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* Mayo Clinic Scottsdale, Scottsdale, Arizona;
American Museum of Natural History, New York, New York;
Department of Immunology and Rheumatology, Merck Research Laboratories, Rahway, New Jersey; and
Department of Respiratory Medicine, Second Hospital of Zhejiang University School of Medicine, Hangzhou, People’s Republic of China
Correspondence: J. J. Lee, Ph.D., Division of Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Samuel C. Johnson Medical Research Building—Research, Mayo Clinic Scottsdale, 13400 E. Shea Boulevard, Scottsdale, AZ 85259. E-mail: jlee{at}mayo.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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to bind receptors on purified mouse eosinophils, it was only able to induce significant eosinophil migration in a mixed splenocyte population and was unable to induce migration of highly purified eosinophils. Collectively, these observations reveal physiologically relevant distinctions in mechanisms mediating human and mouse eosinophil migration that potentially reflect evolutionary disparities between these species.
Key Words: eotaxin • TARC • KC • MIP-1 • RANTES
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The use of mouse models to dissect pathways of allergic inflammation and determine the roles of individual cell types and chemokines/chemokine receptors in allergic pathophysiology has increased dramatically. The ability of these models to develop antigen-induced recruitment of lymphocytes and eosinophils [3 ], increase antigen-specific immunoglobulins, and increase T-helper cell type 2 cytokines and chemokines [4 5 6 ], coupled with the ease of genetic manipulation in these animals (for example, see ref [7 ]), has led to the widespread use of mice as models of human allergic inflammation. The expanded use of mouse models has thus necessitated a more thorough examination of chemokine-eosinophil interactions in mice in order to gain a better understanding of how these responses may differ from those responses in humans.
Chemokines are a family of small peptides (8–14 kD) produced by many cells involved in allergic inflammation, including endothelial cells [8
, 9
], epithelial cells [10
], fibroblasts [11
], smooth muscle [12
, 13
], monocytes/macrophages [14
], T cells [15
], mast cells [16
], and eosinophils [15
]. Chemokines act by binding to cells through G-protein-coupled seven-transmembrane receptors on leukocytes, which promote an array of cellular events associated with vectorial movement including integrin activation, cytoskeletal reorganization, degranulation, and the generation of oxygen radicals and bioactive lipids [17
]. Human eosinophils have been shown to express the chemokine receptors CCR1 [18
], CCR3 [18
, 19
], CXCR2 [20
], and CXCR4 [21
]. In turn, several chemokines have been shown to elicit the migration of human eosinophils through several CC-chemokine receptors including eotaxin-1 [22
], -2 [23
], and -3 [24
]; monocyte-chemotactic protein (MCP)-2 [25
], -3 [26
], and- 4 [27
]; regulated on activation, normal T cell expressed and secreted (RANTES) [28
]; macrophage inflammatory protein (MIP)-1 [29
]; interleukin (IL)-8 [20
]; and macrophage-derived chemokine (MDC) [30
]. In contrast, mouse eosinophils have only been shown to express CCR1 and CCR3 [31
]. Accordingly, only mouse eotaxin-1 [32
] and -2 [33
] (a mouse orthologue of eotaxin-3 has not been identified to date) and MIP-1 [31
] have been shown to have effects on mouse eosinophil migration.
Nominal amounts of data characterizing mouse eosinophil migration in response to chemokines exist as a result of logistical constraints associated with the availability of peripheral blood eosinophils in sufficient numbers and purity for experimental manipulation. As a result, the available data are not comprehensive and are found as part of several independent studies, making it difficult to assess the relative potencies of the chemokines examined. In this study, IL-5 transgenic mice were used to generate pure (>98%) populations of peripheral blood eosinophils to investigate the ability of several chemokines to induce migration in vitro. These data demonstrate that although mouse eosinophils express CCR1 and CCR3, only responses to CCR3 agonists were significant using purified eosinophils. CCR1 ligands MIP-1 and RANTES were unable to elicit migration of purified eosinophils. Chemokines presumed to bind to CCR4/CCR8 [m thymus and activation-regulated chemokine (TARC) and m MIP-1ß] and CXCR2 (m KC) also demonstrated abilities to elicit purified eosinophil migration. Moreover, the response to TARC was accompanied by an increase in intracellular calcium flux, suggesting the presence of an additional chemokine receptor on mouse eosinophils.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and eotaxin were purchased from Perkin Elmer Life Sciences (Boston, MA). HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), PIPES (piperazine-N,N'-bis[2-ethane-sulfonic acid]), EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-bis[ß-aminoethyl ether]tetraacetic acid), digitonin, Percoll, and glucose were purchased from Sigma Chemical Co. (St. Louis, MO). Fura-2 acetoxymethyl ester was purchased from Molecular Probes (Eugene, OR). Fetal calf serum (FCS), Hank’s balanced salt solution (HBSS), and Dulbecco’s phosphate-buffered saline (PBS) were purchased from Gibco Life Technologies (Rockville, MD). Anti-CD45 and -CD90 antibody conjugated magnetic beads were obtained from Miltenyi Biotech (Auburn, CA).
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Isolation of mouse eosinophils
We have developed methods to isolate eosinophils to greater than 98% purity from the IL-5-expressing transgenic line NJ.1638 [34
]. Constitutive expression of IL-5 in these mice leads to a marked increase in circulating total white blood cells as well as a compositional shift to nearly 60% eosinophils. As a consequence, greater than 5 x 108 eosinophils can be recovered from 2 ml blood. Cells were routinely obtained from NJ.1638 mice by collecting 300–400 µl tail blood from each of four mice into 5 ml PBS containing 2% heparin. The blood was then layered onto a single-step Percoll gradient [60% Percoll (
=1.084), 1xHBSS, 15 mM HEPES (pH 7.4)] and was centrifuged (45 min, 2000 g, 4°C). The buffy coat containing lymphocytes and eosinophils was removed and washed twice in PBS containing 2% FCS. The cell pellet was then subject to brief hypotonic lysis in order to disrupt any contaminating red blood cells, then washed and resuspended in PBS containing 2% FCS. Eosinophils were subsequently isolated by removing the contaminating lymphocytes using a magnetic cell-separation system (Miltenyi Biotech), according to the manufacturer’s recommendations. Briefly, B cells and T cells were removed by positive selection following incubation with antibody conjugated magnetic beads specific for CD45-R (B220) and CD90 (Thy 1.2), which bind B cells and T cells, respectively. Recovered cells were washed twice, resuspended in RPMI 1640, and maintained at a concentration of 1 x 107 cells/ml at 4°C. The purity of the recovered cells was determined by visual examination of Wright’s stained cytospin preparations. Cell viability was determined each day by trypan blue exclusion.
Splenocyte isolation
Splenocytes were isolated from nontransgenic littermates. Briefly, spleens were removed and placed in RPMI-1640 media containing 2% FCS. Single cell suspensions were obtained by passing a suspension of splenocytes repeatedly through a 22-gauge needle and 40 µm filter. Viability of the cells was >95% as determined by trypan blue exclusion.
Receptor-binding assays
Binding of 125I-labeled chemokines (typically a total of 2x104 cpm) to intact mouse eosinophils (typically 5x105) at 31°C for 125I-eotaxin, or room temperature for 125I-labeled MIP-1, was performed in the presence of varying concentrations of unlabeled ligands essentially as described [18
].
Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of chemokine receptor RNA
Total RNA was isolated from purified eosinophils using Trizol reagent (Gibco Life Technologies) according to the manufacturer’s protocol. Purified RNA was stored at -80°C until reverse transcribed. Reverse transcription and the generation of PCR amplicons representing individual mouse chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR8, CXCR2, and CXCR4) were performed as described previously by Fischer et al. [35
]. Total RNA that was not reverse transcribed served as the negative control in the PCR amplification. PCR products were visualized on a 1.2% agarose gel following staining with ethidium bromide.
Transmigration assay
The migration of purified eosinophils or splenocytes in response to various chemokines in vitro was investigated using 5 µm polycarbonate membrane Transwell inserts in 24-well tissue-culture polystyrene plates (Costar, Corning, NY). The inserts were preincubated with media (RPMI 1640 containing 5% FCS) for 1 h. Media was removed, and cells (1x106; purified eosinophils included 30 ng/ml recombinant mouse IL-5), in a total volume of 200 µl media, were placed into the inserts. The inserts were then placed into the wells containing 500 µl media alone or media containing chemokines. The plates were incubated at 37°C and 5% CO2 for 90 min. Assay conditions including cell number, FCS, and IL-5 concentrations and incubation time were optimized using mouse eotaxin-1 and mouse RANTES.
The number of cells that had migrated in these Transwell assays was determined as the sum of the cells in the lower chamber plus the cells that had migrated but remained attached to the bottom of the Transwell insert. Cells that had migrated through the insert and were in the lower chamber were collected into a microcentrifuge tube. The cells that had migrated to the lower chamber, but remained attached to the insert, were recovered by initially removing the cells in the upper chamber, as well as any remaining suspension (by wiping with a cotton-tipped applicator). The attached cells were then recovered by placing the bottom of the inserts into 500 µl ice-cold PIPES buffer containing 5 mM EDTA and tapping the plates lightly. The cells displaced from the membrane were added to the corresponding microcentrifuge tubes containing lower chamber cells and were counted using a Coulter particle counter (Model Z1, Beckman/Coulter, Fullerton, CA). Differential counts for splenocyte migration were determined by counting Wright’s stained cytospin slides (Cytospin 3, Shandon Scientific, Pittsburg, PA) counting 
300 cells. Cell migration is expressed as a migration index, which is the ratio of the number of cells migrating in reponse to chemokine relative to the number of cells migrating in response to media alone.
Intracellular calcium measurements
Eosinophils (1x106 cells/ml) were loaded with 2 µM Fura-2 in RPMI 1640 containing 2% FCS and were incubated in the dark for 1 h at 37°C. Cells were washed twice in assay buffer (HBSS containing 1 mM CaCl2), resuspended at 1 x 106 cells/ml, and used within 1 h. The cytoplasmic-free Ca2+ concentration of continually stirring cells (2x106/2 ml) in response to various chemokines was measured using a Hitachi F-4500 spectrofluorometer with excitation at 340 nm and 380 nm and emission at 510 nm. Data are presented as a ratio of fluorescence at 340 nm and 380 nm. Maximal and minimal values were obtained by lysing the cells with 20 µM digitonin and chelation with 10 mM EGTA, respectively.
Alignment and phylogenetic analysis
Nucleotide and amino acid sequences were obtained from Genbank using published accession numbers. Amino acid sequences were aligned using CLUSTAL with a gap penalty of 5.0 [36
]. In addition, Blossum and Pam and Gonnet amino acid weight matrices were used in the different alignments. Alignments with the various amino acid weight matrices did not differ drastically, so we report only those results using the Blossum weighting matrix. Once the amino acid alignments were obtained, the nucleic acid sequences were adjusted to reflect the insertion of gaps in the amino acid alignments [37
]. Because the gaps that were introduced into the nucleic acid sequences cover more than a single base, we recorded these gapped regions as single characters as in DeSalle and Brower [38
]. Mouse B-lymphocyte chemoattractant was used as an out-group gene to polarize the relationships of the chemokines comprising the remaining in-group genes. The presented phylogenetic analyses were performed with the aligned sequences using a gap penalty of 5. Trees derived from alignments using a wide range of gap penalties (5, 25, 50, 100) were relatively consistent. The nucleic acid characters and the recorded gaps were analyzed together using a single matrix and jackknife resampling analysis, with 1000 sampling replicates, to assess the robustness of the phylogenetic analysis using the program PAUP [39
].
Statistical analysis
Data presented are the means ± SE. Statistical analysis was performed on parametric data using t-tests with differences between the means considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and eotaxin-1 demonstrate that receptors for these chemokines are expressed on these cells and bind their cognate ligands with an approximate equal affinity (IC50 
1 nM; Fig. 2B
).
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in a transmigration assay. Mouse eotaxin-2 was the most potent chemokine examined, reaching a >17-fold increase in maximal response at 30 nM (Fig. 3A
). [We observed that the mouse eotaxin-2 obtained from R&D Systems had minimal activity compared with mouse eotaxin-2 obtained from PeproTech. Therefore, the studies that make a quantitative assessment of eotaxin potency (Figs. 3A
and 4
) were conducted with the PeproTech chemokines.] Surprisingly, the CCR1 agonists m MIP-1 and m RANTES had no significant effects on the migration of purified eosinophils at any concentration (0.1–300 nM). However, in contrast to the studies using purified eosinophils, m MIP-1 demonstrated a nominal effect on eosinophil migration in studies using a mixed population of leukocytes derived from the spleen (Fig. 3B) . In comparison, regardless of the leukocyte population used (i.e., pure eosinophils or mixed leukocytes), RANTES failed to elicit eosinophil migration (Fig. 3)
. Significantly, MIP-1 and RANTES induced significant migration of specific mononuclear subpopulations, demonstrating the functionality of these chemokines in this assay system.
Non-CCR3 binding chemokines induce eosinophil migration
Several additional chemokine ligands shown to act through other receptors were similarly tested. Eighteen chemokines originally were screened at concentrations of 30 nM and 100 nM. The rank order of potencies was identical at both doses, and thus the results at 100 nM were arbitrarily chosen and are shown in Figure 4 . Chemokines thought to bind to CCR3 (eotaxins and human MCP-4), with the exception of human eotaxin-3, showed the greatest responses in the transmigration assay. Moreover, only three other chemokines (m TARC, m MIP-1ß, and m KC) showed significant activity (more than twofold). The baseline migration for all assays presented was consistently approximately 1% of total cells.
TARC induces a calcium flux in purified mouse eosinophils
The ability of m TARC, m MIP-1ß, and m KC to bind and signal specifically through eosinophil cell surface receptors was assessed indirectly by measuring intracellular calcium changes following exposure to these chemokines. Mouse eotaxin-1, mouse eotaxin-2, and m TARC caused immediate and significant increases in eosinophil intracellular calcium levels (Fig. 5
). In contrast, m MIP-1ß and m KC failed to induce a significant calcium flux. Similar results were observed at 24, 48, and 72 h post isolation for all chemokines examined.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60% identity), a mouse orthologue of human eotaxin-3 has yet to be identified. Although CCR3 has also been identified on human mast cells, basophils, and T cells, the only mouse leukocyte shown to express CCR3 is the eosinophil [42
]. In vitro migration assays using purified mouse eosinophils demonstrated that mouse eotaxin-1 reached half-maximal responses at similar doses to eotaxin-2; however, the maximal responses to mouse eotaxin-2 were higher relative to eotaxin-1. The increased response of mouse eosinophils to eotaxin-2 compared with eotaxin-1 parallels previous studies of human eosinophils noting a similar pattern of chemokine responsiveness [41
], but is in contrast to a recent study characterizing mouse eotaxins using mixed splenocytes from IL-5 transgenic mice, which demonstrates eotaxin-1 to be more potent than eotaxin-2 [33
]. These differences are likely attributable to the cell populations studied. The data presented here and the studies on human eotaxins used eosinophils that were purified to >98%, whereas the conflicting study [33
] used a population of cells that were only 30–40% eosinophils. Human MCP-4 was also a potent chemokine, exhibiting significant activity toward mouse eosinophils and consistent with its demonstrated activity for human eosinophils [27
]. This is not unexpected given that MCP-4 shares high sequence identity with mouse and human eotaxin-1 and binds CCR3. Human eotaxin-3 is the only chemokine of this group not to exert effects on mouse eosinophil migration. However, because human eotaxin-3 has only been reported to induce human eosinophil migration at a high dose (1 µM) [24
], the lack of effects at the nanomolar concentrations used in this study is not surprising.
The data presented here demonstrate that although MIP-1 can bind to purified mouse eosinophils, this chemokine is unable to elicit migration of these cells. This apparent paradox is reflective of the literature that abounds with conflicting data concerning the ability of MIP-1 to induce human eosinophil migration [18
, 29
, 43
44
45
46
] or eosinophil recruitment in mouse models [6
, 31
, 47
, 48
]. In the case of human eosinophils, the study by Sabroe et al. [45
], examining eosinophils from multiple donors, provides an explanation to resolve this issue by demonstrating that MIP-1 responses are only observed in 20% of individuals. The studies in mice have been particularly problematic because these in vitro studies have been performed on eosinophils whose purity varied from 30% to 99%, and so possible indirect effects of MIP-1 on other cell types may have influenced eosinophil migration. This hypothesis is supported by the present study in which MIP-1 has no effects on purified eosinophils, but exerts some effect on eosinophils when they are studied as a mixed population of leukocytes. The sensitivity of this effect is apparently significant, because even in a mouse eosinophil assay system, comparable with the one used here, Post and colleagues [31
] demonstrated that MIP-1 elicits a small (approximately two- to threefold) increase in eosinophil migration. The reason for this discrepancy is unclear, but may be associated with the use of an eosinophil population of lower purity (85–92%) or a subtle phenotypic difference in eosinophils isolated from transgenic mice with different circulating IL-5 levels. This issue may also complicate the interpretation of in vivo studies that support MIP-1 as an important chemokine in eosinophil recruitment as indirect effects of MIP-1, as the mechanism influencing tissue eosinophil accumulation, cannot be eliminated [6
, 47
, 48
].
RANTES is a potent human eosinophil chemoattractant [49 ] that can bind to CCR1 [50 ] and CCR3 [18 , 19 ], but appears to exert its effects on eosinophils primarily through CCR3 [51 ]. However, RANTES displayed no chemotactic activity on mouse eosinophils using two different assay systems. This conclusion is supported by in vivo studies demonstrating that intradermal injection of eotaxin, but not RANTES, in the mouse skin recruits eosinophils selectively [47 ], and transgenic overexpression of RANTES in the mouse lung recruits neutrophils selectively, but not eosinophils [52 ]. It is interesting that mouse RANTES is unable to displace 125I-eotaxin binding on mouse CCR3 transfected cells (B.L.D., unpublished results), suggesting that the loss of chemotaxis induced by mouse RANTES is a consequence of the inability to bind mouse CCR3. The reason for this disparity is unknown, but likely reflects evolutionarily significant selective pressures in one or both species.
The ability of mouse KC, MIP-1ß, and TARC to induce eosinophil migration in these assays suggests that additional chemokine receptors may represent unique pathways for eosinophil migration and function. The biology of KC, in particular, has been well characterized, and it is known to bind through the chemokine receptor CXCR2 [53 ]. The detection of CXCR2 mRNA in the eosinophils used in this study suggests that signaling through this receptor is responsible for the migration induced by KC. It is interesting that human eosinophils are known to express CXCR2 when they are cultured in IL-5 [51 ], and thus the possibility exists that the eosinophils used in our studies express functional CXCR2 receptors as they were isolated from transgenic mice with elevated, circulating IL-5 levels (400 pg/ml). Nonetheless, because circulating IL-5 levels also increase during allergic inflammation, CXCR2 expression may represent an alternate/additional pathway facilitating eosinophil recruitment in vivo.
TARC and MIP-1ß are the least characterized chemokines that demonstrated activity on mouse eosinophils. MIP-1ß has been characterized as a ligand for CCR5 [54 ], and the detection of mRNA for this receptor in mouse eosinophils may explain the activity of this chemokine in these studies. The receptor for TARC mediating the migration of eosinophils remains problematic as there is conflicting evidence for TARC as a CCR8 ligand [55 56 57 ] but strong evidence for TARC as a CCR4 ligand [58 ]. Garlisi et al. [55 ] reported that TARC bound to CCR8 receptors in transfected cells only at very high (i.e., nonphysiologically relevant) concentrations (>250 nM) to compete with the CCR8 ligand I-309. However, in natural killer (NK) cells, TARC was able to compete away I-309 with an IC50 of 1.6 nM [57 ]. This may reflect a difference in transfected versus primary cells or a difference in cell type receptor usage (T cells vs. NK cells vs. eosinophils). In either case, no previous evidence exists indicating that these chemokines are eosinophil chemoattractants; instead, both chemokines have been implicated in the responses of T cells during allergic, inflammatory responses [59 , 60 ].
It is interesting that m TARC, but not m KC or m MIP-1ß, induced a significant calcium flux in purified eosinophils. An increased intracellular calcium flux is commonly associated with chemokine receptor signaling, but there are many studies that demonstrate chemokine function occurring in conjunction with low or no calcium flux in various cell types including eosinophils [19
, 30
, 56
]. Several reasons could account for this occurrence including calcium-independent signal transduction pathways utilized by various chemokine receptors or receptor expression levels being too low to evoke a measurable calcium flux within the sensitivity of the assay. In the case of CCR3, a recent study indicates that chemokine binding, intracellular calcium flux, and migration responses are not coupled tightly [43
]. Furthermore, given that all of the eotaxin chemokines studied to date bind to eosinophils with similar affinities (Kd 1–5 nM) [18
, 23
, 24
] but exhibit different efficacies and potencies in terms of migration, raises the question of how a chemokine receptor distinguishes between various ligands and modulates the intracellular signaling pathways necessary to achieve graded responses.
An important point to consider in the interpretation of these studies is the possibility that IL-5 has uncoupled or desensitized some of the receptor binding or signaling responses, which occur normally in eosinophils. This would explain why, even with strong binding of MIP-1 to the eosinophils, the binding fails to result in migration. Furthermore, the expression of chemokine receptors such as CCR2 and CCR5 may represent "decoy" receptors for ligands like RANTES and MIP-1, which can bind chemokine but do not elicit intracellular signaling or eosinophil migration. This has been demonstrated previously on dendritic cells and monocytes exposed continuously to the cytokine IL-10 [61
].
The tertiary structural homology between chemokines ultimately predicts their binding and signaling properties. However, primary sequence similarities also provide useful data for the determination of relationships between proteins. The ability of evolutionarily diverse chemokines outlying the large eosinophil chemotactic clade (mouse TARC and mouse KC) to induce eosinophil migration supports the hypothesis that multiple chemokine signaling pathways have evolved. The commonality of chemokines with eosinophil agonist activities in humans and mice has broad implications and suggests that these chemotactic mechanisms likely predate the evolution of the uniquely eosinophil hematopoietic lineage. For example, CCR3 receptor-ligand interactions represent the dominant signaling pathway leading to eosinophil recruitment in at least two extant mammalian orders (i.e., Rodentia and Primata), suggesting that the advent of CCR3-mediated eosinophil chemotaxis occurred before the major radiation of mammalian orders 75–100 million years ago [62 ], and thus potentially prior to the evolution of the mammalian eosinophil hematopoietic lineage.
These studies demonstrate that significant differences exist with regard to the control of eosinophil migration by chemokines between humans and mice and suggest that additional chemokine/receptor interactions may regulate eosinophil migration. These differences stress the importance of increased characterization of the mouse as a model of human diseases. The greater understanding of the mouse as a model system will no doubt provide invaluable insights regarding eosinophils and their effector functions in disease.
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ACKNOWLEDGEMENTS |
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Received September 17, 2001; revised February 4, 2002; accepted February 6, 2002.
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(U49513), mouse MIP-3ß (AF059208), human RANTES (M21121), mouse RANTES (M77747), and mouse MIP-1