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The Journal of Immunology, 2002, 168: 4756-4763.
Copyright © 2002 by The American Association of Immunologists

IL-16 Promotes Leukotriene C4 and IL-4 Release from Human Eosinophils via CD4- and Autocrine CCR3-Chemokine-Mediated Signaling1

Christianne Bandeira-Melo*, Kumiya Sugiyama*, Lesley J. Woods*, Mojabeng Phoofolo*, David M. Center{dagger}, William W. Cruikshank{dagger} and Peter F. Weller2,*

* Department of Medicine, Harvard Thorndike Laboratories, Charles A. Dana Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and {dagger} Pulmonary Center, Boston University School of Medicine, Boston, MA 02118


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human eosinophils are potential sources of inflammatory and immunomodulatory mediators, including cysteinyl leukotrienes, chemokines, and cytokines, which are pertinent to allergic inflammation. We evaluated the means by which IL-16, a recognized eosinophil chemoattractant, might act on eosinophils to affect their capacity to release leukotriene C4 (LTC4) or their preformed stores of chemokines (eotaxin, RANTES) or Th1 (IL-12) or Th2 (IL-4) cytokines. IL-16 dose dependently (0.01–100 nM) elicited new lipid body formation, intracellular LTC4 formation at lipid bodies, and priming for enhanced calcium ionophore-activated LTC4 release. IL-16 also elicited brefeldin A-inhibitable, vesicular transport-mediated release of preformed IL-4, but not IL-12, from eosinophils. CD4 is a recognized IL-16R, and accordingly anti-CD4 Fab, soluble CD4, and a CD4 domain 4-based IL-16 blocking peptide inhibited the actions of IL-16 on eosinophils. Although CD4 is not G-protein coupled, pertussis toxin inhibited IL-16-induced eosinophil activation. IL-16 actions were found to be mediated by the autocrine activity, not of platelet-activating factor, but rather of endogenous CCR3-acting chemokines. IL-16 induced the rapid vesicular transport-mediated release of RANTES. The effects of IL-16 were blocked by CCR3 inhibitors (met-RANTES, anti-CCR3 mAb) and by neutralizing anti-eotaxin and anti-RANTES mAbs, but not by platelet-activating factor receptor antagonists (CV6209, BN52021). RANTES and eotaxin each enhanced LTC4 and IL-4 (but not IL-12) release. Therefore, IL-16 activation of eosinophils is CD4-mediated to elicit the extracellular release of preformed RANTES and eotaxin, which then in an autocrine fashion act on plasma membrane CCR3 receptors to stimulate both enhanced LTC4 production and the preferential release of IL-4, but not IL-12, from within eosinophils.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-16 is a cytokine initially identified as a chemoattractant for CD4+ T cells and subsequently shown to be a chemoattractant for other CD4+ cells, including eosinophils (1, 2). IL-16 can be formed by varied cell types including CD4+ and CD8+ T cells, B cells, mast cells, dendritic cells, epithelial cells, fibroblasts, and eosinophils (1, 3, 4). IL-16 may have roles in diverse immunologic responses (1). In forms of allergic inflammation, including asthma, IL-16 has been found to be prominent. IL-16 was increased in the bronchial mucosa and sputum of patients with atopic asthma (5, 6) and in the bronchial mucosa and bronchoalveolar lavage fluids after segmental allergen challenge (7, 8). Likewise, IL-16 increases in the nasal mucosa of patients with allergic rhinitis after experimental or seasonal allergen exposures (9, 10). In murine models of asthma, administration of anti-IL-16 mAb prevented the production of Ag-specific IgE (11), and administration of an IL-16 blocking peptide inhibited Ag-induced airway hyperresponsiveness (12).

IL-16 may participate in other forms of inflammation, including inflammatory bowel disease (13), in which eosinophils may be a source of IL-16 (14). The functions of IL-16 in these varied forms of immunologically mediated diseases may be complex. For instance, IL-16 can inhibit MLR- and anti-CD3-activated lymphocyte responses (15, 16), can participate in dendritic cell-T cell interactions (4, 17), can enhance pro-inflammatory cytokine release from monocytes (18), and can inhibit IL-5 production by Ag-stimulated T cells in atopic subjects (19).

