Eosinophils but Not of Neutrophils Stimulates Effector Functions of Human Interaction with Secretory Component

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Interaction with Secretory Component Stimulates Effector Functions of Human Eosinophils But Not of Neutrophils

This information is current as Youichi Motegi and Hirohito Kita of September 23, 2021. J Immunol 1998; 161:4340-4346; ; http://www.jimmunol.org/content/161/8/4340 Downloaded from References This article cites 49 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/161/8/4340.full#ref-list-1

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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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Interaction with Secretory Component Stimulates Effector Functions of Human Eosinophils But Not of Neutrophils1

Youichi Motegi and Hirohito Kita2

Eosinophils and their products are important in the pathophysiology of allergic inflammation in mucosal tissues. Secretory component bound to IgA mediates transepithelial transport of IgA and confers increased stability on the resultant secretory IgA; however, the effect of secretory component on the biologic activity of IgA is unknown. Here, we report that secretory IgA and secretory component preferentially activate human eosinophils. When eosinophils were stimulated with immobilized secretory IgA, degranulation and superoxide production were two- to threefold greater than when stimulated with serum IgA. In contrast, neutrophils responded similarly to secretory IgA and serum IgA. Flow cytometric analysis showed that eosinophils bound to purified secretory component. The binding of 125I-labeled secretory component was inhibited by unlabeled secretory component or secretory IgA but not by serum IgA. Superoxide production by eosinophils stimulated with cytokines or IgG was enhanced synergistically by immobilized secretory component; secre- Downloaded from tory component showed no effect on neutrophil activation. Finally, anti-CD18 mAb abolished eosinophil superoxide production stim- ␤ ulated with secretory IgA or secretory component but not with serum IgA, suggesting a crucial role for 2 integrins in eosinophil interactions with secretory IgA or secretory component. Thus, secretory component plays important roles in activating eosinophil functions but not neutrophil functions. This preferential interaction between secretory component and eosinophils may provide a novel mechanism to regulate mucosal tissue inflammation. The Journal of Immunology, 1998, 161: 4340–4346. http://www.jimmunol.org/ osinophils play important roles in the pathophysiology of Secretory IgA (S-IgA)3 is the main Ab secreted by the mucosa allergic diseases, such as bronchial asthma and allergic rhi- of the airways, gastrointestinal tract, and other mucosal tissues E nitis, and in host immunity to parasitic infections (reviewed (reviewed in Ref. 8). A high proportion of Ig-producing cells in the in Ref. 1). During such inflammatory reactions, eosinophils release mucosal lymphoid tissue is committed to the IgA isotype, and the toxic granule proteins, which act as cytotoxins on a variety of cell majority of IgA in mucosa is derived from local synthesis (9). types, including tracheal epithelial cells, pneumocytes, and parasites Receptors for the Fc portion of IgA (Fc␣R) have been established (reviewed in Ref. 1). In humans, eosinophils are tissue-resident cells on a variety of cell types, including lymphocytes (10, 11), mono- with Ͼ99% residing in tissues, especially in the mucosal tissues (2). cytes/macrophages (12, 13), neutrophils (14, 15), and eosinophils by guest on September 23, 2021 In healthy individuals, eosinophils are normally found only in the (16). The importance of IgA in mucosal immunity is further sup- intestine (2). In specimens from patients with bronchial asthma, eo- ported by the presence of secretory component (SC) in S-IgA (17). Epithelial cells express the polymeric Ig receptor, which binds and sinophils and released toxic granule proteins are found in the damaged mediates the transepithelial transport of IgA from the basolateral epithelium of bronchial tissues (3–5). In specimens from patients with side of the cells into the lumen (8, 18). The SC is a soluble pro- intestinal parasitic diseases, with allergic gastroenteritis or with celiac teolytic cleavage fragment of the extracellular domain of mem- disease, eosinophils are detected in large numbers in the lamina pro- brane-bound polymeric Ig receptor (19). Addition of SC to IgA pria of the small and large intestines (1). Furthermore, eosinophil in- confers increased stability on the resultant S-IgA by helping to filtration and degranulation are also observed in nasal epithelium of hold the IgA monomers together (20) and by masking proteolytic patients with upper respiratory allergy (6) and in biliary tracts of pa- sites from proteases present in mucosal secretions (21); however, tients with obstructive hepatic diseases (7). Thus, the association be- the effect of the addition of SC on the biologic activity of IgA is tween eosinophils and mucosal tissues in both healthy individuals and unknown. Recent studies showed that SC did not modify the abil- patients led us to speculate on the specific role(s) of eosinophils in ity of IgA to mediate hemagglutination inhibition and to neutralize mucosal inflammation and/or immunity. influenza virus in vitro (22). The SC consists of five Ig-like domains (23); this structure is used commonly as a cell surface receptor and an adhesion molecule. Furthermore, S-IgA immobilized onto Sepharose 4B beads potently stimulates eosinophil degranulation (24). These observations lead us to hypothesize that SC directly interacts with human eosinophils and plays an important role in activation of this leu- Department of Immunology, Mayo Clinic and Mayo Foundation, Rochester, MN 55905 kocyte. In this report, we demonstrate a unique function of SC as Received for publication April 6, 1998. Accepted for publication June 17, 1998. a costimulatory molecule for effector functions of human eosino- The costs of publication of this article were defrayed in part by the payment of page phils. This interaction between SC and eosinophils may provide a charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Grants AI 34577 and AI 34486 from the National Institutes of Health and by the Mayo Foundation. 3 Abbreviations used in this paper: S-IgA, secretory IgA; Fc␣R, receptor for the Fc 2 Address correspondence and reprint requests to Dr. Hirohito Kita, Department of portion of IgA; DCS, defined calf serum; EDN, eosinophil-derived neurotoxin; GM, Immunology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail ad- granulocyte-macrophage; HSA, human serum albumin; PE, phycoerythrin; SC, se- dress: [email protected] cretory component.

novel mechanism to regulate the magnitude of mucosal tissue eosinophil degranulation, the concentration of an eosinophil granule pro- inflammation. tein, eosinophil-derived neurotoxin (EDN), in the sample supernatants was measured by double Ab competition assay using radioiodinated EDN, rabbit anti-EDN Ab, and burro anti-rabbit IgG, as previously reported (27). Materials and Methods Total cellular EDN was measured in supernatants from cells lysed with Reagents 0.5% Nonidet P-40 detergent. To quantify neutrophil degranulation, the concentration of elastase in the sample supernatants was measured by col- Human S-IgA was obtained from Organon Teknika-Cappel (Malvern, PA) orimetric assay (28, 29). Twenty microliters of samples or serial dilutions or Accurate Chemical & Scientific (Westbury, NY). Purified human serum of elastase standards were mixed with 130 ␮l of HBSS with 10 mM IgA and IgG were purchased from ICN Pharmaceuticals (Costa Mesa, CA). HEPES in 96-well culture plates, and 50 ␮l of 2.5 mM elastase substrate Purified human SC was kindly provided by Dr. Michael W. Russell (Uni- (MeOSuc-Ala-Ala-Pro-Val-pNA) (30) was added. Reaction mixtures were versity of Alabama, Birmingham, AL) (25). The IgG fraction of goat anti- Ј incubated at 37°C for 1 h. After incubation, the absorbance at 405 nm in human SC Ab and phycoerythrin (PE)-conjugated F(ab )2 fragments of each well was measured with a microplate reader (Thermomax; Molecular donkey anti-goat IgG Ab were purchased from INCSTAR (Stillwater, MN) Devices), and the elastase concentration was calculated using an elastase and Jackson Immunoresearch Laboratories (West Grove, PA), respectively. ␣ standard curve made in the same assay. Total cellular elastase was mea- mAb against human Fc R (My43, mouse IgM) and control mAb (anti-ox sured in supernatants from cells lysed with 0.5% Nonidet P-40 detergent. erythrocytes, mouse IgM) were generously provided by Dr. Li Shen (Dart- All experiments were performed in duplicate. mouth Medical School, Hanover, NH) (13). Mouse anti-human CD18 mAb (mouse IgG1) and purified mouse IgG1, as a control, were purchased from Becton Dickinson (San Jose, CA) and ICN Pharmaceuticals, respectively. Superoxide anion generation Recombinant human granulocyte-macrophage (GM)-CSF was purchased from Genzyme (Cambridge, MA). Recombinant human IL-5 was gener- Generation of superoxide by eosinophils and neutrophils was measured by superoxide dismutase-inhibitable reduction of cytochrome c, as previously ously provided by Schering-Plough. Human serum albumin (HSA) and Downloaded from defined calf serum (DCS) were from Sigma Chemical (St. Louis, MO) and reported (31, 32). Flat-bottom 96-well tissue culture plates (Costar) were HyClone Laboratories (Logan, UT), respectively. Human neutrophil elas- blocked with 2.5% HSA. Freshly isolated eosinophils or neutrophils were tase and elastase substrate (MeOSuc-Ala-Ala-Pro-Val-pNA) were pur- suspended in HBSS with 10 mM HEPES, 0.1% HSA, and 100 ␮M cyto- 5 chased from Calbiochem-Novabiochem (La Jolla, CA). chrome c (Sigma) at 5 ϫ 10 cells/ml. Cell suspension (100 ␮l) was added to the wells and stimulated with 100 ␮l of Ig-coated Sepharose 4B beads Cell preparation suspended in HBSS with 10 mM HEPES and 0.1% HSA (bead-cell ratio, 1:10). In some experiments, Ig or SC immobilized onto tissue culture plates

