Augmentation of LTC4 synthesis in human eosinophils caused by CD3-stimulated Th2-like cells in vitro

Nilda M. Muñoz1, Gijs A. van Seventer2, Roshanak T. Semnani2, and Alan R. Leff1,3

1 Section of Pulmonary and Critical Care Medicine, Department of Medicine, and 3 Committees on Clinical Pharmacology and Cell Physiology, Division of the Biological Sciences, and 2 Department of Pathology, University of Chicago, Chicago, Illinois 60637


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We assessed the effect of anti-CD3-stimulated secretion of cultured human Th1- and Th2-like cells on leukotriene C4 (LTC4) secretion in isolated human eosinophils. T helper (Th) cell subsets were generated from human naive CD4+ T cells cocultured with irradiated human transformed B cells and either recombinant human interleukin (rhIL)-1beta plus rhIL-6 plus rhIL-12 for Th1-like cells or rhIL-1beta plus rhIL-6 plus rhIL-4 for Th2-like cells. Coincubation of eosinophils with 1:5 dilution of Th2-supernatant (Sup) caused an increase in LTC4 secretion caused by 0.1 µM formyl-Met-Leu-Phe and 5 µg/ml cytochalasin B from 921 ± 238 to 3,067 ± 1,462 pg/106 eosinophils (P < 0.01). Th1-Sup at the same dilution had no augmenting effect on stimulated secretion of LTC4 in eosinophils despite substantial concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant. Dilution of Th1-Sup caused increased LTC4 that returned to baseline after immunoabsorption of GM-CSF, suggesting the presence of a possible inhibitory factor. We demonstrate that pretreatment of eosinophils with 1:5 dilution of Th2-Sup but not of Th1-Sup causes substantial augmentation of LTC4 secretion in vitro and establishes that human Th2 cells cause direct augmentation of LTC4 secretion within 15-30 min of exposure.

T helper cells; cytokine; T helper type 1-like cells; immunodepletion; leukotriene C4


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE INTERACTION BETWEEN Th2 cells and eosinophils is presumed to be a major mechanism in the pathogenesis of allergic inflammation (2-5, 11, 14, 29) and airway hyperresponsiveness (5, 8, 11, 28). These distinct subsets of CD4+ T helper (Th) cells, Th1 and Th2 phenotypes, have been defined in the murine system based on their cytokine secretion profiles after antigen stimulation (10, 27). In rodent models, Th2 cells have been shown to cause induction of airway hyperresponsiveness by a mechanism that is not replicated by murine Th1 cells. This induction of airway hyperresponsiveness in rodents also has been shown to be independent of interleukin (IL)-4 (3, 4). The same authors have shown that exposure to IL-5, a cytokine produced by Th2 but not by Th1 cells, for 24 h also causes increased expression of 5-lipoxygenase-activating protein and translocation of this enzyme to the nucleus (6). This was associated with an enhanced capacity for cysteinyl leukotriene synthesis after stimulation with calcium ionophore A-23187.

We have shown previously that short exposure (<30 min) of eosinophils to exogenous IL-5 causes cytosolic phospholipase A2 (cPLA2) phosphorylation but not translocation of cPLA2 to the nucleus (30). This short exposure was nonetheless associated with augmented production of leukotriene C4 (LTC4) after exogenous stimulation with formyl-Met-Leu-Phe, which caused translocation of cPLA2 to the nuclear membrane (24, 30, 31).

We hypothesized that these previously established interactions of exogenously administered cytokine could be replicated in vitro by short-term coincubation of supernatant from activated human Th2-like cells with freshly isolated human eosinophils. The objectives of this investigation were 1) to determine whether human Th2 cells interacted directly as activating or augmenting cells over the short time (<30 min) with human eosinophils in isolated cell systems (i.e., independently of other cells or mediators), 2) to determine whether stimulated Th2 (and Th1) cells secreted cytokines in concentrations sufficient to cause eosinophil secretion, and 3) to determine whether murine models of these interactions were relevant to human Th2 cell-eosinophil interactions.

As for rodents (10), similar distinctions in T cell populations have been established in human atopic subjects, and the Th2 cell has been implicated as the Th cell subtype predominantly involved in both atopy and human asthma (4, 14). However, CD4+ T cell populations consisting of Th1 and Th2 subsets are less explicitly defined in humans. There also has been no prior demonstration of direct stimulation of human eosinophils by Th2 cells in isolated systems.

