Comparison of cytokine production in cultures of whole human blood and purified mononuclear cells.

In order to examine inter- and intra-individual variations in cytokine production, blood was collected from 48 healthy subjects on each of 4 occasions separated by 4 weeks. Whole blood (diluted 1:10) and mononuclear cell (MNC) cultures were stimulated for 24 h with either concanavalin A (Con A) or bacterial lipopolysaccharide (LPS) and the concentrations of IL-1alpha, IL-1beta, IL-2, TNF-alpha, IL-10 and IFN-gamma in the culture medium measured. There were highly significant inter-individual variations in the production of each of the cytokines measured. However, the level of the production of each cytokine appeared to be characteristic of an individual. There were significant correlations between production of each cytokine in whole blood and MNC cultures. It is concluded that there is significant inter-individual variation in cytokine production which is unaffected by time or by the stimulus used to elicit cytokine production, and that whole blood cultures can be used instead of MNC cultures to measure cytokine production.     

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.     

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.     

TNFalpha production in whole blood cultures from healthy individuals

Tumor necrosis factor alpha (TNFalpha) is a major mediator of inflammatory responses and also plays a prominent role in bridging the innate and adaptive phases of immunity. In the present work we attempted to study TNFalpha production in endotoxin-stimulated blood of healthy individuals, and the inter-individual variability in TNFalpha production. For this study, we used diluted whole blood stimulated with lipopolysaccharide (LPS). The levels of the pro-inflammatory cytokine TNFalpha were measured by ELISA and by the L929 cytotoxicity bioassay in 16 and 18 healthy donors, respectively. There were highly significant inter-individual variations in the induced TNFalpha production. It is worth noting that there was no difference in sensitivity between ELISA and the cytotoxicity L929 bioassay. We concluded that whole blood culture is a sensitive method to determine the pro-inflammatory cytokine production in response to endotoxin stimuli in a relevant physiologic milieu. Our data indicate that this method provides appropriate information about the state of cellular immunity of the individual.     

Estrogen regulates cytokine production and apoptosis in PMA-differentiated,macrophage-like U937 cells

We have investigated the effects of sex steroids, estradiol (E2), and testosterone (T) on the synthesis of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) in phorbol-myristate-acetate (PMA)-differentiated human monoblastic U937 cells. The ability of both hormones to modulate the viability and programmed cell death of macrophage-like PMA-differentiated U937 cells was also inspected. E2 increased TNF-alpha synthesis, whereas T had no effect on the production of this cytokine. The combination of E2 and its antagonist tamoxifen or ICI-182,789 completely abolished the induction of TNF-alpha, while combination of T and its antagonist Casodex (CSDX) did not significantly affect TNF-alpha production by U937 cells. Exposure of cells to E2 resulted in a dose-dependent decrease of IL-10 synthesis, while again T did not show any detectable effect. In addition, E2 induced a significant increase of apoptosis in macrophage-like U937 cells and this increase was inhibited by the simultaneous addition of either tamoxifen or ICI-182. In contrast, T alone or in combination with CSDX did not modify apoptotic rates of U937 cells. This evidence, taken together, suggests that estrogens, but not androgens, exert a pro-inflammatory action through the modulation of TNF-alpha and IL-10, and regulate the immune effector cells by the induction of programmed cell death.     

Stimulated cytokine production correlates in umbilical arterial and venous blood at delivery

BACKGROUND: Umbilical venous blood is easy to obtain after delivery, and thus has been commonly used in many studies for cytokine analysis. Our aim was to evaluate whether or not induced cytokine production differs after stimulation in umbilical artery and vein whole blood samples, using two different stimulation protocols. The effect of such stimulation on fetal and maternal blood was also evaluated. METHODS: Blood samples from umbilical artery (UA) and vein (UV), and from the mother were collected from 23 women after delivery at term. Concentrations of cytokines (IL-4, IFN-gamma, IL-6 and TNF-alpha) were measured in plasma and whole blood after PMA/ConA and PMA/ionomycin stimulation. RESULTS: Both in maternal and in fetal samples, cytokine concentrations in unstimulated plasma samples were lower than in stimulated samples, except for IL-4 after PMA/ConA stimulation. UA and UV showed similar, average cytokine levels after stimulation and the correlations were high (r=0.68-0.95). Cytokine concentrations were clearly higher in umbilical blood than in maternal blood after stimulation, but not in plasma. Correlations between maternal and umbilical samples after stimulation were generally low (r<0.41). IFN-gamma was not detectable in unstimulated plasma samples. The production of IL-4 and IFN-gamma was more intense after PMA/ionomycin stimulation than after PMA/ConA stimulation. INTERPRETATION OF THE RESULTS: Concentrations of the cytokines examined are similar in blood from the UA and UV. For IL-4 and IFN-gamma, the stimulant used has a significant effect on the level of cytokine expression, and interestingly, the effect is more pronounced on the fetal than on the maternal side.     

Effect of blood storage conditions on leucocyte intracellular cytokine production

Intracytoplasmic detection of leucocyte cytokines has become a powerful tool for the characterisation of cytokine-producing cells in heterogeneous cell populations, however the effect of specimen storage conditions is unknown. The aim of this study was to determine the effect of whole blood stored at room temperature (RT) or 4 degrees C, on intracellular cytokine production by T cells and monocytes. In cell cultures stored at RT or 4 degrees C for 24h, significant changes in several leucocyte cytokines/chemokines were shown compared to blood cultures stimulated at time=0. There was a significant decrease in IL-2, IL-4 and TNFalpha production by CD4+ T cells in blood cultures stored at RT but an increase in IL-2 in cultures at 4 degrees C. There was a significant decrease in TGFbeta production by CD4+ and CD8+ T cells in cultures kept at RT or 4 degrees C. There was a significant increase in MCP-1 and MCP-3 production by monocytes in blood cultures kept at RT or 4 degrees C. There was a decrease in IL-12 production by monocytes in cultures kept at 4 degrees C, whereas IL-10 production was decreased at RT and increased in cultures kept at 4 degrees C. Blood stored at 4 degrees C showed less immunomodulatory changes than blood kept at RT although overall a possible Th1 bias at 4 degrees C.     

Differential effects of copper and zinc on human peripheral blood monocyte cytokine secretion

The addition of copper and zinc salts to human peripheral blood leukocytes cultured in complete medium containing endotoxin and fetal calf serum stimulated tumor necrosis factor (TNF) secretion in a concentration-dependent manner. The secretion of interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) was inhibited by copper under the same culture conditions, while zinc stimulated IL-1 beta secretion in a concentration-dependent manner and had no effect on leukocyte IL-6 release. Both copper and zinc induced increases in TNF mRNA (54 and 14%, respectively) when compared to cells cultured in complete medium alone. In serum-free, low endotoxin medium (less than 6 pg/ml), both copper and zinc failed to stimulate either TNF or IL-1 beta secretion. Under the same conditions the addition of lipopolysaccharide (LPS), at concentrations above 0.01 micrograms/ml, induced a concentration-dependent release of both cytokines. When either copper or zinc were combined with 0.01 micrograms/ml LPS, a synergistic stimulation of TNF secretion resulted. IL-1 beta secretion, unlike TNF, was not synergistically stimulated by combining metals and LPS in serum-free medium. Combining copper and zinc with inhibitors of TNF secretion, transforming growth factor beta, prostaglandin E2, and plasma alpha-globulins, resulted in a reduction of the suppressive effects of each of these agents. This study suggests that the trace metals copper and zinc may play important and possibly distinct roles in regulating leukocyte secretion of TNF, IL-1 beta, and IL-6.     

