Regulation of the production of immune interferon and cytotoxic T lymphocytes by interleukin 2

The activation of alloantigen-specific cytotoxic T lymphocyte precursors is dependent upon the presence of both macrophages and helper T cells or regulatory molecules derived from these facilitative cells. Three biochemically distinct helper factors have been identified: interleukin 1 (macrophage-derived), Interleukin 2 (T cell derived), and immune interferon. All 3 factors are found in supernatants of mixed lymphocyte cultures (MLC), however, the removal of macrophages from these cultures completely ablates the production of these factors as well as the induction of cytotoxic T lymphocytes (CTL). The addition of IL 2 to these macrophage-depleted MLC restores the ability of responder T cells to: 1) bypass the requirement for macrophage soluble function, 2) produce immune interferon, and 3) generate CTL. The kinetics and dose response of immune interferon production in response to IL 2 correlates with the generation of CTL. The production of immune interferon as well as the generation of CTL requires T cells, alloantigen, and IL2. Furthermore, the induction of CTL by IL2 was neutralized by the addition of anti-immune interferon. These data suggest that: 1) the regulation of immune interferon production is based on a T to T cell interaction mediated by IL 2, and 2) immune interferon production may be required for IL 2 induction of CTL. These findings are consistent with the hypothesis that the induction of CTL involves a linear cell-factor interaction in which IL 1 (macrophage-derived) stimulates T cells to produce IL 2, which in turn stimulates other T cells to produce immune interferon and become cytotoxic.     

Interferon-gamma reduces macrophage-suppressive activity by inhibiting prostaglandin E2 release and inducing interleukin 1 production

Normal peritoneal M phi of C3H/HeN mice were able to suppress lymphocyte proliferation in a dose-dependent fashion when added to Con A-pulsed spleen cell cultures. However, M phi-suppressive activity could be partially or completely reduced by in vitro pre-exposure to nonimmune IFN-alpha or immune recombinant IFN-gamma. For both IFN-alpha and IFN-gamma, reduction of M phi suppression was marginal at 10(1) U/ml and became highly significant at 10(2) to 10(3)/ml. The ability of IFN-alpha and IFN-gamma to modulate M phi suppression appears to be related to distinct mechanisms. In fact, impairment of M phi suppression by IFN-alpha occurred in parallel to the decrease of M phi capacity to produce PGE2 and the oxygen intermediate O2-, two molecules responsible for M phi-suppressive activity. In contrast, M phi exposed to IFN-gamma showed only impairment of PGE2 production, whereas O2- release was not significantly affected. Furthermore, at variance with IFN-alpha, IFN-gamma directly stimulated M phi to synthesize and release IL 1, a monokine known to promote lymphocyte proliferation.     

Regulation of immune responses by prostaglandin E2

PGE(2), an essential homeostatic factor, is also a key mediator of immunopathology in chronic infections and cancer. The impact of PGE(2) reflects the balance between its cyclooxygenase 2-regulated synthesis and 15-hydroxyprostaglandin dehydrogenase-driven degradation and the pattern of expression of PGE(2) receptors. PGE(2) enhances its own production but suppresses acute inflammatory mediators, resulting in its predominance at late/chronic stages of immunity. PGE(2) supports activation of dendritic cells but suppresses their ability to attract naive, memory, and effector T cells. PGE(2) selectively suppresses effector functions of macrophages and neutrophils and the Th1-, CTL-, and NK cell-mediated type 1 immunity, but it promotes Th2, Th17, and regulatory T cell responses. PGE(2) modulates chemokine production, inhibiting the attraction of proinflammatory cells while enhancing local accumulation of regulatory T cells cells and myeloid-derived suppressor cells. Targeting the production, degradation, and responsiveness to PGE(2) provides tools to modulate the patterns of immunity in a wide range of diseases, from autoimmunity to cancer.     

The induction of nonspecific T suppressor lymphocytes by prostaglandin E1

Suppressor cell activity is induced by the addition of prostaglandin E₁ to normal mouse spleen cells (NSC). When this population is added to fresh NSC, it suppresses the primary in vitro antibody response to DNP-LPS. This suppressor cell induction requires a brain-associated thy-1 antigen-bearing cell. No role for a nylon-wool-adherent cell could be demonstrated. Suppression occurs in the absence of T cells in the responding population. The addition of indomethacin to prevent endogenous synthesis of prostaglandins suggests that prostaglandininduced suppressor activity does not require further prostaglandin synthesis in the suppressor population but does require such synthesis in the responding population.     

