Leukotrienes: positive signals for regulation of gamma-interferon production

Leukotrienes B4, C4, and D4 were capable of replacing the helper cell or interleukin 2 requirement for gamma-interferon (IFN gamma) production by Lyt-1-,2+ cells from C57BL/6 mouse spleen cells at leukotriene concentrations as low as 0.002 microM. An antioxidant inhibitor (butylated hydroxyanisole) of lipoxygenase metabolism of arachidonic acid suppressed IFN gamma production. The suppression was significantly reversed by leukotriene C4, which further suggests that leukotrienes and possibly other substances produced by the lipoxygenase pathway of arachidonic acid metabolism play an important role in the regulation of IFN gamma production. All of these events may be related to activation of guanylate cyclase activity, since cyclic GMP also significantly reversed the suppressor effects of butylated hydroxyanisole in IFN gamma production. The leukotriene help for IFN gamma production was independent of DNA synthesis or cellular proliferation. The data are consistent with the hypothesis that lipxoygenase products of arachidonic acid metabolism may play a role in the mediation of interleukin 2 help in IFN gamma production. Cells that are rich sources of leukotrienes, then, should play important roles in positive regulation of lymphokine production.     

Differential effects of interferon-alpha and interferon-gamma on interleukin 1 secretion by monocytes

We examined the effect of interferon (IFN)-alpha and IFN-gamma on the ability of human monocytes to secrete interleukin 1 (IL 1). IFN-alpha directly induced IL 1 secretion by monocytes. IFN-gamma did not induce any IL 1. IFN-gamma-stimulated monocyte supernatants were also negative for pyrogenic activity. However, IFN-gamma greatly enhanced the amount of IL 1 secreted when monocytes were stimulated by lipopolysaccharide or Staphylococcus aureus, even at concentrations which by themselves did not induce IL 1. IFN-alpha did not enhance IL 1 secretion induced by other stimuli. IFN-gamma enhanced IL 1 secretion by priming monocytes to be more sensitive to an IL 1-inducing stimulus. However, IFN-gamma does not enhance IL 1 induced by all stimuli, because there was no enhancement of IL 1 induced by PMA. Thus, IFN-alpha and IFN-gamma have very distinct roles in the induction and enhancement of IL 1 by monocytes.     

Interleukin 1 and phorbol 12-myristate 13-acetate induce collagenase and PGE2 production through a PKC-independent mechanism in chondrocytes.

In the present study we demonstrate that interleukin 1 (IL 1) and phorbol 12-myristate 13-acetate (PMA) stimulate collagenase production by bovine chondrocytes in monolayer culture. Since it has been well established that PMA stimulates protein kinase C (PKC), we examined whether IL 1 and PMA also stimulate PKC in chondrocytes. In agreement with other studies, PMA induced the translocation of PKC, reflecting PKC activation by PMA. In contrast, IL 1 did not induce the translocation of PKC. Both IL 1 and PMA stimulated the release of [14C]arachidonic acid from chondrocyte phospholipids, suggesting that both agents stimulate phospholipase A2 (PLA2). Concomitantly, IL 1 and PMA also induced a pronounced increase in the production of PGE2. Pre-incubation of chondrocytes with staurosporine, a PKC inhibitor, did not affect the stimulation of collagenase production by IL 1 and only minimally that induced by PMA. Similarly, high concentrations of staurosporine did not inhibit prostaglandin E2 (PGE2) production induced by IL 1 or PMA. These data show that IL 1 and PMA stimulate the PLA2 pathway and collagenase production, however, these processes can occur in the absence of PKC activation.     

