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.

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.

Characterization of neutrophils and macrophages from ex vivo-cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry.

Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis.

Microwave-assisted synthesis and characterization of hydrophilically functionalized polygalacturonic acid.

Microwave-assisted synthesis of a new class of polymeric surfactants based on polygalacturonic acid (PGA) is presented. PGA is water-insoluble and not surface-active under normal conditions. Single-step hydrophilic modification of PGA with taurine (2-aminoethane sulfonic acid) renders it surface-active. The modification can be carried out either using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCl) as a coupling agent or using microwave irradiation without a catalyst. Microwave irradiation significantly shortens reaction times and eliminates the need for a coupling agent. In all cases, functionalization was confirmed using 1H NMR, FTIR spectroscopy and elemental analysis. PGA-SO3 exhibits surface-active properties comparable to commercial surfactants, Triton X-100 and sodium lauryl sulfate. Detailed cytotoxicity evaluation performed using human dermal fibroblast (HDF) and human leukemic (HL-60) cell lines indicate that PGA-SO3 is not toxic even at 20 fold higher concentrations. These polymeric surfactants synthesized from PGA with no demonstrable cytotoxicity have the potential for serving as 'greener' alternatives to common petrochemical-based surfactants.

Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation.

Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted E-twenty-six (ETS) factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils.