Decreased expression of Fc gamma RIII mRNA in leukemic granulocytes.

Morphologically mature granulocytes from patients with chronic myeloid leukemia show significant impairment in their ability to internalize aggregated IgG, a ligand that is rapidly phagocytosed by normal human granulocytes. With a view to understand the molecular basis of this defect, normal and leukemic granulocytes were examined for the steady-state levels of mRNA for Fc gamma RIII, a membrane-associated receptor that initially binds and traps the IgG-opsonized antigens. Northern blot analyses revealed that the level of the specific mRNA in CML granulocytes was between 0.08 and 0.69 times that seen in the normal granulocytes. This could be one of the contributory factors for the observed endocytic defect in the leukemic granulocytes.

Decreased expression of both Fc gamma RII and Fc gamma RIII mRNA in leukemic granulocytes.

Morphologically mature granulocytes from patients with chronic myeloid leukemia exhibit a defect in internalization of heat-aggregated IgG. In this study, we investigate the status of the steady-state levels of the mRNA for the two receptors for IgG, Fc gamma RII and Fc gamma RIII, as a step towards understanding the molecular basis of the defect and in turn the discordant maturation of the leukemic cells. Our data show that the mRNA for both receptors is lower in the leukemic cells relative to the normal cells. This may be one of the causes for the defective endocytosis.

Fc gamma RII and Fc gamma RIII on normal leukaemic granulocytes: a flow cytometry and northern analysis.

Morphologically mature granulocytes from patients with chronic myeloid leukaemia (CML) exhibit a defect in internalization of heat-aggregated IgG. In order to investigate this defect at the molecular level and, in turn, the discordant maturation of these granulocytes, we compared the expression of Fc gamma RII and Fc gamma RIII, the two receptors for IgG on the surface of granulocytes, in normal and CML samples. Our flow cytometric data show that the number of granulocytes expressing Fc gamma RIII is lowered in CML patients to near half that in normal individuals, with a simultaneous decrease in the steady-state levels of the mRNA for Fc gamma RIII. Mean fluorescence intensity (MFI), an indicator of the number of receptors per cell, varies widely for Fc gamma RIII in normal individuals whereas it is more localized and lowered in granulocytes from CML patients. The number of granulocytes positive for Fc gamma RII is also significantly lowered in CML samples compared to the normals, which exhibit a wide variation in the number of cells positive for the receptor, even though their nRNA levels do not vary much. The CML granulocytes, in general, exhibit lowered levels of the steady-state mRNA for Fc gamma RII. The MFI for the surface expression of Fc gamma RII is only marginally different between the two cell types. Our data indicate that the morphologically homogeneous population of CML granulocytes actually consists of at least two types of cells, one which expresses the Fc receptors and one which does not.

Protein kinase C isoforms in normal and chronic myeloid leukemic neutrophils. Distinct signal for PKC alpha by immunodetection on PVDF membrane, decreased expression of PKC alpha and increased expression of PKC delta in leukemic neutrophils.

Neutrophils from patients with Chronic Myeloid Leukemia (CML) exhibit defects in several functions. They also show altered phosphorylation-dephosphorylation patterns of several proteins on stimulation with phorbol myristate acetate--a direct activator of protein kinase C (PKC). Since PKC mediates several functions of the neutrophil, in this study we investigate the PKC isoform profile and subcellular distribution in normal and CML neutrophils in an attempt to elucidate their role in CML signalling. Our results show the presence of PKC alpha, betaI, betaII and delta in both the cell types. A distinct and clear signal was obtained for PKC alpha, the isoform reported to be absent or present in very low amounts in normal neutrophils. In addition, PKC alpha was present in significantly lower levels in CML neutrophils while the PKC delta isoform was found in significantly higher amounts in the CML cytosol as compared to that in normal cells. PKC alpha, betaI, betaII and delta isoforms could not be detected in the nucleus of unstimulated normal and CML neutrophils. The altered levels of PKC alpha and delta may be one of the causes for the defects in function exhibited by the leukemic cells.

Differential phosphorylation in normal and leukemic granulocytes in response to phorbol 12-myristate 13-acetate.

Granulocytes from the peripheral blood of patients with chronic myeloid leukemia (CML) exhibit a number of functional defects. To explore the relationship of these aberrations to signal transduction, granulocytes from normal subjects and CML patients were labelled with 32Pi, stimulated with phorbol myristate acetate (PMA) and the phosphoproteins (Pps) in the unstimulated and stimulated cells analyzed by 2D-SDS-PAGE followed by autoradiography. Results show that there are six distinct reproducibly phosphorylated proteins referred to as Pp1-Pp6 identifiable in the basal patterns of the resting granulocytes. Amongst these, Pp1 and Pp5 are more intensely phosphorylated and Pp3 is very faint or absent in unstimulated CML cells, relative to the normal granulocytes. On stimulation of normal cells with PMA, Pp1, Pp3, Pp4 and Pp6 exhibit distinct patterns of phosphorylation-dephosphorylation. In the CML cells, however, Pp1 and Pp4 are unresponsive to PMA. We conclude that PKC-mediated functions involving Pp1, Pp3 and Pp4 are most probably defective in CML cells.

Circulating immune complexes in chronic myeloid leukemia: turbidimetric measurement and two-dimensional electrophoretic analysis.

A 3.75% polyethylene glycol -6000 mediated turbidimetric method was used for the estimation of levels of circulating immune complexes (CIC) in sera of normal human subjects and patients with chronic myeloid leukemia (CML). In terms of equivalents of heat-aggregated gamma globulin (eq.HAGG mg/ml serum), the mean levels of CIC were: 5.69 mg/ml (SD 4.78) in 81 normal human subjects; 22.63 mg/ml (SD 9.86) in 52 untreated CML patients; 7.61 mg/ml (SD 5.74) in 54 CML patients in remission; and 21.70 mg/ml (SD 8.15) in 18 CML patients in relapse. High CIC levels, thus, showed a significant association with the pretreatment -- and relapse -- status of the disease (p less than 0.001 and p less than 0.001, respectively). Though the levels decreased during remission, they were still significantly above the mean level in the controls (p less than 0.05). The CIC level-disease status relationship was clearly evident in the serial studies on 19 CML patients who donated serum samples prior to treatment as well as during remission. Two-dimensional electrophoretic analysis of the CIC samples revealed the presence of a polypeptide (mol. wt approx. 37,000 and pI approx. 5.8) in 10 out of 18 CIC samples from the untreated CML patients. Such a moiety was not detected in six CIC samples from normal subjects. The association of this polypeptide with CML gains support from the observation that in 5 CML patients, this moiety was present in the CIC samples obtained prior to treatment but absent in the samples subsequently obtained during remission.

Differential phosphorylation--cause for defective internalization of aggregated IgG by chronic myeloid leukemic granulocytes?

Granulocytes from the peripheral blood of patients with chronic myeloid leukemia (CML) are known to exhibit a defect in internalization of aggregated IgG relative to normal cells. As this aberration may arise due to defective transmembrane signalling, this study was undertaken to analyze alterations, if any, in protein phosphorylation between the two cell types. Normal and CML granulocytes were labeled with 32P-sodium orthophosphate and then stimulated with aggregated IgG. The phosphoproteins in the unstimulated and stimulated cells were analyzed by 2D-SDS-PAGE followed by autoradiography. The results show that there are five distinctly identifiable, reproducibly phosphorylated proteins referred to as Pp1-Pp5. In the unstimulated normal cells, Pp1 is less phosphorylated than Pp3, while in CML cells, Pp1 is more intense than Pp3. On stimulation of normal cells, with aggregated IgG, intensity of Pp1 increases while that of Pp3 decreases. In CML cells this response is reversed. We conclude that one of the causes for the defective internalization of IgG by CML granulocytes may probably be the observed differences in the phosphorylation of the proteins under study.

Chronic myeloid leukemic granulocytes exhibit decreased adhesion to fibronectin.

Extracellular matrix components are known to regulate the process of egress of granulocytes from the bone marrow and their emigration to inflammatory sites. In this study we have compared the adhesion of normal and chronic myeloid leukemic granulocytes to fibronectin. Adhesion of granulocytes from leukemic patients to all the different concentrations of fibronectin assessed was significantly lower than that of normal individuals. This observation suggests that the decreased adhesion of leukemic cells to fibronectin may be responsible for their early egress from the bone marrow and their delayed emigration to the sites of inflammation.

Actin mRNA is not lowered in chronic myeloid leukemic granulocytes.

Chronic myeloid leukemic (CML) granulocytes exhibit defects in several functions, some of which have been associated with changes in the expression of cell surface molecules, actin reorganization and lowered levels of total cellular actin. In this study, we show by northern blotting that the steady-state level of mRNA for actin is not decreased in the CML granulocyte. Our data suggest that the lowered levels of actin protein in the leukemic granulocyte may be due to altered control at the translational/post-translational step, rather than at the level of transcription/post-transcription, implicated in the regulation of expression of the surface molecules, Fc gamma RII, Fc gamma RIII and alkaline phosphatase.

