The chemokines CXCL8 and CXCL12: molecular and functional properties, role in disease and efforts towards pharmacological intervention

Abstract

Chemokines are an indispensable component of our immune system through the regulation of directional migration and activation of leukocytes. CXCL8 is the most potent human neutrophil-attracting chemokine and plays crucial roles in the response to infection and tissue injury. CXCL8 activity inherently depends on interaction with the human CXC chemokine receptors CXCR1 and CXCR2, the atypical chemokine receptor ACKR1, and glycosaminoglycans. Furthermore, (hetero)dimerization and tight regulation of transcription and translation, as well as post-translational modifications further fine-tune the spatial and temporal activity of CXCL8 in the context of inflammatory diseases and cancer. The CXCL8 interaction with receptors and glycosaminoglycans is therefore a promising target for therapy, as illustrated by multiple ongoing clinical trials. CXCL8-mediated neutrophil mobilization to blood is directly opposed by CXCL12, which retains leukocytes in bone marrow. CXCL12 is primarily a homeostatic chemokine that induces migration and activation of hematopoietic progenitor cells, endothelial cells, and several leukocytes through interaction with CXCR4, ACKR1, and ACKR3. Thereby, it is an essential player in the regulation of embryogenesis, hematopoiesis, and angiogenesis. However, CXCL12 can also exert inflammatory functions, as illustrated by its pivotal role in a growing list of pathologies and its synergy with CXCL8 and other chemokines to induce leukocyte chemotaxis. Here, we review the plethora of information on the CXCL8 structure, interaction with receptors and glycosaminoglycans, different levels of activity regulation, role in homeostasis and disease, and therapeutic prospects. Finally, we discuss recent research on CXCL12 biochemistry and biology and its role in pathology and pharmacology.

Keywords: CXCL12; CXCL8; GPCR; atypical chemokine receptor; glycosaminoglycan.

Fig. 1

Primary sequence and 3D structures of CXCL8 and its receptors CXCR1, CXCR2, and ACKR1. A Primary sequence of human CXCL8 after translation, including the signal peptide and B 3D ribbon structure of the mature CXCL8 monomer as determined by NMR spectroscopy. All secondary structural elements are indicated in the amino acid sequence and the 3D structure. The four conserved cysteine residues and the disulfide bonds connecting them for stabilization of the CXCL8 structure are indicated in gray. The CXC motif can be found immediately COOH-terminally of the ELR motif. The three β-strands forming a β-sheet are colored in green. The COOH-terminal α-helix and the small 3₁₀ helix are depicted in blue. The last two amino acids of the signal peptide (Glu-2 and Gly-1) can also be part of the mature CXCL8 protein due to alternative cleavage of the signal peptide. Underlined amino acid residues are implicated in GAG binding. C Dimer structure of CXCL8 in solution as determined by NMR spectroscopy. Monomer units are colored in orange and green. The CXCL8(6–77) 3D structure was drawn from PDB accession code 1IL8. D 3D structure of CXCR1 as determined by solid-state NMR spectroscopy and drawn from PDB accession code 2LN [34]. E Cryo-electron microscopy structure of monomeric CXCL8-activated human CXCR2 in complex with the Gα_i protein, drawn from PDB accession code 6LFO. A similar 3D structure of dimeric CXCL8 activating CXCR2 can be found with PDB accession code 6LFM [35]. F X-ray diffraction heterotetramer structure of two NH₂-terminal ectodomains (forming a helix) of ACKR1 (DARC) binding each to two molecules of the receptor binding domain of Plasmodium vivax Duffy binding protein (DBP-RII), drawn from PDB accession code 4NUV. DBP-RII monomers are indicated in orange and green. DARC monomers are depicted in purple and blue. A DBP-RII∶DARC heterotrimer structure, where a single ACKR1 ectodomain binds two DBP-RIIs, can be found with PDB accession code 4NUU [40]

