Additively with Eotaxin Factor for Eosinophils and Functions Cutting

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 allergen-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.).

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 eosinophil 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. 1B). 5-HT Pharmingen, San Diego, CA). The bound Ab were detected using the chemi- (100 nM) was found to be selective for eosinophils and did not luminescent peroxidase substrate Super Signal Ultra (Pierce, Rockford, IL). induce migration of neutrophils in Transwell chamber chemo- Ϯ Ϯ Animal preparation and superfusion of the vascular bed with 5-HT taxis assays (18 5% (control) vs 20 4% (5-HT) of total cells Ϫ7

added) while, C5a (10 M), which was used as a positive con- http://www.jimmunol.org/ The ability of 5-HT to alter the flux of human eosinophil rolling in postcapillary venules of New Zealand White rabbits was examined by intravital micros- trol, was found to induce migration of both eosinophils and copy (IVM) (12). Briefly, between 6 and 10 h after IL-1␤ stimulation (i.p.), neutrophils (data not shown). portions of the exposed rabbit mesentery were superfused with 5-HT (50 nM) or vehicle alone using a constant flow infusion syringe pump. CFDA-labeled 5-HT2A receptor antagonists inhibit 5-HT-induced eosinophil human eosinophils (0.2–0.5 ϫ 107 cells/ml) were administered into the can- nulated side branch of the superior mesenteric artery after superfusion with chemotaxis in vitro 5-HT had ensued, and their ability to roll in postcapillary venules was deter- We tested the ability of the 5-HT2A receptor antagonists CYP, mined. Data are expressed as rolling fraction (RF; percentage of rolling eosinophils in the total number of eosinophils passing through a segment of a given ketanserin, and pirenperone as well as a CCR3 receptor antag-

blood vessel per injection). The ability of CYP and DPM to alter the flux of onist (DPM) to block human eosinophil migration in response by guest on September 26, 2021 5-HT and eotaxin-induced eosinophil rolling was determined by incubating to 5-HT or eotaxin stimulation. CYP, ketanserin, and piren- ␮ the eosinophils ex vivo with CYP or DPM (10 M) for 15 min at room tem- perone blocked eosinophil chemotaxis in response to 5-HT perature before injecting into the rabbit mesentery. (p ϭ 0.05, 0.05, 0.09, respectively; Fig. 1C). However, ketan- Murine model of allergic airway inflammation serin and pirenperone, but not CYP, were found to antagonize Pulmonary eosinophilia was induced in wild-type (WT) and EotϪ/Ϫ mice, bred the CCR3 receptor in radioligand binding assays, suggesting on the SVEV background (kindly provided by Dr. M. Rothenberg, Children’s that CYP can interact with 5-HT2A and not CCR3 (data not

