CD4+ T Cell and Eosinophil Adhesion Is Mediated by Specific ICAM-3 Ligation and Results in Eosinophil Activation

Ivor S. Douglas, Alan R. Leff and Anne I. Sperling
J Immunol March 15, 2000, 164 (6) 3385-3391; DOI: https://doi.org/10.4049/jimmunol.164.6.3385

Abstract

T cells and eosinophils, which are found in close proximity in asthmatic lungs, express many surface receptors that are counterligands. These data suggest that direct interactions between these cell types could play an important role in regulating airway inflammation in asthma. We examined the effect of selective adhesion between counterligands on human eosinophils and CD4+ T cells to determine 1) the existence of specific adhesive interactions and 2) if augmented specific adhesion to CD4+ T cells also caused augmented secretion of leukotriene C4 (LTC4) from eosinophils. A new method for binding of human CD4+ T cells to microwell plates was developed, which allowed for specific quantitative assessment of eosinophil adhesion to individual CD4+ T cells in culture. Adhesion of CD4+ T cells to eosinophils was minimal in unstimulated cells but increased after activation of T cells by PMA. Augmented adhesion was regulated substantially through binding of ICAM-3 and only minimally by ICAM-1. We further evaluated whether this specific adhesion up-regulated stimulated secretion of LTC4 from eosinophils. Adhesion with CD4+ T cells augmented eosinophil secretion of LTC4 caused by FMLP plus cytochalasin. Blockade of ICAM-3, as well as ICAM-1, inhibited completely the augmented secretion of eosinophil LTC4. We demonstrate that eosinophils and CD4+ T cells are capable of ligand-specific adhesion that is mediated predominantly by ICAM-3 ligation and that this binding causes augmented eosinophil secretion.

CD4+ T cells and eosinophils are regulatory and effector immune cells that are implicated in the airway inflammatory responses characterizing bronchial asthma. The Th2 subset of CD4+ T cells secretes cytokines, including GM-CSF, IL-4, and IL-5 that are proinflammatory in a spectrum of diseases including asthma, allergic rhinitis, and atopic eczema. Many of these cytokines also have direct effects on eosinophil maturation, migration, and survival (1, 2, 3). While Th2 cell-specific cytokines alone have no effect on eosinophil secretion of bronchoactive eicasonoids, they substantially augment, or prime, eosinophil secretion in the presence of other activators (4).

Recent investigations have shown that molecular adhesion between integrins on human eosinophils to vascular endothelial cells and/or airway wall matrix, which occurs during eosinophil migration, also primes stimulated secretion of leukotriene C4 (LTC4)3 from eosinophils independent of cytokines (5). The augmented secretion of eosinophil LTC4 caused by ligation to soluble fibronectin in vitro is sufficient to cause augmented contraction of explanted human airways (6). This augmented contractile response is blocked selectively and completely by mAb directed against very-late Ag-4 (VLA-4), which is a specific ligand to fibronectin, or by 5-lipoxygenase inhibition of cysteinyl leukotriene synthesis (6).

Prior investigations also indicate that ligation between β1 integrins and VCAM-1 and β2 integrin and ICAM-1 at the endothelial cell surface primes human eosinophil secretion (5). This ICAM-mediated ligation in other cell types results in “outside-in signaling” and important second-messenger phosphorylation events, cytoskeletal rearrangements, and up-regulation of cellular activation status (7, 8). Adhesion-primed eosinophil activation has been demonstrated to result from increased translocation of the 85-kDa cytosolic phospholipase A2 to the nuclear membranes, with subsequent augmented secretion of the potent proinflammatory cysteinyl leukotriene, LTC4 (9).

Activated CD4+ T cells and eosinophils have been demonstrated to be in close proximity in the airway submucosa from biopsies of atopic asthmatics (10). These data raise the possibility that these cell types interact directly (11). Therefore, we surveyed the potential adhesive counterligands shared by eosinophils and CD4+ T cell and examined the possible specific adhesive interactions between these two cell types. A new method was developed that allowed for highly specific quantitative assessment of the degree of adhesion and that also avoided homologous interactions among T cells and eosinophils. This study demonstrates that activated CD4+ T cells adhere to human eosinophils through a specific receptor-mediated interaction. In particular, we find a unique role of ICAM-3 (CD50) in this response. The ICAM-3-mediated adhesion of the eosinophils to activated human CD4+ T cells is independent of detectable cytokine production. Finally, we demonstrate that specific adhesion causes substantial augmentation of eosinophil secretion of LTC4 that also is predominantly regulated by ICAM-3 but also affected by ICAM-1.