Eosinophils were purified from peripheral blood obtained from normal vol- were used as stimuli instead of Ig-coated Sepharose 4B beads. In these http://www.jimmunol.org/ unteers as described previously (26). The percentage of eosinophils in pe- experiments, 100 ␮l of cell suspensions and 100 ␮l of HBSS with 10 mM Ͻ ripheral blood leukocytes of these donors was 5%. Briefly, heparinized HEPES and 0.1% HSA were added to the wells precoated with Ig or SC. venous blood was layered onto 1.085 g/ml Percoll (made in PIPES buffer IL-5 (1 ng/ml) or GM-CSF (0.1 ng/ml) was added to the wells to examine (25 mM PIPES, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose, the effects of cytokines on eosinophil superoxide production stimulated pH 7.4)) and centrifuged at 2000 rpm in Beckman CS-6KR (Beckman with immobilized SC. When the effects of mAb to Fc␣R and CD18 were Instruments, Fullerton, CA) for 30 min. Plasma, mononuclear cells, and tested, eosinophils were pretreated with 1/40 dilutions of anti-Fc␣R mAb Percoll layers were removed, and erythrocytes were lysed by osmotic (My43) or control Ab, 10 ␮g/ml anti-CD18 mAb, or 10 ␮g/ml mouse IgG1 shock. The remaining eosinophil/neutrophil pellet was mixed with anti- for 15 min at room temperature before the addition of Ig-coated Sepharose CD16-bound immunomagnetic beads (Miltenyi Biotec, Sunnyvale, CA) 4B beads. Immediately after the addition of the cells and stimulus, the and incubated for 1 h. The cells were then separated using a magnetic cell absorbance at 550 nm in each well was measured with a microplate reader separation system (MACS; Miltenyi Biotec). The eluate was collected, and (Thermomax; Molecular Devices, Menlo Park, CA), followed by repeated by guest on September 23, 2021 cell number and eosinophil purity were determined. Neutrophils were iso- readings for 3 h. Between absorbance measurements, the plate was incu- lated using a gradient material, 1-Step Polymorphs (Accurate Chemical & bated at 37°C. Superoxide anion generation was calculated with an extinc- Scientific), following the procedure recommended by the manufacturer. tion coefficient of 21.1 ϫ 103 cmϪ1 MϪ1 for reduced cytochrome c at 550 nm Ͼ The purities of eosinophil and neutrophil preparations were 97%. All the and was expressed as nanomoles of cytochrome c reduction per 1 ϫ 105 cells. isolation procedures were performed at 4°C or on ice. Preparation of stimuli Flow cytometry analysis of SC binding Human S-IgA, serum IgA, and SC immobilized onto cyanogen bromide- Binding of SC to eosinophils and neutrophils was examined by flow cy- activated Sepharose 4B beads (Sigma) or tissue culture plates were used as tometry. Purified eosinophils or neutrophils (1 ϫ 106 cells) were suspended stimuli for eosinophils and neutrophils. Sepharose 4B beads were coupled in RPMI 1640 medium supplemented with 25 mM HEPES, 1% BSA, and ␮ to S-IgA or serum IgA, at a concentration of 5 mg protein/ml of packed 0.1% NaN3, and incubated with or without 30 g/ml SC for 90 min on ice. beads, as previously described (24). To immobilize Ig or SC to tissue Cells were washed twice with PAB buffer (PBS containing 0.1% NaN3 and culture plates, 96-well flat-bottom tissue culture plates (Costar, Cambridge, 0.5% BSA, pH 7.4), and stained with 20 ␮g/ml goat IgG anti-human SC ␮ ␮ Ј MA) were coated with 50 l of indicated concentrations of S-IgA, serum Ab for 30 min on ice, followed by 5 g/ml PE-conjugated F(ab )2 fragment IgA, or SC diluted in PBS (pH 7.4) overnight at 4°C. Plates were then of donkey anti-goat IgG for 30 min on ice. The cells were then washed with blocked with 100 ␮l of DCS or 2.5% HSA diluted in PBS for2hat37°C. PAB buffer and fixed with 1% paraformaldehyde in PBS (pH 7.4). The After blocking, the wells were washed twice with 200 ␮l of PBS before fluorescence intensity of individual cells was measured with a FACScan use. In some experiments, the wells were coated with 50 ␮lof0.5␮g/ml (Becton Dickinson, Mountain View, CA), and analyzed by Becton Dick- human serum IgG overnight at 4°C before being subsequently coated with inson lysis II software. 50 ␮l of SC or HSA (as a control) diluted at 10 ␮g/ml in PBS for2hat 37°C. These plates were blocked with 100 ␮l of 2.5% HSA and washed 125 twice with 200 ␮l of PBS before use. Binding of I-labeled SC Degranulation assay SC was radioiodinated by IODO-GEN Iodination Reagent (Pierce, Rock- ford, IL) according to the procedure recommended by the manufacturer. Degranulation of eosinophils and neutrophils was induced by Ig immobi- SC (63 ␮g) was labeled with 500 ␮Ci of 125I, and the specific activity of lized onto Sepharose 4B beads as described previously with minor modi- labeled protein was 605 Ci/mmol. The binding of 125I-labeled SC to eo- fications (24). Briefly, 96-well round-bottom tissue culture plates (Costar sinophils was measured according to a method described previously for No. 3799) were blocked with 50 ␮l of 2.5% HSA in PBS (pH 7.4) at 37°C cytokines with minor modifications (33). Eosinophils (7 ϫ 105 cells/sam- for 2 h. After incubation, the wells were washed three times with PBS. ple) suspended in 50 ␮l of binding medium (RPMI 1640 medium supple-