Membrane-associated interleukin 1 activity on human U937 tumor cells: stimulation of PGE2 production by human chondrosarcoma cells

Membrane-associated interleukin 1 (IL 1) activity was induced on the human macrophage tumor cell line, U937, by pretreatment with phorbol myristic acid (PMA). Incubation of PMA-treated, paraformaldehyde-fixed U937 cells with the murine cell line D10.G4.1 in the presence of concanavalin A caused an increase in DNA synthesis as measured by the uptake of tritiated thymidine. Paraformaldehyde-fixed U937, not pretreated with PMA, showed little or no activity. A rabbit polyclonal antibody directed against human IL 1 neutralized all membrane-associated IL 1-like activity, as measured by the inhibition of D10.G4.1 cell proliferation. PMA-treated U937 caused a pronounced enhancement of PGE2 production from a human chondrosarcoma cell line, SW-1353. Membrane-associated IL 1 induced a more potent PGE2 response than did a maximal concentration of soluble IL 1. Rabbit antihuman IL 1 neutralized membrane-bound IL 1 induction of PGE2. The data presented here raise the possibility that membrane-bound IL 1 may play a primary role in the pathophysiology of the inflammatory disease process.     

gamma-Interferon enhances the secretion of arachidonic acid metabolites from murine peritoneal macrophages stimulated with phorbol diesters

Macrophage activation in vivo has been associated with qualitative and quantitative alterations in the release and metabolism of arachidonic acid. In the present study, we examined the effect of in vitro macrophage activation with recombinant gamma-interferon (IFN-gamma) on arachidonic acid secretion induced by exposure to a variety of stimulating agents. Secretion stimulated by challenge with unopsonized zymosan, insoluble immune complexes, the calcium ionophore A23187, or combinations thereof was unaltered in IFN-gamma-treated macrophages. However, when phorbol diesters active as tumor promoters were employed as challenge agents, arachidonate secretion was enhanced as much as 10-fold over that seen in nonactivated controls. The enhanced secretory response to PMA was detectable as early as 1 hr after exposure to IFN-gamma, reached a maximum within 3 to 6 hr, and subsequently declined to control levels even in the continued presence of the agent. Treatment with IFN-gamma did not alter the pattern of individual metabolites produced by macrophages challenged with either zymosan or PMA. Finally, the sensitivity to phorbol diesters was also increased by treatment with IFN-gamma (ED50 reduced from 35 ng/ml to 4 ng/ml). Thus, IFN-gamma could prime macrophages for a substantially amplified response to phorbol esters. Because the cellular mediator of PMA action has been identified as a Ca++, phospholipid-dependent protein kinase, a role for this enzyme in macrophage functional development is indicated.     

Suppression of tumor promoter phorbolmyristate acetate-induced chromosome breakage by antioxidants and inhibitors of arachidonic acid metabolism

To gain insight into the mechanism of formation of chromosomal aberrations by the tumor promoter phorbolmyristate acetate (PMA) in human lymphocytes, we investigated the effect of antioxidants and inhibitors of arachidonic acid metabolism. Among the antioxidants bovine erythrocyte CuZn superoxide dismutase, glutathione peroxidase, mannitol (a scavenger of hydroxyl radicals), butylated hydroxytoluene and butylated hydroxyanisole were anticlastogenic while catalase and dimethylfuran (a scavenger of singlet oxygen) were inactive. These results show that the induction of aberrations by PMA occurs via indirect action, i.e. the intermediacy of superoxide and hydroxyl radicals. The following inhibitors of arachidonic acid metabolism were strongly anticlastogenic: the cyclo-oxygenase inhibitors indomethacin and flufenamic acid and the lipoxygenase inhibitor BN1015. Imidazole, nordihydroguaiaretic acid BN 1048 and 5,8,11,14-eicosatetraynoic acid were moderately active. The inhibitor of phospholipase A₂, fluocinolone acetonide, was also anticlastogenic.

We conclude that the oxidative metabolism of arachidonic acid is involved in the induction of chromosomal aberrations by PMA in human lymphocytes. However, because of the limited selectivity of these drugs, it is not yet possible to identify unambiguously the step(s) in the arachidonic acid cascade responsible for PMA clastogenicity.     