We had previously shown that IL-16 was a potent (ED50 ~ 10-12 M) chemoattractant for human eosinophils and that this activity was dependent on CD4 expressed by eosinophils (2). IL-16, however, did not appear to enhance other responses of eosinophils, such as their capacity for enhanced leukotriene C4 (LTC4)3 formation or their capacity to "degranulate," as assessed by fluid phase assays of granule-derived arylsulfatase B release (2). Just as there is an increasing recognition of the varied activities of IL-16, there has been an increased understanding of the complexities of eosinophil cell biology and functioning in varied immunologic responses. Eosinophils may have functions in immune responses extending beyond their conventional "degranulation"-based effector responses, and these may include functioning as APCs to promote Th2 CD4+ responses (20, 21). Eosinophils are now recognized to contain preformed stores of diverse cytokines and chemokines within their cytoplasmic granules (22). Thus, in addition to their distinctive cationic proteins, eosinophil granules contain chemokines (e.g., eotaxin (23) and RANTES (24, 25)) and cytokines with disparate and potentially opposing activities, notably including the prototypical Th2 cytokine IL-4 (26, 27, 28) and the Th1 cytokine IL-12 (29, 30, 31). The regulated release of these preformed cytokines occurs not by exocytotic fusion of granules with the plasma membranes to extrude all granule contents, but rather by selective processes based on vesicular mobilization and transport of these granule-derived cytokines (32, 33, 34).

We have now applied more sensitive, recently developed investigative methods (32, 33) to evaluate whether IL-16 can promote LTC4 formation by eosinophils and whether IL-16 can participate in the regulated and potentially differential release of chemokines or cytokines that are preformed within human eosinophils. Our data demonstrate that IL-16 can activate specific responses of human eosinophils and in a CD4-dependent manner elicit the release of RANTES and eotaxin from eosinophils, which then function as CCR3-mediated autocrine activators to enhance eosinophil eicosanoid formation, and a selective release of specific cytokines from eosinophil granule stores.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Eosinophil purification

Peripheral blood was obtained with informed consent from 16 normal donors, and eosinophils were isolated as described (32). Briefly, after citrate-anticoagulated blood was mixed with 6% dextran-saline (MacGaw, Irvine, CA) to facilitate erythrocyte sedimentation, the leukocyte-enriched plasma was overlaid onto Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) and centrifuged at 250 x g for 20 min. Granulocyte-enriched cell pellets were collected, washed at 4°C with calcium- and magnesium-free HBSS (HBSS-/-), and depleted of erythrocytes by hypotonic saline lysis. Eosinophils were negatively selected using the MACS system (Miltenyi Biotec, Auburn, CA) with anti-CD16 immunomagnetic beads. The viability of freshly isolated cells was >95% (by trypan blue exclusion) and eosinophil purity was >99% (by HEMA3 staining, Fisher Scientific, Pittsburgh, PA). Purified cell suspensions were adjusted to 1 x 106 or 15 x 106 cells/ml in RPMI 1640 medium containing 0.1% endotoxin-free OVA (Sigma-Aldrich, St. Louis, MO) for use in fluid- or gel-phase assays, respectively.

Lipid body induction, staining, and enumeration

Eosinophil suspensions (106/ml) were incubated (37°C) with IL-16 (0.01–100 nM; R&D Systems, Minneapolis, MN) or medium alone for 1 h and then cytocentrifuged (350 rpm, 4 min) onto glass slides. Cytospin slides, while still moist, were fixed with 2% paraformaldehyde in HBSS-/-, rinsed in 0.1 M cacodylate buffer (pH 7.4), stained in 1.5% OsO4 (30 min), rinsed in dH2O, immersed in 1% thiocarbohydrazide (5 min), rinsed with 0.1 M cacodylate buffer, restained with 1.5% OsO4 (3 min), rinsed, and then dried and mounted (35). Lipid bodies were enumerated by light microscopy with a x100 objective lens in 50 consecutively scanned cells. For each condition there was a unimodal distribution of lipid body numbers per eosinophil without wide variations.