Cells were washed once with RPMI 1640 medium containing 25 mM mented with 25 mM HEPES, 1% BSA, and 0.1% NaN3, pH 7.4) were HEPES and 0.1% HSA and resuspended in the same medium at 5 ϫ 105 incubated with 50 ␮l of 12.5 nM 125I-labeled SC in the presence or absence cells/ml. Aliquots (100 ␮l) of cell suspension were added to the tissue of a serial dilution of unlabeled SC, S-IgA, or IgA for2honarotating culture plates. Cellular degranulation was initiated by adding 100 ␮lof table at 4°C. Cells were then overlayered onto 200 ␮l of DCS and centri- Sepharose 4B beads coated with S-IgA or serum IgA (bead-cell ratio, fuged at 3000 rpm for 3 min. Pellets were frozen on dry ice and cut away

1:20). After incubation for4hat37°C in a 5% CO2 atmosphere, cell-free from the supernatant. Radioactivity in the pellets was measured in a gamma supernatants were collected and kept at Ϫ20°C until assayed. To quantify counter (Packard, Downers Grove, IL). 4342 EOSINOPHIL ACTIVATION BY SECRETORY COMPONENT

FIGURE 1. Degranulation of eosinophils and neutrophils induced by FIGURE 2. Superoxide production by eosinophils and neutrophils stim- S-IgA and serum IgA. Isolated eosinophils or neutrophils were incubated ulated with S-IgA or serum IgA. The wells of 96-well tissue culture plates with medium alone (Ⅺ), Sepharose 4B beads coated with S-IgA (f)or were coated with various concentrations of S-IgA or serum IgA and serum IgA (o) for 4 h. Eosinophil degranulation was quantified by mea- blocked with DCS. Isolated eosinophils or neutrophils were added to the suring the concentration of EDN in the cell-free supernatant by RIA. Neu- wells, and superoxide production was measured by the reduction of cyto- trophil degranulation was quantified by measuring the concentration of chrome c as described in Materials and Methods. The results of superoxide elastase in the cell-free supernatant by enzymatic assay. Data are expressed production at 120 min of incubation are summarized. Data are presented as significant differences ,ء .significant differ- mean Ϯ SEM from four independent experiments ,ء .as mean Ϯ SEM from six independent experiments ence (p Ͻ 0.001) compared with eosinophils incubated with serum IgA. (p Ͻ 0.05) compared with cells incubated with the same concentrations of Downloaded from serum IgA.

Statistical analysis at the suboptimal concentrations ( p Ͻ 0.05 at 1 ␮g/ml). Alto- Ϯ All results are shown as mean SEM from the numbers of experiments gether, these ﬁndings suggest that S-IgA stimulates eosinophil indicated. The statistical signiﬁcance of the differences was assessed with functions more potently than serum IgA. In contrast, S-IgA stimulates