Materials and Methods

Isolation of human eosinophils

Peripheral blood eosinophils were isolated from mildly atopic donors by negative immunomagnetic selection as modified from the method of Hansel et al. (12). Briefly, 120 ml whole blood was withdrawn from the antecubital vein and placed into containers containing heparin. Blood was separated by centrifugation through 1.089 g/ml Percoll (Sigma-Aldrich, St. Louis, MO), and the supernatant and the mononuclear cells at the interface were discarded. Erythrocytes in the granulocyte pellet were lysed by hypotonic shock, and the remaining cells were washed and resuspended in HBSS with 0.2% BSA (Sigma-Aldrich) before automated cell counting using a Coulter counter (Beckman Coulter, Fullerton, CA). Relative neutrophil percentage was calculated by differential counts of Wright-Giemsa-stained cytospin preparations. The granulocytes were incubated with anti-CD16 mAb-coated beads (Miltenyi Biotech, Auburn, CA) at 4°C for 30 min and then passed through a 1 × 10-cm column packed with steel wool and held within a 0.6 Tesla MACS magnet (Becton Dickinson, Mountain View, CA). Cells were eluted with a further 30-ml aliquot of HBSS/0.2% BSA. Neutrophils were retained in the magnetized steel wool, while eosinophils passing through the column were collected, washed, counted, and assessed for purity as above. Eosinophil purity of >99% and viability >98% by trypan blue exclusion was routinely obtained. Eosinophils were prepare fresh for each experiment described below.

CD4+ T cell isolation

CD4+ T cells were isolated from random, leukopheresed units by a method modified from that previously described (13). Briefly, monocytes were separated through a Ficoll gradient, density = 1.077 g/cm3 (Sigma-Aldrich), washed, and counted. The cells were incubated overnight at 4°C on a disk rotator with a combination of Abs (a generous gift provided by Dr. Gijs van Seventer, University of Chicago) designed to deplete all non-CD4+ T cells and prepared from mouse ascites that included anti-human mAbs to HLA class II (IVA12), glycophorin (10F7), CD19 (FMC63), CD14 (63D3), CD11b (NIH11b-1), CD16 (3G8), and CD8 (B9.8) (13). Subsequently, cells were pelleted, washed, counted, and incubated with Dynal beads precoated with anti-mouse IgG mAb (final dilution, 40 beads/cell). After incubation, CD4+ T cells were separated by negative immunomagnetic selection using a custom-designed rare earth magnet to a purity of >99% as confirmed by flow cytometry. CD4+ T cells were suspended in complete RPMI (RPMI 1640 without calcium, 10% heat-inactivated FCS, 104 U/ml penicillin, 10 mg/ml streptomycin, and 25 μg/ml amphotericin B/ml) with 20% DMSO, and viability was confirmed by trypan blue exclusion before staged freezing of aliquots to ensure viability upon thawing. After freezing, the cells were stored in −70°C until thawed for experiments.

Immunofluorescense staining and flow cytometry

Surface expression of adhesion molecules on eosinophils and T cells was analyzed by flow cytometry. For these experiments, 106 eosinophils or CD4+ T cells were resuspended in ice-cold FACS buffer (PBS containing 0.01% sodium azide and 0.5% BSA). Cells were incubated with saturating concentrations (10 μg/ml) of directly FITC-conjugated mouse anti-human mAbs CD11a (G25.2; Immunotech, Westbrook, ME), CD11b (Bear 1; Immunotech), CD18 (L130; Immunotech), ICAM-1 (84H1O; Immunotech), or CD2 (B-E2; Biosource, Camarillo, CA). Some samples were stained with either unconjugated anti-ICAM-3 (KS128; Dako, Carpinteria CA), LFA-3 (TS2/9; Endogen, Cambridge, MA), ICAM-2 (BT-1; Immunotech), CD11d (a generous gift of ICOS, Seattle, WA), or isotype control IgG1 (Becton Dickinson, San Jose, CA) mAbs and developed with directly conjugated FITC goat anti-mouse Ig (Becton Dickinson). After washing in FACS buffer, cells were fixed in 0.5% formaldehyde overnight at 4°C before analysis on a FACScan flow cytometer (Becton Dickinson) using CellQuest software. Appropriate isotype controls were prepared with FITC-conjugated mouse IgG1.

Fluorometric labeling

CD4+ T cell viability after gentle thawing in RPMI 1640 was >90% by trypan blue exclusion. CD4+ T cells were labeled for 1 h at 37°C in the dark by incubating with 15 μM, 4-chloromethyl-7-hydroxycoumarin (Cell Tracker Blue (CTB); absorption, 353 nm; emission, 466 nm), and eosinophils were labeled with 5 μM, 4-chloromethyl-tetramethyl-rhodamine (Cell Tracker Orange (CTO); absorption, 541 nm; emission, 565 nm). These membrane-permeant probes undergo esterase hydrolysis upon entering cells. Stable intracellular fluorochrome labeling is achieved by a GST-mediated reaction to produce membrane-impermeant conjugates. Validation assays confirmed optimal dilutions of CD4+ T cells and eosinophils to be 106 cells/ml of fluorochrome solution (see Results). Cells were washed and pelleted twice in calcium-free RPMI 1640 to remove nonbound fluorochrome and resuspended in appropriate dilutions in complete RPMI for the cell-adhesion assay.

Adhesion assay

Opaque black-walled, transparent-based 96-well plates (Costar, Cambridge, MA) were incubated overnight at 4°C with 4 μg/ml anti-CD4 mAb (QS4120; Calbiochem, Cambridge, MA) in carbonate buffer, pH 8.5, blocked for 2 h with 2% BSA in PBS at 37°C, rinsed with Tween 20 in PBS, and equilibrated with RPMI 1640. CTB-labeled CD4+ T cells (5 × 105 cells/200 μl RPMI 1640) were bound as a monolayer to each well by centrifugation (500 × g) at 4°C. Plate fluorescence readings of the discrete emissions spectrum for the CTB-labeled CD4+ T cells were performed as described below before and after washing to calculate percent CD4+ T cell binding. Where indicated, CD4+ T cells were incubated with PMA 10 ng/ml for 45 min at 37°C (14, 15). Buffer aspiration and immediate, repeated, vigorous washes with fresh buffer terminated PMA activation and removed nonadherent CD4+ T cells. Subsequently, CTO-labeled eosinophils (5 × 105 cells/well) were allowed to adhere to the T cell monolayers for 45 min at 37°C by passive sedimentation. The formation of a CD4+ T cell monolayer and uniform eosinophil settling was monitored by inverted-stage microscopy. In some experiments, eosinophils were suspended in buffer with saturating concentrations (10 μg/ml) of anti-ICAM-1, anti-ICAM-2, and/or anti-ICAM-3 before introduction into the wells. At the end of the predetermined adhesion period, nonadherent eosinophils were removed by submersion and inversion of the 96-well plate for 10 min in a tank of HBSS with calcium (16). This permitted uniform, 1 × g sedimentation of nonadherent cells. Wells were refilled with 200 μl buffer, and appropriate plate blanks and negative controls were prepared. Fresh aliquots of CTO-labeled eosinophils (5 × 105/200 μl/well) were included after the washing step as positive controls. Plate fluorescence readings of the discrete emissions spectra of the two fluorochromes were performed with a CytoFluor II Fluorescence MultiWell Plate Reader (PerSeptive Biosystems, Framingham, MA). Filters for CTO-labeled eosinophils were 530 nm (excitation) and 590 nm (emission) with 50 readings per well. Filters for CTB-labeled CD4+ T cells were 360 nm (excitation) and 460 nm (emission) with 30 readings per well. Average emissions from wells prepared in triplicate for each intervention were expressed as relative fluorescence intensity. Readings were performed before and after washing to calculate relative eosinophil:CD4+ T cell adhesion.

LTC4 assay

In parallel experiments, CD4+ T cells were bound to 96-well plates as described above. A total of 5 × 105 eosinophils per well were added and allowed to adhere for 45 min at 37°C by passive sedimentation. At the end of adhesion, 10−6 M FMLP (Sigma-Aldrich) and 5 μg/ml cytochalasin B (Sigma-Aldrich) were added to wells to activate all eosinophils. We have previously demonstrated that this results in maximal secretion of LTC4 and granular proteins of eosinophils (17). Activation was terminated after 30 min by plate centrifugation with low centrifugal force (500 rpm) for 5 min at 4°C. Supernatants were collected and immediately frozen for later analysis. Supernatant from nonactivated eosinophils and from wells containing buffer alone served as controls. When indicated, the cells were preincubated with saturating concentrations of anti-ICAM-1 and anti-ICAM-3 mAbs. LTC4 was measured in the supernatants by colorimetric competition assay according to the manufacturer’s directions (Cayman Chemicals, Ann Arbor, MI).

GM-CSF and IL-5 ELISA

Sandwich ELISAs kits from PharMingen (San Diego, CA) were used to measure GM-CSF (sensitivity, 0.2 ng/ml) and IL-5 (sensitivity, 0.1 ng/ml) in assay supernatants from nonactivated and PMA-up-regulated adhesion assays. ELISAs were preformed and developed with avidin peroxidase, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and N,N-dimethyl formamide/SDS (Sigma) according to PharMingen’s protocol. Colorimetric quantifications of cytokines were analyzed with Softmax software using a Thermomax Microplate-reader with a 405-nm filter (Molecular Devices, Sunnyvale, CA).

