The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, N.
Right arrow Articles by Vignali, D. A. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, N.
Right arrow Articles by Vignali, D. A. A.
The Journal of Immunology, 2007, 178: 4214-4221.
Copyright © 2007 by The American Association of Immunologists, Inc.

Non-Cell Autonomous Expression of TNF-{alpha}-Converting Enzyme ADAM17 Is Required for Normal Lymphocyte Development1

Nianyu Li2,*, Kelli Boyd{dagger}, Peter J. Dempsey3,{ddagger} and Dario A. A. Vignali4,*

* Department of Immunology and {dagger} Animal Resource Center, St. Jude Children’s Research Hospital, Memphis, TN 38105; and {ddagger} Pacific Northwest Research Institute, Seattle, WA 98122 and Department of Medicine, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TNF-{alpha} converting enzyme (TACE; ADAM17), a member of the ADAM (a disintegrin and metalloprotease) family of metalloproteases, has been shown to cleave a wide variety of cell surface proteins of immunological importance. Due to the broad expression of TACE and the early postnatal lethality of TACE-deficient mice, it has been difficult to assess the role of TACE in lymphocyte development. Indeed, it is not known whether hemopoietic and/or nonhemopoietic expression of TACE is required for normal lymphocyte development. In the current study, we analyzed the lymphoid system of tace{Delta}Zn/{Delta}Zn mice and tace{Delta}Zn/{Delta}Zn bone marrow RAG1–/– recipients. Our results clearly show that nonlymphocyte expression of TACE is required for normal lymphocyte development and lymphoid organ structure. Lack of TACE function resulted in a partial block in T cell development at the double-negative 4:double-positive transition in the thymus, a loss of B cell development/maturation in the spleen, and a lack of B cell follicle and germinal center formation in the spleen. Thus, TACE serves as a lymphocyte extrinsic factor that is essential for normal T development and peripheral B cell maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tumor necrosis factor-{alpha} converting enzyme (TACE)5 is a member of the ADAM (a disintegrin and metalloprotease) family of type I transmembrane, Zn2+-dependent metalloproteases that are involved in ectodomain cleavage or "ectodomain shedding" of a diverse group of transmembrane proteins (see Refs. 1, 2, 3 for review). Many TACE substrates have now been identified, including TNF-{alpha}, TNF-{alpha} receptor, CD30, CD40, CD44, L-selectin, VCAM, Notch, ligands of epidermal growth factor receptor (EGFR) family, and lymphocyte-activation gene 3 (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Previous studies have suggested that the outcome of TACE cleavage is highly substrate dependent. Lack of TACE cleavage has been shown to influence cell function both intrinsically (such as TNF-{alpha} receptor, CD44, L-selectin, and Notch) (7, 8, 9, 11, 12) and extrinsically (such as TNF-{alpha} and EGFR family ligands) (7, 10, 12).

Despite the identification of a large number of TACE substrates by in vitro analysis, the physiological function of TACE in vivo has been difficult to determine due, in large part, to the early postnatal lethality of tace{Delta}Zn/{Delta}Zn mice (these mice lack the Zn2+-binding domain of TACE and thus lack enzymic activity) (7, 12). The reason for this early lethality is not completely understood, although the most profound phenotype identified in new-born tace{Delta}Zn/{Delta}Zn mice is due to the lack of EGFR ligand cleavage and functional EGFR signaling (7, 12). This phenotype includes a failure of eyelids to fuse normally during embryonic development, a disorganized distribution and structure of hair follicles in the skin (both observed in TGF{alpha}- and EGFR-deficient mice) (7, 12), delayed or impaired maturation of epithelial cells in a variety of organs such as intestine and lung (all observed in EGFR-deficient mice) (14, 15), and thickened and misshapen heart valves and defects in the branching morphogenesis of the lung (both observed in heparin-binding EGF- and EGFR-deficient mice) (16, 17, 18).

Due to this complex phenotype, the physiological role of TACE in lymphocyte development and function has not been clearly defined. In humans, TACE mRNA has been shown to be highly expressed in the thymus and spleen (7). High TACE protein levels have been detected in T cells, suggesting that TACE may play an important role in T cell development and/or function (7). To study the effect of TACE-mediated cleavage, several groups have generated mice with noncleavable TACE substrates. By generating knock-in mice with noncleavable L-selectin and noncleavable TNF-{alpha}, two recent studies have shown that shedding of membrane proteins was important for Ag-stimulated lymphocyte migration and for secondary lymphoid organ architecture such as primary B cell follicle formation (19, 20). However, it has been difficult to predict the outcome of TACE deficiency on specific ligand receptor complexes such as the TNF-{alpha}-TNFR pathway when both ligand and receptor are potential TACE substrates (7). In addition, TACE is not the only sheddase that can cleave molecules of immunological importance and, in many instances, multiple and distinct sheddase activities can cleave the same substrate. For example, TACE-independent TNF-{alpha} release has been observed in TACE-deficient cells (4) and the matrix metalloprotease MMP-7 can cleave TNF-{alpha} under some experimental conditions (7, 21). Therefore, it has been difficult to clearly attribute TACE-mediated cleavage to these phenotypes. In addition, given that TACE is responsible for the cleavage of many cell surface proteins other than L-selectin and TNF-{alpha}, it is important to determine first whether TACE has other effects on lymphocyte function. Importantly, it is still not clear whether hemopoietic and/or nonhemopoietic expression of TACE is required for normal lymphocyte development, which is the focus of this study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

tace{Delta}Zn/{Delta}Zn mice were generated by deleting the zinc binding domain of TACE through homologous recombination as described (12). tace{Delta}Zn/+ mice were originally generated using the hybrid 129 R1 ES cell line and were maintained on a mixed (C57BL/6 x 129) background (12). In the current study, tace{Delta}Zn/+ mice obtained from J. Peschon (Amgen) had been back-crossed several times onto the C57BL/6 background and, in initial heterozygous mating studies, showed a decrease in the number of tace{Delta}Zn/{Delta}Zn pups at P1. We sought to improve the number and viability of tace{Delta}Zn/{Delta}Zn null pups by back-crossing these original heterozygous tace{Delta}Zn/+ mice three times onto the 129S3 strain background (22). Primers for the genotyping of ADAM 17 were designed with the forward primer outside of the targeting sequence using 5'-GGTATGTGATAGGTGTAATGTGG-3' and two different reverse primers, 5'-TGGTCACCGCTCACAGCTA-3' for sequences in exon 11 of wild-type TACE and 5'-GAGCCCAGAAAGCGAAGGAG-3' for the NEO sequence. This tace{Delta}Zn/+ mouse colony has been maintained by intercrossing for >3 years. On this strain background, ~25% of the tace{Delta}Zn/{Delta}Zn null pups survive to adulthood. All experiments were conducted using postweaned (>4 wk of age) tace{Delta}Zn/{Delta}Zn mice and control sex- and age-matched wild-type littermates unless otherwise stated. All bone marrow transfers were conducted using specific pathogen-free (Helicobacter-free) RAG1–/– mice (where RAG1 is recombination-activating gene 1). Mice were maintained in a temperature-controlled room with a 12-h/12-h light/dark cycle and were provided with ad libitum access to standard laboratory chow and water. All procedures were approved by the animal care and use committees of St. Jude Children’s Research Hospital and Pacific Northwest Research Institute in accordance with National Institutes of Health guidelines for the care and use of animals.

Flow cytometry

Single cell suspensions from the thymus and spleens were generated by teasing through a 70-µm strainer (BD Biosciences). RBC in splenocytes were lysed with Gey’s solution. The cells were stained with the following fluorochrome-conjugated Abs (BD Pharmingen): TCRbeta (clone H57-597), CD45R/B220 (clone RA3-6B2), CD45 (clone 30-F11), CD4 (clone RM4-4), CD8{alpha} (clone 53-6.7), CD44 (clone IM7), CD62L/L-selectin (clone MEL-14), CD25/IL-2R (clone 7D4), CD21 (clone 7G6), CD23 (clone B3B4), IgM (clone RM60.2), IgD (clone 11-26), CD43 (clone S7), and CD19 (clone 1D3). Flow cytometry was performed in a FACSCalibur (BD Biosciences) device and data were analyzed with FlowJo software (Tree Star).

Purification of thymic epithelial cells

Thymic epithelial cells were prepared as described previously (23, 24). Briefly, five young adult C57BL/6 thymi (3 wk old) were digested for 30 min with collagenase/DNase in RPMI 1640 with 2% FBS. In the last 5 min, EDTA was added to 10 mM. Cells were then teased through a 40 µM falcon filter and then separated on a discontinuous Percoll density gradient. The thymic epithelial cell-enriched fraction was collected and stained with CD45-FITC and G8.8-allophycocyanin (provided by C. Benoist, Joslin Diabetes Center, Harvard Medical School, Boston, MA), and the thymic epithelial cells were sorted by gating on the CD45–G8.8+ population.

Real-time PCR

For PCR, the specific primers and fluorogenic probe for murine ADAM17 were designed using Primer Express 1.5 (Applied Biosystems) and synthesized by the St Jude Hartwell Center for Biotechnology and Bioinformatics (Memphis, TN). The sequences for the TACE primers and probe are 5'-TGGGACACAATTTTGGAGCA-3' (forward primer), 5'-CCTCCTTGGTCCTCATTTGG-3' (reverse primer), and FAM-CATGACCCTGATGGGCTAGCAGAATGTG-TAMRA (probe). The primers were designed to span introns and control against the amplification of genomic DNA. The beta-actin mRNA primer/probe set (Applied Biosystems) was used to normalize differences in the amount of total RNA in each sample. RNA from FACS-purified thymocyte subpopulations (CD4–CD8–, CD4+CD8+, and CD4+, CD8+) and thymic epithelial cells was purified using TRIzol (Invitrogen Life Technologies) according to the manufacturer’s instructions. The relative quantities of mRNA were calculated from standard curves and normalized to actin mRNA.

ELISA

EDTA-treated plasma was obtained from adult tace{Delta}Zn/{Delta}Zn and wild-type age- and sex-matched littermates and stored at –20°C. Thawed plasma was diluted 1/10 according to the manufacturer’s protocol before measuring soluble TNF receptors using specific soluble TNFR1 and soluble TNFR2 ELISAs (R&D Systems).

Preparation of bone marrow chimeras

Bone marrow cells were harvested from the femurs and tibiae of tace{Delta}Zn/{Delta}Zn mice or tace wild-type littermates. Bone marrow cells were then depleted of T cell, B cell, and NK cells (biotinylated Abs against CD3, CD4, CD8, B220, and pan-NK) using streptavidin-coupled paramagnetic beads (autoMACS; Miltenyi Biotec). RAG1–/– recipient mice were irradiated with 450 rad from a cesium source. After irradiation, 3 x 106 bone marrow cells from tace wild-type or tace{Delta}Zn/{Delta}Zn mice were transferred to recipients via tail vein injection. Bone marrow reconstitution was confirmed 6 wk after reconstitution and animals were analyzed 2–6 wk later.

Histologic analysis and immunohistochemistry

Spleens from mice were fixed in 10% neutral buffered formalin overnight and then embedded in paraffin. The tissue was sectioned at 4 µm, stained with H&E, and examined by light microscopy. Immunohistochemistry was performed on paraffin-embedded sections. Anti-CD3 (Santa Cruz Biotechnology) or anti-B220 (BD Pharmingen) Abs were used to stain for T or B cells. Germinal centers were stained using peanut agglutinin (PNA; Vector Laboratories).