Reduced prostaglandin E2 (PGE2) receptors on atopic T lymphocytes

We have recently demonstrated that atopic T lymphocytes have decreased sensitivity to prostaglandin E2 (PGE2). In order to determine whether this decreased sensitivity was reflected at the receptor level, we have employed a radioligand binding assay utilizing [3H]PGE2. We have demonstrated a single specific reversible binding site for [3H]PGE2 on normal T cells (N = 10) with a mean KD (+/-SD) of 32.2 (+/-25.0) nM, a binding capacity of 20.2 (+/-13.0) pM, and a mean of 1004 (+/-118) receptors per cell. Atopic T cells (N = 10) were also found to have a single specific binding site for [3H]PGE2 with a mean KD of 24.9 (+/-17.8) nM, a binding capacity of 7.1 (+/-10.1) pM, and a mean of 372 (+/-61) receptors per cell. These radioligand binding studies were correlated with functional studies in the same subjects. Phytohemagglutinin-stimulated protein synthesis ([3H]leucine uptake) was suppressed in a dose-dependent fashion by PGE2 (10(-6)-10(-12) M). The maximal effect of PGE2 on normal T cells was 10(-6) M PGE2 with an IC50 of 10(-12) M. Atopic T cells responded quantitatively less than normal T cells to PGE2. Further, the maximum suppression of protein synthesis by PGE2 occurred at 10(-6) M with an IC50 of 10(-10) to 10(-11) M. These studies suggest that part of the decreased sensitivity of atopic T cells to PGE2 may result from a reduction in PGE2 binding sites.     

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.     

Polypeptide growth factors augment interleukin 1-induced release of prostaglandin E2 by rheumatoid arthritis synovial cells in vitro

When stimulated with increasing amounts of interleukin 1 beta (IL 1 beta) rheumatoid arthritis (RA), as compared with osteoarthritis (OA), synovial cells grown in RPMI plus fetal bovine serum (FBS), released significantly more prostaglandin E2 (PGE2) (p less than 0.05; paired t test, two-tailed). PGE2 release by IL 1 beta-stimulated RA synovial cells grown for 14 days in serum-free RPMI was significantly less than that released by the same cells grown in medium plus 10% FBS (p less than 0.03; two-tailed). Since these data suggest that growth factors present in FBS may augment the effects of IL 1 beta, experiments were conducted to study the influence of four polypeptide growth factors--transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), on IL 1 beta-induced release of PGE2 by cultured RA synovial cells. Both EGF and bFGF significantly enhanced IL 1 beta-induced release of PGE2 (p less than 0.05; paired t test, one-tailed), while PDGF was synergistic with IL 1 beta, significantly increasing release of PGE2 by these cultured cells (p less than 0.02; two-tailed). No such effect was seen when TGF-beta was added to the culture medium. Taken together, these data lend support to the concept that within the synovial micro-environment small quantities of individual growth factors may potentiate the effects of IL 1 beta to amplify intra-articular inflammation.     

Regulation of NK cell activity by prostaglandin E2: the role of T cells

The influence of T cells on the production of prostaglandins (PGE2) and on PGE2-mediated regulation of natural killer (NK) activity was studied. Supernatants from peripheral blood mononuclear cells (PBMC) and from PBMC depleted of T cells ((PBMC)-T), both of which had been incubated in plastic petri dishes overnight, contained similar amounts of PGE2, as detected by radioimmunoassay and by their potential to inhibit NK activity of peripheral blood mononuclear cells in a 51Cr release assay with K 562 cells as the target population. However, the NK activity of PBMC was inhibited significantly more strongly (P less than 0.005) by PGE2-containing supernatants than was the NK activity of (PBMC)-T. In further assays, in which synthetic PGE2 in concentrations of 10(-4) and 10(-5)M was added, a significant inhibition of NK activity was observed in PBMC populations (P less than 0.05), but not in (PBMC)-T. Thus, T cells did not seem to be involved in the control of PGE2 production, but their presence was necessary for PGE2-mediated inhibition of NK activity.     

Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2

Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE(2) biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathione S-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE(2) generation. Arg(110), a residue well conserved in the microsomal GSH S-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A(2) allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE(2) biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer.     

