Expression Of Mo1 Research Articles

Mb1, a Plasma Membrane Antigen Selectively Expressed by U-937 Cells

To examine the plasma membrane characteristics of an immature monocytic cell capable of proliferation, we have developed a murine monoclonal antibody that identifies an antigen, Mb1, found on the surface of U-937. In immunofluorescence analyses, Mb1 is not expressed by peripheral blood monocytes (freshly isolated, lymphokine-activated, or cultured for seven days), neutrophils, or any other circulating element. It is also absent on human bone marrow mononuclear cells, including the CFU-GM. Among a series of malignant cells from 50 patients with acute myeloid leukemia (including 22 with monocytic or myelomonocytic leukemia), no Mb1 expression was detected. Continuous human cell lines of B or T cell origin were also negative, as were the myeloid lines HL-60 and K562. Apart from U-937, which uniformly expresses Mb1 in high antigen density, only KG-1 (a myeloblastic line) exhibits Mb1 in low antigen density. Exposure of U-937 to phorbol diester (TPA) under conditions that induce features of macrophage differentiation (including the expression of Mo1) results in a significant reduction in Mb1 expression. Mb1 expression is also reduced as a result of culture of U-937 in medium containing anti-Mb1 antibody (antigenic modulation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled immunoprecipitates, Mb1 appears to be a dimeric protein with an estimated molecular weight of 80 kd (43 kd under reducing conditions). Antigenic activity on U-937 is destroyed by treatment with trypsin or papain but is regenerated after 24 hours' culture in enzyme-free medium. Mb1 is a constituent plasma membrane protein of U-937, and its degree of expression relates to the state of cellular differentiation.

Association Between Gelatinase Release and Increased Plasma Membrane Expression of the Mo1 Glycoprotein

Mo1, a glycoprotein heterodimer (gp 155,95) that functions as an adhesion promoting molecule and as the C3bi receptor of human myeloid cells, is expressed in increased amounts in the plasma membrane after exposure of polymorphonuclear leukocytes (PMNs) to various stimuli. Previous studies have suggested that secondary granules represent an intracellular pool of Mo1 that, upon degranulation, fuse with the plasma membrane resulting in a tenfold increase in surface expression of Mo1. To determine the intracellular location of Mo1, we monitored Mo1 expression by immunofluorescence and compared it to the release of myeloperoxidase (MPO, a marker for the primary granules), vitamin B12 binding protein (B12BP, secondary granules), and gelatinase (gelatinase-containing organelles) following exposure to various stimuli. Human neutrophils stimulated with 20 mmol/L fluoride for 16 minutes exhibited a twofold increase in Mo1 expression and gelatinase release but no enhanced release of primary or secondary granular contents. In a similar fashion, incubation of cells at 37 degrees C for five minutes with 7.5 X 10(-9) to 10(-6) mol/L N-formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in significant increases in both surface Mo1 expression (three- to fivefold) and gelatinase release (five- to eightfold) without significant release of either MPO or B12BP. In addition, both the fluoride and FMLP experiments demonstrated that Mo1 up-modulation alone is not sufficient to activate superoxide (O2-) production. These data indicate that at least one intracellular storage pool of Mo1 is the gelatinase-containing organelles and that their fusion with the plasma membrane results in increased expression of Mo1 on the cell surface.

Effect of an anti-Mo1 MAb on ozone-induced airway inflammation and airway hyperresponsiveness in dogs.

Ozone inhalation causes neutrophil migration into the airway and airway hyperresponsiveness in dogs. The leukocyte adhesion molecule Mo1 (CD11b/CD18) is a heterodimeric glycoprotein the expression of which is necessary for neutrophil adhesion to endothelium. To evaluate the contribution of Mo1 to ozone-induced neutrophil influx and airway hyperresponsiveness, six dogs were treated intravenously with an Anti-Mo1 monoclonal antibody (3.75 mg/kg in normal saline) that binds to both human and canine Mo1, or the diluent alone, 1.5 h before inhaling ozone (3 ppm for 30 min), or dry air. Airway responses to doubling doses of inhaled acetylcholine (ACh) were measured before and after inhalation of ozone. Neutrophil influx was assessed by bronchoalveolar lavage (BAL) performed after the second ACh inhalation. Treatment with anti-Mo1 prevented the ozone-induced influx of neutrophils into BAL. After diluent and inhaled dry air, the neutrophil count in BAL was 1.49 +/- 1.26 (SE) x 10(4) (5.0% of total cells). After diluent and inhaled ozone, the neutrophil count increased to 7.27 +/- 3.22 (SE) x 10(4) (22.6% of total cells) (P < 0.05). After anti-Mo1 and inhaled ozone, the neutrophil count was 1.48 +/- 0.62 (SE) x 10(4) (8.5% of total cells). Treatment with anti-Mo1 also significantly reduced the number of eosinophils in BAL after ozone. Ozone-induced ACh airway hyperresponsiveness was not prevented by treatment with anti-Mo1. These results indicate that expression of Mo1 is necessary for ozone-induced neutrophil migration into the airway lumen.