Statistical analysis

All data are represented as mean ± SE. Statistical analysis was performed using the Student’s t test. Values were accepted as being statistically significantly different if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Determining the role of TACE in lymphocyte development

The goal of this study was to determine whether cell autonomous or non-cell autonomous expression of TACE was required for T and B cell development. This was determined by analyzing TACE-deficient (tace{Delta}Zn/{Delta}Zn) mice or tace{Delta}Zn/{Delta}Zn bone marrow RAG1–/– recipients (in which the nonhemopoietic cells would be TACE wild type). By maintaining tace{Delta}Zn/{Delta}Zn mice on a mixed (C57BL/6 x 129) background, we were able to generate a subpopulation of tace{Delta}Zn/{Delta}Zn mice that survived beyond weaning to adulthood (see Materials and Methods). These adult tace{Delta}Zn/{Delta}Zn mice displayed many of the characteristic features previously associated with TACE deficiency, including open eyelids at birth, stunted and curly vibrissae, perturbed hair coats, and reduced body weights (R. W. Gelling, S. M. Fitzgerald, P. O. Lim, S. Al-Noori, J. Raycraft, A. Pardini, G. J. Morton, K. Ogimoto, M. W. Schwartz, and P. J. Dempsey, unpublished observations). Although some mice survive, they have reduced fat mass and increased energy expenditure. The frequency of surviving adult nulls is ~1:10 based on the expected Mendelian ratio. Of the nulls born ~25% survive to adulthood with 50% dying between postnatal day 1 (P1) and postnatal day 2 (P2) (presumably due to heart and/or lung defects) and 25% dying before weaning due to severe wasting.

We first verified the phenotype of TACE deficiency by assessing the cleavage of two well-known TACE substrates, TNFRs and L-selectin (CD62L). Soluble TNFR (sTNFR) ectodomains are readily detected in adult mouse plasma. The dramatic reduction in sTNFR2 levels and, to a lesser extent, the reduction in sTNFR1 levels in plasma from adult tace{Delta}Zn/{Delta}Zn mice confirmed that these mice lack TACE proteolytic activity (Fig. 1A). Interestingly, the plasma levels of sTNFR1 were only decreased by ~50% in tace{Delta}Zn/{Delta}Zn mice compared with their wild-type littermates, suggesting that other sheddases besides TACE may be involved in TNFR1 ectodomain cleavage in vivo.


Figure 1
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 1. Decrease of plasma-soluble TNF-{alpha} receptor concentration in tace{Delta}Zn/{Delta}Zn mice but not in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice. A and B, Plasma TNF-{alpha} receptor I (A) and II (B) concentration from tace{Delta}Zn/{Delta}Zn mice or tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice were measured by ELISA. Each bar chart represents the mean ± SE for each group. Student’s t test was performed with the p values shown. wt, wild type. C, Splenocytes of wild-type (wt) or tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice were activated with 1 µM PMA for 3 h. T cells were stained for surface L-selectin and analyzed by flow cytometry. FACS histograms were representative of six individual mice. D, TACE mRNA levels in purified thymocyte subpopulations (CD4–CD8–, CD4+CD8+, and CD4+, CD8+) and thymic epithelial cells were determined by real-time PCR. tace wild-type (wt) (set to 1.0) and tace{Delta}Zn/{Delta}Zn fibroblasts (no detectable signal) were used as positive and negative controls, respectively. The relative quantities of mRNA were calculated from standard curves and normalized to actin mRNA. Results are the mean ± SE of three independent experiments.

 
Because both TACE and most TACE substrates are broadly expressed, we then asked whether TACE activity on lymphocytes was required for soluble TNF-{alpha} receptor production. Bone marrow from either TACE deficient (tace{Delta}Zn/{Delta}Zn) or TACE wild-type (tace+/+) littermates were used to reconstitute irradiated RAG1–/– mice. Due to the fragility of the few tace{Delta}Zn/{Delta}Zn mice that survive to adulthood, the reciprocal experiment (wild-type bone marrow into irradiated tace{Delta}Zn/{Delta}Zn mice) is not technically feasible. Plasma sTNFR1 levels in both bone marrow-reconstituted groups were identical (Fig. 1A). Likewise, plasma sTNFR2 levels in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted mice were not considerably different from TACE wild-type bone marrow-reconstituted mice (Fig. 1B). However, given that the plasma concentrations of both sTNFRs were significantly higher in the tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted mice compared with their tace{Delta}Zn/{Delta}Zn counterparts, these data suggest that TACE activity on nonlymphoid cells was predominantly responsible for soluble TNF-{alpha} receptor production. To verify that the T cells in these mice do lack TACE activity, we assessed their ability to cleave L-selectin following PMA treatment (12). Although this ability was intact in wild-type bone marrow recipients, CD4+ T cells from tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice clearly lacked PMA-induced L-selectin cleavage (Fig. 1C), confirming the reconstitution of tace{Delta}Zn/{Delta}Zn T cells in RAG1–/– mice.

T cell development is impaired in tace{Delta}Zn/{Delta}Zn mice but not in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice

To assess whether there is a role for nonhemopoietic expression of TACE in modulating T cell development, we first had to assess TACE expression in the thymus. We purified thymic epithelial cells by FACS and compared their levels of TACE expression to that of different thymocyte populations. The data show that double-negative (DN; CD4–CD8–) thymocytes express ~2-fold more TACE mRNA than double-positive (DP; CD4+CD8+) and single-positive (SP; CD4+ or CD8+) thymocyte populations, while thymic epithelial cells express nearly 3-fold more TACE mRNA compared with most thymocyte populations (Fig. 1D).

We next assessed the role of TACE mediated-cleavage in T cell development. tace{Delta}Zn/{Delta}Zn mice had significantly less total thymocytes (~5-fold reduction) compared with wild-type controls (Fig. 2A). Although the number of DN thymocytes was normal, the number of DP and SP thymocytes was significantly reduced (Fig. 2A). These alterations resulted in a reduction in the percentage of DP thymocytes and a compensatory increase in the DN and SP percentages (Fig. 2B). The cause of this decrease is unclear but could be due to a deficiency in T cell development, increased apoptosis, or reduced proliferation at the DN4-DP transition. To further investigate the specific stage of thymocyte development that was blocked in tace{Delta}Zn/{Delta}Zn mice, the DN1-DN4 subcompartments of early T cell development were identified by CD25 and CD44 staining. This analysis clearly showed that early T cell development in tace{Delta}Zn/{Delta}Zn mice is normal, because the number and percentage of DN1-DN4 thymocytes was comparable to that of wild-type mice (Fig. 2C). These data clearly demonstrate a requirement for TACE in T cell development at the DN4-DP transition.


Figure 2
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 2. Thymocyte development is impaired in tace{Delta}Zn/{Delta}Zn mice but not in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice. A and D, Calculation of the absolute numbers of thymocytes within each developmental stage is shown. Data represent the mean thymocyte + SE for each group. B and E, Thymocytes from wild-type (wt) and tace{Delta}Zn/{Delta}Zn mice (B) or wild-type and tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice (E) were stained with CD4/CD8 and analyzed by flow cytometry. Percentages of CD4/CD8 DN, CD4/CD8 DP, CD4 SP, and CD8 SP cells in total thymocytes were calculated. Each bar chart represents the mean + SE for each group. C and F, Thymocytes were stained for CD4/CD8 DN thymic developmental markers (CD25 and CD44) and analyzed by flow cytometry. Analysis was performed by a quadrant gate set up to obtain percentages of each developmental stage from DN1 to DN4. Each bar chart represents the mean ± SE for each group of six mice.

 
To investigate whether this partial block in T cell development was due to a lack of TACE on hemopoietic or nonhemopoietic cells, bone marrow from tace wild-type or tace{Delta}Zn/{Delta}Zn littermates was used to reconstitute RAG1–/– mice that lack T and B cells (25). Interestingly, T cell development was completely normal with the number and percentage of thymocyte subsets in the tace{Delta}Zn/{Delta}Zn bone marrow recipients, compared with that of their wild-type littermate controls (Fig. 2, D–F). These data clearly demonstrate that the requirement for TACE activity in T cell development is non-cell autonomous.

Given that T cell development in tace{Delta}Zn/{Delta}Zn mice is partially blocked in the thymus, it was not surprising that these mice had a ~5-fold decrease of T cell numbers in the spleen (Fig. 3A). In contrast, a normal number and percentage of T cells was observed in tace{Delta}Zn/{Delta}Zn bone marrow recipients (Fig. 3D). The splenic CD4:CD8 T cell ratio was normal in tace{Delta}Zn/{Delta}Zn mice, suggesting both subsets are equally affected (Fig. 3C). Taken together, these results demonstrate that loss of TACE expression in thymocytes has no effect on T cell development. Importantly, our data also show that nonhemopoietic expression of TACE is required for normal T cell development.


Figure 3
View larger version (50K):
[in this window]
[in a new window]

  		FIGURE 3. Decrease of T cell and B cell numbers in spleens of tace{Delta}Zn/{Delta}Zn mice but not in those of tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice. A and D, Splenocytes from wild-type (wt) and tace{Delta}Zn/{Delta}Zn mice (A) or wild-type and tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice (D) were stained with B cell and T cell markers and calculated for absolute number. Each bar chart represents the mean ± SE for each group of six mice. B and E, Splenocytes from wild-type (wt) and tace{Delta}Zn/{Delta}Zn mice (B) or wild-type and tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice (E) were stained with B220/TCR-beta and analyzed by flow cytometry. All cells were gated on lymphocytes. Percentages of cells were labeled in each individual window. C and F, Splenocytes from wild-type and tace{Delta}Zn/{Delta}Zn mice (C) or wild-type (wt) or tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice (F) were stained with CD4/CD8 and analyzed by flow cytometry. All cells were gated on lymphocytes. Percentages of cells were labeled in each individual window.

 
B cell development was blocked in the spleen in tace{Delta}Zn/{Delta}Zn mice but not in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice

Interestingly, as was seen for T cells, the total B cell number in the spleen was decreased ~5-fold in the tace{Delta}Zn/{Delta}Zn mice but not in tace{Delta}Zn/{Delta}Zn bone marrow RAG1–/– recipients (Fig. 3, A and D). To examine whether these mice have a defect in B cell development, the numbers of pro-B cells (CD19+CD43+IgM–IgD–), pre-B cells (CD19+CD43–IgM–IgD–), immature B cells (CD19+CD43–IgM+IgD–), and mature B cells (CD19+CD43–IgD+) in the bone marrow of tace{Delta}Zn/{Delta}Zn and wild-type mice were determined. Essentially, the numbers of all B cell populations examined were normal in tace{Delta}Zn/{Delta}Zn mice and tace{Delta}Zn/{Delta}Zn bone marrow recipients compared with their wild-type counterparts (Fig. 4, A and D), suggesting no defect in bone marrow B cell development.


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 4. B cell development is impaired in tace{Delta}Zn/{Delta}Zn mice but not in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice. A and D, Femur bone marrow was collected from wild-type (wt) and tace{Delta}Zn/{Delta}Zn mice (A) or wild-type or tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice (D). Cells were stained with markers for different B cell developmental stages and analyzed by flow cytometry. B, C, E, and F, Splenocyte developmental stages were characterized by CD21/CD23/B220 (B and E) or IgM/IgD/B220 (C and F). The percentage of each fraction is indicated in each histogram. The absolute cell number was calculated and represented as the mean ± SE for each group of three mice.

 
B cell precursors emigrate from the bone marrow and mature in the spleen (26). Therefore, we next examined B cell development in the spleen. Transitional B cells, follicular B cells, and marginal zone B cells were segregated based on differential CD21 and CD23 expression. There was a significant reduction in both the percentage (~3-fold) and number (~10-fold) of transitional B cells (CD23–CD21–) in the spleens of tace{Delta}Zn/{Delta}Zn mice (Fig. 4B). Although there was a slight increase of the percentage of follicular B cells (CD23+CD21+), their absolute numbers were clearly reduced. Curiously, CD23 expression was also increased ~4.5-fold on tace{Delta}Zn/{Delta}Zn follicular B cells. Given that CD23 has recently been shown to be a substrate for ADAM10/Kuzbanian (27), it is conceivable that it may also be a target for TACE. The percentage of tace{Delta}Zn/{Delta}Zn marginal zone B cells (CD23–CD21+) was normal but there was a ~4-fold reduction in their absolute number (Fig. 4B). Consistent with these observations the percentage and the absolute number of F-III B cells (IgMhighIgDlow) was drastically decreased, with F-III B cells representing early transitional B cells (type 1 transition or T1) and marginal zone B cells (28). Taken together, these data suggest that B cell development may be blocked as early as T1, which represent the most recent emigrants from the bone marrow. Therefore, not surprisingly the numbers of B cells at all developmental stages were drastically decreased in tace{Delta}Zn/{Delta}Zn mice (Fig. 4, B and C). Similar to T cell development, B cell development in the spleens of tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice was normal (Fig. 4, D–F). These results suggest that TACE expression on nonhemopoietic cells in the spleen is required for normal B cell development.

