Granulocytic differentiation of human NB4 promyelocytic leukemia cells induced by all-trans retinoic acid metabolites.

The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid (18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRA and all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required to differentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

Inhibitory effects of areca nut extract on expression of complement receptors and fc receptors in human neutrophils.

BACKGROUND: Chewing of areca quid increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the phagocytic activity of human neutrophils. This in vitro study investigates the effects of ANE on complement- and antibody-opsonized phagocytosis by neutrophils. Expression of complement receptors, Fc receptors, and F-actin in ANE-treated neutrophils is also analyzed. METHODS: The viability of ANE-treated neutrophils was determined using the propidium iodide staining method. The possible effects of ANE on the expression of complement receptors and Fc receptors were examined using an immunofluorescence staining method followed by flow cytometry and confocal laser scanning microscopy. The phagocytic activity of neutrophils against complement or immunoglobulin (Ig)G-opsonized fluorescent beads was analyzed using flow cytometry. Expression of F-actin was determined using confocal laser scanning microscopy. RESULTS: ANE significantly inhibited the production of complement receptors (CR1, CR3, and CR4) and Fc receptors (FcγRII and FcγRIII) in a concentration-dependent manner. Treatment of neutrophils with ANE significantly impaired their ability to phagocytose fluorescent beads. ANE also inhibited phagocytosis of fluorescent beads that were opsonized by complement or IgG. Moreover, expression of F-actin was inhibited after ANE treatment. CONCLUSIONS: ANE inhibits the complement- and IgG-mediated neutrophil phagocytosis that may result from reduction of the expression of complement receptors, Fc receptors, and F-actin formation after ANE treatment. The findings suggest that areca nut chewing may jeopardize the defensive functions of neutrophils and affect periodontal health.

Activation of leukocyte beta2 integrins by conversion from bent to extended conformations.

We used negative stain electron microscopy (EM) to examine the conformational changes in the ectodomains required for activation of the leukocyte integrins alpha(X)beta(2) and alpha(L)beta(2). They transitioned between a bent conformation and two extended conformations in which the headpiece was in either a closed or an open state. Extended integrins exhibited marked flexibility at the alpha subunit genu and between integrin epidermal growth factor-like (I-EGF) domains 1 and 2. A clasp to mimic juxtamembrane association between the integrin alpha and beta subunits stabilized the bent conformation strongly for alpha(X)beta(2) and less so for alpha(L)beta(2). A small molecule allosteric antagonist induced the extended, open headpiece conformation. A Fab known to activate beta(2) integrins on leukocytes induced extension, and a Fab reporter of activation bound only after extension had been induced. The results establish an intimate relationship between extension of beta(2) integrins and their activation in immune responses and leukocyte trafficking.

AP-1 regulates the basal and developmentally induced transcription of the CD11c leukocyte integrin gene.

The p150,95 integrin (CD11c/CD18) mediates leukocyte/endothelium interactions during inflammatory reactions and certain CTL-target interactions, and is also a receptor for fibrinogen, LPS, and the complement component iC3b. CD11c/CD18 is expressed primarily on cells of the myeloid lineage and activated B lymphocytes, and is an important diagnostic marker for hairy cell leukemia. To identify the transcription factors and cis-acting elements involved in the regulated expression of CD11c/CD18 during myeloid cell differentiation and B lymphocyte activation, we have performed structural and functional analysis on the CD11c gene promoter. Electrophoretic mobility shift assays identified an AP-1 binding site (AP1-60) within the proximal promoter region and evidenced differences in the pattern of the Fos family members bound to the AP1-60 element in undifferentiated and differentiated myeloid cells, as well as between B lineage-derived cells. The involvement of the AP1-60 element in DNA-protein interactions was confirmed by means of in vivo footprinting experiments, and its functionality was demonstrated by trans activation of the CD11c promoter by c-Jun. Site-directed mutagenesis of AP1-60 greatly reduced the basal CD11c promoter activity in myeloid and B cells. Furthermore, mutations at AP1-60 inhibited the induction of the CD11c promoter activity during the PMA-triggered U937 cell differentiation, pointing out a key role for the AP-1 transcription factor complex in both the basal and the developmentally regulated expression of the p150,95 leukocyte integrin. The involvement of AP-1 in the transcription of the CD11c gene raises the possibility of altering leukocyte integrin expression by pharmacologic means and will greatly contribute to the characterization of the intracellular signals controlling the expression of leukocyte adhesion molecules.

Enhancement of peritoneal leukocyte function by granulocyte colony-stimulating factor in rats with abdominal sepsis.

OBJECTIVE: To investigate the therapeutic effects of granulocyte colony-stimulating factor (G-CSF) on the functional activities of circulating and peritoneal neutrophils during intra-abdominal sepsis. DESIGN: Placebo, controlled study, using a rat model of intra-abdominal sepsis. SETTING: Animal research facility. SUBJECTS: Male specific pathogen-free Sprague-Dawley rats. INTERVENTIONS: Abdominal sepsis was produced in rats by cecal ligation and puncture. The control animals received a sham operation. G-CSF (subcutaneous injection at 50 microg/kg) or vehicle (100 microL of 5% dextrose) treatment was initiated at 1 hr after cecal ligation and puncture or sham operation and repeated at 12-hr intervals thereafter. MEASUREMENTS AND MAIN RESULTS: Six hours after cecal ligation and puncture, CD11b/c and CD18 expression on circulating neutrophils was significantly up-regulated when compared with those in the sham operated control animals. Peritoneal neutrophils exhibited a further up-regulation of these adhesion molecules than did the circulating neutrophils. A sustained up-regulation of CD11b/c and CD18 was found in peritoneal neutrophils even at 24 hrs after cecal ligation and puncture. G-CSF treatment increased CD11b/c expression on circulating neutrophils in 6-hr sham-operated rats, but did not further up-regulate CD11b/c or CD18 expression on circulating or peritoneal neutrophils in cecal ligation and puncture rats. Phagocytic activities of circulating neutrophils assessed by uptake of fluorescent latex microspheres were lower in 24-hr cecal ligation and puncture rats when compared with the sham-operated controls. G-CSF treatment prevented this inhibition. Furthermore, G-CSF enhanced the phagocytic activities of peritoneal neutrophils in both 6- and 24-hr cecal ligation and puncture rats when compared with those of the vehicle-treated animals. Spontaneous hydrogen peroxide generation by circulating neutrophils was increased in 6-hr cecal ligation and puncture rats, but not in 24-hr cecal ligation and puncture rats. Peritoneal neutrophils exhibited an inhibition of phorbol myristate acetate-stimulated hydrogen peroxide generation. G-CSF treatment did not up-regulate neutrophil hydrogen peroxide generation. CONCLUSIONS: Circulating and peritoneal neutrophils exhibit marked polymorphism in their functional activities during the host response to abdominal sepsis. G-CSF treatment significantly enhanced the phagocytic function of both circulating and peritoneal neutrophils which may be one mechanism underlying its protective effect in abdominal sepsis.

The neuronal glycoprotein telencephalin is a cellular ligand for the CD11a/CD18 leukocyte integrin.

Many leukocyte functions depend on interactions between the leukocyte-specific beta2 integrins CD11/CD18 and their ligands, the intercellular adhesion molecules (ICAMs). Telencephalin (TLN) is a novel member of the Ig superfamily expressed in the central nervous system. The NH2-terminal five Ig-like domains of TLN show the highest homology with the Ig domains of ICAM-1, ICAM-2, ICAM-3, and LW (ICAM-4), the known cellular ligands for CD11a/CD18. Here, we demonstrate that TLN interacts with CD11a/CD18. Peripheral blood T cells, Jurkat T cells, and B lymphoblastoid cells bound to immunopurified recombinant human TLN proteins. This adhesion was through CD11a/CD18 and was significantly inhibited by an Ab to CD11a/CD18. Reciprocally, TLN-transfected L cells also bound to purified CD11a/CD18. Recombinant TLN proteins comprising either the first five Ig domains (TLN(1-5)) or the entire extracellular portion (TLN(1-9)) showed binding to CD11a/CD18. We conclude that TLN is a novel neuronal cell adhesion molecule that may be important in integrin-mediated cell-cell interactions in the central nervous system, and that the CD11a/CD18-dependent recognition site of human TLN is located within the NH2-terminal five domains of this molecule.