Student’s paired t test. http://www.jimmunol.org/ neutrophil functions similarly to or less potently than serum IgA. Results Binding of SC to eosinophils Effects of S-IgA and serum IgA on degranulation of eosinophils and neutrophils The presence of SC in S-IgA, but not in serum IgA, is notable (reviewed in Ref. 8). Therefore, we hypothesized that SC binds to Previous studies suggested that both neutrophils (13–15) as well as eosinophils and activates their effector functions. To test this hy- eosinophils (16) possess Fc␣R. Therefore, we examined whether pothesis, we first examined whether eosinophils are able to bind to S-IgA and serum IgA induce effector functions of these two cell SC by flow cytometry. Purified eosinophils were incubated with or types. Purified eosinophils and neutrophils were stimulated with

without SC, and SC bound to the cells was detected by anti-SC Ab by guest on September 23, 2021 Sepharose 4B beads coated with S-IgA or serum IgA, and degran- followed by fluorochrome-conjugated secondary Ab. As shown in ulation was assessed by measuring the amounts of EDN (for eo- Figure 3, in the absence of SC, minimal amounts of SC were sinophils) and elastase (for neutrophils) in the supernatants. As detected on the surface of eosinophils. In the presence of SC, the shown in Figure 1, serum IgA-beads induced both EDN release binding was greatly increased, suggesting eosinophils do bind to from eosinophils and elastase release from neutrophils, consistent SC. The geometric mean fluorescence intensity of samples stained with the expression of Fc␣R on these two cell types (13–16). S- with control Ab, anti-SC Ab (without SC), and anti-SC Ab (with IgA-beads released approximately twice the amount of EDN compared with those stimulated with serum IgA-beads ( p Ͻ 0.001). In contrast, there were no significant differences in elastase released from neutrophils stimulated with S-IgA-beads and serum IgA- beads. Thus, S-IgA is likely a more potent secretagogue than serum IgA for eosinophils but not for neutrophils. Next, we examined whether another function of eosinophils, i.e., superoxide production, is stimulated with S-IgA. By using the same detection system, the superoxide production assay also al- lows direct comparison of eosinophils and neutrophils. Purified cells were stimulated with serial dilutions of S-IgA or serum IgA immobilized onto tissue culture wells. As shown in Figure 2, left, both S-IgA and serum IgA induced superoxide production from eosinophils in a concentration-dependent manner. The stimulatory effects of these Ig reached a plateau at 3 or 10 ␮g/ml. Striking differences were found in the amounts of superoxide released; S- IgA induced significantly larger amounts of superoxide production FIGURE 3. Flow cytometric analyses of SC binding to eosinophils. Iso- than serum IgA at the optimal concentrations ( p Ͻ 0.05). At 10 lated eosinophils were incubated with or without SC (30 ␮g/ml) for 90 min ␮g/ml, eosinophils stimulated with S-IgA produced ϳ2.5-fold on ice. After washing, the cells were stained with goat IgG anti-human SC (final concentration, 20 ␮g/ml), followed by PE-conjugated F(abЈ) frag- more superoxide compared with those stimulated with serum IgA. 2 ments of donkey anti-goat IgG (final concentration, 5 ␮g/ml). The cells In neutrophils, both S-IgA and serum IgA induced superoxide pro- were then washed, fixed with 1% paraformaldehyde, and analyzed by flow duction in a concentration-dependent manner; however, there was cytometry. Controls indicated by shaded area are the cells incubated with no difference between the magnitudes of neutrophil responses to Ј goat IgG (isotype-matched control), followed by PE-conjugated F(ab )2 S-IgA and to serum IgA at the optimal concentrations (Fig. 2, fragments of donkey anti-goat IgG. Histograms of one of three experiments right). S-IgA induced less superoxide production than serum IgA showing similar results are presented. The Journal of Immunology 4343

FIGURE 4. Binding of 125I-labeled SC to eosinophils and inhibition by unlabeled competitors. Isolated eosinophils were incubated with 12.5 nM of 125I-labeled SC in the presence or absence of the indicated concentrations of unlabeled SC, S-IgA or serum IgA for 2 h. After incubation, eosinophils were separated from unbound 125I-labeled SC by centrifugation, and radioactivity in the cell pellets was measured by a gamma counter. Results are presented as means from two experiments.

