Chemokine Binding to Tenascin-C Influences Chemokine-Induced Immune Cell Migration

Abstract

Tenascin-C (TNC) is a complex glycoprotein of the extracellular matrix (ECM) involved in a plethora of (patho-)physiological processes, such as oncogenesis and inflammation. Since chemokines play an essential role in both disease processes, we have investigated here the binding of TNC to some of the key chemokines, namely CCL2, CCL26, CXCL8, CXCL10, and CXCL12. Thereby, a differential chemokine-TNC binding pattern was observed, with CCL26 exhibiting the highest and CCL2 the lowest affinity for TNC. Heparan sulfate (HS), another member of the ECM, proved to be a similarly high-affinity ligand of TNC, with a Kd value of 730 nM. Chemokines use glycosa-minoglycans such as HS as co-receptors to induce immune cell migration. Therefore, we assumed an influence of TNC on immune cell chemotaxis due to co-localization within the ECM. CCL26- and CCL2-induced mobilization experiments of eosinophils and monocytes, respectively, were thus performed in the presence and the absence of TNC. Pre-incubation of the immune cells with TNC resulted in a 3.5-fold increase of CCL26-induced eosinophil chemotaxis, whereas a 1.3-fold de-crease in chemotaxis was observed when monocytes were pre-incubated with CCL2. As both chemokines have similar HS binding but different TNC binding affinities, we speculate that TNC acts as an attenuator in monocyte and as an amplifier in eosinophil mobilization by impeding CCL2 from binding to HS on the one hand, and by reinforcing CCL26 to bind to HS on the other hand.

Investigating Chemokine-Matrix Networks in Breast Cancer: Tenascin-C Sets the Tone for CCL2

“…De Laporte et al showed that TNC interacts with multiple growth factors, e.g., from the platelet-derived growth factor family or the fibroblast growth factor family, promiscuously via its 5th FNIII repeats domain [55], whilst Loustau et al (2022) showed that TNC binds to CXCL12 and CCL21 predominantly via its FNIII domains TN3-5 [17,43,56]. In an accompanying paper, we demonstrate that purified recombinant TNC binds to purified recombinant CCL2 in solution using isothermal fluorescence titration (IFT) with a Kd of 2.33 +/− 0.39 µM [57]. When immobilizing recombinant TNC on an SPR-chip and using CCL2 as ligand, a Kd of 10.5 µM was found for this interaction (Figure S2A,B).…”

“…Moreover, the setup of the assay a key consideration. Compared to the setup described in [57], where human monocytes were used as migrating cells and the impact of the interaction of CCL2 and TNC in five-fold molecular excess in solution was investigated, less cells could be detected on the lower side of the membrane when CCL2 was pre-incubated with TNC compared to CCL2 alone. These data highlight a cell-and context-specific effect of TNC on CCL2-mediated migration.…”

“…A companion article [57] identifies the binding of TNC to CCL2, showing the Kd of the interaction to be in a similar concentration range as the interaction of CCL2 with its known co-receptors GAGs/proteoglycans [78][79][80]. In contrast, the affinity of CCL2 for the GPCR CCR2, which is the main signaling receptor for CCL2, is higher (0.77 nM [81]).…”

“…fluorescence titration (IFT) with a Kd of 2.33 +/− 0.39 µM [57]. When immobilizing recombinant TNC on an SPR-chip and using CCL2 as ligand, a Kd of 10.5 µM was found for this interaction (Figure S2A,B).…”

“…The following protein combinations were used for in vitro studies based on protein availability: transmission electron microscopy: mTNC and mCCL2, surface plasmon resonance: hTNC and hCCL2, transwell migration assays: hTNC, hFN and mCCL2 and mBMM activation assays: hTNC, hFN and mCCL2. A comparison between the sequences of hTNC and mTNC is described in [57]. The QC of recombinantly expressed hTNC and mTNC was performed via SDS-PAGE and Commassie blue staining as well as an adhesion assay with fibroblasts (Figure S1A-E).…”