Differential rates of cytokine production and apoptosis in venipuncture and finger-stab derived blood cultures

The collection of finger-stab (FS) blood is a convenient and non-invasive method of rapidly acquiring human blood and is becoming increasingly popular for use in human biomonitoring studies. This study compared whole blood (WB) and peripheral blood mononuclear cell (PBMC) cultures derived from venipuncture (VP) and FS blood, to determine whether they respond similarly under culture conditions. The rates of spontaneous- and radiation-induced apoptosis and pro-inflammatory cytokine production were monitored over 72 h in each of four culture conditions. In non-irradiated WB cultures, the spontaneous rate of apoptosis was significantly lower in cultures from FS-derived blood than from VP-derived blood. However, FS- and VP-derived cultures responded similarly to radiation-induced apoptosis. PBMC cultures, regardless of the source, were the most responsive to radiation. When the levels of pro-inflammatory cytokines were measured, a significant time-dependent increase in TNF-alpha, IL-6 and IL-1beta production was observed in FS-derived cultures, but not in VP-derived cultures. While VP and FS blood cultures were found to respond similarly to radiation-induced apoptosis, there was a significant difference in the rate of spontaneous apoptosis in non-irradiated WB cultures and in the in situ production of pro-inflammatory cytokines between VP- and FS-derived blood cultures.     

Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in monocytes.

Flow cytometry has become a powerful technique to measure intracellular cytokine production in lymphocytes and monocytes. Appropriate inhibition of the secretion of the produced cytokines is required for studying intracellular cytokine expression. The aim of this study was to compare the capacity of cytokine secretion inhibitors, monensin and brefeldin A, in order to trap cytokine production (interleukin-1 beta [IL-1beta], IL-6, tumor necrosis factor-alpha [TNF-alpha]) within peripheral blood monocytes. A two-color flow cytometric technique was used to measure intracellular spontaneous and lipopolysaccharide (LPS)-stimulated IL-1beta, IL-6, and TNF-alpha production in monocytes (CD14+) of whole blood cultures. The viability of monensin-treated monocytes was slightly lower than that of brefeldin A-inhibited monocytes, as measured with propidium iodide (PI). The percentage of IL-6 and TNF-alpha-producing monocytes after 8 h of culture without stimulation revealed significant lower values for monensin-treated than for brefeldin A-treated monocytes. The percentages for stimulated cells did not differ. The spontaneous intracellular production in molecules of equivalent soluble fluorochrome units (MESF) of IL-1beta, IL-6, and TNF-alpha after 8 h of culture was higher in brefeldin A than in monensin-inhibited monocytes. The LPS-stimulated intracellular production of IL-1beta, IL-6, and TNF-alpha was increased in brefeldin A-inhibited monocytes. In conclusion, for flow cytometric determination of intracellular monocytic cytokines (IL-1beta, IL-6, and TNF-alpha), brefeldin A is a more potent, effective, and less toxic inhibitor of cytokine secretion than monensin.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Migration of neutrophils across endothelial monolayers is stimulated by treatment of the monolayers with interleukin-1 or tumor necrosis factor-alpha

To study the effects of the cytokines IL-1 and TNF-alpha on the transendothelial migration of neutrophils, human umbilical vein endothelial cells (HUVEC) were grown to confluence on connective tissue prepared from human amniotic membrane. Pretreatment of HUVEC-amnion cultures with rIL-1 beta (7.5 ng/ml) or rTNF-alpha (5 ng/ml) for 4 h resulted in rapid migration of from 20 to 50% of subsequently added neutrophils across the endothelial monolayer. In contrast, only 3 +/- 3% of added neutrophils penetrated the HUVEC monolayer in the absence of any stimulus. The number of neutrophils that migrated across cytokine-treated HUVEC was similar to the number that traversed untreated monolayers in response to gradients of FMLP; in addition, it was only 35% less than the number of neutrophils that migrated in response to leukotriene B4. No consistent additive effect was seen when migration was induced by both cytokine pretreatment of the HUVEC and a chemotactic gradient. The number of neutrophils that migrated across IL-1-treated cultures was proportional to the number added over the range of 2.5 x 10(5) to 4 x 10(6) neutrophils. When used at optimal concentrations, IL-1 and TNF-alpha were equally effective in stimulating neutrophil migration; no additive effect was seen when HUVEC were pretreated with optimal doses of both cytokines together. Direct addition of IL-1 or TNF-alpha to a 1-h migration assay had no effect on neutrophil adhesion to or migration across HUVEC, either in the presence or absence of a chemotactic gradient. Stimulation of neutrophil transendothelial migration in this system did not appear to be caused by adsorption of cytokine by the amniotic tissue, nor was it due to contamination of the cytokine preparations by LPS. These results suggest that IL-1 and TNF-alpha, generated at sites of inflammation, may act upon the endothelium to promote emigration of neutrophils from the vasculature.     

