Assaying Chemokines and Chemotaxis

The EuroSciCon [1] meeting on Assaying Chemokines and Chemotaxis took place on 8th June 2007, and was held at the Biopark, Welwyn Garden City, Hertfordshire, UK. The aim of this meeting was to discuss the most recent advances in the growing field of chemokine research. The meeting was chaired by James Pease (Imperial College, London, UK) who began the meeting by giving a brief history on the discovery of chemokines, including how chemokines were identified originally, the current nomenclature of chemokines and their corresponding receptors and the emerging role of chemokines in proinflammatory diseases. The speakers focused on the role of chemokines in leukocyte migration, how blocking leukocyte migration impacts inflammatory diseases and new techniques used in the identification and synthesis of chemokines and the cells that express them.

Finding chemokines

The quantification of chemokines and the identification of those cells that express chemokines is the first step in studying the effects of these small molecules in inflammation. Alastair Hay (Almac Sciences, UK) presented a method for the chemical synthesis of purified chemokines for use in bioassays, which is independent of bacterial or mammalian expression systems. These chemokines can be subjected to site-specific modifications to allow the addition of tags, such as AlexaFluors and biotin. Tuc Ahmad (Meso Scale Discovery, UK) provided details of a multiplex assay based upon electrochemiluminescence technology that allowed the accurate analysis and quantification of up to ten chemokines from a sample as small as 25 µl. This is a highly sensitive assay that can be customized and used for numerous fluids, including serum, blood, bronchoalveolar lavage and tears.

The identification of the correct population of cells is important for the study of the impact of certain chemokines. Hilary Sandig (Kings College, London, UK) told us about a novel flow cytometry based assay, the gatedautofluorescence/forward scatter (GAFS) assay that helped identify eosinophils based on changes in cell shape in response to prostoglandin D₂ (PGD₂). Using this technique, it was possible to screen alternative agonists for the PGD₂ receptor CRTH2. This assay was shown to be sensitive, quick and cheap, as it does not require antibodies, and can be used to identify neutrophils, basophils and monocytes, as well as eosinophils.

Accurate and fast screening of potential factors influencing chemotactic function of inflammatory cells is a valuable and potent tool to have. Charlotte Weller (Imperial College, London, UK) presented work on the identification of mast cell chemoattractants in the allergic rhinitis murine model. Using a 96-well plate Boyden chamber-based assay from Neuro Probe Inc., it was possible to perform high-throughput screening and assessment of small molecular attractants produced by activated mast cells.

Leukocyte migration & chemotaxis

Chemokines are well documented to be involved in the recruitment of leukocytes to sites of inflammation. In order for the responding leukocytes to participate in the inflammatory response, they must first interact with endothelial cells and then pass through this barrier into the inflamed tissue. We heard presentations from two speakers that addressed each of these steps. Dorian Haskard (Imperial College, London, UK) presented his work on the role of chemokines in leukocyte–endothelial cell adhesion. This study used the in vitro parallel-plate flow adhesion assay that provided real-time imaging to identify the processes involved in leukocyte interactions with endothelial cells, and allowed every cell that passed through the detector to be measured under flow conditions. Using leukocytes taken from patients with systemic lupus erythematosus, this technique was used to study whether auto-antibodies associated with this disease and chemokines associated with inflammation influenced the capture of lymphocytes or lymphocyte function. These data revealed that antibodies increased the adhesion of leukocytes to an endothelial cell line, and that this adhesion required E-selectin and the expression of the chemokine IL-8. It was proposed that chemokines are required for the adhesion cascade.

To address the next step in leukocyte recruitment, Mattieu-Benoit Voisin (Imperial College, London, UK) presented work on the role of chemokines on leukocyte transmigration through the vessel wall. Using an in vivo cremasteric inflammation model and intravital microscopy that allowed for live imaging, they were able to identify mechanisms involved in chemokine-induced leukocyte transmigration. Migration through venule walls requires the leukocytes to pass through three barriers: endothelial cells, pericytes and the basement membrane expressing laminins. They identified that there were areas of low laminin expression and that these areas were preferential for leukocyte transmigration. Using their model of ischemia:reperfusion injury to the cremaster muscle, Voisin et al. identified that these areas of low expression increased in size after injury. They also identified that the damaged cells produced the chemokines KC and monocyte chemotactic protein-1 after injury, which increased leukocyte adhesion. A key point is that adhesion molecules were also upregulated on neutrophils, thus aiding speedy recruitment to the site of damage.