Chronic myeloid leukemic granulocytes exhibit reduced and altered binding to P-selectin: modification in the CD15 antigens and sialylation.

Granulocytes from patients with chronic myeloid leukemia (CML) egress from the bone marrow before they are functionally mature and exhibit delayed emigration to sites of infection. To understand these defects, we have compared the binding of normal and CML granulocytes to the 293 cell line transfected with cDNA for P-selectin. The CML granulocytes show significantly reduced binding to P-selectin relative to normal cells. The binding of normal granulocytes to P-selectin was significantly reduced when the cells were treated with anti CD15, polyclonal anti-P-selectin, monoclonal anti-P-selectin (G1) antibodies or neuraminidase. On average, only 8% of the CML granulocyte population bound to P-selectin. The antibodies and neuraminidase were ineffective in inhibiting the binding of this population of leukemic cells. These data show that the morphologically mature CML granulocytes consist of a heterogeneous population of cells, some of which do not bind to P-selectin and others which adhere to the molecule via modified sugars.

Heterogeneity in the expression of Fc gammaRIII in morphologically mature granulocytes from patients with chronic myeloid leukemia.

Morphologically mature granulocytes from patients with chronic myeloid leukemia exhibit a defect in receptor mediated endocytosis of FITC-conjugated heat-aggregated IgG. In our earlier studies, we have shown that transcripts for Fc gammaRII and Fc gammaRIII are lowered in CML granulocytes, while no such trend was seen for CD11b, CD18 and actin, the other molecules involved in this process. We have also shown by flow cytometry that the number of granulocytes expressing the Fc receptors on their surface is lowered in CML patients. In this report, we show that the lowered steady state level of the mRNA for the Fc receptors is due to their faster degradation and not due to any defect in transcription. A study of the expression of mRNA for Fc gammaRIII in morphologically mature CML granulocytes by in situ hybridization showed that there is heterogeneity in the expression of this receptor, with some cells positive for the message and some not. These results suggest that the Fc gammaRIII transcript reaches a stable RNA pool in only some of the granulocytes, whereas in the rest of the cells, it is probably degraded after it is transcribed and is therefore not detected. The Fc gammaRIII is probably one of the first antigens to be lost from the leukemic granulocyte surface during the transition of the disease from the chronic phase to the accelerated phase and finally to the blast crisis.

Use of polyclonal anti-myeloperoxidase antibody in myeloid lineage determination.

This study reports the production of a rabbit polyclonal antibody to myeloperoxidase (MPO) and its use in ascertaining the myeloid lineage of blasts in leukaemia. Comparison of the immunocytochemical stain using the anti-MPO antibody with the routine cytochemical methodology showed that the former was more sensitive. In all subtypes of acute myeloid leukaemia (AML; 72 patients, M1-M6) greater number of MPO positive blast cells were observed by immunocytochemistry, the highest being in the promyelocytic leukaemia. It was also extremely specific for cells of the myeloid lineage as it did not react with blasts from acute lymphoblastic (50 patients) and megakaryoblastic leukaemias (1 patient). In addition, it proved most useful for the lineage determination of blasts from patients with undifferentiated acute leukaemias (AUL) and those with chronic myeloid leukaemia in blast crisis (CML-BC). Out of 8 patients of AULs, 6 were classified as acute myeloblastic leukaemia due to their reactivity to the anti-MPO antibody. Similarly, out of 12 patients of chronic myeloid leukaemia in blast crisis, blasts from 8 showed reactivity to this antibody and thus could be identified as belonging to the myeloid lineage and/or of the mixed blast crisis type.

Adhesion of neutrophils to fibronectin: role of the cd66 antigens.