Evidence for the involvement of tyrosine kinases in the locomotory responses of human neutrophils

Erbstatin, a recently described inhibitor of tyrosine kinases, has been used to examine the potential role of tyrosine phosphorylation in human neutrophil locomotion. Preincubation of human neutrophils with erbstatin inhibited both spontaneous and directed migration induced by chemotactic factors such as formylmethionylleucylphenylalanine (fMet-Leu-Phe) and leukotriene B4. The decreased migratory responses were correlated with an inhibition of adherence of neutrophils to serum-coated surfaces. Erbstatin did not, however, affect the adherence of human neutrophils to uncoated surfaces. These results indicated that the inhibitory effects of erbstatin were specific and not due to a generalized alteration of the surface of human neutrophils. To elucidate the mechanism of the inhibitory effect of erbstatin on adherence properties, the expression of the leukocyte integrin Mo1 was studied. Erbstatin induced a small but significant increase in the expression of Mo1, but decreased the stimulation of the expression of Mo1 elicited by fMet-Leu-Phe. These results suggest that mechanisms in addition to alteration of the number of surface integrins are involved in the inhibition of neutrophil adherence and locomotion by erbstatin.

Pertussis Toxin Selectively Interferes With the Responses of the HL-60 Human Promyelocytic Cell Line to Dimethylsulfoxide

AbstractThe effects of pertussis toxin (PT) on the growth and dimethylsulfoxide (Me2SO4)-induced differentiation of the HL-60 human promyelocytic leukemia cell line were tested. Cell growth was quantified by direct cell counts. Cell differentiation was estimated by measuring the expression of myeloid-specific cell-surface antigens (Mo-1 and fMet-Leu-Phe [fMLP] receptors), the ability of the cells to produce superoxide anions on stimulation with fMLP, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA), and by monitoring the level of expression of messenger RNA (mRIMA) for tumor necrosis factor α (TNFα). By itself, PT did not affect the proliferation of HL-60 cells in serum-containing medium. In contrast, PT (but not its B-oligomer) dose-dependently inhibited the Me2SO4-induced expression of Mo-1, fMLP receptors, and the oxidative responses to the chemotactic factor and to A23187, but not to PMA. The addition of Me2SO4 induced a significant increase in the steady-state levels of TNFα mRNA, and this effect was strongly inhibited by PT. Finally, the bacterial toxin did not reverse the block of cell division that follows the addition of Me2SO4. These results provide evidence for the involvement of a PT substrate (presumably a guanine nucleotide-binding protein) in the regulation of the maturation of the excitation-response coupling sequence in human myeloid cell precursors and show that the regulation of cell division and maturation of HL-60 cells are under distinct sets of control mechanisms.

Pertussis toxin selectively interferes with the responses of the HL-60 human promyelocytic cell line to dimethylsulfoxide

The effects of pertussis toxin (PT) on the growth and dimethylsulfoxide (Me2SO4)-induced differentiation of the HL-60 human promyelocytic leukemia cell line were tested. Cell growth was quantified by direct cell counts. Cell differentiation was estimated by measuring the expression of myeloid-specific cell-surface antigens (Mo-1 and fMet-Leu- Phe [fMLP] receptors), the ability of the cells to produce superoxide anions on stimulation with fMLP, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA), and by monitoring the level of expression of messenger RNA (mRNA) for tumor necrosis factor alpha (TNF alpha). By itself, PT did not affect the proliferation of HL-60 cells in serum-containing medium. In contrast, PT (but not its B-oligomer) dose-dependently inhibited the Me2SO4-induced expression of Mo-1, fMLP receptors, and the oxidative responses to the chemotactic factor and to A23187, but not to PMA. The addition of Me2SO4 induced a significant increase in the steady-state levels of TNF alpha mRNA, and this effect was strongly inhibited by PT. Finally, the bacterial toxin did not reverse the block of cell division that follows the addition of Me2SO4. These results provide evidence for the involvement of a PT substrate (presumably a guanine nucleotide-binding protein) in the regulation of the maturation of the excitation-response coupling sequence in human myeloid cell precursors and show that the regulation of cell division and maturation of HL-60 cells are under distinct sets of control mechanisms.

