Multistep IgE Mast Cell Desensitization Is a Dose- and Time-Dependent Process Partially Regulated by SHIP-1

    RESEARCH ARTICLE | MARCH 15 2023 Multistep IgE Mast Cell Desensitization Is a Doseand Time-Dependent Process Partially Regulated by SHIP-1 Ather Adnan; ... et. al J Immunol (2023) 210 (6): 709–720. https://doi.org/10.4049/jimmunol.2100485 Related Content Nonspecic Desensitization, Functional Memory, and the Characteristics of SHIP Phosphorylation following IgE-Mediated Stimulation of Human Basophils J Immunol (July,2006) Characterization of RANTESand Aminooxypentane-RANTESTriggered Desensitization Signals Reveals Differences in Recruitment of the G Protein-Coupled Receptor Complex J Immunol (September,1999) P2Y6 Receptors Require an Intact Cysteinyl Leukotriene Synthetic and Signaling System to Induce Survival and Activation of Mast Cells J Immunol (January,2009) Downloaded from http://journals.aai.org/jimmunol/article-pdf/210/6/709/1587522/ji2100485.pdf by Universidad de Granada.- Biblioteca user on 11 July 2023 Multistep IgE Mast Cell Desensitization Is a Doseand Time-Dependent Process Partially Regulated by SHIP-1 Ather Adnan,* ,† Shree Acharya,*Leila A. Alenazy,* ,‡ Leticia de las Vecillas,* ,§ Pedro Giavina Bianchi, { Matthieu Picard, ‖ Lucia Calbache-Gil, #, ** Salvador Romero-Pinedo, #, ** Ana Clara Abad´ ı´a-Molina, #, ** William Kerr, †† Chiara Pedicone, †† Jun Nagai,*Eleanor Hollers,*Daniel Dwyer,*and Mariana Castells* Multistep mast cell desensitization blocks the release of mediators following IgE crosslinking with increasing doses of Ag. Although its in vivo application has led to the safe reintroduction of drugs and foods in IgE-sensitized patients at risk for anaphylaxis, the mechanisms of the inhibitory process have remained elusive. We sought to investigate the kinetics, membrane, and cytoskeletal changes and to identify molecular targets. IgE-sensitized wild-type murine (WT) and FceRIahumanized (h) bone marrow mast cells were activated and desensitized with DNP, nitrophenyl, dust mites, and peanut Ags. The movements of membrane receptors, FceRI/ IgE/Ag, actin, and tubulin and the phosphorylation of Syk, Lyn, P38-MAPK, and SHIP-1 were assessed. Silencing SHIP-1 protein was used to dissect the SHIP-1 role. Multistep IgE desensitization of WT and transgenic human bone marrow mast cells blocked the release of b-hexosaminidase in an Ag-specific fashion and prevented actin and tubulin movements. Desensitization was regulated by the initial Ag dose, number of doses, and time between doses. FceRI, IgE, Ags, and surface receptors were not internalized during desensitization. Phosphorylation of Syk, Lyn, p38 MAPK, and SHIP-1 increased in a doseresponse manner during activation; in contrast, only SHIP-1 phosphorylation increased in early desensitization. SHIP-1 phosphatase function had no impact on desensitization, but silencing SHIP-1 increased b-hexoxaminidase release, preventing desensitization. Multistep IgE mast cell desensitization is a doseand time-regulated process that blocks b-hexosaminidase, impacting membrane and cytoskeletal movements. Signal transduction is uncoupled, favoring early phosphorylation of SHIP-1. Silencing SHIP-1 impairs desensitization without implicating its phosphatase function. The Journal of Immunology, 2023, 210: 709720. Multistep IgE desensitization is achieved by dividing an activating Ag dose into multiple suboptimal doses and delivering the suboptimal doses sequentially at fixed time intervals, which blocks cell activation and inhibits the release of mediators, making mast cells refractory to optimal Ag (13). Mimicking this in vitro inhibitory process, human desensitization protocols have protected patients allergic to antibiotics, chemotherapy, mAbs, and other drugs and foods when reintroduced to their IgE-sensitizing allergens, preventing anaphylaxis and increasing their life expectancy and quality of life (49). Despite the proven safety and efficacy in thousands of highly allergic patients, breakthrough reactions and anaphylaxis are unpredictable, occurring in 25% of desensitization protocols, making the procedures high risk and hampering universal application (10). The perception that desensitization protocols remain empiric has spurred an impetus for a better understanding of the underlying inhibitory mechanisms and the pursuit of molecular targets. IgE mast cell activation is a complex and well-studied process initiated by crosslinking of IgE-sensitized FceRI receptors by multimeric Ags, which induces intracellular signaling and the acute and delayed release of inflammatory mediators (11,12). *Division of Allergy and Immunology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; † Texas A&M Health Science Center, College of Medicine, Houston, TX; ‡ Division of Allergy and Clinical Immunology, Department of Medicine, College of Medicine, King Saud University, Riyadh, Saudi Arabia; x Department of Allergy, Marqu´ es de Valdecilla University Hospital Instituto de Investigaci´ on Marques de Valdecilla, Santander, Spain; { Clinical Immunology and Allergy Division, School of Medicine, Universidade de S˜ ao Paulo, R. Prof. Artur Ramos Sao Paulo, Brazil; ‖ Division of Allergy and Clinical Immunology, Department of Medicine, Hˆ opital Maisonneuve-Rosemont, Universit´ edeMontr ´ eal, Montreal, QC, Canada; # Unidad de Inmunolog´ ıa, IBIMER, CIBM, Universidad de Granada, Granada, Spain; **Departamento de Bioqu´ ımica y Biolog´ ıa Molecular III e Inmunolog´ ıa, Facultad de Medicina, Universidad de Granada, Granada, Spain; and †† Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY ORCIDs: 0000-0001-9640-9916 (A.A.); 0000-0003-4969-5678 (L.d.l.V.); 0000-00033108-5804 (M.P.); 0000-0002-3147-3535 (A.C.A.-M.); 0000-0002-4720-7135 (W.K.); 0000-0002-9486-1746 (C.P.); 0000-0003-4720-5065 (J.N.); 0000-0002-5958-3090 (E.H.); 0000-0001-6451-0163 (M.C.). Received for publication May 25, 2021. Accepted for publication December 22, 2022. This work was supported by a postresidency contract “Wenceslao Lopez-Albo”provided by the Instituto de Investigacion Marques de Valdecilla-IDIVAL (Santander, Spain) (L.d.l.V.). L.C.-G. and S.R.-P. are Ph.D. students in the Official Doctoral Program in Biomedicine of the University of Granada. The research performed by L.C.-G., S.R.-P., and A.C.A.