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.

Chemiluminescence response of granulocytes from elite athletes during recovery from one or two intense bouts of exercise.

In this study nine elite athletes each participated in three different 24- h trials, as follows: (1) complete bed rest (REST), (2) one bout of exercise at 1515 hours (ONE-EX), (3) two exercise bouts, one at 1100 hours and one at 1515 hours (TWO-EX-3 h), and (4) two exercise bouts, one at 0800 hours and one at 1515 hours (TWO-EX-6 h). Exercise was performed on a cycle ergometer with 10 min of warm-up and then 65 min at an exercise intensity of 75% of maximum oxygen uptake (VO(2max)). The polymorphonuclear neutrophil (PMN) counts increased consistently in response to exercise, and more in trial TWO-EX-3 h than in the two other exercise trials (P < 0.01). The respiratory burst of PMN was measured as chemiluminescence (CL), obtained with phorbol myristate (PMA) and serum-opsonised zymosan (SOZ) as stimulators. Exercise triggered the CL response for a defined number of PMN, significantly above baseline (REST) values (P < 0.05) for ONE-EX and TWO-EX-3 h, but not for TWO-EX-6 h. The strongest response was observed for TWO-EX-3 h, but the difference between exercise procedures was not significant. However, as a novel approach, a comparison was made using total oxidative potentials per litre of blood, as obtained by combining CL values and PMN numbers. TWO-EX-3 h yielded significantly higher values than the other experimental treatments. Thus, by this measure the total oxidative potential of PMN x l(-1) blood remains at a higher level with short intervals between exercise bouts (i.e. 3 h instead of 6 h), possibly due to a combined effect of cell number increase and the priming state of PMN. This may suggest that for intensive training twice a day, a recovery phase of 5-6 h is preferable. The elevation in cell number is best explained by a combined effect of catecholamines and cortisol. Growth hormone is one probable candidate as a stimulator of CL, but other molecular participants that respond to exercise may exert roles as either stimulators or inhibitors of CL.

Separation of human lymphocytes from citrated blood by density gradient (NycoPrep) centrifugation: monocyte depletion depending upon activation of membrane potassium channels.

Routine one-step centrifugation procedures (Lymphoprep = LP, Percoll) commonly used for separation of blood cells split the cells into two major fractions. After centrifugation the mononuclear cells (MNC = monocytes and lymphocytes) are located on the top of the separation fluid, whereas erythrocytes and granulocytes have sedimented to the bottom. We now show that a relatively pure lymphocyte suspension can be obtained by one-step centrifugation of citrated blood by using NycoPrep (NP = iohexol), a nonionic X-ray contrast agent. With this gradient medium also the monocytes pass to the bottom, leaving lymphocytes on the top. In parallel separations with LP, which contains Ficoll and a fully dissociated sodium salt of a contrast medium, the results were as usual, i.e. approximately 70-85% lymphocytes and 30-15% monocytes in the top fraction. The monocyte depletion with NP depended upon the use of citrated (ACD) blood and a proper balance of density and osmolality of the gradient medium, and was enhanced by 20 min preincubation with CaCl2 at room temperature. Monocyte depletion could not be obtained with LP. Under optimal conditions (density 1.075 g/ml, osmolality 280-300 mOsm/kg), the monocyte admixture amounted to approximately 1 (0-2)%, in separations with buffy coat samples. For freshly drawn blood, it was necessary to slightly modify the NP solution. The monocyte depletion was counteracted by blockers of K+ channels or by KCl in the cell suspension. Following incubation in NP of Percoll-separated cells, an enhanced release of K+ was observed. The results are interpreted as follows: NP mediates the opening of K+ channels of MNC, which leads to efflux of K+, accompanied with associated anions (Cl-). This reduces the osmolality inside the cells which therefore expel water to maintain osmotic equilibrium. In this regard it appears that monocytes are more sensitive than lymphocytes, their density therefore increasing more, so that they are able to pass the density barrier otherwise exerted by the gradient medium.

Bioactive cytidine deaminase, an inhibitor of granulocyte-macrophage colony-forming cells, is massively released in fulminant meningococcal sepsis.

