells can produce normal levels of Abin vitro, they are unable to class switch efficiently in vivo due to decreased T cell help.

Discussion

Members of the ADAM family regulate a wide range of functions, including cell migration, proliferation and adhesion(32). ADAM10, in particular, has been recently shown to be critical for lymphocyte development through initiation of the canonical Notch signaling pathway(17, 18). Here we report that ADAM10 is essential for the maintenance of splenic and LN architecture during responses to T-dependent antigens. A decrease in antigen specific IgG production is seen both with respect to serum levels and IgGASCs, indicating that PC differentiation is influenced. Cells producing high affinity antibodies were particularly affected, consistent with a defect in GC reactions and markedly repressed FDC-reticula. Moreover, while ADAM10 deficient B cells are able to class switch normally when stimulated in vitro, ADAM10B−/− mice have defective generation of Tfh cells. Therefore, the defective humoral response in ADAM10−/− mice results from impaired B cell activation in vivo.

There are currently two clear substrates for ADAM10 in B cells – Notch and CD23(17, 18, 29). The role of notch signaling in mature B cell function is controversial. While some studies have clearly shown a role for Notch in Ab production, others fail to identify such a role(27, 33). In order to assess the role of Notch signaling in Ab production, we overexpressed Notch2ICDin a B cell specific manner. Here we show that restoration of Notch2 signaling in ADAM10B−/− mice is not sufficient to rescue Ab production or germinal center formation. Consistent with our findings, in vivo studies of B cell specific RBP-J κ deficient mice, a key mediator of notch signaling, revealed no defects in Ab production(33). Moreover, a recently published report demonstrated that B cell specific Notch2 deficient mice have normal GC formation(34). Studies have shown that Notch2ICD and Notch1ICD have redundant functions. Our results, therefore, suggest that the phenotype observed in ADAM10B−/− mice is not dependent Notch1 nor Notch2 signaling(35). However, the role of Notch1 in B cell biology should be more thoroughly studied and these studies are in progress. In vitro studies have demonstrated that Notch signaling enhances B cell activation(27)and support GC B cell survival(28). While Notch signaling enhances humoral responses in vitro, decreased Notch signaling does not explain the phenotype observed in ADAM10B−/− mice. Since Notch signaling also enhances CD21 expression, this finding also suggests that CD21 is not involved in the ADAM10B−/− phenotype. In addition, unlike CD21/CD35−/− mice, ADAM10B−/− mice had impaired affinity maturation and failed to mount an Ab response comparable to controls following immunization with large antigen dosages (i.e. 1mg) (36).

Another B cell expressed ADAM10 substrate is CD23(29). We have recently demonstrated that CD23 surface expression is significantly enhanced after deletion of ADAM10 in B cells(18). Defects observed in ADAM10B−/− could result from CD23 overexpression. Previous studies with CD23Tg mice, however, demonstrated that elevated CD23 levels resulted in inhibition of IgE and IgG1 production, while other classes were not significantly affected. Furthermore, the defect was only evident after boosting(20, 37). On the other hand, in ADAM10B−/−mice, all classes of IgG were influenced and this defect was observed after just one injection. Moreover, when immunize with NP-KLH, CD23Tg mice had Ig levels comparable to that of wild type. In contrast, ADAM10B−/−mice showed diminished levels of both low and high affinity anti-NP antibodies. These results indicate that elevated CD23 levels do not explain the phenotype of ADAM10B−/− mice. It is likely that B cells express another ADAM10 substrate that has yet to be described and/or that ADAM10-mediated trans-cleavage is responsible(14).

In lymphoid organs, the correct positioning of cells, such as B cells and T cells is dictated by the local production of chemokines by stromal cells as well as the coordinated expression of chemokine receptors by migrating cells(5). The positioning of these cells, as well as the migration during an immune response are critical for adequate interaction between them(38). Our results demonstrate that while B cell expressed ADAM10 is dispensable for proper cellular positioning during LN and spleen development, ADAM10 is critical for the maintenance of the correct position of cells after immune stimulation. TNF-alpha is another ADAM10 substrate and it has been implicated in the formation of primary B cell follicles and germinal centers. Unlike ADAM10B−/− mice, TNF-alpha deficient mice have disrupted splenic architecture prior to immunization(39). The phenotype of ADAM10B−/− mice is also different to that seen with LT-deficient mice, as LT-deficient mice lack peripheral LN and have severely disrupted splenic architecture. Moreover, draining and non-draining lymph nodes from immunized mice had comparable levels of LT and TNF in wild types and ADAM10B−/− mice (results not shown).

