chemokine receptors and ligands

Chemokines are highly conserved cytokines that control the movement of cells around the body. All 45 members of the chemokine family can be divided into 4 subfamilies depending on their structure and presence of cysteine motifs (the CC chemokines, CXC CX3C and XC) and are recognised by chemokine receptors, G-protein coupled receptors with 7 transmembrane domains predominantly found on the surface of leukocytes. Upon binding of the chemokine to a cognate chemokine receptor, the receptor undergoes a conformational change exposing the highly conserved DRYLAIV domain. The exposed motif binds to neighbouring G-proteins, kick-starting a signal transduction cascade which will ultimately cause the cell to polarise and initiate migration.

Some chemokines are considered pro-inflammatory and their production is drastically increased during an immune response to recruit inflammatory leukocytes to sites of infection or tissue damage (CCL2, CCL3, CCL5, CXCL1, CXCL2 and CXCL8). Homeostatic chemokines, on the other hand, are involved in controlling migration of cells during normal processes to tissue maintenance or development. These include CCL19, CCL20, CCL21, CCL27, CXCL12 and CXCL13. In the context of inflammation, the mammalian chemokine family evolved to ensure that numerous inflammatory chemokines are expressed simultaneously at infected or damaged sites.

Chemokine receptor redundancy

There are several pathogens that have evolved the ability to either degrade or secrete their own ‘decoy’ chemokines to avoid being detected by the immune system. By expressing many different chemokines and chemokine receptors, the immune system can rely on other signals to correctly move to the site of infection, even if the pathogen has managed to disable a few. This has resulted in apparent redundancy (and extreme confusion) in the inflammatory chemokine system, where several ligands bind and activate each receptor and each receptor displays marked promiscuity of ligand binding. This is most noticeable in monocytes, the most evolutionarily ancient cell type involved in innate immunity, which are capable of responding to the widest range of chemokines. Although redundancy, promiscuity and overlapping gene expression patterns in the chemokine system protects from possible pathogen-induced disruption of chemotactic signals, this has hindered research on the individual role of each inflammatory chemokine. Indeed, even after 20 years of studies and millions of pounds in investments, no pharmacological antagonist for inflammatory chemokine receptors has been licensed for use in treating inflammatory conditions.

Chemokine receptors

Chemokine receptors share very high levels of homology across different species, suggesting a very ancient origin. While fruit flies, sea urchins and sea squirts have no identifiable chemokines or chemokine receptors,more complex organisms, such as chickens (G.gallus), frogs (X. tropicalis), zebrafish (D.rerio) and pufferfish (F.rubripes) all share chemokine receptors which have very high similarities to their human counterparts. For example, human CCR7, the chemokine receptor present on dendritic cells which allows them to migrate to the lymph node, shares 82% of its sequence with chicken CCR7, 80% with frog and 64% with Zebrafish. By tracking the mutations of these conserved chemokine receptors back through time, some groups have placed the origins of ancestral chemokine receptors between 650 and 564 million years ago, at the emergence of the vertebrate lineage. Indeed, this period is marked by the evolution of neural crest tissue, a closed circulatory system, blood-based oxygen transport, and a hematopoietic system- all of which require some sort of controlled primitive cell migration.

Chemokine receptor homology

Not only are the chemokine receptors highly conserved across different species, but they are also very similar to each other and spaced peculiarly across our genome. There are two major clusters of CC chemokine genes and two of CXC genes, plus numerous non-clustered or mini-cluster genes of both types, in both the mouse and human genomes. An explanation for this chromosomal arrangement is found in the evolutionary forces that have shaped the genome: over the course of evolution, gene duplication has been a common event, affecting most gene families. Once duplication occurs, the two copies can evolve independently and develop specialized functions.

While this has resulted in a complex system with several fail-safe mechanisms, it has made it almost impossible for researches to uncover the role of an individual receptor. Gene duplication has resulted in different chemokine receptors with high homology and similar structure (for example, CCR2 shares 51% of sequence with CCR1, 78% with CCR3 and 71% with CCR5), which makes it hard to develop specific antibodies for flow cytometry or immunohistochemistry which do not cross-react with other chemokine receptors. In addition, their ‘clustering’ in the genome makes it impossible to create compound KO mice, as their proximity does not allow for genetic recombination during crossing over events (this is especially true for CC inflammatory chemokine receptors CCR1, CCR2, CCR3 and CCR5, all found on the same 170kb tight cluster on murine chromosome 9).

Chemokine receptors in leukocytes

Furthermore, expression of chemokine receptors in leukocytes is not static, but fluctuates depending on activation, differentiation state and localisation. For example, differentiation of monocytes into macrophages is achieved by exposure to CSF-1, which is secreted by epithelial cells exposed to cytokines such as IL-1 or TNFa. Not only does this terminal differentiation alter the behaviour of the monocyte by inducing an inflammatory state characterised by elevated phagocytic rates, proteolytic activity and up-regulation of pro-inflammatory surface markers, but chemokine receptors expressed on the surface also change as differentiation progresses (CCR2 expression is shut down, while CCR1 and CCR5 are upregulated).

