Dendritic Cell Migration Mast Cell-Associated TNF Promotes

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Mast Cell-Associated TNF Promotes Dendritic Cell Migration Hajime Suto, Susumu Nakae, Maki Kakurai, Jonathon D. Sedgwick, Mindy Tsai and Stephen J. Galli This information is current as of September 29, 2021. J Immunol 2006; 176:4102-4112; ; doi: 10.4049/jimmunol.176.7.4102 http://www.jimmunol.org/content/176/7/4102 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Mast Cell-Associated TNF Promotes Dendritic Cell Migration1

Hajime Suto,2*† Susumu Nakae,2* Maki Kakurai,* Jonathon D. Sedgwick,3‡ Mindy Tsai,* and Stephen J. Galli4*

Mast cells represent a potential source of TNF, a mediator which can enhance dendritic cell (DC) migration. Although the importance of mast cell-associated TNF in regulating DC migration in vivo is not clear, mast cells and mast cell-derived TNF can contribute to the expression of certain models of contact hypersensitivity (CHS). We found that CHS to FITC was significantly impaired in mast cell-deficient KitW-sh/W-sh or TNF؊/؊ mice. The reduced expression of CHS in KitW-sh/W-sh mice was fully repaired by local transfer of wild-type bone marrow-derived cultured mast cells (BMCMCs), but was only partially repaired by transfer of TNF؊/؊ BMCMCs. Thus, mast cells, and mast cell-derived TNF, were required for optimal expression of CHS to FITC. We found that the migration of FITC-bearing skin DCs into draining lymph nodes (LNs) 24 h after epicutaneous administration of FITC in naive mice was significantly reduced in mast cell-deficient or TNF؊/؊ mice, but levels of DC migration in these mutant mice increased to greater than wild-type levels by 48 h after FITC sensitization. Mast cell-deficient or TNF؊/؊ mice also exhibited Downloaded from significantly reduced migration of airway DCs to local LNs at 24 h after intranasal challenge with FITC-OVA. Migration of FITC-bearing DCs to LNs draining the skin or airways 24 h after sensitization was repaired in KitW-sh/W-sh mice which had been -engrafted with wild-type but not TNF؊/؊ BMCMCs. Our findings indicate that mast cell-associated TNF can contribute signif icantly to the initial stages of FITC-induced migration of cutaneous or airway DCs. The Journal of Immunology, 2006, 176: 4102–4112. http://www.jimmunol.org/ ontact hypersensitivity (CHS)5 is a cutaneous allergic indicates that some haptens, including FITC, can induce CHS re- flammatory response mediated by contact allergen- (i.e., sponses that are dependent on the activity of Th2 cells and which, C hapten-) specific T cells (1). A critical initial step in sen- for optimal expression, also require TNF (4–6). The potential sitization for expression of CHS is the migration of hapten-bearing roles of TNF in the sensitization phase of such responses include Langerhans cells (LCs), immature dendritic cells (DCs) located in enhancing LC or dermal DC migration (3, 7–9), as well as the the epidermis, and/or dermal DCs, into draining lymph nodes promotion of LC maturation (7). However, the important cellular (LNs) (2, 3). After completing their maturation in the LNs, the source(s) of TNF in CHS to FITC are not known. mature hapten-bearing DCs then present the Ags to naive T cells, Although mast cells represent a significant potential source of by guest on September 29, 2021 leading to induction of contact allergen-specific memory T cells in TNF (10, 11), the extent to which mast cell-associated TNF might the sensitization phase of the response (1, 2). In the subsequent contribute to the DC migration associated with the sensitization elicitation phase of CHS responses, re-exposure to the cognate phase of CHS is not yet clear. For example, studies performed by hapten results in the recruitment of allergen-specific T cells and several groups using genetically mast cell-deficient mice have other, Ag-nonspecific leukocytes to the site of cutaneous hapten shown that, under certain (but not all) experimental conditions, challenge. These recruited leukocytes then orchestrate a local in- mast cells can be required for optimal expression of CHS (12–14). flammatory response (1). In one such model, CHS to 2,4,6-trinitro-1-chlorobenzine (TNCB), Classically, CHS was regarded as a Th1/type 1 cytotoxic (Tc1) mast cell-derived TNF was required for the optimal expression of cell-mediated immune response (1). However, recent evidence in- the response at sites of hapten challenge (13). In another model, CHS to oxazolone (Ox) in ethanol, mast cell-deficient mice exhib- *Department of Pathology, Stanford University School of Medicine, Stanford, CA ited impaired migration of LCs from the epidermis during an 18-h 94305; †Atopy Research Center, Juntendo University, Tokyo, Japan; and ‡DNAX period after initial exposure to the hapten at the time of sensitiza- Research, Palo Alto, CA 94304 tion (14). Finally, Jawdat et al. (15) reported that the IgE- and Received for publication September 26, 2005. Accepted for publication January specific Ag-dependent activation of skin mast cells can promote 19, 2006. the migration of LCs to local LNs. Taken together, such studies The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance raise the possibility that one mechanism by which mast cells, and with 18 U.S.C. Section 1734 solely to indicate this fact. mast cell-associated TNF, might contribute to the development of 1 This work was supported by U.S. Public Health Service Grants (to S.J.G.) AI-23990, CHS responses is by promoting LC/DC migration from the skin at CA-72074, and HL-67674. DNAX Research is supported by Schering-Plough Re- sites of hapten exposure in the sensitization phase of the response. search Institute. In the present study, we found that both the optimal migration of 2 H.S. and S.N. contributed equally to this study. FITC-bearing DCs to local LNs during the first 24 h after the 3 Current address: Lily Research Laboratories, Eli Lilly, Indianapolis, IN 46285. epicutaneous application of the hapten, and the optimal expression 4 Address correspondence and reprint requests to Dr. Stephen J. Galli, Department of of CHS to FITC, required mast cells and mast cell-associated TNF. Pathology, Stanford University School of Medicine, L-235, 300 Pasteur Drive, Stan- ford, CA 94305-5324. E-mail address: [email protected] Moreover, mast cells and mast cell-associated TNF also were re- 5 Abbreviations used in this paper: CHS, contact hypersensitivity; LC, Langerhans quired for optimal induction of airway DC migration to thoracic cell; DC, dendritic cell; LN, lymph node; Tc1, type 1 cytotoxic T cell; TNCB, 2,4,6- LNs within 24 h of intranasal administration of FITC-OVA to trinitro-1-chlorobenzine; Ox, oxazolone; PMN, polymorphonuclear leukocyte; BM- CMC, bone marrow-derived cultured mast cell; i.d., intradermally; i.n., intranasally; mice. However, neither mast cells nor TNF were absolutely re- WT, wild type. quired for DC migration in response to FITC, and equivalent levels

of T cell sensitization, as reflected in T cell proliferation induced slides and fixed in cold acetone for 20 min. Specimens were washed in PBS in response to Ag challenge in vitro, were detected in either mast for 20 min three times and incubated in PBS containing 3% BSA (blocking cell-deficient or TNF-deficient mice at 6 days after epicutaneous solution) at room temperature for 1 h. After blocking, the specimens were incubated with PE anti-mouse I-A/I-E (M5/114.15.2; eBioscience) at 4°C sensitization to FITC. overnight, then washed with PBS containing 0.2% Tween 20 (Sigma-Al- drich) at room temperature for 1 h. The number of epidermal LCs were Materials and Methods counted by fluorescence microscopy in 10 randomly selected areas of epi- ϫ Mice dermal sheets ( 400). Completely TNF-deficient mice (C57BL/6J-TNFϪ/Ϫ mice), as well as mice Skin LC/DC migration to draining LNs expressing membrane TNF but lacking the ability to release a secreted TNF ⌬ ⌬ Naive mice were treated with 2% FITC (left ear) or vehicle alone (right form (C57BL/6J-memTNF / mice), were generated from C57BL/6 em- Ϫ Ϫ ear) as described above. Twenty-four, 48, or 72 hours after FITC treatment, bryonic stem cells (16, 17). FcR␥ / mice on the C57BL/6 background ϩ/ϩ W/W-v mice were killed for harvesting of submaxillary and brachial LNs on both were obtained from Taconic Farms. WBB6F1-Kit and -Kit mice Ϫ Ϫ Ϫ Ϫ the left (FITC-treated) and right (vehicle alone) sides, and a single-cell and C57BL/6J-TNFR1 / and -TNFR2 / mice were obtained from The suspension was prepared as described (19). After incubation with anti- Jackson Laboratory. Mast cell-deficient KitW-sh/W-sh mice on the C57BL/6 background were generously provided by Dr. P. Besmer (Molecular Biol- CD16/CD32 mAb (2.4G2; BD Pharmingen), cells were incubated with PE anti-mouse I-Ab (M5/114.15.2; eBioscience) and allophycocyanin anti- ogy Program, Memorial Sloan-Kettering Cancer Center and Cornell Uni- ϩ versity Graduate School of Medical Sciences, New York, NY) (18); the mouse CD11c (N418; eBioscience). The proportion (percentage) of FITC cells among 5000 7-aminoactinomycin D-negative, I-Abϩ ϩ KitW-sh/W-sh mice used herein were backcrossed in our laboratory for two to , CD11c cells three generations onto the C57BL/6J background. Female 6- to 10-wk-old was determined on a FACSCalibur (BD Biosciences) using CellQuest soft- mice were used in all experiments. All mice were housed at the Animal ware (BD Biosciences). Care Facilities at Stanford University Medical Center (Stanford, CA) kept Downloaded from under standard temperature, humidity, and timed lighting conditions, pro- Lung DC migration vided mouse chow and water ad libitum, and sacrificed by CO2 inhalation; Mice were treated with 20 ␮l ϫ 5 (total 100 ␮l) of 10 mg/ml FITC- all experiments were performed in compliance with the “Guide for the Care conjugated OVA (FITC-OVA) (20) intranasally (i.n.) (21). At 24 h after and Use of Laboratory Animals” prepared by the Institute of Laboratory FITC-OVA inhalation, submaxillary or thoracic LNs were collected and Animal Resources, National Research Council, and published by the Na- single-cell suspensions were prepared (19). Cells were incubated with bi- tional Academy Press (revised 1996) and the Stanford University Com- otin anti-33D1 and allophycocyanin-anti-mouse CD11c mAbs after FcR

mittee on Animal Welfare. blocking and then incubated with PE-streptavidin (BD Pharmingen). The http://www.jimmunol.org/ proportion of FITCϩ cells among 5000 7-aminoactinomycin D-negative, FITC-induced CHS CD11cϩ33D1ϩ cells was determined by FACS as described above. To analyze both DC migration in response to a hapten and the subsequent development and expression of CHS to that hapten at the same anatomical Depletion of skin LCs/DCs site, we developed in preliminary experiments a protocol in which both Depletion of skin LCs/DCs were performed as described elsewhere (22). sensitization and challenge for CHS could be performed using the ear Briefly, 0.2 g of corticosteroid cream (0.05% clobetasol-17-propionate; ␮ pinna. Mice were sensitized with a total of 80 l of 2% FITC isomer-I Glaxo) per mouse (or vehicle as a control) was applied topically to both (FITC; Sigma-Aldrich) in a vehicle consisting of acetone-dibutylphthalate sides each ear, daily for up to 5 days. After 1–5 days (see Fig. 3A) or 4 days ␮ (1:1) administered to both ears (20 l to each side of each ear). Five days (see Fig. 3B), epidermal sheets or draining LNs were harvested, respec- ␮ b after sensitization with FITC, mice were challenged with a total of 40 l tively, for quantification of LC number after anti-I-A mAb staining in the by guest on September 29, 2021 ␮ ϩ ϩ ϩ of vehicle alone to the right ear (20 l to each side) and 0.5% FITC to the epidermal sheets and for assessment of FITC MHCII CD11c DCs in ␮ left ear (20 l to each side). Each mouse was housed in a separate cage to the LNs. prevent contact with other mice after FITC challenge. Ear thickness was measured before and at multiple intervals after FITC challenge with an FITC-specific LN cell proliferation engineer’s microcaliper (Ozaki). Some mice from each group were killed at 24 h after FITC or vehicle challenge for histological analysis to quantify Mice were sensitized with 2.0% FITC on both left and right ears as de- numbers of mast cells or polymorphonuclear leukocytes (PMNs) in tolu- scribed above. Six days after FITC treatment, submaxillary LNs were col- idine blue-stained or H&E-stained, respectively, 4-␮m sections of ear skin. lected and pooled, single-cell suspensions were prepared, and the LN cells Cells were quantified by light microscopy at ϫ400, by observers not aware were cultured in the presence or absence of 40 ␮g/ml FITC at 37°C for of the identity of the individual specimens. Mast cells and PMNs were 72 h. Proliferation was assessed by pulsing with 0.25 ␮Ci [3H]thymidine counted in the entire area of dermis present in a strip of skin extending (Amersham Bioscience) for 6 h, harvesting the cells using a Harvester 96 6.6 Ϯ 0.3 mm in length from the base to the tip of the ear pinna (repre- Mach IIIM (Tomtec), and measuring incorporated [3H]thymidine using the senting 1.25 Ϯ 0.07 mm2 of dermis); mast cells also were counted sepa- Micro ␤ System (Amersham Bioscience). rately in 10 randomly selected areas of the dermis (ϳ0.625 mm2 in total area) in the central part of the ear pinna (representing the area in which Quantification of numbers of DCs resident in the lung by FACS bone marrow-derived cultured mast cells (BMCMCs) had been injected intradermally (i.d.) in the BMCMC-engrafted KitW-sh/W-sh mice). All data Single-cell suspensions of lungs were prepared as described elsewhere are expressed as mast cells per mm2 of dermis. (23). Lung DCs were stained with FITC-anti-mouse CD11c (N418; eBio- science), PE-anti-mouse I-Ab (M5/114.15.2; eBioscience), and biotin-anti- Preparation of BMCMCs and local mast cell engraftment of mouse 33D1 (33D1; eBioscience) after FcR blocking. The cells were then incubated with allophycocyanin-streptavidin (BD Pharmingen) and 7-ami- mast cell-deficient mice ϩ noactinomycin D-negative, I-Ab highCD11chigh33D1 cells were counted BMCMCs were obtained by culturing bone marrow cells in WEHI-3-con- on a FACSCalibur (BD Biosciences) using CellQuest software (BD ditioned medium (containing IL-3) for 4–6 wk, at which time Ͼ98% of the Biosciences). cells were identified as mast cells by toluidine blue staining and by flow cytometry analysis. For mast cell reconstitution studies, BMCMCs were Quantification of numbers of lung mast cells injected i.d. (1.3 ϫ 106 cells in 40 ␮l/ear for studies of LC/DC migration Single-cell suspensions of lungs were prepared as described above and and CHS) or i.v. (1.0 ϫ 107 cells for studies of lung DC migration) in 4- mast cells were quantified after 2 ϫ 105 to 6-wk-old KitW/W-v mice or KitW-sh/W-sh mice. The mice were used for DC of these cells were cytospun onto migration experiments 6 wk (in the setting of CHS) or 8 wk (in airway glass slides. The glass slides were air-dried overnight, fixed in methanol for studies) after transfer of BMCMCs. 5 min, and then incubated with blocking buffer (Protein Block Serum-Free, Ready-to-Use; DakoCytomation) at room temperature for 1 h. After re- LC/DC migration: LC/DC release from epidermis. moval of blocking buffer, the specimens were incubated with rhodamine- conjugated avidin (Vector Laboratories) in DakoCytomation Ab diluent Naive mice were treated with 2% FITC (left ear) or vehicle alone (right with background-reducing components (DakoCytomation) at room tem- ear) as described above. Twenty-four hours after FITC treatment, mice perature for 4 h. The specimens were then washed five times in PBS at were killed and ear skin was harvested and incubated in PBS containing 20 room temperature for 30 min each time. Rhodamine-positive cells (i.e., mM EDTA at 37°C for 2 h. Epidermal sheets then were collected on glass mast cells) were counted by fluorescence microscopy. 4104 MAST CELL TNF PROMOTES DC MIGRATION

Statistics sites (Fig. 1D). By contrast, the responses elicited by hapten chal- W-sh/W-sh The Student t test (two-tailed), paired or unpaired, as appropriate, was used lenge in the congenic mast cell-deficient Kit mice devel- for statistical evaluation of the results; unless otherwise specified, all re- oped only slightly, albeit significantly, greater swelling than the sults are presented as mean ϩ or Ϯ SEM. control reactions elicited by vehicle (Fig. 1A) and exhibited significantly reduced numbers of PMNs compared with the corre- Results sponding values in the WT mice (Fig. 1D). TNFϪ/Ϫ mice on the Optimal expression of CHS responses to FITC is both mast cell C57BL/6 background also exhibited weak CHS responses to FITC, and TNF dependent with minimal levels of tissue swelling (Fig. 1A) and significantly Wild-type (WT) C57BL/6 mice developed robust CHS responses lower than WT levels of PMN infiltration (Fig. 1D). W-sh/W-sh to FITC, as assessed by tissue swelling at the site of hapten chal- As shown in Fig. 1, B and C, the ear pinnae of Kit mice, lenge (Fig. 1A), and also exhibited infiltration of PMNs at these which were the sites of sensitization and challenge for CHS, were profoundly mast cell-deficient (containing no detectable dermal mast cells), whereas levels of mast cells at this site in the TNFϪ/Ϫ mice were statistically indistinguishable from those in the WT animals. In the central part of the ear pinnae, mast cell counts per mm2 of dermis were nearly identical in WT and TNFϪ/Ϫ mice (Fig. 1B), whereas, over the entire length of the ear pinnae, numbers of mast cells per mm2 were slightly lower in the TNFϪ/Ϫ mice

than in the WT mice, a difference that did not achieve statistical Downloaded from signiﬁcance (Fig. 1C). To determine whether the abnormalities in CHS in KitW-sh/W-sh mice reﬂected the lack of mast cells in these animals, as opposed to other consequences of their c-kit mutations, we tested KitW-sh/W-sh mice that had been selectively engrafted with mast

cells in the ear pinnae. We found that engraftment with WT http://www.jimmunol.org/ BMCMCs fully restored CHS reactivity in KitW-sh/W-sh mice, as judged by tissue swelling over the course of the reaction (Fig. 1A) or by PMN infiltration at 24 h after hapten challenge (Fig. 1D). However, KitW-sh/W-sh mice that had been engrafted with TNFϪ/Ϫ BMCMCs exhibited only partial reconstitution of both the tissue swelling response associated with CHS to FITC (to ϳ50% of the WT level) (Fig. 1A) and only minimal enhancement of the PMN infiltration associated with the response compared with the levels observed in mast cell-deficient KitW-sh/W-sh mice (Fig. 1D). by guest on September 29, 2021 Engraftment of KitW-sh/W-sh mice with TNFϪ/Ϫ BMCMCs resulted in numbers of mast cells in the central part of the ear dermis (the site injected with BMCMCs) that were statistically indistinguishable from those in WT mice or in KitW-sh/W-sh mice that had been engrafted with WT BMCMCs (Fig. 1B). As expected, when measured over the entire length of the ear pinna, numbers of mast cells per mm2 of dermis were substantially lower in the WT BMCMC- or TNFϪ/Ϫ BMCMC-engrafted KitW-sh/W-sh mice than in the WT or TNFϪ/Ϫ mice; mast cells per mm2 also were slightly FIGURE 1. Mast cells and TNF are required for the optimal expression lower in the KitW-sh/W-sh mice that had been engrafted with WT as of FITC-induced CHS responses. A, Tissue swelling responses to vehicle opposed to TNFϪ/Ϫ BMCMCs, but this difference was not statis- Ϫ Ϫ or FITC in FITC-sensitized C57BL/6 WT mice, TNF / mice, mast cell- tically significant (Fig. 1C). W-sh/W-sh W-sh/W-sh deficient Kit mice, and Kit mice that had been selectively Taken together, these findings show that, under the conditions Ϫ/Ϫ repaired of their mast cell deficiency by the i.d. injection of WT or TNF tested in our experiments, mast cells, and mast cell-associated BMCMCs. The first and second numbers in parentheses show total number TNF, are required for optimal expression of CHS to FITC. These of mice in each group in all measurements up to 24 h and at all intervals after 24 h, respectively; the numeral after # is the total number of inde- results are in accord with those of Biedermann et al. (13), who pendent experiments performed that included mice in that group. Ear swell- showed that mast cell-derived TNF contributes to optimal expres- ing responses are shown for vehicle-treated right ears and FITC-challenged sion of CHS reactions to TNCB. left ears. ϩ, ϩϩ, ϩϩϩ ϭ p Ͻ 0.05, Ͻ 0.01, or Ͻ0.001 vs corresponding ϭ p Ͻ 0.001 or Ͻ0.0001 vs Mast cells, and mast cell-associated TNF, are required for ءءءء ,ءءء ;h values for vehicle-treated ears 24 ϭ p Ͻ 0.05, Ͻ 0.01, or optimal DC migration to LNs during the first 24 h of the ءءءء ,ءء ,ء ;h values for KitW-sh/W-sh mice 24 Ͻ0.0001 for the 24 h comparisons shown in parentheses; NS (p Ͼ 0.05). sensitization phase of CHS to FITC B–D, Four mice from each group shown in A were killed for assessment of Skin LCs can firmly adhere to keratinocytes through interactions 2 mast cell numbers, expressed as number per mm of dermis, in the central of adhesion molecules such as E-cadherin (24). After epicutaneous part of the ear pinna (B, Central Site) or for numbers of mast cells (C, exposure to haptens, such adhesion molecule expression is down- Entire Ear) or PMNs (D) in a strip of FITC-challenged skin extending from ϭ p Ͻ 0.05, 0.01, or regulated and LCs can then migrate from the epidermis. We found ءءءء ,ءء ,ء .the base to the tip of the ear pinna Ͻ0.0001 vs corresponding values for WT mice; ††, ††† ϭ p Ͻ 0.01 or that the numbers of LCs in epidermal sheets were significantly Ͻ0.001 vs corresponding values for KitW-sh/W-sh ϩ WT BMCMCs mice; reduced (by Ͼ30%) 24 h after epicutaneous application of FITC to ‡‡ ϭ p Ͻ 0.01 vs corresponding values for TNFϪ/Ϫ or KitW-sh/W-sh ϩ the ear pinnae of WT C57BL/6 mice, compared with the corre- TNFϪ/Ϫ BMCMCs mice; NS (p Ͼ 0.05). sponding values after vehicle treatment (Fig. 2A). In accord with The Journal of Immunology 4105 these findings, application of the sensitizing dose of FITC to WT deplete epidermal LCs at sites of FITC application. We found that C57BL/6 mice also resulted in the appearance of many FITCϩ epicutaneous treatment of the skin with corticosteroids (ϳ0.2gof DCs in the local LNs, whereas LNs draining the contralateral sites corticosteroid cream (0.05% clobetasol-17-propionate) per that had been challenged with vehicle contained very few FITCϩ mouse), topically to both sides of the ear skin once per day for 1–5 DCs (Fig. 2, B and C). consecutive days (22) resulted in nearly complete depletion of skin In mice exposed epicutaneously to high levels of FITC, some LCs by 3 days after initiation of corticosteroid treatment (Fig. 3A). FITC can enter the lymphatic or systemic circulation and thereby At 24 h after application of FITC to the ear skin of corticosteroid- reach DCs already present in the LNs (25). To assess whether the or vehicle-treated mice on day 3 after initiation of treatment, there levels of FITC used in this study might influence the numbers of were significantly fewer FITCϩ DCs in draining LNs of the LC/ FITCϩ DCs we detected in the LNs draining sites of FITC appli- DC-corticosteroid-depleted vs vehicle-treated mice (Fig. 3B). cation, we used an approach described by Grabbe et al. (22) to Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Depletion of epidermal LCs by topical treatment with a corticosteroid is associated with reduced numbers of FITCϩ DCs in the draining LNs after epicutaneous application of FITC. A, Corticosteroid cream was applied to both sides of the ear skin as described in Materials FIGURE 2. Mast cells and TNF are required for optimal migration of and Methods. Before beginning treatment with corticosteroids (day 0) and FITC-bearing LCs/DCs to draining LNs at sites treated epicutaneously at the time intervals shown after initiation of corticosteroid treatment, the with FITC. A, At 24 h after FITC or vehicle treatment, ear skin epidermal numbers of I-Abϩ LCs in epidermal sheets were counted after their staining sheets and submaxillary and brachial LNs were collected as described in with an anti-I-Ab mAb. Data show the average percentage Ϯ SEM of LCs Materials and Methods. The number of I-Abϩ LCs in epidermal sheets vs the corresponding baseline value on day 0 (100%); n ϭ 3 mice/group. were counted in C57BL/6 WT mice (n ϭ 6), TNFϪ/Ϫ mice (n ϭ 6), mast B, At 4 days after initiation of daily corticosteroid (or vehicle) treatment, cell-deficient KitW-sh/W-sh mice (n ϭ 6), and KitW-sh/W-sh mice that had been 2.0% FITC (on skin of left ear) or vehicle alone (on skin of right ear) were selectively repaired of their mast cell deficiency by the i.d. injection of WT applied epicutaneously. At 24 h after FITC or vehicle treatment, the drain- (n ϭ 6) or TNFϪ/Ϫ (n ϭ 6) BMCMCs. B and C, The proportion (percent- ing LNs were collected and the proportion (percentage) of I-Ab high age) of all I-Ab high CD11chigh cells that were FITCϩ (i.e., FITCϩ DCs) CD11chigh cells which were FITCϩ (i.e., FITCϩ DCs) was determined by were determined by FACS analysis in C57BL/6 WT mice (n ϭ 23), FACS analysis as shown in Fig. 2, B and C. Results shown are represen- TNFϪ/Ϫ mice (n ϭ 11), mast cell-deficient KitW-sh/W-sh mice (n ϭ 16), and tative of data obtained from individual mice in two independent experi- KitW-sh/W-sh mice that had been selectively repaired of their mast cell de- ments, each of which gave similar results; data are from one mouse which ficiency by the i.d. injection of WT (n ϭ 8) or TNFϪ/Ϫ (n ϭ 6) BMCMCs. had not been treated with corticosteroids (A) and two mice which had been B, Representative flow cytometry data for individual mice (numbers ϭ treated with corticosteroids (B and C). Based on comparison to the mean percent of FITCϩ cells among I-Ab high CD11chigh DCs). Shaded areas rep- level of fluorescence intensity of FITC on MHCIIϩCD11cϩ DCs in the resent LNs draining vehicle-treated ears; solid lines represent LNs draining LNs draining the FITC-treated ears in mice which had not received corti- FITC-treated ears. A and C, data are mean ϩ SEM of values for individual costeroid treatment, the level of fluorescence intensity of FITC on mice; ϩϩ, ϩϩϩ, ϩϩϩϩ ϭ p Ͻ 0.01, 0.001 or Ͻ0.0001 vs correspond- MHCIIϩCD11cϩ DCs in the LNs draining FITC-treated ears in mice ing values for vehicle-treated ears (A) or vs LNs draining vehicle-treated which had received corticosteroid treatment was significantly reduced (per- ϭ p Ͻ 0.05 or Ͻ0.0001 vs FITC-treated ear skin (A)or cent of FITCϩ DCs in LNs (mean Ϯ SEM) draining ear skin not treated ءءءء ,ء ;(ears (C vs LNs draining FITC-treated ears (C)ofKitW-sh/W-sh mice; ‡, ‡‡, ‡‡‡‡ ϭ with corticosteroids ϭ 43.4 Ϯ 2.5% (n ϭ 6) vs corticosteroid-treated ear p Ͻ 0.05, Ͻ0.01, or Ͻ0.0001 vs FITC-treated ear skin (A)orvsLNs skin ϭ 2.3 Ϯ 1.3 (n ϭ 6), p Ͻ 0.0001). In the mice in the experiments draining FITC-treated ears (C)ofTNFϪ/Ϫ mice; and ¶, ¶¶, ¶¶¶¶ ϭ p Ͻ shown in B, the LC number (mean Ϯ SEM) was, for ear skin not treated 0.05, Ͻ0.01, or Ͻ0.0001 vs FITC-treated ear skin (A) or vs LNs draining with corticosteroids: 590 Ϯ 34 cells/mm2 (n ϭ 6), and, for corticosteroid- FITC-treated ears (C)ofKitW-sh/W-sh mice ϩ TNFϪ/Ϫ BMCMCs. treated ear skin: 19.2 Ϯ 7.2 (n ϭ 6), p Ͻ 0.0001. 4106 MAST CELL TNF PROMOTES DC MIGRATION

FIGURE 4. CHS responses to FITC, and FITC-induced DC migration, in WBB6F1 WT mice, mast cell- deficient KitW/W-v mice and WT BMCMC-engrafted KitW/W-v mice. A, Ear swelling responses for vehicle- treated right ears and FITC-challenged left ears. The first and second numbers in parentheses show total number of mice in each group used for all measurements up to 24 h and at all intervals after 24 h, respectively; the numeral after # is the total number of independent experiments performed using mice in that group. ϩϩ, ϩϩϩϩ ϭ p Ͻ 0.01 or Ͻ0.0001 vs corresponding 24 h ϭ p Ͻ 0.001 ءءءء ,ءءء ;values for vehicle-treated ears or Ͻ0.0001 for the 24 h comparisons shown in brackets; NS ϭ not significant (p Ͼ 0.05). B, Migration of FITCϩ DCs was assessed, and the data expressed, as in Fig. 2, ϭ p Ͻ 0.05 or Ͻ0.01 vs corresponding ءء ,ء .B and C values from LNs draining the FITC-treated ears of KitW/ W-v mice). For all individual groups of mice, the values for LNs draining the FITC-challenged vs corresponding vehicle-treated sites were significantly different at the p Ͻ 0.001 level. Downloaded from

The simplest interpretation of all of our ﬁndings is that most or KitW/W-v mice were repaired in animals that had undergone local all of the FITCϩ DCs, which we detected in draining LNs after selective repair of their cutaneous mast cell deﬁciency (Fig. 4).

epicutaneous application of FITC to the ear pinnae, were those TNF can express biological functions in either its membrane- http://www.jimmunol.org/ which originally had been resident in the skin as LCs or dermal associated or its secreted form (17) and via interactions with cells DCs and then migrated to the LNs, rather than DCs originally bearing TNFR1 and/or TNFR2 (31). The findings shown in Fig. 5 resident in the LNs, which acquired FITC via the systemic distri- indicate that C57BL/6J-memTNF⌬/⌬ mice, which are defective in bution or local lymphatic drainage of the hapten. their ability to produce the secreted form of TNF, are able to sus- The migration of LCs 24 h after application of FITC, as reflected tain apparently normal levels of DC migration to LNs within the in the reduced numbers of LCs in epidermal sheets, was pro- first 24 h or FITC application, and that, in this setting, TNF acts via foundly impaired in mast cell-deficient KitW-sh/W-sh mice and, to a TNFR1 to a significantly greater extent than via TNFR2. somewhat lesser extent, in TNFϪ/Ϫ mice (Fig. 2A). Consistent Taken together, our findings in the FITC-induced model of CHS with these data, numbers of FITCϩ DCs in draining LNs 24 h after show that, under the conditions tested in our experiments, mast by guest on September 29, 2021 hapten application also were markedly reduced in mast cell-defi- cells, and mast cell membrane-associated TNF, were required for cient KitW-sh/W-sh or TNFϪ/Ϫ mice (by mean values, in comparison optimal migration of hapten-bearing cutaneous DCs to local drain- to the corresponding levels in WT mice, of 49 or 54%, respec- ing LNs within the first 24 h of initial application of the hapten, as tively, Fig. 2, B and C). well as for optimal expression of the elicitation phase of the CHS Local transfer of BMCMCs did not significantly influence the response to this hapten. In contrast, detectable, albeit quite weak, number of LCs in the epidermis at the site of mast cell engraftment (LC numbers per mm2 (mean Ϯ SEM) were, for KitW-sh/W-sh mice: 533 Ϯ 14 (n ϭ 5), for KitW-sh/W-sh mice ϩ WT BMCMCs: 474 Ϯ 31 (n ϭ 3) and, for KitW-sh/W-sh mice ϩ TNFϪ/Ϫ BMCMCs: 486 Ϯ 25.3 (n ϭ 3)). However, we found that local engraftment of KitW-sh/W-sh mice with WT BMCMCs fully restored WT levels of hapten-induced release of LCs from the epidermis and appearance of FITCϩ DCs in draining LNs, as assessed 24 h after hapten application (Fig. 2). By contrast, KitW-sh/W-sh mice that had been engrafted with TNFϪ/Ϫ BMCMCs exhibited significantly lower levels of both hapten-induced release of LCs from epidermis and numbers of FITCϩ DCs in local LNs (Fig. 2). Indeed, numbers of ϩ W-sh/W-sh FITC DC in draining LNs from Kit mice that had been FIGURE 5. Membrane-associated TNF and TNFR1 are important for Ϫ/Ϫ engrafted with TNF BMCMCs were only slightly higher than optimal skin DC migration in response to the application of FITC. The Ϫ Ϫ those in KitW-sh/W-sh or TNF / mice, differences that did not migration of FITCϩ DCs was assessed, and the data expressed, as in Fig. 2, B achieve statistical significance (Fig. 2, B and C). and C. The proportion of I-Ab high CD11chigh FITCϩ cells (i.e., % FITCϩ DCs) Ϫ Ϫ Two types of mast cell-engrafted genetically mast cell-deficient was determined by FACS analysis in C57BL/6 WT mice, TNF / mice, ⌬/⌬ Ϫ/Ϫ Ϫ/Ϫ mice can now be used for studies of mast cell function in vivo: memTNF , TNFR1 , and TNFR2 mice. The numbers in parentheses W-sh/W-sh W/W-v show total number of mice in each group; the numeral after # is the total C57BL/6-Kit mice (26, 27) and WBB6F1-Kit mice ϩ number of independent experiments performed using mice in that group. Data (28–30). We found that both the migration of FITC DCs to drain- p Ͻ 0.01 vs corresponding ,ءء .are mean ϩ SEM of values for individual mice ing LNs within 24 h of initial sensitization with FITC, and the values for LNs draining the FITC-treated ears of TNFϪ/Ϫ mice; ‡, p Ͻ 0.05 vs expression of CHS reactions to FITC in sensitized animals, were corresponding values for LNs draining the FITC-treated ears of TNFR1Ϫ/Ϫ W/W-v also markedly reduced in WBB6F1-Kit mast cell-deficient mice. For all individual groups of mice, the values for LNs draining the mice in comparison to values in the congenic WT (i.e., WBB6F1- FITC-challenged vs corresponding vehicle-treated sites were significantly ϩ/ϩ Ͻ Kit ) mice; moreover, both of these abnormalities in WBB6F1- different at the p 0.001 level. The Journal of Immunology 4107

CHS reactions to this hapten occurred in the virtual absence of These results indicate that the initial impairment of DC migra- mast cells or in mice that completely lacked TNF (Fig. 1). Simi- tion to local LNs observed in the absence of mast cells or TNF larly, some migration of FITCϩ DCs from the skin to local drain- eventually resolves, presumably by the engagement of mast cell- ing LNs was elicited within 24 h of FITC application in mast cell- or TNF-independent mechanisms. To assess whether the delay in or TNF-deficient mice, or in mice that contained only mast cells DC migration observed in mast cell-deficient or TNFϪ/Ϫ mice re- that were unable to produce TNF (Fig. 2). sulted in significantly impaired expansion of populations of hap- Thus, while our results clearly show that mast cells and mast ten-specific memory T cells, we harvested draining LNs 6 days cell-associated TNF can significantly enhance both the FITC-in- after FITC sensitization and examined the proliferation induced in duced migration of hapten-bearing DCs upon initial hapten expo- such LN cells in the presence of FITC. We found essentially iden- sure and the robust expression of CHS upon subsequent hapten tical levels of FITC-specific LN T cell proliferation in cells derived challenge of the sensitized mice, neither mast cells nor TNF are from wild-type, KitW-sh/W-sh,orTNFϪ/Ϫ mice (Fig. 6C). Similarly, absolutely required for either the FITC-induced DC migration no differences among the responses of wild-type, KitW-sh/W-sh,or (Figs. 2 and 4) or expression of CHS at sites of hapten challenge TNFϪ/Ϫ mice were detected when FITC-induced proliferation was in sensitized mice (Figs. 1 and 4). Nakae et al. (9) previously measured in draining LN T cells obtained from these mice at 5 reported that DC migration from the skin to draining LNs was days after the onset of sensitization with FITC, in experiments in impaired in TNFϪ/Ϫ mice at 24 h after epicutaneous application of which various numbers of T cells were challenged with any of FITC, but that the levels of DC migration in TNFϪ/Ϫ mice later multiple different concentrations of FITC (data not shown). caught up with those observed in wild-type mice. We found that Thus, under the conditions of FITC sensitization examined, a Ϫ Ϫ TNF / or KitW-sh/W-sh mice, which showed significantly impaired deficiency in mast cells or TNF delayed the migration of hapten- Downloaded from DC migration at 24 h after FITC treatment, exhibited significantly bearing DCs to the local LNs but this did not significantly influ- increased levels of DCs in the draining LNs, compared with cor- ence the expansion of hapten-specific memory T cells during the responding levels in wild-type mice, at 48 h after FITC treatment sensitization phase of FITC-induced CHS, at least as assessed 5 or (Fig. 6, A and B). At 72 h after FITC treatment, mice of all three 6 days after the initial application of hapten. genotypes (WT, TNFϪ/Ϫ,orKitW-sh/W-sh) exhibited very similar The mechanism(s) by which mast cell-associated TNF can in-

levels of FITC-bearing DC in the draining LNs (Fig. 6, A and B). ﬂuence the early stages of DC migration remain to be elucidated. http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Delayed DC migration and normal FITC- specific LN responses in KitW-sh/W-sh and TNFϪ/Ϫ mice. The migration of FITCϩ DCs at 48 and 72 h after FITC treatment was assessed, and the data expressed, as in Fig. 2, B and C. A and B, The proportion of I-Ab high CD11chigh FITCϩ cells (i.e., % FITCϩ DCs) was determined by FACS analysis in C57BL/6 wild-type (n ϭ 5), KitW-sh/W-sh (n ϭ 5) and TNFϪ/Ϫ (n ϭ 5) mice. A, Representative FACS data from one mouse of each group are shown. The upper (in bold) and lower numbers are the percentage of FITCϩ cells among the I-Ab high CD11chigh cells derived from LNs draining FITC- vs vehicle-treated ears, respectively. Shaded areas represent LNs draining vehicle-treated ears; solid lines represent LNs draining FITC-treated ears. B, Columns (Ⅺ, cells from LNs draining vehicle-treated ears; f, cells from LNs draining FITC-treated ears) show the mean ϩ SEM of values for individual mice. †, p Ͻ 0.05, ††, p Ͻ 0.005 vs corresponding values for LNs draining the FITC-treated ears of wild-type mice. For all individual groups of mice, the values for LNs draining the FITC-challenged vs corresponding vehicle-treated sites were significantly different at the p Ͻ 0.001 level. C, FITC-specific LN T cell proliferation responses in C57BL/6 wild-type (n ϭ 5), KitW-sh/W-sh (n ϭ 5), and TNFϪ/Ϫ (n ϭ 5) mice; data are shown as mean Ϯ SEM. 4108 MAST CELL TNF PROMOTES DC MIGRATION

FIGURE 7. CHS responses to FITC, and FITC-induced DC migration, in C57BL/6 WT and C57BL/6-FcR␥Ϫ/Ϫ mice. A, Ear swelling responses for vehicle-treated right ears and FITC-challenged left ears. The ﬁrst and second numbers in parentheses show total number of mice in each group used for all measurements up to 24 h and at all intervals after 24 h, respectively; the numeral after # is the total number of independent experiments performed using mice in that group. Data for WT C57BL/6 mice are from Figs. 1A and 2C. B, Migration of FITCϩ DCs was assessed, and the data expressed, as in Fig. p Ͻ0.01 vs corresponding 24 h values for wild-type ,ءء ;A, ϩϩϩ, ϩϩϩϩ ϭ p Ͻ 0.001 or Ͻ0.0001 vs corresponding 24 h values for vehicle-treated ears .2 (WT) mice; NS, (p Ͼ 0.05). B, ϩϩϩϩ ϭ p Ͻ 0.0001 vs corresponding values for vehicle-treated ears. Downloaded from

We considered the possibility that, in CHS to FITC, as in CHS to mice used in our studies of FITC-induced CHS (Fig. 9, A and B). Ox administered in ethanol (14), activation of mast cells via the In accord with our ﬁndings in the CHS model, the appearance of FcR␥ chain can contribute to the ability of mast cells to enhance FITCϩ DCs in the local (i.e., thoracic) LNs 24 h after airway the early stages of DC migration. We found that FcR␥ chainϪ/Ϫ challenge with hapten was signiﬁcantly impaired, in comparison to

mice did exhibit a defect in their ability to express tissue swelling values in WT mice, in mice deficient in mast cells, TNF or TNFR1, http://www.jimmunol.org/ during the elicitation phase of CHS to FITC (Fig. 7A), a result that and in mast cell-deficient mice engrafted with TNFϪ/Ϫ BMCMCs, is in accord with our findings with FcR␥ chainϪ/Ϫ mice in CHS to but not in mice defective in their ability to produce secreted TNF Ox (14). However, we detected no impairment of the ability of (i.e., memTNF⌬/⌬ mice) or lacking TNFR2, or in mast cell-defi- FcR␥ chainϪ/Ϫ mice to exhibit FITC-induced migration of FITCϩ cient mice engrafted with WT BMCMCs. In accord with findings DCs to draining LNs in the first 24 h of the sensitization phase of reported in our prior work (26, 35, 36), the numbers of mast cells the response (Fig. 7B). Taken together, these results indicate that in the lungs of naive mast cell-deficient mice (i.e., mice not chal- the requirements for FcR␥ chain (and, by implication, Ab) func- lenged i.n. with FITC-OVA) which had received i.v. transfer of tion in various components of the sensitization and elicitation BMCMCs were significantly higher than those in naive WT mice Ϫ Ϫ phases of CHS may vary, depending on the specific hapten tested (Fig. 9C). Moreover, in naive FcR␥ chain / mice, the lungs con- by guest on September 29, 2021 and/or on other conditions of the experiment. tained a percentage of I-Abϩ33D1ϩCD11cϩ DCs (among all lung The same also appears to be true with respect to the roles of cells) which were significantly greater than those in naive WT mast cells in the expression of CHS responses. Studies using ge- mice (Fig. 9D). netically mast cell-deficient KitW/W-v mice have shown that, with some hapten/vehicle combinations, mast cells significantly contribute to the expression of various aspects of the responses (12– Discussion 14). By contrast, with other hapten/vehicle combinations, no sig- We found that, in naive mice, mast cells and mast cell-associated nificant contributions of mast cells have been detected (32, 33). TNF were required for optimal migration of hapten-bearing DCs These findings suggest that the roles of mast cells in influencing from the skin or airways to local LNs within the first 24 h of different aspects of CHS responses may vary significantly depend- epicutaneous or airway administration of FITC or FITC-OVA, re- ing on the hapten and/or conditions of sensitization and challenge spectively. We also found that migration of FITCϩ DCs 24 h after tested. application of FITC or FITC-OVA was normal in mice which were defective in their ability to secrete TNF, strongly indicating that Mast cells, and mast cell-associated TNF, are required for the membrane-associated form of TNF fully or largely accounted optimal DC migration to LNs during the first 24 h after i.n. for the TNF-dependent effects on DC migration in these settings. administration of FITC-OVA Mast cells and mast cell-associated TNF also were required for To examine possible effects of mast cells and mast cell-associated optimal expression of the tissue swelling and neutrophil infiltration TNF on DC migration in a different context, we administered at sites of FITC challenge in sensitized mice. However, in this FITC-OVA to mice i.n. We used an Ab to 33D1 as a DC marker, model of CHS, significantly greater numbers of FITCϩ DCs were rather than an Ab to MHC class II, to help to identify FITCϩ detected in draining LNs 48 h after epicutaneous application of airway DCs (34). We found that the percentage of FITCϩ DCs in FITC in mast cell- or TNF-deficient mice than in WT mice. These ϩ/ϩ the thoracic LNs of FITC-OVA-treated WBB6F1-Kit (WT) findings indicate that, at the later stages of the sensitization pro- mice 24 h after intranasal application of FITC-OVA was signifi- cess, the engagement of mast cell- and/or TNF-independent mech- cantly higher than the corresponding values for mast cell-deficient anisms can contribute significantly to the migration of hapten- W/W-v WBB6F1-Kit mice, and that the systemic engraftment of bearing DCs from the skin to local LNs. Moreover, the delay in KitW/W-v mice with mast cells by i.v. administration of WT BM- migration of FITCϩ DCs from the skin to local LNs in the absence CMCs permitted these mice to express a response which was sta- of mast cells or TNF was not associated with reduced levels of tistically indistinguishable from that of the WT mice (Fig. 8). proliferation upon Ag challenge of T cells obtained from draining We also examined the migration of FITCϩ airway DCs in re- LNs 5 or 6 days after the onset of sensitization. Thus, the defect in sponse to i.n. administration of FITC-OVA in the same strains of the expression of CHS at sites of FITC challenge in sensitized The Journal of Immunology 4109

sitization phase of acquired immune responses, multiple mechanisms probably can contribute to mast cell activation in such settings. Bryce et al. (14) showed that Ag nonspecific IgE-Fc⑀RI signals, as well as mast cells, significantly promoted the egress of epidermal LCs when mice were sensitized with 2% Ox in ethanol. Demeure et al. (46) reported that mosquito saliva can directly induce mast cell degranulation in vitro and in vivo, in an apparently IgE- and specific Ag-independent manner, and that such mast cell activation can promote the migration of leukocytes, including skin DCs, to draining LNs. Although Demeure et al. (46) did not for- mally rule out a role for Ag-independent effects of IgE on mast cells in this model, their study showed that mosquito saliva can directly or indirectly promote the mast cell-dependent enhancement of DC migration. FIGURE 8. Mast cells are required for optimal DC migration from the We found that the migration FITCϩ DCs to local LNs during the airways to thoracic LNs in response to intranasal administration of FITC- first 24 h after application of FITC or FITC-OVA was not signif- OVA. A and B, At 24 h after intranasal administration of FITC-OVA, icantly diminished in mice which lacked expression of FcR␥. thoracic and submaxillary LNs were separately collected from WBB6F1- ϩ ϩ Thus, in these skin or airway models, mast cells apparently exert Kit / (WT) mice, KitW/W-v mice and KitW/W-v mice after i.v. transfer, 8 wk before the experiment, of 1.0 ϫ 107 congenic WT BMCMCs. Then, the their effects on DC migration independently of activation by either Downloaded from ϩ ϩ ⑀ ␥ proportion (percent) of FITC cells among the 33D1 CD11chigh DCs was IgE/Fc RI or IgG1/Fc RIII signaling. Although we have no data determined by FACS. A, Representative FACS data from one mouse of proving that FITC or FITC-OVA induced FcR␥-independent mast each group. The upper (in bold) and lower numbers are the percentage of cell activation in our experiments, and, if so, by what mecha- FITCϩ cells among the thoracic and submaxillary LNs, respectively. nism(s) this occurred, there are a number of possibilities which can Shaded areas, submaxillary LNs; bold lines, thoracic LNs. B, Columns (Ⅺ, be investigated. submaxillary LNs; f, thoracic LNs) show the average ϩ SEM proportion For example, the vehicle used for administration of FITC con- http://www.jimmunol.org/ ϩ ϩ high (percent) of FITC cells among the 33D1 CD11c DCs in mice of each sists of acetone and the dialkyl phthalate, dibutylphthalate. A 1:1 group. The numbers in parentheses represent the total number of mice in mixture of acetone and dibutylphthalate can induce the migration each group; the numeral after # is the total number of independent exper- of F4/80 Agϩ and macrophage C-type lectinϩ macrophages from p Ͻ ءءءء iments performed using mice in that group. , 0.0001 vs corre- ␤ sponding values for thoracic LNs from Kitϩ/ϩ (WT) mice or from KitW/W-v the mouse dermis by a mechanism which is at least in part IL-1 mice which had been engrafted with congenic ϩ/ϩ (WT) BMCMCs. For dependent (47). Perhaps the vehicle-induced local production of all individual groups of mice, the values for thoracic vs the corresponding cytokines (e.g., by keratinocytes, which can produce TNF and submaxillary LNs were significantly different at the p Ͻ 0.0001 level. other proinflammatory mediators; Ref. 1), can contribute to mast cell activation independently of FcR␥-dependent signals. Alterna- tively, in our airway model, FITC-OVA might first activate alve- by guest on September 29, 2021 mice may have solely reflected defects in mast cell- and/or TNF- olar macrophages to produce mediators which can then induce associated effector function at these sites. FcR␥-independent mast cell activation. Finally, dialkyl phthalates The findings that mast cells, and mast cell-associated TNF, can not only can enhance the Ag- and IgE-dependent activation of rat make important contributions to the migration of hapten-bearing basophilic leukemia cells, but they also can directly increase con- DCs during the initial stages of the sensitization phase of acquired, centrations of cytosolic calcium ions in the cells and, at high con- T cell-dependent immune responses are in accord with prior ob- centrations, weakly promote degranulation independently of IgE servations indicating that mast cells, and mast cell-derived TNF, and specific Ag (48). However, these represent only a few of the can promote the migration of other hemopoietic cell types, includ- many possible direct or indirect mechanisms of mast cell activa- ing neutrophils, monocytes, and T cells, in the context of both tion which could contribute to our observation that mice lacking innate and acquired immune responses (13, 37–39). Our observa- FcR␥ exhibited no apparent defects in their ability to express mast tions are also consistent with work with other haptens demonstrat- cell-dependent enhancement of DC migration in response to FITC. ing defects in both DC migration and the expression of CHS in Moreover, in attempting to understand how mast cell function is TNFϪ/Ϫ mice (4, 9). elicited in this setting, it is important to recognize that the form of Although our experiments show that mast cell-associated TNF mast cell TNF which contributes to FITC-induced, mast cell-de- is necessary for optimal DC migration in response to FITC in the pendent enhancement of DC migration appears to be the mem- two model systems tested, we have not excluded the possibilities brane-associated form of the cytokine. Perhaps relatively subtle that other mast cell-derived mediators also can contribute to these changes in the expression of the membrane-associated form of responses or have important roles in other settings associated with TNF by mast cells at sites of hapten challenge are sufficient to DC migration. Mast cells represent a rich source of a diverse array permit the elicitation of mast cell-dependent enhancement of DC of biologically active mediators, including histamine and many migration. Possibly, even constitutive levels of expression of cytokines and chemokines (40, 41), many of which have potential membrane-associated TNF are sufficient to allow mast cells to pro- effects on DC migration, maturation, or function (7, 42–45). And mote DC migration. In that case, initiation of DC migration by Jawdat et al. (15) showed that activation of skin mast cells with hapten may require a local signal other than an alteration of levels IgE and specific Ag can enhance epidermal LC migration by of mast cell-associated TNF. mechanisms that are, at least in part, both histamine and H2R Even though FcR␥-dependent signals were not essential for the dependent. Accordingly, it is possible that histamine and/or other enhancement of skin (or airway) DC migration, expression of mast cell-derived mediators, as well as TNF, may also have sig- FcR␥ was required for the optimal local expression of CHS re- nificant effects on DC migration during responses to FITC. sponses upon subsequent FITC challenge of sensitized mice. This Just as multiple mast cell-derived mediators have the potential may have reflected an important role for FcR␥ in either the sen- to influence DC migration, maturation, or function during the sensitization or elicitation phases of CHS to FITC. For example, FcR␥ 4110 MAST CELL TNF PROMOTES DC MIGRATION

FIGURE 9. Mast cells, TNF, and TNFR1 are required for optimal DC migration from airways to thoracic LNs upon i.n. administration of FITC-OVA. A and B, Airway DC migration was assessed as in Fig. 7 for WT mice (n ϭ 21), KitW-sh/W-sh mice (n ϭ 10), TNFϪ/Ϫ mice (n ϭ 18), KitW-sh/W-sh mice, which had been engrafted i.v. with WT (n ϭ 14) or TNFϪ/Ϫ (n ϭ 10) BMCMCs, memTNF⌬/⌬ mice (n ϭ 10), TNFR1Ϫ/Ϫ mice (n ϭ 10), TNFR2Ϫ/Ϫ mice (n ϭ 14), and FcR␥Ϫ/Ϫ mice (n ϭ 10). A, Columns (Ⅺ, submaxillary LNs; f, thoracic LNs) show the average ϩ SEM proportion (percent) of FITCϩ cells among the p Ͻ ,ءءءء .33D1ϩCD11chigh DCs in mice of each group 0.0001 vs corresponding values for thoracic LNs from WT mice; †††, p Ͻ 0.001 vs corresponding values for thoracic LNs from KitW-sh/W-sh mice that had been engrafted i.v. with WT BMCMCs. For all individual groups of mice, the values for thoracic vs the corresponding submaxillary LNs were signiﬁcantly different at the p Ͻ 0.0001 level. Data show the pooled results from at least three independent Downloaded from experiments. B, Representative FACS data are shown for one mouse from each genotype. The upper (in bold) and lower numbers are the percentages of FITCϩ cells in the thoracic and submaxillary LNs, respectively; shaded areas, submaxillary LNs; bold lines, thoracic LNs. C, The proportion (percent) of I-Ab high33D1ϩCD11chigh resident http://www.jimmunol.org/ DCs in single-cell suspensions of lungs (prepared as described in Materials and Methods) from naive mice (i.e., mice that had not been challenged i.n. with FITC-OVA) p Ͻ ,ءءء .(was assessed by FACS (n ϭ 5 for each group 0.001 vs corresponding values for wild-type (WT) mice. D, The number of resident lung mast cells was determined among 2 ϫ 105 lung cells from naive mice (as described in p Ͻ ,ءءءء .(Materials and Methods)(n ϭ 8 for each group 0.0001 for values for KitW-sh/W-sh mice vs corresponding values for any other group; ††††, p Ͻ 0.0001 vs corre- by guest on September 29, 2021 sponding values for WT mice.

(on DCs and/or other cell types) may be required for key steps in Nevertheless, our data indicate that mast cell-associated TNF DC maturation or function, and/or for the optimal orchestration of has the potential to contribute to the development of acquired im- the elicitation phase of the response. mune responses involved in host defense or immunological disor- The findings that mast cell-associated TNF can significantly en- ders. One might even speculate that some of the clinical benefit of hance DC migration from the skin or airways in mice, as well as anti-TNF therapy in humans reflects suppression of either “effec- contribute to the effector phase of CHS responses, offer a new tor/proinflammatory” or “immunoregulatory” functions of mast perspective on a large body of work implicating TNF, and specif- cell-derived TNF. Anti-TNF therapy has been reported to be help- ically mast cell-derived TNF, in a wide variety of mouse models of ful in rheumatoid arthritis (54), Crohn’s disease (55), and asthma disease, and, by extension, in the corresponding human disorders. (56), each of which has been associated with increased numbers Thus, in mice, mast cell-derived TNF can contribute to the devel- and/or activation of mast cells at sites of disease (57–65). In such opment of the pathology associated with certain models of sepsis disorders, anti-TNF treatment may confer benefit in part by antag- (37, 49–51), cutaneous granuloma formation (52), skin vasculitis onizing effector functions of TNF derived from mast cells, as well and glomerulonephritis (53), and CHS (13). In such settings, it is as from the many other potential cellular sources of this cytokine. generally thought that mast cell-derived TNF mediates “proinflam- Although the work reported herein indicates that anti-TNF treat- matory” effects at sites of disease. ment also has the potential to interfere with an “immunoregula- However, our data indicate that mast cell-associated TNF also tory” role of mast cell-associated TNF, the literature does not re- can promote DC migration during the early stages of the sensiti- veal a clear picture regarding the importance of TNF in the zation phase of acquired immune responses, and thereby may con- sensitization phase of CHS. On the one hand, anti-TNF Ab treat- tribute to the development of such responses. The actual extent to ment in the sensitization phase of a model of Ox-induced CHS in which mast cell-associated TNF can contribute to immune sensi- mice almost completely suppressed DC migration to local LNs, tization in various settings will have to be established on a case- and this was associated with attenuated inflammation in the elic- by-case basis. For example, in the FITC-induced CHS model in- itation phase (66). Neutralization of TNF in the elicitation phase vestigated herein, we so far have not detected a defect in immune also can suppress the development of CHS reactions to TNCB in sensitization in the absence of either mast cells or TNF, at least as mice (67). In contrast, normal levels of Ag-specific proliferation can be assessed by quantifying Ag-induced proliferation of drain- were observed in LN T cells obtained from TNFϪ/Ϫ mice sensi- ing LN T cells obtained 5 or 6 days after application of FITC. tized with TNCB, and mice that received TNCB-sensitized T cells The Journal of Immunology 4111

Ϫ Ϫ from either WT or TNF / mice developed very similar CHS 1983. Defective elicitation of delayed-type hypersensitivity in W/Wv and SI/SId responses upon subsequent hapten challenge (9). mast cell-deficient mice. J. Immunol. 131: 2687–2694. 13. Biedermann, T., M. Kneilling, R. Mailhammer, K. Maier, C. A. Sander, We found that the levels of Ag-specific proliferation in T cells G. Kollias, S. L. Kunkel, L. Hultner, and M. Rocken. 2000. Mast cells control isolated from draining LNs 5 or 6 days after FITC application were neutrophil recruitment during T cell-mediated delayed-type hypersensitivity re- Ϫ/Ϫ actions through tumor necrosis factor and macrophage inflammatory protein 2. nearly identical in WT, mast cell-deficient, and TNF mice (Fig. J. Exp. Med. 192: 1441–1452. Ϫ/Ϫ Ϫ/Ϫ 6C and data not shown). Thus, as in TNF or IL-1 mice, 14. Bryce, P. J., M. L. Miller, I. Miyajima, M. Tsai, S. J. Galli, and H. C. Oettgen. which exhibited a transient defect in DC migration after epicuta- 2004. 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