Lymphoid organ structure and germinal center formation were impaired in tace{Delta}Zn/{Delta}Zn mice but not in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice

Splenic white pulp is highly structured and organized into three major compartments: the periarteriolar lymphoid sheaths that contain mainly T cells, primary B cell follicles consisting of recirculating B cells, and the marginal zone in which lymphocytes enter the white pulp area. TNF-{alpha}, a key TACE substrate, is important for maintaining this structure. TNF-{alpha} knockout mice do not have primary B cell follicles and PNA-positive marginal zone metallophilic macrophages (29, 30). Interestingly, a noncleavable TNF-{alpha} knock-in could partially rescue the formation of the continuous layer of PNA-positive marginal zone metallophilic macrophages but not the formation of primary B cell follicles (20). With this in mind, we asked what effect TACE deficiency has on splenic white pulp architecture. Consistent with the phenotype of TNF-{alpha} knockout and noncleavable TNF-{alpha} knock-in mice, no B cell follicle formation was observed in tace{Delta}Zn/{Delta}Zn mice (Fig. 5). The primary B cells in the spleens of tace{Delta}Zn/{Delta}Zn mice formed a typical ring-like structure around T cell area (Fig. 5). Due to the abnormality in T/B cell development, the loss of B cell and T cell segregation was also apparent in the white pulp in tace{Delta}Zn/{Delta}Zn mice (Figs. 4B and 5). TNF-{alpha} is also required for splenic germinal center formation and a noncleavable TNF-{alpha} knock-in partially restores the establishment of germinal centers. In tace{Delta}Zn/{Delta}Zn mice, however, we did not observe any germinal center formation in the spleen (Fig. 5).


Figure 5
View larger version (106K):
[in this window]
[in a new window]

  		FIGURE 5. B cell development is impaired in tace{Delta}Zn/{Delta}Zn mice but not in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice. Spleen sections from wild-type (wt), tace{Delta}Zn/{Delta}Zn, or bone marrow-reconstituted RAG1–/– mice were used for immunohistochemical analysis of B cells (B220; brown stain), T cells (CD3; brown stain), and germinal centers (PNA; brown stain). Original magnification: x10 for large pictures (bars, 100 µm) and x40 for insets (bars, 50 µm).

 
Consistent with our observations above, primary B cell follicle formation and germinal center formation were both normal in all tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice, demonstrating that TACE expression in B cells is not required for the maintenance of splenic architecture and germinal center formation. The immunohistochemical characterization of the entire splenic lymphoid tissue correlates with the immunocytologic findings and clearly demonstrates the presence of B cells, T cells, and germinal centers in similar numbers and sizes compared with the control. However, TACE expression on nonlymphocyte populations was clearly required for B cell development.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our results clearly show that TACE activity on nonhemopoietic cell populations was required for normal T cell development in the thymus, suggesting that TACE requirement was non-T cell autonomous. What might be the possible target of this TACE activity? Of all the currently identified TACE substrates, Notch (and its ligands) is the only one that had been shown to be crucial for T cell development (31, 32). However, while this might appear to be the most likely target for nonhemopoietic TACE activity, we cannot rule out the possibility that other, as yet unknown substrates play a significant role in lymphocyte development. For instance, an alternative hypothesis is that loss of TACE activity in nonhemopoietic cells results in the development of aberrant stromal cells that indirectly block lymphocyte development due to aberrant thymic or splenic architecture or a block in stromal cell development.

Two ADAM family proteases Kuzbanian (ADAM10) and TACE have been implicated in Notch signaling (8, 33, 34). To initiate Notch signaling, they need to be cleaved by Kuzbanian and/or TACE (8, 33, 34). In addition, Kuzbanian will also regulate Notch signaling by releasing Notch ligands by cell surface cleavage (35, 36). In Caenorhabditis elegans, Kuzbanian (SUP-17) and TACE (ADM-4) are functionally redundant with regard to Notch receptor cleavage (37). However, in mice only the Kuzbanian-deletion mutant had a similar phenotype to that of Notch-deficient mice during development (38). A recent study showed that dominant-negative Kuzbanian transgenic mice have a partial T cell development block at the DN stage, a phenotype very similar to that of Lck-conditional Notch-1–/– mice (39, 40). It has also been suggested that this partial block in T cell development is due to the lack of Notch-1 cleavage on T cells (39). In this study we showed that tace{Delta}Zn/{Delta}Zn mice have a strikingly similar phenotype to that of the Lck-conditional Notch-1–/– mice and the dominant-negative Kuzbanian transgenic mice, suggesting that TACE may also be involved in Notch signaling. However, surprisingly T cell development was normal in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice, suggesting that Notch in tace{Delta}Zn/{Delta}Zn T cells is functioning normally. This observation raises the possibility that TACE is involved in the release of Notch ligands from thymic stromal cells and that this is required for normal T cell development. A possible candidate might include Delta1, which has been shown to be a target of TACE (41). A direct link between TACE and Notch ligands could be derived from colocalization or coimmunoprecipitation experiments. However, although informative these approaches have limitations. Colocalization would not confirm whether there is functional shedding and would not be useful if trans-shedding was occurring. Furthermore, given that TACE is ubiquitously expressed, it is unlikely to be informative. Coimmunoprecipitation would be more definitive but has not been demonstrated even for well-characterized TACE substrates in primary cells.

