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Age-Induced Reprogramming of Mast Cell Degranulation MyTrang Nguyen, Amy J. Pace and Beverly H. Koller This information is current as J Immunol 2005; 175:5701-5707; ; of October 3, 2021. doi: 10.4049/jimmunol.175.9.5701 http://www.jimmunol.org/content/175/9/5701

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Age-Induced Reprogramming of Mast Cell Degranulation1

MyTrang Nguyen, Amy J. Pace, and Beverly H. Koller2

Mast cell degranulation can initiate an acute inflammatory response and contribute to the progression of chronic diseases. Alteration in the cellular programs that determine the requirement for mast cell degranulation would therefore have the potential to dramatically impact disease severity. Mast cells are exposed to increased levels of PGE2 during inflammation. We show that although PGE2 does not trigger the degranulation of dermal mast cells of young animals, in older mice, PGE2 is a potent mast cell stimulator. Intradermal administration of PGE2 leads to an EP3 receptor-dependent degranulation of mast cells, with the number of degranulated cells approaching levels observed in IgE- and Ag-treated controls. Taken together, these studies suggest that the ability of PGE2 to initiate mast cell degranulation changes in the aging animal. Therefore, elevated PGE2 levels might provide an important pathway by which mast cells are engaged to participate in inflammatory responses in the elderly patient. The Journal of Immunology, 2005, 175: 5701–5707. Downloaded from ctivated mast cells undergo degranulation, a series of The identification of PGE2 in rat inflammatory exudates led to the biological and morphological changes that lead to secre- initial proposal of a role for PGs in inflammation (14), and this idea tion of inflammatory mediators from cytoplasmic gran- was supported by the observation of increased levels of PGE in A 2 ules. Large numbers of mast cells are present in the dermis and, numerous types of experimental inflammation models and clinical when activated, release a number of stored inflammatory media- studies. The observation that PGE2 was a potent vasodilator led to ␣ tors, including histamine, TNF- , IL-1, IL-4, IL-6, IL-8, IL-13, the more specific theory that the ability of PGE2 to alter protein platelet-activating factor, and leukotriene C4 (1). Although tradi- plasma extravasation was an indirect consequence of its actions on http://www.jimmunol.org/ tionally mast cells are associated with acquired immune responses, vascular smooth muscle (15), and this contention was supported by where the cells are activated by the binding of Ag-IgG or -IgE the observation that injection of PG into human skin induced dose- complexes to FcRs or the recognition of Ags by Fc⑀RI-bound IgE, dependent erythema (16, 17). However, other reports suggested they can also be activated by the presence of certain microorgan- that PGE2 alone was able not only to elicit erythema, but also to isms as well as C3a and C5a, generated by nonspecific activation alter vascular permeability. Kaley et al. (18) demonstrated that of the complement system. A large body of evidence supports a PGE1 was equipotent with bradykinin and histamine in causing role for mast cells in innate immune responses (2, 3), including vascular leakage in rat skin; however, other studies indicated that response to tissue injury and activation through TLR signaling in the increase in vascular permeability might not be due to a direct by guest on October 3, 2021 response to LPS and peptidoglycan (4). It has also been shown that action of PGE2, but was perhaps secondary to endogenous hista- mast cells are required for normal levels of edema and leukocyte mine release (19). High doses of PGE2 produced only small infiltration in response to PMA, an agent that induces acute in- changes in vascular permeability in guinea pig or rabbit skin (20– flammation (5). In these models, mast cell degranulation is pre- 22), also suggesting that PGs do not contribute to edema by a sumed to be independent of engagements of the FcRs. More re- direct effect on blood vessel permeability. cently, a role for mast cells has been documented in chronic The realization that PGE2 could modulate the function of most diseases, such as inflammatory arthritis, allergic encephalomyeli- inflammatory cells raised the possibility that PGE2 acted through a tis, and asthma (6–8). number of different mechanisms to modulate acute inflammation: The ability of PGE2 to modulate mast cell function has been indirectly through its activation of tissue leukocytes, and directly noted for many years, and the ability of PGE2 to both enhance mast through its actions on the vasculature. Furthermore, the discovery cell degranulation and inhibit the release of mast cell mediators has that PGE2 could activate a family of cell surface receptors raised been reported (9–13). PGE levels increase during tissue injury 2 the possibility that the proinflammatory actions of PGE2 might be and infection and in chronically inflamed lesions. PGE2 is impor- specific to only a single receptor or a subset of receptors (23). tant as a mediator of acute inflammation, which is characterized by PGE2 mediates its effects by binding to specific cell surface, G changes in the caliber and permeability of the microvasculature, protein-coupled receptors, of which there are four subtypes, desig- the exudation of fluid and plasma proteins, and the migration of nated EP1,EP2,EP3, and EP4 (24). The diverse actions of PGE2 are leukocytes, predominantly neutrophils, into the injured area (1). believed to be related to the fact that EP receptors couple to various

