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The Journal of Immunology, 2002, 169: 2303-2312.
Copyright © 2002 by The American Association of Immunologists

Quantitative Differences in Chemokine Receptor Engagement Generate Diversity in Integrin-Dependent Lymphocyte Adhesion1

Daniele D’Ambrosio2,*, Cristina Albanesi{dagger}, Rosmarie Lang*, Giampiero Girolomoni{dagger}, Francesco Sinigaglia* and Carlo Laudanna{ddagger}

* BioXell, Milan, Italy; {dagger} Istituto Dermopatico dell’Immacolata, Istituto del Ricovero e Cura a Carattere Scientifico, Rome, Italy; and {ddagger} Section of General Pathology, Department of Pathology, University of Verona, Verona, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemokines control the specificity of lymphocyte homing. Numerous chemokines have been identified but the significance of redundancy in chemokine networks is unexplained. Here we investigated the biological significance of distinct chemokines binding to the same receptor. Among CCR4 ligands, skin vessels endothelial cells present C-C chemokine ligand (CCL) 17 but not CCL22 consistent with CCL17 involvement in T lymphocyte arrest on endothelial cells. However, CCL22 is much more powerful than CCL17 in the induction of rapid integrin-dependent T cell adhesion on VCAM-1 under conditions of physiological flow. The dominance of CCL22 over CCL17 extends to other CCR4-mediated phenomena such as receptor desensitization and internalization and correlates with the peculiar kinetics of CCR4 engagement by the two ligands. A similar phenomenological pattern is also shown for CXC chemokine ligand 9 and CXC chemokine ligand 11, which share binding to CXCR3. Our analysis shows how quantitative variations in chemokine receptor expression level and ligand engagement may alter the selectivity of integrin-dependent lymphocyte adhesive responses, suggesting a mechanism by which chemokine networks may either generate or break the specificity of lymphocyte subset recruitment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The functions of the immune system rely on an integrated guidance system for the appropriate distribution of its cellular components to various tissues. Recruitment of blood-borne leukocytes into tissues involves a multistep process of leukocyte-endothelial interactions, which culminates in integrin-dependent arrest on endothelial cell surface as a prerequisite for subsequent diapedesis (1, 2). Tethering and rolling precede the firm adhesion of circulating leukocytes and are essential to slow leukocyte motion, thus facilitating microenvironmental sampling and subsequent interactions with proadhesive chemokines presented by endothelial cells (3, 4). Tethering and rolling of leukocytes on vessel walls are primarily mediated by specialized selectins and mucins (5, 6), although {alpha}4 integrins, namely {alpha}4{beta}1 (very late Ag-4) and the mucosal homing receptor {alpha}4{beta}7 have been shown to support tethering and rolling (7). Chemokines, which generate heterotrimeric Gi protein-dependent signaling pathways, are physiological activators of rapid integrin-dependent lymphocyte arrest on endothelial cells (8, 9, 10, 11).

Once extravasated, leukocytes navigate through complex chemoattractant gradients by integrating conflicting chemotactic signals within tissues (8, 12). The complexity of the chemokine system appears well suited to convey the large body of information needed to guide the navigation of leukocytes from the blood stream to their final destination within tissues (13, 14). However, the promiscuity and redundancy in ligand-chemokine receptor interactions and the intricate pattern of chemokine receptor expression on leukocytes highlight a scarcely understood level of complexity.

To gain novel insights into chemokine-mediated control of lymphocyte recruitment, we analyzed the microenvironmental presentation of CCR4 ligands C-C chemokine ligand (CCL)3 17 and CCL22 and compared their capacity to trigger rapid integrin-dependent lymphocyte adhesion under conditions of physiological flow as well as their ability to trigger other CCR4-mediated phenomena. Paradoxically, we found that the chemokine CCL17, found on the endothelium, is not the higher potency CCR4 agonist. Interestingly, the dominance of CCL22 over CCL17 correlates with distinct kinetics of CCR4 engagement. By using human Th1 or Th2 cells expressing different levels of the chemokine receptors CCR4 and CXCR3, we find that quantitative differences in chemokine receptor level and agonistic potency of the ligands are two distinct parameters, which are integrated at the cellular level to generate diversity in lymphocyte subset proadhesive responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of polarized human Th lymphocytes and L1.2-human CCR4 (hCCR) transfectants

Human Th cell lines were generated as previously described by stimulation of CD8 T cell-depleted cord blood mononuclear cells with 2 µg/ml PHA (Wellcome, Beckenham, U.K.) in the presence of various combinations of cytokines and anti-cytokine Abs (15). Th1 cells were generated by the addition of 5 ng/ml IL-12 (Hoffmann-LaRoche, Nutley, NJ) and 200 ng/ml neutralizing anti-IL-4 Ab (BD PharMingen, San Diego, CA). Th2 cells were generated by the addition of 10 ng/ml IL-4 (BD PharMingen) and 2 µg/ml neutralizing anti-IL-12 Abs 17F7 and 20C2 (Hoffmann-LaRoche). The cells were cultured in complete medium (RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 5% FetalClone (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 1 mM sodium pyruvate, and 100 U/ml penicillin-streptomycin). On day 3, the cultures were washed and expanded in complete medium with addition of 100 U/ml IL-2 (Hoffman-LaRoche). Mouse L1.2 pre-B cells were cultured in complete medium at 1 x 106/ml (5 x 106) and were transfected with 3 µg of full length hCCR4 cDNA (a gift from S. Bhakta, Roche Bioscience, Palo Alto, CA) and 20 µg of Lipofectamine reagent (Life Technologies, Gaithersburg, MD). Forty-eight hours after transfection, cells were cultured in complete medium supplemented with 0.8 mg/ml G418 (Life Technologies). L1.2 stably expressing hCCR4 were sorted by FACS by staining with anti-hCCR4 Ab 1G1 (BD PharMingen).

Cell surface staining

Cells were washed in FACS buffer (50 mM phosphate, 150 mM NaCl, pH 7.4; 1% FetalClone; 0.05% sodium azide) and incubated with anti-hCCR4 (BD PharMingen) or anti-hCXCR3 (R&D Systems, Minneapolis, MN) for 30 min on ice, washed, and analyzed by FACScan flow cytometry (BD Biosciences, San Jose, CA).

Analysis of intracellular calcium mobilization

Indo1-acetoxymethyl ester loading was performed by incubating the cells (5 x 106/ml) in buffer A (HBSS with 10 mM HEPES) with 2.5 µM Indo1-acetoxymethyl ester and 0.05% w/v F-127 Pluronic (Molecular Probes, Eugene, OR) at 37°C for 30 min. The incubation was prolonged for 30 min after the addition of an equal volume of buffer B (HBSS with 10 mM HEPES and 5% FCS). Cells were washed twice in buffer B, resuspended at 1 x 106/ml, and analyzed by FACS before and after stimulation with the indicated chemokines (Dictagene, Epalinges, Switzerland). Data were recorded every 0.5 s as the relative ratio of fluorescence emitted at 395 and 480 nm after excitation at 364 nm. Mean 395/480 fluorescence ratio (y-axis) vs time (x-axis) were calculated using FlowJo analysis software (BD Biosciences). Fold induction of intracellular Ca2+ concentration ([Ca2+]i) was calculated by dividing the peak fluorescence ratio (395 nm:480 nm) of stimulated cells by the peak fluorescence ratio of unstimulated cells. Percentage of cross-desensitization = 100 - [(x - 1/y - 1) x 100], where x represents the fold induction of [Ca2+]i of secondary stimulation and y represents the fold induction of [Ca2+]i of primary stimulation.

Immunohistochemistry

Punch biopsies of normal skin from healthy individuals (n = 2), chronic atopic dermatitis (n = 3), and psoriasis lesions (n = 3) were embedded in OCT, snap-frozen in liquid nitrogen, and stored at -80°C until sectioning. Cryostatic sections of 4 µm were fixed in 5% paraformaldehyde for 10 min, treated with 0.3% hydrogen peroxide to quench endogenous peroxidase activity, and incubated with normal horse serum (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) for 20 min. Double immunostainings were performed with anti-CCL22 mAb (clone 272D; 5 µg/ml) (20) or rabbit anti-hCCL17 Ab (12 µg/ml) (PeproTech, Rocky Hill, NJ) and anti-Factor VIII/von Willebrand factor (vWF) (1/25) (DAKO, Glostrup, Denmark). Single stainings for CCL22, CCL17, and Factor VIII/vWF were also performed; to verify the specificity of the Abs used, sections were subsequently incubated with mouse or rabbit total serum (Vector Laboratories) as controls for the anti-CCL22 or anti-CCL17 Abs, respectively, and with purified mouse IgG1 (BD Biosciences) as controls for the anti-Factor VIII/vWF Abs. Avidin-biotin-peroxidase or avidin-biotin-phosphatase activities were revealed with 3-amino-9-ethylcarbazole and Blue Vector (Vector Laboratories), respectively. For each biopsy, 10 sections were stained with each Ab, and positive cells were evaluated in four adjacent fields at a magnification of x200.

Analysis of chemokine receptor internalization and ligand dissociation rates

Internalization of CCR4 was analyzed by incubating 1 x 106/ml Th2 cells in the absence or presence of various concentrations of CCL17 or CCL22 for the indicated time at 37°C or 4°C. In selected experiments, the cells were incubated for 2 h at 37°C with 10 µg/ml of pertussis toxin (Sigma). Subsequently, Th2 cells were washed twice in ice cold FACS buffer and stained with Ab to CCR4 or an isotype-matched control and analyzed by FACS. Percentage of receptor internalization were (%) = [(x - k)/(y - k)] x 100, where x represents mean fluorescence intensity of CCR4 staining after internalization, y represents mean fluorescence intensity of CCR4 staining before internalization, and k represents mean fluorescence intensity of isotype control. For evaluation of ligand-binding dissociation rates, L1.2-hCCR4 transfectants were washed and resuspended in binding buffer (125 nM NaCl, 25 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA, pH 7.0) and incubated for 2 h on ice. For each assay point, 1 x 106 cells in 0.1 ml of binding buffer were incubated on ice in the presence of 250 pM human recombinant 125I-labeled CCL17 or 125I-labeled CCL22 (specific activity, 2000 Ci/mmol; Amersham Pharmacia Biotech, Little Chalfont, U.K.). After 2 h, the cells were washed twice and resuspended in 1 ml of ice cold binding buffer. At various times, the cells were washed twice in ice cold binding buffer and lysed in 2% SDS, and radioactivity was counted with a gamma counter. Nonspecific binding was calculated by addition of a 500-fold molar excess of unlabeled CCL17 or CCL22.

Analysis of lymphocyte adhesion under flow

VCAM-1 was engineered as an IgG fusion protein using human IgG1 CH2-CH3 domains onto which the extracellular domains of human VCAM-1 was fused (kindly provided by Drs. U. Gubler and L. Renzetti, Hoffman-LaRoche). VCAM-1 sequence was from aa 1–696 ending 2 residues before the transmembrane domain. The construct was expressed in Drosophila cells, purified by affinity chromatography from lysates, and stored at -80°C. Recombinant extracellular domain of human VCAM-1 (native VCAM-1) purified from Chinese hamster ovary cells (R&D Systems) was also used with essentially the same results. Before use, VCAM-1-IgG fusion protein (0.5 mg/ml) was dialyzed against PBS containing 1% {beta}-octyl glucoside. Microcap glass capillary tubes (100 µl capacity; Drummond Scientific, Broomall, PA) were coated for 16 h at 4°C with 20 µl of human VCAM-1 at 2000 sites/µm2. Site densities per square micrometer of immobilized VCAM-1 were calculated using a 125I-labeled anti-human IgG1 H chain mAb, as previously described (16). Before use, tubes were washed and cocoated with 20 µl of 2 µM chemokines for 60 min. After a washing with PBS, the behavior of interacting Th1 and Th2 lymphocytes was recorded on S-VHS videotape (Panasonic, Verona, Italy) and analyzed frame by frame, as described (17). Th1 or Th2 cells were resuspended at a concentration of 1.5 x 106/ml and injected by applying a wall shear stress of 2 dyne/cm2 with the help of a digital syringe pump. After 2–3 min needed to establish laminar flow, single areas of 0.2 mm2 were recorded for at least 30 s. Interactions (rolling, arrest, or both) of 1 s were considered significant and were scored. Lymphocytes that remained firmly adherent for 10 s were considered fully adherent (11).

Quantification of chemokine immobilization

Sections (10 mm long) of 100-µl Microcap glass capillary tubes were coated with human VCAM-1 at 2000 sites/µm2, as described above. Human recombinant 125I-labeled CCL17 and 125I-labeled CCL22 (specific activity, 2000 Ci/mmol; Amersham Pharmacia Biotech) were reconstituted at 100 µCi/ml in PBS. A labeled/unlabeled (1/100) mixture of CCL17 and CCL22 was made containing 5 pmol of 125I-labeled chemokines in 100 µl of PBS. Ten microliters of chemokine mixture (corresponding to 50 pmol of chemokine) were added to the capillary tubes to cocoat a 10-mm-long section. For the competition binding assay, radioactive CCL17 was mixed with nonradioactive CCL22 or vice versa (corresponding to 100 pmol of total chemokines). After variable incubation times at room temperature, the tubes were washed with 10 ml of PBS at a flow rate of >10 dyne/cm2. Radioactivity bound to the tubes was quantified with a gamma counter and transformed in number of molecules per square micrometer. Background binding to glass in the absence of VCAM-1 was calculated for both chemokines and was subtracted from the binding in the presence of immobilized VCAM-1. The number of molecules of each chemokine specifically immobilized by one molecule of VCAM-1 was finally calculated.

Statistical analysis

SDs were calculated and statistical significance was assessed by the paired two-tailed Student t test. Values of p < 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distinct patterns of microenvironmental presentation of CCL17 and CCL22

Recent data have suggested a critical role for the chemokine receptor CCR4 in recruitment of cutaneous memory T cells into the inflamed skin (18, 19, 20, 21). Because CCL17/TARC and CCL22/MDC have been identified as two distinct ligands of CCR4 (22, 23), we wished to determine their expression pattern in the inflamed human skin by analysis of lesional skin from atopic dermatitis and psoriasis patients. In both psoriasis and atopic dermatitis, expression of CCL17 was detected on endothelial cells of the majority of vessels (85%) present in both the superficial and reticular dermis and on tissue-infiltrating cells with the morphology of dendritic cells (Fig. 1Go and data not shown). In sharp contrast, in both skin disorders, CCL22 was not detected on endothelial cells, although it was detectable on tissue-infiltrating dendritic cells (Fig. 1Go and data not shown). Neither CCL17 nor CCL22 could be detected in normal skin (Ref. 20 and data not shown). These results indicate a distinct pattern of microenvironmental presentation for CCL17 and CCL22 in inflamed skin, with CCL17 but not CCL22 being specifically presented by endothelial cells. Notably, this distinct pattern of chemokine presentation was not markedly different in diseases as diverse as psoriasis and atopic dermatitis, suggesting that the depicted tissue distribution of CCL17 and CCL22 could be a general feature of these chemokines.



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  		FIGURE 1. CCL17, but not CCL22, is localized on endothelial cells of inflamed human skin. Immunohistochemical analysis of human skin biopsies was obtained from psoriasis (A–D) or atopic dermatitis (E–H) patients. Anti-CCL17 Ab (red staining) marks intensely the endothelial cells of the majority of the vessels in the papillary dermis of psoriasis (C) and atopic dermatitis (G), as assessed by costaining for Factor VIII (blue staining; D and H). A number of CCL22-positive cells (red staining) are present in psoriasis (A) as well as in atopic dermatitis (E) lesions. Differently from CCL17, CCL22 expression was not found in Factor VIII+ endothelial cells (blue; B and F). To test the specificity of the anti-CCL17 and anti-CCL22 Abs, after Factor VIII stainings (shown in the insets of C and G), sections were incubated with rabbit and mouse serum, respectively, whereas purified mouse IgG1 were used as control for the anti-Factor VIII Ab (A and G). Neither CCL17- nor CCL22-positive cells are detected in both dermis and epidermis of normal skin from healthy individuals (not shown). Bars, 30 µm.

 
CCL17 and CCL22 chemokines differentially promote {alpha}4 integrin-dependent adhesion of human Th2 cells under conditions of physiological flow

The results reported above suggested that CCL17 and CCL22 binding to CCR4 could act at distinct locations, with CCL17 being uniquely devoted at capturing leukocytes from the blood stream. The role of CCL17 and CCL22 may not be restricted to regulating T cell recruitment to the skin given that CCR4 expression is found on activated T cells, Th2 cells, and regulatory T cells (24, 25, 26), suggesting the involvement of this receptor in different phases of T cell localization. This scenario fostered us to compare the ability of CCL17 and CCL22 to promote rapid integrin-dependent adhesion under conditions of physiological flow. Given the possibility to generate large numbers of human CD4+ Th cells with a well-characterized profile of chemokine and adhesion receptors, we initially used in vitro-generated human Th2 cells, which express high levels of CCR4 as well as {alpha}4 integrins (Fig. 2GoA) (25, 27). Because {alpha}4 integrins have been shown to support transient adhesive interactions, namely tethering and rolling due to their microvillous distribution (28), we were able to investigate the interactions of Th2 cells on immobilized {alpha}4{beta}1 integrin ligand VCAM-1, under physiological flow conditions. However, in the absence of chemokines, VCAM-1 poorly supported transient adhesion of Th2 cells even at high site density (data not shown). Because CCR4 is highly expressed on Th2 cells (Fig. 2GoA), we analyzed the effect of coimmobilizing CCL17 or CCL22, the two ligands of CCR4, with VCAM-1. The two chemokines coimmobilized with VCAM-1 induced a powerful up-regulation of tethering and rolling, rapidly followed by firm adhesion of Th2 cells (Fig. 2GoB). Although both chemokines were similarly efficient inducers of tethering and rolling, CCL22 was much more efficient than CCL17 in triggering conversion from rolling to firm adhesion of Th2 cells on VCAM-1 (Fig. 2GoB). Importantly, neutralizing anti-CCR4 Abs were able to completely inhibit the adhesive interactions observed with CCL17 or CCL22 but not CXCL12 (Fig. 2GoC). These findings confirm the recently reported rapid up-regulation of {alpha}4{beta}1 integrin-dependent lymphocyte tethering and rolling by chemokines (29). Most importantly, they demonstrate that the two CCR4-sharing chemokines CCL17 and CCL22 elicit qualitatively distinct types of integrin-mediated interactions under conditions of physiological flow.



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  		FIGURE 2. Rapid induction of rolling and firm adhesion of human Th2 cells on VCAM-1 by CCL17 or CCL22 under flow. A, Surface expression of chemokine receptors CCR4 and CXCR3 and integrins {alpha}4, {alpha}E, {beta}1, and {beta}7 on human Th2 cells. Shown are representative FACS profiles of Th2 cells stained with Abs specific for chemokine receptors CCR4 and CXCR3 and integrins {alpha}4, {alpha}E, {beta}1 and {beta}7 (thick lines) or isotype control Abs (dashed lines). Geometric mean fluorescence intensity (GMFI) values are indicated in each quadrant. B, VCAM-1 immobilized at 2000 sites/µm2 supports tethering/rolling and firm adhesion of Th2 cells in response to 2 µM coimmobilized chemokines. CCL17 and CCL22 triggers marked up-regulation of tethering/rolling and robust sticking of Th2 cells. CCL22 induces more sticking than CCL17. Values are the average number ± SD of tethering/rolling ({square}) or firmly adherent ({blacksquare}) cells during 30 s calculated from 12 distinct 0.2-mm2 areas of the capillary tube from 5 experiments. A significant difference between CCL17 and CCL22 induction of Th2 cell sticking is shown (*, p < 0.001). C, Chemokine receptor CCR4 is required for tethering/rolling and firm adhesion of Th2 cells induced by CCL17 and CCL22 coimmobilized with VCAM-1. Th2 cells were incubated for 15 min at room temperature with 5 µg/ml anti-hCCR4 Ab (1G1; BD PharMingen) or isotype-matched control and then assayed in their ability to adhere under flow to VCAM-1 in the presence of coimmobilized CCL17, CCL22, or CXCL12. Values are average number ± SD of tethering/rolling ({square}) or firmly adherent ({blacksquare}) cells during 30 s from eight distinct 0.2-mm2 areas of the capillary tube from two experiments. A significant reduction of Th2 cell rolling and sticking by anti-CCR4 Ab is shown (*, p < 0.001).

 
Chemokine binding to immobilized VCAM-1

To verify that the differences observed were not due to a different degree of chemokine immobilization on VCAM-1, we next quantified the number of molecules of CCL17 or CCL22 immobilized in presence of purified human VCAM-1. Various concentrations of radiolabeled CCL17 or CCL22 were incubated in the presence of immobilized VCAM-1 and the number of bound chemokine molecules was quantified. This experiment revealed a high number of CCL17 and CCL22 molecules that can specifically bind to one molecule of integrin ligand (Fig. 3Go). Binding was rather rapid in that it was detectable within 15 min, reaching a plateau within 30 min. Saturation was reached at chemokine concentration of 5 µM for both CCL17 and CCL22 (data not shown). Interestingly, VCAM-1 was capable of absorbing CCL17 more efficiently than CCL22 (Fig. 3GoA). The specificity of these interactions was confirmed by the inability of immobilized albumin and human IgG to bind the chemokines (Fig. 3GoA) and by the ability of CCL17 to displace more efficiently CCL22 binding to VCAM-1 (Fig. 3GoB). These data show for the first time that VCAM-1 is able to specifically bind chemokines and suggest that endothelial integrin ligands can act as highly efficacious chemokine-presenting molecules. Similar results have been observed with mucosal addressin cell adhesion molecule-1, and further studies are in progress to characterize these interactions (D. D’Ambrosio and C. Laudanna, unpublished results). Importantly, these experiments show that CCL17 is immobilized ~2- to 3-fold more efficiently than CCL22, indicating that the lower efficiency of CCL17 in triggering rapid arrest of Th2 cells under flow is not due to reduced presentation of this chemokine.



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  		FIGURE 3. VCAM-1 specifically binds CCL17 and CCL22. A, CCL17 binds more efficiently than CCL22 to immobilized VCAM-1. Chemokines display background binding to glass. In contrast, chemokine binding to BSA or human IgG (H-IgG) is negligible. BSA (1 mg/ml) and human IgG (5 mg/ml) were immobilized to glass capillary tubes as for the extracellular domain of VCAM-1 alone (native VCAM-1) or fused to human IgG (VCAM-1-Fc). Values shown indicate the average number ± SD of chemokine molecules bound in 60 min to an area of 1 µm2 obtained from three experiments performed in triplicate. A significant binding of CCL17 and CCL22 on immobilized VCAM-1 is shown (*, p < 0.01; **, p < 0.0005). B, CCL17 and CCL22 compete for binding to VCAM-1. CCL22 binding to VCAM-1 is displaced more efficiently by CCL17 (CCL22 + CCL17) than CCL17 binding to VCAM-1 is displaced by CCL22 (CCL17 + CCL22). Values indicate the average number ± SD of chemokine molecules bound (in 60 min) to one molecule of immobilized VCAM-1 obtained from two experiments performed in triplicate. A significant displacement between CCL17 and CCL22 in binding to immobilized VCAM-1 is shown (*, p < 0.05; **, p < 0.005).

 
Role of chemokine receptor expression level in rapid adhesion triggering

The expression level of chemokine receptors on memory/effector T cells is highly heterogeneous, and it has been suggested to be a critical parameter regulating rapid lymphocyte adhesion under flow (17). To evaluate how variations in the expression levels of chemokine receptors influenced the efficiency of integrin-dependent adhesion triggered by CCL17 and CCL22, we took advantage of in vitro-derived human Th1 cells. Th1 cells express levels of {alpha}4 integrins similar to Th2 cells but poorly express CCR4 (Fig. 4GoA). When the interactions of Th1 cells on immobilized VCAM-1 in the presence of coimmobilized CCL17 or CCL22 were analyzed under flow, we found that coimmobilization of CCL17 with VCAM-1, although able to induce a moderate up-regulation of Th1 cell rolling, was ineffective in triggering the complete transition from rolling to firm adhesion (Fig. 4GoB). By contrast, CCL22 consistently triggered a remarkable level of firm adhesion of Th1 cells (Fig. 4GoB). Importantly, on Th1 cells expressing low levels of CCR4, the difference between CCL17 and CCL22 in triggering integrin-dependent firm adhesion becomes an all or none phenomenon (Fig. 4GoB). Our data indicate that the agonistic potency of the chemokine cooperates with the expression level of the receptor to quantitatively and qualitatively regulate integrin-dependent lymphocyte adhesion.



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  		FIGURE 4. Effect of chemokine receptor expression level on the efficiency of chemokine-induced rapid integrin-dependent lymphocyte adhesion. A, Surface expression of chemokine receptors CCR4 and CXCR3 and integrins {alpha}4, {alpha}E, {beta}1, and {beta}7 on human Th1 cells. Shown are representative FACS profiles of Th1 cells stained with Abs specific for chemokine receptors CCR4 and CXCR3 and integrins {alpha}4, {alpha}E, {beta}1, and {beta}7 (thick lines) or isotype control Abs (dashed lines). Geometric mean fluorescence intensity (GMFI) values are indicated in each quadrant. B, Tethering/rolling and firm adhesion of human Th1 cells on VCAM-1. CCL17 does not trigger stable adhesion to VCAM-1, whereas rolling is effectively induced. In contrast, CCL22 efficiently induces rolling and stable adhesion. Values are average number ± SD of rolling or firm adherent cells during 30 s from 14 distinct 0.2-mm2 areas of the capillary tube from six experiments. A significant difference between CCL17 and CCL22 induction of Th1 cell sticking is shown (*, p < 0.0005).

 
Relative efficiencies of integrin-dependent lymphocyte arrest induced by CCL22 vs CCL17

Taken together, our data suggest that CCL22 is more potent than CCL17 at promoting integrin-dependent arrest of lymphocytes, a conclusion that is consistent with previous analysis of CCR4 binding affinity of the two ligands (22, 23) and their ability to trigger CCR4-mediated signaling. However, because it is conceivable that different chemokines may be presented on endothelial cells at different densities, it was of interest to investigate whether variations in the density of a presented chemokine could compensate for intrinsic differences among ligands. To this purpose, we assayed the interactions of Th1 cells on VCAM-1 in the presence of decreasing amounts of CCL22. The usage of Th1 cells expressing low CCR4 was instrumental to test the critical role of chemokine density in suboptimal conditions of chemokine receptor expression. Under these conditions, coimmobilization of 2 µM CCL17 together with VCAM-1 resulted in a marked induction of Th1 cell tethering and rolling with only few arrested cells (Fig. 5Go). In contrast, 2 µM CCL22 triggered firm adhesion of more cells (Fig. 5Go). Analysis of the spectrum of adhesive interactions triggered by decreasing amounts of immobilized CCL22 revealed that only when it was immobilized at a concentration ~200-fold lower than that of CCL17 did the two chemokines behave similarly by promoting almost exclusively rolling (Fig. 5Go). It should be added that given the ~2- to 3-fold higher efficiency of CCL17 vs CCL22 binding to VCAM-1 (Fig. 3Go), the actual quantitative difference in potency between the two CCR4 ligands might even be underestimated.



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  		FIGURE 5. Relative efficiencies of integrin-dependent lymphocyte adhesion induced by CCL22 vs CCL17. CCL22 immobilized at very low dose is still more efficient than CCL17 in triggering full adhesion of Th1 cells to VCAM-1. CCL17 was immobilized at 2 µM. CCL22 was immobilized at the indicated doses. Values indicate the average number ± SD of rolling or firm adherent cells observed during 30 s from 10 separate 0.2-mm2 areas of the capillary tube from two experiments. A significant difference between CCL17 and CCL22 induction of Th1 cell sticking is shown (*, p < 0.05; **, p < 0.005).

 
Differential CCR4 desensitization and internalization triggered by CCL17 and CCL22

Our analysis of the efficiency of induction of rapid adhesion by CCL17 and CCL22 indicated a remarkable level of dominance of CCL22 over CCL17. This seemed somewhat surprising given the relatively small difference of CCR4 binding affinity between the two ligands (22, 23). Thus, we performed a quantitative evaluation of calcium mobilization, desensitization, and internalization of CCR4, which are not directly related to integrin activation in response to CCL17 or CCL22 but provide a more direct measurement of the agonistic potency of the two chemokines. CCL22 consistently induced more robust calcium mobilization in Th2 cells (Table IGo and data not shown). Moreover, stimulation of Th2 cells with CCL22 followed by stimulation with a 10- or 20-fold molar excess of CCL17 or vice versa demonstrated an absolute dominance of CCL22 over CCL17 in the ability to desensitize CCR4 (Table IGo). Because it is theoretically possible that CCL22 binds to an unidentified receptor in addition to CCR4, we have extended our analysis to mouse L1.2 pre-B cells transfected with hCCR4 receptor (L1.2-hCCR4). L1.2 cells transfected with hCCR4 receptor but not parental L1.2 cells mobilized calcium in response to CCL17 or CCL22 (Table IGo and data not shown). As seen with Th2 cells, CCL22 was dominant over CCL17 in desensitizing calcium mobilization in L1.2-hCCR4 even at a 20-fold lower concentration (Table IGo).


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  		Table I. Magnitude of intracellular calcium mobilization and cross-desensitization of CCL17 and CCL22 in Th2 cells and L1.2hCCR4 transfectantsa

 
Ligand-induced chemokine receptor desensitization involves receptor phosphorylation by G protein-coupled receptor kinases, which facilitate binding of {beta}-arrestins and promote receptor sequestration and internalization (30). Given the hierarchy of CCR4 desensitization induced by CCL17 and CCL22, we investigated how the two ligands elicited CCR4 internalization. Human Th2 cells were incubated at 37°C for various times with CCL17 or CCL22, and the cell surface CCR4 expression was monitored by Ab staining. Strikingly, CCL22 promoted a rapid and potent down-modulation of CCR4 expression, whereas CCL17 had a minimal effect (Fig. 6GoA). CCL22 elicited a marked CCR4 down-modulation after 5 min (Fig. 6GoA). A dose-dependent increase in CCR4 internalization was observed, with CCL22 being effective at a concentration as low as 6.2 nM, whereas CCL17 was poorly effective even at a 20-fold higher concentration (125 nM) (Fig. 6GoA). Similar findings were observed using L1.2-hCCR4 cells (data not shown). To confirm that the reduction of CCR4 staining intensity was due to sequestration/internalization of CCR4 rather than binding competition, we performed incubations at 4°C to inhibit receptor internalization. At 4°C, no significant down-regulation of CCR4 expression was observed with either CCL17 or CCL22 (Table IIGo). To evaluate the signaling requirements for ligand-induced internalization of CCR4, we analyzed the effects of inhibiting inhibitory G{alpha} subunit (Gi protein) signaling using pertussis toxin. Internalization of CCR4 induced by CCL22 was unaffected by pertussis toxin (Fig. 6GoB) showing that this is independent of Gi signaling pathways. In contrast, pertussis toxin treatment completely inhibited the adhesion of lymphocytes under flow (data not shown). These data indicate that the hierarchy of dominance of CCL22 over CCL17 extends beyond the induction of rapid integrin-dependent adhesion to diverse CCR4-mediated phenomena.



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  		FIGURE 6. CCR4 internalization by and dissociation rates of CCR4 ligands CCL17 and CCL22. A and B, Down-regulation of cell surface expression level of CCR4 was measured by anti-CCR4 Ab staining and FACS analysis of Th2 cells. Geometric (Geo) mean fluorescence intensity values are indicated in each quadrant. A, Th2 cells were incubated for 5 min at 37°C in the presence or absence of the indicated concentration of CCL17 or CCL22. B, Th2 cells were incubated for 120 min at 37°C in the presence or absence of 125 nM CCL17 or CCL22 and where indicated (bottom) were pretreated with 10 µg/ml pertussis toxin (+PT). C, Dissociation rates of 125I-labeled CCL17 and 125I-labeled CCL22 bound to L1.2-hCCR4 transfectants at 4°C. Data represent the percentage ± SD of radioactive counts of each radiolabeled chemokine that remained specifically bound to L1.2-hCCR4 cells at different time point relative to time 0, which is taken as 100%. Data are representative of two experiments performed in triplicate.

 

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  		Table II. CCR4 down-modulation induced by CCL17 or CCL22 in Th2 cellsa

 
CCL17 and CCL22 display different kinetics of dissociation from CCR4

Previous data have shown that CCL17 and CCL22 bind to CCR4 with different affinities (22, 23), with CCL22 exhibiting ~2- to 3-fold higher affinity than CCL17. Thus, higher CCR4 affinity could result in higher receptor occupancy by CCL22 relative to CCL17, and this may help establish a functional hierarchy between these chemokines. However, it is evident that the dose range over which CCL22 exhibited clear dominance over CCL17 went well above a 3-fold difference (Tables IGo and IIGo and Fig. 5Go). Thus, the reduced efficiency of CCL17 in triggering receptor desensitization and internalization as well as rapid integrin-dependent adhesion under flow is unlikely to be explained only on the basis of a lower number of receptors occupied.

This raised the possibility that differences in the modality of interaction of CCL17 or CCL22 with CCR4 could contribute to establish the observed phenomenological diversity. Activation of signaling pathways is critically dependent on triggering the active conformation of a threshold number of receptors and/or the duration of single ligand/receptor interactions (31, 32). To evaluate the kinetics of ligand/receptor interaction, we compared time-dependent dissociation of CCL17 or CCL22 bound to CCR4. L1.2-hCCR4 cells were incubated with 250 pM radiolabeled CCL17 or CCL22, and after removal of nonbound chemokine the amount of radioactivity that remained associated to the cells was quantified at different times. At time 0, we found ~1700 molecules of CCL22 vs ~500 molecules of CCL17 specifically bound per cell, consistent with the reported higher affinity of CCL22 for CCR4 (23). Surprisingly, this experiment revealed a faster rate of dissociation of CCL22 vs CCL17 (Fig. 6GoC). Similar results were obtained using human Th2 cells (data not shown). These findings indicate that CCL22 and CCL17 bind to CCR4 with different kinetics, raising the possibility that this difference could, at least in part, explain the observed phenomenological diversity.

The regulatory role of receptor expression level and agonistic potency as a general paradigm of chemokine-induced integrin-dependent rapid lymphocyte adhesion

We finally wished to test whether the observations made with CCR4 and its ligands represent a general paradigm of regulation of integrin-dependent rapid lymphocyte adhesion by chemokines. To this end, we analyzed the adhesive interactions of Th1 or Th2 cells on immobilized VCAM-1 in response to CXCL9 and CXCL11, which bind specifically to the CXCR3 receptor that is preferentially expressed on Th1 cells (Figs. 2GoA and 4A). Previous work has shown that CXCL11 displays higher affinity and agonistic potency than CXCL9 for CXCR3 (33, 34). Analysis of intracellular calcium mobilization in response to CXCL9 and CXCL11 confirmed that CXCL11 is dominant over CXCL9 on human Th1 cells as well as on human CXCR3-transfected L1.2 cells (data not shown). In flow adhesion assays, both chemokines induced a marked up-regulation in the number of tethering/rolling and firmly adherent Th1 cells on VCAM-1 (Fig. 7GoA). CXCL11 was consistently more efficient than CXCL9 in the conversion of rolling into firmly adherent Th1 cells (Fig. 7GoA). When analysis was performed on Th2 cells, CXCL11 was still able to elicit a significant number of interactions, a fraction of which consisted of firmly adherent cells (Fig. 7GoB). In contrast, CXCL9 was able to induce moderate rolling but poor arrest of Th2 cells (Fig. 7GoC). Overall, these data depict a pattern of interactions induced by CXCL9 and CXCL11 that mirrors that seen with CCL17 and CCL22.



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  		FIGURE 7. The efficiency of CXCL9 and CXCL11 triggering of rapid adhesion of Th1 and Th2 cells on VCAM-1 under flow correlates with agonistic potency and the expression level of CXCR3 expression. VCAM-1 supports, upon chemokine triggering, tethering, rolling, and firm adhesion of both Th1 and Th2 cells. A, CXCL9 and CXCL11 trigger marked up-regulation of tethering/rolling of Th1 cells. CXCL9 and particularly CXCL11 trigger robust sticking of Th1 cells. Values are the average number ± SD of rolling or firm adherent cells during 30 s from 14 separate 0.2-mm2 areas of the capillary tube from three experiments. A significant difference between CXCL9 and CXCL11 induction of Th2 cell sticking is shown (*, p < 0.005). B, On Th2 cells, CXCL9 and CXCL11 induce tethering/rolling. However, CXCL9 fails to induce sticking, whereas CXCL11 triggers a significant level of firm adhesion of Th2 cells. Values are the average number ± SD of rolling or firm adherent cells during 30 s from 11 separate 0.2-mm2 areas of the capillary tube from three experiments. A significant difference between CXCL9 and CXCL11 induction of Th2 cell sticking is shown (*, p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The concerted action of chemokines and adhesion molecules controls the process of lymphocyte vascular recognition and extravasation. Different chemokines involved in tissue-specific recruitment of various leukocyte subsets show redundancy by sharing the same receptor. Chemokine redundancy could be a way to increase robustness of the system (35), but it may also constitute a physiologically important aspect of its specificity. Here, we investigated a possible meaning of chemokine network redundancy by quantitative analysis and comparison of CCL17 and CCL22 expression pattern and ability to trigger rapid integrin-dependent T cell adhesion under flow.

First, we documented that CCL17 but not CCL22 is expressed on vascular endothelium in distinct skin inflammatory conditions in vivo (Fig. 1Go). Moreover, we showed a more efficient immobilization on VCAM-1 of CCL17 vs CCL22. Notably, CCL22 but not CCL17 is sensitive to CD26 proteolytic degradation, indicating that CCL17 may be a more stable ligand adept at presentation by endothelial cells (36, 37, 38). Together, these results suggest that CCL17 but not CCL22 is devoted to trigger vascular recognition of CCR4 expressing cells such as cutaneous memory T cells or Th2 cells.

Starting from these observations, we wished to verify whether CCL17 was the most efficient proadhesive agonist for CCR4-expressing lymphocytes. To this end, we used in vitro-derived human Th1 and Th2 cells as a model system. These cells proved ideal for our analysis because large numbers can be generated in vitro and exhibit distinctive profiles of chemokine receptors. To simplify our analysis, we took advantage of the fact that endothelial vascular ligand VCAM-1 can support, upon chemokine triggering, lymphocyte tethering, rolling, and arrest under physiological flow, likely due to subsecond induction of {alpha}4{beta}1 integrin clustering induced by chemokine receptor signaling (29). In accordance with this study, we found that immobilization of CCL17 or CCL22 promotes pertussis toxin-sensitive transient tethering and rolling of T cells on VCAM-1 (data not shown). The involvement of integrin {alpha}4{beta}1 in our setting was confirmed by the inhibitory effect of blocking Abs (data not shown).

Data obtained with adhesion assays under flow showed striking differences in the ability of chemokines to trigger firm arrest. In Th1 cells (which express low level of CCR4), CCL22 but not CCL17 induced full arrest. The level of chemokine receptor expression was suggested to be a relevant parameter for induction of lymphocyte arrest under flow conditions (39). However, the striking difference between CCL22 and CCL17 cannot be explained simply in terms of lower receptor occupancy, because in Th2 cells, which express high levels of CCR4, CCL22 was still much more efficient than CCL17 in triggering firm adhesion. Furthermore, CCL22 was remarkably more powerful than CCL17 in triggering CCR4 desensitization and internalization, indicating that the dominance of CCL22 over CCL17 extends well beyond the induction of integrin activation to also affect Gi-independent signaling pathways. These differences prompted us to investigate the possible molecular basis by evaluating how the two ligands interact with the receptor. Our findings that the higher affinity ligand CCL22 dissociates from the receptor more rapidly than CCL17 suggest that differential kinetics of chemokine receptor binding may trigger qualitatively different biological responses.

An intriguing possibility is that the frequency of association/dissociation of ligand-receptor complexes is a critical parameter regulating the activation of certain intracellular signaling pathways. This idea is supported by recent data showing that the frequency more than the amplitude of intracellular signaling events controls the specificity and magnitude of gene expression (40, 41) and calmodulin-dependent protein kinase II activation (42). Another possibility is that the conditions of flow impose a tight temporal restrain for a productive signal-generating chemokine receptor engagement on lymphocytes that rapidly flow and contact vessel walls. Coimmobilization of chemokines with VCAM-1 has been reported to be necessary for triggering T cell rolling and adhesion under flow (29). The kinetic parameter inherent to the conditions of flow may help establish a "velocity threshold" for chemokine receptor occupancy that is required for adhesion triggering by the ligand. In this scenario, the efficiency of lymphocyte adhesion under flow would critically depend on the kinetics of chemokine receptor occupancy. Thus, it would be predicted that chemokines with faster rates of receptor association/dissociation, as in the case of CCL22 binding to CCR4, could more easily achieve a kinetic receptor occupancy threshold required for induction of integrin-dependent adhesion under flow. Regardless of the exact mechanism, our data suggest that a different modality of CCR4 engagement by CCL22 induces a quantitatively and/or qualitatively different signal transduction leading to phenomenological diversity.

To investigate whether these findings could be generalized to another chemokine ligand/receptor system, we also analyzed the activities triggered by two distinct ligands of CXCR3, a receptor that, opposite to CCR4, is expressed at higher levels on Th1 cells. CXCL9 and CXCL11 have been reported to exhibit distinct binding characteristics and agonistic potencies with respect to CXCR3, with CXCL11 being the dominant ligand (34, 43, 44). Furthermore, a recent study has reported the selective expression of the low potency CXCL9 on HEV from inflamed tissue (45). When CXCL9 and CXCL11 were analyzed for their ability to elicit rapid integrin-dependent adhesion and calcium mobilization on Th1 and Th2 cells (Fig. 7Go and data not shown), the pattern of induced phenomena mirrored that seen with CCL17 and CCL22, with CXCL11 being the most potent agonist and CXCL9 almost unable to trigger arrest of Th2 cells. Again, differences in receptor binding affinity allow a remarkable level of diversification in signaling and biological responses.

Overall, our data show that lymphocytes presented with different chemokines binding to same receptor display completely different biological responses depending on the modality of receptor engagement and the level of receptor expression. Importantly, our data indicate that the most potent chemokine not only increases the absolute number of adherent lymphocytes, but also triggers the arrest of different lymphocyte subtypes. In this scenario, a quantitative difference becomes a qualitative change. Notably, the most potent chemokines seem not to be presented to circulating lymphocytes. This result seems paradoxical, given that one could expect that the critical conditions imposed by the flow require the most potent chemokine to be presented on vascular endothelium. We propose a possible interpretation for these findings. Although presented less efficiently, the most potent chemokine may elicit proadhesive responses in different lymphocyte subtypes expressing high as well as low level of chemokine receptor (Fig. 8Go). Thus, by presenting the less potent chemokine, the immune system adopts a useful strategy to achieve the selective recruitment of cells expressing only a high level of a given chemokine receptor, which corresponds to a peculiar functional phenotype. In this context, it is significant that CCL17, the less potent CCR4 ligand, is bound more efficiently to VCAM-1 but still does not trigger significant adhesion of CCR4low Th1 lymphocytes. In contrast, CCL22 seems so powerful that, even if bound at ~20-fold lower concentrations, it is still able to trigger full arrest of CCR4low Th1 lymphocytes. From the viewpoint of selectivity, CCL22 could be considered a dangerous chemokine. Interestingly, expression of all three CXCR3 ligands (CXCL9, CXCL10, and CXCL11) was detected on the endothelium of atherosclerotic plaques (46), which are considered the result of a chronic, rather nonspecific, inflammatory process. Thus, our findings underscore a potential mechanism of "diversity breaking" in the immune system, based on expression on the microvasculature of chemokines able to elicit full proadhesive responses in leukocyte subtypes expressing either low as well as high levels of a specific chemokine receptor (Fig. 8Go).



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  		FIGURE 8. Model for how quantitative variations in chemokine potency and receptor level may be integrated to control the specificity of lymphocyte subset recruitment. In our model, endothelial display of a low potency chemokine such as CCL17 will result in selective recruitment of a lymphocyte subset expressing low levels of receptor, thus achieving a high degree of specificity in lymphocyte subset recruitment. By contrast, endothelial display of a high potency chemokine such as CCL22 should promote recruitment of lymphocyte subsets expressing both high and low levels of receptor, potentially breaking the diversity of lymphocyte subset recruitment.

 
In conclusion, our work indicates that the modality of receptor engagement, level of chemokine receptor expression, and chemokine presentation are not independent parameters regulating the exquisite specificity of lymphocyte vascular recognition. Instead, it appears they are quantitatively and functionally integrated at the cellular level to achieve a global threshold of signals necessary to induce rapid integrin activation. In this context, our data illuminate the simple elegance of chemokine-controlled immune regulation in which a subtle shift in chemokine potency may generate or break the specificity of lymphocyte subset recruitment.


   Acknowledgments
 
We thank members of BioXell for their critical comments and discussions.


   Footnotes
 
1 This work was supported by European Network for the Investigation of the Pathogenesis of Psoriasis Grant ERBFMRXCT98-0205 (to F.S.); European Grant SILENT QLG-1CT-1999-01036 (to D.D. and F.S.); Cofinanziamento Il Ministero dell’Istruzione, dell’Università e della Ricerca and University of Verona, Progetto Sanità; 1996/97, Fondazione Cassa di Risparmio, Ministero della Sanità (ricerca finalizzata), Consiglio Nazionale delle Ricerche; and a grant from the Italian Association for Cancer Research, 2001 (to C.L.). Back

2 Address correspondence and reprint requests to Dr. Daniele D’Ambrosio, BioXell, Via Olgettina 58, Milan, Italy I-20132. E-mail address: daniele.dambrosio{at}bioxell.com Back

3 Abbreviations used in this paper: CCL, C-C chemokine ligand; hCCR, human CCR; [Ca2+]i, intracellular Ca2+ concentration; vWF, von Willebrand factor; CXCL, C-X-C chemokine ligand. Back

Received for publication March 13, 2002. Accepted for publication June 26, 2002.


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