LTC4 measurements

After samples were taken for lipid body enumeration, cell suspensions (106/ml) were washed in HBSS-/-, resuspended in 1 ml of HBSS containing calcium and magnesium, and then stimulated with 0.1 µM A23187 (Sigma-Aldrich) for 15 min (37°C). Reactions were stopped on ice, cell suspensions were centrifuged (500 x g for 10 min; 4°C), and supernatants were assayed for LTC4 by enzyme immunoassay (EIA) (2) (sensitivity, <7.8 pg/ml) (Cayman Chemicals, Ann Arbor, MI). Intracellular formation of LTC4 within eosinophils embedded in an agarose matrix was evaluated as described previously using carbodiimide fixation of newly formed LTC4 before its immunofluorescent localization with an Alexa488-labeled (Molecular Probes, Eugene, OR) rat anti-LTC4/LTD4/LTE4 mAb (clone 6E7; Sigma-Aldrich) (35).

EliCell assay for detection of released IL-4, IL-12, and RANTES

The EliCell assay, a gel-phase dual-Ab capture and detection assay based on microscopic observations of individual viable cells, was performed as detailed (32) to enumerate the proportions of eosinophils releasing preformed cytokines or chemokines (IL-4, IL-12, or RANTES) and to electronically quantitate (in arbitrary units x 106) the average relative amounts of each cytokine released extracellularly. Biotinylated goat polyclonal Abs against IL-4, IL-12, and RANTES (each at 20 µg/ml; R&D Systems) were used as capturing Abs and paired with Alexa546-labeled mouse anti-IL-4, anti-IL-12, and anti-RANTES mAb (400 µl of 10 µg/ml; R&D Systems) to detect released IL-4, IL-12, and RANTES, respectively. Alexa546 labeling was performed per a protocol from Molecular Probes. Controls to ascertain the specificity of extracellular immunodetection of these three cytokines and to confirm that the detected cytokines were not from the intracellular pool were performed. No IL-4, IL-12, or RANTES staining were found either when Alexa546-labeled mouse IgG1 was used as a nonimmune isotype control or when the biotinylated capture Abs (necessary to immobilize cytokines at their extracellular sites of release) was substituted with a biotinylated irrelevant control Ab. As evaluated by LIVE/DEAD fluorescent assay (Molecular Probes), eosinophil viability at the end of assays was >80%.

Stimuli and treatments

Eosinophils were stimulated with IL-16 (0.01–100 nM; R&D Systems), RANTES, or eotaxin (6 nM; R&D Systems) for time periods ranging from 5 min to 3 h. For inhibitor studies, cells were pretreated for 30 min with pertussis toxin (PTX) (20 ng/ml; Calbiochem, La Jolla, CA), recombinant soluble CD4 (sCD4) (50 ng/ml; R&D Systems), Fab of anti-CD4 (clone OKT4; American Type Culture Collection, Manassas, VA) and anti-HLA class I (clone W6/32; Sigma-Aldrich) mAbs (1 µg/ml), anti-CCR3 mAb (clone 61828.111; R&D Systems), or isotype control rat IgG2a at 10 µg/ml (BD PharMingen, San Diego, CA), met-RANTES (60 nM; R&D Systems), the platelet-activating factor (PAF) receptor antagonists CV6209 and BN52021 (10 µM; Biomol, Plymouth Meeting, PA), brefeldin A (BFA) (0.1 and 1 µg/ml; Biomol), or their vehicles, as indicated. Alternatively, cells were co-incubated with 10 µg/ml of anti-eotaxin (clone 43911.11) and anti-RANTES (clone 21445.1) mAb (each from R&D Systems) or with their matching nonimmune mouse IgG1. Inhibitors were prepared in RPMI 1640 containing 0.1% of endotoxin-free OVA. PTX was prepared in DMSO, and the final DMSO concentration was <0.01% and had no effect on eosinophils. A peptide from domain 4 of human CD4, peptide 2:H-Q346CLLSDSG(353)-amide, and a scrambled control peptide 3:H-DLGQSLSC-amide, were purchased from Research Genetics (Huntsville, AL) (36).