Cytidine deaminase (CDD) catalyzes the hydrolytic deamination of cytidine, which thereby is converted to uridine. CDD is found in serum and different tissues, with particularly high concentrations in polymorphonuclear neutrophils (PMN). We measured the CDD levels in plasma from patients with systemic meningococcal disease. Thirty-seven patients had significantly higher plasma levels of CDD than did 29 healthy control subjects (P=.0001). CDD levels in plasma or serum increased from a median of 96 ng/mL in healthy control subjects to medians of 168 ng/mL in patients without persistent shock (n=23; P=.001) and 422 ng/mL in patients with fulminant meningococcal septicemia (n=14; P=.0001). In most patients with fulminant septicemia, CDD levels in plasma increased during the first 3-53 h after the initiation of therapy (P=.003). CDD alone had no immediate harmful effect when injected into mice during a 4-day period. CDD may modulate the stimulatory effect of colony-stimulating factors on PMN in patients.

Growth inhibition of granulocyte-macrophage colony-forming cells by human cytidine deaminase requires the catalytic function of the protein.

Previous studies have indicated that cytidine deaminase (CDD) is a potent growth inhibitor of granulocyte-macrophage colony-forming cells (GM-CFC). In this study, we have undertaken molecular cloning and purification of recombinant human CDD to elucidate the growth regulatory potential and mechanism behind the growth suppressive effect. The purified protein had a specific activity of 1.35 x 10(5) U/mg and a Km value of 30 micromol/L. In the GM-CFC assay, the recombinant protein was shown to reduce colony formation to 50% at 16 pmol/L concentration. Similarly, as was observed with CDD derived from granulocyte extract, the effect depended on the presence of thymidine (>/= 4 x 10(-5) mol/L). These results imply that CDD is an extremely potent inhibitor of GM-CFC and that no additional factor from the granulocyte extract is required for the growth inhibitory effect. Modification of CDD by truncation from the C-terminal end, or by amino acid substitution of an active site glutamate residue, eliminated both the enzyme activity and the growth regulatory potential of CDD. Furthermore, CDD from Escherichia coli was found to be even more effective than human CDD in growth suppression of GM-CFC, with 10-fold higher inhibitory activity corresponding to a 10-fold higher enzymatic activity. Taken together, these results show that the catalytic nucleoside deaminating function of the protein is essential for the growth suppressive effect of CDD. Most probably, CDD exerts growth inhibition by depleting the cytidine and deoxycytidine pool required for DNA synthesis, as addition of deoxycytidine monophosphate, which is not a substrate for CDD, neutralizes the inhibiting effect.

The effect of strenuous exercise, calorie deficiency and sleep deprivation on white blood cells, plasma immunoglobulins and cytokines.

Moderate exercise appears to stimulate the immune system, but there is good evidence that intense exercise can cause immune deficiency. In the present study the authors examined the effect of continuous physical exercise (35% of VO2 max), calorie deficiency and sleep deprivation on the immune system of young men participating in a 5-7 days military training course. There was a two-three fold increase of neutrophils from day 1, the values remained high and decreased slightly at the end of the course. Monocyte counts also increased with a pattern similar to that of neutrophils. Eosinophils decreased to 30% of control and lymphocyte numbers decreased by 30-40%. All the major subgroups (CD4 T cells, CD8 T cells, B cells, NK cells) were reduced. Neutrophil function, as tested by measuring chemotaxis, was significantly stimulated during the first days of the course, in particular in the group with the lowest calorie intake. The mitogenic response of lymphocytes to PHA and Con A was variable, ranging from stimulation during one course to no effect in another course. Serum levels of immunoglobulins decreased significantly during the course. IgG was reduced by 6-7%, IgA by 10-20% and IgM by 20-35%. The authors found no changes of interleukin 1, 2 and 4 during the course, but a (12-20%) reduction (P less than 0.01) of interleukin 6, and an increase (P less than 0.01) of granulocyte-macrophage colony stimulating factor. Altogether the results from the ranger course present a mixed-up picture. The non-specific phagocyte-related immunity was enhanced. On the other hand, the data indicate that even a moderate physical activity, around the clock, caused significant suppression of a number of parameters reflecting the status of the specific, lymphocyte-related immunity. It is noteworthy, however, that there was no significantly increased infection rate during the course or in the first 4-5 weeks thereafter.

