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. 2005 Jul 15;106(2):436-43.
doi: 10.1182/blood-2005-02-0489. Epub 2005 Mar 31.

CXCL9 inhibits eosinophil responses by a CCR3- and Rac2-dependent mechanism

Patricia C Fulkerson  1 , Hongyan Zhu, David A Williams, Nives Zimmermann, Marc E Rothenberg
Affiliations

CXCL9 inhibits eosinophil responses by a CCR3- and Rac2-dependent mechanism

Patricia C Fulkerson et al. Blood. .

Abstract

Recently, inhibitory cytokine pathways for leukocyte chemoattraction and activation have been identified, but there is little insight into the operational mechanisms except for models that rely on simple receptor antagonism. We have previously identified the existence of a murine eosinophil inhibitory pathway mediated by the CXC chemokine ligand 9 (CXCL9, Mig [monokine induced by interferon-gamma]) that impressively blocks eosinophil chemoattraction and function, but the mechanism has remained elusive. We now demonstrate that Mig's inhibitory action extends beyond receptor antagonism alone. Notably, in addition to inhibiting eotaxin-induced filamentous actin (F-actin) formation and chemoattraction, Mig potently blocks platelet activating factor (PAF)- and leukotriene B4 (LTB4)-induced responses. Remarkably, Mig-treated eosinophils display an abnormal F-actin assembly in the absence of agonist stimulation. Additionally, Mig pretreatment inhibits eotaxin-induced activation of the Rho-guanosine triphosphatase (GTPase) Rac, and Rac2-deficient eosinophils demonstrate an impaired transmigration and actin polymerization response to eotaxin stimulation. Furthermore, Mig was unable to inhibit eotaxin-induced responses in Rac2-deficient eosinophils. Finally, using CCR3 gene-targeted cells, Mig's inhibitory activity is demonstrated to be mediated by CC chemokine receptor 3 (CCR3). Thus, by altering agonist-induced signaling and abrogating cytoskeletal reorganization by a Rac2-dependent mechanism, Mig markedly inhibits eosinophil responses to diverse stimuli. These results establish evidence that distinct chemokines can use CCR3 to induce opposing signals in eosinophils.

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Figures

Figure 1.
Figure 1.
Mig inhibits eosinophil transmigration toward the non-CCR3 ligands PAF and LTB4 in vitro. Eosinophil transmigration following pretreatment with buffer or Mig is shown. Data represent mean ± SD of eosinophils that migrated toward PAF 10 nM (A) or LTB4 1 nM (B). *P < .05.
Figure 2.
Figure 2.
Mig inhibits agonist-induced actin polymerization. (A) Eosinophils were treated with 10 nM eotaxin-1 (♦), 40 nM Mig (▪ and dashed line), or 40 nM Mig and 10 nM eotaxin-1 (▴) for the indicated period of time. Cells were fixed and stained with NBD-phallacidin. Relative F-actin content is expressed as the ratio of the mean channel fluorescence between eotaxin- and media alone–stimulated cells. A representative experiment is shown (n = 3). (B) Mean (± SD) percent inhibition of 10 nM PAF-induced F-actin polymerization in eosinophils in the presence of 2 to 200 nM Mig (n = 3 experiments). The analysis was performed following 10 seconds of chemokine exposure. *P < .05. (C) Actin localization was determined by fluorescence microscopy. Cells were fixed and stained with rhodamine-labeled phalloidin after stimulation for 10 seconds with buffer alone (resting), eotaxin-1 (10 nM), Mig (200 nM), or eotaxin-1 and Mig. Images were acquired with a fluorescence microscope equipped with a deconvolution system driven by Openlab software. Results are representative of 4 experiments.
Figure 3.
Figure 3.
Mig inhibits agonist-induced Rac activation and Rac2 is required for Mig's inhibitory activity. (A) Eosinophil lysates were used for affinity precipitation with 5 μg PAK-PBD for 60 minutes at 4°C. Active Rac-GTP precipitated by PAK-PBD was separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted for pan-Rac, followed by enhanced chemiluminescence (ECL) detection. In the bottom panel, aliquots of lysates were immunoblotted and probed for Rac to confirm equal protein expression. Representative blots are shown (n = 3). (B) Wild-type (WT, ♦) and Rac2-deficient (▪; KO indicates knock out) eosinophil transmigration toward eotaxin-2 is shown. Data represent mean ± SD of eosinophils that migrated toward eotaxin-2 (0-10 nM). A representative experiment is shown (n = 3). P = .03 between wild-type and Rac2–/– at 1 and 10 nM based on paired Student t test. (C) Wild-type (□) and Rac2-deficient (▪) eosinophil transmigration toward eotaxin-2 is shown following pretreatment with buffer, eotaxin-2 (Etx2; 5 nM), or Mig (0.8-40 nM). Data represent mean ± SD of eosinophils that migrated toward eotaxin-2 (1 nM). A representative experiment is shown (n = 3). *P < .05 when compared with pretreatment of buffer alone. (D) Wild-type (solid lines) or Rac2-deficient (dashed lines) eosinophils were treated with 12 nM eotaxin-1 (♦), or 40 nM Mig and 12 nM eotaxin-1 (•) for the indicated period of time. Cells were fixed and stained with NBD-phallacidin. Relative F-actin content is expressed as the ratio of the mean channel fluorescence between eotaxin- and media alone–stimulated cells.
Figure 4.
Figure 4.
Mig does not inhibit agonist-induced calcium mobilization. Calcium transients in purified murine eosinophils were measured by labeling cells with Fura-2 AM, and monitoring immediately following stimulation with 50 nM eotaxin-1, 25 nM eotaxin-2 (A-B), and 10 nM PAF (C). Prestimulation with Mig (200 nM) has no effect on eotaxin- (B) or PAF-induced calcium mobilization (C). Data are representative of 3 experiments.
Figure 5.
Figure 5.
Mig requires CCR3 expression for inhibitory activity. (A) The dose-dependent binding of Mig to the surface of wild-type (♦) and CCR3-deficient (▪) cells is shown. Data represent mean ± SD percent mean channel fluorescence (chemokine binding) compared with no chemokine for 3 independent experiments combined. Staining with control antibody (dashed lines) is shown. *P < .05. (B) Mig does not inhibit CCR3-deficient eosinophil chemotaxis toward PAF. Cells were allowed to transmigrate following pretreatment with buffer or Mig. Data represent mean ± SD of eosinophils that migrated toward PAF (10 nM). *P < .05. The results are representative of 3 experiments. (C) Mig does not inhibit PAF-induced actin polymerization in CCR3-deficient eosinophils. Wild-type (dashed line) and CCR3-deficient (solid line) eosinophils were treated with PAF (10 nM, ▪), or Mig (200 nM) and PAF (♦) for the indicated period of time. Cells were fixed and stained with NBD-phallacidin. Relative F-actin content is expressed as the ratio of the mean channel fluorescence between eotaxin- and media alone–stimulated cells. A representative experiment is shown (n = 3).
Figure 6.
Figure 6.
Mig alters CCR3 receptor-ligand interaction. (A) Binding of 10 nM biotinylated eotaxin-1 (b-Etx1, black line) without (A) or with 200 nM eotaxin-2 (B) to the surface of eosinophils compared with control chemokine (biotinylated JE, b-JE, filled histogram) is shown. Representative histogram from 3 experiments is shown. (C) Binding of eotaxin-1 (black line) with or without 500 nM Mig (dashed line) is shown. A representative histogram is shown (n = 3). (D) Percent eotaxin-1 binding in the presence of 20 to 2000 nM Mig compared with eotaxin-1 alone is shown. Data represent mean ± SD of mean channel fluorescence of eotaxin-1 binding in the presence of Mig compared with eotaxin-1 alone (n = 3 experiments).
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