-M. was supported and funded by the Plan Estatal de Investigaci´ on Cient´ ıfica y T´ ecnica y de Innovaci´ on 20132016, ISCIII-Subdirecci´ on General de Evaluaci´ on y FomentodelaInvestigaci ´ on, Ministerio de Econom´ ıa y Competitividad, Spain (Grants PI16/01642 and PI10/01096), Plan Propio de la Universidad de Granada, Spain 2014 (Grant PP2014.01). Address correspondence and reprint requests to Dr. Mariana Castells, Brigham and Women’s Hospital, 60 Fenwood Road, Room 5002N, Boston, MA 02115. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: 3AC, 3a-aminocholestane; Der p or Dp, Dermatophagoides pteronyssinus;hBMMC,FceRI chain humanized bone marrowderived mast cell; HSA, human serum albumin; ITAM, immunoreceptor tyrosine-based activation motif; LAMP-1, lysosomal-associated membrane protein 1; mBMMC, murine bone marrowderived mast cell; NP, nitrophenyl; SHIP1, SH2-containing inositol phosphatase 1; Tg, transgenic; WT, wild type. This article is distributed under The American Association of Immunologists, Inc., Reuse Terms and Conditions for Author Choice articles. Copyright © 2023 by The American Association of Immunologists, Inc. 0022-1767/23/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.2100485 The Journal of Immunology Downloaded from http://journals.aai.org/jimmunol/article-pdf/210/6/709/1587522/ji2100485.pdf by Universidad de Granada.- Biblioteca user on 11 July 2023 Phosphorylation of the b-andg-chain immunoreceptor tyrosinebased activation motifs (ITAMs) provides docking for upstream molecular players, leading to downstream signal transmission by second messengers, mobilization of intracellular calcium with extracellular calcium influx, and cytoskeletal movements led by actin and tubulin, whereas FceRIIgEAg complexes are internalized (13,14). We and others have shown that this well-orchestrated machinery is halted by desensitization. Bone marrowderived mast cells (BMMCs) sensitized with DNP-IgE and desensitized to an optimal dose of DNP administered as 11 sequential suboptimal doses cannot mobilize calcium, do not release granule mediators, do not generate PGs and leukotrienes from membrane arachidonic acid, and do not produce cytokines (3, 15). Although models of basophil IgE desensitization have provided some insight (16), how mast cells engage in this inhibitory pathway is poorly understood. The fundamental questions of what amount of Ag can start the desensitization process, how to increase the doses without inducing signal transduction, and the time requirements have not been defined. Intriguingly, signal transduction is impaired during the final steps of desensitization when optimal Ag is reached, indicating that the initial steps have diverted activation partners. Although Syk phosphorylation is required to initiate signal transduction, recent data suggest that phosphatases such as SH2-containing inositol phosphatase 1 (SHIP-1) associate with the FceRIIgEAg complex and compete with Syk when Ag doses are delivered in quick sequence in contrast to more prolonged times (1719). Quick Ag delivery leads to SHIP-1 activation and inhibitory signaling, indicating that the time between doses can dictate outcomes. This is consistent with the notion that the propagation of signal requires kinetic proofreading with enough receptor/ ligand dwell time to generate activated receptor complexes that can progress to distal responses. Rapidly dissociating ligands are less effective at generating distal responses. Whether SHIP interferes with signal propagation by reducing activation time is not known. The generation of signaling membrane domains with activation partners has been demonstrated, but the membrane arrangements during desensitization have not been defined (21), and the potential participation of immunoreceptor tyrosine-based inhibition motifs bearing inhibitory receptors such as gp49B1, which associate with phosphatases SHP-1, SHP-2, and SHIP-1 and dephosphorylate FceRI ITAMs, is not known (22, 23). Although internalization of FceRIIgEAg complexes is not required for signal transduction at low Ag (13), it remains controversial whether internalization can initiate desensitization and if tubulin and actin can provide cytoskeletal support to the inhibitory state (1, 2). Here, we reproduce an in vitro model of IgE activation and desensitization (3, 15) using wild-type murine (WT mBMMCs) and FceRIa-chain humanized mast cells (transgenic [Tg] hBMMCs) and provide the initial effective dose, dose progression, and time intervals for optimal Ag-specific desensitization, including inhalant and food allergens. We follow surface membrane and intracytoplasmic changes, including actin and tubulin movements and phosphorylation of Syk, Lyn, p38-MAPK, and SHIP-1, to assess the uncoupling of signal transduction. We show enhanced SHIP-1 phosphorylation during the early steps of desensitization, not associated with its phosphatase function. By silencing SHIP-1, desensitization is impaired, uncovering the first molecular target of multistep IgE desensitization. Materials and Methods Mice Male WT BALB/c 812-wk-old mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Tg BALB/c mice expressing the human FceRIa(gift from Jean-Pierre Kinet, Beth Israel Deaconess Medical Center, Boston, MA) (24, 25) were bred at the Dana-Farber Cancer Institute Animal Resource Facility. Briefly, male mFceRIa −/− mice carrying the transgene encoding hFceRI were bred to females as described previously (26). Mice were euthanized by CO 2 asphyxiation. Mast cell cultures Tg BMMCs carrying the a-chain of the high-affinity IgE receptor FceRI (Tg hBMMCa) and murine BMMCs (mBMMCs) were harvested from 812-wk-old WT murine femurs and cultured for 416 wk in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillinstreptomycin, 0.1 mM MEM nonessential amino acids (all from SigmaAldrich), and 10 ng/ml of IL-3 (3). IL-3 was obtained from supernatants of 293T cells expressing murine IL-3 (27) and from commercial sources (R&D Systems and PeproTech). All animal work was approved by the Dana-Farber Cancer Institute Animal Care and Use Committee (IACUC 2016N000552). WT mBMMCs (4 ×10 6 cells/ml) were sensitized with anti-DNP IgE (clone SPE-7; Sigma-Aldrich) at 1 mg/ml (0.25 mg/10 6 cells/ml) overnight at 37◦C. Tg hBMMCs were incubated overnight with anti-nitrophenyl (anti-NP) IgE (0.5 mg/10 6 cells/ml, clone JW8/1; Bio-Rad Laboratories) at 37◦Corwith concentrated human serum diluted at 20% in RPMI (4 ×10 6 cells/ml) for 1824hat37 ◦C. Cells were then washed twice and resuspended at 1 ×10 6 cells/50 ml RPMI without IL-3 and distributed into tubes at 37◦C for activation or desensitization. WT mBMMCs (50 ml/tube containing either 0.5 ×10 6 or 1 ×10 6 mBMMCs) were challenged with 50 mlofDNP-humanserum albumin (DNP-HSA) and Tg hBMMCs with NP-BSA (Sigma-Aldrich) at 20 pg/ml or 200 pg/ml (1 ng, 10 ng) and, for control, with 50 mlofHSAor BSA at 20 pg/ml or 200 pg/ml (1 ng, 10 ng) or with dust mites and peanut allergens and, after 10 min in 37◦C, placed on ice. LAD2 cells LAD2 cells were obtained from the National Institutes of Health (28)andcultured at 1 ×10 6 in StemPro34 serum-free medium with the StemPro media supplement (catalog no. 10639011; Life Technologies), L-glutamine (catalog no. G7513; Sigma-Aldrich), penicillin-streptomycin, and recombinant human stem cell factor (100 ng/ml, catalog no. 300-07; PeproTech). Cells were sensitized with human myeloma plasma IgE (Athens Research and Technology) at 5 mg/ml overnight at 37◦C with 5% CO 2 . Cells were then washed once with sterile HBSS and resuspended in warmed StemPro34 media (Life Technologies) (supplemented with StemPro nutrient supplement; Life Technologies), 2 mM L-glutamine (Sigma-Aldrich), 100 ng/ml recombinant human stem cell factor (PeproTech), 1% penicillin-streptomycin) at a concentration of 0.5 ×10 6 cells/ml and used in activation and desensitization experiments at 1 ×10 6 cells/ml. Human serum Informed consent was obtained from human subjects (institutional review board approval no. 2020P002043). Sera from one dust mite allergic individual (donor 1) and one peanut and dust mite allergic individual (donor 2) were concentrated by centrifugation at 4000 ×gfor 1 h in 100 kDa centrifugal filter units (Merck Millipore) and stored at −20◦C and used for sensitization of Tg hBMMCa. Donors 1 and 2 had positive skin test results, and serum allergenspecific IgE titers were determined by ImmunoCAP (Phadia). After concentration, donor 1 total IgE was 1127 ng/ml, and donor 2 total IgE was 545 ng/ml. Before concentration, donor 1 had Dermatophagoides pteronyssinus (Der p or Dp)-specific IgE of 40 kUA/L, and donor 2 had a Der p-specificIgEof 1.64 kUA/L and a peanutspecific IgE of 18.4 kU/L. Mast cell activation Dermatophagoides pteronyssinus and peanut. Tg hBMMCs were stimulated with Der p extract 30,000 AU/ml (Hollister-Stier, Spokane, WA) and/or peanut extract (1 mg/ml) (Greer, Lenoir, NC) in RPMI. Tg hBMMCs were activated as described for WT mBMMCs (Fig. 1A, Supplemental Fig. 1, and Supplemental Tables IIV). DNP and NP. WT mBMMCs (50 ml/tube containing either 1 ×10 6 or 0.5 × 10 6 BMMCs) were challenged with 50 ml of DNP-HSA and Tg hBMMCs with 4-hydroxy-3-NP-BSA (Sigma-Aldrich) at 20 pg/mlor 200 pg/ml (1 ng, 10 ng, respectively). A negative control, 50 ml of culture medium 150 mlofHSAorBSAat20pg/ml or 200 pg/ml (1 ng, 10 ng, respectively), was used. After incubation for 10 min at 37◦C, cells were placed on ice. For doseresponse experiments, Ags were diluted in RPMI 1640 culture medium. Doseresponse experiments were performed by adding 50 ml of varying Ag dilutions to 50 ml of cell suspension for 10 min at 37◦C before the suspension was placed on ice. 710 MULTISTEP MAST CELL IgE DESENSITIZATION: ROLE OF SHIP-1 Downloaded from http://journals.aai.org/jimmunol/article-pdf/210/6/709/1587522/ji2100485.pdf by Universidad de Granada.- Biblioteca user on 11 July 2023 Mast cell desensitization Dermatophagoides pteronyssinus and peanut. Tg hBMMCawere desensitized to Der p and peanut Ags in incremental steps using the protocols described in Fig. 1A, Supplemental Fig. 1, and Supplemental Tables IIV and based on a previously established protocol (15). For desensitization experiments with Der p and peanut Ags (Fig. 2B, 2C), the 11-step protocol in Supplemental Fig. 1A was used. Fig. 2A, 2B, and 2C used serum from donor 2, whereas experiments for Fig. 2D, 2E, and 2F used serum from donor 1. The target dose for activation in Fig. 2D, 2E, and 2F was 2.5 AU/ml. The protocols for experiments in Fig. 2D and 2E involved varying the number of total steps to achieve desensitization (Supplemental Fig. 1, Supplemental Tables I and II). The starting concentration of 0.02 AU/ml (Fig. 2D, 2F) was based on a doseresponse curve showing that 0.02 AU/ml did not elicit significant activation (Fig. 2A) and was similar to 0.01 AU/ml (Fig. 2E). The protocol for Fig. 2D2F is the eight-step protocol (Supplemental Fig. 1, Supplemental Table II) (i.e., the fold increase 2×protocol; Supplemental Fig. 1, Supplemental Table I). Stated 0s show unstimulated cells. For specificity experiments, cells were sensitized with donor 2 serum (dust mite and peanut allergic) and desensitized to Der p Ag and challenged with peanut Ag or desensitized to peanut AgandchallengedwithDerpAg. DNP and NP. WT mBMMCs or Tg hBMMC (50 ml/tube containing either 0.5 ×10 6 or 1 ×10 6 mBMMCs) were stimulated with increasing concentrations of DNP-HSA at 10-min intervals at 37◦C in 11 incremental doses, starting at 1 pg and reaching 1 ng (or 10 ng) as previously described (7) (Supplemental Table I), and placed on ice. The same method was used for anti-NP IgE sensitization (0.5 mg/10 6 cells/ml) and activation with 10 ng (20 pg/ml) of NP-BSA or control BSA at 10 ng (20 pg/ml) for 10 min. For desensitization, the above protocol of 11 incremental doses was used (starting dose 10 pg, targeting 10 ng total). The total time was 110 min for desensitization. Cell viability was assessed by trypan blue dye exclusion. Eleven-step activation and desensitization. For experiments evaluating activation, 11 tubes were used, each containing 50 ml/tube of either 0.5 ×10 6 or 1×10 6 mBMMCs. Each tube received a single dose of Ag in 50 ml, representing each of the 11 steps. Cells were incubated at 37◦C for 10 min, placed on ice, and processed for b-hexosaminidase release assay or flow cytometry. For experiments evaluating each step of the desensitization, 11 tubes were used (50 ml/tube containing either 0.5 ×10 6 or 1 ×10 6 mBMMCs), the first tube received one dose only, the second tube received two doses (the first dose and, 10 min later, the second dose), and each tube received one more dose than the previous