Data analysis

All data are expressed as mean ± SE for each group. Paired t tests were used to analyze difference between groups. Repeated measures ANOVA was used where more than one comparison was made. Statistical significance was claimed when p < 0.05.

Results

T cells and eosinophils express receptor/counterligand adhesion molecules

T cells and eosinophils were assessed for their surface expression of β2 integrin adhesion molecules CD11a/CD18, CD11b/CD18, and CD11d/CD18 and their counterligands, ICAM-1, ICAM-2, and ICAM-3. Peripheral blood eosinophils expressed CD11a/CD18 and CD11b/CD18, but not CD11d, while T cells expressed only CD11a/CD18 (Fig. 1⇓A). In contrast, T cells expressed all three ICAM molecules (ICAM-1, ICAM-2, and ICAM-3), while eosinophils only expressed ICAM-3 (Fig. 1⇓B). Expression of these adhesion molecules did not change significantly after PMA activation (Fig. 1⇓, A and B). From these data, we identified several potential molecular adhesive receptor-ligand pairs that could interact to induce T cell-eosinophil adhesion. T cell CD11a/CD18 could interact with eosinophil ICAM-3, eosinophil CD11a/CD18 could interact with ICAM-1, ICAM-2, or ICAM-3 on T cells, and eosinophil CD11b/CD18 could interact with ICAM-1 on T cells.

FIGURE 1.
FIGURE 1.

Eosinophil and CD4+ T cell-surface ligands. Representative flow cytometric histograms from FACScan performed on freshly isolated human eosinophils (PBE). CD4+ T cells were either untreated (control) or PMA activated as described in the text. Cells were fluorochrome-labeled by incubation with pertinent mAbs. Cell-surface expression was defined as shift in mean fluoresence activity compared with appropriate isotype controls. A, Integrin adhesion molecule expression on eosinophils and T cells; B, Ig superfamily adhesion molecule expression on eosinophils and T cells. Shaded histograms, control IgG Abs; open histograms (superimposed), fluorochrome-labeled anti-cell adhesion molecule Abs. The histograms are from a representative experiment (n = 3).

Activation of CD4+ T cells induces adhesion of eosinophils

To determine whether T cells and eosinophils are capable of direct and specific ligation, we developed a plate-based photofluorometric adhesion assay. In this assay, binding of CD4+ T cells to anti-CD4 mAb-coated wells was used to produce nonconfluent, but uniformly distributed, single-cell layers as confirmed by inverted-stage fluorescence microscopy (Fig. 2⇓A). We confirmed CD4+ T cell binding to be optimal (91.4 ± 6.83%; n = 3) at a density of 5 × 105 cells/well by direct measurements before and after washing. Visual inspection confirmed that at this density the CD4+ T cells were not in direct contact with each other, thus minimizing the effects of contact-mediated intercellular signaling. To up-regulate adhesion-molecule activation states on the T cells, some wells were treated with PMA (10 ng/ml) for 45 min at 37°C (Fig. 2⇓B; (18). Because PMA modulates T cell cytoskeletal function, the increased spreading seen in Fig. 2⇓B confirmed CD4+ T cell activation by PMA. To determine the ability of T cells to adhere to eosinophils, freshly isolated peripheral blood eosinophils were added to the T cell monolayers and incubated together for 45 min. The plate was then washed by a 1 × g sedimentation method. As described in Materials and Methods, the entire plate was first completely immersed in a buffer tank and then inverted in the tank without surfacing the plate. The nonadherent eosinophils were allowed to “fall” out of the well for 10 min. The optimal time for inversion and sedimentation was determined to be 10 min after the method of St. John et al. (16). We validated inversion-sedimentation time by placing CTO-labeled eosinophils in wells in the absence of prebound CD4+ T cells. The reduction in relative fluorescence intensity was measured at 1-min intervals until fluorescence plateaued at background levels. Using this method, we found that after equal loading of eosinophils (Fig. 2⇓, C and D), the eosinophils adhered to both the nonactivated and activated CD4+ T cells (Fig. 2⇓, E and F). However, adhesion to activated CD4+ T cells was substantially greater than for nonactivated CD4+ T cells.

FIGURE 2.
FIGURE 2.

Eosinophil:CD4+ T cell adhesion (×4, inverted stage microscopy). Nonconfluent CD4+ T cells are bound to flat-bottom wells (A). PMA activation causes cytoskeletal rearrangements and cell-spreading vs control (B). Eosinophils are allowed to adhere to nonactivated (C) and PMA-activated CD4+ T cells (D). Eosinophils adhesion to PMA CD4+ T cells (F) is marked in comparison to nonactivated controls (E). Arrows indicate adherent eosinophils; arrow-heads indicate CD4+ T cells.