Experiments thus were performed using cells isolated from human subjects and cultured in vitro. Using a technique previously developed in our laboratory for culture of Th1- and Th2-like cells derived from "naive" CD4+ T lymphocytes obtained from the peripheral blood of normal donors, we first examined the role of anti-CD3-stimulated human Th cell subtypes in causing augmented secretion of eosinophils. In further studies, we immunodepleted Th cytokines with specific monoclonal antibodies (MAbs) to determine their potential augmenting effects on eosinophil secretion. Our study is the first demonstration in vitro that there is a direct interaction between human Th2 (but not Th1)-like cells, causing substantial augmentation of stimulated eosinophil synthesis of cysteinyl leukotriene.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of Human Resting Naive CD4+ Th cells

Peripheral blood mononuclear cells were isolated from buffy coats of nonatopic donors (United Blood Services and Life-Source; Chicago, IL) by Ficoll gradient centrifugation. The naive CD4+ Th cells were purified by subjecting the treated cells to negative immunomagnetic selection as described previously (13, 22, 26).

Generation of Human Th1- and Th2-Like Cells Derived From Naive CD4+ Th Cells

Purified naive CD4+ Th cells (106 cells) obtained from eight different donors were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 20 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin and were transferred onto microplate wells coated overnight with 0.5 ml of 1 µg/ml humanized anti-CD3 MAb (OKT3 CDR) (1, 22). Irradiated (4,000 rad) 106 Epstein-Barr virus-transformed human B cells (JY-EBV cells) were added to wells containing naive CD4+ Th cells, as a source of accessory cells (25). The initial stimulation was performed by addition of the combination of exogenous recombinant human (rh) IL-1beta plus rhIL-6 plus rhIL-12 (rhIL-1/6/12) or rhIL-1beta plus rhIL-6 plus rhIL-4 (rhIL-1/6/4) to cell cultures. The addition of rhIL-1/6 (both 2.5 U/ml) has been shown to increase the cell survival and expansion rates without affecting either Th1- or Th2-like cytokine secretion (22, 26). rhIL-12 (1 ng/ml) and rhIL-4 (1 ng/ml) were used for differentiation of Th cells into Th1- and Th2-like cells, respectively, as described previously (25). For all cell fractions, viability was confirmed by propidium iodide staining (see RESULTS). Fresh culture medium was added as needed between cell stimulations, and after 7 days, the expanded cells were restimulated under the same conditions as for initial stimulation. The supernatant was collected from each well and assayed for cytokines 48 h later using specific cytokine ELISAs (see below). Aliquots of supernatants were frozen at -20°C until used in specific protocols (see below). These supernatants were randomly utilized for all subsequent experimental interventions.

Preparation of Th Cell Supernatant for Incubation With Eosinophils

To determine the concentration of Th cell supernatant (Th-Sup) causing maximal augmentation of stimulated eosinophil secretion, serial dilutions of supernatants from activated Th1- and Th2-like cells (see above) were performed. Eosinophils were then pretreated with increasing dilutions of Th-Sup for 30 min at 37°C and activated with 0.1 µM formyl-Met-Leu-Phe plus 5 µg/ml cytochalasin B (fMLP/B) (15, 18, 20) for an additional 30 min. The perfusate then was assayed for LTC4 release (see below). These studies demonstrated that 1:5 dilution for Th2-Sup and 1:10 dilution for Th1-Sup were sufficient for substantial release of LTC4, and these concentrations were used in all subsequent studies (see below).

Isolation of Human Peripheral Blood Eosinophils

Eosinophil purification was performed by a modification of the method of Hansel et al. (12). Peripheral blood (120 ml) was obtained from 21 mildly atopic donors in accordance with the approved institutional protocol. With this immunomagnetic separation technique, the purity of eosinophils was always >99% as confirmed by cell differential counting using Diff-Quik-stained cytoslides. The purified eosinophils were washed with Hanks' balanced salt solution (HBSS) before each experimental intervention. All experimental procedures were carried out on ice to prevent eosinophil activation. Viability was confirmed by using the trypan blue exclusion dye.

