The Journal of Immunology Disturbed Follicular Architecture in B Cell A Disintegrin and Metalloproteinase (ADAM)10 Knockouts Is Mediated by Compensatory Increases in ADAM17 and TNF-a Shedding Lauren Folgosa,*,† Hannah B. Zellner,† Mohey Eldin El Shikh,‡,1 and Daniel H. Conrad†,1 B cell A disintegrin and metalloproteinase 10 (ADAM10) is required for the development and maintenance of proper secondary lymphoid tissue architecture; however, the underlying mechanism remains unclear. In this study, we show disturbances in naive lymph node architecture from B cell–specific ADAM10-deficient mice (ADAM10B2/2) including loss of B lymphocyte/T lymphocyte compartmentalization, attenuation of follicular dendritic cell reticula, excessive collagen deposition, and increased high endothe- lial venule formation. Because TNF-a signaling is critical for secondary lymphoid tissue architecture, we examined compensatory changes in ADAM17 and TNF-a in ADAM10B2/2 B cells. Surprisingly, defective follicular development in these mice was associated with increased rather than decreased TNF-a expression. In this article, we describe an increase in TNF-a message, mRNA stability, soluble protein release, and membrane expression in ADAM10B2/2 B cells compared with wild type (WT), which coincides with increased ADAM17 message and protein. To assess the mechanistic contribution of excessive TNF-a to abnormal lymphoid architecture in ADAM10B2/2 mice, we performed a bone marrow reconstitution study. Rectification of WT architecture was noted only in irradiated WT mice reconstituted with ADAM10B2/2 + TNF knockout bone marrow because of normalization of TNF-a levels not seen in ADAM10B2/2 alone. We conclude that ADAM17 overcompensation causes excessive TNF-a shedding and further upregulation of TNF-a expression, creating an aberrant signaling environment within B cell cortical regions of ADAM10B2/2 lymph nodes, highlighting a key interplay between B cell ADAM10 and ADAM17 with respect to TNF-a homeo- stasis. The Journal of Immunology, 2013, 191: 5951–5958. disintegrin and metalloproteinases (ADAMs) are a fam- We have shown it is critical in Notch2-mediated marginal zone ily of zinc-dependent proteinases known to be involved B cell development and CD23-mediated regulation of allergic A in ectodomain cleavage and regulated intramembrane diseases (4, 5). Lastly, although we have previously reported that proteolysis of transmembrane proteins. Of all of the ADAMs, B cell ADAM10 is required for maintenance of proper secondary ADAM10 and ADAM17, commonly referred to as TNF-a con- lymphoid tissue architecture, formation of germinal centers, as verting enzyme, are known to be most closely related with regard well as optimal class-switched Ab (Ig) production, the underlying to structure and share many overlapping substrate specificities (1, mechanism was unclear (6). 2). Classically, ADAM17 is thought to orchestrate inflammatory TNF-a is a key proinflammatory cytokine, which exists as a 26- responses as the principle, physiologic sheddase of pro–TNF-a; kDa transmembrane protein (mTNF-a) before it is shed from the however, ADAM10 can also cleave membrane TNF-a (mTNF-a) surface as a 17-kDa soluble molecule (sTNF-a) (7). Tristetraprolin when ADAM17 is not present (3). In addition, ADAM10 is crucial (TTP), also known as ZFP36, is a low-abundance cytosolic zinc for functional and phenotypic maturation of the immune system. finger protein induced by LPS and is critical for mRNA degradation of multiple mRNA targets including TNF-a (8, 9). TTP-deficient mouse models portray the downstream consequences of increased *Center for Clinical and Translational Research, Virginia Commonwealth University, a † TNF- mRNA stability including inflammatory arthritis, autoim- Richmond, VA 23298; Department of Microbiology and Immunology, Virginia a Commonwealth University, Richmond, VA 23298; and ‡Experimental Medicine munity, and cachexia (10, 11). In addition, B cell–TNF- has been and Rheumatology, William Harvey Research Institute, Queen Mary University of implicated in the functional decline of aging B cells where in- London, Charterhouse Square campus, London EC1M 6BQ, United Kingdom creased TNF-a production is inversely correlated with response to 1 M.E.E.S. and D.H.C. are co-senior authors. stimulation in vitro by LPS. Interestingly, aging B cells additionally Received for publication August 1, 2013. Accepted for publication October 16, 2013. exhibit increased TTP, which causes reduced optimal class-switched This work was supported by National Institute of Allergy and Infectious Diseases/ Ab production by downregulating E47 and activation-induced cy- National Institutes of Health Grant RO1AI18697, American Asthma Foundation Grant tidine deaminase. The paradoxical increase of both TTP and TNF-a 11-0094, a bridge grant from the Virginia Commonwealth University School of Medi- cine, and cytometry support from the Massey Cancer Center Core (Grant P30 CA16059). in unstimulated B cells from old mice may reflect increased TNF-a Address correspondence and reprint requests to Dr. Daniel H. Conrad, Department of transcription by these B cells to overcome elevated TTP, thus Microbiology and Immunology, Virginia Commonwealth University, 1217 E. Marshall placing them in a preactivated state that is less susceptible to sub- Street, 419 MSB, Richmond, VA 23298. E-mail address: [email protected] sequent stimulation (12). The online version of this article contains supplemental material. The role of TNF-a in maintaining proper secondary lymphoid Abbreviations used in this article: ADAM, A disintegrin and metalloproteinase; tissue architecture is indisputable, and ADAM10 also seems to ADAM10B2/2, B cell–specific ADAM10-deficient mouse; B/T, B lymphocyte/ T lymphocyte; FDC, follicular dendritic cell; HEV, high endothelial venule; KO, be involved in this maintenance. Both B cell–specific ADAM10- B2/2 knockout; LN, lymph node; LT, lymphotoxin; MMP, matrix metalloproteinase; deficient mice (ADAM10 ) and global TNF-a–deficient mice mTNF-a, membrane TNF-a; qPCR, real-time quantitative PCR; sTNF-a, soluble mol- exhibit disorganized follicular dendritic cell (FDC) networks, ecule TNF-a; TSA, tyramide signal amplification; TTP, tristetraprolin; WT, wild type. aberrant germinal centers, and lack of splenic B cell follicles (13). Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 Furthermore, using B cells that express a noncleavable form of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302042 5952 EXCESSIVE TNF-a IN ADAM10KO mTNF-a showed that adequate levels of B cell–produced sTNF-a T20936; Molecular Probes) and one of the following Ig: anti-mouse FITC- was critical for maintaining secondary architecture in the lymph conjugated B220 or PE-conjugated B220 (Biolegend). Kit reagents were node (LN), spleen, and Peyer’s patches, and for IgG production prepared according to manufacturer’s protocol, and tyramide amplification using the “Peroxidase Labeling assay” was performed with the following against T-dependent Ags (14). Although it is clear that regulation modifications: cells were incubated with blocking reagent (10 mganti- of B cell TNF-a is required for proper follicular architecture and mouse unlabeled CD16/32 [2.4G2]) for 15 min; stained with biotin anti- B cell function, the role of TNF-a cleaving enzymes (ADAM10 mouse TNF-a primary Ab (Biolegend); and after tyramide labeling, cells and ADAM17) has yet to be explored. were washed twice and stained with anti-mouse B220 (see earlier) for 30 min and examined on a BD Canto Flow analyzer; data analysis was with In this article, we investigate the hypothesis that compensatory FCS Express, v. 4. overexpression of ADAM17 after B cell–specific ADAM10 de- letion mediates excessive TNF-a levels in ADAM10B2/2 mice, Bone marrow reconstitution ultimately providing the mechanism underpinning the aberrant Bone marrow cells were isolated as previously described with the following secondary lymphoid tissue architecture in these mice. modifications (16). In brief, two femurs and two tibias from each mouse (WT [CD45.2], ADAM10B2/2 [CD45.2], or TNF-aKO) were centrifuged, RBCs lysed with ACK Lysing Buffer (Quality Biological), bone marrow Materials and Methods cells counted, and 5 million cells were i.v. injected. For 50/50 mixtures, Mice such as ADAM10B2/2 + TNFKO, 100 ml/2.5 million cells from each were All mice were housed in the Virginia Commonwealth University Molecular used to prepare the final injection mixture. Medicine Research Building Barrier Facility in accordance with institu- B6-Ly5.2/Cr (CD45.1) congenic mice from National Cancer Institute/ tional and National Institutes of Health guidelines. All animal care and National Institutes of Health were pretreated 5 d before irradiation with 100 mg/l (concentration 0.01%) enrofloxacin (Baytril) in sterile water. experimental protocols were approved by the Virginia Commonwealth Uni- m versity Institutional Animal Care and Use Committee. C57BL/6 ADAM10B2/2 CD45.1 mice were anesthetized using a 100 l i.p. injection of ketamine/ (CD19-cre+) mice were generated as previously described and compared xylazine in PBS at a dose of 80 and 8 mg/kg, respectively. This was fol- 2 a lowed by two doses of 550 cGy irradiation, separated by a 2-h rest period, with littermate wild type (WT) controls (CD19-cre ) (4). TNF- knockout 137 (TNF-aKO) mice were purchased from Jackson Laboratory (no. 005540, using an MDS Nordion Gammacell 40 research irradiator with a [ Cs] B6.129S-Tnf) for use in bone marrow reconstitution. Healthy