Leukocyte adhesion: reconceptualizing chemokine presentation by glycosaminoglycans

Recruitment of immune cells from the vasculature relies on the presentation of glycosaminoglycan-bound chemokines on the luminal side of vascular endothelial cells. However, the current model of chemokine–glycosaminoglycan interactions, and its implications for receptor interactions, remains poorly developed. We propose a refined ‘Chemokine Cloud’ model, arguing that chemokines are not presented to leukocytes bound to glycosaminoglycans, but rather, in solution while sequestered within the hydrated glycocalyx. We posit that glycosaminoglycans provide an immobilized chemokine depot maintaining a ‘cloud’ of ‘solution-phase’ chemokines within the glycocalyx, and that it is this soluble form of any given chemokine that interacts with leukocyte-bound receptors. Our proposition clarifies certain anomalies associated with the current model of chemokine–glycosaminoglycan interactions, with implications for the design of blockers of chemokine function

Elucidating the CXCL12/CXCR4 Signaling Network in Chronic Lymphocytic Leukemia through Phosphoproteomics Analysis

, and is thought to be due to enhanced survival signaling responses to environmental factors that protect CLL cells from spontaneous and chemotherapy-induced death. Although normally associated with cell migration, the chemokine, CXCL12, is one of the factors known to support the survival of CLL cells. Thus, the signaling pathways activated by CXCL12 and its receptor, CXCR4, were investigated as components of these pathways and may represent targets that if inhibited, could render resistant CLL cells more susceptible to chemotherapy.To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells. While some of the survival pathways activated by CXCL12 in CLL are known, including Akt and ERK1/2, this approach enabled the identification of additional signaling targets and novel phosphoproteins that could have implications in CLL disease and therapy. In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4), is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed. Additionally, heat shock protein 27 (HSP27), which mediates anti-apoptotic signaling and has previously been linked to chemotherapeutic resistance, was detected in a subset (∼25%) of CLL patients cells examined.Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets. PDCD4 also represents a previously unknown signaling target of chemokine receptors; therefore, these observations increase our understanding of alternative pathways to migration that may be activated or inhibited by chemokines in the context of cancer cell survival

A critical role for lymphatic endothelial heparan sulfate in lymph node metastasis

Abstract Background Lymph node metastasis constitutes a key event in tumor progression. The molecular control of this process is poorly understood. Heparan sulfate is a linear polysaccharide consisting of unique sulfate-modified disaccharide repeats that allow the glycan to bind a variety of proteins, including chemokines. While some chemokines may drive lymphatic trafficking of tumor cells, the functional and genetic importance of heparan sulfate as a possible mediator of chemokine actions in lymphatic metastasis has not been reported. Results We applied a loss-of-function genetic approach employing lymphatic endothelial conditional mutations in heparan sulfate biosynthesis to study the effects on tumor-lymphatic trafficking and lymph node metastasis. Lymphatic endothelial deficiency in N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in sulfating nascent heparan sulfate chains, resulted in altered lymph node metastasis in tumor-bearing gene targeted mice. This occurred in mice harboring either a pan-endothelial Ndst1 mutation or an inducible lymphatic-endothelial specific mutation in Ndst1. In addition to a marked reduction in tumor metastases to the regional lymph nodes in mutant mice, specific immuno-localization of CCL21, a heparin-binding chemokine known to regulate leukocyte and possibly tumor-cell traffic, showed a marked reduction in its ability to associate with tumor cells in mutant lymph nodes. In vitro modified chemotaxis studies targeting heparan sulfate biosynthesis in lymphatic endothelial cells revealed that heparan sulfate secreted by lymphatic endothelium is required for CCL21-dependent directional migration of murine as well as human lung carcinoma cells toward the targeted lymphatic endothelium. Lymphatic heparan sulfate was also required for binding of CCL21 to its receptor CCR7 on tumor cells as well as the activation of migration signaling pathways in tumor cells exposed to lymphatic conditioned medium. Finally, lymphatic cell-surface heparan sulfate facilitated receptor-dependent binding and concentration of CCL21 on the lymphatic endothelium, thereby serving as a mechanism to generate lymphatic chemokine gradients. Conclusions This work demonstrates the genetic importance of host lymphatic heparan sulfate in mediating chemokine dependent tumor-cell traffic in the lymphatic microenvironment. The impact on chemokine dependent lymphatic metastasis may guide novel therapeutic strategies

Structures of atypical chemokine receptor 3 reveal the basis for its promiscuity and signaling bias

Both CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are activated by the chemokine CXCL12 yet evoke distinct cellular responses. CXCR4 is a canonical G protein–coupled receptor (GPCR), whereas ACKR3 is intrinsically biased for arrestin. The molecular basis for this difference is not understood. Here, we describe cryo-EM structures of ACKR3 in complex with CXCL12, a more potent CXCL12 variant, and a small-molecule agonist. The bound chemokines adopt an unexpected pose relative to those established for CXCR4 and observed in other receptor-chemokine complexes. Along with functional studies, these structures provide insight into the ligand-binding promiscuity of ACKR3, why it fails to couple to G proteins, and its bias toward β-arrestin. The results lay the groundwork for understanding the physiological interplay of ACKR3 with other GPCRs

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t

CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested the receptor may exist as a dimer or oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means we confirm that CXCR4 can exist as a mixture of monomers, dimers and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t— a small, drug-like isothiourea derivative — rapidly destabilizes the oligomeric structure, while AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state

Natural Plant Sugar Sources of Anopheles Mosquitoes Strongly Impact Malaria Transmission Potential

An improved knowledge of mosquito life history could strengthen malaria vector control efforts that primarily focus on killing mosquitoes indoors using insecticide treated nets and indoor residual spraying. Natural sugar sources, usually floral nectars of plants, are a primary energy resource for adult mosquitoes but their role in regulating the dynamics of mosquito populations is unclear. To determine how the sugar availability impacts Anopheles sergentii populations, mark-release-recapture studies were conducted in two oases in Israel with either absence or presence of the local primary sugar source, flowering Acacia raddiana trees. Compared with population estimates from the sugar-rich oasis, An. sergentii in the sugar-poor oasis showed smaller population size (37,494 vs. 85,595), lower survival rates (0.72 vs. 0.93), and prolonged gonotrophic cycles (3.33 vs. 2.36 days). The estimated number of females older than the extrinsic incubation period of malaria (10 days) in the sugar rich site was 4 times greater than in the sugar poor site. Sugar feeding detected in mosquito guts in the sugar-rich site was significantly higher (73%) than in the sugar-poor site (48%). In contrast, plant tissue feeding (poor quality sugar source) in the sugar-rich habitat was much less (0.3%) than in the sugar-poor site (30%). More important, the estimated vectorial capacity, a standard measure of malaria transmission potential, was more than 250-fold higher in the sugar-rich oasis than that in the sugar-poor site. Our results convincingly show that the availability of sugar sources in the local environment is a major determinant regulating the dynamics of mosquito populations and their vector potential, suggesting that control interventions targeting sugar-feeding mosquitoes pose a promising tactic for combating transmission of malaria parasites and other pathogens

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation due to a lack of mechanistic understanding. Specifically, CXCL4 (Platelet Factor 4, PF4) has no established receptor that explains its function. Here, we use biophysical, in vitro, and in vivo techniques to determine the mechanism underlying CXCL4-mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars on proteoglycans within the endothelial extracellular matrix, resulting in increased adhesion of leukocytes to the vasculature, increased vascular permeability, and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulfation confers selectivity onto chemokine localization. These findings present mechanistic insights into chemokine biology and provide future therapeutic targets