Ana Cristina G. Grodzki

FcεRI -INDUCED ACTIVATION BY LOW ANTIGEN CONCENTRATIONS RESULTS IN NUCLEAR SIGNALS IN THE ABSENCE OF DEGRANULATION

High affinity IgE receptor (FcvarepsilonRI)-induced activation of mast cells results in degranulation and generation of leukotrienes and cytokines. FcvarepsilonRI-induced mast cell activation was analyzed at a single cell basis using a rat basophilic leukemia (RBL-2H3) cell line transfected with a reporter plasmid containing three tandem NFAT (nuclear factor of activated T cells) binding sites fused to enhanced green fluorescent protein (GFP). Surprisingly, with this sensitive detection system, there is activation of IgE sensitized cells at concentrations of antigen as low as 10pg/ml, which was 10-fold lower than was detected by degranulation. There were differences in signaling pathways leading to degranulation compared to NFAT-mediated gene activation. Both signaling to NFAT activation and degranulation required Syk and calcineurin. However inhibitors of the phosphatidylinositol 3-kinase pathway blocked degranulation but did not NFAT activation. The results also indicate that NFAT was activated at lower intracellular signals compared to degranulation. Therefore, FcvarepsilonRI activation can result in nuclear signals in the absence of the release of mediators.

Identification and Isolation of Rat Bone Marrow-derived Mast Cells Using the Mast Cell-specific Monoclonal Antibody AA4

SUMMARY Previous studies of mast cell maturation, structure, and function have been hampered by the lack of mast cell-specific markers. In this study, using a well-characterized mast cell-specific monoclonal antibody, MAb AA4, mast cells from rat bone marrow in various stages of maturation were isolated and characterized. The very immature mast cells, which have not been previously described, contained few granules and would not be recognized as mast cells by standard cytological methods. Pure populations of mast cells were isolated from the bone marrow using MAb AA4-conjugated magnetic beads. The same stages of maturation were observed in the isolated mast cells as were seen in the unfractionated bone marrow. All of these cells were immunopositive for the α-subunit of Fc∊RI, IgE, and c-kit, confirming their identity as mast cells. By direct counting of immunolabled cells and by flow cytometry, approximately 2.4% of the cells in the bone marrow are mast cells. Staining with toluidine blue and berberine sulfate, as well as RT-PCR of the cells, indicates that these cells are connective tissue-type mast cells. The use of immunological methods for identification of mast cell precursors should facilitate the study of these cells. (J Histochem Cytochem 49:219–228, 2001)

Mast cell repopulation of the peritoneal cavity: contribution of mast cell progenitors versus bone marrow derived committed mast cell precursors

BackgroundMast cells have recently gained new importance as immunoregulatory cells that are involved in numerous pathological processes. One result of these processes is an increase in mast cell numbers at peripheral sites. This study was undertaken to determine the mast cell response in the peritoneal cavity and bone marrow during repopulation of the peritoneal cavity in rats.ResultsTwo mast cell specific antibodies, mAb AA4 and mAb BGD6, were used to distinguish the committed mast cell precursor from more mature mast cells. The peritoneal cavity was depleted of mast cells using distilled water. Twelve hours after distilled water injection, very immature mast cells could be isolated from the blood and by 48 hours were present in the peritoneal cavity. At this same time the percentage of mast cells in mitosis increased fourfold. Mast cell depletion of the peritoneal cavity also reduced the total number of mast cells in the bone marrow, but increased the number of mast cell committed precursors.ConclusionsIn response to mast cell depletion of the peritoneal cavity, a mast cell progenitor is released into the circulation and participates in repopulation of the peritoneal cavity, while the committed mast cell precursor is retained in the bone marrow.

Immunomagnetic isolation of rat bone marrow-derived and peritoneal mast cells.

Mast cells are difficult to purify from heterogeneous cell populations and to preserve, especially for pre-embedding immunostaining at the ultrastructural level. We have developed a technique that permits the isolation of a pure population of mast cells suitable for immunocytochemical studies. A rat mast cell-specific monoclonal antibody (MAb AA4) conjugated to tosylactivated Dynabeads 450 was used to immunomagnetically separate mast cells from rat bone marrow and peritoneal cell suspensions. Approximately 85% of the mast cells were recovered in the positive population that comprised virtually pure mast cells. After microwave fixation, morphological examination showed that the cells were intact and retained their ultrastructural detail. Mast cells in all stages of maturation were immunolabeled with a panel of antibodies after immunomagnetic separation. The combination of immunomagnetic separation followed by immunostaining should prove useful for the study of mast cell maturation and for the characterization of other specific cell types that are present in tissues in only limited numbers.

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O2 and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O2) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O2 versus 5% O2 indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O2 significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O2 decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.

Differential expression of integrin subunits on adherent and nonadherent mast cells

Mast cell progenitors arise in bone marrow and then migrate to peripheral tissues where they mature. It is presumed that integrin receptors are involved in their migration and homing. In the present study, the expression of various integrin subunits was investigated in three systems of adherent and nonadherent mast cells. Mesentery mast cells, freshly isolated bone marrow-derived mast cells (BMMC) and RBL-2H3 cells grown attached to tissue culture flasks are all adherent mast cells and peritoneal mast cells, and cultured BMMC and RBL-2H3 cells grown in suspension represent nonadherent mast cell populations. Pure populations of mast cells were immunomagnetically isolated from bone marrow, mesentery and peritoneal lavage using the mast cell-specific monoclonal antibody AA4. By immunomicroscopy, we could demonstrate that all of these mast cells expressed alpha 4, alpha 5, alpha 6, beta 1 and beta 7 integrin subunits. The expression of the alpha 4 integrin subunit was 25% higher in freshly isolated mesentery mast cells and BMMC. Consistent with the results obtained by immunomicroscopy, mesentery mast cells expressed 65% more mRNA for the alpha 4 integrin subunit than peritoneal mast cells. In vitro studies were also conducted using the rat mast cell line RBL-2H3. RBL-2H3 cells grown attached to the tissue culture flasks or as suspension cultures expressed the same integrin subunits identified in bone marrow, mesenteric and peritoneal mast cells ex vivo. Similarly, the expression of alpha 4 integrin was higher in adherent cells. Therefore, alpha 4 integrins may play a critical role in the anchorage of mast cells to the extracellular matrix in bone marrow and in peripheral tissues.

