Eosinophil priming by cytokines: from cellular signal to in vivo modulation.

Eosinophils play an important role in the effector phase of allergic inflammation. This review will focus on the conversion of the unprimed eosinophil phenotype in the peripheral blood of normal individuals to the primed phenotype found in the peripheral blood and tissues of allergic patients, a phenomenon called priming. Recent data on the signals initiated after cytokine receptor activation on eosinophils will be reviewed.

Mechanisms involved in eosinophil migration. Platelet-activating factor-induced chemotaxis and interleukin-5-induced chemokinesis are mediated by different signals.

Eosinophils play an important role in the pathogenesis of allergic diseases such as allergic asthma. Eosinophil migration in vitro can be divided into directed migration, or chemotaxis, and random migration, or chemokinesis. Here, we studied intracellular signals involved in eosinophil migration in vitro induced by platelet-activating factor (PAF) and interleukin-5 (IL-5), applying a Boyden chamber assay. Migration induced by PAF (10(-11)-10(-6) M) largely consisted of chemotaxis with some chemokinesis, whereas IL-5 (10(-12)-10(-8) M) induced chemokinesis only. Eosinophils were depleted from intracellular and extracellular Ca2+ to study the role of Ca2+ as a second messenger. Ca2+ depletion did not change PAF-induced chemotaxis, however, IL-5-induced chemokinesis was inhibited. Interestingly, PAF, but not IL-5, induced changes in [Ca2+]i. This rise originated mainly from internal stores. Inhibition of protein kinase A by H-89 and protein kinase C by GF 109203X had no effect on both forms of eosinophil migration. Addition of the protein kinase inhibitor staurosporine significantly inhibited IL-5-induced chemokinesis. Inhibition of tyrosine kinases by herbimycin A completely blocked IL-5-induced chemokinesis. PAF and IL-5-induced actin polymerization was studied to compare migratory responses with a migration-associated intracellular response. Ca2+ depletion significantly enhanced PAF-induced (10(-8) M) actin polymerization, whereas IL-5-induced actin polymerization was not influenced. Addition of staurosporine led to an increase in F-actin. Subsequent addition of PAF or IL-5 resulted in an additive increase in F-actin content. In summary, both forms of eosinophil migration are protein kinase A and protein kinase C independent. In contrast to PAF-induced chemotaxis, Il-5-induced chemokinesis was found to be completely Ca2+ and tyrosine kinase dependent.

Effects of nedocromil sodium on in vitro induced migration, activation, and mediator release from human granulocytes.

Using the allergen-induced late-phase asthmatic reaction as a working model, we studied the activity of certain inflammatory cells and their reaction to nedocromil sodium. The processes that were examined in vitro included the following: the chemotaxis of purified neutrophils and eosinophils, the early steps of neutrophil and eosinophil activation, and the release of mediators from these cells. Nedocromil sodium strongly inhibited neutrophil mobilization caused by four chemotactic factors (zymosan activated serum, N-formyl-methionyl-leucyl-phenylalanine platelet-activating factor [PAF], and leukotriene B4 [LTB4] and eosinophil mobilization caused by two factors (PAF and LTB4). In vitro treatment of eosinophils from normal subjects with picomolar concentrations of interleukin-3, interleukin-5, or granulocyte-macrophage colony stimulating factor increased the chemotactic responsiveness toward PAF and LTB4 and induced a chemotactic responsiveness toward N-formyl-methionyl-leucyl-phenylalanine and neutrophil activating factor/interleukin-8. The zymosan activated serum-induced chemotactic responsiveness remained unaltered. Nedocromil sodium inhibited the cytokine-primed chemotactic responsiveness to the various chemotaxins, not the influence of the cytokines on the cells. Activation of granulocytes, as measured by Ca2+ influx, was not inhibited by nedocromil sodium. Mediator formation in eosinophils was modified only slightly. These results suggest that inhibiting the mobilization of inflammatory cells in the lung tissue may be an important action of nedocromil sodium. Therefore these effects may be relevant to the treatment of asthma given the role of airway inflammation in this disease process.

Upregulation of formyl-peptide and interleukin-8-induced eosinophil chemotaxis in patients with allergic asthma.

