The phosphatidylinositol 3-kinase/protein kinase B signaling pathway is activated by lipoteichoic acid and plays a role in Kupffer cell production of interleukin-6 (IL-6) and IL-10

Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-alpha levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-alpha, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.     

Organ injury and cytokine release caused by peptidoglycan are dependent on the structural integrity of the glycan chain

Several studies have implicated a role of peptidoglycan (PepG) as a pathogenicity factor in sepsis and organ injury, in part by initiating the release of inflammatory mediators. We wanted to elucidate the structural requirements of PepG to trigger inflammatory responses and organ injury. Injection of native PepG into anesthetized rats caused moderate but significant increases in the levels of alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, and bilirubin (markers of hepatic injury and/or dysfunction) and creatinine and urea (markers of renal dysfunction) in serum, whereas PepG pretreated with muramidase to digest the glycan backbone failed to do this. In an ex vivo model of human blood, PepG containing different amino acids induced similar levels of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-8, and IL-10, as determined by plasma analyses (enzyme-linked immunosorbent assay). Hydrolysis of the Staphylococcus aureus cross-bridge with lysostaphin resulted in moderately reduced release of TNF-α, IL-6, IL-8, and IL-10, whereas muramidase digestion nearly abolished the ability to induce cytokine release and IL-6 mRNA accumulation in CD14⁺ monocytes compared to intact PepG. However, additional experiments showed that muramidase-treated PepG synergized with lipopolysaccharide to induce TNF-α and IL-10 release in whole blood, despite its lack of inflammatory activity when administered alone. Based on these studies, we hypothesize that the structural integrity of the glycan chain of the PepG molecule is very important for the pathogenic effects of PepG. The amino acid composition of PepG, however, does not seem to be essential for the inflammatory properties of the molecule.     

Interaction of IL-1 beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) in human T cells activated by murine antigens.

We used a mixed leucocyte culture between human T cells and irradiated murine splenocytes which allowed us to distinguish between cytokine production from the responder and stimulator cells by the use of species-specific assays for mRNA up-regulation. Using this model of T cell activation by antigen, we studied the effects of human antigen-presenting cell-derived cytokines IL-1 beta, IL-6 and TNF-alpha on the activation of human T cell subsets. We show in this system that exogenously added IL-1 beta, IL-6 and TNF-alpha induces IL-2 receptor (R) up-regulation and IL-2 production, and proliferation by both CD4+ and CD8+ cells. The addition of IL-1 beta induces IL-6 mRNA, and anti-IL-1 antibodies or an IL-1R antagonist protein completely suppresses IL-6 and TNF-alpha supported proliferation. Similarly, addition of IL-6 or TNF-alpha induces up-regulation of IL-1 beta mRNA. However, anti-IL-6 and anti-IL-6R antibodies only partially block proliferation supported by IL-1 beta. These findings suggest that IL-6 and TNF-alpha will induce IL-2R up-regulation/IL-2 secretion via the induction of IL-1 beta production.     

人疱疹病毒6型导单细胞产生IL—10

目的 探讨人疱疹病毒６型（ＨＨＶ－６）感染的免疫发病机理。方法 用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）和双抗体夹心酶联免疫吸附试验（ＥＬＩＳＡ）检测白细胞介素＾－１０（ＩＬ－１０）等细胞因子的产生。结果 ＨＨＶ－６诱导单核细胞表达和产生ＩＬ－１０，细胞因子ｍＲＮＡ动力学研究发现ＴＮＦ－α（肿瘤坏死因子）、ＩＬ－１β及ＩＬ－６随ＩＬ－１０ｍＲＮＡ积累而减少，用抗人ＩＬ－１０单克隆抗体阻断ＨＨＶ－６诱     

Cytokine induction by purified lipoteichoic acids from various bacterial species--role of LBP, sCD14, CD14 and failure to induce IL-12 and subsequent IFN-gamma release

We have recently shown that highly purified lipoteichoic acid (LTA) represents a major immunostimulatory principle of Staphylococcus aureus. In order to test whether this translates to other bacterial species, we extracted and purified LTA from 12 laboratory-grown species. All LTA induced the release of TNF-alpha, IL-1beta, IL-6 and IL-10 in human whole blood. Soluble CD14 (sCD14) inhibited monokine induction by LTA but failed to confer LTA responsiveness for IL-6 and IL-8 release of human umbilical vein endothelial cells (HUVEC). In a competitive LPS-binding protein (LBP) binding assay, the IC(50) of the tested LTA preparations was up to 3,230-fold higher than for LPS. LBP enhanced TNF-alpha release of human peripheral blood mononuclear cells (PBMC) upon LPS but not LTA stimulation. These data demonstrate a differential role for the serum proteins LBP and sCD14 in the recognition of LPS and LTA. Different efficacies of various anti-CD14 antibodies against LPS vs. LTA-induced cytokine release suggest that the recognition sites of CD14 for LPS and LTA are distinct with a partial overlap. While the maximal achievable monokine release in response to LTA was comparable to LPS, all LTA induced significantly less IL-12 and IFN-gamma. IL-12 substitution increased LTA-inducible IFN-gamma release up to 180-fold, suggesting a critical role of poor LTA-inducible IL-12 for IFN-gamma formation. Pretreatment with IFN-gamma rendered galactosamine-sensitized mice sensitive to challenge with LTA. In conclusion, LTA compared to LPS, are weak inducers of IL-12 and subsequent IFN-gamma formation which might explain their lower toxicity in vivo.     

HIV-1 induces IL-10 production in human monocytes via a CD4-independent pathway

In HIV-infected patients, increased levels of IL-10, mainly produced by virally infected monocytes, were reported to be associated with impaired cell-mediated immune responses. In this study, we investigated how HIV-1 induces IL-10 production in human monocytes. We found that CD14(+) monocytes infected by either HIV-1(213) (X4) or HIV-1(BaL) (R5) produced IL-10, IL-6, tumor necrosis factor-alpha (TNF-alpha), and to a lesser extent, IFN-gamma. However, the capacity of HIV-1 to induce these cytokines was not dependent on virus replication since UV-inactivated HIV-1 induced similar levels of these cytokines. In addition, soluble HIV-1 gp160 could induce CD14(+) monocytes to produce IL-10 but at lower levels. Cross-linking CD4 molecules (XLCD4) with anti-CD4 mAbs and goat anti-mouse IgG (GAM) resulted in high levels of IL-6, TNF-alpha and IFN-gamma but no IL-10 production by CD14(+) monocytes. Interestingly, neither anti-CD4 mAbs nor recombinant soluble CD4 (sCD4) receptor could block IL-10 secretion induced by HIV-1(213), HIV-1(BaL) or HIV-1 gp160 in CD14(+) monocytes, whereas anti-CD4 mAb or sCD4 almost completely blocked the secretion of the other cytokines. Furthermore, HIV-1(213) could induce IL-10 mRNA expression in CD14(+) monocytes while XLCD4 by anti-CD4 mAb and GAM failed to do so. As with IL-10 protein levels, HIV-1(213)-induced IL-10 mRNA expression in CD14(+) monocytes could not be inhibited by anti-CD4 mAb or sCD4. Taken together, HIV-1 binding to CD14(+) monocytes can induce CD4-independent IL-10 production at both mRNA and protein levels. This finding suggests that HIV induces the immunosuppressive IL-10 production in monocytes and is not dependent on CD4 molecules and that interference with HIV entry through CD4 molecules may have no impact on counteracting the effects of IL-10 during HIV infection.     

Spontaneous cytokine production and its effect on induced production

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) by peripheral-blood B lymphocytes, T cells, CD8(-) T cells, CD8(+) T cells, CD3(-) CD16/56(+) lymphocytes (natural killer [NK] cells), CD3(+) CD16/56(+) lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-alpha production occurred most frequently, followed in descending order by IFN-gamma, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-alpha; for 12 of these patients, TNF-alpha was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-alpha and IFN-gamma, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.     