G proteins to affect different inﬂammatory mediators. EP2 and EP4

receptors are coupled to Gs and activate adenylate cyclase, leading to

Department of Genetics, University of North Carolina, Chapel Hill, NC 27599 increased levels of intracellular cAMP. EP1 receptor activation is as- 2ϩ Received for publication May 19, 2005. Accepted for publication August 16, 2005. sociated with increases in intracellular Ca ; however, its coupled G The costs of publication of this article were defrayed in part by the payment of page protein remains unidentiﬁed (23). EP3 is unique among the prostanoid charges. This article must therefore be hereby marked advertisement in accordance receptors, in that multiple isoforms of the receptor are produced from with 18 U.S.C. Section 1734 solely to indicate this fact. the EP3 gene. EP3 isoforms couple to Gi,Gs,orGq to mediate the 1 This work was supported by National Institutes of Health Grant 1-R01-HL68141- regulation of adenylate cyclase; however, in most systems extensively 01-03 (to B.H.K.) and Cystic Fibrosis Foundation Grant Koller00Z0 (to B.H.K.). studied, Gi predominates (25, 26). 2 Address correspondence and reprint requests to Dr. Beverly H. Koller, Department of Medicine, University of North Carolina, Chapel Hill, NC 27599. E-mail address: PGE has been shown to enhance IL-6 production by rat mast [email protected] peritoneal cells (27), and additional studies using rat peritoneal

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 5702 AGE-INDUCED MAST CELL DEGRANULATION REPROGRAMMING cells and bone marrow mast cells (BMMCs)3 have indicated that stained with toluidine blue. Ear biopsies treated with PGE and IgE were although PGE alone cannot induce mast cell degranulation, ad- fixed in 0.1 M sodium cacodylate buffer for4hatroom temperature and ministration of PGE in addition to IgE/Ag-mediated activation re- returned to 4°C overnight for additional fixing. Samples were then pro- cessed in the automated Reichert Lynx EM tissue processor. Specimens sults in synergistically increased activation of BMMCs and en- were embedded in Spurr’s resin and polymerized overnight at 70°C. Sec- hanced IL-6 production. Pharmacological studies have indicated tions (1 ␮m thick) were cut and stained with 1% Toluidine Blue-O in 1% that these increases in mast cell degranulation and IL-6 production sodium borate for light microscopy. Mast cells were identified according to are mediated through the EP or EP receptors (28), and other their cytoplasmic granules. They were considered normal if Ͻ10% of the 1 3 granules exhibited fusion, moderate if 10–50% of the granules exhibited investigations, using EP receptor-deficient BMMCs, have con- fusion, or extensively degranulated if Ͼ50% of the cytoplasmic granules cluded that EP3 is the primary mediator for these responses, at least exhibited fusion and extrusion from cells. in these immature cells (13). In the present study we examine the effects of PGE on inflam- Statistical analysis mation in vivo. We show that PGE can induce cutaneous inflam- Data are presented as the mean Ϯ SEM. Statistical analysis was performed mation in mice, and that this response is mast cell dependent. Us- using two-sample Student’s t test for unequal variances or single-factor ing EP receptor-deficient mice, we show that the EP receptor ANOVA, where indicated. The p values reported have been adjusted using 3 the Bonferroni method to account for multiple comparisons. expressed by mast cells has the predominant role in PGE-induced inflammation. We also show that the extent of this inflammation Results varies between strains of inbred mice and that the age of the mouse PGE induces cutaneous inflammation in mice is the most important factor determining the response of the mast 1 We first examined the ability of s.c. application of PGE and PGE cell to PGE2. 2 1 to induce an inflammatory response. To monitor this response, we Downloaded from Materials and Methods injected mice with Evan’s Blue dye, which binds to serum proteins Mice and allows changes in vascular permeability to be monitored by determining the levels of dye in a tissue. Wild-type 129/SvEv mice The generation of mice deficient in EP ,EP,EP, and EP receptors has 1 2 3 4 received an intradermal injection of PGE in the right ear, whereas been previously reported (29–32). Mast cell-deficient mice (WBB6F1/J- KitW/KitW-v; 8–12 wk old) were purchased from The Jackson Laboratory. the left ear was injected with vehicle (PBS). Mice were killed 0.5 h All mice used were bred and maintained in specific pathogen-free animal later, an ear tissue sample was removed, and the difference in the http://www.jimmunol.org/ facilities at University of North Carolina in accordance with the institu- amount of dye in the tissue from the PGE-treated and vehicle- tional animal care and use committee guidelines. treated ears was determined (data not shown). Using this protocol,

