Cutting Edge: Serotonin Is a Chemotactic Factor for Eosinophils and Functions Additively with Eotaxin This information is current as Stefen A. Boehme, Francisco M. Lio, Lyudmila Sikora, of September 26, 2021. Terlika S. Pandit, Karine Lavrador, Savita P. Rao and P. Sriramarao J Immunol 2004; 173:3599-3603; ; doi: 10.4049/jimmunol.173.6.3599 http://www.jimmunol.org/content/173/6/3599 Downloaded from References This article cites 21 articles, 5 of which you can access for free at: http://www.jimmunol.org/content/173/6/3599.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE JOURNAL OF IMMUNOLOGY CUTTING EDGE Cutting Edge: Serotonin Is a Chemotactic Factor for Eosinophils and Functions Additively with Eotaxin1 Stefen A. Boehme,*† Francisco M. Lio,† Lyudmila Sikora,* Terlika S. Pandit,* Karine Lavrador,† Savita P. Rao,* and P. Sriramarao2* Elevated levels of serotonin (5-hydroxytryptamine, 5-HT) demonstrate the importance of the eotaxin-CCR3 interaction are observed in the serum of asthmatics. Herein, we dem- in allergic asthma, but they also suggest that other eosinophil onstrate that 5-HT functions independently as an eosino- chemoattractants that function through different receptors are phil chemoattractant that acts additively with eotaxin. likely to be involved in selectively attracting eosinophils to re- 5-HT2A receptor antagonists (including MDL-100907 spiratory tissue. and cyproheptadine (CYP)) were found to inhibit 5-HT- 5-Hydroxytryptamine (5-HT, serotonin) is one of the most Downloaded from induced, but not eotaxin-induced migration. Intravital extensively studied neurotransmitters of the CNS that is also microscopy studies revealed that eosinophils roll in re- present in constituents of the immune system. It is an impor- tant inflammatory mediator that is released by mast cells upon sponse to 5-HT in venules under conditions of physio- IgE cross-linking and has recently been shown to play a role in logical shear stress, which could be blocked by pretreating the pathophysiology of asthma (8). Increased levels of free eosinophils with CYP. OVA-induced pulmonary eosino- 5-HT are present in the plasma of symptomatic asthmatic pa- http://www.jimmunol.org/ philia in wild-type mice was significantly inhibited using tients compared with asymptomatic subjects (9), and recent CYP alone and maximally in combination with a CCR3 studies have also demonstrated that 5-HT can induce lung fi- receptor antagonist. Interestingly, OVA-induced pulmo- broblasts to produce eotaxin (10). Although these studies sug- ؊/؊ nary eosinophilia in eotaxin-knockout (Eot ) mice was gest a role for 5-HT in allergic asthma, a direct effect of 5-HT in inhibited by treatment with the 5-HT2A but not CCR3 mediating eosinophil recruitment/chemotaxis relative to the receptor antagonist. These results suggest that 5-HT is a function of eotaxin has not been determined. In the present potent eosinophil-active chemoattractant that can func- study, we have investigated the role of 5-HT to function di- tion additively with eotaxin and a dual CCR3/5-HT2A rectly as an eosinophil-specific chemoattractant. by guest on September 26, 2021 receptor antagonist may be more effective in blocking al- lergen-induced eosinophil recruitment. The Journal of Materials and Methods Immunology, 2004, 173: 3599–3603. Eosinophil isolation Eosinophils were purified from the peripheral blood of allergic donors (11). For the in vivo experiments, eosinophils were fluorescently labeled with carboxy- osinophils play a prominent proinflammatory role in fluorescein diacetate (CFDA, Invitrogen, Carlsbad, CA) (12). airway allergic inflammation including the pathogene- Synthesis of the CCR3 antagonist N-{1(S)-[4-(3,4- E sis of asthma (1, 2, 3). This inflammatory role is medi- dichlorobenzyl)piperazin-1-yl-methyl]-2-methylpropyl}-4- ated by lipid mediators, cytokines, and toxic granule proteins methylbenzamide dihydrochloride salt (DPM) released by activated eosinophils (4). Several studies have dem- DPM was synthesized following the protocol described in patent EP 0 903 349 onstrated a critical role for eotaxin in the selective recruitment A2. This antagonist has a Ki value of 62 nM for the human CCR3 receptor and of eosinophils following allergen challenge (5–7). In experi- Ͼ10 ␮M for the 5-HT2 receptors (data not shown). mental studies of allergic asthma, treatment of allergen-chal- Chemotaxis assays lenged mice with an anti-eotaxin Ab resulted in a 56% inhibi- Boyden chamber assay. The ability of 5-HT to induce eosinophil migra- Ϫ Ϫ tion of pulmonary eosinophil influx (7). Eot / mice