Flow Cytometry

Two-color flow cytometry was used to assess the purity of the isolated naive human CD4+ T cells using the following MAbs: CD45RA-phycoerythrin (PE; clone B-C15, Biosource; Camarillo, CA); CD45RO-FITC (clone UCHL-1, Caltag; San Francisco, CA); and CD3-FITC and CD4-PE (Biosource) as described previously (22, 26). This method was used for all CD4+ T-cell isolations having mixed populations to discriminate clearly and to obtain discretely identifiable groups of positive cells, i.e., CD45RA.

Sandwich ELISA

The concentrations of endogenously secreted cytokines from stimulated Th subsets were measured by sandwich ELISA (sELISA). MAb pairs (PharMingen; San Diego, CA) were utilized to quantify human IL-4, IL-5, IL-13, interferon-gamma (IFN-gamma ), tumor necrosis factor-alpha (TNF-alpha ), and granulocyte-macrophage colony-stimulating factor (GM-CSF). This modified assay is highly sensitive and more accurate in the determination of endogenously secreted cytokines than the original method that had been derived (26). For determination of IL-2, MAb directed against IL-2 (clone B-G5, Biosource) was used as capture antibodies, and IL-2 rabbit polyclonal antibody (Genzyme; Cambridge, MA) was used to detect bound cytokine. The biochemical assay was conducted using the standard sELISA procedure as previously described (26). Samples and standards were loaded (50 µl/well) into 96-well microplates and analyzed in duplicate. The absorbance at 412 nm was determined using Molecular Devices Vmax 96-well plate reader. The final concentrations of secreted cytokines from collected Th subset supernatants were calculated using the Immunofit software. All MAbs were specific for their respective cytokines, and cross-reaction between epitopes for GM-CSF, IL-5, or any other cytokine did not occur.

Enzyme Immunoassay

The concentration of LTC4 secreted during eosinophil activation was analyzed by competitive enzyme immunoassay (Cayman Chemical; Ann Arbor, MI) using a method that we have reported previously (16, 17, 19). The density measurements were made on a microplate absorbance spectrophotometer at 405 nm (Molecular Devices; Menlo Park, CA). The final concentrations were calculated from standard curves fitted by four-parameter analysis (Softmax v2.01 software, Molecular Devices), and data are expressed as picograms per million eosinophils (pg/106 eosinophils).

Immunodepletion

Microplate wells were coated in duplicate with saturating concentrations (5 µg/ml) of MAbs directed against GM-CSF, IL-5, IL-13, or TNF-alpha overnight at 4°C before 30-min blockade of nonspecific binding with 1% BSA in PBS, pH 7.4. Briefly, the treated wells were washed with PBS containing 0.05% Tween 20, pH 7.5, and stored at -20°C until later use in cytokine-depletion studies. From an initial volume of 1 ml, an aliquot of 500 µl (n = 4 undepleted Th2-Sup) was saved, and the remaining 500 µl/ml of Th2-Sup were transferred to wells coated with anti-GM-CSF. The soluble GM-CSF was immunoabsorbed for 2 h, and the GM-CSF-depleted Th2-Sup (500 µl) was saved for subsequent experiments (see below). A separate aliquot (undepleted) from the same donor was exposed to anti-IL-5-coated plates, and the IL-5-depleted Th2-Sup was saved as for GM-CSF-depleted supernatant. The exposure of Th2-Sup to MAb-coated wells was interchanged to deplete the soluble cytokines singly (GM-CSF, IL-5, IL-13, TNF-alpha ) or in combination (GM-CSF/IL-5, IL-5/GM-CSF). Control and cytokine-depleted Th2-Sup were used to pretreat the eosinophils before activation (see below) and determination of LTC4 secretion. As controls, eosinophils were exposed to IgG-coated plates, and experiments were performed as for immunodepleted cytokines.

In separate studies (n = 4 undepleted Th1-Sup), microplate wells were coated with anti-GM-CSF as above, and undepleted Th1-Sup was pipetted onto the treated wells. Identical experiments were performed as for immunodepleted Th2-Sup. Because significant amounts of soluble GM-CSF were detected after stimulation from both Th1 and Th2 subsets, this experiment was conducted to determine whether GM-CSF secreted by activated Th1-like cells could cause augmented secretion of LTC4.