FIGURE 5. Effects of immobilized SC on superoxide production by eo- Downloaded from sinophils and neutrophils stimulated with GM-CSF and IL-5. A, The wells of 96-well tissue culture plates were coated with (closed symbols) or without (open symbols) 10 ␮g/ml SC and blocked with HSA. Isolated eosin- SC) were 77.2 Ϯ 9.2, 76.7 Ϯ 9.3, and 116.2 Ϯ 13.0, respectively Ϯ ϭ Ͻ ophils (left) or neutrophils (right) were added to the wells, and incubated (mean SEM, n 3, p 0.01 between “without SC” and in the presence or absence of 0.1 ␮g/ml GM-CSF. Kinetics of superoxide “with SC”). production was measured by the reduction of cytochrome c as described in The specificity of SC binding to eosinophils was further exam- Materials and Methods. One of four experiments showing similar results is http://www.jimmunol.org/ 125 ined by using I-labeled SC. Eosinophils were incubated with presented. B, The wells of 96-well tissue culture plates were coated with 125I-labeled SC in the presence or absence of serial dilutions of (closed columns) or without (open columns) 10 ␮g/ml SC and blocked unlabeled SC, S-IgA, or IgA. As shown in Figure 4, when eosin- with HSA. Isolated eosinophils or neutrophils were added to the wells and ophils were incubated with 12.5 nM 125I-labeled SC, substantial incubated in the presence or absence of 0.1 ng/ml GM-CSF or 1 ng/ml IL-5 amounts of bound SC were detected. This binding of 125I-labeled for 120 min. Superoxide production was measured by the reduction of Ϯ SC was inhibited by unlabeled SC in a concentration-dependent cytochrome c. Data are presented as mean SEM from four independent -significant differences (p Ͻ 0.01) compared with eosin ,ءء .experiments manner, indicating specific binding of SC to eosinophils. Further- ophils incubated without SC. more, the binding of 125I-labeled SC was also inhibited by unlabeled S-IgA in a concentration-dependent manner albeit not as by guest on September 23, 2021 potently as unlabeled SC. In contrast, binding of 125I-labeled SC was not inhibited by unlabeled serum IgA. The findings described above suggest that SC acts synergistically with other agonists (e.g., cytokines) to stimulate eosinophil Effects of SC on eosinophil activation function. As shown in Figure 6, this concept was further tested by coimmobilizing SC and IgG onto the wells of tissue culture plates. GM-CSF activates effector functions of both eosinophils (34, 35) By itself, immobilized IgG induced superoxide production from and neutrophils (36). Furthermore, IL-5 enhances various func- both eosinophils and neutrophils. Eosinophil superoxide produc- tions of eosinophils, such as degranulation, helminthotoxicity, and tion stimulated with IgG was enhanced strikingly by SC coimmo- survival, and it is implicated in the pathophysiology of allergic bilized to the wells (107 Ϯ 18% of enhancement, mean Ϯ SEM, diseases (reviewed in Ref. 37). GM-CSF and IL-5 can also induce superoxide production from eosinophils (38). Thus, we examined whether binding of SC to eosinophils leads to enhancement of their effector functions when stimulated with these cytokines. Eosino- phils and neutrophils, as a control, were added to wells coated with SC in the presence or absence of cytokines; superoxide production was measured. As shown in Figure 5A, left, immobilized SC by itself did not induce eosinophil superoxide production up to 120 min; GM-CSF, 0.1 ng/ml, by itself induced moderate superoxide production; SC and GM-CSF worked synergistically and induced considerable superoxide production. In contrast, as shown in Fig- ure 5A, right, GM-CSF induced minimal superoxide production from neutrophils, and immobilized SC did not enhance superoxide FIGURE 6. Effects of SC coimmobilized with IgG on superoxide pro- production. Figure 5B summarizes five experiments with a similar duction by eosinophils and neutrophils. The wells of 96-well tissue culture experimental design. Although immobilized SC by itself did not plates were incubated with PBS or 0.5 ␮g/ml human serum IgG overnight. induce superoxide production from eosinophils, immobilized SC After removal of solutions, the wells were subsequently coated with 10 ␮g/ml SC (f) or HSA (Ⅺ) for 2 h. The wells were then blocked with 2.5% strikingly enhanced the eosinophil response to GM-CSF by 80% Ͻ HSA. Isolated eosinophils or neutrophils were added to the wells and (mean of five experiments, p 0.01). Likewise, eosinophil super- incubated for 120 min. Superoxide production was measured by the oxide production stimulated with 1 ng/ml IL-5 was enhanced by reduction of cytochrome c as described in Materials and Methods. Data ,ء .immobilized SC by 92% (mean of five experiments, p Ͻ 0.01). In are presented as mean Ϯ SEM from three independent experiments contrast, immobilized SC did not affect neutrophil superoxide pro- significant differences (p Ͻ 0.05) compared with eosinophils incubated duction either in the presence or absence of GM-CSF. without SC. 4344 EOSINOPHIL ACTIVATION BY SECRETORY COMPONENT