This coordinated regulation of chemokine receptor expression and altered functional responsiveness during differentiation adds even more complexity to the chemokine system, as a pharmacological antagonist might only be effective at blocking migration at a specific temporal window in the life of a leukocyte.

Inhibiting Chemokine receptors

Monocytes are thought of as the effector cells in the pathogenesis of RA15, a chronic inflammatory disease characterised by massive infiltration of immune cells in synovial tissue and synovial fluid mediated by chemokines and adhesion molecules16. Since their numbers in affected joints usually correlates with clinical signs and symptoms, several clinical trials were set in place to block monocyte recruitment to inflamed joints by inhibiting the chemokine receptors responsible: CCR2, CCR5 and CCR1. Although CCR2 and CCR5 receptor blockade had shown positive results in RA animal models, using chemokine antagonists to block these receptors was not effective in patients affected by RA. In vivo and in vitro studies had also shown a positive correlation between blocking CCR1 ligands and inhibition of chemotaxis and reduction of synovial inflammation, but clinical trials using CCR1 antagonist gave conflicting results, with some groups reporting a modest trend towards clinical improvement and others reporting no efficacy at all.

CCR1 was also shown to have a role in the pathology of multiple sclerosis. Inhibiting CCL3, a ligand for CCR1, prevented the development of both acute and relapsing paralytic disease and infiltration of monocytes into the CNS28. Deletion of CCR1 was also shown to be protective in model of multiple sclerosis in mice and a non-peptide agonist of CCR1 BX471 was effective against EAE, a rat model of acute multiple sclerosis. Due to these initial promising results, the compound BX471 entered a clinical trial in 2004 for multiple sclerosis, and, although no safety concerns emerged, it was stopped after the clinical Phase II study failed to show a reduction in the number of new inflammatory CNS lesions.

Chemokine receptor clinical trials

Several reasons have been proposed as to why clinical trials blockading chemokine receptors have not worked, ranging from off target effects to redundancy of the target.

Initially, it had been suggested that the redundancy in the chemokine system could have explained the failed trails using CCR2 or CCR5 antagonist, as both CCR2 and CCR5 share common ligands. However, another trial in which both chemokine receptors were blocked simultaneously showed no inhibition of monocyte recruitment to the inflamed joint, possibly indicating that other chemokine receptors are at play. In addition, lack of efficacy of treatment by CCR5 antagonists could be explained by inhibition of regulatory T-cell (Treg) recruitment to the site of inflammation. T-regs also use CCR5 to migrate and are responsible for suppressing the immune response by scavenging IL-2 (necessary for proliferation of activated leukocytes) and releasing anti-inflammatory IL-10 and TGF-b. By preventing recruitment of Tregs to the inflamed joint, excessive inflammation might counter-balance the positive effect of reduced monocyte recruitment.

Antagonist dosage might have been also been to blame for the failed CCR1 blockade trials, as a group using a daily 10mg dose CCR1 antagonist MLN389 showed no clinical efficacy, while another group reported clinical improvements after administration of 300mg of another CCR1 antagonist CP-481715 every eight hours. These results suggest that CCR1 blockade may be sufficient to inhibit monocyte recruitment to the synovial compartment only in the presence of high levels of receptor occupancy27. However, off target effects have been considered as the potential reason behind the modest protection observed at very high antagonist concentrations. At such high levels (almost 1g of antagonist/day), there was sufficient drug present to cross-react effectively with other GPCRs, even those for which the compound had low affinity. It is therefore possible that the protective effects reported by the group could have been mediated through these other GPCRs. This was the case with Compound 1, a highly potent human CCR1 inhibitor which could cross react with other GPCRs including adenosine A and dopamine D232. The compound was not developed further as it was discovered that the protective effects were not mediated by CCR1 inhibition but by affecting dopamine, which is known to attenuate T-cell functions and secretion of Th1 d that are involved in the pathophysiology of Multiple Sclerosis.

Disease Heterogeneity

Multiple Sclerosis (MS) is an example of an extremely heterogeneous disease which consists of at least for distinct patterns of neuron demyelination. These different stages are characterised by different leukocytes and driven by different chemokine receptors. CCR1, CCR2, CCR5 and CXCR3 have all been implicated in the development and progress of MS3. Thus, similarly to RA, depending on the expression and activation of these receptors in patients with the disease, targeting more than one receptor might be necessary to show any efficacy in a clinical trial.

Although studies have suggested it is possible to alter monocyte recruitment pharmacologically, the examples elucidated so far underline the complexity of the chemokine system and the vast ramification of consequences which can arise by downregulating the expression of a single receptor. Before any attempt can be made to treat inflammatory conditions by targeting critical chemokine=ligand combinations, it is necessary to fully understand and determine how the various chemokine receptors and their ligands interact with each other and which one (or combinations of) is necessary for the correct functioning of a specific leukocyte subset. A radical new approach will be required to fully elucidate the role of each inflammatory chemokine receptor before effective pharmacological intervention can be considered.


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1st Dec 2021 Robin Bartolini PhD Candidate, University of Glasgow

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