Experimental Interventions

Effect of Th1- and Th2-Sup on stimulated eosinophil secretion. Purified human eosinophils (2.5 × 105 eosinophils) from five different donors were resuspended in 100 µl of HBSS, pelleted by centrifugation and pretreated with either 200 µl of 1:5 dilution of 1) culture medium alone, 2) culture medium with rhIL-1/6/4, no Th2-Sup, 3) culture medium with rhIL-1/6/12, no Th1-Sup, 4) Th2-Sup alone, or 5) Th1-Sup alone for 30 min at 37°C. Eosinophils then were activated with 0.1 µM fMLP/B. Prior investigations have validated this method and concentration of fMLP/B, which causes metabolic burst activity (20), maximal secretion of eosinophil granular protein (19, 21), bronchoactive metabolites (17-19), and maximal eosinophil chemotaxis (15) without inducing cytoxicity (17-19). At the end of the incubation period, the reaction was terminated by centrifugation (Eppendorf 5415C, Brinkman Instrument; Westbury, NY) at 13,500 rpm for 2 min. The activated supernatant was saved for determination of LTC4 secretion (see above).

Because 1:5 dilution of Th1-Sup had no augmentatory effect on stimulated eosinophil secretion of LTC4, serial dilutions of 1:10 and 1:25 were performed using Th1-Sup obtained from four donors. Experiments were performed in an identical manner as above, and the activated supernatant was saved for determination of LTC4 secretion (see above).

Effect of cytokine depletion from Th1- and Th2-Sup on stimulated eosinophil LTC4 secretion. Isolated eosinophils were pretreated for 30 min at 37°C with Th-Sup after depletion of secreted Th1- or Th2-like cell cytokines (see above and RESULTS). Experiments then were conducted in an identical manner as for either undepleted Th1- or Th2-Sup, and stimulated supernatant from the reaction tubes was collected for later analysis of LTC4 secretion (see above). As a control, eosinophils were exposed to isotype IgG-coated microplate wells and activated with either buffer control or fMLP/B for all groups (n = 8 eosinophil isolations).

Effect of exogenous recombinant human Th cytokines on stimulated eosinophil LTC4 secretion. Isolated eosinophils from four different donors were pretreated with exogenous recombinant human cytokines, i.e., rhGM-CSF, rhIL-5, rhIFN-gamma , and rhTNF-alpha at concentrations similar to those of endogenously secreted cytokines measured from CD3-stimulated Th-like cells. Activation was elicited with either fMLP/B or buffer control as above, and the supernatant from all experimental interventions was collected and stored at -70°C until later analysis for LTC4 secretion.

Data Analysis

All data are expressed as the means ± SE. Final data analysis was performed using ANOVA to determine differences between cytokine depletion paradigms. As indicated by the F statistic for particular experimental interventions, collated data were analyzed further by Fisher's least protected difference test. When comparisons of paired data within a single experimental protocol were made, Bonferroni correction was applied after paired Student's t-test. Statistical significance was claimed at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Validation and Viability of Isolated Naive CD4+ T Cells and Eosinophils

The isolation of mature, unstimulated naive Th cells derived from peripheral blood CD4+ T cells by immunomagnetic separation technique was reproducible in all cell preparations. Immunostaining of surface markers of the naive CD4+ T cells demonstrated >95% CD3+ CD4+ T cells. Staining for CD45RA (naive CD4+ T cells) and CD45RO (memory CD4+ T cells) showed no CD45RA-RO+ "memory/effector" T cells after purification by negative immunomagnetic separation. Isolated populations were consistently >95% CD45RA+RO- naive T cells, with only a very minor population (<5%) expressing low levels of CD45RO, and were >98% viable as determined by propidium iodide staining. Viability of eosinophils after experimental interventions remained >99% as assessed by trypan blue exclusion analysis.

Differentiation and Validation of Naive Th Subsets and Activation Markers

The differentiated cultured Th1- and Th2-like cells derived from naive CD4+ T cell-anti-CD3 MAb cross-linking revealed a similar pattern of cytokine production as previously reported (22, 26). In these differentiated cultured human CD4+ Th subsets, IL-5 was solely secreted by Th2-like cells, whereas IFN-gamma and TNF-alpha were predominantly produced from Th1-like cells. Both Th1- and Th2-like cells secreted GM-CSF at similar concentrations. Secretory IL-13 was present in significantly greater quantities in Th2-Sup, whereas IL-2 concentration was significantly greater in Th1-Sup. Unlike the murine model (10), endogenous IL-4 was undetectable in the Th-Sup of anti-CD3 MAb-stimulated human Th cells. However, activation of these in vitro differentiated T cells with a combination of phorbol 12-myristate 13-acetate and ionomycin generated a more than twofold release of secretory IL-4 from Th2-like cells compared with that from Th1-like cells.

Effect of 1:5 Dilution of Th1- and Th2-Sup on Stimulated Eosinophil Secretion

Activated secretion of LTC4 was augmented substantially only for eosinophils (n = 5 donors) pretreated with 1:5 dilution of Th2-Sup (Fig. 1). LTC4 secretion caused by fMLP/B activation was 921 ± 238 vs. 28.9 ± 9 pg/106 eosinophils for cells pretreated with buffer alone. There was no difference in stimulated secretion for eosinophils obtained from the same donors after pretreatment with 1:5 dilution of Th1-Sup (1,050 ± 319 pg/106 eosinophils, P = NS). However, exposure of eosinophils to 1:5 dilution of Th2-Sup caused a more than threefold augmentation of stimulated LTC4 secretion to 3,067 ± 1,462 pg/106 eosinophils (P < 0.01 vs. fMLP/B alone, no Th-Sup, or fMLP/B plus Th1-Sup; Fig. 1).


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Fig. 1.   Effect of 1:5 dilution of Th1 (Th1-Sup) and Th2 supernatant (Th2-Sup) on isolated human eosinophils. Eosinophils (eos) were pretreated with either Th1- or Th2-Sup for 30 min before activation with 0.1 µM formyl-Met-Leu-Phe plus 5 µg/ml cytochalasin B (fMLP/B). Leukotriene C4 (LTC4) secretion was augmented substantially in stimulated cells exposed to Th2-Sup. There was no difference between LTC4 secretion caused by fMLP/B alone (no Th1- or Th2-Sup) and eosinophils treated with Th1-Sup before stimulation with fMLP/B. LTC4 secretion was measured by enzyme immunoassay (see MATERIALS AND METHODS). Data are means ± SE.

In separate studies using eosinophil aliquots from the same donors, addition of culture medium containing either rhIL-1/6/12 used to generate Th1-like cells or rhIL-1/6/4, which was used to generate Th2-like cells, did not augment secretion of LTC4 in activated human eosinophils in the absence of Th-Sup (P = NS for all comparisons vs. fMLP/B plus buffer control alone, no Th-Sup; Fig. 2).


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Fig. 2.   Effect of recombinant human (rh) cytokines used to cause T helper (Th) cell subset differentiation on eosinophil secretion. There was no difference between cells activated by fMLP/B alone vs. eosinophils pretreated with interleukin (IL)-1beta plus rhIL-6 plus rhIL-12 (rhIL-1/6/12) for Th1 cell differentiation or rhIL-1beta plus rhIL-6 plus rhIL-4 (rhIL-1/6/4) for Th2 cell differentiation before activation with fMLP/B. Data are means ± SE.

Effect of Serial Dilution of Th1-Sup on Stimulated Eosinophil Secretion of LTC4

Because 1:5 dilution of Th1-Sup had neither a direct nor a priming effect on the stimulated eosinophil secretion (see Fig. 1), three different Th1-Sup were diluted 1:5, 1:10, and 1:25, and the effect on fMLP/B-induced LTC4 secretion was assessed (n = 4 eosinophil donors). Stimulated secretion of LTC4 was substantially decreased to 213 ± 53 pg/106 eosinophils for eosinophils treated with concentrated Th1-Sup vs. 982 ± 107 pg/106 eosinophils for buffer-treated eosinophils (P < 0.01; Fig. 3). Stimulated LTC4 secretion in eosinophils pretreated with 1:5 dilution of Th1-Sup was comparable to secretion from control (untreated) cells (1,096 ± 269 pg/106 eosinophils, P = NS). By contrast, a >1.8-fold increase to 1,813 ± 228 pg/106 eosinophils was obtained for stimulated eosinophils exposed to 1:10 dilution of Th1-Sup (P < 0.01 vs. 1:5 Th1-Sup and control). At 1:25 dilution there was no further effect on secretion of LTC4 caused by fMLP/B activation (P = NS vs. 1:10 Th1-Sup; Fig. 3).


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Fig. 3.   Effect of serial dilution of Th1-Sup on stimulated eosinophil secretion. Augmentation of LTC4 synthesis caused by fMLP/B activation occurred in Th1-Sup after progressive dilution. Increased LTC4 synthesis was maximal at 1:10 dilution; secretion was unchanged in eosinophils exposed to 1:25 dilution of Th1-Sup. undil, Undiluted. Data are means ± SE.