FIGURE 8. Effects of anti-CD18 mAb on eosinophil superoxide production induced by immobilized SC. The wells of 96-well tissue culture plates were coated with 10 ␮g/ml of SC and blocked with HSA. Eosino- FIGURE 7. Effects of anti-Fc␣R mAb and anti-CD18 mAb on eosinophils were added to the wells; preincubated with medium alone (none), 10 phil superoxide production induced by S-IgA or serum IgA. The wells of ␮g/ml anti-CD18 mAb, or 10 ␮g/ml mouse IgG1 (control); and stimulated 96-well tissue culture plates were blocked with HSA. Eosinophils were for 60 min with 0.1 ng/ml GM-CSF. Superoxide production was measured preincubated in the wells with medium alone, anti-Fc␣R mAb (clone by the reduction of cytochrome c as described in Materials and Methods. My43, mouse IgM, 1/40 dilution of hybridoma supernatants), control mAb Data are presented as mean Ϯ SEM from three independent experiments. signiﬁcant differences (p Ͻ 0.01) compared with control. Downloaded from ,ءء -anti-ox erythrocytes, mouse IgM, 1/40 dilution of hybridoma superna) tants), 10 ␮g/ml anti-CD18 mAb (mouse IgG1), or 10 ␮g/ml control Ig (mouse myeloma IgG1) for 15 min at room temperature and stimulated for 60 min with Sepharose 4B beads coated with S-IgA or serum IgA. Super- ␣ oxide production was measured by the reduction of cytochrome c as de- and 2), and an anti-Fc R inhibits the eosinophil response (Fig. 7). scribed in Materials and Methods. Data are presented as mean Ϯ SEM These results are consistent with previous observations showing from four independent experiments. Signiﬁcant differences compared with that both eosinophils and neutrophils express Fc␣R (13–16), bind