Interleukin 1 regulation of interleukin 2 and interferon-gamma gene activation in a human leukemic HSB.2 subclone

The role of interleukin 1 (IL1) in causing IL2 and interferon-gamma (IFN-gamma) production and their associated gene activation has been studied in a human leukemic HSB.2 subclone. One of the subclones, HSB.2-C5B2 clone #28, which produced only low levels of IL2 and IFN-gamma when stimulated with phytohemagglutinin (PHA) or with a combination of phorbol-myristate-acetate (PMA) and Ca2+ ionophore, ionomycin, showed marked IL2 and IFN-gamma production in the presence of IL1. The augmentation by IL1 was observed in both dot and Northern blot analysis, indicating that IL1 regulates lymphokine genes at the pretranslational level. The kinetics of IL2 and IFN-gamma production were essentially similar for both lymphokines except that there was a faster accumulation of IFN-gamma mRNA than IL2 mRNA. In contrast to the IL2 and IFN-gamma gene activation, IL2 receptor (Tac/p55 antigen) expression was induced directly by IL1 itself as with PMA in this subclone. In these studies, IL1 action was not replaced by the drugs raising intracellular cyclic AMP (cAMP). Taken collectively, these experiments support the notion that IL1 does not trigger IL2 gene activation per se, but amplifies the preactivated lymphokine genes initiated by PMA and ionomycin, whereas IL1 can activate the IL2 receptor gene without any other known signal requirements.     

Effect of prolactin on the secretion of milk casein: metabolism of arachidonic acid

Mammary gland fragments were incubated in the presence of prolactin and arachidonic acid which stimulate casein secretion. The effects of these stimuli in the presence of agents that influence arachidonic acid metabolism were investigated. Chloroquine, a blocker of phospholipase A2 activity, decreased prolactin but not arachidonic acid stimulation of casein secretion. Phospholipase A2 markedly stimulated casein secretion. Nordihydroguaiaretic acid (NDGA), an antioxidant that inhibits lipoxygenase, blocked the stimulating effect of prolactin and arachidonic acid. Ultrastructural studies indicated that phospholipase A2-induced stimulation of secretion was comparable to that of prolactin but that arachidonic acid-induced stimulation did not involve the same Golgi membrane modifications. These studies suggest that prolactin and phospholipase A2 stimulate secretion by a common way, and that arachidonic acid interferes with secretion by metabolic products of the lipoxygenase pathway.     

One-signal requirement for interferon-gamma production by human large granular lymphocytes

This laboratory has been investigating IFN-gamma gene expression by highly purified human large granular lymphocytes (LGL) and T cells. We report here that within 1 hr after interleukin 2 (IL 2) treatment of freshly isolated human LGL, IFN-gamma mRNA can be detected, with IFN-gamma protein in the culture medium within 4 to 6 hr of treatment. CD3- Leu-11+ LGL require only a single signal for IFN-gamma production because phytohemagglutinin (PHA), phorbol myristate acetate (PMA), IL 2, or ionomycin can each independently induce IFN-gamma production. In addition, PHA and ionomycin (but not IL 2) show significant synergy with PMA as a stimulus to LGL. In contrast, CD3+ T cells require two stimuli for high levels of IFN-gamma production, and not only are PMA plus ionomycin or PHA synergistic, but in addition, IL 2 and PHA demonstrate some synergy. Furthermore, we have found by fractionation of peripheral blood lymphocytes that IL 2-induced IFN-gamma production is associated with the LGL population and not T cells. These results indicate that with certain stimuli, LGL may be the predominant source of IFN-gamma from peripheral blood lymphocytes.     

Studies on the mechanism by which melittin stimulates insulin secretion from isolated rat islets of Langerhans

Incubation of isolated rat islets of Langerhans with melittin resulted in a dose-dependent stimulation of insulin secretion with half the maximal response occurring at 4 micrograms/ml melittin. The effect of melittin on insulin secretion was dependent on extracellular calcium, was inhibited by the phospholipase A2 inhibitor quinacrine and by the lipoxygenase inhibitor nordihydroguaiaretic acid. Stimulation of insulin secretion by melittin was associated with a calcium-dependent loss of [3H]arachidonic acid from phospholipids in islet cells prelabelled with [3H]arachidonic acid. Analysis of the islet phospholipids involved in this response revealed that the [3H]arachidonic acid was released predominantly from phosphatidylcholine. These results suggest that melittin may stimulate insulin secretion by activating phospholipase A2 in islet cells, causing the release of arachidonic acid from membrane phospholipid. The results are consistent with suggestions that the subsequent metabolism of arachidonic acid via the lipoxygenase pathway may be involved in regulating the insulin secretory response.     

Regulation of B cell tolerance by macrophage-derived mediators: antagonistic effects of prostaglandin E2 and interleukin 1

A role for prostaglandins in the mechanism of B cell tolerance induction in normal adult mouse spleen cells was examined. Two inhibitors of the cyclooxygenase pathway of arachidonic acid metabolism, indomethacin and acetylsalicylic acid, abrogated hapten-specific B cell tolerance induction by trinitrophenyl-human gamma-globulin. Tolerance was fully restored by the addition of prostaglandin E2 (PGE2) at a concentration of greater than or equal to 6 nM. T cell-depleted spleen cells produced comparable amounts of PGE2 in culture, indicating that the tolerance promoting activity of PGE2 occurred with physiologically relevant concentrations. Depletion and reconstitution experiments indicated that macrophages in the spleen cell preparations completely accounted for both PGE2 production and the effects of indomethacin and acetylsalicylic acid on B cell tolerance induction. The macrophage product interleukin 1 (IL 1) was also found to alter B cell susceptibility to tolerance induction. Thus, human IL 1 containing monocyte supernatants and purified IL 1 were found to interfere with B cell tolerance induction when added to macrophage- and T cell-depleted splenic B cells. Tolerance was restored in such cultures by the addition of 10 nM PGE2. These experiments demonstrate that within mixed lymphoid populations macrophages through the release of mediators modulate B cell susceptibility to tolerance induction.     

Effects of immune complexes on production by human monocytes of interleukin 1 or an interleukin 1 inhibitor

The objective of these studies was to examine the ability of phorbol myristic acetate (PMA), Fc fragments, and various forms of immune complexes to induce the production by human monocytes of factors stimulatory to chondrocytes or thymocytes. All of these materials were prepared free of detectable contamination with bacterial lipopolysaccharides (LPS) at the level of less than 0.1 ng/ml. Supernatants and lysates from stimulated human monocytes were assayed for their ability to induce collagenase production in cultured rabbit articular chondrocytes or to augment mitogen-induced proliferation of murine thymocytes. The activity detected by these assays exhibited an m.w. of approximately 15,000, and electrophoretic heterogeneity in the pH ranges of 5 to 5.5 and 6.5 to 7.0, characteristics of human interleukin 1 (IL 1) or IL 1-like factors. Monocytes cultured with 2 ng/ml LPS produced chondrocyte and thymocyte stimulatory factors. PMA, Fc fragments, and soluble, precipitated, particulate, or adherent immune complexes were inactive in stimulating the monocytes. However, complement fixation by precipitated immune complexes did generate activity capable of inducing monocytes to synthesize and secrete chondrocyte and thymocyte stimulatory factors. Adherent immune complexes and PMA were biologically active, as evidenced by induction of superoxide generation in the human monocytes. Supernatants from monocytes cultured on adherent immune complexes contained a factor inhibitory to chondrocyte and thymocyte responsiveness. This factor had a m.w. approximately 22,000 and appeared to inhibit specifically IL 1 stimulation, not interleukin 2 stimulation or cell proliferation. It was concluded that PMA, Fc fragments, and various forms of immune complexes in the absence of complement do not induce IL 1 production in human monocytes. However, complement fixation by immune complexes does lead to activation of monocytes to produce IL 1. Monocytes cultured on adherent immune complexes produce an IL 1 inhibitor.     