Statistical analysis

Data were expressed as means ± SD. Percentage inhibition with antagonists was calculated in comparison with stimulated increases in lipid body numbers, LTC4 production, or IL-4 release above baselines. Statistical comparisons were done by ANOVA followed by Newman-Keuls Student’s test. Differences were considered significant when p < 0.05. Correlation coefficients evaluating lipid body numbers vs quantities of LTC4 released were determined from the means of four different experiments by linear regression with significance (F test) at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-16 primes for enhanced LTC4 production and elicits lipid body formation in eosinophils

IL-16 very effectively primed eosinophils for increased LTC4 release in response to a submaximal 0.1 µM concentration of calcium ionophore A23187. Prestimulation of eosinophils for 1 h with IL-16 dose dependently elicited increases in A23187-induced LTC4 production (Fig. 1GoB). At 100 nM, IL-16-primed eosinophils released ~6-fold as much LTC4 as did eosinophils challenged with A23187 alone. Because the formation of lipid bodies and LTC4 synthesis at these organelles provides a basis for this enhanced capacity for LTC4 release (35), we analyzed the effect of IL-16 on the number of cytoplasmic lipid bodies. Resting eosinophils contained ~9.4 ± 2.3 lipid bodies (mean ± SD, n = 10). IL-16 dose dependently induced the formation of new lipid bodies withineosinophils, doubling their initial numbers (Fig. 1GoA). The increased quantities of LTC4 generated by eosinophils primed with increasing concentrations of IL-16 correlated highly with the increased numbers of lipid bodies (r = 0.89; p < 0.05; n = 4), in accord with similar correlations observed previously with other specific chemoattractants (i.e, PAF, eotaxin, eotaxin-2, eotaxin-3, and RANTES) (35, 37, 38, 39). In the absence of A23187 activation, eosinophils stimulated for 1 h with IL-16 only (100 nM) did not release extracellular quantities of LTC4 sufficient to be detectable by EIA (data not shown), but they did synthesize new LTC4 immunodetectable at lipid bodies within 33 ± 7% (mean ± SD, n = 3) of IL-16-stimulated eosinophils (Fig. 2GoD).



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  		FIGURE 1. Dose responses of IL-16-induced lipid body formation (A), priming for enhanced LTC4 production (B), and IL-4 release (C and D) from human eosinophils at 1 h. Lipid bodies are expressed as the mean number enumerated in 50 consecutive eosinophils. LTC4 quantities released by 106 eosinophils in 1 ml after 15-min activation with 0.1 µM A23187 were assayed by EIA in cell supernatants. IL-4 release, detected in solid-phase EliCell assays, was expressed both as the percentage of eosinophils exhibiting extracellularly released immunofluorescent detected IL-4 (D) and as the average of fluorescence intensities (in arbitrary units x 106) for immunoreactive IL-4 around 50 individual eosinophils (C). Results are means ± SD from four donors. * and **, p < 0.05 and p < 0.01, respectively, compared with nonstimulated eosinophils.

 


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  		FIGURE 2. IL-16 elicits release of IL-4 and RANTES, but not of IL-12, from eosinophils and stimulates LTC4 formation at lipid bodies within eosinophils. Phase contrast (left) and fluorescent (right) microscopic images of identical fields of eosinophils within a solid-phase matrix are shown. Anti-IL-4 (A), anti-IL-12 (B), and anti-RANTES (C) immunoreactive sites in red and anti-LTC4 (D) in green are overlaid on phase-contrast images to facilitate their extracellular localization for cytokines/chemokines around nonpermeabilized eosinophils or for intracellular LTC4 within fixed and permeabilized eosinophils. Images show representative eosinophils stimulated for 3 h with 100 nM IL-16 (bottom) or medium alone (top). Bar, 5 µm.