Although Lck-conditional Notch-1–/– mice, dominant-negative Kuzbanian transgenic mice, and tace{Delta}Zn/{Delta}Zn mice all had a block in early T cell development (39, 40), it appeared to occur at different stages. Thymocyte development was blocked at the DN4:DP transition in tace{Delta}Zn/{Delta}Zn mice, at DN3-DN4 in Lck-conditional Notch-1–/– mice, and at an early DN2 stage in dominant negative Kuzbanian transgenic mice. Taken together, the simplest hypothesis based on these observations and our data is that TACE regulates T cell development at the DN4:DP transition by controlling Notch ligand cleavage on thymic epithelial cells, while Kuzbanian/ADAM10 primarily regulates Notch cleavage on T cells between DN2 and DN4. However, why these events are required at different stages of T cell development remains to be clarified.

Our data also showed that TACE expression was required for B cell development/maturation in the spleen. This block occurred as early as the T1 stage. Several TACE target proteins, such as Notch and several members of TNF-{alpha} family, have been shown to play important roles in B cell development (42, 43). However, the phenotype of mice lacking these proteins does not completely phenocopy the pattern of B cell development in mice that lack TACE. Therefore, it is not clear whether this phenotype was due to the effect of an unknown TACE substrate or to the synergistic effect of several TACE substrates, suggesting that further study is required.

In the spleens of tace{Delta}Zn/{Delta}Zn mice, B cell follicle and germinal center formation was severely impaired. This phenotype was very similar to that of TNF-{alpha} and TNF receptor-1 knockout mice, suggesting that TNF-{alpha} cleavage was crucial for TNF-{alpha} function (29, 30). The loss of B cell follicle formation was consistent with a previous analysis of noncleavable TNF-{alpha} knock-in mice (20). However, there were some differences in the phenotypes of these mice. The noncleavable TNF-{alpha} knock-in partially rescued the establishment of germinal centers, which were seen in tace{Delta}Zn/{Delta}Zn mice. The loss of TACE activity would be predicted to not only perturb TNF-{alpha} signaling at the level of ligand presentation but also at the level of functional receptors, and this may explain the more severe phenotype observed in tace{Delta}Zn/{Delta}Zn mice. Alternatively, there may be an additional, as yet unknown TACE substrate that is required for these key events. Furthermore, we observed normal B cell follicle and germinal center formation in tace{Delta}Zn/{Delta}Zn bone marrow-reconstituted RAG1–/– mice, clearly indicating that these TACE-dependent events occur on non-B cell populations.

In conclusion, we have shown that TACE plays an important role in lymphocyte development. Early T cell development, splenic B cell maturation, B cell follicle organization, and germinal center formation were all impaired in tace{Delta}Zn/{Delta}Zn mice. Interestingly, all of these defects were restored in tace{Delta}Zn/{Delta}Zn bone marrow reconstituted RAG1–/– recipients, clearly demonstrating that TACE activity on lymphocytes was not required. However, we cannot rule out the possibility that lymphocyte-expressed substrates are being cleaved by TACE in trans, which has recently been shown to occur in some instances in ADAM10-mediated cleavage (44). These data demonstrate that nonlymphocyte, non-cell autonomous expression of TACE is required for normal T cell development, peripheral B cell maturation, and the maintenance of splenic architecture.


   Acknowledgments
 
We are very grateful to Peter Ong Lim, Sarah M. Fitzgerald, and Yao Wang for technical assistance, Richard Cross for flow cytometry analysis, and Mike Nash for autoMACS. We thank Jacques Peschon and Roy Black (Amgen) for supplying the original tace{Delta}Zn/+ mice and for their helpful advice and support, and Christophe Benoist and Natasha Asinovski for generously providing G8.8-allophycocyanin.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI-39480, Cancer Center Support CORE Grant CA-21765, funds from the American Lebanese Syrian Associated Charities (to D.A.A.V.), and National Institutes of Health Grants DK59778 and DK63363 and funds from the Crohns and Colitis Foundation of America (to P.J.D.). Back

2 Current Address: Department of Investigative Toxicology, Amgen Incorporated, 1201 Amgen Court West, Seattle, WA 98119. Back

3 Current address: Department of Pediatrics and Department of Molecular and Integrative Physiology, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, MI 48109. Back

4 Address correspondence and reprint requests to Dr. Dario Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. E-mail address: dario.vignali{at}stjude.org Back

5 Abbreviations used in the paper: TACE, TNF-{alpha}-converting enzyme; ADAM, a disintegrin and metalloprotease 17; DN, double negative; DP, double positive; EGFR, epidermal growth factor receptor; PNA, peanut agglutinin; sTNFR, soluble TNFR; SP, single positive; T1, type 1 transition (B cells). Back