Granulocyte chemiluminescence response to serum opsonized zymosan particles ex vivo during long-term strenuous exercise, energy and sleep deprivation in humans.

The chemiluminescence response of granulocytes to serum opsonized zymosan particles (SOZ) ex vivo was investigated during two ranger training courses lasting 7 days with continuous moderate physical activities corresponding to about 32% of maximal oxygen uptake or 35000 kJ.24 h-1, with energy deficiency (energy supply 0-4000 kJ.24 h-1), and less than 3-h sleep during the 7 days. Significant granulocytosis in combination with a lymphopenia in peripheral blood was observed during the whole course. A priming of the granulocytes for accentuated chemiluminescence response to SOZ was observed during the first days of the course with a maximal increase on day 3 in course A (+35% of control response) and on day 1 in course B (+12%). Thereafter, reduced responses to SOZ compared to control values (-28% and -21% in course A and B) were observed. In course A, a group (n = 8) receiving 5000 kJ.24 h-1 of additional energy, showed a more pronounced priming (maximum +57% versus +21% of control response) during the first days. In course B, all the cadets had 3 h of organised rest/sleep on day 5, and a second priming of the chemiluminescence response was observed on the subsequent 2 days. These data indicated that moderate, continuous, predominantly aerobic physical activities for 1-3 days around the clock primed the production of reactive oxygen species in granulocytes. This priming may be beneficial for, for example, host defence against micro-organisms, but may also contribute to inflammatory damage to normal tissues such as muscle, tendons and joints during exercise. However, when the moderate exercise continued for several more days, a down-modulation of the granulocyte response was observed. The findings of this study further support the possibility that moderate physical activity stimulates immunity, while more extreme duration of the same activities may result in a down-modulation of non-specific (and specific) immunity.

Effect of in vivo corticosterone and acute food deprivation on rat resident peritoneal cell chemiluminescence after activation ex vivo.

Adrenoglucocorticoid regulation of rat peritoneal monocyte/macrophage function was studied by exposing rats to corticosterone (CS) in the drinking water, and to fast (48 h). Production of reactive oxygen metabolites was measured by luminol amplified chemiluminescence (CL) in preparations of peritoneal cells activated by serum treated zymosan (STZ). Administration of CS which led to an increase in plasma CS from 31 (controls) to 46 ng mL-1, reduced CL (per cell) by 31%. Fast, which did not change plasma CS or ACTH, also had an inhibitory effect on CL (-25%), while the combination of CS administration and fast strongly inhibited the CL (-89%), indicating that plasma CS and fast reduced CL in a synergistic way. Similar effects on cell number were observed: CS-administration, fast and the combination reduced macrophage numbers (-13, -19.7 and -55%), while no significant effect was observed on the number of monocytes. The effect of adrenalectomy (adx) was studied in another series of experiments; adx induced no significant change in peritoneal leucocyte number or composition, while cells from adx animals had significantly higher chemiluminescence reaction than cells from sham operated animals. CS substitution in adx animals reduced CL by 30% while sham operated animals had 49% lower CL in adx. The data from adx animals also suggest that endogenous levels of CS are inhibitory for CL, but the results are not conclusive for the effect of very low doses of CS since other mechanisms than elimination of CS could prime the chemiluminescence reaction after adx. In conclusion, a moderate elevation of CS after systemic administration in vivo reduced the total number of mononuclear phagocytes in rat peritoneum, reduced the relative number of macrophages compared with monocytes, and suppressed the function of monocytes/macrophages by reducing the production of reactive oxygen molecules in activated cells. Furthermore, the effect of corticosterone was also dependent on the physiological situation, since the effects of fast and corticosterone were synergistic.

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.

Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality.