To quantitate the adhesion found in the T cell/eosinophil assay, the eosinophils were fluorescently dyed with the membrane permeant dye, CTO. A standard curve of the eosinophils produced highly reproducible readings on a fluorescence plate reader (Fig. 3⇓). The measurements were found to be highly sensitive and accurate (r2 = 0.99). The CTO-labeled eosinophils were used to measure the percent adhesion to the CD4 monolayers by reading each plate before and after inversion washing. By this method, percent adhesion of eosinophil to CD4+ T cells could be derived for each well. Eosinophil adhesion to PMA-activated CD4+ T cells was demonstrated to be maximal at 45 min (46.1% at 45 min vs 11.9% at 10 min; p < 0.05) (Fig. 4⇓). In contrast, adhesion did not increase significantly over baseline in wells containing non-PMA-activated CD4+ T cells (9.33% at baseline vs 15.9% at 45 min). Adhesion time was thus standardized to 45 min in all subsequent experiments.

FIGURE 3.
FIGURE 3.

Standardization of adhesion. Performance characteristics of the assay were determined by doubling dilutions of “unknown” numbers of CTO-labeled eosinophils, calibrated against a standard curve generated from dilutions of known cell numbers.

FIGURE 4.
FIGURE 4.

Time-course of CTO-labeled eosinophil adhesion to CD4+ T cell monolayer. Representative adhesion time course for eosinophils adhering to CD4+ T cells under basal and PMA-activated conditions. Percent adhesion was calculated at predetermined time points. Optimal adhesion was at 45 min. Cellular disengagement was seen as reduced adhesion beyond 45 min. These data are representative of three independent experiments.

PMA activation of CD4+ T cells greatly augmented by eosinophil adhesion. In eight separate experiments, eosinophil adhesion to nonactivated CD4+ T cells was 21.9 ± 4.90% vs 48.4 ± 4.29% for PMA-up-regulated CD4+ T cells; p < 0.001 (Fig. 5⇓).

FIGURE 5.
FIGURE 5.

Eosinophil adhesion to CD4+ T cells. Percentage adhesion of eosinophils to quiescent and PMA-activated CD4+ T cells was determined by plate fluorometry. Cells were coincubated with saturating concentrations (10 μg/ml) of the indicated mAbs or with vehicle control. The cells were allowed to adhere for 45 min.

Receptor specificity in eosinophil/T cell adhesion

In further studies, we used blocking mAbs to ICAM-1, ICAM-2, and ICAM-3 to determine their involvement in T cell-eosinophil interactions. Coincubation of cells with anti-ICAM-3 mAbs resulted in substantial, but not complete, inhibition, of eosinophil adhesion to 33.26 ± 3.67% (p < 0.01 vs buffer control; Fig. 5⇑). In contrast, neither anti-ICAM-1 mAb or anti-ICAM-2 substantially inhibited intercellular binding to either quiescent or PMA-up-regulated CD4+ T cells (p = NS). Coincubation with all of the anti-ICAM Abs together did not result in greater adhesion blockade than anti-ICAM-3 alone (data not shown).

Adhesion of eosinophils to CD4+ T cells results in significant priming of LTC4 secretion by FMLP

LTC4 secretion cause by FMLP plus cytochalasin B was significantly augmented to 2022 ± 276.8 pg/ml when the eosinophils were adherent to CD4+ T cells vs 1455 ± 259.3 pg/ml for nonadherent, activated eosinophils (p < 0.05; Fig. 6⇓). To evaluate the specificity of the adhesive priming effect, we tested the ability of anti-ICAM-3 mAb to block stimulated eosinophil secretion. Adhesion-mediated augmentation of activated LTC4 secretion was inhibited below control levels to 1092 ± 439.2 after anti-ICAM-3 blockade vs 2022 ± 276.8 for buffer-treated controls (p = 0.045; Fig. 6⇓). Coincubation with anti-ICAM-1 resulted in a attenuation of augmented LTC4-release to control level (1552 ± 258.1; p = NS). Coincubation with both anti-ICAM-1 and anti-ICAM-3 did not result in LTC4 secretion inhibition that was greater than anti-ICAM-3 alone. (Data not shown).

FIGURE 6.
FIGURE 6.

Augmented LTC4 secretion by eosinophils adherent to CD4+ T cells. Secretion of LTC4 in the supernatants of FMLP-eosinophils adherent to CD4+ T cell monolayers was determined by colorimetric competition assay. Cells were coincubated with saturating concentrations of the indicated mAbs (10 μg/ml) or vehicle control.

CD4+ T cell secretion of cytokines caused by activation or adhesion did not cause the augmented secretion of LTC4 from eosinophils. Both GM-CSF and IL-5 were below threshold levels of detection in triplicate samples from three separate eosinophil donors and below that necessary to augment stimulated eosinophil secretion in vivo (19).

Discussion

We have demonstrated specific in vitro eosinophil adhesion to PMA-activated CD4+ T cells that is substantially regulated by ICAM-3, which is expressed both on eosinophils and CD4+ T cells (Fig. 5⇑). The biological importance of this intercellular adhesion process is implied by the augmented cysteinyl leukotriene release that results from this adhesion interaction (Fig. 6⇑.)

Eosinophils secrete granular proteins, cysteinyl leukotrienes and cycloxygenase products, and oxygen radicals in response to agonists such as FMLP and platelet activating factor. Adhesion to matrix proteins and vascular, interstitial, and airway epithelial cell-surface adhesion proteins, predominantly by ICAM-1 and VCAM-1, primes eosinophils for subsequent activated secretion of preformed and phospholipid-derived inflammatory mediators (5). We have previously demonstrated that adhesion interactions in the chemokine-mediated transpulmonary migration of eosinophils through the asthmatic airway sequentially prime eosinophils for secretion (20). In this study, we have also found that direct binding of eosinophils to PMA-activated CD4+ T cells augments FMLP-induced eosinophil stimulation of activated cysteinyl leukotriene (LTC4) secretion.

The photoflurometric adhesion assay used in these studies to evaluate intercellular adhesion is unlike previously described eosinophil:epithelial cell adhesion assays. We have established a sensitive method for creating a nonconfluent monolayer of CD4+ T cells to model intercellular leukocyte adhesion. Binding of T cells via CD4 provided a unique anchoring protein for the assay, which allowed high levels of selectivity and minimizes nonspecific eosinophil binding. CD4 is expressed in high density on Th cell surfaces but is not expressed on quiescent eosinophils (21). Unlike CD3 ligation, which is a well-characterized T cell-activating signal (22), CD4+ T cell binding did not cause detectable secretion of cytokines that activate eosinophils. The undetectable cytokine production suggests that the CD4+ T cell layers were generated without causing nonspecific CD4+ T cell activation, although further studies would be required to fully rule out this possibility.

The current study was designed to consider the potential for biologically important eosinophil:CD4+ T cell adhesive interactions independent of accompanying cytokine effects. We have implicated ICAM-3 as an important, but not exclusive, regulator of CD4+ T cell:eosinophil adhesion because incubation with anti-ICAM-3 does not completely inhibit adhesion. ICAM-1 also was not shown to contribute substantially to CD4+ T cell:eosinophil adhesion. It is noted that ICAM-3 is expressed on both eosinophil and T cell surfaces (Fig. 1⇑, A and B). In contrast, ICAM-1 is exclusively expressed on T cells in this model. Thus, this investigation does not clarify if functional ICAM-3 on either or both cell types is responsible for intercellular adhesion. We have not elucidated the contribution of other probable counterligands, notably αLβ2 or the more recently described αDβ2, in this process. Notably, αDβ2 is described to have a relatively higher affinity for ICAM-3 than ICAM-1 (23). However, we found no detectable expression of αD on eosinophils by flow cytometry.

Skubitz and coworkers have suggested that ICAM-3 is a potentially important coligand for human neutrophil αMβ2 (Mac-1; CD11b/CD18) adhesion to HUVEC (24). Additionally, preincubation of neutrophils with the anti-ICAM-3 Ab inhibits up-regulated cell-surface expression of αMβ2, as well as L-selectin (CD62-L) shedding on subsequent activation. This highlights the potential for ICAM-3-mediated outside-in signaling as an explanation for enhanced eosinophil:CD4+ T cell adhesion, which is incompletely inhibited with anti-ICAM-3 mAb alone. In contrast, ICAM-3 costimulation in conjunction with CD3 crosslinking has been demonstrated to activate both resting and activated T cells (25).

The significant augmentation of LTC4 secretion by eosinophils adherent to CD4+ T cells suggests a potential priming phenomenon resulting from the adhesion process. Our results were recorded in the absence of detectable concentrations of the key regulatory and proinflammatory cytokines, IL-5 and GM-CSF. These data are consistent with other studies suggesting adhesion-mediated priming of eosinophils following ligation to fibronectin (26). It is impressive that a relatively small attenuation of ligand-specific adhesion causes substantial inhibition of eosinophil secretion of LTC4 (Fig. 6⇑). While anti-ICAM-1 causes a minimal decrease in adhesion, augmented secretion was blocked completely. Anti-ICAM-3, which caused substantial blockade of CD4+ T cell adhesion to eosinophils, caused blockade of eosinophil secretion of LTC4 to substantially below control level (Fig. 6⇑). We conclude that ligand-specific adhesion between CD4+ T cells and eosinophils is associated with augmented activated secretion of LTC4 from eosinophils.

Acknowledgments

We thank Dr. Gijs van Seventer (Department of Pathology) and Julie Auger (Cancer Research Center Flow Cytometry Facility, University of Chicago) for reagents, advice, and thoughtful direction; Anja Herrnreiter and Diane Meyer for technical assistance; and Nancy Trojan for administrative support.

Footnotes

  • ↵1 This work was supported by National Heart Lung and Blood Institute Grant HL-46368 and National Heart Lung and Blood Institute Specialized Center of Research Grant IP50HL56399 and by Glaxo Wellcome. A.I.S. is a fellow of the Parker B. Francis Foundation for Pulmonary Research.

  • ↵2 Address correspondence and reprint requests to Dr. Alan R. Leff, Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC 6076, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address: aleff{at}medicine.bsd.uchicago.edu

  • ↵3 Abbreviations used in this paper: LTC4, leukotriene C4; VLA-4, very-late Ag-4; CTB, Cell Tracker Blue; CTO, Cell Tracker Orange.

  • Received June 8, 1999.
  • Accepted January 13, 2000.

References

  1. ↵
    Corrigan, C. J., A. B. Kay. 1992. T cells and eosinophils in the pathogenesis of asthma. Immunol. Today 13: 501
    OpenUrlCrossRefPubMed
  2. ↵
    Kay, A. B.. 1996. TH2-type cytokines in asthma. Ann. NY Acad. Sci. 796: 1
    OpenUrlCrossRefPubMed
  3. ↵
    Koenderman, L., T. van der Bruggen, R. C. Schweizer, R. A. Warringa, P. Coffer, E. Caldenhoven, J. W. Lammers, J. A. Raaijmakers. 1996. Eosinophil priming by cytokines: from cellular signal to in vivo modulation. Eur. Respir. J. 22: (Suppl.):119s
    OpenUrl
  4. ↵
    Weller, P. F., K. Lim. 1997. Human eosinophil-lymphocyte interactions. Mem. Inst. Oswaldo. Cruz. 92: (Suppl. 2):173
    OpenUrlCrossRefPubMed
  5. ↵
    Nagata, M., J. B. Sedgwick, H. Kita, W. W. Busse. 1998. Granulocyte macrophage colony-stimulating factor augments ICAM-1 and VCAM-1 activation of eosinophil function. Am. J. Respir. Cell Mol. Biol. 19: 158
    OpenUrlCrossRefPubMed
  6. ↵
    Anwar, A. R., G. M. Walsh, O. Cromwell, A. B. Kay, A. J. Wardlaw. 1994. Adhesion to fibronectin primes eosinophils via α4β1 (VLA-4). Immunology 82: 222
    OpenUrlPubMed
  7. ↵
    Lub, M., Y. van Kooyk, C. G. Figdor. 1995. Ins and outs of LFA-1. Immunol. Today 16: 479
    OpenUrlCrossRefPubMed
  8. ↵
    Feldhaus, M. J., J. M. Kessel, G. A. Zimmerman, T. M. McIntyre. 1998. Engagement of ICAM-3 activates polymorphonuclear leukocytes: aggregation without degranulation or β2 integrin recruitment. J. Immunol. 161: 6280
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Sano, A., N. M. Munoz, H. Sano, J. Choi, X. Zhu, B. Jacobs, A. R. Leff. 1999. Inhibition of cPLA2 translocation and leukotriene C4 secretion by fluticasone proprionate in exogenously activated human eosinophils. Am. J. Respir. Crit. Care Med. 159: 1903
    OpenUrlCrossRefPubMed
  10. ↵
    Bradley, B. L., M. Azzawi, M. Jacobson, B. Assoufi, J. V. Collins, A. M. Irani, L. B. Schwartz, S. R. Durham, P. K. Jeffery, A. B. Kay. 1991. Eosinophils, T-lymphocytes, mast cells, neutrophils, and macrophages in bronchial biopsy specimens from atopic subjects with asthma: comparison with biopsy specimens from atopic subjects without asthma and normal control subjects and relationship to bronchial hyperresponsiveness. J. Allergy Clin. Immunol. 88: 661
    OpenUrlCrossRefPubMed
  11. ↵
    Azzawi, M., B. Bradley, P. K. Jeffery, A. J. Frew, A. J. Wardlaw, G. Knowles, B. Assoufi, J. V. Collins, S. Durham, A. B. Kay. 1990. Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am. Rev. Respir. Dis. 142: 1407
    OpenUrlCrossRefPubMed
  12. ↵
    Hansel, T. T., I. J. De Vries, T. Iff, S. Rihs, M. Wandzilak, S. Betz, K. Blaser, C. Walker. 1991. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145: 105
    OpenUrlCrossRefPubMed
  13. ↵
    Semnani, R. T., T. B. Nutman, P. Hochman, S. Shaw, G. A. van Seventer. 1994. Costimulation by purified intercellular adhesion molecule 1 and lymphocyte function-associated antigen 3 induces distinct proliferation, cytokine and cell surface antigen profiles in human “naive” and “memory” CD4+ T cells. J. Exp. Med. 180: 2125
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Petruzzelli, L., L. Maduzia, T. A. Springer. 1998. Differential requirements for LFA-1 binding to ICAM-1 and LFA-1-mediated cell aggregation. J. Immunol. 160: 4208
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Haverstick, D. M., H. Sakai, L. S. Gray. 1992. Lymphocyte adhesion can be regulated by cytoskeleton-associated, PMA-induced capping of surface receptors. Am. J. Physiol. 262: C916
    OpenUrlAbstract/FREE Full Text
  16. ↵
    St John, J. J., D. J. Schroen, H. T. Cheung. 1994. An adhesion assay using minimal shear force to remove nonadherent cells. J. Immunol. Methods 170: 159
    OpenUrlCrossRefPubMed
  17. ↵
    White, S. R., M. E. Strek, G. V. Kulp, S. M. Spaethe, R. A. Burch, S. P. Neeley, A. R. Leff. 1993. Regulation of human eosinophil degranulation and activation by endogenous phospholipase A2. J. Clin. Invest. 91: 2118
    OpenUrlCrossRefPubMed
  18. ↵
    Tominaga, T., K. Sugie, M. Hirata, N. Morii, J. Fukata, A. Uchida, H. Imura, S. Narumiya. 1993. Inhibition of PMA-induced, LFA-1-dependent lymphocyte aggregation by ADP ribosylation of the small molecular weight GTP binding protein, rho. J. Cell Biol. 120: 1529
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Munoz, N. M., A. Herrnreiter, G. A. van Seventer, R. T. Semnani, D. Mayer, A. R. Leff. 1998. Generation of soluble factors from cultured human Th2-like cells augment LTC4 secretion in human eosinophils. Am. J. Respir. Crit. Care Med. 157: A395
    OpenUrl
  20. ↵
    Munoz, N. M., D. Mayer, A. Hernreiter, K. Hamann, S. P. Neeley, A. R. Leff. 1999. Augmentation of eosinophil degranulation of LTC4 secretion by integrin-mediated cell adhesion. Am. J. Physiol. Lung Cell Mol. Physiol. 277: L802
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Hossain, M., Y. Okubo, S. Horie, M. Sekiguchi. 1996. Analysis of recombinant human tumour necrosis factor-α-induced CD4 expression on human eosinophils. Immunology 88: 301
    OpenUrlCrossRefPubMed
  22. ↵
    Bluestone, J. A., R. Khattri, G. A. van Seventer. 1998. Fundamental immunology accessory molecules. M. William E. Paul, ed. Fundamental Immunology 4th Ed.531 Lippincott, Williams & Wilkins, Baltimore, MD.
  23. ↵
    Van der Vieren, M., H. Le Trong, C. L. Wood, P. F. Moore, T. St. John, D. E. Staunton, W. M. Gallatin. 1995. A novel leukointegrin, αdβ2, binds preferentially to ICAM-3. Immunity 3: 683
    OpenUrlCrossRefPubMed
  24. ↵
    Skubitz, K. M., K. D. Campbell, A. P. Skubitz. 1997. CD50 monoclonal antibodies inhibit neutrophil activation. J. Immunol. 159: 820
    OpenUrlAbstract
  25. ↵
    Hernandez-Caselles, T., G. Rubio, M. R. Campanero, M. A. del Pozo, M. Muro, F. Sanchez-Madrid, P. Aparicio. 1993. ICAM-3, the third LFA-1 counterreceptor, is a co-stimulatory molecule for both resting and activated T lymphocytes. Eur. J. Immunol. 23: 2799
    OpenUrlCrossRefPubMed
  26. ↵
    Munoz, N. M., K. F. Rabe, S. P. Neeley, A. Herrnreiter, X. Zhu, K. McAllister, D. Mayer, H. Magnussen, S. Galens, A. R. Leff. 1996. Eosinophil VLA-4 binding to fibronectin augments bronchial narrowing through 5-lipoxygenase activation. Am. J. Physiol. 270: L587
    OpenUrlAbstract/FREE Full Text
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The Journal of Immunology: 164 (6)
The Journal of Immunology
Vol. 164, Issue 6
15 Mar 2000
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