Effect of Cytokine Depletion From Th1- and Th2-Sup on Stimulated Eosinophil LTC4 Secretion

The relative contribution of Th2 cytokines in augmenting eosinophil secretion caused by fMLP/B activation was assessed sequentially after immunodepletion in samples obtained from eight different donors. Depletion of GM-CSF by immunoabsorption caused a >40% decrease in LTC4 secretion caused by fMLP/B activation, to 1,832 ± 729 pg/106 eosinophils (P < 0.05 vs. undepleted Th2-Sup; Fig. 4). However, this level of stimulated secretion still was significantly greater than that obtained for eosinophils not exposed to Th2-Sup (921 ± 238 pg/106 eosinophils; P < 0.01). Pretreatment of eosinophils with IL-5-depleted Th2-Sup caused an even greater attenuation of fMLP/B-stimulated secretion, to 1,366 ± 614 pg/106 eosinophils [P < 0.001 vs. fMLP/B-activated Th2-Sup alone; P = NS vs. fMLP/B-stimulated cells not exposed to Th2-Sup (control); Fig. 4].


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Fig. 4.   Depletion of secreted cytokines from Th2-Sup by immunoabsorption method. Microplate wells were precoated with monoclonal antibodies (MAb) directed against human granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, IL-13, and tumor necrosis factor-alpha (TNF-alpha ; see MATERIALS AND METHODS), and cytokines were depleted selectively. Depletion of IL-5 and GM-CSF caused substantial attenuation of augmented eosinophil secretion by fMLP/B activation. By contrast, depletion of IL-13 and TNF-alpha from Th2-Sup had no attenuating effect on augmented LTC4 secretion. IgG-coated microplate wells were used as control for buffer and fMLP/B-activated cells. Data are means ± SE.

In contrast, depletion of IL-13 did not affect eosinophil secretion caused by fMLP/B activation. For eosinophils treated with undepleted Th2-Sup, fMLP/B-induced secretion of LTC4 was 3,027 ± 1,462 vs. 3,657 ± 2,237 pg/106 eosinophils after IL-13 depletion. TNF-alpha -depleted Th2-Sup also had no effect in LTC4 secretion (2,477 ± 1,056 pg/106 eosinophils; P = NS vs. undepleted Th2-Sup).

With use of an aliquot from the same eosinophil isolations, LTC4 secretion caused by fMLP/B activation was 981 ± 107 vs. 8.6 ± 1.1 pg/106 eosinophils for control (IgG1 isotype plus buffer; P < 0.001; Fig. 5). For cells treated with undepleted 1:10 dilution of Th1-Sup, release of LTC4 increased significantly to 1,818 ± 228 pg/106 eosinophils (P < 0.05 vs. undepleted Th1-Sup plus fMLP/B). By contrast, absorption of soluble GM-CSF from 1:10 dilution of Th1-Sup attenuated the augmented LTC4 secretion caused by fMLP/B to near basal level (Fig. 5).


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Fig. 5.   Immunodepletion of GM-CSF from 1:10 dilution of Th1-Sup by immunoabsorption method. Release of LTC4 from stimulated eosinophils pretreated with Th1-Sup decreased significantly to near basal level. IgG-coated microplate wells were used as control for buffer and fMLP/B-activated cells. Data are means ± SE.

Effect of Combined Immunodepletion of Th2-Sup Cytokines

Maximal inhibition of Th2-Sup augmentation of eosinophil secretion was achieved by depletion of IL-5 alone (see Fig. 4). Depletion of either IL-5 or GM-CSF caused a decrease in augmented eosinophil secretion by nearly threefold, and there was no further inhibition of augmented secretion by combined immunodepletion of both secretory IL-5 plus GM-CSF (Fig. 6). Interchanging the sequence of immunoabsorption, GM-CSF followed by IL-5, had no significant effect on LTC4 secretion (Fig. 6). Activated secretion of LTC4 was 1,318 ± 448 pg/106 eosinophils for eosinophils treated with Th2-Sup from which IL-5 was depleted (Fig. 5); this did not differ significantly for eosinophils treated with supernatant from which both GM-CSF and IL-5 were depleted (Fig. 6).


