calcimycin and Immunologic-Deficiency-Syndromes

Polymorphonuclear granulocytes (PMN) play a key role in host defense against microbial infections. After severe trauma PMN show cellular dysfunctions including chemotactic migration, phagocytosis, and bacterial killing. In these settings the contribution of the cellular maturation stage compared to functional activities has not been investigated. Polymorphonuclear granulocytes are potent producers of lipid mediators via the 5-lipoxygenase (5-LO) pathway (leukotrienes, LTs) which exert important proinflammatory and immunoregulatory activities. We analyzed leukotriene generation from PMN-fractions (N = 23) of 15 polytrauma patients in comparison to 17 healthy donor cell fractions and correlated this lipid mediator release to the hematopoietic maturation stage of respective PMN. Polymorphonuclear granulocytes were isolated from EDTA-anticoagulated peripheral blood employing a one step procedure based on a discontinuous double Ficoll-gradient. Cells (5 x 10(6)/500 microl phosphate-buffered saline) were stimulated for 20 min at 37 degrees C with 1 microM Ca-ionophor A23187 in the presence of 1 mM Ca++ and 0.5 mM Mg++. Leukotrienes were analyzed by reversed-phase HPLC. Expression of 5-lipoxygenase (5-LO) was additionally determined by Western blot. Maturation stage of PMN was quantitated by Pappenheim-staining of cell smears. After polytrauma the generation of leukotrienes from PMN was individually diminished. Synthesis of enzymatically formed metabolites (LTB4, OH-LTB4 and COOH-LTB4) was concomitantly reduced. The decresaed leukotriene synthesis strongly correlated (r2 = 0.907, P < 0.0001) to the occurrence of immature PMN (mostly band cells). The expression of 5-lipoxygenase in PMN fractions consisting mainly of band cells was decreased. Our results provide evidence that posttraumatic granulocyte dysfunction is partly due to immature functional cell capacities.

Topics: Adolescent; Adult; Arachidonate 5-Lipoxygenase; Blotting, Western; Calcimycin; Calcium Signaling; Cell Differentiation; Cellular Senescence; Chromatography, High Pressure Liquid; Enzyme Induction; Female; Humans; Immunologic Deficiency Syndromes; Inflammation Mediators; Ionophores; Leukotriene B4; Leukotrienes; Male; Middle Aged; Multiple Trauma; Neutrophils

To investigate whether ADP-ribosylation of proteins by cholera toxin could influence B cell activation, B cells were incubated with the A subunit of cholera toxin. Ionomycin acted synergistically to induce B cell proliferation with the A subunit of cholera toxin but not with cAMP-enhancing agents or with the B subunit of cholera toxin, suggesting that the synergistic effect of the A subunit was mediated via ADP-ribosylation and not via cAMP elevations or ganglioside GM1 binding. Indeed, inhibitors of ADP-ribosylation blocked the synergistic effect. Unlike anti-Ig, B cell proliferation stimulated by LPS or by the combination of the A subunit and ionomycin was observed in protein kinase C (PKC)-depleted B cells. However, neither the A subunit nor ionomycin enhanced B cell proliferation stimulated by low dose LPS, suggesting that the A subunit plus ionomycin stimulated an activation pathway distinct from the LPS-stimulated pathway. Additionally, unlike LPS, the A subunit plus ionomycin did not stimulate B cells in vitro to secrete Ig. IL-4 acted synergistically with the A subunit to induce B cell proliferation to the same extent as it did with anti-Ig; unlike the anti-Ig plus IL-4 synergy, however, the A subunit plus IL-4-mediated synergy persisted in PKC-depleted B cells. Taken together, our data suggest that cholera toxin A subunit-catalyzed ADP-ribosylation modifies a non-Gs protein involved in the activation of B cells, either through a novel pathway or at a point distal to the activation of PKC along the anti-Ig-stimulated pathway.