PGE-induced ear edema we found that PGE2 or PGE1 alone is sufficient to elicit a measurable change in vascular permeability in mouse dermis, and that Mice were injected i.v. with 0.5% Evan’s Blue dye (Sigma-Aldrich) in PBS at a concentration of 10 ml/kg body weight. Mice were then anesthetized, this change was similar using either PGE2 or PGE1 (data not ␮ ␮ shown). and 20 lofPGE1 (0.5 g) was injected intradermally into the right ear, whereas the left ear was injected with an equal amount of PBS. One-half hour after intradermal injection, mice were killed by CO2, and ears were PGE induced edema formation in EP receptor-deficient mice cut off close to the base of the ear. Ear biopsies were incubated in 1 ml of by guest on October 3, 2021 formamide at 55°C for 48 h. The absorbance of the formamide extracts To determine whether the observed inflammation was the result of were measured at 610 nm for quantification of serum protein extravasation. the specific action of PGE2 and to define the receptor(s) mediating this action, we examined the impact of loss of each of the four Passive cutaneous anaphylaxis PGE2 receptors on this response. We first examined the roles of the Passive cutaneous anaphylaxis was performed as described previously two Gs protein-coupled PGE2 receptors, EP2 and EP4, in PGE- (33). Briefly, animals were lightly anesthetized, and the right ears were mediated plasma extravasation. Comparison of age-matched 129/ injected intradermally with 20 ng of murine monoclonal anti-DNP IgE SvEv mice and congenic EP Ϫ/Ϫ animals failed to support a role diluted in 20 ␮l of PBS. The left ears were injected with 20 ␮l of PBS. 2 Twenty-four hours later, animals were injected i.v. with 100 ␮l of 0.9% for this receptor in PGE-induced edema formation (data not PBS containing 100 ␮g of DNP-albumin and 1% Evan’s Blue dye. Ani- shown). Similarly, no significant difference was observed in the Ϫ/Ϫ mals were killed 90 min after i.v. injection, and ears were cut off close to response of EP4 animals compared with littermate controls the base of the ear and incubated in 1 ml of formamide at 54°C for 48 h. (data not shown). Activation of both the EP1 and the EP3 PGE Quantitative analysis of formamide extracts was determined by measuring 2ϩ the absorbance of Evan’s Blue at 610 nm with a spectrophotometer. receptors can lead to an increase in intracellular Ca ; thus, these receptors are likely candidates for mediating the proinflammatory

Bone marrow transplantation actions of PGE. The EP1 mutation was introduced into an ES cell ϩ/ϩ Ϫ/Ϫ line established from DBA/1J embryos and has been maintained EP3 and EP3 animals served as donors for mast cell-deficient (W/ WV) mice. Bone marrow transplantation was performed as previously de- on this genetic background (30). Comparison of wild-type DBA/1J scribed (34). Briefly, femurs from donors were flushed with 4 ml of PBS Ϫ/Ϫ and congenic EP1 mice indicated a similar level of induced to obtain bone marrow cell suspensions. Bone marrow cell suspensions edema formation in both groups of mice, suggesting that PGE were filtered through Miracloth (22- to 25-␮m pores; Calbiochem-Nova- biochem). Cell suspensions were washed twice with PBS and resuspended activation of the EP1 receptor did not play a part in this response in 0.5 ml of PBS. Recipients then received whole bone marrow cells by tail (Fig. 1A). Two congenic EP3-deficient mouse lines have been gen- vein injection. Transplanted mice were fed normal mouse chow without erated, one on the 129/SvEv background and a second on the antibiotic supplementation. Seven months after transplantation, reconsti- C57BL/6 genetic background. PGE-induced edema formation was tuted mice were injected intradermally with PGE in the ears to examine 1 significantly attenuated in both of these lines compared with wild- the formation of edema, as described above. ϭ Ϫ/Ϫ type controls (Fig. 1B; p 0.0003 for EP3 vs wild-type ϭ Ϫ/Ϫ Histological analysis C57BL/6; Fig. 1C; p 0.006 for EP3 vs wild-type 129/SvEv; ϩ/ϩ Ϫ/Ϫ by Student’s t test). Ear tissue from EP3 and EP3 animals was harvested and fixed in 10% formalin. Tissue was embedded in paraffin, and 5-␮m sections were PGE fails to induce edema in mast cell-deficient mice