Regulation of interleukin 2 synthesis by cAMP in human T cells

T cell activation requires two initial signals that first lead to the expression of interleukin 2 (IL 2) receptors and the initiation of IL 2 synthesis and then to T cell proliferation. Jurkat T lymphoma cells have been shown to be a good model for studying IL 2 synthesis because these cells also require two signals for activation. The first signal can be provided by the lectin phytohaemagglutinin (PHA), and the second one by the phorbol ester, 12-o-tetradecanoylphorbol 13-acetate (TPA). The regulation of IL 2 synthesis in Jurkat cells, however, is unclear, and the present study deals with the role of cAMP on IL 2 synthesis. In Jurkat cells, IL 2 synthesis appears to be highly regulated by the activity of adenylate cyclase. This was demonstrated by using different means to increase intracellular cAMP level, namely by using permeant cAMP analogs, using the activator of adenylate cyclase, forskolin, using the activator of the alpha subunit of the stimulatory GTP binding protein cholera toxin, and using inhibitors of phosphodiesterase. In addition, prostaglandins E1 and E2 were shown to bind specifically to Jurkat cells, to induce a rise in intracellular cAMP level, and to markedly decrease IL 2 synthesis. All together, these results suggest that in T lymphocytes, the prostaglandin E2 receptor is linked to adenylate cyclase through a GTP binding protein and regulates the production of IL 2 by controlling the intracellular cAMP level.     

Effects of vitamin E and prostaglandin E2 on expression of CREB1 and CREB2 proteins by human T lymphocytes

Both prostaglandins (PGs) and vitamin E are known to deeply affect immune responses. It is shown here that they both influence T cell-mediated immunity through reciprocal interference on the expression of cyclic-AMP responsive element binding (CREB) family proteins. CREB1 protein of human T lymphocytes was significantly modulated by a brief treatment of 5 to 10 min with PGE2. On the contrary, vitamin E appeared to be ineffective on the CREB1 behavior, while it abolished the PGE2-induced modulation of this protein. The CREB2 protein expression was also affected by PGE2 treatment, but a longer period of incubation (>20 min) was needed to observe these changes. Vitamin E showed a strong enhancing effect on CREB2 that was partially reversed by the subsequent treatment with PGE2. Our results support the idea that there is reciprocal interference between PGE2 and vitamin E on PGE2-induced signals in T lymphocytes. These data are in agreement with the reports concerning different cell systems and experimental conditions.     

Regulation of breathing movements in fetal sheep by prostaglandin E2

In fetal sheep, plasma prostaglandin (PG) E2 concentrations are high, and fetal breathing movements (FBM) occur intermittently, primarily during low-voltage fast electrocortical activity (LVFA). There is evidence suggesting that prostaglandins, specifically PGE2, may regulate FBM. To define the physiological role of PGE2 in regulation of FBM, we infused meclofenamate (0.9 mg X kg-1 X h-1), a prostaglandin synthetase inhibitor, into six fetal sheep to suppress endogenous prostaglandin production. Afterward, PGE2 was added in mean doses of 9, 18, 36, and 90 ng X kg-1 X min-1. Meclofenamate decreased PGE2 concentrations and increased FBM, especially during high-voltage slow electrocortical activity (HVSA). Addition of PGE2 reversed the effects of meclofenamate, increasing PGE2 concentrations and decreasing FBM, especially during HVSA. The response to PGE2 was dose dependent; the overall incidence of FBM and incidences of FBM during HVSA and LVFA were inversely correlated with both the infused PGE2 dose and the mean PGE2 concentration. At higher doses of PGE2, FBM occurred intermittently and only during LVFA; thus PGE2 infusion restored the physiological pattern of FBM. These results indicate that PGE2 regulates FBM by inhibiting FBM during HVSA.     