 
IL-16 elicits IL-4, and not IL-12, release from eosinophils

Eosinophils contain preformed stores of almost two dozen cytokines resident largely within eosinophil-specific granules (22), and these include IL-4 and IL-12, two cytokines with potentially disparate and opposing activities. As detected with a sensitive solid-phase dual-Ab capture and detection assay developed to detect the extracellular release of eosinophil chemokines and cytokines (32), IL-16 stimulated the release of IL-4 from eosinophils. IL-16-stimulated eosinophils showed a punctate pattern of immunoreactive IL-4 released at discrete loci proximate to the cell surface, compatible with a vesicular transport-mediated process of IL-4 release (Fig. 2GoA). No IL-4 staining was found with nonstimulated eosinophils (Fig. 1Go, C and D), when the Alexa546-labeled anti-IL-4 detection Ab was replaced by an Alexa546-labeled isotype nonimmune control (data not shown), or when biotinylated anti-IL-4 capture Ab (which immobilizes IL-4 at its extracellular sites of release) was substituted with a biotinylated irrelevant control (data not shown). The last condition assured that neither intracellular nor membrane-bound IL-4 was being detected in the nonpermeabilized eosinophils. In contrast, IL-16 did not stimulate the release of IL-12 from eosinophils (Fig. 2GoB), another cytokine stored preformed within eosinophils (29, 30, 31), nor did RANTES or eotaxin (data not shown).

CD4 receptors mediate the effects of IL-16 on eosinophils

Because the chemoattractant activity of IL-16 for human eosinophils is mediated by IL-16 engagement of CD4 (2), we evaluated whether IL-16 promotion of LTC4 and IL-4 release was likewise mediated through CD4 expressed on eosinophils. Treatment of eosinophils with either soluble CD4 or neutralizing Fab of OKT4 anti-CD4 mAb (but not with a control mAb to surface-expressed class I MHC protein) blocked lipid body formation, priming for enhanced LTC4 production and IL-4 release from eosinophils stimulated with IL-16 (Fig. 3Go, left panels). Of note, Fab were used because the binding of CD4 with whole OKT4 mAb mimics IL-16 stimulation of eosinophils (2). Thus, IL-16-elicited eosinophil activation was mediated via CD4 expressed by eosinophils.



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  		FIGURE 3. Receptors involved in IL-16-induced activation of eosinophils. A, CD4 mediates the activation of eosinophils by IL-16. As indicated, eosinophils were pretreated for 30 min with inhibitors of IL-16/CD4 interaction (including sCD4 and Fab of OKT4 anti ({alpha})-CD4 Ab (or irrelevant control W6/32 anti-HLA Ab)) or with the G{alpha}i protein inhibitor PTX and then were incubated with IL-16 (100 nM) for 1 h for induction of lipid bodies, priming for enhanced LTC4 production (fluid-phase assays), and IL-4 release (solid-phase matrix assays). B, Involvement of G{alpha}i protein-coupled CCR3 receptors. As indicated, eosinophils were pretreated for 30 min with antagonists of PAF (CV6209 and BN52021) and CCR3 (Met-RANTES (MetR) and anti-CCR3 neutralizing mAb (or isotype control rat IgG2a)) receptors and then were activated with IL-16. Results are means ± SD from three independent assays. + and *, p < 0.05 compared with nonstimulated and IL-16-stimulated eosinophils, respectively. B, Values represent the calculated percentage of inhibition with antagonists in comparison with stimulated increases in lipid body numbers, LTC4 production, and IL-4 release above baselines.

 
The amino acid sequence of the extracellular Ig-like region of CD4 contains four domains designated D1 through D4. Recent studies reported that the binding sites for IL-16 within the CD4 molecule are within the D4 residues (36, 40). To further investigate the specific CD4 domain required for IL-16 binding andeosinophil activation, eosinophils were pretreated with an octapeptide (peptide 2) derived from the D4 region of CD4 molecule. As shown in Table IGo, peptide 2 but not peptide 3 (a control peptide with a random scrambled amino acid sequence) abolished IL-16-induced lipid body formation, enhanced LTC4 production, and IL-4 release. It is of note that neither these peptides nor the Fabof anti-CD4 affected the resting status of nonstimulated eosinophils or the lipid body formation and LTC4 and IL-4 release from RANTES-stimulated eosinophils (data not shown). These data indicate that the effects of IL-16 on eosinophils depend on IL-16 binding to the D4 domain of CD4 expressed by eosinophils.