/p Ͻ 0.01. IgA (14, 24, 40, 41), and can be stimulated through this receptor http://www.jimmunol.org ,ءء ;p Ͻ 0.05 ,ء .isotype-matched control Abs are shown (14, 24, 42). However, eosinophil and neutrophil responses to S- IgA were strikingly different. In eosinophils, S-IgA induced more EDN release and superoxide production than serum IgA; but in n ϭ 3). In contrast, neutrophil superoxide production stimulated neutrophils, S-IgA and serum IgA induced similar elastase release with IgG was not enhanced by coimmobilized SC. and S-IgA induced slightly less superoxide production than serum ␤ IgA. Compatible observations by others demonstrated that the eo- Role of 2 integrins for the effects of SC on eosinophil activation sinophil chemotactic response to IL-8 was enhanced by immobilized S-IgA but not by serum IgA (43). Furthermore, Staphylococ- Recent studies suggest that adhesion molecules, especially ␤ in- by guest on September 23, 2021 2 cus aureus opsonized with serum IgA and S-IgA induced similar tegrins, play critical roles in eosinophil functions stimulated by degrees of respiratory burst in neutrophils (42). Therefore, it is cytokines and lipid mediators (31, 32). Furthermore, degranulation apparent that S-IgA stimulates functions of eosinophils more po- of eosinophils stimulated with immobilized IgG has been com- tently than serum IgA, and that S-IgA and serum IgA stimulate pletely inhibited by either mAb against Fc␥RII receptors (32) or functions of neutrophils similarly. mAb against CD18 (39), suggesting that IgG-mediated eosinophil This stronger stimulatory effect of S-IgA compared with serum functions are controlled by both the Fc␥ receptor and ␤ integrin. 2 IgA on eosinophils is unlikely due to the difference in binding Therefore, to investigate the mechanism of SC on eosinophils, we efficiency of S-IgA and IgA to solid surfaces, such as Sepharose examined the roles of the IgA receptor, Fc␣R, and ␤ integrin in 2 4B beads or plastic plates, because the responses of neutrophils to eosinophil function stimulated with serum IgA and S-IgA. An anti- immobilized S-IgA were similar to or slightly less than those to Fc␣R mAb, My43, inhibits binding of IgA to neutrophils (13). As serum IgA (Figs. 1 and 2). The differences in the potencies of shown in Figure 7A, eosinophil superoxide production stimulated S-IgA and serum IgA cannot be explained by the differences in with serum IgA was significantly inhibited by this anti-Fc␣R mAb IgA isotypes, because eosinophils respond similarly to IgA1 and ( p Ͻ 0.05), suggesting that Fc␣R similar to that on neutrophils is IgA2 (24). Thus, SC, which is bound to S-IgA but not to serum involved in eosinophil activation stimulated with serum IgA. In IgA, may activate eosinophil functions. In fact, we found that bind- contrast, anti-CD18 mAb did not show any effect on eosinophil ing of purified SC to eosinophils was inhibited by S-IgA but not by superoxide production stimulated with serum IgA. As shown in serum IgA (Fig. 4). SC immobilized onto the tissue culture plates Figure 7B, when eosinophils were stimulated with S-IgA, anti- strikingly enhanced eosinophil superoxide production stimulated Fc␣R mAb inhibited superoxide production minimally but signif- with cytokines, such as GM-CSF and IL-5 (Fig. 5), or IgG (Fig. 6); icantly ( p Ͻ 0.05); in contrast, S-IgA-induced superoxide produc- SC did not affect neutrophil superoxide production stimulated with tion was almost abolished by anti-CD18 mAb, which blocked the GM-CSF or IgG. We also noted that SC by itself does not provoke ␤ integrin ( p Ͻ 0.01). Thus, the major difference in eosinophil 2 eosinophil functions in the absence of other agonists. These find- function stimulated with serum IgA and S-IgA is the involvement ings suggest that, in eosinophils, SC acts synergistically with other of ␤ integrin; SC is likely the critical molecule in this difference. 2 agonists, such as cytokine and Ig, and stimulates their effector Consistent with this observation, as shown in Figure 8, eosinophil functions. In contrast, neutrophil function is not affected by SC superoxide production stimulated by immobilized SC and GM- even in the presence of other agonists. Thus, SC likely has a CSF was completely inhibited by anti-CD18 mAb. unique capacity to stimulate eosinophils specifically. Then, how does SC activate eosinophil functions? Our previous Discussion ␤ studies suggest that adhesion molecules, especially 2 integrins, In this study, we found that serum IgA stimulates degranulation play crucial roles in eosinophil activation stimulated with cyto- and superoxide production by eosinophils and neutrophils (Figs. 1 kines or IgG (31, 32). Eosinophil superoxide production stimulated The Journal of Immunology 4345 with these agonists was strikingly enhanced when cells were able unique Ig among the various isotypes because of its restricted ␤ to adhere to substrates through the 2 integrins (31, 32). In this ability to use ancillary effector mechanisms, such as activation ␤ study, we found that 2 integrins were involved in eosinophil su- of complement, phagocytosis, and binding to effector cells (re- peroxide production after stimulation with S-IgA (Fig. 7B) but not viewed in Ref. 56). It would be interesting to know how eosin- with serum IgA (Fig. 7A). Furthermore, eosinophil superoxide pro- ophils interacting strongly with S-IgA may fit into this global ␤ duction on SC-coated plates was also totally dependent on 2 in- paradigm of IgA and mucosal immunology. ␤ tegrins (Fig. 8). Therefore, 2 integrins play critical roles in up- regulation of eosinophil function by SC. Acknowledgments The remaining question is how SC stimulates eosinophil functions through ␤ integrins. Although this issue is currently under We thank Drs. M. W. Russell and L. Shen for providing us with purified 2 ␣ study in our laboratory, we can discuss several possibilities. It is human SC and anti-Fc R mAb, respectively. We also thank Dr. G. J. Gleich for critical reading of the manuscript, Linda H. Arneson for secretarial assis- commonly accepted that integrins in a resting cell do not bind or tance, and Cheryl Adolphson for editorial assistance. bind weakly to their ligands and that cellular activation is required for firm interaction between integrins and their ligands (reviewed ␤ References in Ref. 44). Furthermore, 2 integrins are often promiscuous, binding to various molecules (45). Therefore, the first possibility is that 1. Gleich, G. J., C. R. Adolphson, and K. M. Leiferman. 1992. Eosinophils. In Inflammation: Basic Principles and Clinical Correlates, 2nd Ed. J. I. Gallin, binding of SC may specifically stimulate the avidity of eosinophil I. M. Goldstein, and R. Snyderman, eds. Raven Press, New York, pp. 663–700. ␤ 2 integrins. The second and more likely possibility is that SC may 2. Osgood, E. E. 1954. Number and distribution of human hemic cells. Blood 9:114. ␤ 3. Bousquet, J., P. Chanez, J. Y. Lacoste, G. Barneon, N. Ghavanian, I. Enander, Downloaded from serve as a ligand for 2 integrins. In fact, SC stimulated eosinophil P. Venge, S. Ahlstedt, J. Simony-Lafontaine, P. Godard, and F. B. Michel. 1990. function only when eosinophils were stimulated with cytokines or Eosinophilic inflammation in asthma. N. Engl. J. Med. 323:1033. Ig; this characteristic is similar to stimulus-dependent up-regula- 4. de Monchy, J. G. R., H. F. Kauffman, P. Venge, G. H. Koeter, H. M. Jansen, tion of the ligand-binding capacity of integrins (44). Our Scatchard H. J. Sluiter, and K. de Vries. 1985. 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