Fig. 2

CXCL8-mediated neutrophil attraction to and activation at the inflammatory site. Upon an inflammatory trigger such as bacterial infection, several tissue cells and tissue-resident leukocytes produce CXCL8. CXCL8 establishes a concentration gradient from the production site to the blood vessels guiding neutrophils toward the inflammatory site, where they eliminate the pathogens and resolve acute inflammation. Afterwards, CXCL8 may promote angiogenesis by stimulating endothelial cell proliferation and migration to repair the damaged tissue. CXCL8 activity is regulated by: A The need for immobilization of CXCL8 on endothelial GAGs inhibiting proteolytic degradation and diffusion in the blood stream, establishing and maintaining a concentration gradient toward the inflammatory site. B Removal of non-immobilized CXCL8 from the bloodstream by binding to ACKR1 expressed on erythrocytes, preventing systemic leukocyte activation and providing a chemokine reservoir. C Translocation of CXCL8 from the extracellular matrix to the surface of the endothelial layer (transcytosis), which is controlled by binding to GAGs and endothelial ACKR1. D Synergy of CXCL8 with other chemoattractants to amplify the inflammatory response

Fig. 3

CXCL8 binding to CXCR1 and CXCR2 on neutrophils activates G protein- and β-arrestin-mediated signal transduction pathways. G protein-mediated signaling activates MAPK, PLC, and PI3K pathways leading to neutrophil chemotaxis and effector functions. ROS production was suggested to be specifically linked to activation of PLD after CXCR1, but not CXCR2, activation since CXCL8, but not CXCL1, induced ROS [72]. CXCL8 binding to CXCR1/CXCR2 also leads to desensitization of G protein-mediated signaling, through internalization of the receptors mainly mediated by β-arrestins. Afterwards, receptors can be degraded, recycled to the membrane, or induce an additional round of MAPK or tyrosine kinase signaling

Fig. 4

The production process of CXCL8 after inflammatory stimulation is tightly regulated. In response to an inflammatory trigger, pro-inflammatory cytokines like IL-1 or TNF-α induce the production of CXCL8 by stimulation of their receptors. This induces downstream signaling pathways resulting in activation of NF-κB and the AP-1 complex, which translocate into the nucleus and initiate transcription of the CXCL8 gene and production and release of CXCL8. This process is regulated at different levels. A Specific CXCL8 polymorphisms influence the CXCL8 production levels. B Transcription is only initiated after de-repression of the CXCL8 gene promoter, mediated through association of NF-κB with NF-κB-repressing factor (NRF) and the negative regulatory element (NRE) in the promoter and replacing octamer-1 (OCT-1) by the transcription factor CCAAT/enhancer-binding protein (C/EBP). This recruits the co-activator CREB-binding protein (CBP)/p300 which results in histone hyperacetylation and chromatin remodeling so that AP-1 and NF-κB can activate the gene transcription process. Anti-inflammatory stimuli like IL-10 and TGF-β can block this transcription process. C After production, the labile CXCL8 mRNA needs to be stabilized by a MAP kinase-activated protein kinase 2 (MK2)-dependent AU-rich cis-elements (ARE)-targeted mechanism through activation of the p38 MAPK pathway. This stabilization is promoted by LPS, IL-1, TNF-α, IFN-γ, nitric oxide, and hypoxia (not shown) and repressed by IL-4, IL-10, and glucocorticoids (GC) promoting mRNA degradation. D After mRNA translation, CXCL8 localizes intracellularly in the Golgi apparatus, from where it is secreted (constitutive secretory pathway). E After exocytosis, CXCL8 can be subjected to multiple post-translational modifications like proteolysis with profound effects on its activity

Fig. 5

Post-translational modifications affect the biological activity of CXCL8. Natural CXCL8 has been identified as a partially citrullinated protein and as multiple NH₂-terminally truncated proteoforms (green, red, and gray boxes). Citrullination of CXCL8 on Arg5 significantly reduces its biological activity whereas removal of 5–8 amino acids significantly potentiates the activity of CXCL8 up to almost 30-fold, compared to CXCL8(1–77). Moreover, due to induction of increased neutrophil migration and activation, additional proteases are released which again enhance the proteolytic activation of CXCL8 (positive feedback loop). Further proteolytic cleavage in the ELR motif and nitration have not been reported on natural CXCL8 (yellow boxes) but CXCL8(10–77) could be generated after incubation of CXCL8 with MMP-12. Cleavage in the ELR motif or nitration reduce or abolish the activity of CXCL8 in vitro

Fig. 6

Cutting-edge research progress in the field of CXCL12. The solution NMR structure of the CXCL12 monomer was drawn from PDB accession code 2KEC. BAL fluid broncho-alveolar lavage fluid, GAG glycosaminoglycan, HMGB1 high-mobility group box 1, WHIM syndrome Warts, Hypogammaglobulinemia, Immunodeficiency, and Myelokathexis syndrome