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  		Table I. Inhibitory effect of peptide 2 derived from the D4 domain of CD4 molecule on IL-16-induced eosinophil activation1

 
Involvement of G protein-coupled CCR3 activation in IL-16-initiated eosinophil activation

Although membrane-bound CD4 molecules are not G protein-coupled receptors (GPCRs), we evaluated whether endogenously generated ligands that signal via GPCR might be downstream mediators of responses initiated by IL-16 engagement of CD4. Inhibition of G protein activation by PTX pretreatment blocked IL-16-elicited lipid body formation, priming for enhanced LTC4 production and IL-4 release from eosinophils (Fig. 3Go, left panels). PTX-sensitive GPCRs known to elicit lipid body formation and enhanced eicosanoid synthesis include the PAF receptor (38, 39, 41) and CCR3 (35, 37). Pretreatment with two specific PAF receptor antagonists (CV6209 and BN52021) did not inhibit IL-16-elicited lipid body formation, priming for enhanced LTC4 production, or IL-4 release from eosinophils (Fig. 3Go, right panels). In contrast, IL-16-mediated activation of eosinophils was blocked by pretreatment with either the CCR3 antagonist met-RANTES (42) or a neutralizing anti-CCR3 receptor mAb (but not by an isotype control Ab) (Fig. 3Go, right panels). These findings suggested that actions of IL-16 to enhance LTC4 and IL-4 release from eosinophils might be mediated by endogenous CCR3-acting chemokines.

Eosinophils contain two chemokines, eotaxin and RANTES, that act via CCR3 and are stored preformed within eosinophil granules (23, 24, 25). We have shown that both RANTES and eotaxin induce lipid body formation, prime for enhanced LTC4 production (35), and elicit vesicular transport-mediated IL-4 release (43) from eosinophils via CCR3 activation. With the EliCell assay, we evaluated whether IL-16 elicited release of endogenous, preformed RANTES from eosinophils. Indeed, IL-16 stimulated the rapid release of RANTES that temporally preceded IL-16-induced IL-4 release (Fig. 4Go). Consistently, in kinetic studies with three different donors, IL-16 induced RANTES release as early as 5 min, whereas at least 30 min were needed for detectable IL-4 release, a time course consistent with autocrine intermediary roles for endogenously derived CCR3 agonists. With eosinophils incubated for 1 h with 100 nM IL-16 (n = 3), 57.6% exhibited extracellular RANTES release, and addition of sCD4 or OKT4 Fab anti-CD4 mAb during incubation with IL-16 inhibited the numbers of eosinophils releasing RANTES by 91.8 and 87.6%, respectively (Table IIGo). In a punctate pattern similar to that for IL-4 release, released RANTES also appeared as extracellular focal spots close to eosinophil membrane, again indicating a vesicular transport-dependent secretory process (Fig. 2GoC). Interestingly, exogenous eotaxin also induced the rapid release of endogenous RANTES (Fig. 4Go, left panel). The RANTES-releasing activity of eotaxin was fully inhibited by PTX and a CCR3-blocking mAb and was partially inhibited by met-RANTES (Table IIGo). These inhibitors of RANTES release, however, did not inhibit IL-16-induced release of RANTES.



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  		FIGURE 4. IL-16-induced RANTES release precedes IL-4 release. Time courses of RANTES (left panel) and IL-4 (right panel) release from eosinophils stimulated with medium, eotaxin, RANTES, or IL-16. With EliCell assays, RANTES and IL-4 released extracellularly from eosinophils embedded in an agarose matrix were captured with anti-RANTES or anti-IL-4 bound to a gel matrix and detected with Alexa546-labeled anti-RANTES or anti-IL-4 mAb, respectively. Results were expressed as the percentages ± SD of triplicates from a single experiment of eosinophils exhibiting extracellularly released RANTES or IL-4 (300 cells counted). Data are representative of one of three replicate experiments.

 

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  		Table II. Involvement of CD4 and CCR3 receptors on IL-16-induced RANTES release from eosinophils1

 
To confirm the involvement of endogenously eosinophil-derived CC chemokines (RANTES and eotaxin) on IL-16-mediated activation of eosinophils, neutralizing anti-RANTES and anti-eotaxin mAbs were co-incubated with IL-16-stimulated eosinophils. Both mAbs inhibited IL-16-elicited lipid body formation, enhanced LTC4 production, and IL-4 release from eosinophils (Fig. 5Go), indicating that released chemokines were acting as autocrine activators. In addition, IL-16-induced release of RANTES was inhibited 34.3% by exogenous anti-eotaxin mAb and 0% by a controlmAb (data not shown). Thus, IL-16 was directly mobilizing both RANTES and eotaxin from eosinophils for their extracellular release and autocrine activities. That IL-16 was eliciting the release of these chemokines by vesicular transport-mediated secretion was supported by findings with BFA, a vesicle formation inhibitor. Pretreatment with BFA blocked IL-16-induced RANTES release and enhanced LTC4 production and IL-4 release (Fig. 6Go).



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  		FIGURE 5. Endogenous eosinophil-derived RANTES and eotaxin mediate IL-16-induced activation of eosinophils. As indicated, IL-16-stimulated eosinophils were cotreated with neutralizing anti ({alpha})-eotaxin or anti-RANTES mAb or with isotype control mouse IgG1. After 1 h, formation of new lipid bodies, priming for enhanced LTC4 production (fluid-phase assay), and IL-4 release (gel-phase assay) were analyzed. Results are means ± SD from three independent assays. + and *, p < 0.05 compared with nonstimulated and IL-16-stimulated eosinophils, respectively. Dotted lines represent basal levels.

 


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  		FIGURE 6. Vesicular transport mediates IL-16-induced RANTES and IL-4 release from eosinophils. Eosinophils were pretreated for 30 min with an inhibitor of vesicle formation, BFA, and then were stimulated with 100 nM IL-16 or 6 nM RANTES. After 1 h, RANTES release, priming for enhanced LTC4 production, and IL-4 release were analyzed. Results are means ± SD from three independent assays. + and *, p < 0.01 compared with nonstimulated and IL-16-stimulated eosinophils, respectively. Dotted lines represent basal levels.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The capabilities of eosinophils to contribute to varied immunologic and inflammatory responses within tissues may be dependent on the activation of mechanisms that enhance their formation of eicosanoids, notably including LTC4, and their regulated release of varied granule-stored proteins, which include not only their cardinal cationic proteins but also varied chemokines and cytokines with potentially diverse and disparate biologic activities. Because these responses may not be adequately studied in conventional fluid-phase assays, we have developed complementary techniques to evaluate the capacity of individual eosinophils to synthesize LTC4 and to release specific preformed chemokines and cytokines by a physiologic process of vesicular mobilization of granule-derived proteins for their transport and focal release outside of the plasma membrane of eosinophils. With techniques that can evaluate the functional responses of individual eosinophils, we have shown that both RANTES and eotaxin induce lipid body formation (35), prime for enhanced LTC4 production (35), and elicit vesicular transport-mediated IL-4 release (43) from eosinophils via CCR3 activation. Our current results indicate that IL-16 can not only enhance specific responses of eosinophils, but that it can also identify major roles for eosinophil-derived eotaxin and RANTES in functioning as important autocrine intermediators of these IL-16-elicited responses.

IL-16 dose dependently elicited new lipid body formation, intracellular LTC4 formation at lipid bodies, and priming for enhanced calcium ionophore-activated LTC4 release. In our earlier studies with IL-16 (2), lesser concentrations of IL-16, a greater concentration of activating calcium ionophore, and a lack of methods to detect intracellular LTC4 formation contributed to an inability to recognize that IL-16 stimulation of eosinophils could enhance the formation of the 5-lipoxygenase pathway-derived eicosanoid LTC4. IL-16 also elicited BFA-inhibitable, vesicular transport-mediated release of preformed RANTES and IL-4, but not IL-12, from eosinophils. Although studies in CD4-knockout mice indicate that IL-16 may act on monocytes and dendritic cells via as yet unidentified CD4-independent mechanisms (44, 45), with human and mouse T cells CD4 is the recognized IL-16R (1). We previously established that IL-16-elicited chemotaxis of human eosinophils was CD4-dependent (2). In support of a central role for eosinophil-expressed CD4 in functioning as the signal-transducing receptor on eosinophils for IL-16, we demonstrated that anti-CD4 Fab, sCD4, and a CD4 domain 4-based IL-16 blocking peptide inhibited the actions of IL-16 on eosinophils.