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Fig. 6.   Combined immunodepletion of GM-CSF and IL-5. Maximal inhibition of augmented LTC4 secretion was achieved by selective depletion of IL-5 from Th2-Sup (Fig. 4). Combined immunodepletion of IL-5 plus GM-CSF (or vice versa) had no further effect. Data are means ± SE.

Effect of Exogenous Cytokines on Stimulated Eosinophils

To assess the effect of comparable concentrations of exogenously added cytokines on eosinophil secretion (n = 4 eosinophil isolations), further studies were generated in naive cells using concentrations measured in Th-Sup. At 5 ng/ml, rhGM-CSF-treated eosinophils caused a more than twofold increase in stimulated secretion of LTC4, from 920 ± 165 to 2,179 ± 19 pg/106 eosinophils (P < 0.01). Coincubation with 3 ng/ml rhIL-5 increased the stimulated synthesis of LTC4 to 1,632 ± 156 pg/106 eosinophils (P = NS vs. GM-CSF-treated cells; P < 0.05 vs. fMLP/B activated, no rh cytokines). Neither rhTNF-alpha nor rhIFN-gamma , which are secreted predominantly by Th1-like cells, blocked the synthesis of LTC4 caused by fMLP/B activation (Fig. 7).


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Fig. 7.   Effect of exogenous recombinant human cytokines on eosinophil secretion. Application of common cytokine GM-CSF secreted by both CD3-activated Th1- and Th2-like cells caused a >2-fold increase in LTC4 secretion after fMLP/B activation. Comparable eosinophil secretion resulted in cells treated with rhIL-5, the cytokine that is solely secreted by CD3-activated Th2-like cells. Pretreatment with rhTNF-alpha and recombinant human interferon-gamma (rhIFN-gamma ) had neither stimulatory nor augmentatory effect on eosinophil secretion.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The objective of this investigation was to determine whether direct stimulation of Th cells caused eosinophil activation or augmentation of secretion of LTC4. Whereas many prior investigations have implicated the role of Th2 cells in initiating asthma and atopic responsiveness (5, 7, 9, 10, 28), there has been no prior report of direct interaction between Th cells and eosinophils in isolated cell systems. In these studies, we used a method to cause differentiation of CD3-stimulated peripheral human CD4+ lymphocytes into Th1- and Th2-like subsets (22). These cells demonstrated a clear pattern of cytokine secretion profiles, which indicated their similarity to Th cell subsets as differentiated in vivo (14, 23, 29). However, because IL-3 was not secreted by Th2-like subsets in this system, its relative activity in endogenous cell-cell interactions could not be assessed. A further hypothesis of this investigation was that secretion from the Th2 cell subset would cause substantially greater augmentation of eosinophil secretion than Th1 cells and that the difference could be related to the cytokine secretion profiles obtained in isolated cell systems in vitro.

Our data indicate that the supernatant from CD3-activated Th1-like cells had neither a direct stimulatory nor augmentatory effect on eosinophil secretion. However, supernatant from CD3-activated Th2-like cells caused a threefold augmentation of LTC4 release vs. sham control or medium containing rhIL-1/6/4 to cause differentiation of Th2 cells or rhIL-1/6/12 to generate Th1-like cells (Fig. 2). Accordingly, Th2 cells appear to lack the capacity to activate directly eosinophil secretion but have a unique capacity to augment the stimulated secretion of LTC4.

In further studies, we found that the augmentation of stimulated secretion of eosinophils caused by pretreatment with Th2-Sup was primarily related to secretion of IL-5 and secondarily to GM-CSF (Fig. 4). By contrast, a decrease in augmented eosinophil secretion caused by CD3-stimulated Th2-like cells was not observed for eosinophils treated with immunodepleted TNF-alpha and/or IL-13. The system we used allowed us to isolate the augmentatory stimulus. Th1- and Th2-like cells were cultured de novo, and eosinophils were isolated from peripheral blood. This allowed us to assess whether there was a direct interaction and physiologically relevant relationship between Th-Sup priming of eosinophil secretion for concentrations of cytokines generated from CD3-stimulated Th cells. Although Th1- and Th2-like cells produced similar quantities of soluble GM-CSF, our data indicate that secreted cytokines from CD3-activated Th2-like cells are predominantly capable of causing direct augmentation of human eosinophil secretion in the absence of other intermediary cells or amplification responses.

It is important, however, to consider some limitations of our findings. The system we used to provide stimulus isolation in vitro involves a preparation of Th cells and eosinophils in an environment that may not replicate the in vivo situation in human asthma, and interactions with other cells and/or tissues cannot occur. However, the Th subsets that we used closely reflected the cytokine secretion profiles of Th1- and Th2-like cells from both mouse (3, 4, 8, 27) and human systems (14, 22, 26). Furthermore, specific interactions that occur solely between Th cells and eosinophils cannot be assessed accurately in the presence of other inflammatory cells or matrix (2, 21). Another unanswered question is why GM-CSF, which was secreted in comparable quantities from both Th1- and Th2-like cells, caused apparent augmentation of eosinophil secretion only for eosinophils treated with Th2 cell supernatant. rhGM-CSF substantially augments eosinophil secretion when given exogenously in the absence of Th cells (16). Prior investigations in rodents have indicated that augmented airway responsiveness is elicited only by Th2 cells and not by Th1 cells (3, 4). This increase in airway hyperresponsiveness was shown to be independent of IL-4, but no mechanism for the selective augmentation of airway hyperresponsiveness by Th2 cells vs. Th1 cells in mice was elucidated.

In this study, we observed an inhibitory effect in Th1-Sup that could account for Th2 selectivity in augmenting eosinophil secretion. Although we were unable to identify the soluble factor(s) associated with apparent Th1-Sup inhibition of stimulated eosinophil secretion, we demonstrated a substantial increase in LTC4 secretion by serial dilution of Th1-Sup (Fig. 3). We further investigated whether exogenous application of cytokines secreted by either activated Th1- or Th2-like cells could mimic the augmenting effect on eosinophil secretion observed in Th2-Sup-treated eosinophils. Pretreatment of naive eosinophils with either rhGM-CSF or rhIL-5 in concentrations determined from Th-Sup caused comparable augmentation of LTC4 secretion (Figs. 4 and 7). Other endogenously secreted cytokines from Th subsets did not affect the synthesis of cysteinyl leukotrienes when added exogenously to naive eosinophils (Fig. 7).

Although our data establish a likely role for direct Th2-Sup augmentation of stimulated de novo LTC4 synthesis in eosinophils, we did not identify a physiological trigger for eosinophil activation. The secretory stimulus hence was modeled for control and Th cell supernatant exposure by activation of eosinophils with fMLP/B. This method is well established (17-20), and augmentation of fMLP/B-induced secretion did not occur in control studies after exposure to cytokines, causing Th subset differentiation, or for CD3-stimulated Th1 cells. Hence, secretion caused by fMLP/B reflects cellular synthesis of LTC4 (15, 17-19, 21), and controls were obtained to validate conditions for these specific experiments. Whereas these data did not establish the Th2-like cell as the cell causing direct activation of eosinophil secretion (as contrasted to augmentation of secretion), our data do not exclude the possibility that Th2 cells could trigger eosinophil secretion as well under more physiological circumstances. Although the mechanism by which augmented secretion was elicited by IL-5 and GM-CSF was not examined directly in this study, we have shown previously that rhIL-5 caused increased phosphorylation of cPLA2, the enzyme that hydrolyzes the nuclear membrane phospholipids causing arachidonic acid release, which is the initial step in the synthesis of cysteinyl leukotrienes (24, 30, 31). Because phosphorylation (30) and augmented secretion of LTC4 occur within 30 min (24) after cell activation, we did not examine the effects of this short incubation time on mRNA expression of enzymes in the 5-lipoxygenase pathway.

We conclude that at a physiological concentration of secretion, supernatant (1:5 dilution) from cultured human Th2-like cells, but not from human Th1-like cells, is capable of substantially augmenting the synthesis of LTC4 in isolated, pharmacologically stimulated human eosinophils. This augmentation results predominantly from secretion of IL-5, solely secreted by CD3-activated Th2-like cells, and GM-CSF, a cytokine commonly secreted from both Th subsets.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-46368, Specialized Center of Research Grant HL-56399, National Institute of Allergy and Infectious Diseases Grant 2PO1-AI-20531-06, and by a grant from Glaxo Wellcome.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. R. Leff, Section of Pulmonary and Critical Care Medicine, Dept. of Medicine M6076, Univ. of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: aleff{at}medicine.bsd.uchicago.edu).

Received 1 July 1999; accepted in final form 11 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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