FIGURE 1. Eosinophils respond functionally to 5-HT. A, Migration of eosinophils in response to various concentrations of 5-HT alone or in combination with 50 nM eotaxin was evaluated. The dashed line indicates the background migration with medium alone, while the dotted line indicates migration in response to 50 nM eotaxin. Five high-power fields (hpf) were counted per well (10 hpf/condition) and the average number of cells per hpf Ϯ SEM is shown. This experiment was repeated with three different donors independently and a representative experiment is shown. B, 5-HT is chemotactic but not chemokinetic for eosinophils. Eo- sinophil migration (n ϭ 3 donors) across Transwell membranes was performed with 5-HT only in the upper wells or in the upper and lower wells as indicated. Results are expressed as percent migration of eosinophils Ϯ SE. C, 5-HT2A receptor antagonists inhibit 5-HT-induced eosinophil chemotaxis in vitro. Eosinophil migration in response to 50 nM eotaxin (left panel) or 100 nM 5-HT (right panel) was examined following incubation with CCR3-specific antagonist DPM (300 nM) and the 5-HT2A receptor antagonists CYP, ketanserin, and pirenperone (each at 50 nM). Results are expressed as mean Ϯ SEM of a representative experiment of four separate experiments using different allergic donors. The Journal of Immunology 3601 shown). Eotaxin-mediated eosinophil chemotaxis was not inhibited by CYP (Fig. 1C), but was blocked by DPM (p ϭ 0.01). However, DPM did not inhibit 5-HT-mediated eosinophil chemotaxis (Fig. 1C). Although ketanserin, pirenperone, and CYP bind to 5-HT2A, they are also known to bind to the 5-HT2B and 5-HT2C receptors at a much lower affinity (16, 17). To further confirm the receptor involved in 5-HT-mediated eosinophil migration, the effect of MDL-100907, a highly selective antagonist of the 5-HT2A receptor (14), on 5-HT-mediated eosinophil chemotaxis was investigated using Transwell chambers (Fig. 2A). MDL-100907 significantly inhibited 5-HT-induced, but not eotaxin-induced chemotaxis of human eosinophils. Next, eosinophils from allergic subjects were observed to express the 5-HT2A receptor when analyzed by RT-PCR (Fig. 2B, lanes 2 and 3) as well as Western blot analysis (Fig. 2C). Stimulation with 50 nM eotaxin or 20 ng/ml IL-5 did not further enhance eosinophil 5-HT2A receptor expression (Fig. 2C, Downloaded from lanes 2 and 3, respectively). FIGURE 3. 5-HT-induced eosinophil rolling in vivo is inhibited by CYP. The circulation of CFDA-labeled human eosinophils in postcapillary venules of 5-HT2A antagonist inhibits eosinophil rolling in vivo IL-1␤-stimulated rabbit mesenteric blood vessels during continuous superfu- We next determined the effect of 5-HT and its receptor antag- sion with saline (control) or 50 nM 5-HT (A) or eotaxin (B) was examined by onist on the rolling of CFDA-labeled human eosinophils in in- IVM. To determine the effect of 5-HT2A and CCR3 antagonists on eosinophil rolling, cells were incubated ex vivo with CYP or DPM, respectively, for 15 min flamed blood vessels of the rabbit mesentery by IVM (Fig. 3). http://www.jimmunol.org/ at room temperature before being injected into the rabbit mesentery. Results are Superfusion of the mesentery with 50 nM 5-HT resulted in a Ϯ Ϯ expressed as mean SE of the RF of eosinophils from five different donors 3.5-fold increase in the flux of rolling eosinophils (RF: 16 3% studied in three to five venules per rabbit. (control) vs 56 Ϯ 8% (5-HT); p ϭ 0.004). Pretreatment of eosinophils with CYP (10 ␮M) resulted in near complete inhibition of rolling (p Ͻ 0.003). The rolling of vehicle-treated eosinophils was not affected (data not shown). As a control, superfusion of the mesentery with eotaxin (50 nM) resulted in

a 3-fold increase in eosinophil rolling (RF: 33 Ϯ 5% (eotaxin) by guest on September 26, 2021 vs 11 Ϯ 2% (control) p ϭ 0.01). This eotaxin-induced eosinophil rolling was completely inhibited by DPM (p ϭ 0.002). These results suggest that a functional 5-HT2A receptor is re- quired for multiple steps during 5-HT-induced migration of eosinophils.

5-HT2A antagonist inhibits allergen-induced pulmonary eosinophilia in EotϪ/Ϫ mice and functions additively with a CCR3 antagonist in WT mice To determine whether treatment with the 5-HT2A receptor antagonist CYP had any effect on a pathological eosinophil influx in a murine model of allergic asthma, WT as well as Ϫ Ϫ Eot / mice were sensitized with OVA and then treated with CYP, DPM, or saline (control) before allergen challenge (Fig. 4). OVA sensitization followed by aerosolized allergen chal- Ϫ Ϫ lenge induced significant BAL eosinophilia in Eot / as well as in WT mice. There was a 68% decrease in the total number of Ϫ Ϫ FIGURE 2. 5-HT-induced human eosinophil migration is mediated eosinophils recovered in the BAL fluid from Eot / mice com- through the 5-HT2A receptor expressed by eosinophils. A, MDL-100907 (20 pared with that of WT mice, consistent with previous findings ␮M), a 5-HT2A-specific antagonist, significantly inhibited 5-HT-induced eo- ϭ (5). In the WT mice, i.p. administration of CYP or DPM before sinophil migration (n 4) in an in vitro chemotaxis assay using Transwell Ͼ ϭ chambers, while it had no effect on eotaxin (Eot)-induced chemotaxis. B, Eo- OVA challenge significantly inhibited ( 80%, p 0.01 for sinophils from two allergic subjects express the 5-HT2A receptor. 5-HT2A ex- both inhibitors) pulmonary eosinophilia compared with vehi- pression by eosinophils was determined by RT-PCR (lanes 2 and 3). The cle-treated OVA-challenged mice. Furthermore, a greater inhi- 5-HT2A receptor PCR product observed in these samples as well as in human bition of the eosinophil influx was observed when both antag- brain RNA, which was used as a positive control (lane 4), was 254 bp. Lane 1, onists were used (94% reduction, p ϭ 0.01). Interestingly, in The 100-bp ladder. C, Western blot analysis of 5-HT2A receptor protein ex- Ϫ Ϫ the Eot / mice, CYP was effective in significantly reducing pression by untreated eosinophils from allergic subjects (Eos) or eosinophils ϭ stimulated with 20 ng/ml IL-5 (Eos ϩ IL-5) or 50 nM eotaxin (Eos ϩ Eot) for pulmonary eosinophilia (80%, p 0.04) while the administra- 6 h was performed. HEK 293 cell lysates were used as a negative control. Equal tion of DPM had no effect in reducing early stage eosinophilia amounts of protein were loaded in each lane. under these experimental conditions. When both antagonists 3602 CUTTING EDGE: SEROTONIN: A CHEMOTACTIC FACTOR FOR EOSINOPHILS

