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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 endothelial 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 homeostasis. 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 deletion 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 male and source. After irradiation, mice were reconstituted by i.v. injection of the indicated bone marrow cells as described earlier. After 6 wk of reconsti- female mice aged 6–12 wk were used in all experimentation except bone m marrow reconstitution where only WT female mice ages 6–8 wk were tution, mice were footpad immunized in two ipsilateral paws with 10 g reconstituted with sex-matched bone marrow cells. 4-hydroxy-3-nitrophenylacetyl coupled to keyhole limpet hemocyanin at a ratio of 27:1 (Biosearch Technologies) in 4 mg alum. Draining and B cell isolation, reagents, in vitro activation, and ELISA Spleen was crushed, filtered through 40-mm Nylon Mesh (Fisherbrand), and RBCs lysed with ACK Lysing Buffer (Quality Biological). B cells were isolated by positive selection (B220+) using magnetic beads and following manufacturer’s protocol (Miltenyi). B cells were grown in complete RPMI 1640 medium containing 10% heat-inactivated (56˚C, 30 min) FBS (Gemini Bio-Products, West Sacramento, CA), 2 mM L-glutamine, 50 mg/ml pen- icillin, 50 mg/ml streptomycin, 1 mM sodium pyruvate, 50 mM 2-ME, 13 nonessential amino acids, and 20 mM HEPES buffer (all from Invitrogen, Carlsbad, CA), and stimulated in vitro for 1, 3, or 5 d with 1000 U IL-4 and either 50 mg/ml LPS (LPSs from Escherichia coli 0111:B4; Sigma) or 1.25 mg/ml purified anti-mouse CD40 (no. 102902; Biolegend). Proliferation was assessed after 72 h of growth, and a 24-h pulse of [3H]thymidine, 1 mCi/well (Perkin Elmer, Waltham, MA), was used. Plates were then harvested using a Filtermate cell harvester onto UniFilter-96 GF/C micro- plates (Perkin Elmer) and analyzed using Topcount Plate Counter (Perkin Elmer). Soluble TNF-a from B cell supernatants was determined by mouse quantitative ELISA kit (88-7324-88; eBioscience) according to manu- facturers’ instructions. RT-PCR, quantitative PCR, and Western blotting Total RNA was extracted from naive and stimulated total B cells using TRIzol reagent (Invitrogen) according to manufacturer’s protocol and RNA concentration quantified by a ND-100 NanoDrop spectrophotometer. RNA (400 ng/ml) was reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad). Real-time quantitative PCR (qPCR) was performed with a real-time PCR machine (iQ5; Bio-Rad Laboratories). Primers and probes used for TaqMan qPCR assay (all from Applied Biosystems) were the following: TNF-a (Mm00443258), ADAM10 (Mm00545742), ADAM17 (Mm00456428), TTP/Zfp36 (Mm00457144), matrix metalloproteinase 13 (MMP13; Mm00439491), and 18s (Mm03928990). Fold variation was determined using the ΔΔCt method of analysis (15). Protein lysates were made according to the manufacturer’s protocol using Cell Lysis Buffer (no. 9803; Cell Signaling). Equal amounts of protein were loaded onto Novex NuPAGE 10% Bis-Tris gels (Invitrogen), run for 35 min at 200 V, transferred to nitrocellulose membrane, and equal transfer verified by Ponceau S (Sigma) staining. Blots were probed with primary anti-mouse B2/2 Abs: anti–b-actin peroxidase (no. A3854; Sigma) or anti-ADAM17 (no. FIGURE 1. Naive ADAM10 LNs display abnormal follicular ar- B2/2 2051; Abcam). Goat anti-rabbit IgG, HRP conjugate secondary Ab was chitecture. Compared with WT, ADAM10 mice (A) lack well-devel- used for ADAM17 blots and signal detected with SuperSignal West Pico oped FDC reticula (red, CR1/2) in the B cell follicle (blue, B220), (B) lack Chemiluminescent Substrate (no. 34080; Thermo Scientific). B cell/T cell (red, Thy1.2) segregation with more HEVs (green, peripheral node addressin [pNAD]) in the LN cortex, (C) display more collagen Flow cytometry with tyramide signal amplification (green) deposition in the B cell follicle, and (D) express higher levels of Single-cell suspensions of naive or stimulated B cells were stained using TNF-a (green) than WT mice. Scale bar, 50 mm. Micrographs are rep- tyramide signal amplification (TSA) Kit #26 with HRP streptavidin (no. resentatives of at least three LNs. The Journal of Immunology 5953 nondraining popliteal and axillary LNs were dissected at 14 d postimmu- TNF-a labeling was enhanced using fluorescein-TSA (TSA Plus Fluo- nization and analyzed by immunohistochemistry. rescein System; NEL741001KT; Perkin Elmer). In brief, after quenching endogenous peroxidase using 1% H2O2, anti–TNF-a Ig was applied for Immunohistochemistry, tyramide amplification, and confocal 2 h, washed, then HRP-conjugated secondary Ig was added for 1 h. After microscopy washing, HRP was allowed to catalyze the deposition of fluorescein- labeled tyramide for 10 min, then was washed, mounted, and examined. Ten-micrometer-thick frozen sections were cut from the excised mouse LNs, fixed in absolute acetone, air-dried, and blocked with serum-free Statistical analysis protein block (X0909; Dako). The sections were dual- and triple-labeled The p values were calculated using unpaired two-tailed Student t tests in for FDCs (PE anti-mouse CD21/CD35; 123410; Biolegend), B cells (Alexa GraphPad Prism. Error bars represent the SEM between samples. A p value Fluor 647 anti-mouse/human CD45R/B220; 103226; Biolegend), high en- ,0.05 is considered significant. dothelial venules (HEVs; anti-mouse/human peripheral node addressin; 120804; Biolegend), T cells (rat anti-mouse CD90.2/Thy-1.2-PE; 1750- 09L; Southern Biotech), collagen type 1 (ab21286; Abcam), and TNF-a Results 2 2 (ab34674; Abcam). The Ig concentrations ranged between 5 and 10 mg/ml. C57BL/6 ADAM10B / mice exhibit abnormal LN architecture Sections were mounted with antifade mounting medium, Vectashield (Vector and excessive TNF-a in B cell regions Laboratories), coverslipped, and examined with a Leica TCS-SP2 AOBS confocal laser-scanning microscope. Three lasers were used: argon (488 nm), Our laboratory previously described secondary lymphoid tissue HeNe (543 nm), and HeNe (633 nm; far red emission is shown as pseu- architectural defects in immunized and naive LNs isolated from doblue). Parameters were adjusted to scan at 1024 3 1024 pixel density and C57BL/6 ADAM10B2/2 mice. These defects included improper 8-bit pixel depth. Emissions were recorded in two or three separate channels, and digital images were captured and processed with Leica Confocal, LCS localization of B and T cells, reduced germinal center formation, Lite software, and ImageJ for color separation and quantitative assessment of and a decrease in FDC networks (6). These architecture aberran- immunohistochemistry. cies bore some similarity with secondary lymphoid tissue defects

FIGURE 2. Increased TNF-a surface expression and production in ADAM10B2/2 B cells (A10KO). Naive (A) and 5-d-stimulated with LPS + IL-4 (B). WT (black thin line) and A10KO (black bold line) live B cells were analyzed for coexpression of TNF-a using TSA. Black bars (overlay plots) indicate B cells staining high in mTNF-a, represented in bar graphs (A, B). n = 9 per group, 3 independent studies. (C and D) Supernatants were harvested on day 1, 3, or 5 from WT (white) or A10KO (black) B cell cultures stimulated with LPS + IL-4 (C) or anti-CD40 + IL-4 (D) and sTNF-a determined by ELISA. n =9 per group, three independent studies. *p , 0.05, **p , 0.005. 5954 EXCESSIVE TNF-a IN ADAM10KO noted in early studies of global TNF-a–deficient mice. Specifi- cally, lack of splenic B cell follicles, disorganized FDC networks, and aberrant germinal centers were noted (13). Recent studies using B cells only capable of expressing mTNF-a showed that sTNF-a produced by B cells is required for maintaining secondary architecture in LN, spleen, Peyer’s patches, and for IgG production against T-dependent Ags (14). Taken together, ADAM10 and TNF-a seem to reciprocally interact to maintain proper secondary lymphoid architecture and permit class-switched Ab production. Fig. 1 shows immunohistochemistry analysis of LNs from naive (nonimmunized) WT and ADAM10B2/2 mice. Although our ini- tial studies had indicated a relatively normal architecture in naive ADAM10B2/2 nodes (6), additional analysis revealed that not only are LN FDC networks largely absent and B lymphocyte/ T lymphocyte (B/T) boundaries intermingled (Fig. 1A, 1B), but other abnormalities can also be seen. These changes include excessive collagen deposition, as well as an increase in HEVs, es- pecially within B cell cortical regions (Fig. 1B, 1C). Most striking, however, was the dramatic increase in TNF-a within the B cell regions of ADAM10B2/2 nodes (Fig. 1D). Given the data in Fig. 1 and previous reports of secondary lymphoid tissue architecture abnormalities in TNF-a KOs and those capable of expressing only mTNF-a, it appears that not only subnormal but also excessive TNF-a levels in the LN cortices may lead to disruption of normal follicular architecture. B cells from C57BL/6 ADAM10B2/2 mice exhibit increased expression, stability, and production of TNF-a Given the excessive TNF-a staining in B cell regions of C57BL/6 ADAM10B2/2 LNs (Fig. 1), we further analyzed differences in TNF-a expression and production in B cells purified from both ADAM10B2/2 and WT mice. As can be seen in Fig. 2A and 2B, flow analysis for mTNF-a revealed that both naive and stimulated FIGURE 3. Increased TNF-a gene expression and message stability 2 2 ADAM10B2/2 B cells exhibit increased mTNF-a. Furthermore, in ADAM10B / B cells (A10KO). Naive [(A) checkered, (B) left,(C) ELISA analysis of supernatants from ADAM10B2/2 B cells cul- checkered] or 5-d-stimulated (LPS + IL-4) [(A) stripe, (B) right,(C) stripe] tured with LPS/IL-4 (Fig. 2C) or anti–CD40/IL-4 (Fig. 2D) for 1, B cells were analyzed by qPCR for TNF-a (A, B) and TTP (C) message A 3, or 5 d all showed significantly higher sTNF-a production com- normalized to 18s. Data presented as fold change of A10KO over WT ( ) or fold change of WT over A10KO (C) using the ΔΔCt method of analysis. pared with WT. Absolute RNA quantification for TNF-a normalized to 18s in WT (white) Relative (Fig. 3A) and absolute (Fig. 3B) message analysis by qPCR and A10KO (black) B cells appears in (B). n = 9 per group, three inde- a B2/2 indicated a strong increase in TNF- message in ADAM10 pendent studies. *p , 0.05, ***p , 0.0005. compared with WT B cells poststimulation in vitro. Furthermore, TTP is known to promote TNF-a mRNA degradation (8, 9). We, therefore, analyzed TTP message expression in both WT and quantification revealed that naive ADAM10B2/2 B cells exhibit ADAM10B2/2 B cells and found it to be 2-fold higher in WT significantly increased ADAM17 RNA expression, which increases B cells, which also produce less sTNF-a and mTNF-a (Figs. 2, 3C). further upon stimulation (Fig. 4B). Western blot analysis, fur- The combined results of increased TNF-a message and soluble thermore, showed a 2.3-fold increase in ADAM17 protein levels protein suggest a possible feedback mechanism in which increased in naive ADAM10B2/2 B cells over WT, which also increased upon TNF-a shedding upregulates further TNF-a production. In any stimulation to 5-fold (Fig. 4C, 4D). Upon overexposure of the blots, case, it is clear that TNF-a is increased at both the message and both the precursor and glycosylated forms of ADAM17 were seen protein level in ADAM10B2/2 B cells. (data not shown).

B2/2 In addition, Vandenbroucke et al. (17) recently established that ADAM10 B cells exhibit higher ADAM17 gene and protein MMP13 also cleaves TNF-a at least in intestinal epithelium. qPCR expression analysis of MMP13 in naive B cells, however, showed there was no ADAM17 is known to be the principle sheddase of the membrane- difference between WT and ADAM10B2/2 B cells (ΔΔCt = 1.04; expressed pro–TNF-a. Because ADAM10B2/2 B cells exhibit higher fold change ADAM10B2/2 over WT). Western blot, furthermore, expression and production of TNF-a, we next examined ADAM17 failed to show MMP13 protein in naive WT and ADAM10B2/2 expression and function by analyzing ADAM17 message (Fig. B cells compared with positive control, RAW 264.7 macrophages 4A, 4B) and protein levels (Fig. 4C, 4D) in WT compared with (data not shown). This finding is in agreement with the report ADAM10B2/2 B cells. Although both naive and stimulated that significant levels of MMP13 are not found in B cells (18). ADAM10B2/2 B cells express significantly more ADAM17 mes- Taken together, Figs. 1–4 demonstrate that ADAM10 deletion sage (Fig. 4A, 4B), relative gene expression analysis showed that from B cells results in a compensatory increase in ADAM17 ex- in the naive state, ADAM10B2/2 B cells express two times higher pression and activity, leading to excessive TNF-a cleavage. The ADAM17 compared with WT, which increased to 4-fold higher aberrant signaling environment created by this compensatory ef- expression upon stimulation (Fig. 4A). Similarly, absolute RNA fect is an excellent candidate to explain the abnormal lymphoid The Journal of Immunology 5955

FIGURE 4. Increased ADAM17 message and protein levels in ADAM10B2/2 B cells (A10KO). Naive [(A) checkered, (B) left,(C)] or 5-d-stimulated with LPS + IL-4 [(A) stripe, (B) right, (D)] B cells were analyzed by qPCR and West- ern blotting. (A) Relative ADAM17 expression normalized to 18s presented as fold change of A10KO over WT using the ΔΔCt method of analysis. (B) Ab- solute quantiﬁcation of ADAM17 RNA normalized to 18s. n = 9 mice per group, 3 independent studies. Band densitom- etry of naive (C) and stimulated (D)B cells represents ADAM17 (∼93 kDa, mature form) normalized to actin (∼42 kDa). n = 6 per group total, four independent studies. *p , 0.05, **p , 0.005, ***p , 0.0005.

tissue architecture in our ADAM10B2/2 model and was thus TNF-a staining returned to WT levels. TNF-a staining is further further explored. compared and quantified by mean gray values in multiple equal areas (at least six) in Fig. 5H. Mice reconstituted with ADAM10B2/2 Reconstitution of irradiated C57BL/6 WT with combination B2/2 B2/2 alone as well as ADAM10 + WT CD45.1 exhibit significantly ADAM10 + TNFKO bone marrow rectifies LN follicular 2 2 more TNF-a staining compared with WT and ADAM10B / + abnormalities TNFKO (Fig. 5H). We reason that although B cells from To assess whether TNF-a is involved in the mechanism underly- ADAM10B2/2 mice produce excessive TNF-a, those from the TNFKO B2/2 ing LN tissue abnormalities in ADAM10 mice, we performed mice produce none, thus averaging to a normal, WT range allowing a bone marrow chimera experiment in which irradiated CD45.1 WT proper TNF-a signaling to occur. C57BL/6 mice were reconstituted with one of the following bone marrow combinations: 1) WT (CD45.2) alone; 2) ADAM10B2/2 Discussion (CD45.2) alone; 3) TNF-a–deficient (TNFKO) alone; 4) 50/50 mix This study provides evidence that B cell TNF-a and lymphoid ADAM10B2/2 + WT (CD45.1); or 5) 50/50 mix ADAM10B2/2 + tissue architecture are regulated by the orchestrated interplay be- TNFKO. After 6 wk of reconstitution, mice were bled and analyzed tween ADAM10 and ADAM17. Mezyk-Kopec´ et al. (3) previ- for successful reconstitution, and CD45.2 cells predominated ously described, in mouse embryonic fibroblasts, a compen- (Supplemental Fig. 1). The mice were then footpad immunized satory relationship between ADAM10 and ADAM17 with regard with a T-dependent Ag, 4-hydroxy-3 nitrophenylacetyl coupled to to TNF-a cleavage. In that study, ADAM17-deficient mouse em- keyhole limpet hemocyanin, and draining LNs were assessed by bryonic fibroblasts still exhibited TNF-a cleavage because of a immunohistochemistry 14 d postimmunization (Fig. 5). As ex- compensatory increase in ADAM10 (3). In this study, we show, pected, LNs from WT mice reconstituted with WT (CD45.2) bone in a B cell–targeted model, that in the absence of B cell ADAM10, marrow exhibited normal LN architecture (Fig. 5D) and TNF-a ADAM17 is overexpressed more than WT levels (Fig. 4). This levels (Fig. 5G), which was comparable with nonirradiated WT compensatory change in B cell ADAM17 results in increased TNF-a nodes (Fig. 5A, 5G). Those reconstituted with ADAM10B2/2 shedding, gene expression, mRNA stability, and surface expres- bone marrow (Fig. 5B, 5G), however, had a similar phenotype sion, and thus skews the cytokine environment within B cell follicles to those of our ADAM10B2/2 mice (Fig. 1): loss of B cell/T cell (Figs. 2, 3). Although the absence of B cell sTNF-a is known segregation, decreased FDC networks, and increased TNF-a. to cause LN architecture defects, we demonstrate that excessive Those reconstituted with TNFKO bone marrow alone (Fig. 5C) sTNF-a also dramatically disrupts LN follicular architecture (14). exhibit no TNF-a staining but do still demonstrate FDC networks In addition to B cells, T cells, FDCs, and tingible body mac- because these are resistant to irradiation. Furthermore, reconsti- rophages contribute to TNF-a expression in secondary lymphoid tution with 50/50 mix ADAM10B2/2 + WT CD45.1 still yielded tissues (19–21). Cooperation between these sources is needed to abnormal LN architecture (Fig. 5E) and high TNF-a staining (Fig. maintain TNF-a homeostasis, which is essential for lymphoid 5G) similar to ADAM10B2/2. Thus, the amount of TNF-a made tissue architectural maintenance and ultimately B cell humoral by B cells from this combination is still too high. Interestingly, when responses. Tumanov et al. (14) clearly demonstrated this concept, WT mice were reconstituted with combination ADAM10B2/2 + showing impaired LN organization and B cell Ab production TNFKO bone marrow (Fig. 5F, 5G), LN tissue architecture and postimmunization when either B or T cell TNF-a was inhibited. 5956 EXCESSIVE TNF-a IN ADAM10KO

FIGURE 5. Reconstitution (Reconst) of irradiated WT with ADAM10B2/2 + TNFKO bone marrow restores normal follicular architecture and rectiﬁes structural abnormalities induced by ADAM10B2/2 reconstitution alone. Draining LN sections from nonirradiated WT [(A, G) CD45.1 WT] and irradiated CD45.1 WT reconstituted with ADAM10B2/2 [(B, G) Reconst with ADAM10KO], TNFKO (C), WT CD45.2 [(D, G) Reconst with CD45.2 WT], ADAM10B2/2 + WT CD45.1 [(E, G) Reconst with ADAM10KO and CD45.1 WT], ADAM10B2/2 + TNFKO [(F, G) Reconst with ADAM10KO and TNFKO] were compared in (A–F) with regard to B/T compartmentalization (left column), FDC reticular development (middle column), and TNF-a staining (right column). (A–G) The following stains were used: B cells (blue, B220), T cells (red, Thy1.2), Collagen 1 (green), FDCs (red, CR1/2), and TNF-a (green). White boxes in (A)–(F)(right column) are magniﬁed in (G)(left column), with separation of the TNF-a labeling (middle column) and measurements (histograms, right column). (H) TNF-a expression (mean gray values) in multiple equal areas (at least six) have been calculated and the results expressed as mean 6 SD. Using unpaired t test, the p value between CD45.1 WT and reconstitution with CD45.2 WT is 0.33; CD45.1 WT and reconstitution with ADAM10B2/2 (ADAM10KO) and TNFKO is 0.36; and reconstitution with ADAM10KO and ADAM10KO and CD45.1 WT is 0.2. Micrographs are representative of at least three LNs from three independent experiments. Scale bars, 50 mm (A–F) and 20 mm (G).

Furthermore, therapeutic inhibition of TNF-a with etanercept in- Possible candidates responsible for defects characterizing hibited the maintenance of FDC networks, resulting in decreased ADAM10B2/2 LNs are TNF-a and lymphotoxin (LTa,LTb). These germinal center responses and Ab production (22). Although in- structurally homologous and genetically linked cytokines have sufficient TNF-a levels lend to disorganization and stunted B been studied individually and as double-deficient mouse models, cell function, an example of pathologic overexpression of B cell in an attempt to tease out the contributions of each to secondary TNF-a is seen in B cell chronic lymphocytic leukemia. Leukemic lymphoid tissue microarchitecture development and maintenance B cells overexpress TNF-a, which then acts in an autocrine (25). LTa-deficient mice lack LNs and Peyer’s patches, and ex- fashion to upregulate its own mRNA and protein expression (23). hibit abnormal splenic architecture including loss of B/T segre- Enhanced leukemic cell TNF-a signaling leads to upregulation gation and a complete absence of FDC networks, germinal center of MMP9, a key protease involved in proangiogenic pathways formation, and marginal zone B cells (26, 27). LTa as a soluble needed for cancer metastasis (24). homotrimer (LTa3) is also known to play an integral role in The Journal of Immunology 5957 lymphoid organization including B/T segregation by binding to and ADAM17 levels should be monitored as a direct indication the TNF-a receptor, p55TNFR-1, suggesting that blockade of this of disease susceptibility and severity. Lastly, in this article, we interaction would result in a similar phenotype to ADAM10B2/2 demonstrate that compensatory increases in ADAM17 occur in LNs (28). LTb-deficient mice, however, experience more mild ADAM10B2/2 B cells leading to increased production of TNF-a, disruption as they retain mesenteric and cervical LN development which ultimately underlies architectural aberrancies noted in and maintain B/T segregation, FDC networks, and germinal center ADAM10B2/2 LNs. This finding lends new insight to the discussion formation in spleen (29). More similar to LTa-deficient mice, regarding how B cell TNF-a helps regulate secondary lymphoid TNF-a KOs lack FDC networks in B cell follicles and fail to form tissue organization and how a proper ADAM10/ADAM17 ratio is germinal centers postimmunization (13, 30, 31). Furthermore, it is needed to ultimately control TNF-a signaling. known that without TNF-a or its receptor p55TNFR-1, B cell follicles and FDC networks fail to form in peripheral LNs, Peyer’s Acknowledgments patches, and spleen; however, the effects of B cell TNF-a overex- We thank Lee Dean, Rebecca K. Martin, and Julie Farnsworth for technical pression are unreported (13, 32). assistance, and Dr. Suzanne Barbour for helpful comments on the manu- a a Because p55TNFR-1 binds both TNF- and LT 3, it is rea- script. sonable to conclude that excessive TNF-a could outcompete LTa3 for binding to this receptor, resulting in noted defects in B/T segregation. Furthermore, it has been shown that local cytokine Disclosures factors such as TNF-a contribute to HEV neogenesis (33). Ex- The authors have no financial conflicts of interest. cessive B cell TNF-a could, therefore, explain the induction of increased cortical HEV neogenesis resulting in increased T cell References recruitment via CCL21/CCR7 interactions and ultimate aberran- 1. Edwards, D. R., M. M. Handsley, and C. J. Pennington. 2008. The ADAM cies in B/T segregation (Fig. 1). Alternatively, Blobel et al. (34) metalloproteinases. Mol. Aspects Med. 29: 258–289. 2. Le Gall, S. M., P. Bobe´, K. Reiss, K. Horiuchi, X. D. Niu, D. 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