Received for publication July 25, 2006. Accepted for publication January 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Black, R. A.. 2002. Tumor necrosis factor-{alpha} converting enzyme. Int. J. Biochem. Cell Biol. 34: 1-5. [Medline]
  2. Black, R. A., J. R. Doedens, R. Mahimkar, R. Johnson, L. Guo, A. Wallace, D. Virca, J. Eisenman, J. Slack, B. Castner, et al 2003. Substrate specificity and inducibility of TACE (tumour necrosis factor {alpha}-converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity. Biochem. Soc. Symp. : 39-52.
  3. Killar, L., J. White, R. Black, J. Peschon. 1999. Adamalysins: a family of metzincins including TNF-{alpha} converting enzyme (TACE). Ann. NY Acad. Sci. 878: 442-452. [Medline]
  4. Hansen, H. P., S. Dietrich, T. Kisseleva, T. Mokros, R. Mentlein, H. H. Lange, G. Murphy, H. Lemke. 2000. CD30 shedding from Karpas 299 lymphoma cells is mediated by TNF-{alpha}-converting enzyme. J. Immunol. 165: 6703-6709. [Abstract/Free Full Text]
  5. Contin, C., V. Pitard, T. Itai, S. Nagata, J. F. Moreau, J. Dechanet-Merville. 2003. Membrane-anchored CD40 is processed by the tumor necrosis factor-{alpha}-converting enzyme: implications for CD40 signaling. J. Biol. Chem. 278: 32801-32809. [Abstract/Free Full Text]
  6. Garton, K. J., P. J. Gough, J. Philalay, P. T. Wille, C. P. Blobel, R. H. Whitehead, P. J. Dempsey, E. W. Raines. 2003. Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-{alpha}-converting enzyme (ADAM 17). J. Biol. Chem. 278: 37459-37464. [Abstract/Free Full Text]
  7. Black, R. A., C. T. Rauch, C. J. Kozlosky, J. J. Peschon, J. L. Slack, M. F. Wolfson, B. J. Castner, K. L. Stocking, P. Reddy, S. Srinivasan, et al 1997. A metalloproteinase disintegrin that releases tumour-necrosis factor-{alpha} from cells. Nature 385: 729-733. [Medline]
  8. Brou, C., F. Logeat, N. Gupta, C. Bessia, O. LeBail, J. R. Doedens, A. Cumano, P. Roux, R. A. Black, A. Israel. 2000. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol. Cell 5: 207-216. [Medline]
  9. Buxbaum, J. D., K. N. Liu, Y. Luo, J. L. Slack, K. L. Stocking, J. J. Peschon, R. S. Johnson, B. J. Castner, D. P. Cerretti, R. A. Black. 1998. Evidence that tumor necrosis factor {alpha} converting enzyme is involved in regulated {alpha}-secretase cleavage of the Alzheimer amyloid protein precursor. J. Biol. Chem. 273: 27765-27767. [Abstract/Free Full Text]
  10. Lee, D. C., S. W. Sunnarborg, C. L. Hinkle, T. J. Myers, M. Y. Stevenson, W. E. Russell, B. J. Castner, M. J. Gerhart, R. J. Paxton, R. A. Black, et al 2003. TACE/ADAM17 processing of EGFR ligands indicates a role as a physiological convertase. Ann. NY Acad. Sci. 995: 22-38. [Medline]
  11. Nagano, O., D. Murakami, D. Hartmann, B. De Strooper, P. Saftig, T. Iwatsubo, M. Nakajima, M. Shinohara, H. Saya. 2004. Cell-matrix interaction via CD44 is independently regulated by different metalloproteinases activated in response to extracellular Ca2+ influx and PKC activation. J. Cell Biol. 165: 893-902. [Abstract/Free Full Text]
  12. Peschon, J. J., J. L. Slack, P. Reddy, K. L. Stocking, S. W. Sunnarborg, D. C. Lee, W. E. Russell, B. J. Castner, R. S. Johnson, J. N. Fitzner, et al 1998. An essential role for ectodomain shedding in mammalian development. Science 282: 1281-1284. [Abstract/Free Full Text]
  13. Li, N., Y. Wang, K. Forbes, K. M. Vignali, B. S. Heale, P. Saftig, D. Hartmann, R. Black, J. J. Rossi, C. Blobel, P. J. Dempsey, et al 2006. Metalloproteases regulate T cell proliferation and effector function via LAG-3. EMBO J. 26: 494-504.
  14. Miettinen, P. J., J. E. Berger, J. Meneses, Y. Phung, R. A. Pedersen, Z. Werb, R. Derynck. 1995. Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376: 337-341. [Medline]
  15. Sibilia, M., E. F. Wagner. 1995. Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 269: 234-238. [Abstract/Free Full Text]
  16. Jackson, L. F., T. H. Qiu, S. W. Sunnarborg, A. Chang, C. Zhang, C. Patterson, D. C. Lee. 2003. Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling. EMBO J. 22: 2704-2716. [Medline]
  17. Iwamoto, R., S. Yamazaki, M. Asakura, S. Takashima, H. Hasuwa, K. Miyado, S. Adachi, M. Kitakaze, K. Hashimoto, G. Raab, et al 2003. Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function. Proc. Natl. Acad. Sci. USA 100: 3221-3226. [Abstract/Free Full Text]
  18. Shi, W., H. Chen, J. Sun, S. Buckley, J. Zhao, K. D. Anderson, R. G. Williams, D. Warburton. 2003. TACE is required for fetal murine cardiac development and modeling. Dev. Biol. 261: 371-380. [Medline]
  19. Venturi, G. M., L. Tu, T. Kadono, A. I. Khan, Y. Fujimoto, P. Oshel, C. B. Bock, A. S. Miller, R. M. Albrecht, P. Kubes, et al 2003. Leukocyte migration is regulated by L-selectin endoproteolytic release. Immunity 19: 713-724. [Medline]
  20. Ruuls, S. R., R. M. Hoek, V. N. Ngo, T. McNeil, L. A. Lucian, M. J. Janatpour, H. Korner, H. Scheerens, E. M. Hessel, J. G. Cyster, et al 2001. Membrane-bound TNF supports secondary lymphoid organ structure but is subservient to secreted TNF in driving autoimmune inflammation. Immunity 15: 533-543. [Medline]
  21. Haro, H., H. C. Crawford, B. Fingleton, K. Shinomiya, D. M. Spengler, L. M. Matrisian. 2000. Matrix metalloproteinase-7-dependent release of tumor necrosis factor-{alpha} in a model of herniated disc resorption. J. Clin. Invest. 105: 143-150. [Medline]
  22. Strunk, K. E., V. Amann, D. W. Threadgill. 2004. Phenotypic variation resulting from a deficiency of epidermal growth factor receptor in mice is caused by extensive genetic heterogeneity that can be genetically and molecularly partitioned. Genetics 167: 1821-1832. [Abstract/Free Full Text]
  23. Derbinski, J., A. Schulte, B. Kyewski, L. Klein. 2001. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat. Immunol. 2: 1032-1039. [Medline]