tube up to the last tube, which received 11 doses added sequentially at 10-min intervals at 37◦C for a total of 110 min. 3 a -Aminocholestane. For selected experiments, mBMMCs were activated and desensitized in the presence of 3a-aminocholestane (3AC), a selective inhibitor of SHIP-1, to block phosphatase activity. The 1 ×10 6 cells condition was incubated with 10 mM, 15 mM, or 20 mMof3ACat37 ◦C(17). After 30 min, cells were washed and resuspended with 50 ml medium and activated or desensitized with 1 ng DNP-HSA or 1 ng HSA control. LAD2 cells were used to assess the effect of 3AC in activation and desensitization of human mast cells. A concentration of 15 mM 3AC or vehicle (200 proof ethanol) was added to 1.5 ×10 6 cells and incubated for 30 min at 37◦C. The cells were washed with sterile HBSS, resuspended in warmed StemPro34 media (prepared as above), and split into three tubes (0.5 million cells in 50 ml) for control, activation, and desensitization conditions. A quantity of 2 ml of lysosomalassociated membrane protein 1 (LAMP-1) Ab (clone H4A3; BioLegend, San Diego, CA) was added to each tube to assess activation and desensitization. The control cells then received 50 ml of warmed media with a final BSA dose of 1mg. The activation cells received 50ml of warmed media with a final goat anti-human myeloma IgE (polyclonal human; Chemicon) dose of 1 mg. Control and activation conditions were kept at 37◦C for 10 min before being placed on ice. The desensitization group was given anti-IgE in warmed media through 11 steps of 10-min incubations each at 37◦Cforafinal dose of 1 mg anti-IgE in 50 ml of warmed media (dose increments match Supplemental TableI,withtheonlychangebeingthefinal dose of 1 mg). After the final desensitization step, cells were washed, resuspended in FACS buffer, and collected on the FACSCanto II flow cytometer. Percentage degranulation was reported for the activation and desensitization groups on the basis of gating around 1% of LAMP-1-positive cells in the control condition. Calcium ionophore. Calcium ionophore (A23187; Sigma-Aldrich) was added to the cell suspension to reach a total volume of 100 ml and a concentration of 8mM. BMMCs were then activated or desensitized, and b-hexosaminidase was quantified. b-Hexosaminidase release assay. Activated or desensitized BMMCs were placed on ice, then centrifuged, and cell pellets were lysed by resuspension in culture medium containing 0.5% Triton X-100. b-Hexosaminidase release was assessed by a kinetic microplate ELISA reader (Molecular Devices, Sunnyvale, CA), and percentage release was calculated as described previously (3). Western blot analysis Primary Abs used, including SHIP-1 (catalog no. 2728; Cell Signaling Technology); phospho-SHIP-1 (Tyr1020) Ab (catalog no. 3941; Cell Signaling Technology); phospho-Src/Lyn (Tyr416) Ab (catalog no. 2101; Cell Signaling Technology); phospho-Syk (Tyr525/526) Ab (catalog no. 2711; Cell Signaling Technology); phospho-p38 MAPK (Thr180/Tyr182) Ab (catalog no. 9211; Cell Signaling Technology); b-actin Ab (catalog no. 4967; Cell Signaling Technology), and goat anti-rabbit IgG-HRP conjugate (catalog no. 1706515; Bio-Rad Laboratories) BMMCs (1 ×10 6 cells/condition), were lysed on ice for 20 min in radioimmunoprecipitation assay lysis buffer (100120 ml/1 ×10 6 cells). Radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology) was supplemented with 4 mM PMSF 10 ml/ml protease inhibitor mixture, 2 mM sodium orthovanadate, and 10 ml/ml phosphatase inhibitor cocktails A and B (Roche, Santa Cruz Biotechnology). Protein lysates were resuspended with LDS sample buffer 4×(Novex) containing 2.5% 2-ME heated at 70◦C for 10 min, and proteins were separated by NuPAGE 412% Bis-Tris gels and transferred to a polyvinylidene difluoride membrane or subjected to SDS-PAGE on a 412% polyacrylamide gel and transferred to a nitrocellulose membrane (Invitrogen). Membranes were blocked with 3% nonfat dry milk for 1 h at room temperature and probed with primary Abs (1:500 SHIP-1, 1:1,000 p-SHIP-1, 1:500 p-Src/Lyn, 1:300 p-Syk, 1:20,000 p-p38 MAPK, and 1:100,000 b-actin) in 3% BSA/nonfat dry milk overnight at 4◦C, washed, and probed with 1:2,000 HRP-conjugated goat anti-rabbit IgG for 46 h at room temperature. Signal detection was performed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposed to film. Densitometry and relative expression of bands was quantified using ImageJ software. Microscopy Light microscopy with toluidine blue staining. The cytoplasmic and granular morphology of 46-wk-old mature WT mBMMCs and 68-wk-old mature Tg hBMMCs was assessed by toluidine blue in previously sensitized cells with anti-DNP IgE and anti-NP IgE, which were activated or desensitized with 1 ng and 10 ng of DNP-HSA or NP-BSA and negative control HSA or BSA. After desensitization or activation, cells were washed and resuspended in cold PBS, transferred onto poly-L-lysine-coated round coverslips for 20 min at 4◦C, and fixed with 4% paraformaldehyde in PBS for 10 min at 4◦C. After three PBS washes, cells were mounted using an aqueous mounting medium (15% w/v polyvinyl alcohol, 33% v/v glycerol, 0.1% azide). Fluorescence microscopy. mBMMCs sensitized with anti-DNP IgE (1 mg/ml) were challenged and desensitized with DyLight Fluor 649-conjugated DNP (23) in a dark room using 500,000 cells/condition. Because of detection limitations, DNP was 5 ng (10 pg/ml), and the desensitization protocol was adjusted at 5×concentrations (Supplemental Table I). Cells were desensitized with 11 steps starting at 5 pg and targeting 5 ng. After activation or desensitization, cells were placed on ice and centrifuged to remove the supernatant. Cells were washed twice and resuspended in 200 ml cold PBS plus 200 ml of paraformaldehyde 4% during 15 min at room temperature, washed twice with cold PBS, and resuspended in 400 ml of PBS. A quantity of 200 ml (250,000 cells) was transferred to a 12-mm round coverslip for cytospin (10 min at 500 rpm). Coverslips were then transferred to a 24-well plate, cell side facing up, and 500 ml of blocking buffer (10% BSA) was added to each well for 30 min at room temperature. After washing,cellswereincubatedfor12hwith5ml of each Ab (PE anti-mouse LAMP-1 [0.2 mg/ml], red; FITC anti-mouse c-Kit [0.5 mg/ml], green; mouse Fc block purified rat anti-mouse CD16/CD32 [0.5 mg/ml]) in 500 