3 Abbreviations used in this paper: BMMC, bone marrow mast cell; SMB, selected Mast cells are an important constituent of the dermis, and we have mixed background. previously shown that PGE can potentiate degranulation of The Journal of Immunology 5703

sible explanation for these ﬁndings is that the PGE-stimulated EP3 receptor is required for normal development or function of mast cells. To test this, we examined the number and morphology of the Ϫ/Ϫ ϩ/ϩ mast cells in the EP3 and EP3 mice. No difference in the Ϫ/Ϫ number or histological appearance of dermal mast cells in EP3 mice was noted (data not shown), suggesting that the EP3 receptor is not required for normal mast cell development; however, this does not rule out a possible alteration in the ability of these cells to degranulate and release mediators capable of altering vascular Ϫ/Ϫ physiology. To determine whether the EP3 mast cells can degranulate and stimulate edema formation, we examined edema formation induced by passive anaphylaxis in the two populations of animals. No signiﬁcant decrease in edema resulting from passive Ϫ/Ϫ anaphylaxis was observed in the EP3 animals. This suggests Ϫ/Ϫ that the mast cells of EP3 mice are mostly normal in both number and function (Fig. 2). Two possible models can be proposed to explain the lack of V Ϫ/Ϫ response to PGE in the W/W and EP3 mice. First, it is possible that PGE acts directly on EP3 receptors present on mast cells. Downloaded from Alternatively, EP3 might bring about the release of mediators by other cells, which then act on the mast cells, causing them to degranulate and release mediators that increase vascular permeability. To distinguish between these two possibilities, W/WV mice

were reconstituted with either wild-type or EP3-deficient mast cells. As expected, reconstitution of W/WV mice with wild-type http://www.jimmunol.org/ mast cells significantly restored the dermal response to PGE1 (Fig. 3; p ϭ 0.0012, by Student’s t test with Bonferroni correction), albeit not to the level seen in the congenic wild-type animals. This is likely to reflect incomplete reconstitution of the dermal mast cell population (35, 36). In contrast, reconstitution of W/WV mice with

bone marrow from EP3-deﬁcient mice did not result in a signiﬁcant increase in PGE-mediated edema above that observed in W/WV

animals (Fig. 3), suggesting that PGE acts directly on EP3 receptors on mast cells. by guest on October 3, 2021

Induction of cutaneous inflammation by PGE increases in older mice Large differences have been noted between mouse strains in the FIGURE 1. PGE1-induced edema formation in EP receptor-deficient ϭ inflammatory response elicited by various stimuli and, in particu- mice. A, Loss of the EP1 (n 5) receptor has no effect on the level of PGE2-induced inflammation. Wild-type and EP1-deficient mice exhibited lar, in the role of arachidonic acid metabolites in this response similar responses to vehicle, and although treatment with PGE2 caused an (37). In addition, immune responses can change in magnitude over increase in inflammation, this increase was not significantly different be- the lifetime of the animal. We therefore sought to determine tween the wild-type and deficient mice. B and C, The EP3 receptor appears whether the magnitude of the response to intradermal application to play a role in PGE2-induced inflammation. Examination of EP3-deficient mice on both the C57BL/6 and 129/SvEv backgrounds reveals reduced ϭ edema in response to PGE1.EP3-deficient mice (n 12) demonstrate a significantly reduced level of inflammation compared with both C57BL/6 (p ϭ 0.0003; n ϭ 14) and 129/SvEv (p ϭ 0.006; n ϭ 15) wild-type mice. .p Յ 0.001 ,ءء ;p Յ 0.01 ,ء

BMMCs (13). To determine whether mast cells play a part in PGE- mediated edema formation, we treated mast cell-deficient (W/WV) mice with PGE. We show that the response in the mast cell-deficient mice was significantly reduced compared with that in wild- type mice (Fig. 3; p ϭ 9 ϫ 10Ϫ5). This response in the W/WV mice was not significantly higher in the PGE-treated ear than in the vehicle-treated control. This suggests that the PGE-induced edema is dependent either directly or indirectly on the presence of mast cells in the dermis. ϩ/ϩ Ϫ/Ϫ FIGURE 2. Normal degranulation of EP3 and EP3 mast cells. ϩ/ϩ ϭ Ϫ/Ϫ ϭ PGE-induced edema in mast cell-deficient mice reconstituted EP3 (n 10) and EP3 (n 11) mice exhibit increased inflammation ϩ/ϩ Ϫ/Ϫ in response to IgE stimulation compared with the response to vehicle. No with EP3 and EP3 mast cells ϩ/ϩ Ϫ/Ϫ difference in edema formation was noted between EP3 and EP3 Ϫ/Ϫ PGE-induced edema appears to be largely dependent on the ex- mice, suggesting that mast cells from EP3 mice are degranulating nor- ϭ pression of EP3 receptors and the presence of mast cells. A pos- mally (p 0.731, by two-sample Student’s t test with unequal variances). 5704 AGE-INDUCED MAST CELL DEGRANULATION REPROGRAMMING

mediators released upon mast cell degranulation. Histological examination of mast cells from C57BL/6 young and old mice revealed no difference in the number of mast cells (data not shown). To determine whether there was a difference in the release of inflammation mediators or in the response of the tissues to the released mediators, we examined passive anaphylaxis in young and old C57BL/6 mice, directly comparing this to the change in responsiveness to PGE. Young (2-mo-old) and old (6-mo-old) mice were loaded overnight with monoclonal IgE to DNP. The next day, serum proteins were labeled with Evan’s Blue, and mice were treated with an intradermal injection of Ag in one ear and with PGE in the other. Unlike PGE, IgE mediated a robust response in the young mice: the tissue extravasation was 3 times that induced by PGE (Fig. 5). Similar to PGE, IgE/Ag-mediated mast cell de- FIGURE 3. PGE1 induced edema in mast cell-deficient mice reconsti- granulation and edema formation was increased in the older mice. ϩ/ϩ Ϫ/Ϫ tuted with EP and EP mast cells. Recipient mice were exposed to Ϫ5 3 3 Although this increase was significant (Fig. 5; p ϭ 3.6 ϫ 10 ), 9 Gy of ionizing radiation. Within3hofirradiation of recipient mice, bone the increase represented a change of ϳ30% over that in wild-type marrow cells were obtained from donors, and recipients then received whole bone marrow cells by tail vein injection. Reconstituted mice were animals. In comparison, edema formation in response to PGE was Ϫ5 increased by Ͼ200% in the older animals ( p ϭ 4 ϫ 10 ). Downloaded from injected intradermally with PGE1 in the ears to examine the formation of edema. Edema formation in PGE-treated, mast cell-deficient mice was sim- We next determined whether the temporal difference between ilar to that in mast cell-deficient mice treated with PBS and was signifi- mouse strains in the development of responsiveness to PGE was cantly different from the level of inflammation observed in wild-type mice easily explained by differences in the accumulation and/or matu- ϭ Ϫ/Ϫ ء ( , p 0.00009). Mast cell-deficient mice reconstituted with EP3 mast ration of mast cells in the various mouse lines. To address this cells exhibited levels of inflammation similar to mast cell-deficient mice point, we measured cutaneous anaphylaxis in 2- and 4- to 5-mo-old and controls treated with PBS. Mast cell-deficient mice reconstituted with ϩ/ϩ 129/SvEv and C57BL/6 mice. All mice received an intradermal http://www.jimmunol.org/ EP3 mast cells had significantly increased levels of inflammation compared with both mast cell-deficient mice (#, p ϭ 0.0012) and mast cell-deficient mice injection of anti-DNP monoclonal IgE into the pinna of the left ear. Ϫ/Ϫ ϩ ϭ reconstituted with EP3 mast cells ( , p 0.0004). Values are shown as the Twenty-four hours later, mice received an i.v. injection of Evan’s mean Ϯ SEM. Samples were compared using two-sample Student’s t test for Blue containing anti-DNP and an intradermal injection of PGE in unequal variances, and p values were adjusted for multiple comparisons using the right ear, and edema formation was monitored. As expected Bonferroni’s correction. and consistent with the results shown in Fig. 4A, both these mouse strains displayed only a small increase in edema formation in response to PGE at 2 mo of age (Fig. 4B). Again, consistent with the of PGE1 differed between inbred strains of mice, and if these re- experiments shown in Fig. 4A, a robust response to PGE was ob- by guest on October 3, 2021 sponses differed in younger and older animals. We examined the served in 4-mo-old C57BL/6 mice, whereas 129/SvEv mice con- following inbred mouse lines: 129/SvEv, C57BL/6, DBA1/J, and tinued to respond poorly (Fig. 4B). In contrast, the responses of DBA/2J. In addition, we examined a recombinant inbred line de- 129/SvEv and C57BL/6 mice to IgE/Ag did not differ significantly rived from 129/Ola, C57BL/6, and DBA/2. This recombinant in- at either 2 or 4–5 mo of age. Thus, the delayed development of bred line provides a genetic background permissive to loss of the responsiveness to PGE2 in 129/SvEv mice vs C57BL/6 mice can- PGE EP4 receptor. For simplicity, we refer to this line as selected not easily be explained by a difference between these strains in mixed background (SMB). Injection of PGE in 6-wk-old mice re- either the number of dermal mast cells or the response of the tissue sulted in a small, but measurable, increase in vascular permeability to inflammatory mediators released by mast cells. in all five mouse lines. No change in the magnitude of this response was observed in 3-mo-old 129/SvEv, SMB, or DBA/2J mice (Fig. 4). In contrast, a significant increase in this response Degranulation of mast cells by PGE and IgE in young and was observed in 3-mo-old C57BL/6 ( p ϭ 0.003) and DBA1/J old mice ( p ϭ 0.02) mice compared with that in 1.5-mo-old mice. At 6 mo of age, the responses of all mouse strains were higher than those in We next determined whether there was a correlation between the the 6-wk-old and 3-mo-old animals. To determine whether this inflammatory response to PGE and mast cell degranulation. Young Ͼ response continued to increase as the mice aged, we examined (2-mo-old) and old ( 6-mo-old) mice received an i.v. injection of 1-year-old mice from three of the strains, C57BL/6, SMB, and 20 ng of monoclonal IgE. The next day, the mice received an 129/SvEv, and found additional increases in the vascular perme- intradermal injection of IgE on the right ear and PGE on the left ability of these mice. Analysis of the responses obtained for each ear. After 90 min, the mice were killed, the pinna was harvested, strain were evaluated by ANOVA and were found to be signifi- and mast cell degranulation was evaluated, as described by Takai cantly different over time (C57BL/6, p ϭ 0.0007; SMB, p ϭ et al. (33). As shown in Fig. 6 and consistent with the small change 0.0063; 129/SvEv, p ϭ 6 ϫ 10Ϫ6; DBA1/J, p ϭ 7 ϫ 10Ϫ7; in vascular permeability, few mast cells responded to PGE in the DBA2/J, p ϭ 0.0147). These results suggest that as mice age, their young animals. In contrast, IgE resulted in 60% of the mast cells vascular permeability in response to PGE1 increases, and this ef- degranulating either completely or partially in these animals. In fect is not strain dependent. older animals, however, the percentage of mast cells degranulated by injection of PGE approached that observed with IgE. Thus, Comparison of PGE and passive anaphylaxis in young and although the tissue mast cell of the 8-wk-old mouse is virtually old mice unresponsive to PGE2, the addition of this lipid mediatory alone in This increased inflammation in older mice could result from an the aged mouse is sufficient to initiate degranulation of Ͼ50% of increase in mast cell size or number or from an increase in the dermal mast cells. The Journal of Immunology 5705

FIGURE 5. Comparison of edema induced by passive anaphylaxis and PGE in young and old mice. Mice were treated with DNP-IgE mAb, and 24 h later, cutaneous passive anaphylaxis was induced by intradermal injection of Ag (f). At the same time the other ear of the mouse received an intradermal injection of PGE (Ⅺ). The responses of two populations of mice, 2-mo-old animals (n ϭ 9) and animals Ͼ6moofage(n ϭ 10), were examined in the same experiment. The mice were killed, and extravasation of plasma proteins into the tissue was quantitated by extraction of dye with Downloaded from formamide and spectrophotometric analysis of extracts at 610 nm. The data shown are the mean OD, and error bars indicate the SEM.

Discussion

Soon after its isolation, the inﬂammatory actions of PGE2 were http://www.jimmunol.org/ investigated by direct injection of this lipid into skin (16, 17).

Although the proinﬂammatory actions of PGE2 were generally observed, reports differed on the ability of PGE2 alone to induce all the cardinal signs of inﬂammation. For example, although PGE2 was generally observed to sensitize the tissue to pain and cause

erythema, the ability of PGE2 to mediate changes in vascular permeability was not observed in all systems. A number of studies

showed that PGE2 acted primarily to amplify edema formation initiated by other inflammatory mediators, such as histamine (18, by guest on October 3, 2021 19). Our studies provide some clarification of these results. First, similar to earlier studies, we found that PGE alone can induce edema formation. However, this action is dependent on the presence of mast cells in the tissue, because no protein extravasation was observed in the mast cell-deficient W/WV mice. This observation is consistent with a model in which PGE alone has limited ability to alter the permeability of the postcapillary venules, but, rather, acts by stimulating tissue mast cells to release bioactive

mediators, such as platelet-activating factor, leukotriene C4, and histamine. These mediators, either alone or in synergy with PGE2, change the permeability of these vessels. The variable previous

FIGURE 4. Induction of cutaneous inflammation by PGE1 is elevated in reports on the proinflammatory acts of PGE2 could reflect the dif- older mice. A, Selected mixed background (129/Ola, C57BL/6, and ference in the number of mast cells present in the tissues studied DBA/2) and mice from the inbred lines 129/SvEv, C57BL/6, DBA1/J, and and/or, as discussed below, in the age of the subjects or animals in DBA2/J were evaluated at 1.5, 3, 6, and 12 mo for PGE -induced inflam- 1 which these studies were conducted. mation. Mice were injected i.v. with 0.5% Evan’s Blue dye and then anes- ␮ ␮ The availability of mice lacking specific receptors provides a thetized and given 20 lofPGE1 (0.5 g) intradermally into the right ear and 20 ␮l of PBS into the left ear. One-half hour after intradermal injec- useful tool for determining the contributions of individual EP re- tion, mice were killed, and ear biopsies were incubated in formamide for ceptors to the physiological actions of PGE. We have used this 48 h. Serum protein extravasation was measured by absorbance reading approach in this study to examine the mechanism by which PGE (610 nm) of the formamide extracts. Values shown represent the change in OD (610 nm) between PBS- and PGE-treated ears. Although inflammation -p ϭ 5-mo-old 129/SvEv and C57BL/6 mice. Mice received an intradermal in ,ء) levels were similar in all strains at 1.5 mo of age, the C57BL/6 0.003) and DBA/1J (#, p ϭ 0.02) mice had significantly increased inflam- jection of DNP-IgE mAb into the pinna of the left ear, and 24 h later, mice mation at 3 mo compared with 1.5 mo of age. ANOVA of the edema received an i.v. injection of Evan’s Blue containing anti-DNP (C). At the formation readings of each strain at the time points shown revealed that same time the right ear was treated with an intradermal injection of PGE there was a significant difference among these values, and vascular per- (B). A pronounced difference in the responses of 129/SvEv and C57BL/6 ,ء) meability in the animals of each strain appeared to increase over time mice to PGE was observed, particularly in the 4- to 5-mo-old animals Ϫ (C57BL/6, p ϭ 0.0007; SMB, p ϭ 0.0063; 129/SvEv, p ϭ 6 ϫ 10 6; p ϭ 1.83 ϫ 10Ϫ6;#,p ϭ 2.2 ϫ 10Ϫ7). In contrast, the inflammation Ϫ DBA1/J, p ϭ 7 ϫ 10 7; DBA2/J, p ϭ 0.0147). B and C, Comparison of observed after treatment with Ag did not differ between the two strains at IgE/Ag- and PGE-induced cutaneous inflammation in 2- and 4- to either age (p Ͼ 0.5; n ϭ 8 for each group). 5706 AGE-INDUCED MAST CELL DEGRANULATION REPROGRAMMING

sitive to regulation of mediators such as PGE and that these pathways provide a means by which to participate in the innate immune response. We have found that the amount of edema induced by subdermal injection of PGE was dependent on the age of the mice tested. Inflammation was minimal in very young mice, but inflammation increased significantly with age in mice from all genetic backgrounds examined. There are a number of explanations for this increased inflammation. First, it is possible that the number of mature mast cells might increase with age in mice. Hart et al. (38) have observed an age-dependent increase in dermal mast cell numbers in BALB/c mice; however, this increase was not observed in mice on a C57BL/6 genetic background. We also failed to observe a change in mast cell number in C57BL/6 mice. A second possible explanation is that the level of mediators released by mast cells increases as they age. This would be consistent with the observation that vascular permeability was also greater when the mast FIGURE 6. Comparison of the percentage of degranulated mast cells cells of older mice were triggered by IgE. Harada et al. (39), who after induction of passive anaphylaxis or treatment with IgE in young and have also examined the potentiation of passive anaphylaxis in old mice. Mice were treated with monoclonal IgE to DNP. The following young and old mice, found that cataract Shionogi, diazepam sen- Downloaded from day, mice received an intradermal injection of PGE in the left ear and of sitive, and C57BL/6J mice exhibit an increase in passive anaphy- DNP in the right ear. Ninety minutes later, the tissue was harvested, and the number of mast cells undergoing degranulation was determined. The data lactic shock that is age dependent. However, it is unlikely that this shown represent the mean number of extensively degranulated mast cells in alone can account for the dramatic change in the response to PGE PGE-treated ears of young mice (Ⅺ), PGE-treated ears of old mice (o), in the old and young animals, because the IgE response was en- IgE-treated ears of young (s), and IgE-treated ears of old mice (f) per hanced by only 30%, whereas the response to PGE increased square millimeter. n ϭ 3 for each group of mice. Ͼ200-fold. These results suggest that the increased inflammation http://www.jimmunol.org/ observed is unlikely to be due to a generalized increase in mast cell number, size, or the mediators released upon degranulation. mediates edema formation. We report that plasma protein extrav- Another possible explanation for the increase in inflammation asation in response to PGE is not observed in animals lacking the with age is that the degranulation of the mast cell does not change

EP3 receptor. The involvement of EP3 in this response together with age, but, rather, other changes in the tissue occur that increase with the results of previous studies both from our laboratory (13) the sensitivity of the tissue to the release of mast cell mediators. and others (28) indicate that EP3 could alter mast cell function and Again, this explanation is not consistent with the modest change in suggest that perhaps the PGE-induced edema formation was me- the response of the older mice in the passive anaphylaxis model. by guest on October 3, 2021 diated in part through its actions on resident mast cells. We show A more likely explanation is that as the dermal mast cells ma- V by reconstitution of W/W mice that EP3 receptor expression is ture, the program regulating mast cell responses to various stimuli necessary for PGE-mediated plasma extravasation. However, our changes, and this reprogramming may begin to favor activation by studies differ in one important aspect from our previous studies non-FcR pathways. The mast cell may then become more sensitive with bone marrow-derived cultures of mast cells. Our current stud- to increased innate inﬂammatory mediators and less dependent on ies suggest that PGE alone is sufﬁcient to mediate the degranula- an active adaptive immune system. This interpretation is consistent tion of mast cells. with the dramatically increased number of degranulated mast cells

Previously, using BMMCs, we had shown that although PGE2 present in the PGE-treated tissue of older mice compared with 2ϩ acting through the EP3 receptor could increase intracellular Ca younger mice. in BMMCs, EP3 activation alone was not sufficient to mediate the A role for mast cells has been demonstrated in numerous in- degranulation of BMMCs (13). PGE2 activation of the EP3 recep- flammatory diseases. Many of these diseases, including, most re- tor did, however, augment degranulation and cytokine production cently, arthritis and atherosclerosis, are more frequent in older by BMMCs. Our findings were consistent with previous pharma- populations, and nonsteroidal anti-inflammatory drugs are com- cological studies of rat peritoneal mast cells, which were shown monly used to treat these disorders. It is intriguing to speculate that not to degranulate in response to PGE alone, although these cells the efficacy of nonsteroidal anti-inflammatory drugs in these pa- did produce cytokines without additional signals (27). There are a tients might in part reflect the inhibition of mast cell deregulation number of possible explanations for the differential actions of PGE via this PGE2/EP3 pathway. in vivo and in vitro. First, it is possible that PGE elicits the production of another mediator(s) in the in vivo model that, together Acknowledgments with PGE, activates the mast cell. Such mediators would not be We thank B. Bagnell and V. Madden for assistance with histology and available in the in vitro systems. We previously reported that EP3 microscopy, and J. Ledford and J. Hartney for helpful discussions of the was able to degranulate BMMCs treated with PMA (13). Thus, the manuscript. production of a mediator in vivo in response to PGE that is capable of activating protein kinase C could synergize with PGE in trig- Disclosures gering mast cell degranulation. The authors have no financial conflict of interest. Alternatively, it is possible that the response of a mature con- nective tissue mast cell to PGE differs from that of an immature References BMMC or from the response of peritoneal mast cells. It is inter- 1. Metcalfe, D. D., D. Baram, and Y. A. Mekori. 1997. Mast cells. Physiol. Rev. 77: 1033–1079. esting to speculate that as the mast cell matures within various 2. Galli, S. J., M. Maurer, and C. S. Lantz. 1999. Mast cells as sentinels of innate tissue compartments, it acquires characteristics that make it sen- immunity. Curr. Opin. Immunol. 11: 53–59. The Journal of Immunology 5707

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