BACKGROUND: The cytokine granulocyte-macrophage colony-stimulating factors, interleukin-3 and interleukin-5, are important modulators of eosinophilia and eosinophil function. In particular, eosinophil chemotaxis is very sensitive to cytokine priming. METHODS: We evaluated chemotactic responses of eosinophils from patients with allergic asthma. These cells exhibited a primed phenotype as deduced from enhanced responses toward formyl-methionyl-leucyl-phenylalanine and platelet-activating factor and a decreased responsiveness toward granulocyte-macrophage colony-stimulating factor. Bronchoprovocation of patients with allergic asthma with allergen was performed as a possible means to enhance in vivo priming. RESULTS: Indeed, eosinophils isolated 3 hours after allergen challenge exhibited a more pronounced primed phenotype, which was reflected by an induction of responsiveness towards interleukin-8. Eosinophil responses induced by platelet-activating factor, formyl-methionyl-leucyl-phenylalanine, complement fragment C5a, interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor were not significantly altered after allergen challenge. CONCLUSION: These data provide further evidence that eosinophils are already primed in the peripheral blood of individuals with allergic asthma. This is most likely due to the presence of circulating cytokines in the peripheral blood of those individuals. This in vivo priming results in selective upregulation and downregulation of responses toward various chemotaxins, which may be released in the lungs during allergic inflammation.

Inhibition of cytokine-primed eosinophil chemotaxis by nedocromil sodium.

BACKGROUND: Eosinophil influx into the lung tissue is considered to be relevant for the pathogenesis of asthma. Various chemotactic factors may be responsible for this influx. Recently it has been demonstrated that the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) are present in the circulation of individuals with allergic asthma. These cytokines have the capacity to modulate chemotactic responses of eosinophils toward platelet-activating factor, formyl-methionyl-leucyl-phenylalanine, (FMLP) and neutrophil-activating factor (NAF)/IL-8, but not toward complement fragment C5a (C5a). Here the effect of nedocromil sodium on the chemotactic response of eosinophils from allergic asthmatic individuals and from normal donors preincubated with GM-CSF or IL-3 toward FMLP, NAF/IL-8 was evaluated. RESULTS: Nedocromil sodium inhibited the chemotactic response toward FMLP and NAF/IL-8 of GM-CSF primed eosinophils approximately 60% (inhibitory concentration of 50% [IC50] approximately 1 to 10 nmol/L), whereas these responses of IL-3 primed eosinophils was completely inhibited (IC50 approximately 1 nmol/L). CONCLUSIONS: The chemotactic responses toward C5a were inhibited by nedocromil sodium at higher concentrations than were required in the priming studies (IC50 approximately 10 to 100 nmol/L). Nedocromil sodium (0.1 mumol/L) was also effective in inhibiting the chemotactic response toward FMLP (10 nmol/L) of eosinophils isolated from the circulation of patients with allergic asthma 3 hours after allergen challenge. These findings might explain in part the antiinflammatory action of nedocromil sodium.

The involvement of eosinophils in the patch test reaction to aeroallergens in atopic dermatitis: its relevance for the pathogenesis of atopic dermatitis.

Atopic dermatitis (AD) is considered a T-cell mediated disease. Activated T-cells, mainly of the CD4-subtype, are abundantly present in lesional AD skin. Although not many intact eosinophils are present, deposits of eosinophil derived major-basic-protein (MBP) and eosinophil-cationic-protein (ECP) suggest eosinophil involvement. After patch testing AD patients with aeroallergens, an eczematous reaction develops after 24-48 hr at the site of application. This patch test reaction shows macroscopic resemblance to lesional AD skin and does not take place in normal individuals, asthma and allergic rhinitis patients. Lymphocytes together with eosinophils infiltrate into the dermis 2-6 hr after allergen application. Twenty-four to forty-eight hours after patch testing, eosinophils are in an activated state since they release ECP (being EG2-positive). At this point in time eosinophils have also infiltrated the epidermis. Here they are EG2-negative. Forty-eight to seventy-two hours after patch testing the eczematous reaction decreases. This coincides with disappearance of eosinophils from both the dermis and the epidermis; then, a dendritic staining pattern can be observed in the epidermis with anti-eosinophil peroxidase. Thus, eosinophils infiltrate the dermis and epidermis after patch testing AD patients with aeroallergens and release part of their granular constituents. Recent in vitro investigations revealed that eosinophils from the circulation of AD patients react more powerfully in in vitro test systems such as chemiluminescence, chemotaxis and endothelial adherence and transmigration. It is very likely that this activated (= primed) state is caused by the influence of lymphocyte-derived cytokines like IL-3, IL-5 and GM-CSF, since activated lymphocytes in the circulation (and tissue) may release these cytokines. The primed state of the eosinophils may facilitate tissue infiltration. The subsequent activation of eosinophils within the tissue leading to mediator release and the function of these mediators need to be further elucidated. The close similarity between the cellular events after a patch test reaction to aeroallergens in AD patients and those present in lesional AD skin suggests that the patch test reaction may be a helpful in vivo model to study the pathogenesis of AD. The prominent involvement of lymphocytes and eosinophils in this reaction also suggests some similarity with late phase reactions (LPR) observed in the skin after intracutaneous allergen challenge.