ml of PBS and then washed twice with ice-cold PBS. Images were collected using a KEYENCE BZ-X700 fluorescence microscope (Keyences Corporation). Actin and tubulin confocal microscopy. After activation or desensitization, WT BMMCs were washed and transferred onto poly-D-lysine (Sigma-Aldrich)- coated chamber slides (Nunc) for 20 min at 4◦C. For F-actin staining, cells were fixed with BD Cytofix/Cytoperm solution (BD Biosciences) for 20 min, washed three times with 0.05% PBS-saponin (Merck), incubated with phalloidin-tetramethylrhodamine isothiocyanate following supplier instructions (Sigma-Aldrich) in 0.05% PBS-saponin for 40 min at 4◦C, and washed twice with 0.02% PBS-saponin and PBS. For a-tubulin staining, cells were washed and incubated with 0.2% PBS-Tween 20 (Acros Organics) with 5% goat serum (Sigma-Aldrich) and purified rat anti-mouse CD16/CD32 (BD Biosciences). Then, cells were washed with 0.2% PBS-Tween 20 and incubatedwithanti-a-tubulin (B5-1-2; Sigma-Aldrich) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) Ab in 0.2% PBS-Tween 20 containing 1% BSA. Slides were washed twice with 0.2% PBS-Tween 20 and PBS, and coverslips with DAPI mounting medium were added (Vector Laboratories). A63×objective on a Nikon A1 microscope was used, and the fluorescence intensity was measured as mean Gray value of maximum-projection images using ImageJ software. Four experiments were done as described previously The Journal of Immunology 711 Downloaded from http://journals.aai.org/jimmunol/article-pdf/210/6/709/1587522/ji2100485.pdf by Universidad de Granada.- Biblioteca user on 11 July 2023 (29), and 75100 BMMCs per condition were analyzed. b-Hexosaminidase was assessed for each condition to validate activation and desensitization. Flow cytometry Mast cell activation was assessed by measuring LAMP-1 (CD107a) surface expression. LAMP-1 (CD107a) surface expression on Tg hBMMCawas determined by adding 5 mg/ml APC anti-mouse CD107a (clone 1D4B; BioLegend) or 5 mg/ml allophycocyanin rat IgG1a, k(isotype control, BioLegend) to 50 ml of BMMC suspension before activation or desensitization (Supplemental Table II). Tg hBMMCawere placed on ice 10 min after stimulation and washed with ice-cold FACS buffer (PBS 1×containing 0.5% BSA and 0.1% sodium azide). Cells were then washed with ice-cold FACS buffer and stained for 30 min on ice: for Tg hBMMCs, 2.5 mg/ml PE antihuman IgE (clone MHE-18; BioLegend) or 2.5 mg/ml PE mouse IgG1, k (isotype control; BioLegend) and 10 mg/ml FITC anti-human FceRIa (clone AER-37; BioLegend) or 10 mg/ml FITC mouse IgG2b, k(isotype control, BioLegend); for WT mBMMCs, 5 mg/ml APC anti-mouse CD107a (clone 1D4B; BioLegend) or 5 mg/ml APC rat IgG1a, k(isotype control; BioLegend), 5 mg/ml FITC anti-mouse IgE (clone RME-1; BioLegend) or 5 mg/ml FITC rat IgG1, k(isotype control; BioLegend) and 2 mg/ml PE anti-mouse FceRIa(clone MAR-1; BioLegend) or 2 mg/ml PE anti-mouse LILRB4(cloneH1.1)or2mg/ml PE Armenian hamster IgG (isotype control; BioLegend). Activated, desensitized, or HSA-treated mBMMCs were first washed and resuspended in ice-cold 1×PBS containing 0.5% BSA and 0.05% sodium azide (FACS buffer) and then incubated for 10 min on ice (4◦C) with either anti-FcgRI/II mAb (eBioscience) or 1 mg/ml anti-mouse CD16/32 (TruStain fcX, FcgRIII; BioLegend) to block Fcgreceptors. Cells were then washed with ice-cold FACS buffer and incubated on ice with FITC anti-mouse FceRIaAb clone MAR-1 0.5 mg/ml, Brilliant Violet 510 anti-mouse CD117 (c-Kit) Ab clone 2B8 0.2 mg/ml, PerCP/cyanine 5.5 anti-ERK1/2 phospho-Thr202/Tyr204 Ab clone 6B8B69 (BioLegend), PE mouse anti-SHIP-1 clone 32/SHIP-1 0.2 mg/ml (BD Biosciences), rabbit anti-Syk (tyrosine protein kinase SYK, spleen tyrosine kinase) (Pacific Blue) clone D3Z1E 2 mg/ml (US Biological Life Sciences), and allophycocyanin phospho-Syk (Tyr348) mAb (clone moch1ct) 5 ml(0.06mg)/test (eBioscience), or with the recommended isotype controls (FITC Armenian hamster IgG isotype control Ab, clone HTK888, 0.5 mg/ml), Brilliant Violet 510 rat IgG2b, kisotype control Ab (clone RTK4530, 0.2 mg/ml), PerCP/cyanine 5.5 mouse IgG2a, kisotype control Ab (clone MOPC-173, 0.2 mg/ml) (BioLegend), PE mouse IgG1, kisotype control (clone MOPC-21, 0.2 mg/ml) (BD Biosciences), mouse IgG1 kisotype control (clone P3.6.2.8.1, 0.2 mg/ml), APC (eBioscience) for 30 min at 4◦C. Samples were read on a BD FACSCanto (BD Biosciences) or BD LSR Fortessa (BD Biosciences) flow cytometer using FACSDiva acquisition software, and data were analyzed in FlowJo analysis software (BD Biosciences, Ashland, OR). SHIP-1 knockdown with siRNA Prior to sensitization, BMMCs were electroporated with an Amaxa Nucleofector II according to the manufacturer’s instructions (Amaxa; Lonza). BMMCs were divided into four groups: nonelectroporated (NE), blank electroporation (B), electroporation with anti-SHIP-1 siRNA (S) (anti-SHIP1 siRNA 7), or electroporation with scrambled negative control siRNA (NC). The anti-SHIP1 siRNA used was against mouse inositol polyphosphate-5-phosphatase D (Mm_Inpp5d_7 FlexiTube siRNA) with detected transcripts: NM_001110192 (4937 bp), NM_001110193 (4757 bp), NM_010566 (4940 bp). For all groups, 1.2 ×10 6 FIGURE 1. Desensitization of mBMMCs induces significantly less b-hexosaminidase and maintains granule integrity. (A) Desensitization protocol adapted from Sancho-Serra et al. (3). After overnight sensitization with anti-DNP IgE, 11 doses of DNP-HSA were delivered sequentially to 1 ×10 6 BMMCs at 10-min intervals until the target dose of 1 ng for DNP-HSA was reached. For activation and HSA control, single doses were delivered to 1 ×10 6 BMMCs in separate tubes. (B) WT mBMMCs were sensitized with anti-DNP IgE (1 mg/ml). Clockwise from the top left: activated (Act) with 1 ng DNP-HSA; desensitized (Desens) to 1 ng DNP-HSA; treated with 1 ng HSA or untreated (resting). Representative images of n56 independent experiments with toluidine blue at ×100 magnification. (C) WT mBMMCs were sensitized with 0.25 mg anti-DNP IgE (per 10 6 cells/ml). The percentage of b-hexosaminidase release was calculated at each step of the 11 steps of the desensitization and corresponding activation doses. For desensitization, each step represents an additional number of doses delivered every 10 min up to 11 doses in 110 min, and for activation, each step is a single dose. The positive control was cells activated with a single 1-ng DNP-HSA dose (Act), and the negative control was 1 ng HSA, and processed at 10 and 110 min. Mean with SEM of n55 independent experiments. **p<0.01 , ****p<0.0001. 