Activation of human monocytes by interleukin 2: Role of T lymphocytes

The effect of interleukin 2 (IL 2) on the capability of human monocytes to secrete reactive oxygen species triggered via Fc-γ receptor (Fc-γ R) function had been investigated by measurement of chemiluminescence (CL). IL 2 did not activate highly purified (hp) monocytes to respond to Fc-γ R mediated phagocytic stimulation with an enhanced respiratory burst activity unless low numbers of T cells had been co-cultured with hp monocytes. Supernatants from IL 2 treated PBMC contained interferon-γ (IFN-γ) and monocyte activating factor (MAF) activity. The secretion of both cytokine activities was strongly enhanced by cooperative function of monocytes. The correlation of IL 2 induced secretion of IFN-γ and MAF activity was striking, however, monoclonal antibody (mAb) anti-human IFN-γ failed to abrogate IL 2 stimulated and lymphocyte dependent monocyte activation. Although IL 2 had no direct monocyte activating effect, pretreatment of hp monocytes with IL 2 led to monocyte priming: subsequent co-culture with autologous control T cells enhanced the monocyte Fc-γ R mediated CL response. The priming of monocytes by IL 2 was dependent on the interaction of IL 2 with the monocytic IL 2 receptor as shown by inhibition experiments with anti IL 2 R monoclonal antibody. Thus the IL 2 driven monocyte/T-cell interaction leads to an increased Fc-γ R mediated monocytic respiratory burst activity and to the secretion of a soluble MAF activity, but there were no detectable amounts of IFN-γ.     

Regulation of macrophage tumor necrosis factor production by prostaglandin E2

We have studied the role of prostaglandin E2 on the modulation of tumor necrosis factor by immunologically elicited and lipopolysaccharide treated murine macrophages. Indomethacin, a potent inhibitor of prostaglandin E2 production, caused a dose dependent augmentation of lipopolysaccharide induced tumor necrosis factor production (2-3 fold at 10(-7) molar). Tumor necrosis factor was released into the extracellular environment and no activity was found to be associated with membrane or cytosolic fractions. Prostaglandin E2 added to the lipopolysaccharide treated cultures suppressed tumor necrosis factor in a dose dependent manner. In these studies, 10(-7) molar PGE2 reduced tumor necrosis factor production to basal levels. These data suggest that PGE2 may be a potent autoregulatory factor that dramatically influences tumor necrosis factor production.     

Up-regulation of prostaglandin E2 binding and of prostanoid release in rabbits producing antibodies against prostaglandin E2

German Giant rabbits successfully immunized against prostaglandin (PG) E2 as shown by a rise in antibody titers developed gastric mucosal lesions. Enzymatically dispersed gastric mucosal cells of these animals had a significantly enhanced production of PG E2 and PG I2 as measured by specific radioimmunoassays. This may be explained by an increased supply with endogenous arachidonic acid (as indicated by an enhanced phospholipase A2/LAT ratio) and by a higher activity of the subsequent PG forming enzymes (as indicated by a more effective stimulation of PG production by exogenous arachidonic acid). Gastric mucosal plasma membranes of immunized rabbits had significantly higher PG E2 binding capacity (108 +/- 9 fmol/mg protein) than those of nonimmunized rabbits (72 +/- 5 fmol/mg protein). The ligand affinity was not affected by immunization. Neither histamine-stimulated 14C-amino-pyrine uptake of isolated parietal cells as a marker for acid production nor its inhibition by PG E2 were influenced by receptor up-regulation. The increased eicosanoid release can be regarded as an endogenous defense mechanism against increased mucosal vulnerability caused by PG E2 scavenging. The potential role of PG E2 receptor up-regulation in support of this process remains to be established.     

Calcitonin gene-related peptide inhibits interleukin 2 production by murine T lymphocytes.

Evidence from the literature suggests that the nervous and the immune systems closely interact via neuromediators, which affect the immune system, and cytokines, which control nerve cell growth and activity. Calcitonin gene-related peptide (CGRP) is a neuropeptide that has been identified in numerous tissues including immune organs and inhibits the proliferation of spleen cells. We investigated whether CGRP altered the function of T lymphocytes. We present evidence that CGRP induces a dose-dependent cAMP accumulation in interleukin 2-producing TH1 cells and inhibits their production of interleukin 2. These effects are prevented by CGRP8-37, a CGRP antagonist that is missing the first 7 amino acids. This CGRP-mediated inhibition of interleukin 2 production is accompanied by a decrease in interleukin 2 mRNA accumulation. CGRP also inhibits the accumulation of mRNA coding for tumor necrosis factor-alpha and -beta and interferon-gamma. Thus, we have identified one mechanism by which CGRP inhibits the proliferation of spleen cells.     

Impairment of blood-cerebrospinal fluid barrier properties by retrovirus-activated T lymphocytes: reduction in cerebrospinal fluid-to-blood efflux of prostaglandin E2

The choroid plexus epithelium forms the interface between the blood and the CSF. In conjunction with the tight junctions restricting the paracellular pathway, polarized specific transport systems in the choroidal epithelium allow a fine regulation of CSF-borne biologically active mediators. The highly vascularized stroma delimited by the choroidal epithelium can be a reservoir for retrovirus-infected or activated immune cells. In this work, new insight in the implication of the blood-CSF barrier in neuroinfectious and inflammatory diseases is provided by using a differentiated cellular model of the choroidal epithelium, exposed to infected T lymphocytes. We demonstrate that T cells activated by a retroviral infection, but not non-infected cells, reduce the transporter-mediated CSF-to-blood efflux of organic anions, in particular that of the potent pro-inflammatory prostaglandin PGE2, via the release of soluble factors. A moderate alteration of the paracellular permeability also occurs. We identified the viral protein Tax, oxygenated free radicals, matrix-metalloproteinases and pro-inflammatory cytokines as active molecules released during the exposure of the epithelium to infected T cells. Among them, tumour necrosis factor and interleukin 1 are directly involved in the mechanism underlying the decrease in some choroidal organic anion efflux. Given the strong involvement of CSF-borne PGE2 in sickness behaviour syndrome, these data suggest that the blood-CSF barrier plays an important role in the pathophysiology of neuroinflammation and neuroinfection, via changes in the transport processes controlling the CSF biodisposition of PGE2.     

Idiotype-specific T lymphocytes. I. Regulation of antibody production by idiotype-specific H-2-restricted T lymphocytes

Cytotoxic T lymphocytes (CTL) specific for MOPC-104E myeloma cells of BALB/c origin could be induced in BALB/c, (BALB/c X BALB.B)F1, and (BALB/c X BALB.K)F1 mice. (BALB/c X BALB.B)F1 CTL activity specific for MOPC-104E was effectively inhibited by anti-H-2d but not by anti-H-2b alloantiserum. However, the activity was hardly blocked by specific anti-idiotypic antibodies to MOPC-104E. For further analysis of the recognition of idiotype on target cells by CTL, the effect of those lymphocytes on anti-dextran B1355S antibody-producing B lymphocytes, which have a cross-reactive idiotype to MOPC-104E, was investigated. Lymphocytes from the CTL population did inhibit antibody production by dextran-immune spleen cells, but those from the CTL population specific for irrelevant myeloma cells (MOPC-167) did not. The (BALB/c X BALB.K)F1 CTL population suppressed the antibody production of BALB/c but not of BALB.K. This indicates that F1 cells can preferentially see H-2 antigens of immunizing myeloma cells on target B lymphocytes. The inhibition of antibody production was antigen specific and was only restricted to the PFC that were inhibitable by anti-idiotypic antibodies. The surface phenotypes of the cells that inhibited the antibody production were Thy-1+, Lyt-1-, Lyt-2+, and I-J-. These results strongly suggest that CTL specific for MOPC-104E recognize self H-2 antigens simultaneously with idiotypic determinants on B lymphocytes. Possible immunoregulatory roles of idiotype-specific CTL on antibody production systems are also suggested.     

The activation of human fibroblast prostaglandin E production by interleukin 1

We have examined the induction of prostaglandin E2 (PGE2) release from fibroblasts by human interleukin 1 (IL-1). A number of fibroblast cell lines appear to respond to IL-1 in a fashion similar to that seen with synovial fibroblast cultures. Using the Gin-1 primary fibroblast cell line, the earliest time where a significant increase in PGE2 release can be detected is 2 hr. Thereafter PGE2 appears to increase dramatically, with levels after 5 hr increased over 50-fold above baseline. IL-1 appears to directly induce the increase in PGE2 since removal of other proteins from culture medium does not affect induction. PGE2 induction by IL-1 also does not require cell proliferation. The induction appears to involve the synthesis of new protein since the enhanced release can be completely blocked by addition of actinomycin D or cycloheximide. Arachidonic acid mobilization in cells does not appear to be altered following IL-1 addition. However, the ability to convert arachidonic acid to PGE2 is increased following 5 hr of culture with IL-1. While increasing the release of PGE2, the addition of phorbol esters, alone or in combination with calcium ionophores, does not mimic the protein synthesis-dependent increase seen with IL-1. Taken together these results suggest that IL-1 induction of fibroblast PGE2 involves the synthesis of new protein or proteins involved in the conversion of free arachidonic acid to PGE2.