tion was tested using the Boyden chamber assay (11). In some experiments, demonstrated a 70% reduction of eosinophils in bronchoalveo- eosinophils were preincubated with cyproheptadine (CYP), ketanserin, pirenperone, or DPM for 15 min before the chemotaxis assay. lar lavage (BAL)3 fluid 18 h postallergen challenge (5), while Ϫ/Ϫ ϳ Transwell chamber assay. Eosinophils isolated from different allergic do- eotaxin receptor CCR3 mice exhibited an 60% decrease nors (n ϭ 4) were preincubated with MDL-100907 (13, 14) (kindly pro- in pulmonary eosinophilia postchallenge (6). These studies vided by Dr. H. Y. Huang, Columbia University, New York, NY) at a *La Jolla Institute for Molecular Medicine, San Diego, CA 92121; and †Neurocrine Bio- 2 Address correspondence and reprint requests to Dr. P. Sriramarao, Division of Vascular sciences, Inc., San Diego, CA 92121 Biology, La Jolla Institute for Molecular Medicine, 4570 Executive Drive, San Diego, CA 92121. E-mail address: [email protected] Received for publication May 12, 2004. Accepted for publication July 19, 2004. 3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; %-HT, 5-hydroxytryp- The costs of publication of this article were defrayed in part by the payment of page charges. tamine (serotonin); CFDA, carboxyfluorescein diacetate; DPM, N-{1(S)-[4-(3,4-dichlo- This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. robenzyl)piperazin-1-yl-methyl]-2-methylpropyl}-4-methyl benzamide dihydrochloride Section 1734 solely to indicate this fact. salt; CYP, cyproheptadine; IVM, intravital microscopy; RF, rolling fraction; WT, wild 1 This work was supported by National Institutes of Health Grant AI 35796 and California type; hpf, high-power field. Tobacco-Related Disease Research Program Grant 10 RT-0171 (to P.S.). Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 3600 CUTTING EDGE: SEROTONIN: A CHEMOTACTIC FACTOR FOR EOSINOPHILS concentration of 20 ␮M for 20 min or with medium alone before addition Hospital Medical Center, Cincinnati, OH) (5, 15). In brief, mice were sensi- (2 ϫ 105/well) to Matrigel-coated (200 ␮g/ml) Transwell chambers. Cul- tized by two i.p. injections of 50 ␮g OVA in 200 ␮l of alum (Pierce). Nonsen- ture medium alone or medium containing 5-HT (100 nM) or eotaxin (50 sitized mice received 200 ␮l of alum alone. Ten days after the second injection, nM) was added to the lower chamber. In certain experiments, 5-HT was CYP (0.2 mg/mouse), DPM (2 mg/mouse), or both were administered (i.p.) to added to only upper or both upper and lower chambers and incubated at different groups of mice (n ϭ 3 mice/group). Fifteen minutes after injection of 37°C for 4 h, after which the chambers were removed and the number of the inhibitor, mice were exposed to three inhalations (30 min each) of aerosol- migrated cells was quantitated and expressed as a percentage of total cells ized OVA (10 mg/ml in 0.9% sterile saline) at 1-h intervals. Nonsensitized mice added to the well. received an i.p. injection of vehicle and aerosol challenge of saline only. One hour after the last aero-allergen challenge, mice were sacrificed and processed RT-PCR for BAL fluid collection to determine eosinophil counts (15). Amplification of the gene for 5-HT2A was performed by RT-PCR of eosino- phil RNA isolated from human allergic subjects. Eosinophil reverse transcrip- tase product underwent two rounds of PCR amplification using the following Results conditions: 15 s at 94°C,30sat57°C, and 45 s at 72°C for 30 cycles. The Eosinophils respond functionally to 5-HT sequence of the primers was as follows: 5-HT2A receptor external sense primer, Ј Ј Ј 5-HT alone was found to induce migration of human eosino- 5 -CTATAGGTCAGCCTTTTCACG-3 and external antisense primer, 5 - Ϫ6 GCCTTCCACAGTTGCCACG-3Ј. Two microliters of the first PCR was phils in a dose-dependent manner, which was maximal at 10 used as a template for the second round. The internal nested primer sequences M (Fig. 1A). Furthermore, 5-HT, at various concentrations, were as follows: 5-HT2A receptor internal nested sense primer, 5Ј-TATAT had an additive effect on eosinophil chemotaxis when tested in Ј Ј TCAGTGTCAGTACAAGG-3 and internal nested antisense primer, 5 - combination with 50 nM eotaxin. As a positive control, 50 nM CCTATCACACACAGCTACC-3Ј. All primer pairs spanned an intron. eotaxin alone also induced eosinophil migration, consistent Downloaded from Western blot analysis with our previous studies (11). The migration induced by Western blot analysis was conducted using an anti-5-HT2A receptor Ab (BD 5-HT was chemotactic and not chemokinetic (Fig.