Topics: Adenosine Diphosphate Ribose; Animals; B-Lymphocytes; Calcimycin; Cholera Toxin; Chromatography, High Pressure Liquid; Cyclic AMP; Drug Synergism; Female; Immunologic Deficiency Syndromes; Interleukin-4; Ionomycin; Lymphocyte Activation; Male; Mice; Mice, Inbred CBA; Mice, Inbred DBA; Signal Transduction; X Chromosome

We have identified a patient with a number of neutrophil dysfunctions. The patient was a female baby who lived for 8 months. During her life, she developed severe bacterial infections and showed omphalitis, impaired wound healing, and a pronounced leukocytosis. She was not a patient with leukocyte adhesion deficiency, because all leukocyte CD18 complex proteins were expressed at normal levels. Yet, neutrophil polarization and chemotaxis to platelet-activating factor, leukotriene B4, or formyl-methionyl-leucyl-phenylalanine (FMLP) were completely absent. We found a strong defect in actin polymerization in response to chemotactic stimuli, but only a retarded or even normal reaction with other stimuli. This indicates that the cellular dysfunctions were not due to an intrinsic defect in actin metabolism. Instead, the regulation of actin polymerization with chemotactic stimuli seemed to be defective. We concentrated on FMLP-induced responses in the patient's neutrophils. Functions dependent on activation of complement receptor type 3, such as aggregation or adherence to endothelial cells, were normally induced. Binding to serum-coated coverslips was normal in cell number; however, spreading was not observed. Exocytosis from the specific granules was readily induced. In contrast, FMLP failed to induce a respiratory burst activity or degranulation of the azurophil granules. FMLP induced a normal increase in free intracellular Ca2+, but a decreased formation of diglycerides (especially the 1-O-alkyl,2-acyl compounds). Thus, we have described a patient whose neutrophils show a severe defect in functional activation via chemotaxin receptors, resulting in a selective absence of NADPH oxidase activity, exocytosis from the azurophil granules, and actin polymerization. Our findings show that actin polymerization for neutrophil spreading and locomotion is regulated differently from that for phagocytosis. Also, the release of azurophil and specific granule contents is clearly shown to be regulated in a different way.

Topics: Actins; Antigens, CD; Calcimycin; Calcium; CD18 Antigens; CD4 Antigens; CD8 Antigens; Cell Adhesion; Cell Aggregation; Chemotaxis, Leukocyte; Cytochalasin B; Endothelium, Vascular; Female; Humans; Immunologic Deficiency Syndromes; In Vitro Techniques; Infant, Newborn; Kinetics; Leukocyte Count; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxygen Consumption; Platelet Activating Factor; Reference Values; Sepsis; Syndrome; T-Lymphocyte Subsets

In our previous report, we demonstrated that the functions of phagocytes and lymphocytes were defective in patients with systemic lupus erythematosus (SLE). In an attempt to further clarify the defective mechanisms of these cells, 25 active SLE, 10 bronchial asthma patients (BA) on corticosteroids and 25 age and sex-matched normal individuals were investigated for the expression of membraneous C3b receptors, ionophore-induced 45Ca(2+)-uptake, mitochondrial potentials and phagocytic activity of neutrophils. We found decreased expression of C3b receptors on SLE PMN in both resting (37.2 +/- 3.7% of the normal controls) and FMLP-stimulated (68.3 +/- 7.1% of the normal controls) conditions, whereas the C3b receptor expression on BA-PMN receiving long-term steroid treatment was not different from normal controls. This suggests that the defective phagocytosis of SLE PMN is in the recognition, but not in the ingestion phase because of the normal function of Ca(2+)-influx and mitochondrial activity in SLE PMN. On the other hand, hyporesponsiveness to PHA stimulation (stimulation index: 127.4 +/- 46.3 in SLE vs. 311.2 +/- 30.4 in normals, p = 0.0077) was a distinct cell-mediated immune abnormality in our SLE patients. We measured the membrane potential of individual cells using 3,3'-dihexyloxacarbocyanin and found hyperpolarization in resting SLE lymphocytes. However, the membrane polarization of SLE lymphocytes became lower than that of normal cells after PHA stimulation for 3 days. A similar tendency was also found in Na(+)-K(+)-dependent ATPase activity in SLE lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Asthma; Calcimycin; Calcium; Disease Susceptibility; Female; Humans; Immunologic Deficiency Syndromes; Infections; Lupus Erythematosus, Systemic; Lymphocyte Activation; Lymphocytes; Membrane Potentials; Middle Aged; Neutrophils; Phagocytosis; Receptors, Complement; Receptors, Complement 3b; Sodium-Potassium-Exchanging ATPase

