Chemokines are essential for guiding cell migration. Atypical chemokine receptors (ACKRs) contribute to the cell migration process by binding, internalizing and degrading local chemokines, which enables the formation of confined gradients. ACKRs are heptahelical membrane spanning molecules structurally related to G-protein coupled receptors (GPCRs), but seem to be unable to signal through G-proteins upon ligand binding. ACKR4 internalizes the chemokines CCL19, CCL21, and CCL25 and is best known for shaping functional CCL21 gradients. Ligand binding to ACKR4 has been shown to recruit β-arrestins that has led to the assumption that chemokine scavenging relies on β-arrestin-mediated ACKR4 trafficking, a common internalization route taken by class A GPCRs. Here, we show that CCL19, CCL21, and CCL25 readily recruited β-arrestin1 and β-arrestin2 to human ACKR4, but found no evidence for β-arrestin-dependent or independent ACKR4-mediated activation of the kinases Erk1/2, Akt, or Src. However, we demonstrate that β-arrestins interacted with ACKR4 in the steady-state and contributed to the spontaneous trafficking of the receptor in the absence of chemokines. Deleting the C-terminus of ACKR4 not only interfered with the interaction of β-arrestins, but also with the uptake of fluorescently labeled cognate chemokines. We identify the GPCR kinase GRK3, and to a lesser extent GRK2, but not GRK4, GRK5, and GRK6, to be recruited to chemokine-stimulated ACKR4. We show that GRK3 recruitment proceded the recruitment of β-arrestins upon ACKR4 engagement and that GRK2/3 inhibition partially interfered with steady-state interaction and chemokine-driven recruitment of β-arrestins to ACKR4. Overexpressing β-arrestin2 accelerated the uptake of fluorescently labeled CCL19, indicating that β-arrestins contribute to the chemokine scavenging activity of ACKR4. By contrast, cells lacking β-arrestins were still capable to take up fluorescently labeled CCL19 demonstrating that β-arrestins are dispensable for chemokine scavenging by ACKR4.

CD4+ T cell repopulation of the gut is rarely achieved in HIV-1–infected individuals who are receiving clinically effective antiretroviral therapy. Alterations in the integrity of the mucosal barrier have been indicated as a cause for chronic immune activation and disease progression. In this study, we present evidence that persistent immune activation causes impairment of lymphocytes to respond to chemotactic stimuli, thus preventing their trafficking from the blood stream to peripheral organs. CCR6+ and CXCR3+ Th cells accumulate in the blood of aviremic HIV-1–infected patients on long-term antiretroviral therapy, and their frequency in the circulation positively correlates to levels of soluble CD14 in plasma, a marker of chronic immune activation. Th cells show an impaired response to chemotactic stimuli both in humans and in the pathogenic model of SIV infection, and this defect is due to hyperactivation of cofilin and inefficient actin polymerization. Taking advantage of a murine model of chronic immune activation, we demonstrate that cytoskeleton remodeling, induced by okadaic acid, restores lymphocyte migration in response to chemokines, both in vitro and in vivo. This study calls for novel pharmacological approaches in those pathological conditions characterized by persistent immune activation and loss of trafficking of T cell subsets to niches that sustain their maturation and activities.

Chemokine synergy-inducing molecules are emerging as regulating factors in cell migration. The alarmin HMGB1, in its reduced form, can complex with CXCL12 enhancing its activity on monocytes via the chemokine receptor CXCR4, while the form containing a disulfide bond, by binding to TLR2 or TLR4, initiates a cascade of events leading to production of cytokines and chemokines. So far, the possibility that the CXCL12/HMGB1 heterocomplex could be maintained in chronic inflammation was debated, due to the release of reactive oxygen species. Therefore, we have assessed if the heterocomplex could remain active in Rheumatoid Arthritis (RA) and its relevance in the disease assessment. Monocytes from RA patients with active disease require a low concentration of HMGB1 to enhance CXCL12-induced migration, in comparison to monocytes from patients in clinical remission or healthy donors. The activity of the heterocomplex depends on disease activity, on the COX2 and JAK/STAT pathways, and is determined by the redox potential of the microenvironment. In RA, the presence of an active thioredoxin system correlates with the enhanced cell migration, and with the presence of the heterocomplex in the synovial fluid. The present study highlights how, in an unbalanced microenvironment, the activity of the thioredoxin system plays a crucial role in sustaining inflammation. Prostaglandin E2 stimulation of monocytes from healthy donors is sufficient to recapitulate the response observed in patients with active RA. The activation of mechanisms counteracting the oxidative stress in the extracellular compartment preserves HMGB1 in its reduced form, and contributes to fuel the influx of inflammatory cells. Targeting the heterocomplex formation and its activity could thus be an additional tool for dampening the inflammation sustained by cell recruitment, for those patients with chronic inflammatory conditions who poorly respond to current therapies.

At clinical concentrations, lidocaine significantly inhibits CXCR4 signalling. The results presented shed new insights on the molecular mechanisms governing the inhibitory effect of lidocaine on cell migration.

Review on synergistic activities induced by heterocomplexes formed with chemokines.