Antibody Purification: Ion-Exchange Chromatography

Ion exchange chromatography techniques are the focus of this chapter and they showcase the power of this method for the purification of proteins and monoclonal antibodies. The technique is powerful and can separate biomolecules that have minor differences in their net charge, e.g., two protein molecules differing by a single charged amino acid. Given the amphoteric character of proteins the pH of the solution is important in the determination of the type of ion exchanger used. Immunoglobulins, although they can be purified by either cation or anion exchange chromatography, are most frequently purified by anion exchange with DEAE resins. The purification of the rabbit IgG fraction from serum using a DEAE column is detailed as well as the purification of IgG from ascitic fluid using FPLC, from loading to elution of the purified and concentrated protein.

Mis-trafficking of endosomal urokinase proteins triggers drug-induced glioma nonapoptotic cell death

5-Benzylglycinyl-amiloride (UCD38B) is the parent molecule of a class of anticancer small molecules that kill proliferative and nonproliferative high-grade glioma cells by programmed necrosis. UCD38B intracellularly triggers endocytosis, causing 40–50% of endosomes containing proteins of the urokinase plasminogen activator system (uPAS) to relocate to perinuclear mitochondrial regions. Endosomal “mis-trafficking” caused by UCD38B in human glioma cells corresponds to mitochondrial depolarization with the release and nuclear translocation of apoptotis-inducing factor (AIF) followed by irreversible caspase-independent cell demise. High-content quantification of immunocytochemical colocalization studies identified that UCD38B treatment increased endocytosis of the urokinase plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) into the early and late endosomes by 4- to 5-fold prior to AIF nuclear translocation and subsequent glioma demise. PAI-1 was found to comparably relocate with a subset of early and late endosomes in four different human glioma cell lines after UCD38B treatment, followed by caspase-independent, nonapoptotic cell death. Following UCD38B treatment, the receptor guidance protein LRP-1, which is required for endosomal recycling of the uPA receptor to the plasmalemma, remained abnormally associated with PAI-1 in early and late endosomes. The resultant aberrant endosomal recycling increased the total cellular content of the uPA–PAI-1 protein complex. Reversible inhibition of cellular endocytosis demonstrated that UCD38B bypasses the plasmalemmal uPAS complex and directly acts intracellularly to alter uPAS endocytotic trafficking. UCD38B represents a class of small molecules whose anticancer cytotoxicity is a consequence of causing the mis-trafficking of early and late endosomes containing uPAS cargo and leading to AIF-mediated necrotic cell death.

A Novel Carboline Derivative Inhibits Nitric Oxide Formation in Macrophages Independent of Effects on Tumor Necrosis Factor α and Interleukin-1β Expression

Neuropathic pain is a maladaptive immune response to peripheral nerve injury that causes a chronic painful condition refractory to most analgesics. Nitric oxide (NO), which is produced by nitric oxide synthases (NOSs), has been implicated as a key factor in the pathogenesis of neuropathic pain. β-Carbolines are a large group of natural and synthetic indole alkaloids, some of which block activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), a predominant transcriptional regulator of NOS expression. Here, we characterize the inhibitory effects of a novel 6-chloro-8-(glycinyl)-amino-β-carboline (8-Gly carb) on NO formation and NF-κB activation in macrophages. 8-Gly carb was significantly more potent than the NOS inhibitor NG-nitro-l-arginine methyl ester in inhibiting constitutive and inducible NO formation in primary rat macrophages. 8-Gly carb interfered with NF-κB–mediated gene expression in differentiated THP1-XBlue cells, a human NF-κB reporter macrophage cell line, but only at concentrations severalfold higher than needed to significantly inhibit NO production. 8-Gly carb also had no effect on tumor necrosis factor α (TNFα)–induced phosphorylation of the p38 mitogen-activated protein kinase in differentiated THP1 cells, and did not inhibit lipopolysaccharide- or TNFα-stimulated expression of TNFα and interleukin-1β. These data demonstrate that relative to other carbolines and pharmacologic inhibitors of NOS, 8-Gly carb exhibits a unique pharmacological profile by inhibiting constitutive and inducible NO formation independent of NF-κB activation and cytokine expression. Thus, this novel carboline derivative holds promise as a parent compound, leading to therapeutic agents that prevent the development of neuropathic pain mediated by macrophage-derived NO without interfering with cytokine expression required for neural recovery following peripheral nerve injury.

Introduction to the purification of antibodies.

Antibodies are a powerful and essential tool in scientific laboratories being used in an array of applications such as immuno-histochemistry, immunobloting, immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). The different sources for antibodies include polyclonal antisera from immunized animals and monoclonal antibodies from cells in culture or from ascites in animals. Both polyclonal and monoclonal antibodies have their advantages, and or disadvantages, but in general the production of monoclonal antibodies is more time consuming and requires tissue culture facilities and skills. The use of either monoclonal or polyclonal antibodies in some of the applications may require that the antibody is in a purified form. They can be purified by a variety of methods described in the next few chapters. The availability of commercially available kits primarily designed for the purification of IgG and IgM classes of antibodies derived from all common animal species should also be mentioned.