Regulation of IFN-gamma induction in human peripheral blood cells by exogenous and endogenously produced interleukin 2

Interferon (IFN)-gamma production, stimulated by the addition of exogenous interleukin (IL) 2, T cell mitogens, or tuberculin purified protein derivative (PPD) was studied in cultures of separated human mononuclear cells or unseparated peripheral blood leukocytes (PBL). IFN-gamma was induced by the addition of IL 2 to cultures of otherwise unstimulated cells. The minimal concentration of exogenous IL 2 required to cause a reproducible stimulation of IFN-gamma was about 10 U/ml, i.e., approximately 50 times the minimal concentration required to stimulate proliferation in an IL 2-dependent murine cytotoxic T cell line. Approximately 500 to 1000 IL 2 U/ml were required to produce maximal stimulation of IFN-gamma production in otherwise unstimulated cultures. Monoclonal antibody anti-Tac, specific for an epitope associated with the IL 2 receptor (IL 2 R), inhibited IFN-gamma induction by exogenous IL 2 less strongly than induction by phytohemagglutinin (PHA) or concanavalin A (Con A). The highest degree of inhibition was exerted by anti-Tac on IFN-gamma production stimulated with PPD. Stimulation of IFN-gamma induction by exogenous IL 2 and the inhibitory action of anti-Tac on IFN-gamma production were also seen in cultures of irradiated (2000 R) cells. Treatment of cells with subinducing doses of Con A or phorbol myristate acetate increased IFN-gamma induction by exogenous IL 2. Taken together, the data suggest that endogenously generated IL 2 is a major mediator of IFN-gamma induction in PBL cultures stimulated with antigens or T cell mitogens.     

Basic fibroblast growth factor and interleukins 4 and 6 stimulate the release of IFN-gamma by individual NK cells.

Both the secretory and cytotoxic activity of natural killer (NK) cells are known to be regulated by such cytokines as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In the present study we have used the reverse hemolytic plaque assay to investigate either the direct effects of the protein kinase activator, phorbol myristate acetate (PMA), or exposure to recombinant human interleukins 2, 4, and 6 (IL-2, IL-4, and IL-6) tumour necrosis factor alpha (TNF-alpha) and basic fibroblast growth factor (bFGF) on the release of IFN-gamma by individual, immunoidentified NK cells isolated from peripheral blood. This sensitive immunoassay was adapted and coupled with immunocytochemistry not only to immunophenotype and enumerate cells secreting IFN-gamma in a given cell population, but also to quantify the amount of this cytokine released per individual cell. These studies have confirmed mononuclear cells with the morphology of large granular lymphocytes and the immunophenotype of CD3-/CD16+ NK cells to be the predominant source of spontaneously released IFN-gamma in vitro. In contrast to this, fewer than 2% of the CD3+ T cells secreted detectable levels of this cytokine during the assay, irrespective of the stimulus applied. Whilst TNF-alpha had no significant effect on IFN-gamma release by NK cells, a 6-hr exposure to IL-2 or PMA stimulated an increase in the amount secreted per single cell. Furthermore, bFGF and interleukins 4 and 6 elicited a marked, dose-dependent stimulation of IFN-gamma secretion by this cell type. However, exposure to these cytokines did not alter the number of cells capable of releasing detectable levels of IFN-gamma during the assay. These studies demonstrate that (i) both the spontaneous and stimulated release of IFN-gamma by NK cells can be visualized and quantified at the single-cell level using this sensitive immunoassay, and (ii) bFGF and interleukins 2, 4, and 6, but not TNF-alpha, are potent stimulants of IFN-gamma secretion by CD3-/CD16+ NK cells.     

T cell receptor triggering induces responsiveness to interleukin 1 and interleukin 2 but does not lead to T cell proliferation

The antigen-like activity of monoclonal antibodies directed at the T3-Ti antigen receptor complex of human T lymphocytes was employed to study activation requirements of resting T cells. Efficient antigen recognition (signal 1) by T lymphocytes requires multimeric antigen receptor triggering because under appropriate experimental conditions soluble ligands do not produce this initial signal for T cell activation. The latter leads to receptiveness for both interleukin 1 (IL 1) and interleukin 2 (IL 2). Importantly, induction of proliferation requires an additional signal (signal 2), namely IL 1, which appears to be required to enable optimal secretion of IL 2. In contrast, presensitized T lymphocytes do not require IL 1 for IL 2 production. In this case, antigen receptor oligomerization is in itself sufficient to induce IL 2 receptor expression, and IL 2 secretion as well.     

