Generation of stable advective-diffusive chemokine gradients in a three-dimensional hydrogel

Physiologic chemoattractant gradients are shaped by diffusion, advection, binding to an extracellular matrix, and removal by cells. Previous in vitro tools for studying these gradients and the cellular migratory response have required cells to be constrained to a 2D substrate or embedded in a gel devoid of fluid flow. Cell migration in fluid flow has been quantified in the absence of chemoattractant gradients and shown to be responsive to them, but there is a need for tools to investigate the synergistic, or antagonistic, effects of gradients and flow. We present a microfluidic chip in which we generated precisely controlled gradients of the chemokine CCL19 under advective-diffusive conditions. Using torque-actuated membranes situated between a gel region and the chip outlet, the resistance of fluid channels adjacent to the gel region could be modified, creating a controllable pressure difference across the gel at a resolution inferior to 10 Pa. Constant supply and removal of chemokine on either side of the chip facilitated the formation of stable gradients at Péclet numbers between −10 and +10 in a collagen type I hydrogel. The resulting interstitial flow was steady within 0.05 μm s−1 for at least 8 h and varied by less than 0.05 μm s−1 along the gel region. This method advances the physiologic relevance of the study of the formation and maintenance of molecular gradients and cell migration, which will improve the understanding of in vivo observations

CCR2 Acts as Scavenger for CCL2 during Monocyte Chemotaxis

<div><h3>Background</h3><p>Leukocyte migration is essential for effective host defense against invading pathogens and during immune homeostasis. A hallmark of the regulation of this process is the presentation of chemokines in gradients stimulating leukocyte chemotaxis via cognate chemokine receptors. For efficient migration, receptor responsiveness must be maintained whilst the cells crawl on cell surfaces or on matrices along the attracting gradient towards increasing concentrations of agonist. On the other hand agonist-induced desensitization and internalization is a general paradigm for chemokine receptors which is inconsistent with the prolonged migratory capacity.</p> <h3>Methodology/Principal Findings</h3><p>Chemotaxis of monocytes was monitored in response to fluorescent CCL2-mCherry by time-lapse video microscopy. Uptake of the fluorescent agonist was used as indirect measure to follow the endogenous receptor CCR2 expressed on primary human monocytes. During chemotaxis CCL2-mCherry becomes endocytosed as cargo of CCR2, however, the internalization of CCR2 is not accompanied by reduced responsiveness of the cells due to desensitization.</p> <h3>Conclusions/Significance</h3><p>During chemotaxis CCR2 expressed on monocytes internalizes with the bound chemoattractant, but cycles rapidly back to the plasma membrane to maintain high responsiveness. Moreover, following relocation of the source of attractant, monocytes can rapidly reverse their polarization axis organizing a new leading edge along the newly formed gradient, suggesting a uniform distribution of highly receptive CCR2 on the plasma membrane. The present observations further indicate that during chemotaxis CCR2 acts as scavenger consuming the chemokine forming the attracting cue.</p> </div

First international consensus on the methodology of lymphangiogenesis quantification in solid human tumours

The lymphatic system is the primary pathway of metastasis for most human cancers. Recent research efforts in studying lymphangiogenesis have suggested the existence of a relationship between lymphatic vessel density and patient survival. However, current methodology of lymphangiogenesis quantification is still characterised by high intra- and interobserver variability. For the amount of lymphatic vessels in a tumour to be a clinically useful parameter, a reliable quantification technique needs to be developed. With this consensus report, we therefore would like to initiate discussion on the standardisation of the immunohistochemical method for lymphangiogenesis assessment

Post-translational control of chemokines: a role for,decoy receptors?

Copyright © 2004 Elsevier B.V. All rights reserved.It is well-established that chemokines play a critical role in the orchestration of inflammation and immunity. Interactions between chemokines and their receptors are essential for the homing of specific subsets of leukocytes to their functional microenvironments. They also influence other diverse biological processes such as development, leukocyte activation, Th1/Th2 polarisation, tumour metastasis, angiogenesis, and HIV pathogenesis. However, despite their importance, only now are we beginning to understand the complex regulation brought to bear on these molecules. In this review, we discuss a number of these key chemokine regulators that exert their influence once these proteins have been synthesised. We examine (i) chemokine storage, release, and presentation, (ii) protease regulation, (iii) viral manipulation of host chemokines, and (iv) natural mammalian receptor antagonists. Principally, the growing evidence for a role for decoy receptors in the chemokine system is discussed. In particular, the potential decoy function of the 'silent' pro-inflammatory chemokine receptor D6 is described alongside two other candidate decoy receptor molecules, DARC, and CCX-CKR. Dissecting the biological and pathological function of these chemokine controllers will lead to a deeper understanding of chemokine regulation, and may reveal novel strategies to therapeutically modify the chemokine system.Iain Comerford and Robert J.B. Nibbshttp://www.elsevier.com/wps/find/journaldescription.cws_home/506020/description#descriptio

Chemokine transport dynamics and emerging recognition of their role in immune function

Leukocyte migration is critically important during all protective and pathological immune and inflammatory responses. Chemokines play fundamental roles in this process, and chemokine concentration gradients stimulate the directional migration of leukocytes. The formation and regulation of these gradients is poorly understood. These are complex processes that depend on the specific properties of each chemokine and interactions between physical, biological and biochemical processes, including production, diffusion, advection, scavenging, post-translational modification, and extracellular matrix (ECM) binding. While some of these mechanisms have been investigated in isolation or limited combinations, more integrative research is required to provide a quantitative knowledge base that explains how chemokine gradients are established and maintained, and how cells respond to, and modify, these gradients

Regulation of chemotactic networks by 'atypical' receptors

Directed cell migration is a fundamental component of numerous biological systems and is critical to the pathology of many diseases. Although the importance of secreted chemoattractant factors in providing navigational cues to migrating cells bearing specific chemoattractant receptors is now well-established, how the function of these factors is regulated is not so well understood and may be of key importance to the design of new therapeutics for numerous human diseases. While regulation of migration clearly takes place on a number of different levels, it is becoming clear that so-called 'atypical' receptors play a role in scavenging, or altering the localisation of, chemoattractant molecules such as chemokines and complement components. These receptors do this through binding and/or internalising their chemoattractant ligands without activating signal transduction cascades leading to cell migration. The atypical chemokine receptor family currently comprises the receptors D6, DARC and CCX-CKR. In this review, we discuss the evidence from in vitro and in vivo studies that these receptors play a role in regulating cell migration, and speculate that other orphan receptors may also belong to this family. Furthermore, with the advent of gene therapy on the horizon, the therapeutic potential of these receptors in human disease is also considered.Iain Comerford, Wendel Litchfield, Yuka Harata-Lee, Robert J.B. Nibbs, Shaun R. McCol