  24. Anderson, M. S., E. S. Venanzi, L. Klein, Z. Chen, S. P. Berzins, S. J. Turley, H. von Boehmer, R. Bronson, A. Dierich, C. Benoist, D. Mathis. 2002. Projection of an immunological self shadow within the thymus by the aire protein. Science 298: 1395-1401. [Abstract/Free Full Text]
  25. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, V. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68: 869-877. [Medline]
  26. Nagasawa, T.. 2006. Microenvironmental niches in the bone marrow required for B-cell development. Nat. Rev. Immunol. 6: 107-116. [Medline]
  27. Weskamp, G., J. W. Ford, J. Sturgill, S. Martin, A. J. Docherty, S. Swendeman, N. Broadway, D. Hartmann, P. Saftig, S. Umland, et al 2006. ADAM10 is a principal ‘sheddase’ of the low-affinity immunoglobulin E receptor CD23. Nat. Immunol. 7: 1293-1298. [Medline]
  28. Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, M. C. Lamers, R. Carsetti. 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med. 190: 75-89. [Abstract/Free Full Text]
  29. Pasparakis, M., L. Alexopoulou, V. Episkopou, G. Kollias. 1996. Immune and inflammatory responses in TNF {alpha}-deficient mice: a critical requirement for TNF {alpha} in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J. Exp. Med. 184: 1397-1411. [Abstract/Free Full Text]
  30. Matsumoto, M., S. Mariathasan, M. H. Nahm, F. Baranyay, J. J. Peschon, D. D. Chaplin. 1996. Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers. Science 271: 1289-1291. [Abstract]
  31. Robey, E. A., J. A. Bluestone. 2004. Notch signaling in lymphocyte development and function. Curr. Opin. Immunol. 16: 360-366. [Medline]
  32. Maillard, I., T. Fang, W. S. Pear. 2005. Regulation of lymphoid development, differentiation, and function by the Notch pathway. Annu. Rev. Immunol. 23: 945-974. [Medline]
  33. Rooke, J., D. Pan, T. Xu, G. M. Rubin. 1996. KUZ, a conserved metalloprotease-disintegrin protein with two roles in Drosophila neurogenesis. Science 273: 1227-1231. [Abstract]
  34. Mumm, J. S., E. H. Schroeter, M. T. Saxena, A. Griesemer, X. Tian, D. J. Pan, W. J. Ray, R. Kopan. 2000. A ligand-induced extracellular cleavage regulates {gamma}-secretase-like proteolytic activation of Notch1. Mol. Cell 5: 197-206. [Medline]
  35. Bland, C. E., P. Kimberly, M. D. Rand. 2003. Notch-induced proteolysis and nuclear localization of the Delta ligand. J. Biol. Chem. 278: 13607-13610. [Abstract/Free Full Text]
  36. LaVoie, M. J., D. J. Selkoe. 2003. The Notch ligands, Jagged and Delta, are sequentially processed by {alpha}-secretase and presenilin/{gamma}-secretase and release signaling fragments. J. Biol. Chem. 278: 34427-34437. [Abstract/Free Full Text]
  37. Jarriault, S., I. Greenwald. 2005. Evidence for functional redundancy between C. elegans ADAM proteins SUP-17/Kuzbanian and ADM-4/TACE. Dev. Biol. 287: 1-10. [Medline]
  38. Hartmann, D., B. de Strooper, L. Serneels, K. Craessaerts, A. Herreman, W. Annaert, L. Umans, T. Lubke, I. A. Lena, K. von Figura, P. Saftig. 2002. The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for {alpha}-secretase activity in fibroblasts. Hum. Mol. Genet. 11: 2615-2624. [Abstract/Free Full Text]
  39. Manilay, J. O., A. C. Anderson, C. Kang, E. A. Robey. 2005. Impairment of thymocyte development by dominant-negative Kuzbanian (ADAM-10) is rescued by the Notch ligand, {delta}-1. J. Immunol. 174: 6732-6741. [Abstract/Free Full Text]
  40. Wolfer, A., A. Wilson, M. Nemir, H. R. MacDonald, F. Radtke. 2002. Inactivation of Notch1 impairs VDJbeta rearrangement and allows pre-TCR-independent survival of early {alpha} beta lineage thymocytes. Immunity 16: 869-879. [Medline]
  41. Dyczynska, E., D. Sun, H. Yi, A. Sehara-Fujisawa, C. P. Blobel, A. Zolkiewska. 2007. Proteolytic processing of {delta}-like 1 by ADAM proteases. J. Biol. Chem. 282: 436-444. [Abstract/Free Full Text]
  42. Mackay, F., P. Schneider, P. Rennert, J. Browning. 2003. BAFF AND APRIL: a tutorial on B cell survival. Annu. Rev. Immunol. 21: 231-264. [Medline]
  43. Cancro, M. P.. 2004. Peripheral B-cell maturation: the intersection of selection and homeostasis. Immunol. Rev. 197: 89-101. [Medline]
  44. Janes, P. W., N. Saha, W. A. Barton, M. V. Kolev, S. H. Wimmer-Kleikamp, E. Nievergall, C. P. Blobel, J. P. Himanen, M. Lackmann, D. B. Nikolov. 2005. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 123: 291-304. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
K. Horiuchi, T. Kimura, T. Miyamoto, K. Miyamoto, H. Akiyama, H. Takaishi, H. Morioka, T. Nakamura, Y. Okada, C. P. Blobel, et al.
Conditional Inactivation of TACE by a Sox9 Promoter Leads to Osteoporosis and Increased Granulopoiesis via Dysregulation of IL-17 and G-CSF
J. Immunol., February 15, 2009; 182(4): 2093 - 2101.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
R. W. Gelling, W. Yan, S. Al-Noori, A. Pardini, G. J. Morton, K. Ogimoto, M. W. Schwartz, and P. J. Dempsey
Deficiency of TNF{alpha} Converting Enzyme (TACE/ADAM17) Causes a Lean, Hypermetabolic Phenotype in Mice
Endocrinology, December 1, 2008; 149(12): 6053 - 6064.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
L. Tian, X. Wu, C. Chi, M. Han, T. Xu, and Y. Zhuang
ADAM10 is essential for proteolytic activation of Notch during thymocyte development
Int. Immunol., September 1, 2008; 20(9): 1181 - 1187.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. M. Briso, O. Dienz, and M. Rincon
Cutting Edge: Soluble IL-6R Is Produced by IL-6R Ectodomain Shedding in Activated CD4 T Cells
J. Immunol., June 1, 2008; 180(11): 7102 - 7106.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, N.
Right arrow Articles by Vignali, D. A. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, N.
Right arrow Articles by Vignali, D. A. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS