Detection of mRNA for Eotaxin-2 and Eotaxin-3 in Human Dermal Fibroblasts and Their Distinct Activation Profile on Human Eosinophils

Abstract

As many new biologically active chemokines have been cloned exploring the genomic DNA sequence database in the vicinity of already known chemokine sequences without demonstrating their natural origin, it is important to transfer findings from in vitro experiments with chemokines into the in vivo situation. With respect to eosinophils and fibroblasts that play an important part in the pathogenesis of allergic and autoimmune diseases, the role of the recently discovered members of the eotaxin family, eotaxin-2 and eotaxin-3, is not really understood. In order to elucidate the origin and biologic potency of the eotaxin family this study was performed. Conventional reverse transcription-polymerase chain reaction analysis was suitable to detect mRNA for eotaxin and eotaxin-3 but not for eotaxin-2 in dermal fibroblasts. In contrast to conventional reverse transcription-polymerase chain reaction, LightCycler analysis revealed that dermal fibroblasts constitutively expressed mRNA not only for eotaxin and eotaxin-3 but also for eotaxin-2. Moreover, with this technique we investigated mRNA expression levels after stimulation of fibroblasts with interleukin-4 and interleukin-4 plus tumor necrosis factor-alpha: the rank order of expression levels within the eotaxin family was eotaxin > eotaxin-3 > eotaxin-2. To address the question of the efficacy of eotaxin-3, we compared its activity with eotaxin, eotaxin-2, monocyte chemotactic protein-3, monocyte chemotactic protein-4, and RANTES in different test systems for eosinophils. The efficacy of the CC chemokines at equimolar concentrations with respect to the chemotactic response of human eosinophils was eotaxin-3 = eotaxin = eotaxin-2 > RANTES > monocyte chemotactic protein-4. The rank order of activity with respect to actin polymerization and release of toxic reactive oxygen species was eotaxin-3 = eotaxin = eotaxin-2 and eotaxin = eotaxin-2 > eotaxin-3 = monocyte chemotactic protein-3 = monocyte chemotactic protein-4 = RANTES, respectively. This study indicated a distinct profile in expression levels of the members of the eotaxin family in dermal fibroblasts. Indeed, all three eotaxin ligands demonstrated activation of human eosinophils with similar efficacies for chemotaxis, cytoskeletal rearrangements, activation of Gi proteins and transients of [Ca2+]i, but a distinct profile of activity with respect to the binding to CCR3 and the release of toxic reactive oxygen species. These findings may help to understand further the role of CC chemokines in fibroblast/eosinophil activation, which is of interest particularly in allergic and autoimmune diseases.

Eotaxin-3 is a natural antagonist for CCR2 and exerts a repulsive effect on human monocytes

“…[12][13][14][15][16] In addition to eosinophils, basophils, and Th2 lymphocytes, which are typically found in allergic inflammations and express CCR3, these infiltrates also contain monocytes that lack CCR3. 17 Eotaxin-3 is expressed in vascular endothelial cells and dermal fibroblasts after interleukin 4 (IL-4) and IL-13 stimulation, 18,19 and in bronchial tissue on allergen challenge. 20,21 In a current report, its relevance for transendothelial migration of eosinophils has been elucidated, 22 whereas the significance of the constitutive expression in the reproductive system and the heart 2 remains to be clarified.…”

CCR3 functional responses are regulated by both CXCR3 and its ligands CXCL9, CXCL10 and CXCL11

“…In contrast, the chemokine receptor CCR3 is expressed on cells implicated in the pathogenesis of allergic inflammation, such as eosinophils, basophils, mast cells and Th2-type T lymphocytes [14][15][16][17]. The major CCR3 ligands are the eotaxin family of CC chemokines, CCL11/eotaxin [18,19], CCL24/eotaxin-2 [20][21][22] and CCL26/eotaxin-3 [23,24], which share low aminoacid sequence identity and are characterized by differences in their potencies and efficacies at CCR3 as well as distinct temporal and spatial expression patterns [21,[24][25][26][27]. In has been suggested that leukocyte recruitment during inflammatory responses may be tightly regulated by chemokines acting as agonists at some receptors and antagonists at others.…”

Eotaxin-3/CC Chemokine Ligand 26 Is a Functional Ligand for CX3CR1

“…Interestingly, CCL26 is stored in Weibel-Palade bodies, the endothelial cell-specific storage granules, and is rapidly released upon secretogenic stimuli (14). Subsequent studies have further shown that IL-4 as well as IL-13 induces CCL26 in a wide variety cells such as fibroblasts, bronchial epithelial cells, airway smooth muscle cells, intestinal epithelial cells, and epidermal keratinocytes (15)(16)(17)(18)(19). Thus, CCL26 is likely to play a major role in the emigration and migration of eosinophils and other CCR3-expressing cells in Th2-shifted conditions.…”