712 MULTISTEP MAST CELL IgE DESENSITIZATION: ROLE OF SHIP-1 Downloaded from http://journals.aai.org/jimmunol/article-pdf/210/6/709/1587522/ji2100485.pdf by Universidad de Granada.- Biblioteca user on 11 July 2023 BMMCs were resuspended in 92 ml of Nucleofector solution kit T. The remaining 8 ml varied, depending on the group: group NE received 8 ml of Nucleofector solution, group B received 8 ml of Nucleofector solution, group S received 8 ml of 1 nM anti-SHIP1 siRNA (catalog no. SI04919726; QIAGEN), and group NC received 8 ml of 1 nM AllStars negative control siRNA (catalog no. SI03650318; QIAGEN). Cells were electroporated using program T-030, except NE cells, incubated for 5 d for maximal SHIP-1 knockdown, and flow cytometry, Western blot, or b-hexosaminidase release assays were performed as described above. Deletion of SHIP-1 in Cre SHIP-1 fl/fl BMMCs Cells were isolated and cultured from bone marrow from Cre SHIP-1 fl/flmice (gift of William Kerr laboratory, SUNY Upstate Medical University, Syracuse, NY) as per WT BMMC protocol in RPMI medium with 10% FBS in 10 ng/ml IL-3 for 4 wk. Mature Cre SHIP1 fl/flBMMCs were incubated with TatCre recombinase (MilliporeSigma) to catalyze the site-specific deletion of SHIP-1 between the two flox sites. A total of 3 million SHP-1 Cre 1 BMMCs were split into two wells of 1.5 ml media (R10/supplemented RPMI, 1% penicillinstreptomycin, 10 ng/ml rmIL3) each, with 2.7 mM TatCre recombinase (EMD Millipore) added to one well. After 24-h incubation at 37◦C, the cells were collected, washed with HBSS, and resuspended in 1.5 ml of warmed media. Cells were then sensitized with 1 mg/ml anti-DNP IgE (clone SPE-7; Sigma-Aldrich) for 48 h at 37◦C before they were collected, washed with HBSS, resuspended in warmed media, and split into tubes for activation, desensitization, and control groups (as described previously; Supplemental Table I). A quantity of 2 ml LAMP-1 was added to each sample for the duration of activation, control, or desensitization. After 10 min on ice, cells were fixed, permeabilized, and stained intracellularly for SHIP-1. Statistical analysis GraphPad Prism 8.0 software was used to perform all statistical tests (GraphPad Software, La Jolla, CA). For all experiments, groups were analyzed using unpaired, two-tailed Student ttests, where *p<0.05, **p<0.01, and ***p<0.001 were used to determine statistical significance. The data shown in all graphs are represented as mean ± SEM. Results Rapid IgE desensitization inhibits b-hexosaminidase release while preserving granule integrity in mouse BMMCs We reproduced the Sancho-Serra et al. and Morales et al. (3, 15) protocol for IgE activation and desensitization of BMMCs with 11 doses of DNP-HAS starting at a concentration of 0.02 ng/ml (Fig. 1AC). Activated cells received one dose of Ag at each step, whereas desensitized cells received the summation of previous steps (Fig. 1A). Desensitized cells remained fully granulated after receiving the target dose of 1 ng in 11 steps, whereas activated cells with the same dose presented extensive degranulation with empty cytoplasm (Fig. 1B). With the first seven doses of activation and desensitization, whereas 9.1% of the target dose was delivered, the percentage of b-hexosaminidase release was not different from the control HSA/BSA (Fig. 1C). From steps 8 to 11, activated cells presented an incremental dose response, whereas desensitized cells remained insensitive to the increments in cumulative doses, indicating uncoupling of signal transduction (Fig. 1C). Suboptimal sequential Ag doses added during the first seven desensitization steps segregated crosslinked FceRI receptors from activation partners. We next examined inhibitory signals generated by changing the starting dose, the interval time between doses, and the dose increase at each step. Transgene hBMMC a desensitization with dust mites and peanut allergens is specific and depends on the starting dose, rate of allergen increases, and time between doses To provide evidence of the universal application of desensitization, we used Tg hBMMCs sensitized with human allergic serum and activated with dust mites and peanut allergens. A doseresponse release FIGURE 2. Desensitization of Tg hBMMCais Ag specific and depends on starting Ag dose, rate of dose increases, and time between steps. Percentage b-hexosaminidase release was calculated in Tg hBMMCasensitized with serum from a dust mite and peanut allergic donor (AC) or a dust mite allergic donor (DF) and activated with Der p or peanut allergens (Supplemental Fig. 1 and Supplemental Tables IIV) at 10-min intervals (AE)orvariabletimeintervals(F). (A)Singledoseresponse activation for Der p allergen with optimal plateau doses. (B) Activation and desensitization to peanut (target dose 1 mg/ml) and Der p (target dose 10 AU/ml) allergens. (C) Der p desensitized hBMMCastimulated with an activating dose of peanut, and peanut desensitized hBMMCastimulatedwithanactivatingdoseofDerptoshowspecificity.(D) Desensitization to Der p 2.5 AU/ml with varying starting concentrations (Supplemental Fig. 1, Supplemental Table II). (E) Desensitization to Der p 2.5 AU/ml with varying concentration increases per step (Supplemental Fig. 1, Supplemental Table I). (F) Desensitization to Der p 2.5 AU/ml with varying time intervals between steps (Supplemental Figure 1, Supplemental Table III). 1 AU 50.0025 mg/ml or 2.5 ng/ml of Derp 1. Mean with SD of four independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The Journal of Immunology 713 Downloaded from http://journals.aai.org/jimmunol/article-pdf/210/6/709/1587522/ji2100485.pdf by Universidad de Granada.- Biblioteca user on 11 July 2023 of b-hexosaminidase was seen in Tg hBMMCs sensitized with human dust mite allergic serum when activated with Der p with an optimal activation at 2.5 AU/ml and a plateau at 10 AU/ml (Fig. 2A). Tg hBMMCs were then sensitized with human serum containing dust mites and peanut IgE and activated and desensitized with dust mites and peanut Ags (Supplemental Fig. 1). Desensitization to Der p and to peanut allergens in 11 doses significantly inhibited b-hexosaminidase release (Fig. 2B) as compared with activation. Specific desensitization was shown because Tg hBMMCs desensitized to dust mites partially inhibited b-hexosaminidase release when activated by peanut allergen and Tg hBMMCs desensitized to peanut partially inhibited activation by dust mite allergens (Fig. 2C). Dust mite Ag Der p was used to establish the optimal starting dose, dose increments, and time between doses (Supplemental Tables IIV). Maximal inhibition of b-hexosaminidase release was observed when the starting concentration was 100 times lower than the target concentration (Fig. 2D), with twofold or less increases per step (Fig. 2E) and when doses were delivered at least 10 min apart (Fig. 2F). We then assessed the dynamic membrane changes associated with desensitization by following LAMP-1 and membrane receptors. Reverse surface expression of LAMP-1, hFceRI a , and hIgE during activation and desensitization LAMP-1 is translocated from the cytoplasm to the surface membrane during activation and is associated with the release of granule mediators (30). We followed LAMP-1 to assess the dynamic membrane changes associated with desensitization. LAMP-1 translocation to the membrane was maximal with 2.5 AU/ml delivered as a single activating dose (Fig. 3A, 3D, lower right panel) and was negligible in cells desensitized to Der p starting at 0.02 AU/ml and reaching 2.5 AU/ml (Fig. 3A, 3D, upper right panel). Increased translocation was seen with increasing starting doses (Fig. 3A), mirroring the release of b-hexosaminidase (Fig. 2D). In contrast, the surface expression of IgE (Fig. 3B) and FceRIa(Fig. 3C) decreased maximally with activation, indicating internalization, and minimally with desensitization, indicating surface membrane retention. Cells desensitized with a starting concentration of 0.02 AU/ml (1% of target) and a target dose of 2.5 AU/ml did not express LAMP-1 and remained hIgE/hFceRIa positive, in contrast to cells activated with 2.5 AU/ml, which expressed LAMP-1 and became hIgE/hFceRIanegative (Fig. 3D and 3E, upper right panel for desensitization and lower right panel for activation). Starting desensitization at a concentration of 0.16 AU/ml (6.4% of target concentration) induced partial expression of LAMP-1 and a reduction in the surface expression of FceRI and IgE (Fig. 3D and 3E, lower left panels), indicating internalization and a decrease in desensitization. The reverse movements of LAMP-1, hIgE, and FceRI during activation and desensitization to the same target dose were consistent with a dynamic association with divergent downstream partners induced by cumulative doses during desensitization versus single doses during activation. To assess the impact of desensitization, we looked at its specificity, the potential depletion of mediators, and membrane receptor changes. FIGURE 3. Surface expression of LAMP-1 is decreased by desensitization while IgE and FceRIaremain at the cell surface. (AC) Surface expression by quantitative flow cytometry analysis (geometric mean fluorescence intensity [MFI]) of LAMP-1, IgE, and FceRIa, respectively, on Tg hBMMCasensitized (or not) with serum from a dust mite allergic subject and subsequently desensitized or activated with Der p 2.5 AU/ml. Desensitization was performed with increasing starting concentrations of Der pasinFig.2E.(Dand E) Dot plot of surface expression of LAMP-1, IgE, and FceRIaon Tg hBMMCasensitized with serum from a dust mite allergic subject and treated with medium, desensitized with Der p 2.5 AU/ml starting at 0.02 AU/ml or 0.16 AU/ml, or activated with Der p 2.5 AU/ml. Mean with SD of n54 experiments, where *p< 0.05, **p<0.01, ***p<0.001. 714 MULTISTEP MAST CELL IgE DESENSITIZATION: ROLE OF SHIP-1 Downloaded from http://journals.aai.org/jimmunol/article-pdf/210/6/709/1587522/ji2100485.pdf by Universidad de Granada.- Biblioteca user on 11 July 2023 Desensitized mBMMCs are refractory to activation and cannot internalize Ags and surface receptors but are not depleted of granule mediators We looked at the movements of activating receptors such as KIT, which has been shown to amplify IgE-mediated signals (31), and inhibitory receptors such as gp49B and PDL-1, which have been shown to compete with IgE-mediated signals (22) to assess membrane changes beyond FceRI. gp49B1, PDL-1, and KIT were internalized during activation (Fig. 4A) but remained at the cell surface during desensitization, in contrast to LAMP-1 but similar to the fate of IgE and FceRI (Fig. 3A3C), indicating that membrane changes during desensitization extended beyond Ag/IgE/FceRI aggregates. Desensitized cells challenged with an optimal Ag dose remained hyporesponsive (Fig. 4B), indicating a prolonged inhibitory state, which was not due to the depletion of mediators, because the response to calcium ionophore A23187 was intact (Fig. 4C). Calcium ionophore A23187 complexes with extracellular calcium and allows intracellular entry, bypassing membrane receptors, through a non-IgE associated mechanism (32). To follow simultaneous membrane changes during desensitization, fluorescent labels were used (Fig. 4D and 4E). LAMP-1 (red) was detected at the cell surface of activated cells, but not on desensitized cells, and KIT (green) was detected in desensitized and resting cells, but not on activated cells (Fig. 4E). DNP-BSA Ag (purple) was internalized during activation but remained at the cell surface in desensitized and resting cells. These opposite changes correlated with changes observed with toluidine blue showing extensive degranulation in activated cells, but not in desensitized cells (Fig. 4D), and with the inhibition of b-hexosaminidase release during desensitization (Fig. 4F). Because membrane changes and the release of mediators depend on actin and tubulin movements, we analyzed the impact of desensitization on actin and tubulin protein polymerization. Tubulin and actin movements are limited in desensitized mBMMCs FceRI activation by multimeric Ags induces an increase in microtubule structures of a-andb-tubulin heterodimers and the disassembly of the cortical F-actin rings (33, 34). We observed the formation of cortical ab-tubulin microtubules in activated cells, which was significantly inhibited in desensitized cells as compared with resting and HSA-activated cells (Fig. 5A, 5C). Concomitant disassembly of FIGURE 4. Desensitization prevents internalization of Ags and surface receptors KIT, gp49B1, and PDL1 and does not deplete mediators in mBMMCs. WT mBMMCs were sensitized with anti-DNP IgE and activated or desensitized with DNP-HAS. (A) Quantitative surface expression (geometric mean fluorescence intensity [gMFI]) of LAMP-1, FceRI, PDL1, GP49B1/LILRB4, and KIT was measured by flow cytometry after activation and desensitization to 1 ng DNP-HSA. NC, negative control (HSA). (B) LAMP-1 expression (gMFI) after activation or desensitization and after desensitized mBMMCs were challenged with an additional dose of 1 ng DNP-HSA. (C) Percentage b-hexosaminidase (b-hex) release after mBMMCs were desensitized and treated with either 1 ng of DNP-HSA or calcium ionophore (CaI). (D) Light microscopy of Tg hBMMCs. Anti-NP IgE-sensitized Tg hBMMCs were activated (Act) or desensitized (Des) with 10 ng of NP-BSA (adapted from Fig. 1A for Ag dose ×10) or treated with HSA and stained with toluidine blue (original magnification ×63). (E)Fluorescence microscopy of mBMMCs sensitized with anti-DNP IgE and activated (Act) and desensitized (Desens) with 5 ng of DyLight Fluor 649-conjugated DNP or HSA. Color legend: red, PE anti-mouse LAMP-1; green, FITC anti-mouse c-Kit; purple, DyLight Fluor 649-conjugated DNP (original magnification ×40). (F) Percentage b-hexosaminidase released after activation or desensitization with 10 ng NP-BSA or 10 ng HSA. *p<0.05, **p<0.01, ***p<0.001. The Journal of Immunology 715 Downloaded from http://journals.aai.org/jimmunol/article-pdf/210/6/709/1587522/ji2100485.pdf by Universidad de Granada.- Biblioteca user on 11 July 2023 cortical F-actin occurred during activation, which was significantly inhibited during desensitization as compared with resting and HSAactivated cells (Fig. 5B, 5D). Desensitization promoted the stabilization of the cytoskeleton, limiting the movements of actin and tubulin without significant disassembly of F-actin and little formation of microtubules, likely contributing to membrane reorganization, which has been previously described and attributed to aberrant actin remodeling (1). Because activation-induced cytoskeletal changes associate with FceRIinduced signal transduction, we investigated the activation and phosphorylation of Syk, p38-MAPK, and SHIP-1 proteins. Phosphorylation of Syk, Lyn, p38-MAPK, and SHIP-1 is increased in activated mBMMCs and decreased in desensitized mBMMCs Aggregation of FceRI receptors with optimal Ag results in upstream phosphorylation of b-andg-chain ITAMs, Lyn activation, docking and phosphorylation of Syk through its two SH2 domains, and activation of enzymes and adapter molecules, leading to extracellular calcium influx and mediator release. Downstream events include the phosphorylation of p38-MAPK, which regulates nuclear transcription factors and the generation of cytokines (12, 35). Syk phosphorylation was found to be dose dependent following single activation doses of DNP-HSA (Fig. 6A). In contrast, when delivering the target dose in sequential increments in desensitization, decreased Syk and p38-MAPK phosphorylation was seen (Fig. 6B). DNP-HSAactivated mBMMCs had high Syk and p38-MAPK phosphorylation, which was significantly reduced in desensitized and HSA-activated cells (Fig. 6C, 6D), suggesting the downstream uncoupling of signal transduction. Surprisingly, SHIP-1, which has been shown to counteract Syk activation (36), was less phosphorylated in desensitization than in activation (Fig. 6E, 6F). To further understand these changes, we looked at the dynamic changes at each of the 11 steps of desensitization and corresponding activation doses. Enhanced phosphorylation of SHIP-1 in the early steps of desensitization. The phosphorylation of Syk, Lyn, p38-MAPK, and SHIP-1 increased with increasing activating doses, which correlated with the observed increases in b-hexosaminidase release (Fig. 7A and Fig. 1A). In contrast, during desensitization, phosphorylation of SHIP-1 was significantly enhanced in the first four steps (Fig. 7B, 7C, and Supplemental Fig. 3A, 3B), whereas Lyn, Syk, and p38-MAPK had no significant increase in phosphorylation. Early phosphorylation of SHIP-1 when b-hexosaminidase release was absent (Fig. 1A) was intriguing, implicating SHIP in the uncoupling of activation signals through an unknown interaction of one of its motifs (18). The NPXY domain functions include proteinprotein associations with adaptor proteins, the cytoskeleton, actin, and tubulin, among others (37). To further understand the role of SHIP-1 in desensitization, we blocked its phosphatase function and silenced the protein. Desensitization does not depend on SHIP-1 phosphatase function and is impaired by SHIP-1 protein silencing. The phosphatase activity of SHIP-1 depletes membrane lipids of phosphatidylinositol (3,4,5)- trisphosphate and prevents the activation of BTK, Akt/PKB, and the influx of extracellular calcium necessary for signal transduction. We wanted to explore if SHIP-1 phosphatase activity could be affected by early SHIP-1 phosphorylation (18) and used 3AC, a cell-permeable steroidal compound that inhibits SHIP-1 catalytic polyphosphatase activity, at doses that have previously been shown not to be cytotoxic (17). Desensitization was not significantly inhibited by the lack of SHIP-1 phosphatase function (Fig. 8A, 8B) in mouse BMMCs or human LAD2 cells, prompting a further look at other SHIP-1 FIGURE 5. a-Tubulin and F-actin movements are halted during desensitization of mBMMCs. (Aand B), Confocal images of mBMMCs stained with anti-tubulin (green) (A) or with Rh-Phalloidin (red) (B)for F-actin detection. High magnification at right. Scale bars, 50 mm. % b-Hex, % b-hexosaminidase; Act, activated mBMMCs; Desens, desensitized mBMMCs; HSA, control IgE-sensitized mBMMCs; R, resting mBMMCs. Representative pictures of one of two independent experiments. (C) Quantification of a-tubulin and (D) F-actin staining by confocal microscopy, analyzed using ImageJ software. Values indicate mean±SEM(n575100). *p<0.05, ***p<0.001. Representative data of one of two independent experiments. 716 MULTISTEP MAST CELL IgE DESENSITIZATION: ROLE OF SHIP-1 Downloaded from http://journals.aai.org/jimmunol/article-pdf/210/6/709/1587522/ji2100485.pdf by Universidad de Granada.- Biblioteca user on 11 July 2023