Sendai virus induces various cytokines in human peripheral blood leukocytes: different susceptibility of cytokine molecules to low pH

Virus infection of cell cultures induces the synthesis of various cytokines which can either inhibit or stimulate virus replication. The Sendai virus induces large quantities of biologically active interferon (IFN-alphan3) in human peripheral blood leukocytes (hPBL) in vitro, as well as many other cytokines. The supernatants of Sendai virus-infected hPBL contained biologically active IFN-alphan3, significant amounts of immunogenic IFN-gamma, monokines (IL-1alpha, IL-beta, TNF-alpha), lymphokines (IL-6, TNF-beta), growth factor (PDGF-AB) and small concentrations of IL-2 and GM-CSF. The analysis of the influence of the Sendai virus inactivation by lowering pH 2.0 on the cytokine concentrations showed that IL-1alpha, TNF-alpha, TNF-beta and IFN-gamma are susceptible to acid conditions, while IFN-alphan3, IL-1beta, IL-6 and IL-2 concentrations remained unchanged.     

Caspase-1-deficient mice have delayed neutrophil apoptosis and a prolonged inflammatory response to lipopolysaccharide-induced acute lung injury

Caspase-1, the prototypic caspase, is known to process the cytokines IL-1beta and IL-18 to mature forms but it is unclear whether, like other caspases, it can induce apoptosis by activation of downstream protease cascades. Neutrophils are known to express caspase-1, to release IL-1beta and to undergo rapid, caspase-dependent apoptosis. We examined apoptosis and IL-1beta production in peripheral blood neutrophils of caspase-1-deficient and wild-type mice. Constitutive apoptosis of caspase-1-deficient neutrophils was delayed compared with wild-type neutrophils and LPS-mediated inhibition of apoptosis was absent, but caspase-1-deficient neutrophils were susceptible to Fas-mediated apoptosis. LPS-stimulated IL-1beta production was absent from caspase-1-deficient neutrophils. To ascertain whether these differences in apoptosis and IL-1beta production would alter the response to acute lung injury, we studied pulmonary neutrophil accumulation following intratracheal administration of LPS. Caspase-1-deficient mice showed increased, predominantly neutrophilic pulmonary inflammation, but inflammation had resolved in both wild-type and deficient animals by 72 h after LPS instillation. IL-1beta production was increased in wild-type lungs but was also detected in caspase-1-deficient mice. We conclude that caspase-1 modulates apoptosis of both peripheral blood and inflammatory neutrophils, but is not essential for IL-1beta production in the lung.     

Involvement of phosphoinositide 3-kinases in neutrophil activation and the development of acute lung injury.

Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-kappaB, and expression of the proinflammatory cytokines IL-1beta and TNF-alpha through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110gamma catalytic subunit of PI3-K. In PI3-Kgamma(-/-) mice, lung edema, neutrophil recruitment, nuclear translocation of NF-kappaB, and pulmonary levels of IL-1beta and TNF-alpha were significantly lower after endotoxemia as compared with PI3-Kgamma(+/+) controls. Among neutrophils that did accumulate in the lungs of the PI3-Kgamma(-/-) mice after endotoxin administration, activation of NF-kappaB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kgamma(+/+) mice. These results show that PI3-K, and particularly PI3-Kgamma, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.     

Immunological parameters of candidiasis in cancer patients

This study attempts to correlate the frequency of infection by Candida with the following immunological factors presented in cancer patients undergoing chemotherapy: neutrophil number; level of exogenous myeloperoxidase (MPO); cytokines IL-12, IL-10, TNF-alpha and IFN-gamma. A total of 31 serum and blood samples were tested for MPO and cytokines concentrations by using the ELISA technique. The level of cytokines and MPO was measured in serum samples and whole blood cell culture (WBC) supernates. We observed that: 1) MPO deficiency seems to be an important risk factor for invasive candidiasis independently of the neutrophil count; 2) the levels of cytokines in serum showed disturbances in cancer-patient group; 3) in vitro production of TNF-alpha, IFN-gamma and IL-10 by WBC is stimulated in cancer patients and to various degrees may be connected with previous Candida colonization or infection. In addition, production of IL-12 is decreased in cancer patients, and Candida spp. Occurrence may have significant influence on its blood concentration.     

In vivo microdialysis sampling of cytokines produced in mice given bacterial lipopolysaccharide

Cytokines are proteins that mediate communication between cells of the immune system as well as certain other non-immune host cells. These proteins are produced by many cell types and they mediate immune and inflammatory responses. However, the direct site analysis of these critical proteins is hampered by the lack of site-specific tools available for such direct measurements. In this study, both in vitro and in vivo microdialysis sampling of different cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], interleukin-6 [IL-6], IL-12p70, and macrophage chemoattractant protein-1 [MCP-1]) was performed. A mouse model of bacterial lipopolysaccharide (LPS) administration and response pattern was used for in vivo studies. Three cytokines, TNF-alpha, IL-6, and MCP-1 were quantified in the serum from mice given LPS. In vivo studies demonstrated the ability to monitor increasing levels of these cytokines (TNF-alpha, IL-6, and MCP-1) via microdialysis probes placed in the peritoneal cavity of mice given LPS. All three cytokines were quantified simultaneously in 15 muL of dialysate using a multiplexed bead-based immunoassay for flow cytometry. The detected dialysate cytokine concentrations varied between 200 pg/mL and 1500 pg/mL for TNF-alpha, between 600 pg/mL and 3000 pg/mL for MCP-1, and between 2700 pg/mL and more than 5000 pg/mL for IL-6. The detected serum cytokine concentrations ranged from 5700 pg/mL to 35,000 pg/mL for TNF-alpha, from 40,000 pg/mL to 65,000 pg/mL for MCP-1, and greater than than 100,000 pg/mL for IL-6. This work demonstrates that microdialysis sampling can be used in vivo to collect temporal profiles of cytokine production.     

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.     

Neutrophils as firemen, production of anti-inflammatory mediators by neutrophils in a mixed cell environment

Neutrophils represent critical components of the innate immune system that bear primary responsibility for phagocytosis and killing of invading pathogens. Following stimulation of human whole blood, robust production of multiple cytokines and cytokine inhibitors occurs. We attempted to define the cell population responsible for the synthesis of different mediators by first stimulating whole blood and then isolating pure populations of granulocytes and monocytes. Monocytes produced mRNA coding for the classic pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6, while mRNA for these cytokines was not detectable in the isolated neutrophils. In contrast, neutrophils produce significant quantities of cytokine inhibitors such as the type 2 TNF soluble receptor and the IL-1 receptor antagonist. Both neutrophils and monocytes produced mRNA coding for IL-8. These data indicate that following stimulation of a mixed cell population the monocytes primarily produce pro-inflammatory mediators while the neutrophils synthesize a significant portion of the anti-inflammatory mediators. The neutrophils may be compared to firemen who bring the resources necessary to put out the flame of acute inflammation.     

Human immunodeficiency virus does not induce interleukin-1, interleukin-6, or tumor necrosis factor in mononuclear cells.

The production of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) by fresh peripheral blood mononuclear cells was evaluated after exposure to human immunodeficiency virus (HIV) or purified recombinant HIV-1 envelope glycoprotein (rgp120). To exclude the role of contaminating endotoxin in this study, all media were subjected to ultrafiltration and reagents contained less than 25 pg of endotoxin per ml by Limulus assay. Under endotoxin-free conditions, no increases in IL-1 beta, IL-6, or TNF-alpha mRNA or protein were detectable in cell cultures exposed to HIV-1, HIV-2, or rgp120 (0.1 to 10 micrograms/ml), as compared with cytokine levels in mock-exposed cultures. However, concentrations of endotoxin (lipopolysaccharide) as low as 0.5 ng/ml induced significant production of mRNA and protein for these three cytokines. Preincubation of mononuclear cells with "shake" HIV-1 preparations and also mock-infected shake preparations prior to lipopolysaccharide stimulation resulted in a two- to threefold increase in IL-1 beta and TNF-alpha production. This priming effect was not observed with rgp120 (0.1 to 10 micrograms/ml) or standard HIV-1 or mock-infected supernatants, suggesting the presence of biologically active material independent of virus in the shake preparations. Our studies indicate that, in the absence of endotoxin, HIV-1, HIV-2, and HIV gp120 do not induce production of IL-1 beta, IL-6, or TNF-alpha by peripheral blood mononuclear cells.     

Role of hypothermia induced by tumor necrosis factor on apoptosis and function of inflammatory neutrophils in mice

Changes in body temperature and cell infiltration, mediated by cytokines including tumor necrosis factor-alpha (TNF-alpha), occur during inflammation, but a role of body temperature on inflammatory responses remains obscure. Intraperitoneal injection of 10% casein to mice resulted in transient hypothermia followed by neutrophil accumulation in peritoneal cavities. Peritoneal TNF-alpha was rapidly raised, and pretreatment of mice with an anti-TNF-alpha antibody promoted temperature restoration and partially inhibited neutrophil accumulation. To investigate direct effects of body temperature on neutrophils, peritoneal or peripheral blood neutrophils were cultured at 35 degrees C or 37 degrees C with or without recombinant murine TNF-alpha (100 ng/ml) or a protein synthesis inhibitor cycloheximide (1 microg/ml). Significant inhibition of spontaneous and TNF-alpha-induced apoptosis was obtained at 35 degrees C compared with 37 degrees C, an effect that was not altered by the addition of cycloheximide. Moreover, phagocytic ability of peritoneal neutrophils was significantly enhanced by incubating them at the lower temperature. These results indicate that mild hypothermia induced by endogenous TNF-alpha has enhancing roles on neutrophil survival and function during peritoneal inflammation.     

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.     

Serum proinflammatory mediators at different periods of therapy in patients with multiple myeloma

Multiple myeloma (MM) is a malignant disease characterized by the clonal proliferation of plasma cells within the bone marrow. Several cytokines have been demonstrated to be involved in the control of growth, progression, and dissemination of MM. We determined serum levels of interleukin-1beta (IL-1beta), soluble interleukin-2 receptor (sIL-2R), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), and C-reactive protein (CRP) in 14 newly diagnosed MM patients. The median age of the patients was 63.4 +/- 10.8 years and all of the patients were stage III (classified according to the Durie-Salmon classification). The same parameters were measured in 15 healthy controls. In addition, we also examined the effects of vincristine-adriamycin-dexamethasone (VAD) therapy on the same parameters and mediators as well as the relationship among the parameters in the same patient groups. The serum concentrations of TNF-alpha, IL-1beta, sIL-2R, IL-6, IL-8, and CRP (18.6 +/- 3.7 pg/mL, 10.1 +/- 2.8 pg/mL, 730 +/- 220 U/mL, 11.4 +/- 3.3 pg/mL, 23.9 +/- 8.3 pg/mL, and 49.9 +/- 19.5 mg/dL, resp) were significantly higher in newly diagnosed MM patients than in healthy controls (P < .0001). All of the parameters were found to be significantly reduced after chemotherapy. In conclusion, we found that after the VAD therapy, the level of these cytokines which are thought to play an important role in the pathogenesis of MM was significantly suppressed. This is the first study demonstrating strong impact of VAD treatment on circulating mediators of sIL-2R and IL-8 levels parameters.     

IFN-gamma is not induced through increased plasma concentrations of interleukin-12/interleukin-18 during human endotoxemia

Endotoxin administration to animals and humans is an accepted experimental model of Gram-negative sepsis, and endotoxin is believed to play a major role in triggering the activation of cytokines. In septic patients, the IL-12/IL-18/IFN-gamma axis is activated and correlates with mortality. Our aim was to investigate the effects of endotoxin administration in humans on the activation of the IL-12/IL-18/IFN-gamma axis. Seven healthy volunteers received E. coli endotoxin (O:113). Hemodynamics, temperature and the course of plasma concentrations of TNF-alpha, IL-1beta, IL-12, IL-18 and IFN-gamma were determined. Endotoxin administration resulted in the expected flu-like symptoms, a temperature of 38.8 +/- 0.3(o)C (p < 0.003), a decrease in mean arterial blood pressure of 14.8 +/- 1.8 mmHg (p < 0.0002) and an increase in heart rate of 27.5 +/- 4.8 bpm (p < 0.002) compared to baseline values. TNF-alpha increased from 16.6 +/- 8.2 to 927 +/- 187 pg/mL (p < 0.003). IL-1beta increased from 8.6 +/- 0.5 to 25.3 +/- 2.0 pg/mL (p < 0.0001). IL-12 showed no significant increase (8.2 +/- 0.2 to 9.3 +/- 0.8 pg/mL, p = 0.13), and all IL-18 measurements remained below the level of detection. In contrast, IFN-gamma showed an increase from 106.6 +/- 57.1 to 152.7 +/- 57.8 (p < 0.005). These results indicate that pathways other than the IL-12/IL-18 axis may induce IFN-gamma production in human endotoxemia.     

The time course of inflammatory cytokine secretion in a rat model of postoperative pain does not coincide with the onset of mechanical hyperalgesia

We characterized the time course of inflammatory cytokine release at the site of injury and in plasma after surgery on the rat tail. Anesthetized Sprague-Dawley rats had a 20 mm long incision made through the skin and fascia of their tails. Control rats were anesthetized, but no incision was made. Blood and tissue samples were taken 2 h and 1, 2, 4, and 8 days after surgery and analysed by ELISA for interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and cytokine-induced neutrophil chemoattractant-1 (CINC-1). In another group of rats, daily behavioral measurements were made of the rats' responses to a blunt noxious mechanical stimulus (4 Newtons) applied to their tails. Primary hyperalgesia developed within 2 h of surgery and lasted for 6 days. The tissue concentrations of IL-1beta, IL-6, and CINC-1 increased within 24 h of surgery, and TNF-alpha concentration increased within 48 h of surgery. Thereafter, cytokine concentrations remained elevated for 4 (IL-1beta and IL-6) to 8 days (CINC-1, TNF-alpha) after surgery. Control animals did not develop hyperalgesia and no changes in cytokines concentrations were detected. Thus, in our model of postoperative pain, secretion of inflammatory cytokines IL-1beta, IL-6, TNF-alpha, and CINC-1 was not essential for the initiation of postoperative hyperalgesia.     

Presence of cytokines in biological preparations.

In vaccines produced in eukaryote cells as well as in commercial medical preparations of leukocyte interferon a number of cytokines such as IL-1beta, IL-6 and TNF-alpha have been detected. Among the vaccines examined in this study the highest level of IL-1beta was demonstrated in inactivated hepatitis A vaccine prepared in the green monkey kidney cell line 4647, that of IL-6 in inactivated rabies vaccine produced in Syrian hamster kidney (SHK) cell culture, and that of TNF-alpha in live poliomyelitis vaccine manufactured in VERO cells. A spontaneous and poliovirus-induced capacity of cell cultures to produce cytokines was detected. The level of cytokines produced depend on the kind of cell culture and the type of virus, a more pronounced effect being generated by types 1 and 2 poliovirus as compared with type 3. The presence of highly active cytokines in virus vaccines and interferon preparations points to the necessity of investigating the influence of the presence of cytokines on the biological activity of these preparations and to the advisability of standardizing and controlling the cytokine content.