Suppression of proinflammatory cytokines and induction of IL-10 in human monocytes after coxsackievirus B3 infection

Coxsackievirus B3 (CVB3) causes acute and chronic myocarditis, which is accompanied by an intense mononuclear leukocyte infiltration. Because myocardial tissue damage may either result from viral infections or from a dysregulated immune response, the susceptibility of human monocytes and macrophages to CVB3 was examined in this study with regard to virus replication, virus persistence, and release of cytokines. Monocytes were infected by CVB3 as shown by the intracellular appearance of plus- and minus-strand viral RNA, which was also capable of persisting for more than 10 days. Fresh monocytes were not permissive for full virus replication whereas monocyte-derived macrophages yielded a low amount of new viruses, which led to cell death. Although CVB3 infection induced the mRNA for the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1, and IL-6, only little cytokine production occurred. When infected monocytes were stimulated in addition by lipopolysaccharides (LPS), cytokine production was partially suppressed. In striking contrast, IL-10 expression was strongly and persistently induced by CVB3 on the mRNA and the protein level. These data show a dysregulated cytokine response in CVB3-exposed human monocytes and macrophages, which is characterized by a suppression of proinflammatory cytokines and a dominance of IL-10. This viral strategy may aid CVB3, causing chronic myocardiopathy.     

Adhesion of Plasmodium Falciparwn-lnfected Erythroeytes to Human Cells and Seeretion of Cytokines (IL-1-β, IL-1RA, IL-6, IL-8, IL-10, TGFβ, TNFα, G-CSF, GM-CSF)

The scientific interest in tbe pbysical interaction of Plasmodium falciparum -infected erytbrocytes with host cells stems from the suggestion ibat excessive binding in the microvasculature leads to severe malaria. Tbe authors studied, therefore, two parasites for their ability to adbere to normal human cells and to induce cytokine production, one parasite lacking a binding capacity (DD2) and one which adhered to CD36⁺ transfected CHO cells (MCAMP). The MCAMP parasites readily bound to platelets and erytbrocytes and to monocytes, polymorphonuclear granulocytes and EBV-transformed B cells as seen by ligbt and electron microscopy. Platelets were frequently attached in large numbers to the infected erythrocyte surface and groups of infected erytbrocytes were sometimes held together by several platelets. Nine out of 17 cytokines tested were found to be secreted into the culture supernatants after 35 h of co-eultures containing monocytes or unfractionated peripheral blood mononuclear cells (PBMC) and parasites (IL-lRA, IL-6, IL-8, IL-10, TGFβ, TNFα, G-CSF, IL-1-β, and GM-CSF). Three additional cytokines were also present in low levels (<200pg/ml, IL-2, IL-4, IFNγ) in tbe culture supernatants after incubation of the cells for 4 days. TNFa, IL-RA, and IL-8 were secreted from polymorphonuclear granulocytes, LGLs and T cells. Platelets and, to a lesser degree, monocytes and T cells secreted large amounts of TGFβ (10–30 ng/ml). Cytokines may participate in tbe patbogenesis but also the suppression of immune responses seen during acute malarial infections.     

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.     

A multidonor ELISPOT study of IL-1 beta,IL-2, IL-4, IL-6, IL-13, IFN-gamma and TNF-alpha release by cryopreserved human peripheral blood mononuclear cells

