Eotaxins (CCL11, CCL24, CCL26)
Eotaxin-1 : C-C motif chemokine 11; CCL11; Chemokine (C-C motif) ligand 11; Eosinophil chemotactic protein; Eotaxin; SCYA11; Small-inducible cytokine subfamily A (Cys-Cys), member 11 (eotaxin); Small-inducible cytokine A11
Eotaxin-2 : C-C motif chemokine 24; CCL24; Chemokine (C-C motif) ligand 24; CK-beta-6; Ckb-6; Eosinophil chemotactic protein 2; MPIF-2; MPIF2; Myeloid progenitor inhibitory factor 2; SCYA24; Small-inducible cytokine subfamily A (Cys-Cys), member 24; Small-inducible cytokine A24
Eotaxin-3 : C-C motif chemokine ligand 26; CCL26; IMAC; MIP-4a; MIP-4alpha; SCYA26; TSC-1
Eosinophils are a subpopulation of granulocytes in the blood, identified by their bilobed nucleus and distinctive cytoplasmic granules staining pink with eosin. Eosinophils or related cells are found in mammals and also in other animals including birds, reptiles, amphibians, fish, and insects. Eosinophil-like cells are also present in invertebrates such as the horseshoe crab and sea cucumber. Eosinophils are part of our host defense system, especially to parasitic helminths, and localized tissue eosinophilia is associated with worm infections. There is also evidence that eosinophils can have a role in fungal and viral infections. Of clinical importance, tissue eosinophilia is a characteristic feature of allergy and asthma, which are thought to represent an aberrant response to otherwise innocuous molecules in the environment. Eosinophils produce an arsenal of cytotoxic molecules in their role in combating parasites. These same molecules can also cause tissue injury and dysfunction in allergy and asthma. Hence, eosinophils have become a major therapeutic target with a focus on the signaling molecules mediating their recruitment in allergy and asthma. Current clinical evidence suggests that eosinophils have a strong association with airway remodeling and disease exacerbations in severe asthma. The attraction of eosinophils as a therapeutic target is that their numbers can be reduced to low numbers (e.g., by corticosteroids) without overt effects on host defense to common pathogens, such as bacteria. This is in contrast to neutrophils, where interference with recruitment severely compromises survival in the face of assault by pathogens in the environment.
The eosinophil was identified by the German scientist, Paul Ehrlich, in 1879. Ehrlich stained blood cells and tissues with aniline dyes and discovered neutrophils, basophils, lymphocytes, mast cells, and eosinophils by their differential staining characteristics. The highly basic cytosolic granules in a small subpopulation of blood cells stained pink with the acid dye eosin, hence “eosinophils.” Ehrlich observed high numbers of eosinophils in the sputum of asthmatic patients and realized that there was a close relationship between eosinophilia and the severity of asthma. Significantly, he proposed the existence of a “material which attracts eosinophils.” He also postulated that eosinophils and neutrophils possessed different “chemotactic irritability” and that eosinophils only migrate to sites where a “specific stimulating substance” is present (Gleich 2013).
The “specific substance” became the subject of many investigations, and several large and small molecules were found to have eosinophil chemoattractant activity, but none of these could account for the selective eosinophil recruitment that can occur in allergic reactions. A breakthrough occurred in 1994 with the description of a small protein produced in vivo with potent and selective chemoattractant activity for eosinophils (Jose et al. 1994). Bronchoalveolar washings were collected from allergen-challenged sensitized guinea pigs in vivo and tested in the skin of unsensitized bioassay guinea pigs, also in vivo. The bioassay animals were preinjected intravenously with radiolabeled eosinophils, and the accumulation of these cells provided a highly sensitive and selective measurement of eosinophil chemoattractant activity in the fluid. Purification and sequencing revealed a CC chemokine which was named “eotaxin,” condensed from eosinophil chemotaxin. The sequence of eotaxin was used to identify the molecule in several species including mouse (Rothenberg et al. 1995) and human (Ponath et al. 1996). Subsequently, chemokines with similar activity but low sequence similarity were discovered and termed eotaxin-2/CCL24 and eotaxin-3/CCL26 (Forssmann et al. 1997; Shinkai et al. 1999). The original eotaxin molecule is now usually referred to as eotaxin-1/CCL11.
Cell-Cell Signaling from Tissue Cells to Circulating Blood Eosinophils
The Th2 cytokine IL-5 has been regarded as chemotactic for eosinophils. The IL-5 receptor is highly expressed on eosinophils, and the cytokine is critically important in eosinophilopoiesis in the bone marrow. IL-5 induces eosinophil migration in Boyden chamber assays in vitro, but this is independent of a chemotactic gradient and is caused by increased random migration or chemokinesis. Experiments in vivo have shown IL-5 to be poor in mediating eosinophil migration into tissues but very effective in releasing the cells from the bone marrow by inducing migration of the cells into sinusoids (Collins et al. 1995). Further evidence was obtained that IL-5 is produced in the sensitized lung on exposure to allergen and travels as a circulating signal in the blood to the bone marrow where it releases mature eosinophils (Humbles et al. 1997). As eosinophil numbers in the blood are normally low, this system ensures efficient recruitment to tissues in response to locally generated eotaxins. Circulating eotaxins can also cause the release of bone marrow eosinophils and synergize with IL-5 to induce this effect by combining chemotactic (eotaxins) and chemokinetic (IL-5) actions (Palframan et al. 1998).
The Eotaxin Receptor CCR3
Several groups simultaneously identified CCR3 as the cell surface receptor for the eotaxins. A member of the class A family of G protein-coupled receptors, CCR3 binds all three eotaxins along with at least another six chemokines with varying potency (CCL5, CCL7, CCL8, CCL13, CCL15, and CCL28) making it one of the most promiscuous chemokine receptors. Quite how CCR3 can interact productively with so many different chemokines is not completely understood, although the highly acidic second extracellular loop containing the EELFEET amino acid motif is likely involved in electrostatic interactions with the predominantly basic chemokines. The interaction of eotaxin-1 with regions of CCR3 has been studied by the use of chimeric receptor constructs (Pease et al. 1998), NMR (Mayer and Stone 2001), and small-molecule agonists which mimic the actions of eotaxin-1 (Wise et al. 2007a). Collectively, the work supports a two-step model of CCR3 activation in which the core of eotaxin-1 interacts with the receptor N-terminus (tethering the ligand to the receptor), and subsequent interactions with the extracellular (Wise et al. 2007b) loops orientate the chemokine such that the N-terminus of eotaxin-1 can insert into an intrahelical pocket inducing receptor activation.
CCR3 antagonists have been developed by several companies. Flow cytometry was employed to measure functional responses of human eosinophils to chemokines (the “GAFS” shape change assay). This showed that a subpopulation of donors had eosinophils that responded to both CCR3 and CCR1 agonists. The GAFS technique was used in the first publication demonstrating a CCR3 antagonist, one that could also antagonize CCR1 (Sabroe et al. 2000). A small molecule CCR3 antagonist was tested in clinical trials in asthma patients but did not demonstrate efficacy (Neighbour et al. 2014). To date, no CCR3 antagonists have reached the market (Pease and Horuk 2014). A potent therapeutic antibody (CAT-213, iCo-008, bertilimumab) that neutralizes eotaxin-1 was developed by Cambridge Antibody Technology (now Medimmune UK) (Main et al. 2006). The antibody has been licensed to iCo Therapeutics and to Immune Pharmaceuticals, for testing in clinical trials.
The eotaxins have been evaluated as therapeutic targets in asthma and in allergic diseases in general (for review see: Ahmadi et al. 2016). They are also used as biomarkers in clinical trials. Eotaxins are thought to be important in diseases of the gastrointestinal tract (Abonia and Rothenberg 2012), and trials are planned in ulcerative colitis and Crohn’s disease. Unexpectedly, eotaxins are implicated in diseases, independently of their action on eosinophils. CCR3 is expressed on endothelial cells in vessels overgrowing the macula in age-related macular degeneration (AMD). Locally produced eotaxins are thought to mediate angiogenesis in the retina in AMD (Takeda et al. 2009). Bertilimumab is being evaluated for the treatment of AMD and other eye diseases. Early studies showed that CCR3 is expressed by microglia in the brain. CCR3 can act as a co-receptor for HIV infection, leading to the suggestion that CCR3 may be involved in the later stages of HIV infection where the brain becomes affected. In support of this, it was shown that HIV can infect microglia in vitro and that this can be suppressed by eotaxin-1 or an antibody to CCR3 (He et al. 1997). Interestingly, there is more recent evidence of a role for circulating eotaxin-1 in neurodegenerative diseases. Villeda et al. 2011 carried out cross-circulation studies between old and young mice. They concluded that circulating levels of eotaxin-1 rise during aging and that this suppresses neurogenesis and impairs cognitive function (Villeda et al. 2011). Increasing levels of eotaxin-1 with age were also observed in human plasma and cerebral spinal fluid, raising interesting possibilities for future therapy. This potentially important emerging field is the subject of a recent review, where the evidence for underlying mechanisms is discussed (Huber et al. 2016).
To conclude, the discovery of the eotaxin family of chemokines proved Ehrlich’s postulate correct, namely that a “material” exists which selectively recruits eosinophils. Targeting the eotaxins or their specific receptor CCR3 in a disease setting has to date proved challenging as with other chemokines, although the involvement of the eotaxins in additional disease areas may drive further progress.
We thank Asthma UK and the Wellcome Trust for supporting our research.
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