Intricacies of redoxome function demonstrated with a simple in vitro chemiluminescence method, with special reference to vitamin B12 as antioxidant.

The homeostatic control of the redox system (the redoxome) in mammalian cells depends upon a large number of interacting molecules, which tend to buffer the electronegativity of cells against oxidants or reductants. Some of these components kill - at high concentration - microbes and by-stander normal cells, elaborated by professional phagocytes. We examined whether a simple, in vitro chemiluminescence set-up, utilizing redox components from human polymorphonuclear neutrophils (PMN) and red blood cells (RBC), could clarify some unexplained workings of the redoxome. PMN or purified myeloperoxidase (MPO) triggers formation of reactive oxygen species (ROS), quantified by light emission from oxidized luminol. Both PMN and RBC can generate abundant amounts of ROS, necessitating the presence of a high-capacity redoxome to keep the cellular electronegativity within physiological limits. We obtained proof-of-principle evidence that our assay could assess redox effects, but also demonstrated the intricacies of redox reactions. Simple dose-responses were found, as for the PMN proteins S100A9 (A9) and S100A8 (A8), and the system also revealed the reducing capacity of vitamin B12 (Cbl) and lutein. However, increased concentrations of oxidants in the assay mixture could decrease the chemiluminescence. Even more remarkable, A9 and NaOCl together stimulated the MPO response, but alone they inhibited MPO chemiluminescence. Biphasic responses were also recorded for some dose-response set-ups and are tentatively explained by a 'balance hypothesis' for the redoxome.

Identification of cytidine deaminase as inhibitor of granulocyte-macrophage colony formation.

Mature human blood granulocytes produce regulatory factors that inhibit colony formation by human and murine granulocyte-macrophage colony-forming cells (GM-CFC). The inhibition of GM-CFC by granulocyte extract (GRE) was strongly enhanced by the addition of thymidine (3 to 6 x 10(-5) M for human cells) and by the presence of fetal calf serum (FCS) in the growth medium. Deoxycytidine and deoxyuridine produced effects similar to those of thymidine, but at higher concentrations (2 to 4 x 10(-4) M). It was further observed that GRE prevented the antiproliferative effects of cytosine arabinoside (Ara-C) and azadeoxycytidine, suggesting that GRE contained cytidine deaminase (CDD) activity, since CDD is known to abolish the effects of these nucleoside analogs. Accordingly, the GRE was tested for and shown to contain an enzymatic activity that converted deoxycytidine to deoxyuridine, confirming the presence of CDD activity in GRE. The GM-CFC inhibition factor was found to copurify with CDD activity during three succeeding chromatographic separations, indicating that CDD was indeed the inhibiting factor itself. This conclusion was further substantiated by gel filtration experiments demonstrating a molecular weight (MW) of approximately 50 kd, which corresponds to the MW previously published for CDD activity. Furthermore, addition of tetrahydrouridine (THU), a known specific inhibitor of CDD, abolished the suppressive effect of GRE on GM-CFC, which independently confirmed the identification of CDD as an inhibitor of GM-CFC. The growth-regulating property of CDD could be explained by depletion of deoxycytidine nucleotides necessary for DNA synthesis or by a direct effect of CDD binding to specific receptors on progenitor cells.

Monocyte-conditioned medium and interleukin 1 induce granulocyte-macrophage colony-stimulating factor production in the adherent cell layer of murine bone marrow cultures.

We have studied how production of colony-stimulating factors (CSF) can be induced in murine long-term bone marrow cultures (LTBMC). We found that the adherent cells, but not the nonadherent cells, of LTBMC synthesized and secreted large amounts of CSF upon stimulation with monocyte-conditioned medium (MCM) from the early phase of monocyte culturing. This CSF induced both granulocyte- and macrophage-containing colonies. Interleukin 1 (IL-1) also induced CSF production by the adherent cells, although not to the same extent as MCM. Medium conditioned by E-rosette-positive lymphocytes could not substitute for MCM. CSF production varied in long-term bone marrow cultures less than two weeks old, but thereafter the amount of CSF obtained was relatively independent of the age of the cultures (2-26 weeks). No correlation was found between the content of granulocyte-macrophage colony-forming cells (GM-CFC) in the nonadherent cell fraction of LTBMC and the ability of the adherent cell layer to produce CSF. These results suggest a two-stage process for CSF synthesis. Monocytes produce a factor(s) that, in a second step, leads to bioassayable levels of CSF in the supernatant of adherent cells in LTBMC.

Receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes are upregulated during prolonged strain and energy deficiency.

VIP receptors on blood mononuclear leucocytes and plasma VIP concentrations were studied during a ranger training course lasting for five days with almost continuous physical activity, and energy deficiency. The maximum binding capacity (Bmax) for the high affinity receptor increased (p less than 0.0005) from 0.71 (SEM = 0.11, N = 10) fmol/million cells to a maximum of 7.33 (SEM = 1.0) fmol/million cells on Day 4. There was no significant change in the dissociation constant (Kd) for the high affinity receptor, and no effect on Kd nor Bmax for the low affinity VIP receptor was detected. Plasma VIP concentration increased (p less than 0.0005) from 8.8 pmol/l (SEM = 0.6) to a maximum of 23.4 (SEM = 1.9) on the second day of the course. However, the highest plasma concentrations were about one order of magnitude lower than the dissociation constant (Kd) for the high affinity VIP receptor on the mononuclear leucocytes. These data indicate that heterologous upregulation of the high affinity VIP receptor on mononuclear blood cells takes place during combined strenuous physical exercise, and calorie deficiency.