eotaxin-CCR3 interaction in allergic inflammation, they also illustrate that other eosinophil chemoattractants that function through different receptors are also likely to be involved in attracting eosinophils to lung tissue since blocking CCR3 and/or eotaxin resulted only in partial inhibition of pulmonary eosinophilia. This study confirms previous findings demonstrating the role eotaxin plays in early-stage eosinophil recruitment to the airway tissue (5) as there was a 67% decrease of eosinophils Ϫ Ϫ in the BAL fluid isolated from OVA-challenged Eot / mice compared with that of WT mice. However, in studies examin- ing WT mice, the 5-HT2A antagonist CYP inhibited eosino- FIGURE 4. CYP inhibits allergen-induced pulmonary eosinophilia in WT Ϫ Ϫ philia to a similar extent as the CCR3 antagonist DPM. Since and Eot / mice and exerts an additive effect with CCR3 receptor antagonists in a murine model of allergic airway inflammation. WT and EotϪ/Ϫ mice were 5-HT has been shown to induce lung fibroblasts to produce sensitized and aerosol challenged with OVA. Fifteen minutes before the three eotaxin (10), this raises the question of whether CYP acts by aerosol challenges with OVA, the indicated cohorts of mice (n ϭ 3) were treated blocking eotaxin production by lung fibroblasts or whether it with either CYP (0.2 mg/mouse) and/or DPM (2 mg/mouse) administered i.p. acts in a more direct manner to inhibit eosinophil recruitment Ϫ Ϫ No adverse behavioral effects were observed in the treated mice compared with by 5-HT. Examination of the Eot / mice challenged with control mice. One hour after the third inhaled OVA challenge, the WT and Ϫ Ϫ aerosolized OVA and treated with CYP showed a profound de- Downloaded from Eot / mice were sacrificed and the number of eosinophils in BAL fluid was enumerated in the presence and absence of 5-HT2A and CCR3 antagonist ad- crease in pulmonary eosinophilia, suggesting that 5-HT can ministration. Results are expressed as the mean Ϯ SEM of the percentage of play an integral role in allergic eosinophil recruitment indepen- eosinophils in BAL fluid of two separate experiments. dent of the involvement of eotaxin. This is underscored by the Ϫ Ϫ lack of effect elicited by DPM treatment in the Eot / mice. Furthermore, treatment of OVA-challenged WT mice with were administered in combination, there was no further reduc- CYP and DPM had a greater inhibitory effect than either an- http://www.jimmunol.org/ tion in pulmonary eosinophilia compared with administration tagonist alone. of CYP alone (78%, p Ͼ 0.01). These results clearly demon- A recent study demonstrated that airway hyperresponsiveness strate a role for 5-HT as a chemoattractant for eosinophils in and eosinophilia can be modulated by histamine and 5-HT2 Ϫ Ϫ allergen-challenged WT as well as Eot / mice. Overall, our receptor antagonists in a mouse model of allergic inflammation studies demonstrate that there is an additive effect in preventing (20). However, the mechanism by which the effects of 5-HT pulmonary eosinophilia when both 5-HT2A and CCR3 antag- were mediated is difficult to interpret in these studies as ketan- onists are used in WT mice and that 5-HT independently plays serin, the 5-HT2 antagonist used in the study, also antagonizes