Adhesion of neutrophils to substrate is initiated by receptor-ligand interactions that induce outside-in signaling. Inside-out signals and lateral interactions between surface molecules further fine tune the response. This study investigates the role of CD66 in adhesion of neutrophils to fibronectin, using domain-mapped monoclonal antibodies to CD66. Neutrophils express CD66a, CD66b, and CD66c on their surface. The neutrophil surface molecules that bind to fibronectin are the alpha(4)beta(1) and alpha(5)beta(1) integrins. Our results show that the monoclonal antibody Kat4c, which recognizes the AB domain of CD66a, b, and c and the polyclonal anti-CD66 (anti-carcinoembryonic antigen), augments neutrophil adhesion to fibronectin, while monoclonal antibodies to the individual CD66 antigens, the Fab fragment of Kat4c, and a mixture of the individual antibodies to CD66 antigens were unable to affect the adhesion. Thus heterodimerization of CD66a, b, and c is required for promoting neutrophil adhesion to fibronectin. The increased adhesion in presence of Kat4c was inhibited by antibodies to the beta(1) and beta(2) integrins. Antibody ligation of CD66 antigens causes their clustering and concomitant coclustering of the alpha(M) subunit of the beta(2) integrin, thereby activating the integrin. The sugar alpha-methyl mannoside inhibited anti-CD66-mediated clustering, indicating that a carbohydrate-lectin interaction may exist between CD66 and alpha(M) integrin. It also reduced the increased adhesion of neutrophils to fibronectin, suggesting that beta(2) integrin activation precedes beta(1) integrin activation. Further, the anti-CD66-mediated adhesion to fibronectin is accompanied by increased localization of Src family kinases (lyn and hck) to the cytoskeleton and an increase in their kinase activity. These results suggest that crosslinking of CD66a, CD66b, and CD66c promotes activation of the beta(2) integrin and in turn an alteration in the affinity of the beta(1) integrin, which enhances the adhesion of neutrophils to fibronectin.

Protein kinase C isoforms in human erythrocytes.

Erythrocytes are the most abundant cells in blood and carry out the vital function of oxygen transport. These cells lack nuclei and do not synthesise new proteins. Their cellular responses are modulated entirely by post-translational modifications in existing proteins. Phosphorylation mediated by protein kinase C (PKC) plays a significant role in red cell physiology. To date PKC alpha and zeta are the only isoforms reported to be expressed in erythrocytes. Upon activation they influence cytoskeletal integrity and erythrocyte functions. In this study we report for the first time the presence of PKC iota and PKC mu in addition to PKC alpha and zeta in human erythrocytes. All isoforms were present only in the cytosolic fraction. PKC alpha was the only isoform that translocated to the membrane upon stimulation with phorbol myristate acetate (PMA). It could thus mediate several of the reported PMA-regulated membrane modifications. Investigations on alterations in PMA-mediated phosphorylation of erythrocyte skeletal components in disorders such as chronic myeloid leukaemia can now focus on PKC alpha, which is the only isoform in erythrocytes, which translocates to the membrane on stimulation of the cells.

Isolation and characterization of membranes from Drosophila melanogaster.

As a prerequisite for examining the membranes of neurological mutants, we have undertaken to fractionate and characterize membranes derived from heads of adult and whole larvae of wild type Canton-S (C-S) Drosophila. Of particular interest to us are membrane fractions rich in putative brain membrane marker enzyme acetylcholinesterase.

Peptide mapping of proteins in gel bands after partial cleavage with acidic cyanogen bromide vapors.

A procedure is described for obtaining peptide maps from microgram quantities of protein in gel bands, after cleavage at the methionyl peptide bonds with vapors of acidic cyanogen bromide (CNBr). Absence of direct contact of the gel pieces with CNBr eliminates the need for extensive equilibration of the gel piece to remove CNBr prior to electrophoresis. The milder conditions lead to partial cleavage of the proteins, yielding larger peptides and thereby reducing the risk of peptide loss during the postelectrophoresis procedures. The "fingerprints" obtained are reproducible and independent of an eightfold change in CNBr concentration.

Plasma membrane proteins from human normal and chronic myeloid leukemic granulocytes: identification and partial characterization of the concanavalin A-binding and detergent resistant proteins.

In this work granulocytes from normal human donors and patients suffering from chronic myeloid leukemia (CML) were externally labeled with 125Iodine, using the Iodogen method. 125Iodine labeled Concanavalin A binding proteins (CBP) and detergent-resistant proteins (DRP) were isolated from the cell lysates and characterized by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D- and 2D-SDS-PAGE). Autoradiographs of the 2D-gels of DRP show seven proteins with Mr 118,000 (spot 1 a), Mr 112,000 (spot 1b), Mr 78,000-85,000 (spot 2), Mr 85,000 (spot 4), Mr 52,000 (spot 3, 3 a and 3 b). Of this set, spot 1 b, 2 and 4 are also present in the autoradiographs of 2D-gels of CBP and, hence, may be considered to be transmembrane components. Spot 4 is expressed more intensely in the normal granulocytes while spots 3 a and 3 b are mainly expressed on the leukemic granulocytes.