Neisseria gonorrhoeae survives within and modulates apoptosis and inflammatory cytokine production of human macrophages

The human‐adapted organism Neisseria gonorrhoeae is the causative agent of gonorrhoea, a sexually transmitted infection. It readily colonizes the genital, rectal and nasalpharyngeal mucosa during infection. While it is well established that N. gonorrhoeae recruits and modulates the functions of polymorphonuclear leukocytes during infection, how N. gonorrhoeae interacts with macrophages present in infected tissue is not fully defined. We studied the interactions of N. gonorrhoeae with two human monocytic cell lines, THP‐1 and U937, and primary monocytes, all differentiated into macrophages. Most engulfed bacteria were killed in the phagolysosome, but a subset of bacteria was able to survive and replicate inside the macrophages suggesting that those cells may be an unexplored cellular reservoir for N. gonorrhoeae during infection. N. gonorrhoeae was able to modulate macrophage apoptosis: N. gonorrhoeae induced apoptosis in THP‐1 cells whereas it inhibited induced apoptosis in U937 cells and primary human macrophages. Furthermore, N. gonorrhoeae induced expression of inflammatory cytokines in macrophages, suggesting a role for macrophages in recruiting polymorphonuclear leukocytes to the site of infection. These results indicate macrophages may serve as a significant replicative niche for N. gonorrhoeae and play an important role in gonorrheal pathogenesis.     

Differential inducibilities of GFAP expression, cytostasis and apoptosis in primary cultures of human astrocytic tumours

Glial fibrillary acidic protein (GFAP) is an astrocytic lineage-specific intermediate filament protein, and its expression or non-expression is inversely correlated with the tumourigenecity of astrocytoma cells. To estimate the GFAP levels of astrocytes in intracranial tumour tissues, we established primary cultures from six astrocytic tumour specimens and used a double-staining flow cytometric method to detect the different levels of GFAP among these primary cultures. Although these primary cultures exhibited the same Matrigel invasiveness, their GFAP expression is inversely related to the rate of cell growth and the histologic grade of the original tumour. Phenylacetate, 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, which are potent inducers of differentiation in various cancer cells, have been examined for their effects on these primary cultures. Cytostasis was more or less caused by these compounds in all six primary cultures, but induction of GFAP was observed only in the primary culture derived from a less malignant astrocytoma specimen having the highest intrinsic GFAP level. Interestingly, this primary culture, but not others, also exhibited increased HRG-α expression after phenylacetate or sodium butyrate treatment. Loss of the inducibility of differentiation-related gene expression could be one of the events involved in the malignant progression of astrocytomas. In addition, the chemotherapeutic agent BiCNU has a killing effect on all six primary culture cells, with LD50 less than 60nM. The underlying mechanism was through the induction of apoptosis in these primary culture cells regardless of their varying malignancies of original tumours. However, unlike colon cancer and leukaemia cells, sodium butyrate could not induce apoptosis within 4 days in these astrocytic tumour cells, indicating that the cell context of different cell types indeed determined the ability of sodium butyrate to induce apoptosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro effects of eicosanoids derived from different 20-carbon Fatty acids on production of monocyte-derived cytokines in human whole blood cultures

INTRODUCTION: Prostaglandins (PG) and leukotrienes (LT) are usually formed from arachidonic acid (e.g. PGE(2), LTB(4), LTC(4)). The anti-inflammatory effects of fish oil may be mediated through the production of alternative PG and/or LT formed from eicosapentaenoic acid (e.g. PGE(3), LTC(5)). This study examines the effects of PG and LT derived from different fatty acid precursors on lipopolysaccharide-induced cytokine production by cultured human whole blood.Methods Human whole blood was diluted 1:5 and incubated for 48h with lipopolysaccharide. PGE and LT were added and the concentrations of inflammatory cytokines in the cell culture supernatants determined. RESULTS: Tumour necrosis factor (TNF)-alpha concentrations were significantly decreased by the addition of PGE. At the maximum concentration used (10(-6)M) TNF-alpha concentration was reduced to 100%, 90% and 70% by PGE(1), PGE(2) and PGE(3) respectively. Likewise, interleukin (IL)-1beta concentration was decreased to 60%, 30% and 40% by 10(-6)M PGE(1), PGE(2) and PGE(3), respectively. IL-6 and IL-10 concentrations were not altered by PG. LTB(4), LTC(4) or LTC(5) did not significantly affect cytokine concentrations. CONCLUSIONS: PGE inhibit lipopolysaccharide-stimulated TNF-alpha and IL-1beta production in human whole blood cultures. PGE(1), PGE(2) and PGE(3) show a similar pattern and magnitude of effect. This suggests that the anti-inflammatory effects of dietary fish oil may not be mediated through a simple substitution of one family of eicosanoids for another.     

Piperine inhibits cytokine production by human peripheral blood mononuclear cells

Piperine, an amide isolated from Piper species (Piperaceae), has been reported to exhibit central nervous system depression, anti-pyretic and anti-inflammatory activity. Immunomodulatory and anti-tumor activity of piperine has been demonstrated in mouse carcinomas. However, there is little information available concerning the effect of piperine on humans. We evaluated the immunopharmacological activity of this compound in human immune cells. Human peripheral blood mononuclear cells (PBMCs) were exposed to piperine, and cell proliferation was determined by the MTS assay. Piperine significantly inhibited phytohemagglutinin-stimulated human PBMC proliferation after exposure for 72 h. This compound inhibited PBMC activity, with an IC(50) of 100.73 ± 11.16 μg/mL. Production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) was measured using an ELISA assay and RT-PCR. Piperine inhibited IL-2 and IFN-γ production in the PBMCs. RT-PCR data indicated that IL-2 and IFN-γ mRNA expression in PBMCs is suppressed by piperine. This compound significantly inhibited the production of these two cytokines by activated PBMCs in a dose-dependent manner. In conclusion, piperine appears to have potential as an immunomodulatory agent for immune system suppression.     

IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.     

Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in splenocytes by Galphai proteins

Heterotrimeric Gi proteins play a role in lipopolysaccharide (LPS) and Staphylococcus aureus (SA) activated signaling leading to inflammatory mediator production. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS and SA. LPS- and heat killed SA-induced cytokine and chemokine production in splenocytes from wild type (WT), Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice were investigated. LPS- or SA-induced production of TNFalpha, IL-6, IFNgamma, IL-12, IL-17, GM-CSF, MIP-1alpha, MCP-1, MIG and IP-10 were significantly increased (1.2 to 33 fold, p<0.05) in splenocytes harvested from Galpha(i2)(-/-) mice compared with WT mice. The effect of Galpha(i) protein depletion was remarkably isoform specific. In splenocytes from Galpha(i1/3) (-/-) mice relative to WT mice, SA-induced IL-6, IFNgamma, GM-CSF, and IP-10 levels were decreased (59% to 86%, p<0.05), whereas other LPS- or SA-stimulated cytokines and chemokines were not different relative to WT mice. LPS- and SA-induced production of KC were unchanged in both groups of the genetic deficient mice. Splenocytes from both Galpha(i2) (-/-) and Galpha(i1/3) (-/-) mice did not exhibit changes in TLR2 and TLR4 expression. Also analysis of splenic cellular composition by flow cytometry demonstrated an increase in splenic macrophages and reduced CD4 T cells in both Galpha(i2) (-/-) and Galpha(i1/3) (-/-) mice relative to WT mice. The disparate response of splenocytes from the Galpha(i2) (-/-) relative to Galpha(i1/3) (-/-) mice therefore cannot be attributed to major differences in spleen cellular composition. These data demonstrate that G(i2) and G(i1/3) proteins are both involved and differentially regulate splenocyte inflammatory cytokine and chemokine production in a highly Gi isoform specific manner in response to LPS and Gram-positive microbial stimuli.     

Seasonal variation in whole blood cytokine production after LPS stimulation in normal individuals

We examined seasonal differences in whole blood cytokine production after endotoxin (LPS) stimulation in 17 healthy individuals from an urban area having normal sleep/wakefulness pattern. We used 500 pg/ml of LPS for incubation period of 4 h to stimulate 100 microl of whole blood of the same subjects in June, September, February, and March. We found no differences in the circulating total WBCs and differentials including monocytes between different seasons. We found during September (autumn) a reduced pro-inflammatory cytokine production in terms of TNF-alpha and IL-6 production compared to the other seasons. We also found a reduced anti-inflammatory cytokine production in June (summer) and September (autumn) in terms of IL-10, TNF-RI and TNF-RII compared to February (winter) and March (spring). Our results suggest that in early summer there is a predominating pro-inflammatory cytokine response which is counterbalanced early in autumn. These results may have significant implications in the determination of reference values, in exploration of immune response and inflammatory disease prevalence between different seasons, in determining LPS tolerance (immunoparalysis) and planning clinical trials and immunomodulary therapies. However, the effect of dark/light exposure differences on the circadian periodicity in the responsiveness of immune cells during different seasons should be further investigated.     

Suppressed cytokine production in whole blood cultures may be related to iron status and hepcidin and is partially corrected following weight reduction in morbidly obese pre-menopausal women

Objective: Assess ex vivo whole-blood (WB) cytokine production and its association with iron status and serum hepcidin in obese versus non-obese women. Determine the change in ex vivo WB cytokine production 6 months after restrictive bariatric surgery in the obese group. Subjects: Seventeen obese (BMI: 46.6 ± 7.9 kg/m²) and 19 non-obese (BMI: 22.5 ± 3.0 kg/m²), pre-menopausal women; frequency matched for hemoglobin, age and race. Measurements: At baseline control and ex vivo stimulated IL-6, IL-10, IL-22, IFNγ, and TNFα from heparinized WB cultures, hemoglobin from finger-stick and transferrin receptor, hepcidin, CRP, IL-6, HOMA-IR from fasted serum samples and anthropometric parameters were assessed in the women. All parameters were reassessed 6-months following restrictive bariatric surgery in the obese women only. Results: Whole blood ex vivo LPS and ZY stimulated production of IL-6, TNFα, and IFNγ was reduced, IL-22 increased, and IL-10 was unaffected in obese compared with the non-obese women. Furthermore, ex vivo stimulated production of IL-6 and TNFα normalized, but IFNγ production remained unchanged with weight loss following restrictive bariatric surgery. In the obese women, serum transferrin receptor (a marker of iron status) and serum hepcidin were correlated with ex vivo stimulated IFNγ production at baseline. Conclusion: Ex vivo LPS and ZY stimulated cytokine production from WB cultures was altered in pre-menopausal women with morbid obesity. Significant weight loss resulted in normalization of some but not all observed alterations. Furthermore, iron status and serum hepcidin were associated with ex vivo LPS and ZY stimulated IFNγ in obesity.     

Differential inhibition of prostaglandin and superoxide production by dexamethasone in primary cultures of rat Kupffer cells

Dexamethasone inhibited the stimulus-induced prostaglandin E2 formation by rat Kupffer cells in primary culture, e.g. after treatment with zymosan, phorbol ester, calcium ionophore A23187, platelet-activating factor or lipopolysaccharide. Prostaglandin E2 production from added free arachidonic acid was not influenced by the hormone. The time course, as well as the partial inhibition of the hormone effect by actinomycin D and cycloheximide, point to the hormone-induced formation of a protein which regulates phospholipase A2. The hormone did not affect the phagocytotic activity of the Kupffer cells. The quantity of [3H]arachidonic acid incorporated into phospholipids was also not altered by dexamethasone. After stimulation with zymosan, [3H]arachidonic acid was liberated from phosphatidylcholine only. Superoxide generation by rat Kupffer cells was induced by zymosan, phorbol ester and, to a much smaller extent, by platelet-activating factor. A23187 and lipopolysaccharide were without effect. In contrast to prostaglandin formation, the generation of superoxide was not influenced by dexamethasone. These results indicate that in cultured rat Kupffer cells prostaglandin formation and superoxide generation are independently triggered processes.