Fluorescently Tagged CCL19 and CCL21 to Monitor CCR7 and ACKR4 Functions

Abstract

Chemokines are essential guidance cues orchestrating cell migration in health and disease. Cognate chemokine receptors sense chemokine gradients over short distances to coordinate directional cell locomotion. The chemokines CCL19 and CCL21 are essential for recruiting CCR7-expressing dendritic cells bearing pathogen-derived antigens and lymphocytes to lymph nodes, where the two cell types meet to launch an adaptive immune response against the invading pathogen. CCR7-expressing cancer cells are also recruited by CCL19 and CCL21 to metastasize in lymphoid organs. In contrast, atypical chemokine receptors (ACKRs) do not transmit signals required for cell locomotion but scavenge chemokines. ACKR4 is crucial for internalizing and degrading CCL19 and CCL21 to establish local gradients, which are sensed by CCR7-expressing cells. Here, we describe the production of fluorescently tagged chemokines by fusing CCL19 and CCL21 to monomeric red fluorescent protein (mRFP). We show that purified CCL19-mRFP and CCL21-mRFP are versatile and powerful tools to study CCR7 and ACKR4 functions, such as receptor trafficking and chemokine scavenging, in a spatiotemporal fashion. We demonstrate that fluorescently tagged CCL19 and CCL21 permit the visualization and quantification of chemokine gradients in real time, while CCR7-expressing leukocytes and cancer cells sense the guidance cues and migrate along the chemokine gradients.

CXCL14 Preferentially Synergizes With Homeostatic Chemokine Receptor Systems

“…Synthetic chemokines (CXCL14, CCL19, CCL21, CXCL13, CCL5, CXCL10 and CXCL11) were chemically synthezised, as previously described ( 30 ). CCL19 Dy69P1 was produced as previously described ( 31 , 32 ). Human CXCL14 conjugated to the fluorochrome Alexa Fluor 647 attached to a C-terminal lysine residue was purchased from Almac Sciences (Edinburgh, United Kingdom).…”

Chemokine Receptor 7 (CCR7) Participates in the Migration of DCs and Has Impact on the Progression of Primary Pterygium

“…However, there have not been any papers studying CCR7 in pterygia yet. Ligands C-C motif ligand 19(CCL19) and ligands C-C motif ligand 21 (CCL21), the two important ligands of CCR7, are found in the paracortical regions of the lymph node, and CCL21 could also be expressed by lymphatic vessels in nonlymphoid tissue [10]. In the ndings of our previous research [11], DCs which populate in the cornea of dry eye migrating to the lymphatic vessels could be affected by CCL21 and CCL19.…”

Engineering of Nanobodies Recognizing the Human Chemokine Receptor CCR7

“…HEK293 cells as well as H1299 cells stably expressing CCR7-HA [15,31] were cultured in DMEM containing 10% FCS (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Lonza, Basel, Switzerland). HEK293 cells were stably transfected with CCR7-YFP2 using TransIT-LT-1 (MirusBio, Madison, WI, USA) as specified by the manufacturer and selection with G418 (Gibco).…”

“…Analysis of chemokine-mediated changes in intracellular free calcium concentrations in H1299-CCR7-HA cells transiently transfected for 24 h with individual Nb-YFP1 clones was performed as previously described [15,33]. Briefly, cells were loaded with fluo-3-AM for 20 min at 37°C, washed and fluorescence changes upon chemokine stimulation were measured on a LSRII flow cytometer (BD Biosciences).…”

“…In particular, Gα-GTP is responsible for reassembly and regeneration of the inactive heterotrimeric G-protein, whereas free βγ-subunits activate phospholipase C and phosphoinositide-3-kinase. Activation of these effector enzymes is followed by activation of both second messengers, including calcium ions from intracellular stores, and different kinases, such as the extracellular signaling regulated kinases Erk-1/2 [13,14,15]. For CCR7, receptor oligomerization was shown to facilitate the interaction with Src kinase, which becomes activated upon chemokine triggering, resulting in tyrosine phosphorylation of the conserved DRY motif of the receptor [13,14,16].…”