CCL21, CCL19 and CXCL13 have been shown to play a key role in inducing and maintaining normal cellular compartments(5). Draining LNs from ADAM10B−/− mice showed increased CCL21 expression. This increase in CCL21 was also confirmed at the protein level by immunohistochemistry. Interestingly, ADAM10B−/− mice had a larger area of CCL21 staining. The larger area of production is consistent with the aberrant T cell localization seen in draining LNs.

FDCs are important for GC development, immunoglobulin class switching, somatic hypermutation and affinity maturation as well as induction of recall responses(40–45). FDCs also secrete CXCL13 and are thus important for recruitment of CXCR5-expressing B cells and T cells into the follicle(46) and are important for the maintenance B- and T cell zones(38). The loss of B cell and T cell segregation and the lack of GCs in the draining secondary lymphoid tissues of ADAM10B−/− mice is thus consistent with a decreased number of activated FDC networks. The changes in architecture correlate with the decrease in Tfh cells. Tfh cells provide B cell help via CD40L within GCs and are thus essential for PC differentiation, memory cell generation and affinity maturation(6). Decrease Tfh cell number leads to decreased CD40L available to stimulate B cells, thus leading to diminished isotype switching. Decreased B cell activation is consistent with the phenotype we observe in the ADAM10B−/− mice and is the likely mechanism for the defect. These findings further support the idea that GC formation and Ab production are secondary to changes in architecture and decreased interaction between cognate B cells and T cells. Additional studies will be required to determine how B cell ADAM10 is functioning to cause this aberrant humoral response.

Secondary lymphoid organs are positioned such that they facilitate immune responses to foreign antigens. In states of chronic inflammation, lymphoid cell aggregates that have a similar organization to secondary lymphoid organs can develop; these structures are known as tertiary lymphoid structures. These structures have been described in patients with atherosclerosis and rheumatoid arthritis(38). Tertiary lymphoid organs have organized B cell compartments, which often include GCs, and T cell compartments, in which antigen-presenting cells can be detected. When tertiary organs develop at sites where autoantigens are continuously being presented, they may lead to unnecessary tissue destruction by facilitating the activation of autoreactive lymphocytes (38). Our results demonstrate that ADAM10 is critical for the structural maintenance of secondary lymphoid organs during active immune responses and thus, suggest a role for ADAM10 in the maintenance of tertiary lymphoid organs. As complete loss of ADAM10 is lethal in utero (47)blockade of ADAM10 would need to be done locally. We have recently shown that intranasal administration of ADAM10 inhibitors reduced symptomology in the mouse asthma model without causing any obvious deleterious activities (48). Thus, it may be possible to use local ADAM10 inhibition in some autoimmune scenarios to decrease autoimmune symptoms.

In conclusion, examination of humoral responses in B cell specific ADAM10 deficient mice has demonstrated that ADAM10 is critical for the maintenance of splenic and lymph node architecture during active immune responses. ADAM10B−/− mice have decreased numbers of Tfh cells, leading to diminished T cell help and impaired GC formation, affinity maturation and class switch recombination. Therefore, B cell expressed ADAM10 is required for proper humoral responses. Thus, ADAM10 is essential for the maintenance of lymphoid structure following antigen challenge.

Supplementary Material

Acknowledgments

We thank J. Tew and R. Smeltz for discussion and Hannah Zellner and Patrick Paez for expert technical assistance.

Footnotes

1

Work supported in part by Grants RO1AI19697 (to DC) and U19AI077435 (to DC), American Heart Association Award 11PRE7600119 (to NC), Fogarty International Research Collaborative Award R03TW007174-01 (to ECB and JC) and the Ministry of Scientific Research, Poland N N401 003635 (to JC). Flow cytometry and imaging studies supported in part by P30 CA16059.

Authorship Contributions

N.C. designed research, did experiments, analyzed data and wrote the manuscript; D.H.C designed research and wrote the manuscript; R.M. designed, carried out and analyzed experiments regarding lymph node and tissue structure; J.C. designed, carried out and analyzed experiments demonstrating ADAM10 expression in germinal centers.; P.P. maintained mouse colony and did ELISA assays; J.F. aided in the analysis of flow cytometry data. E.B. designed and analyzed experiments demonstrating ADAM10 expression in germinal centers. B.M. contributed the N2ICD mouse, which was critical for this analysis.

Conflict of Interest Disclosures

The authors declare that they have no competing financial interests.

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