Effect of VIP on the respiratory burst in human monocytes ex vivo during prolonged strain and energy deficiency.

VIP-stimulated cyclic AMP production and VIP effect on the production of reactive oxygen compounds in human monocytes activated by serum opsonized zymosan (respiratory burst) were studied during a ranger training course lasting for five days with almost continuous physical activity, and deficiency of sleep and energy. Respiratory burst was inhibited and cyclic AMP production was stimulated by VIP on all days. Maximum cyclic AMP production stimulated by VIP (0.1 microM) on the day of control was 148.6% of basal, and 255.3%, 213.8%, 218.9% and 198.7% on Days 1, 2, 3 and 5. Maximum inhibition was observed 20 min after addition of the peptide on the day of control, after 5 min on Days 1, 2 and 3, and after 10 min on Day 5. Inhibition at the 5-min time point was 33.1% on the day of control, and 34.7%, 53.6%, 53.3% and 36.2% on the different days during the training course. The observed increment in VIP effect adds to prior reported data about increased VIP secretion during the training course, and may indicate enhanced physiological significance of VIP during stress.

Binding of vasoactive intestinal polypeptide (VIP) by human blood monocytes: demonstration of specific binding sites.

Vasoactive intestinal polypeptide (VIP) interaction with a 94% pure preparation of monocytes isolated from human peripheral blood was studied by direct binding technique using 3-[125I]tyrosyl-VIP as a tracer ligand. Scatchard analysis of binding data was compatible with two classes of binding sites, one with Kd = 0.25 nM and maximal binding capacity of 16 fmol/10(6) cells, and another one with Kd = 25 nM and maximal binding capacity of 180 fmol/10(6) cells. The binding was time-, temperature-, and pH-dependent and was saturable, reversible, and specific. This study has demonstrated that human monocytes have high affinity/low capacity as well as low affinity/high capacity binding sites for VIP. No specific VIP binding was found in pure preparations of human granulocytes, platelets or erythrocytes.

Separation of lymphocytes, lymphocyte subgroups and monocytes: a review.

Several techniques are now available for isolating lymphocytes from blood and other sources, but no single technique can be considered best for all purposes. In selecting a separation procedure, it is recommended that the procedure be as simple as possible, if otherwise satisfactory. The present paper reviews the most widely used techniques, including the separation of lymphocyte subpopulations and monocytes.

Isolation of human blood monocytes with Nycodenz, a new non-ionic iodinated gradient medium.

Monocytes were separated from human blood with Nycodenz, an iodinated gradient medium. Monocytes have a lower average density than lymphocytes, but because of overlapping efficient separation cannot be achieved on the basis of density differences alone. Thus the isolation procedure was based on the assumption that the low-density fraction of lymphocytes increases its density more than monocytes by expelling water when exposed to an increased osmolarity. Thereby they might pass through a density barrier present initially, whereas the monocytes remain at the top of the gradient layer. Separation fluids with densities ranging from 1.061 to 1.096 g/ml were prepared by mixing Nycodenz with NaCl solutions of various concentrations. EDTA-blood (3 ml) or a leucocyte suspension (2-6 ml) obtained by dextran sedimentation was loaded on 3 ml of separation fluid and centrifuged for 15 min at 1900 rpm. Then the cells in the interface region were collected. At each density level it was possible to obtain an almost pure monocyte suspension (95-98%) by increasing the osmolarity. However, the higher the purity, the lower the monocyte yield. Apparently, the viability of monocytes was not affected, even when subjected to an osmolarity of 600 mosmol. For routine use, it appears that separation fluids with densities from 1.061 to 1.078 g/ml and corresponding osmolarity in the 300 to 410 mosmol range are suitable.

Isolation of lymphocytes, granulocytes and macrophages.

This paper presents the standard procedure for isolating lymphocytes and granulocytes from blood, using the Isopaque-Ficoll technique. A procedure for isolating granulocytes and macrophages from peritoneal fluid is also described.

Bone marrow cell culturing in double diffusion chambers.

A double diffusion chamber technique (DDC) has been established. The bone marrow cells (BMC) were cultured in the peritoneal cavity of mice in DDC consisting of 2 compartments separated from one another by a Millipore membrane. One chamber half contained the mouse bone marrow target cells, and the other half (regulator compartment) either medium (control), spleen cells or BMC. In the controls the BMC proliferated rapidly from day 2, and the cell yield on day 7 was reduced by only 20% when compared with single diffusion chambers. Diffusible factors from spleen cells stimulated the growth of CFU-S and CFU-C in the bone marrow, and increased the number of granulocytes and macrophages harvested in 7 day cultures. Conversely, BMC in the regulator compartment depressed granulopoiesis in the other chamber half.