CXCR2 Antagonist RIST4721 Acts as a Potent Chemotaxis Inhibitor of Mature Neutrophils Derived from Ex Vivo-Cultured Mouse Bone Marrow.

Neutrophils act as critical mediators of innate immunity, which depends on their rapid responses to chemokines followed by their migration towards sites of infection during chemotaxis. Chemokine receptors expressed on the surface of neutrophils mediate chemotaxis by activating contractile machinery as the cells escape from capillary beds and then attack pathogens. Neutrophils also contribute to inflammatory responses, which support pathogen destruction but can lead to acute and chronic inflammatory disorders. CXCR2, a G-protein-coupled chemokine receptor expressed on both myeloid and epithelial cells, is well-characterized for its capacities to bind multiple chemokines, including interleukin-8 and growth-related oncogene alpha in humans or keratinocyte chemokine (KC) in mice. Here we show that a small molecule CXCR2 antagonist termed RIST4721 can effectively inhibit KC-stimulated chemotaxis by neutrophils derived from ex vivo-cultured mouse bone marrow in a potent and dose-dependent manner. Antagonistic properties of RIST4721 are thoroughly characterized, including the maximal, half-maximal and minimum concentrations required to inhibit chemotaxis. Importantly, RIST4721-treated neutrophils exhibit robust phagocytosis and reactive oxygen species production, confirming drug specificity to chemotaxis inhibition. Together our data indicate that RIST4721 acts to inhibit inflammation mediated and potentiated by neutrophils and therefore promises to facilitate treatment of a host of inflammatory conditions.

Quantitatively Assessing the Respiratory Burst in Innate Immune Cells.

The respiratory burst is a rapid cellular consumption of oxygen resulting in abundant production of reactive oxygen species (ROS), most often associated with primary mediators of innate immunity, neutrophils and macrophages. These myeloid cells convert ROS into potent antimicrobial oxidants that efficiently kill pathogens. The respiratory burst also can have destructive consequences, as ROS are well known to support chronic inflammation and aberrant autoimmune responses. Interestingly, ROS perform conflicting roles in the tumor microenvironment; ROS and derived cytotoxic products can destroy cancer cells but also suppress important tumor-fighting functions of T cells or natural killer cells, or yield mutagenized proteins that can promote tumorigenesis or support tumor cell growth. Moreover, high numbers of neutrophils or macrophages in tumors are associated with poor prognosis. Therefore, accurate and quantitative assays to assess the respiratory burst are an important tool for measuring ROS production by neutrophils or cells of the monocyte/macrophage system, each recently identified in the tumor microenvironment. Described are methods to derive mouse or human models of neutrophils or macrophages, which are then used in a detailed assay to quantitatively measure ROS produced by either cell type using luminescence-enhanced reagents and a multi-well platform along with different stimulants that cause rapid ROS production.

Expression Levels of Lamin A or C Are Critical to Nuclear Maturation, Functional Responses, and Gene Expression Profiles in Differentiating Mouse Neutrophils.

Neutrophils mediate critical innate immune responses by migrating to sites of infection or inflammation, phagocytosing microorganisms, and releasing an arsenal of antimicrobial agents, including reactive oxygen species. These functions are shared by other innate immune cell types, but an interesting feature of neutrophils is their hallmark lobulated nuclei. Although why this bizarre nuclear shape forms is still being elucidated, studies of two intermediate filament proteins that associate with the nuclear envelope, lamin A and C, indicate that expression levels of these proteins govern nuclear maturation. These A-type lamins also modulate nuclear stiffness, the loss of which may be critical to the migration of not only neutrophils but also cancer cells that become prone to metastasis. We investigated whether increased expression of either lamin A or C affects neutrophil nuclear morphologic maturation, but more importantly we tested whether overexpression of either lamin also affects neutrophil functional responses, using two mouse myeloid progenitor models that can be induced toward functionally responsive neutrophil-like cells. Collectively, our results demonstrate that overexpression of either lamin A or C not only disrupts nuclear lobulation but also causes aberrant functional responses critical to innate immunity, including chemotaxis, phagocytosis, and reactive oxygen species production. Moreover, the lamin A-overexpressing cells exhibit decreased expression of a critical NADPH oxidase complex factor, gp91phox, and transcriptomic profiling demonstrated differential expression of a number of myeloid differentiation and functional pathway components. Taken together, these data demonstrate that A-type lamin expression levels modulate not only nuclear morphologic features but also gene expression changes as neutrophils mature.

Quantification of Chemotaxis or Respiratory Burst Using Ex Vivo Culture-Derived Murine Neutrophils.

Two critical functional responses of neutrophils are chemotaxis, a response driven by concentration gradients of chemokines released by infected or inflamed tissues, and production of reactive oxygen species (ROS), molecules essential to their capacity to kill pathogens. Assays to accurately test each response have been important to assess efficacies of pharmaceuticals predicted to block recruitment of neutrophils or attenuate their ROS production. Identified antagonists to neutrophil functions may help to reduce tissue damage following inflammation. Described are detailed assays to test these functions, along with steps to generate neutrophils from ex vivo-cultured murine bone marrow that produce robust responses in either assay. The first function protocol details a quantitative assay for chemotaxis that involves culture plates with dual chamber wells that separate cells from a chemokine with small pore-sized membranes. Quantitative measurements of cell numbers in the chemokine-containing chamber are performed with either fluorescence or luminescence detection reagents, which provide signals directly proportional to the numbers of migrated cells. Multiwell plates are used for rapidly testing a variety of conditions and/or chemoattractants. Described in the second function protocol is an assay to measure ROS produced by stimulated neutrophils, again using a multiwell platform for rapid, quantitative measurements of several conditions simultaneously.