Eosinophil migration in atopic dermatitis. I: Increased migratory responses to N-formyl-methionyl-leucyl-phenylalanine, neutrophil-activating factor, platelet-activating factor, and platelet factor 4.

Eosinophil granular protein deposits have been demonstrated in lesional atopic dermatitis skin. This suggests active tissue infiltration of eosinophils. To find an explanation for the tissue influx of eosinophils, eosinophil migration was studied in vitro by means of a microchemotaxis assay. Eosinophils from the circulation of patients with atopic dermatitis showed an altered capacity to respond to chemotactic stimuli in vitro compared with eosinophils from healthy donors. Eosinophils from patients with atopic dermatitis had significantly increased migratory responses toward dose ranges of N-formyl-methionyl-leucyl-phenylalanine, neutrophil-activating factor, platelet-activating factor, and platelet factor 4. Eosinophils from normal individuals did not respond to N-formyl-methionyl-leucyl-phenylalanine and neutrophil-activating factor and responded only slightly to platelet factor 4. The migratory responses toward tumor necrosis factor-alpha and complement factor C5a were identical in both groups. Interleukin-5, an eosinophil-selective cytokine, is a strong modulator of the migratory responses to these chemotaxins in eosinophils from normal donors. A migratory response toward N-formyl-methionyl-leucyl-phenylalanine and neutrophil-activating factor was induced by interleukin-5, whereas the migratory response toward platelet-activating factor and platelet factor 4 was markedly potentiated. In contrast, the response to complement fragment C5a was only slightly influenced. Our findings indicate that the increased migratory responsiveness of eosinophils from patients with atopic dermatitis to various chemotaxins reflects in vivo "priming" of eosinophils, presumably by circulating cytokines such as interleukin-5. This in vivo "priming" is not optimal because it can be further potentiated by renewed contact with interleukin-5.

Modulation of eosinophil chemotaxis by interleukin-5.

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5). We have investigated the modulatory role of IL-5 on N-formyl-methionyl-leucyl-phenylalanine (FMLP), neutrophil-activating factor (NAF/IL-8), platelet factor 4 (PF4), and cytokine-induced chemotaxis of eosinophils from normal individuals. These eosinophils show a small chemotactic response toward PF4 but not to NAF/IL-8 and FMLP. Preincubation of eosinophils with low concentrations of IL-5 caused significantly increased responses toward PF4 and induced a significant chemotactic response toward FMLP and NAF/IL-8. In marked contrast, IL-5 (or IL-3) priming of eosinophils from normal donors resulted in a strong inhibition of GM-CSF-induced chemotaxis. A similar decrease in the chemotactic response toward GM-CSF was observed in eosinophils derived from allergic asthmatic individuals. This finding suggests that the latter eosinophils may have had a prior exposure to IL-5 (or IL-3). Washing of the cells after priming did not abrogate the inhibition of the GM-CSF response. Our data indicate that at low concentrations IL-5 is an important modulator of eosinophil chemotaxis, causing selective upregulation or downregulation of chemotactic responses toward different agents.

In vivo priming of platelet-activating factor-induced eosinophil chemotaxis in allergic asthmatic individuals.

The cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 are important modulators of eosinophilia and eosinophil function. Eosinophil chemotaxis is known to be particularly sensitive for cytokine priming. In the present study, we compared chemotactic responses of eosinophils derived from peripheral blood of allergic asthmatics to responses of eosinophils from peripheral blood of healthy individuals. Eosinophils from allergic asthmatics exhibited a markedly increased sensitivity in their chemotactic response toward platelet-activating factor (PAF) compared with eosinophils from normal donors. In contrast, C5a-induced eosinophil chemotaxis between both groups was similar. This in vivo-primed phenotype could be mimicked in vitro, by preincubating eosinophils from peripheral blood of healthy individuals with picomolar concentrations of either GM-CSF, IL-3, or IL-5. The chemotactic response of eosinophils derived from the circulation of allergic asthmatic patients toward GM-CSF was significantly lower compared with the response of eosinophils of healthy individuals. Our data strongly suggest that release of cytokines may be an important in vivo priming mechanism for eosinophils in the circulation of allergic asthmatic patients. Such an in vivo priming can subsequently result in selective upregulation and downregulation of chemotactic responses toward various chemoattractants release in the lung tissue.

Early activation or "priming" of eosinophils in asthma.

Increased numbers of blood and tissue eosinophils are regularly observed in subjects suffering from bronchial asthma. The eosinophil number in the diseased organ is normally closely associated with the presence of clinical symptoms. Not only the cell number, but also the concentration of eosinophil-granule derived mediators is increased in the diseased organ. In particular toxic proteins released by the eosinophil may be responsible for the allergic inflammatory reaction and the concomitant tissue damage. Our recent investigations have shown that eosinophilic granulocytes from asthmatic individuals have the same phenotype as eosinophils from normal individuals (i.e. with respect to their density distribution pattern and surface receptor expression). In contrast, eosinophils from asthmatic individuals do possess increased metabolic activity (i.e. increased leukotriene C4 (LTC4) generating capacity and migration capacity). This increased metabolic activity is due to the presence of circulating factors, i.e. the cytokines interleukin 3 (IL-3), interleukin 5 (IL-5) and granulocyte-macrophage colony stimulating factor (GM-CSF). These cytokines are demonstrable in the circulation of asthmatic, but not normal individuals; they are synthetized by activated T-lymphocytes. This early activation, called "priming", should be the goal of future pharmacological endeavours in order to achieve more efficient treatment of asthma.

Modulation and induction of eosinophil chemotaxis by granulocyte-macrophage colony-stimulating factor and interleukin-3.

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5. We have investigated the modulatory role of GM-CSF and IL-3 on the platelet-activating factor (PAF)-, neutrophil-activating factor (NAF/IL-8)-, leukotriene B4 (LTB4)-, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-, and human complement factor C5a-induced chemotaxis of eosinophils from normal individuals. These eosinophils show a chemotactic response toward PAF, LTB4, and C5a, but not to NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of GM-CSF caused a significant increase in the response toward LTB4 and induced a significant chemotactic response toward NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of IL-3 also induced a chemotactic response toward NAF/IL-8 and FMLP, and enhanced the PAF-induced chemotaxis response toward C5a was not influenced by both cytokines. Nanomolar concentrations of GM-CSF or IL-3 caused a significant inhibition of the C5a-induced chemotaxis. The LTB4-induced chemotaxis was also significantly inhibited in case of GM-CSF. At these concentrations both GM-CSF and IL-3 acted as chemotaxins for eosinophils were washed after pretreatment with GM-CSF and IL-3 the potentiation of the chemotactic response remained, whereas the inhibitory mode of action disappeared. Our data indicate that at picomolar concentrations the cytokines GM-CSF and IL-3 can modulate eosinophil chemotaxis and at nanomolar concentrations these cytokines can act as chemotaxins for eosinophils.

Inhibition of neutrophil and eosinophil induced chemotaxis by nedocromil sodium and sodium cromoglycate.