a significant role in the eosinophilia observed in the murine the CCR3 receptor. Additionally, another study investigating by guest on September 26, 2021 model of allergic inflammation as demonstrated by the studies bronchospasm in asthmatic humans showed that ketanserin Ϫ Ϫ with Eot / mice. had a protective effect on adenosine-induced bronchoconstric- tion (21). However, the mechanism by which ketanserin elic- Discussion ited these protective effects was not determined. The current In this study, we demonstrate that 5-HT by itself is a potent study using multiple 5-HT2A receptor antagonists in in vitro eosinophil chemoattractant that can act additively with eotaxin. chemotaxis and in vivo rolling studies as well as in a murine Ϫ/Ϫ Furthermore, we demonstrate that pharmacological antago- model of allergic inflammation with WT and Eot mice nism of the 5-HT2A receptor by multiple receptor antagonists clearly shows that the effects of 5-HT are mediated by this re- (CYP, ketanserin, pirenperone, and MDL-100907) blocks ceptor. Although CYP has been shown to have some antagonist 5-HT-mediated chemotaxis in highly controlled in vitro che- effect on the histamine H2 receptor, CYP can block 5-HT-me- motaxis experiments. In addition, CYP was also observed to diated eosinophil chemotaxis in vitro as well as 5-HT-induced block 5-HT-mediated rolling of eosinophils under conditions eosinophil rolling in rabbit postcapillary venules in the absence of physiologic blood flow in postcapillary venules and to inhibit of histamine stimulation. Thus, the effects of CYP observed in Ϫ/Ϫ allergen-induced pulmonary eosinophilia in mice. It is conceiv- the allergen-challenged WT and Eot mice are most likely able that 5-HT provides a gradient to circulating eosinophils due to antagonism of the 5-HT2A receptor. and, through interaction with the 5-HT2A receptor expressed In summary, we demonstrate that 5-HT independently acts by eosinophils, induces a series of signaling events resulting in as a chemoattractant for eosinophils and this effect appears to be rolling (the earliest step of the adhesion cascade) and subse- mediated by the 5-HT2A receptor. Additionally, 5-HT has an quent transmigration of the cells toward the source of the che- additive effect with eotaxin, inducing eosinophil chemotaxis moattractant as observed in the case of other eosinophil active and pulmonary eosinophilia. chemoattractants (11, 18, 19). Overall, these data strongly suggest that the 5-HT2A receptor is responsible for mediating the References chemoattractant effects of 5-HT. 1. Weller, P. F., K. Lim, H. C. Wan, A. M. Dvorak, D. T. Wong, W. W. Cruikshank, These observations are critical for understanding the role of H. Kornfeld, and D. M. Center. 1996. Role of the eosinophil in allergic reactions. Eur. various chemoattractants in orchestrating eosinophil migra- Respir. J. Suppl. 22:109s. 2. DeMonchy, J. G., H. F. Kauffman, P. Venge, G. H. Koeter, H. M. Jansen, tion. Several studies have clearly demonstrated the importance H. J. Sluiter, and K. DeVries. 1985. Bronchoalveolar eosinophilia during allergen in- of CCR3 and eotaxin in regulating eosinophil migration using duced late asthmatic reactions. Am. Rev. Respir. Dis. 131:373. 3. Durham, S. R., and A. B. Kay. 1985. Eosinophils, bronchial hyperreactivity, and late murine models of allergic inflammation (5–7). However, al- phase asthmatic reactions. Clin. Allergy 15:411. though these studies demonstrate the importance of the 4. Weller, P. F. 1991. The immunobiology of eosinophils. N. Engl. J. Med. 324:1110. The Journal of Immunology 3603

5. Rothenberg, M. E., J. A. MacLean, E. Pearlman, A. D. Luster, and P. Leder. 1997. receptor antagonist MDL 100907 as a putative atypical antipsychotic: behavioral, Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue electrophysiological and neurochemical studies. J. Pharmacol. Exp. Ther. 266:684. eosinophilia. J. Exp. Med. 85:785. 14. Kehne, J. H., H. J. Ketteler, T. C. McCloskey, C. K. Sullivan, M. W. Dudley, and 6. Humbles, A. A., B. Lu, D. S. Friend, S. Okinaga, J. Lora, A. Al-Garawi, T. R. Martin, C. J. Schmidt. 1996. Effects of the selective 5-HT2A receptor antagonist MDL N. P. Gerard, and C. Gerard. 2002. The murine CCR3 receptor regulates both the 100907 on MDMA-induced locomotor stimulation in rats. Neuropsychopharmacology role of eosinophil and mast cells in allergen-induced airway inflammation and hyper- 15:116. responsiveness. Proc. Natl. Acad. Sci. USA 99:1479. 15. Broide, D. H., S. Sullivan, T. Gifford, and P. Sriramarao. 1998. Inhibition of pulmo- 7. Gonzalo, J. A., C. M. Lloyd, L. Kremer, C. Finger, C. Martinez-A, M. H. Siegelman, nary eosinophilia in P-selectin and ICAM-1 deficient mice. Am. J. Respir. Cell Mol. M. Cybulsky, and J. C. Gutierez-Ramos. 1996. Eosinophil recruitment to the lung in Biol. 18:218. a murine model of allergic inflammation. The role of T-cells, chemokines and adhe- 16. Barnes, N. M., and T. Sharp. 1999. A review of central 5-HT receptors and their sion receptors. J. Clin. Invest. 98:2332. function. Neuropharmacology 38:1083. 8. Nomura, H., E. Sato, S. Koyama, M. Haniuda, K. Kubo, S. Nagai, and T. Izumi. 17. Wainscott, D. B., V. L. Lucaites, J. D. Kursar, M. Baez, and D. L. Nelson. 1996. 2001. Histamine stimulates alveolar macrophages to release neutrophil and monocyte Pharmacologic characterization of the human 5-hydroxytryptamine 2B receptor: ev- chemotactic activity. J. Lab. Clin. Med. 138:226. idence for species differences. J. Pharmacol. Exp. Ther. 276:720. 9. Cazzola, I., and M. G. Matera. 2000. 5-HT modifiers as a potential treatment of asthma. Trends Pharmacol. Sci. 21:13. 18. DiScipio, R. G., P. J. Daffern, M. A. Jagels, D. H. Broide, and P. Sriramarao. 1999. 10. Sato, E., M. Haniuda, H. Numanami, T. Ushiyama, A. Tsukadaira, S. Takashi, A comparison of C3a and C5a mediated stable adhesion of rolling eosinophil in post- Y. Okubo, and S. Koyama. 2002. Histamine and serotonin stimulate eotaxin produc- capillary venules and transendothelial migration in vitro and in vivo. J. Immunol. tion by a human lung fibroblast cell line. Int. Arch. Allergy Immunol. 128:12. 162:1127. 11. Boehme, S. A., S. K. Sullivan, P. D. Crowe, M. Santos, P. J. Conlon, P. Sriramarao, 19. Baggiolini, M. 1998. Chemokines and leukocyte traffic. Nature 392:565. and K. B. Bacon. 1999. Activation of mitogen-activated protein kinase regulates 20. De Bie, J. J., P. A. Henricks, W.W. Cruikshank G. Hofman, E.H. Jonker, eotaxin-induced eosinophil migration. J. Immunol. 163:1611. F.P. Nijkamp, and A.J. Van Oosterhout. 1998. Modulation of airway hyperrespon- 12. Sriramarao, P., U. H. von Andrian, E. C. Butcher, M. A. Bourdon, and D. H. Broide. siveness and eosinophilia by selective histamine and 5-HT receptor antagonists in a 1994. L-selectin and very late antigen-4 integrin promote eosinophil rolling at phys- mouse model of allergic asthma. Br. J. Pharmacol. 124:857. iological shear rates in vivo. J. Immunol. 53:4238. 21. Cazzola M, G. Assogna, G. Lucchetti, G. Cicchitto, and G. D’Amato. 1990. Effect of 13. Sorensen, S. M., J. H. Kehne, G. M. Fadayel, T. M. Humphreys, H. J. Ketteler, ketanserin, a new blocking agent of the 5-HT2 receptor, on airway responsiveness in Downloaded from C. K. Sullivan, V. L. Taylor, and C. J. Schmidt. 1993. Characterization of the 5-HT2 asthma. Allergy 45:151. http://www.jimmunol.org/ by guest on September 26, 2021