These data indicate a greater, more complex role for chemokines in leukocyte migration than just providing a gradient for targeting leukocytes to sites of inflammation.

Chemokines as targets for therapeutic agents in the treatment of inflammatory disease

Leukocytes can cause considerable damage to tissues during an inflammatory response. If leukocytes are responding to injury or infection then this inflammatory response will be tightly regulated and most likely short lived. However autoimmune diseases, such as asthma, chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis, can induce chronic inflammation that persists for a long time, creating lasting damage to the tissue affected. The prevention of leukocyte recruitment into these sites of inflammation is, therefore, a potential target for therapeutic intervention. A number of presentations were given on this theme of identifying inhibitors of chemokines and their receptors.

Aletta Kraneveld (Utrecht University, The Netherlands) presented her work on a small tripeptide inhibitor for chemokine-induced alveolar damage in COPD. She had published previously that intratracheal exposure to the collagen-derived N-acetyl-Pro-Gly-Pro tripeptide (PGP) tripeptide induced a dose-dependent infiltration of neutrophils into the lungs in the mouse model, and that this recruitment was CXCR mediated. In addition, another tripeptide, Arg-Thr-Arg tripeptide (RTR), acts to bind to PGP sequences and inhibit PGP-mediated neutrophil infiltration. Here, data were presented that showed that PGP was found in the lungs of patients with COPD and healthy smokers, but not asthmatics. PGP also induced lung emphysema in the mouse after exposure to PGP, lipopolysaccharide (LPS) or cigarette smoke. In this model, the application of RTR as short as 15 min before PGP or LPS exposure abolished neutrophil recruitment in PGP- and LPS-induced emphysema. These data provide evidence that PGP may be important in COPD pathogenesis, and that PGP antagonists (e.g., RTR) may prevent or ameliorate the inflammation associated with this disease.

Inhibition of the chemokine/chemokine receptor is one method of inhibiting chemokine function, another protocol utilizes the signaling pathways that are essential for this response. Stephen Ward (University of Bath, UK) presented data that dissected the navigational signals allowing T cells to mount a chemotactic response, specifically responding to CXCL12. Using the broad-spectrum PI3K inhibitor LY294002, it was possible to elucidate that PI3K was a major contributor to CXCL12-induced signaling and migration in freshly isolated human T cells. However, maintenance of these cells ex vivo resulted in the T cells becoming resistant to this inhibitor, suggesting that the role of PI3K in CXCL12-mediated directional migration is context dependent. Further analysis to identify the specific isoform of PI3K relevant to CXCL12-mediated migration showed that the PI3K-γ isoform was a major contributor. These data show that chemokine-induced signaling is a complex process that is dependent on the treatment of the cells studied.

Upon binding of the chemokine ligand to its receptor, the receptor is internalized by the cell, a phenomenon that was exploited by Louise Jopling (UCB Pharma, Cambridge, UK). The internalization of CXCR3 provided an effective pharmacokinetic/pharmacodynamic (PK/PD) model for the study of CXCR3 antagonists. These PK/PD models are important for the identification of biomarkers and are essential to drug discovery. In this case, the use of an animal model allows for the direct comparison of a number of antagonists, as well as the determination of important information, such as the effect of the route of treatment administration, optimal agonist concentration, plasma tolerance and potency.

Conclusion

This was a well-organized meeting that catered well to the target audience. The presenting speakers were from both industry and academia, and the topics that were presented complemented each other well. Some speakers presented cutting-edge research with emphasis on treatment and disease, whereas others provided much needed technical and practical insight into the ever-changing field of chemotaxis research. In summary, this was a highly informative and important meeting.