1. Neutrophils and eosinophils infiltrate the airways in association with the allergen-induced late phase asthmatic reaction. Mobilization of these cells takes place via lipid-like and protein-like chemotactic factors. In this study platelet-activating factor (PAF), leukotriene B4 (LTB4), zymosan-activated serum (ZAS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) were used as illustrative examples of both groups. Chemotaxis was studied in human neutrophils and eosinophils. The inhibitory effects of nedocromil sodium and sodium cromoglycate were evaluated. 2. All chemotactic factors tested attracted neutrophils with the following rank order of activity: ZAS greater than PAF identical to FMLP identical to LTB4. Eosinophils were only mobilized by PAF, LTB4 and ZAS with the following rank order of activity: ZAS greater than PAF greater than LTB4. 3. Nedocromil sodium and sodium cromoglycate were equally active as the PAF antagonist BN 52021 in inhibiting the PAF-induced chemotaxis of neutrophils (IC50 approximately 10(-8) M). Both drugs were also equally active in inhibiting the chemotaxis of neutrophils induced by ZAS (IC50 approximately 10(-7)-10(-6) M), FMLP (IC50 approximately 10(-7) M) and LTB4 (IC50 approximately 10(-6) M). 4. Nedocromil sodium significantly inhibited the chemotaxis of eosinophils induced by PAF (IC50 approximately 10(-6) M) and LTB4 (IC50 approximately 10(-7) M). The inhibitory potency of BN 52021 was similar to that of nedocromil sodium on the PAF-induced chemotaxis of eosinophils. Sodium cromoglycate was incapable of eliciting significant inhibition of these chemotactic responses. However, sodium cromoglycate significantly inhibited the chemotaxis of eosinophils induced by ZAS (IC50 approximately 10(-7) M), whereas nedocromil sodium was ineffective.

Popliteal lymph node enlargement and antibody production in the mouse induced by zimeldine and related compounds with varying side chains.

Structural requirements for induction of draining lymph node responses by the antidepressant drug zimeldine ((Z)-3-(4-bromophenyl)-N,N-dimethyl-3-(3-pyridyl)allylamine) in mice were determined by comparison of its activity with that of metabolites and analogues having different side chains. Mice received 1.0 mg of the compounds into the hind footpad and the popliteal lymph nodes (PLN) were removed 7 days later to determine weight, cell number and antibody production. Compounds with a methylated (Z)-(homo)allylamine side chain induced a marked PLN weight gain in C57BL/6 mice and a significant increase of the PLN cell number and of the IgM and IgG production per 10(6) PLN cells in BALB/c mice. Moderate PLN weight increase, but no significant antibody formation was induced by the doubly demethylated zimeldine metabolite, while compounds without an aliphatic amine or having a saturated side chain lacked significant activity in all assays. The (E)-diastereomer of zimeldine induced significantly less PLN weight gain than zimeldine in C57BL/6 mice, but an equal increase of PLN cell number in BALB/c mice. IgM and IgG responses to the (E)-diastereomer were moderate and absent, respectively. The antihistaminic drug, brompheniramine, having a saturated side chain and a 2-pyridyl ring, induced less PLN weight and cell gain than zimeldine and failed to increase antibody formation. The capacity of the compounds to induce PLN responses appeared not related to their pharmacological potential to inhibit the reuptake of serotonin and noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of platelet-activating factor- and zymosan-activated serum-induced chemotaxis of human neutrophils by nedocromil sodium, BN 52021 and sodium cromoglycate.

1. Inflammatory cells such as eosinophils and neutrophils are thought to contribute actively to the pathogenesis of asthma since they infiltrate into the lung tissue. These cells are mobilized by lipid-like and protein-like chemotactic factors. As illustrative examples of both groups, platelet-activating-factor (Paf) and zymosan-activated-serum (ZAS) were used in this study. The inhibitory effects of nedocromil sodium, the Paf antagonist BN 52021 and sodium cromoglycate on Paf- and ZAS-induced neutrophil chemotaxis were evaluated. 2. All tested drugs inhibited Paf-induced neutrophil chemotaxis with approximately the same potency (IC50 approximately 1 nM). 3. Nedocromil sodium and sodium cromoglycate were equally potent in inhibiting ZAS-induced neutrophil chemotaxis (IC50 = 0.1-1 microM), whereas BN 52021 was considerably less potent (IC30 = 10 microM). 4. To find out whether the drugs tested could inhibit early events in cell activation, their capacity to inhibit Paf- and ZAS-induced cytosolic free Ca2+-mobilization was investigated. BN 52021, at a concentration of 100 microM, completely inhibited Paf-induced Ca2+-mobilization and inhibited ZAS-induced Ca2+-mobilization by about 50%. Nedocromil sodium and sodium cromoglycate were ineffective.

Vulnerability of rat and mouse brain cells to murine hepatitis virus (JHM-strain): studies in vivo and in vitro.