Suppression of interleukin-1 beta and LDL scavenger receptor expression in macrophages by a selective protein kinase C inhibitor.

A human monocytic cell line, THP-1, stimulated with 40 nM phorbol myristate acetate (PMA), differentiated to macrophage-like cells, and exhibited increased expression and release of interleukin-1 beta and expression of acetylated low density lipoprotein (ac-LDL) receptors. A selective inhibitor, MDL 29,152 (4-propyl-5-(4-quinolinyl)-2(3H)-oxazolone) was used to show that this induction required activation of protein kinase C. MDL 29,152 acts in the catalytic domain of protein kinase C and is at least 200-fold selective for protein kinase C over cAMP-dependent protein kinase in THP-1 cells. MDL 29,152 (50 microM) reduced levels of interleukin-1 beta mRNA in PMA-stimulated cells by 76% and eliminated detectable interleukin-1 beta in the media. Flow cytometric analysis showed that 48 h after THP-1 activation, approximately 50% of the cells expressed ac-LDL receptors, while in the presence of 100 microM MDL 29,152, less than 5% of the cells expressed receptors. The relationship between THP-1 differentiation and protein kinase C activation was determined by following the expression of the cell surface antigen MO-1. Expression of MO-1 antigen increases as monocytes differentiate to macrophages. After 48 h of phorbol activation, 90% of the THP-1 population was MO-1-positive; less than 16% of the population was MO-1-positive when 100 microM MDL 29,152 was present. By dual analysis, it was found that within the differentiated, MO-1-positive population, only approximately 50% of the cells also expressed ac-LDL receptors. Based on these findings, we conclude that protein kinase C promotes processes important in THP-1 activation and differentiation to macrophage-like cells including interleukin-1 beta expression and secretion, ac-LDL receptor and MO-1 expression.

GM-CSF Primes and Modulates Neonatal PMN Motility

Neonatal polymorphonuclear leukocytes (PMNs) are deficient in the expression of the adherence protein C3bi (Mo1), and are associated with reduced physiological inflammatory responses. We evaluated the priming and direct stimulating effect of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) on newborn PMN expression of C3bi (Mo1), PMN adherence and PMN aggregation. Cord PMNs were incubated with rhGM-CSF (Amgen 4 x 10(7) U/mg) for 0-15 min, and C3bi surface receptor expression measured by immunofluorescence with CD11b, adherence with nylon wool, and aggregation using a Payton aggregometer. RhGM-CSF (15 min) significantly induced Mo1 expression: 250 pM/L 129.4 +/- 5.3% of C (p less than 0.001) 500 pM/L 141.5 +/- 4.1% (p less than 0.001), 1,000 pM/L 150.2 +/- 1.3% (p less than 0.0001). RhGM-CSF (1,000 pM/L x 5 min) followed by A23187 also primed newborn PMNs for increased Mo1 expression 122 +/- 4.5% of C (p less than 0.001). Additionally, rhGM-CSF (10 min) induced significant PMN adherence 50 pM/L 117.9 +/- 8.3% and 100 pM/L 131.5 +/- 5.7% of C (p less than 0.04). RhGM-CSF additionally primed newborn PMNs (100 pM/L) for increased adherence following A23187 (107.9 +/- 0.6% of C), p less than 0.02. Lastly, rhGM-CSF primed newborn PMNs for increased aggregation following FMLP: 100 pM/L, 15 min, 138.1 +/- 14.1%, p less than 0.0001. Co-incubating murine-antihuman GM-CSF AB 100 micrograms/ml neutralized 86.8 +/- 7.0% of newborn PMN Mo1 up-regulation. These studies demonstrate that rhGM-CSF primes and directly stimulates newborn PMNs for increased in vitro expression of Mo1, adherence, and aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

No Relationship in the Expression of Mo-1 on Monocytes and GP II b-III a on Platelets in Glanzmann's Thrombasthenia

No Relationship in the Expression of Mo-1 on Monocytes and GP II b-III a on Platelets in Glanzmann's Thrombasthenia

Human recombinant granulocyte-macrophage colony-stimulating factor increases cell-to-cell adhesion and surface expression of adhesion-promoting surface glycoproteins on mature granulocytes.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit migration of mature granulocytes and to enhance their antibody-dependent cellular cytotoxicity. We found that human recombinant GM-CSF also enhanced granulocyte-granulocyte adhesion and increased by two- to threefold the surface expression of Mo1 and LeuM5 (P150, 95), two members of a family of leukocyte adhesion molecules (Leu-CAM). Increased Mo1 surface expression occurred within 15 min at 37 degrees C and was maximal at the migration inhibitory concentration of 500 pM. One-half maximal rise in the expression of Mo1 on the cell surface occurred at 5 pM. The chemotactic peptide f-Met-Leu-Phe produced a comparable rise in surface Mo1 with one-half maximal expression occurring at 7 nM. Both GM-CSF and f-Met-Leu-Phe produced optimal granulocyte-granulocyte adhesion at 500 pM and 100 nM, respectively. This adhesion-promoting effect induced by either stimulus was inhibited by a mouse monoclonal antibody directed against Mo1 antigen. These data indicate that GM-CSF promotes cell-to-cell adhesion, presumably through enhanced expression of leukocyte adhesion molecules. This mechanism may explain, in part, the known effects of GM-CSF on the function of mature granulocytes.

Increased expression of an adhesion-promoting surface glycoprotein in the granulocytopenia of hemodialysis.

To identify the mechanisms accounting for hemodialysis-induced granulocytopenia, we undertook quantitative kinetic studies of a granulocyte-adhesion-promoting surface glycoprotein (Mo1). In eight patients undergoing maintenance hemodialysis, there was a fivefold increase in the mean cell-surface expression of Mo1 within 15 minutes after the start of dialysis with a new cuprophane membrane. The peak increase in surface Mo1 coincided with the maximal drop in neutrophil count and with the peak rise in the plasma levels of the complement-activation products C5a desArg and C3a desArg. During dialysis on a membrane being reused for the fifth time, no significant complement activation, no increase in Mo1 expression, and no change in neutrophil count were seen. C5a desArg (but not C3a desArg) induced a comparable increase in Mo1 expression on normal granulocytes in vitro at concentrations similar to those measured in vivo. Chemotactic peptide-induced granulocyte aggregation (a reflection of increased cell-to-cell adhesiveness) was specifically blocked by mouse monoclonal antibodies to Mo1 in vitro. These data suggest that the increased expression of Mo1 on granulocytes in vivo is in part mediated by C5a (and C5a desArg). The quantitative increase in granulocyte-surface Mo1 may provide a mechanism for initiating leukoaggregation, sequestration of granulocytes, and neutropenia during hemodialysis.

P150/95, Third member of the LFA-1/CR3 polypeptide family identified by anti-Leu M5 monoclonal antibody.

Monoclonal antibody (mAb) anti-Leu M5 reacts with a two-chain molecule composed of a 150-kDa alpha subunit noncovalently associated with a 95-kDa beta subunit and probably is specific for an epitope on the 150-kDa alpha chain. This p150/95 antigen is the third member of a family of polypeptides sharing a common 95-kDa beta chain, which includes the lymphocyte function-associated antigen LFA-1 (p177/95) and complement receptor CR3 (Mo1/MAC-1/OKM1; p165/95) antigens. Sequential immunoprecipitation with anti-p95 beta chain mAb specifically removed the antigens detected by anti-LFA-1, anti-CR3 and anti-Leu M5 mAb. Certain patients with recurrent bacterial infections are genetically deficient in expression of the LFA-1 and Mo1 antigens, and have impaired granulocyte function. Granulocytes from a patient with this disease also failed to react with anti-Leu M5. Stimulation of normal granulocytes with f-Met-Leu-Phe, C5a-desArg, or calcium ionophore resulted in increased expression of Mo1 and Leu M5 antigens on the cell surface, but did not significantly increase expression of LFA-1 antigen. In functional assays, anti-Leu M5 did not inhibit T cell-mediated or natural killer cell-mediated cytotoxicity. In addition, anti-Leu M5 neither inhibited the binding of complement-coated particles to CR1 or CR3 nor did it affect the binding of EC3dg to neutrophils (CR4). These studies clearly indicate that the p150/95 antigen recognized by the anti-Leu M5 antibody is a structurally distinct member of the LFA-1/CR3 family.