Interferons as macrophage-activating factors. III. Preferential effects of interferon-gamma on the interleukin 1 secretory potential of fresh or aged human monocytes

Human peripheral blood adherent leukocytes incubated with interferon (IFN) of three different species (alpha, beta, or gamma) show an enhanced potential of IL 1 synthesis and secretion that can be revealed by a second signal provided by endotoxins or Poly IC. We have shown that recombinant IFN-gamma, compared with recombinant IFN-alpha or purified IFN-beta, has preferential effects on IL 1 secretion in fresh monocyte cultures. We have observed a progressive and profound loss of the ability of adherent cell cultures to secrete IL 1 upon aging for 4 to 12 days in vitro. IFN-gamma was found to be more efficient than IFN-alpha or -beta at maintaining (when added at the onset of the cultures) or reversing the loss (when added on the fourth day of culture) of the IL 1 secretory function. These observations suggest that the secretion of IFN-gamma during the course of immune responses may have a critical role in feeding back the cascade of interleukins in a loop of amplification, and may thereby regulate macrophage-T lymphocyte interactions.     

The role of interleukin 1 and interleukin 2 in human T colony formation

We investigated the roles of interleukin 1 (IL1) and interleukin 2 (IL2) on T colony formation by PHA-stimulated peripheral blood lymphocytes (PBL). Purified T cells stimulated by PHA could not generate T colonies as did PBL. Media conditioned by PHA-stimulated PBL (PHA-LCM) contained IL2 and a T colony-promoting activity (TCPA) which induced T colony formation in PHA-stimulated purified T cells. IL2 and TCPA are coeluted in the same peak of 18,000 molecular weight after gel filtration chromatography. Moreover, TCPA present in the PHA-LCM could be absorbed on IL2-sensitive cells which possessed specific receptors for IL2. These results suggest that TCPA and IL2 are related entities. Monocytes or IL1 (a monokine released by activated monocytes) also induced T colony formation in purified T cells. Phorbol myristate acetate (PMA) could replace monocytes in the induction of T colony. Monocytes, IL1, or PMA are known to be crucial requirements for IL2 production by PHA-stimulated T cells. This combined with the fact that IL2 participates in T colony formation suggests that monocytes induce T colony formation through IL2 production.     

Parallel inhibition of neutrophil arachidonic acid metabolism and lysosomal enzyme secretion by nordihydroguaiaretic acid

Arachidonic acid at concentrations from 0.2 to 2.0 × 10?⁶M induces the secretion of lysosomal enzymes from cytochalasin B-treated rabbit neutrophils. These concentrations of arachidonic acid are metabolized primarily to hydroxyeicosatetraenic acids rather than to cyclooxygenase products. A good correlation is observed between the extent of arachidonic acid metabolism and the secretion of lysosomal enzymes. Nordihydroguaiaretic acid (1?10μM) inhibits both lysosomal enzyme secretion and the production of lipoxygenase products by neutrophils.     

Immunologic and molecular characterizations of T cell-derived T cell activating factor

Culture supernatants from several subclones of a human T hybrid line (24A) stimulated with PMA showed co-stimulatory activity in the proliferation of Con A-stimulated murine thymocytes, but did not show any IL 2 activity. Some subclones did not show co-stimulatory activity even when stimulated with PMA, excluding the possibility of a carry-over effect. The factor found in the culture supernatants increased IL 2 production in normal T cells stimulated with a suboptimal concentration of PHA. The factor also induced IL 2 production in a T hybrid clone, T-394.1, when the latter was stimulated with a suboptimal concentration of mitogens, indicating a direct effect by this T cell-derived factor on mitogen-stimulated T cells inducing IL 2 production. This factor also induced the generation of other lymphokines such as BCDF and IFN-gamma. Northern blot analysis showed that the factor induced an increase in mRNA for IL 2 as well as IL 2 receptor. These results indicated that T cells could secrete a factor with IL 1-like activity. However, Northern blot analysis showed that mRNA from a T hybrid clone does not cross-react with cDNA for IL 1 (beta) derived from human monocytes.