This paper briefly reviews commonly used procedures for separation of mononuclear cells (MNC) and granulocytes from human blood with X-ray contrast media as gradient material, and also presents new and modified procedures for leucocyte preparation. Standard techniques for human blood do not always yield satisfactory results with blood from other species. In general pure MNC are easily obtained (top fraction), but often the granulocyte fraction has a low purity, due to contamination with MNC that move to the bottom during centrifugation and contaminate the granulocyte suspension. Obviously the density distribution of MNC differs between species. However, the separation can be improved by fine adjustment of gradient medium osmolality. For this purpose we have used Nycodenz, a non-ionic X-ray contrast medium. A favourable property of Nycodenz solutions is that the osmolality and density can easily be varied over a broad range. The cells react promptly to a change of medium osmolality. In hypertonic medium the cells expel water, shrink, their density increases and they sediment faster, in spite of a smaller radius. Further, the cells may pass what was initially a density barrier. A hypotonic environment has the opposite effect. In the present work we were able to show that a slight change of medium osmolality clearly improved different techniques for separation of leucocyte subgroups. For instance, the Isopaque-Ficoll (IF) technique consistently yielded MNC and granulocytes of high purity with human blood. However, with blood from rabbits, rats and mice the granulocyte suspensions were contaminated by 40-60% MNC. By utilizing Nycodenz, and lowering the osmolality by 10-12 per cent (at constant density--1.077 g/ml) we obtained satisfactory separation of MNC as well as granulocytes with blood from these species. A problem in the routine separation of granulocytes (IF) is a high contamination of erythrocytes (2-5 per cell) in the granulocyte suspension. With a two-layer technique with Nycodenz solutions of different densities it was possible to separate granulocytes almost devoid of erythrocytes, after proper adjustment of osmolality. By appropriate combination of density and osmolality, Nycodenz was a suitable gradient material in other separation procedures as well, e.g. the separation of monocytes and mast cells. To facilitate the use of Nycodenz as a versatile gradient material, a computer program providing recipes for various Nycodenz solutions is included as an appendix.

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.

Human granulopoiesis in vitro--medium-dependent growth regulation by a granulocyte-derived inhibitor.

A number of reports have indicated that mature blood granulocytes produce regulators that inhibit proliferation of progenitor cells in the bone marrow. However, this concept of negative feedback of granulopoiesis is still controversial. To examine whether conflicting results may depend upon the experimental set-up, we have compared colony formation by human bone marrow cells in different growth media. Unmodified McCoy's medium, which in feeder layer cultures supports the formation of large numbers of colonies, was a poor growth medium in cultures supplied with crude or recombinant colony stimulating factor (CSF). The colony formation improved when the medium was supplemented with defined additives. In CMRL 1066 cultures, granulocyte extract (GRE) consistently caused a strong inhibition of colony formation. In contrast, with unmodified McCoy's medium, granulocyte extract enhanced colony formation in a dose-dependent manner. The enhancing effect of granulocyte extract coincided with low colony numbers in the control cultures. The stimulatory effect of granulocyte extract in McCoy's medium, switched to strong inhibition when thymidine, a component of CMRL 1066 medium, was added. The inhibitory and stimulatory activities were found in the same molecular weight fractions (30-60 kD) after gel filtration. Both modulators in granulocyte extract appeared to be independent of monocytes and T lymphocytes in the bone marrow, as shown by removal of these cells with magnetic microspheres coated with specific monoclonal antibodies. The present work shows that regulation of cell proliferation in vitro depends strongly on culture conditions, such as choice of medium. It appears that thymidine acts as co-factor for the inhibitor in granulocyte extract.

Thymidine-dependent effect of granulocyte-derived inhibitor on granulocyte-macrophage progenitor cells (GM-CFC).

We have compared the effect of granulocyte extract (GRE) on proliferation of hemopoietic cells from various sources, using two different cultures media, CMRL 1066 and McCoy 5A. GRE caused a strong (80-90%) inhibition of granulocyte-macrophage colony forming cells (GM-CFC) in cultures with medium CMRL 1066. GM-CFC in human blood and human and mouse bone marrow were equally sensitive to the inhibitor. The inhibitor had a maximal effect in the concentration range corresponding to GRE from 2 x 10(5) to 2 x 10(6) cells per 1 ml culture dish. At higher GRE concentration the inhibition was reduced. GM-CFC from human blood and mouse marrow were suppressed in cultures with McCoy's medium as well, but to a lesser extent than in CMRL 1066 cultures. On the other hand, in cultures with human bone marrow cells (BMC) and McCoy's medium, GRE had no inhibitory effect. CMRL 1066 medium contains a number of components not present in McCoy's medium. In a systematic study where these substances were added one by one to McCoy's medium we found that inhibition by GRE depended upon the presence of thymidine. At a thymidine concentration of 3 x 10(-5) mol/l GRE strongly suppressed GM-CFC in human blood and bone marrow. This thymidine concentration itself had no effect. Other nucleosides or components of the CMRL 1066 did not potentiate the suppressive effect of GRE.

Cytotoxicity and effects of T2-toxin on plasma proteins involved in coagulation, fibrinolysis and kallikrein-kinin system.

The activity of both the coagulation and fibrinolytic systems was markedly depressed 24 h after a sublethal dose of T-2 toxin. T-2 toxin was active as an anticoagulant at low doses, which did not affect the basal state of the animals. The kallikrein-kinin system was also affected by depletion of the prekallikrein, which indicates increased bradykinin levels in plasma. At the same time there was an increased activity of some clinically relevant enzymes in serum, indicating tissue injuries caused by T-2 toxin. All effects observed in this study reached their maximum within 24 h after administration, which corresponds to the time animals usually die when receiving a lethal dose. T-2 toxin does not, however, seem to affect the protease enzymes by reduced protein synthesis, because of early onset of the effects, nor does it act as a trigger itself. The effect of T-2 toxin on plasma protease enzymes is probably secondary to cytotoxic effects in the vascular endothelium.

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.

Colony inhibiting factor in mature granulocytes from normal individuals and patients with chronic myeloid leukemia.

Inhibitory activity in extract from human blood granulocytes was tested on granulocyte-macrophage colony formation in vitro. The inhibition depended on the type of serum used. With mouse BMC and FCS in the cultures, extract corresponding to 2.5 X 10(4) granulocytes/ml reduced the colony number by 35%, and extract from 2 X 10(5) cells caused maximal inhibition (80-90%). With HS and mouse BMC the colony number was reduced by only 11-12%, but stronger inhibition (55%) was observed when the serum concentration was reduced. With both types of sera the total cell number per culture plate was reduced relatively more than the colony number. Human GM-CFC were as sensitive as mouse GM-CFC, and extract from CML granulocytes inhibited less (p less than 0.01) than extract from normal cells. Biochemical studies indicated that the inhibitor is a protein with a molecular weight of 30-60,000. Lactoferrin, a putative inhibitor of CSF production, did not inhibit spontaneous or CSF-induced colony formation in these studies.

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.

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.

Release of growth promotors from mononuclear human blood cells by LPS, lectins and lithium, as tested in the mouse CFU-C assay.

Medium (MCM, 20% human serum), conditioned for 24 h by mononuclear human blood cells, had no colony-forming ability when tested in the mouse CFU-C assay. However, when combined with L-CSF, which predominantly generates macrophage colonies, MCM increased the colony number and size. Even more significant was the increased cellularity with a striking shift towards granulocyte production. Some human sera induced GIF formation without additives, but mostly LPS was required. After heat inactivation, all sera became dependent on LPS to yield an active MCM. Lithium, PHA, Con A and PMW also stimulated GIF formation, which itself depended upon protein synthesis. Heat inactivation of LPS and serum did not reduce their ability, in combination, to yield an active MCM. However, when serum and LPS were mixed before heat treatment, the ability to induce GIF production was abolished, but could be restored by adding intact LPS. This may indicate that LPS exerts its effect by combining with a serum factor yielding a heat-sensitive complex. However, even in the absence of serum, LPS had a stimulatory effect when used in large concentrations, but still the MCM was less active than MCM with serum.