This study was undertaken to clarify the mechanism behind the severely decreased lymphocyte proliferative response upon stimulation with mitogens and antigens seen after allogeneic bone marrow transplantation (BMT) in man. We investigated eight BMT patients and eight controls and found that the proliferative response of patient cells was reduced both when the cells were stimulated with phytohaemagglutinin (PHA) and when they were stimulated with a combination of phorbol myristate acetate (PMA), which is an activator of protein kinase C (PKC), and the calcium ionophore A23187, which irreversibly opens for calcium transport into the cell (median relative responses were 41 and 37%, respectively). However, the PHA-induced increase in the concentration of intracellular free calcium in post-BMT cells was not significantly different from the values found in control cells and the expression of interleukin 2 (IL-2) receptors (CD25) was only slightly decreased. However, the production of IL-2 was severely decreased in patient cells after stimulation with A23187/PMA (median 3541 units), although it was higher than in PHA-stimulated control cells (median 354 units). These results show that a direct activation of PKC by PMA combined with an increase in intracellular free calcium by A23187 cannot overcome the lymphocyte proliferation deficiency in cells from patients after allogeneic BMT. The data suggests that the defect is affecting the diacylglycerol pathway considerably more than the inositol triphosphate pathway.

Topics: Adolescent; Adult; Bone Marrow Transplantation; Calcimycin; Calcium; Cells, Cultured; Child; Female; Humans; Immunologic Deficiency Syndromes; Interleukin-2; Lymphocyte Activation; Male; Phytohemagglutinins; Receptors, Immunologic; Receptors, Interleukin-2; T-Lymphocytes; Tetradecanoylphorbol Acetate

We have selected 11 patients with primary immunodeficiency disorders predominantly affecting T lymphocyte function (four with ataxia-telangiectasia (AT), four with common variable immunodeficiency (CVI) and one each with Wiskott-Aldrich syndrome, hyper-IgE syndrome and combined immunodeficiency) with defective gamma interferon (IFN-gamma) production in vitro. Induction with phytohaemagglutinin showed low interleukin 2 (IL-2) production concomitant with reduced IFN-gamma titres. However the addition of 10 U/ml of rIL-2 to cultures stimulated with staphylococcal enterotoxin B or galactose oxidase failed to restore IFN-gamma production in defective cases. IFN-gamma was titrated by both bioassay and immunoradiometric assay, ruling out the possible release of inactive or altered IFN-gamma molecules. Normal levels of IFN-gamma were found in patients of patients with AT, as well as in two AT and two CVI cases, demonstrating heterogeneity of defects within these syndromes. Soluble inhibitors or cellular suppression of IFN-gamma were not observed in mixing experiments. The possibility that defective interaction between accessory cells and T lymphocytes might account for the poor response to the inducing agents was ruled out as no IFN-gamma was produced using a calcium ionophore--which bypasses this step--in seven patients with absolute IFN-gamma deficiency.

Topics: Ataxia Telangiectasia; Calcimycin; Humans; Hypergammaglobulinemia; Immunologic Deficiency Syndromes; Interferon-gamma; Interleukin-2; Phytohemagglutinins; T-Lymphocytes; Wiskott-Aldrich Syndrome

Topics: Animals; B-Lymphocytes; Calcimycin; Cytochalasin D; Cytochalasins; Drug Synergism; Female; Immunologic Deficiency Syndromes; Lymphocyte Activation; Male; Mice; Mice, Inbred A; Mice, Mutant Strains

We report a girl with severe combined immunodeficiency with functional impairment of both humoral and cellular immunity despite normal numbers of B and T lymphocytes. Immunologic studies revealed hypergammaglobulinaemia, absent migratory response by polymorphonuclear leucocytes to chemoattractants, and diminished lymphocyte proliferative responses to mitogens, antigens and allogeneic leucocytes. However, stimulation of the patient's mononuclear leucocytes with the calcium ionophore, A23187, resulted in a normal proliferative response. We therefore postulate a membrane defect as the basis for immunologic dysfunction in this child.

Topics: Calcimycin; Cell Division; Cell Membrane; Chemotaxis, Leukocyte; Female; Humans; Immunoglobulins; Immunologic Deficiency Syndromes; Infant; Leukocyte Count; Lymphocytes; Neutrophils

Topics: Adult; Agammaglobulinemia; Calcimycin; Female; Humans; Immunoglobulin D; Immunoglobulin E; Immunoglobulin G; Immunoglobulin M; Immunologic Deficiency Syndromes; In Vitro Techniques; Infant; Lymphocyte Activation; Lymphocytes; Male; Receptors, Antigen, B-Cell

Topics: Antigens; Calcimycin; Calcium; Cell Membrane Permeability; Female; Humans; Immunologic Deficiency Syndromes; Infant; Ionophores; Male; Thymus Gland