Utilization of cryopreserved peripheral blood mononuclear cells (PBMCs), rather than fresh ones collected from the same donor on different dates, overcomes the variability in sensitivity of these cells to activation agents. To understand the effect of cryopreservation, frozen PBMCs from eight healthy donors were studied to release T(H)1 or T(H)2 cytokines including IL- 1 beta, IL-2, IL-4, IL-6, IL-13, TNF-alpha and IFN-gamma using ELISPOT assay. The number of spot-forming cells (SFC) was determined using three concentrations of PBMCs (5 x 10(6), 5 x 10(5) and 5 x 10(4) cells/ml). PBMCs from all eight donors were found to retain their functional capacity to release T(H)1 or T(H)2 cytokines after freezing and thawing. When PBMCs were taken in concentrations 5 x 10(6) or 5 x 10(5) cells/ml, the density of IL-1 beta-, IL-2-, IL-6- and TNF-alpha-related spots in a well for most of the donors appeared to be overly high, making SFC quantification either difficult or impossible. To the contrary, PBMCs in concentration 5 x 10(4) produced distinct and quantifiable spots. The density of spots related to IL-4 and IL-13 release appeared to be optimal for SFC quantification when PBMCs were taken in concentration 5 x 10(6) whereas in 5 x 10(5) cells/ml the spot density was very low and absent in 5 x 10(4) cells/ml concentration group. No relationship between release levels of different cytokines was found, except IFN-gamma and IL-2 cytokine indicating that cryopreserved PBMCs with a high IFN-gamma response will likely have a high IL-2 response as well. Our results indicate that a release level of one cytokine may not be reliably predicted by knowing the level of the other. This implies that it is necessary to test cryopreserved PBMCs in a broad range of concentrations to determine one, which will be optimal for producing distinct and quantifiable spots.     

Distribution of IFN-gamma, IL-4 and TNF-alpha protein and CD8 T cells producing IL-12p40 mRNA in human lung tuberculous granulomas

In order to examine the immune response at the site of pathology in tuberculosis, we analysed cytokines present in lung granulomas, their associations with each other and with caseous necrosis as well as the phenotype of the cellular infiltrate. Paraffin-embedded tissue from the lungs of seven patients with pulmonary tuberculosis was analysed by immunohistochemistry and in situ hybridization to detect interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) proteins and IL-12p40 mRNA. All seven patients had granulomas staining positive for IFN-gamma, TNF-alpha and IL-12p40, but only four stained positive for IL-4. Cells with the morphology of lymphocytes, macrophages and giant cells expressed TNF-alpha, IFN-gamma and IL-4 protein. Furthermore, CD68-positive myeloid cells expressed IL-12p40 mRNA, as expected, but a subset of CD3-positive lymphocytes also expressed this mRNA. These lymphocytes producing IL-12p40 also stained positive for CD8 but not CD4. A total of 141 granulomas were scored for the presence or absence of cytokine or necrosis and two major associations were identified. The first association was between IFN-gamma and IL-12, with 76% of granulomas staining positive for both cytokines. Unexpectedly, those granulomas positive for IL-4 were always positive for IFN-gamma. The second association was between TNF-alpha and caseous necrosis, where all necrotic granulomas were TNF-alpha positive. This association was modulated by IL-4. Therefore, heterogeneity of cellular infiltrate and cytokine expression is observed between adjacent granulomas in the same patient.     

Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.

Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.     

Interleukin-8, interleukin-6, and soluble tumour necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus.

The release of interleukin-8 (IL-8), interleukin-6 (IL-6) and the soluble forms of the tumour necrosis factor receptor (sTNF-R) from human pulmonary type II-like epithelial cells (A549) after respiratory syncytial virus (RSV) infection was analysed. RSV infection alone induced a time- and RSV dose-dependent IL-8 and IL-6 release from A549 cells. Furthermore, the soluble form of the TNF-RI was also secreted in a time- and RSV dose-dependent fashion. The soluble TNF-RII was not detected in the cell supernatant of infected epithelial cells. The effect of various cytokines [IL-1 alpha/beta, TNF-alpha/beta, IL-3, IL-6, interferon-gamma (IFN-gamma), transforming growth factor-beta 2 (TGF-beta 2)] and colony-stimulating factors [granulocyte (G)-CSF; granulocyte-macrophage (GM)-CSF] on the IL-8 release from A549 cells was also studied. Our data show that the proinflammatory cytokines IL-1 alpha/beta and TNF-alpha/beta induced an IL-8 release in non-infected A549 cells, and increased the IL-8 release of RSV-infected A549 cells synergistically. In addition, IL-3, G-CSF, IFN-gamma and TGF-beta 2, albeit at high concentrations, induced a low IL-8 release from non-infected A549 cells. The enhanced IL-8 secretion rates were accompanied with elevated cytoplasmic IL-8 mRNA steady state levels, as was shown by Northern blot analysis. Cellular co-culture experiments performed with A549 cells and polymorphonuclear granulocytes or peripheral blood mononuclear cells revealed that increased IL-8 amounts were secreted in the co-culture of non-infected as well as RSV-infected cells. The present study suggests a central role for the airway epithelium during RSV infection with regard to cytokine and cytokine receptor release, resulting in a recruitment and activation of inflammatory and immune effector cells. Our data also suggest that paracrine cytokine networks and cell-cell contact are involved in the regulation of IL-8 secretion within the microenvironment of the bronchial epithelium.     

Ureaplasma urealyticum modulates endotoxin-induced cytokine release by human monocytes derived from preterm and term newborns and adults

We previously observed that Ureaplasma urealyticum respiratory tract colonization in infants with a birth weight of < or =1,250 g was associated with increases in the tracheal aspirate proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) relative to the counterregulatory cytokine IL-6 during the first week of life (A. M. Patterson, V. Taciak, J. Lovchik, R. E. Fox, A. B. Campbell, and R. M. Viscardi, Pediatr. Infect. Dis. J. 17:321-328, 1998). We hypothesized that U. urealyticum alters the host immune response in the presence of a coinflammatory stimulus (e.g., bacterial infection or hyperoxia) by shifting the balance of cytokine expression towards the proinflammatory cytokines. To test this hypothesis, we compared the release of TNF-alpha, IL-8, IL-6, and IL-10 in vitro by unstimulated and U. urealyticum (with or without lipopolysaccharide [LPS])-stimulated human monocytes from adult peripheral blood and from term and preterm cord blood. U. urealyticum alone and in combination with LPS induced concentration- and development-dependent changes in cytokine release. In vitro inoculation with low-inoculum U. urealyticum (10(3) color-changing units [CCU]) (i) partially blocked the LPS-stimulated IL-6 release by all cells and reduced LPS-stimulated IL-10 release by preterm cells, (ii) stimulated TNF-alpha and IL-8 release by preterm cells, and (iii) augmented LPS-stimulated TNF-alpha release in all cells. In preterm cells, high-inoculum U. urealyticum (10(6) CCU) (i) stimulated TNF-alpha and IL-8, but not IL-6 or IL-10, release and (ii) augmented LPS-stimulated TNF-alpha and IL-8 release. High-inoculum U. urealyticum (i) stimulated release of all four cytokines in term cells and IL-8 release in adult cells and (ii) augmented LPS-induced TNF-alpha, IL-10, and IL-8 release in term cells but did not significantly affect LPS-induced cytokine release in adult cells. We speculate that U. urealyticum enhances the proinflammatory response to a second infection by blocking expression of counterregulatory cytokines (IL-6 and IL-10), predisposing the preterm infant to prolonged and dysregulated inflammation, lung injury, and impaired clearance of secondary infections.     

Effects of tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and monocytes on lymphokine-activated killer (LAK) induction from natural killer (NK) cells and T lymphocytes.

Roles of monocytes and cytokines were investigated on LAK induction from T and NK cells. Monocytes augmented more T-LAK induction than did NK-LAK. Expression of IL-1 beta, TNF-alpha and interferon-gamma (IFN-gamma)-mRNA and their cytokine production were superior in NK cells compared with T cells in parallel with their LAK activities. An increase of TNF-alpha, IL-1 beta and IFN-gamma production was induced by co-culturing NK or T cells with autologous monocytes. The augmentation of T cell cytokine production and T-LAK activity by monocytes was more prominent than that of NK cells. TNF-alpha and IL-1 beta were generated 24 h after IL-2 stimulation, and these cytokines were able to almost substitute for monocytes in LAK induction. Conversely, LAK induction was almost completely suppressed by both anti-IL-1 beta and anti-TNF-alpha antibodies, if they were added within 24 h after the start of the LAK induction. IFN-gamma, which was produced at a later stage, scarcely affected LAK induction in spite of the cooperation with TNF-alpha. The results obtained indicate conclusively that the superiority of NK-LAK depends on their superior productivity of both IL-1 beta and TNF-alpha, and that the up-regulation of LAK induction by monocytes is largely due to the enhanced generation of both cytokines.