The pathogenicity and cell tropism of mouse hepatitis virus (MHV-JHM-strain) in the developing mouse (Balb/c) and rat (Wistar and Lewis) brain were analysed. Intracranial infection of Balb/c mice at postnatal day 5 induced a lethal encephalitis in all animals. Of Wistar rats infected at day 2 or 5 after birth, 30 to 70%, respectively, survived. The distribution of viral antigen was studied in frozen brain sections of animals that died after infection; astrocytes were found to be the major virus-infected cell type throughout the central nervous system. More than 75% of the surviving rat pups developed paralysis, but viral antigen was detected in only few brain cells and not in astrocytes. The cell tropism of MHV-JHM was examined further in virus-infected glial cell cultures derived from brains of rats or mice. In the glial cultures derived from Wistar rats, only oligodendrocytes were infected, whereas in cultures derived from mouse or Lewis rat brain viral antigen was detected in both astrocytes and oligodendrocytes. Infection of astrocytes led to the formation of syncytia and degradation of the cytoskeleton. Infected rat oligodendrocytes gradually disappeared from the cultures because of cell death. These phenomena indicate that, besides an indirect autoimmune response triggered by infected astrocytes, direct virus-induced injury to astrocytes or to oligodendrocytes can have a dominant role in the neuropathogenicity of mouse hepatitis virus. The present results underscore the importance of species and developmental stage of experimental animals in the neurotropism and pathogenicity of MHV-JHM.

Inhibitory effects of nedocromil sodium on the in vitro induced migration and leukotriene formation of human granulocytes.

Inflammatory cells, such as neutrophils and eosinophils, are thought to actively contribute to the pathogenesis of asthma since they infiltrate into the lung tissue and may be activated locally to release bronchoconstrictor mediators. In this study we provide evidence that nedocromil sodium is capable of effectively inhibiting the platelet-activating factor (PAF) and zymosan-activated serum (ZAS)-induced chemotaxis of polymorphonuclear granulocytes (PMN) [IC50 approximately 1 nmol/L and 0.1 mumol/L respectively]. The same inhibitory potency was obtained with sodium cromoglycate. Thus, nedocromil sodium may effectively inhibit the mobilisation of inflammatory cells in the lung. Furthermore, nedocromil sodium is capable of inhibiting the formation of the bronchoconstrictor mediator leukotriene-C4 (LTC4) by eosinophils in a concentration-dependent way [IC30 for A23187: 5.6 10(-5) mol/L; IC30 for opsonised zymosan (OZ): 6.3 10(-5) mol/L], whereas this drug is not capable of inhibiting leukotriene-B4 (LTB4) formation by neutrophils. These findings indicate that nedocromil sodium inhibits the release of bronchoconstrictor mediators not only from mast cells but also from eosinophils.

Cellular location of glutamine synthetase and lactate dehydrogenase in oligodendrocyte-enriched cultures from rat brain.

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.

Hydrocortisone stimulates the development of oligodendrocytes in primary glial cultures and affects glucose metabolism and lipid synthesis in these cultures.

Cultures of glial cells were prepared from the brains of one-week-old rat pups. After one day in culture, serum was omitted from the medium and replaced by a combination of growth-stimulating hormones and other factors that enhanced the percentage of oligodendrocytes in the cultures. We investigated the effects of hydrocortisone on the development of oligodendrocytes, on the activities of oligodendrocyte-specific enzymes and on glucose- and lipid-metabolism of the glial cells. Hydrocortisone greatly enhanced the survival of glial cells in culture. The development of galactocerebroside-positive cells and the specific activity of 2',3'-cyclic-nucleotide 3'-phosphodiesterase were stimulated by 50 nM hydrocortisone, whereas these effects were partly reversed at higher concentrations of the hormone. The specific activity of glycerol-3-phosphate dehydrogenase was markedly stimulated by hydrocortisone; 1 microM or higher concentrations of hydrocortisone were required for an optimal effect. The consumption of glucose and the production of lactate were lowered by hydrocortisone whereas the oxidation of [6-14C]glucose to 14CO2 was not affected. Incorporation of [35S]sulfate into sulfolipids was greatly enhanced by hydrocortisone and [14C]incorporation from [1-14C]acetate into cholesterol and fatty acids was also stimulated but to a smaller extent. These results show that hydrocortisone exerts a general trophic function on glial cells in our culture system; enhances the ratio of oligodendrocytes over astrocytes, possibly by directing bipotential progenitor cells to develop into oligodendrocytes; specifically induces glycerol-3-phosphate dehydrogenase in oligodendrocytes.