That IL-16 was not directly acting to enhance eosinophil LTC4 formation and IL-4 release was indicated by the inhibition found with PTX, which catalyzes the ADP ribosylation of certain G protein {alpha}-subunits and uncouples PTX-sensitive G proteins from cell surface receptors (46). Potentially, CD4 on eosinophils might be interacting with the PTX-sensitive CXCR4, which is expressed on eosinophils (47). Alternatively, PTX-sensitive receptors for PAF or for chemokines acting via CCR3 might be involved. Bartemes et al. (41) recently documented a central role for endogenous PAF and its receptor in the augmented functional responses of eosinophils elicited by IL-5 or IgG, and these included enhanced LTC4 formation and correlative increases in lipid body formation. Endogenous PAF generation and signaling through its receptor were not involved in IL-16-mediated signaling, because two PAF receptor antagonists (CV6209 and BN52021) were without effect. Instead, IL-16 actions were mediated by the autocrine activities of endogenous CCR3-acting chemokines. IL-16 induced the rapid vesicular transport-mediated release of RANTES, which temporally preceded later IL-4 release (Fig. 4Go). The effects of IL-16 were blocked by CCR3 inhibitors (met-RANTES and anti-CCR3 mAb) and by neutralizing anti-eotaxin and anti-RANTES mAbs. Both RANTES and eotaxin each enhanced LTC4 and IL-4 (but not IL-12) release.

Thus, IL-16 acts to initiate the release of specific chemokines and cytokines that are stored preformed within eosinophils. Our findings provide insights into the mechanisms that regulate this mobilization and release process. First, as indicated by the finding that exogenous eotaxin also induced the rapid release of endogenous RANTES (Fig. 4Go, left panel; Table IIGo), chemokines acting via CCR3 can enhance the release of additional CCR3 active chemokines from within eosinophils. This could provide a positive feedback loop amplifying the initial IL-16-elicited chemokine release and consequent eosinophil activation. Second, the vesicular transport-mediated process of eosinophil "degranulation" was highly selective. Notably, the IL-16-initiated transport and release of RANTES occurred in vesicles that were not also loaded with IL-4, whose release occurred only later in vesicles formed in response to the CCR3-active chemokines. Additional selectivity in the regulated release of eosinophil-derived cytokines was evident in the release of IL-4, but not of IL-12, by the IL-16- or CCR3 chemokine-activated eosinophils. Because eosinophils also contain preformed IL-16 (3), an ability for further autocrine activation of eosinophils exists, but at present the mechanisms that physiologically elicit IL-16 release from eosinophils have not been defined.

Thus, the capacity of IL-16 to activate specific functional responses of eosinophils has the potential to influence the nature of immunologic responses. IL-16 can direct eosinophils to make new LTC4 and release the chemokines, RANTES, and eotaxin. These chemokines can act in an autocrine fashion on eosinophils via CCR3 and potentially on other cells that express CCR3, including basophils (48), mast cells (49), airway epithelial cells (50), and Th2 cells (51). Moreover, the differential release of IL-4, which can contribute to the polarization toward Th2 differentiation, promote IgE class switching, and stimulate other cellular responses pertinent to allergic inflammation (52) (in contrast to IL-12, which can suppress Th2-type responses and the manifestations of allergic inflammation (53, 54)), provides a means by which IL-16 and CCR3 chemokines may promote specific eosinophil contributions to allergic and other types of inflammation.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI20241, AI22571, AI51645, HL56386, AI35680, and HL32802. Back

2 Address correspondence and reprint requests to Dr. Peter F. Weller, Beth Israel Deaconess Medical Center, DA-617, 330 Brookline Avenue, Boston, MA 02215. E-mail address: pweller{at}caregroup.harvard.edu Back

3 Abbreviations used in this paper: LTC4, leukotriene C4; EIA, enzyme immunoassay; PTX, pertussis toxin; sCD4, soluble CD4; PAF, platelet-activating factor; BFA, brefeldin A; GPCR, G protein-coupled receptor. Back

Received for publication December 20, 2001. Accepted for publication February 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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